TRANSFERRIN BINDING PROTEINS OF PASTEURELLA HAEMOLYTICA AND VACC INES CONTAINING SAME

Abstract: Transferrin binding proteins from Pasteurella haemolytica and nucleic acid molecules encoding the proteins are disclosed. Antibodies against the proteins are disclosed. The invention also relates to vaccines containing the proteins of the invention. The invention also provides methods for identifying substances which affect the binding of transferrin to the proteins and methods for screening for agonists or antagonists of the binding of the proteins and transferrin.

Description: Title: Transferrin Binding Proteins of Pastenrella haemolyttca and Vaccines Containing Same FIELD OF THE INVENTION 5 The invention relates to novel transferrin binding proteins of Pasteurella haemolytica, truncations, analogs, homologs and isoforms thereof, nucleic acid molecules encoding the proteins and truncations, analogs, and homologs of the proteins, vaccines containing the proteins, antibodies against the proteins, and, uses of the protems and nucleic acid molecules 10 BACKGROUND OF THE INVENTION Members of the genus Pasteurella comprise a group of related bacterial species that are important pathogens of ruminants This group includes the species Pasteurella haemolytica which has been classified into two biotypes, A and T, on the basis of sugar utdization, and into 16 serotypes which are recognized on the basis of their somatic antigens (Biberstern, E L et al , 15 1960, Fraser et al , 1982) The T-type strams of P haemolytica, characterized by utilization of trehalose, have been recently reclassified as a new species P trehalosi (Sneath, P H.A et al , 1990) Pneumonic pasteurellosis caused by Pasteurella haemolytica is a major economic problem to the cattle, sheep and goat industries world-wide Shipping fever, a variation of this 20 disease, is a major problem in the cattle industry m North America and is almost exclusively caused by type Al strams of this species (Babiuk, L A and S D Acres, 1984) Serotype A2 is the most prevalent disease-causing type m sheep but other serotypes may be important in sheep and goats (Gilmour and Gilmour, 1991) The related species, Pasteurella trehalosi (formerly know as T-type P haemolytica) is the causitive agent of sephcemia in lambs, a problem plaguing the 5 sheep industry particularly in the United Kingdom Similarly, strains of the related species Pasteurella multocida, are responsible for haemorrhagic sephcemia, a serious infection in cattle and water buffalo, which is particularly serious m South East Asia Vaccination is a desired method of control for pasteurellosis in ruminants but success has been l i mited by the lack of immunizing preparations that induce protection against all 0 d i sease-causmg serotypes, particularly if a vaccme effective for all ruminants is considered K i lled whole cell vaccines elicited inconsistent levels of protection and antibody response i n calves (W i lk i e, B N , 1980) Homologous vaccines containing sodium sa cylate extracts (SSEs) protected sheep against diseases due to serotypes Al, A6 and A9 (Gilmour et al , 1983) but not aga i nst the more epidemic serotype A2 (Fraser et al , 1982) An exotoxin produced by P 5 haemolyt i ca which is specifically lethal to leucocytes and alveolar macrophages from rum i nants (Benson et al , 1978) has shown a lot of promise as a vaccme candidate m protect i on exper i ments i n calves and sheep (13,35) but there is limited protection against heterologous serotypes (33) The inclusion of proteins induced under iron-limited growth conditions mto a vaccine for pasteurellosis in lambs has been implicated in enhanced protection (15) Previous studies have established that the ability of pathogenic bacteria to acquire iron in vivo is a critical factor in their pathobiology (7,11) One mechanism of iron retrieval from the host iron-binding glycoprotein, transferrin, involves direct binding of transferrin by surface receptors on the bacteria and the removal of iron from transferrin and uptake to the cell (21) Schryvers (1992) describes the isolation of transferrin receptor proteins from various bacterial pathogens usmg affinity chromatography The transferrin receptor has been shown to consist of two proteins, called transferπn binding protein 1 or A (Tbpl or TbpA) and transferrin bmding protein 2 or B (Tbp2 or TbpB) The receptor-mediated type of iron uptake has been demonstrated to operate in serotype A bovme strains of P haemolytica (26) Cells of P haemolytica growing in in tro under iron-limi ted conditions express a number of iron-repressible outer membrane proteins (IROMPs) identical to those produced by cells recovered in vivo from mfected sites in animals with pasteurellosis (9J0) Especially prominent among these proteins were those of molecular sizes 100, 77, 70 and 60 Kda (9,10) The 100 Kda protein has been identified as one of the host specific transferrin receptors in bovine isolates (26) while some of the other IROMPs had been suggested as possibly associated with the 100 Kda protein in an iron acquisition receptor complex (26) The role of the IROMPs expressed by P haemolytica from lambs (10) in iron acquisition has not been elucidated, neither is it known if similar proteins are expressed by goat isolates P haemolytica acquires iron from bovme host transferrin by a receptor-mediated type of mechanism The proposal that bacteria with this type of iron acquisition mechanism may be solely dependent upon their surface receptor for iron acquisition in vivo (29) implies that they can only cause disease in those hosts whose transferrin is recognized by their surface receptors P haemolytica has been reported to cause disease in cattle, sheep and goats and accordingly their surface receptors would be expected to recognize these hosts' transferrms Therefore it is important to determine whether sheep and goat isolates also possessed transferrin receptors involved m iron acquisition, to evaluate their specificities for different ruminant transferrms and to determine if there is antigenic relatedness amongst the surface receptors from the different strams causing pneumonic pasteurellosis in cattle, sheep and goats SUMMARY OF THE INVENTION Transferrin receptors were identified in a collection of Pasteurella haemolytica (and P trehalosi ) strams of various serotypes and biotypes (A and T) from cattle, sheep and goats Growth studies, binding studies and affinity isolation experiments demonstrated that these receptors had identical specificities which recognized transferrms from cattle, sheep and goats This indicates that there are conserved regions on the receptor proteins, involved in ligand binding, which are accessible at the cell surface Anhsera prepared against the individual purified receptor proteins (TbpA and TbpB) from a serotype Al strain of P haemolytica demonstrated considerable crossreactivity against receptor proteins from a representative selection of strams The cross-reactivity was also observed against mtact cells indicating that there are conserved immunological epitopes at the 5 cell surface which could serve as targets for the host's immune effector mechanisms The present mventors have cloned, sequenced and expressed tbpA and tbpB genes encodmg the transferrin receptor proteins, TbpA and TbpB (also referred to herein as Tbpl and Tbp2, respectively), from Pasteurella haemolytica Al The genes were organized in an operon arrangement of tbpB- tbpA The tbpB gene was preceded by putative promoter and regulatory 10 sequences, and followed by a 96 base pair mtergenic sequence m which no promoter regions were found, suggestmg that the two genes are coordinately transcribed The deduced ammo acid sequences of the TbpA and TbpB proteins had regions of homology with the corresponding Neisseriα meningi ttd s, N gonorrhoeαe, Hαemophilus i nfluenzαe and Actinobαcillus pleuropneumomαe Lbp and Tbp proteins The mtact tbpB gene was expressed m a T7 expression 15 system and the resultmg recombmant TbpB protem retained the functional bovme transferrin binding characteristics The availability of the recombmant TbpB enabled the mventors to demonstrate its specificity for rummant transferrin, its ability to bind both the C-and N- terminal lobes of bovine transferrin, and its preference for the iron-loaded form of this protem The present mventors also significantly found that vaccmation with a formulation 20 containing P haemolytica TbpA and TbpB provided significant protection agamst experimental bovme pneumonic pasteurellosis Immunization with two doses of TbpB also provides protection Broadly stated, the present mvention provides a purified and isolated nucleic acid molecule compπsmg a sequence encodmg a TbpA protem, or a purified and isolated nucleic acid molecule comprising a sequence encodmg a TbpB protem The TbpA and TbpB proteins bmd 25 rum ant transferrms and function in receptor-mediated iron acquisition by P haemolytica in its rummant hosts The TbpA protem is approximately lOOkDa, and TbpB is approximately 60 kDa In an embodiment of the mvention, the purified and isolated nucleic acid molecules comprise a sequence encodmg a TbpA protem hav g the ammo acid sequence as shown m Figure 30 22 or SEQ ID NO 2, or a sequence encodmg a TbpB protem havmg the ammo acid sequence as shown in Figure 24 or SEQ ID NO 4 hi a preferred embodiment of the mvention, the purified and isolated nucleic acid molecules comprise a sequence encodmg a TbpA protein and having the nucleic acid sequence as shown in Figure 21 or SEQ ID NO 1, or a sequence encodmg a TbpB protem havmg the nucleic acid sequence as shown m Figure 23 or SEQ ID NO 3 35 The mvention also contemplates (a) nucleic acid molecules comprising a sequence encodmg a truncation of TbpA or TbpB which is unique to the protem, an analog or homolog of TbpA or TbpB or a truncation thereof, (herein collectively referred to as "TbpA related proteins" or "TbpB related proteins", respectively), (b) a nucleic acid molecule comprising a sequence which hybridizes under high stringency conditions to the full length nucleic acid encodmg TbpA or TbpB ,havιng the ammo acid sequences as shown in Figures 22 and 24 respectively, or to a TbpA or TbpB related protein, (c) a nucleic acid molecule comprising a sequence which hybridizes under high strmgency conditions to the full length nucleic acid sequence of the tbpA or tbpB genes havmg the sequences as shown m Figures 21 or SEQ ID NO 1, or Figure 23 or SEQ ID NO 3, respectively The invention further contemplates a purified and isolated double stranded nucleic acid molecule contammg a nucleic acid molecule of the invention, hydrogen bonded to a complementary nucleic acid base sequence The nucleic acid molecules of the invention may be mserted into an appropriate expression vector, 1 e a vector which contains the necessary elements for the transcription and translation of the mserted codmg sequence Accordingly, recombmant expression vectors adapted for transformation of a host cell may be constructed which comprise a nucleic acid molecule of the mvention and one or more transcription and translation elements operatively lmked to the nucleic acid molecule The recombmant expression vector can be used to prepare transformed host cells expressing TbpA and/or TbpB, or a TbpA or a TbpB related protem Therefore, the invention further provides host cells contammg a recombmant molecule of the mvention The mvention further provides a method for preparing a novel TbpA or TbpB, and TbpA or TbpB related protems, utilizmg the purified and isolated nucleic acid molecules of the mvention In an embodiment a method for preparing TbpA or TbpB is provided comprising (a] transferrmg a recombmant expression vector of the invention into a host cell, (b) selectmg transformed host cells from untransformed host cells, (c) culturmg a selected transformed host cell under conditions which allow expression of TbpA or TbpB, and (d) isolating the recombmant TbpA or TbpB The mvention further broadly contemplates a purified and isolated TbpA or TbpB which binds to ruminant transferrin, preferably obtained by culturmg a host cell contammg a recombmant expression vector of the mvention In an embodiment of the invention, a purified TbpA or TbpB is provided which has the ammo acid sequence as shown m Figure 22 or Figure 24 respectively The mvention also mcludes truncations of the protem and analogs, homologs, anc isoforms of the protem and truncations thereof (I e , "TbpA or TbpB related protems") The TbpA and TbpB, or TbpA and TbpB related protems of the mvention may be conjugated with other molecules, such as protems, to prepare fusion protems This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion protems The invention further contemplates antibodies havmg specificity agamst an epitope of TbpA or TbpB, or TbpA or TbpB related protems of the mvention Antibodies may be labelled with a detectable substance and they may be used to detect the TbpA or TbpB, or TbpA or TbpB related protems of the mvention m samples The invention also permits the construction of nucleotide probes which are unique to the nucleic acid molecules of the mvention and accordmgly to TbpA or TbpB, or TbpA or TbpB related protems of the mvention. Thus, the invention also relates to a probe comprismg a sequence encodmg TbpA or TbpB, or TbpA or TbpB related protems The probe may be labelled, for example, with a detectable substance and it may be used to select from a mixture of nucleotide sequences a nucleotide sequence coding for a protem which displays one or more of the properties of TbpA or TbpB The invention still further provides a method for identifying a substance which is capable of binding to TbpA or TbpB, or TbpA or TbpB related protems, or an activated form thereof, comprismg reactmg TbpA or TbpB, or TbpA or TbpB related protems, or an activated form thereof, with at least one substance which potentially can bmd with TbpA or TbpB, or TbpA or TbpB related protems, or an activated form thereof, under conditions which permit the formation of complexes between the substance and TbpA or TbpB, or TbpA or TbpB related proteins, or an activated form thereof, and assaymg for complexes, for free substance, for non-complexed TbpA or TbpB or a TbpA or TbpB related protems, or an activated form thereof Substances which potentially can bmd TbpA or TbpB, or TbpA or TbpB related protems, mclude transferrms, particularly rummant transferrins, analogs and derivatives of transferrms and antibodies agamst TbpA and TbpB, or TbpA or TbpB related protems Still further, the invention provides a method for assaying a medium for the presence of an agonist or antagonist of the mteraction of TbpA or TbpB, or TbpA or TbpB related protems, and a substance which binds to TbpA or TbpB, or TbpA or TbpB related protems or an activated form thereof In an embodiment, the method comprises providmg a known concentration of TbpA or TbpB, or TbpA or TbpB related protems, with a substance which is capable of binding to TbpA or TbpB, or TbpA or TbpB related protems and a suspected agonist or antagonist substance under conditions which permit the formation of complexes between the substance and TbpA or TbpB, or TbpA or TbpB related protems, and assaymg for complexes, for free substance, for non-complexed TbpA or TbpB, or TbpA or TbpB related protems In a preferred embodiment of the invention, the substance is a rummant transferrin, analog, derivative or part thereof or an antibody agamst TbpA or TbpB, or TbpA or TbpB related protems Substances which affect expression of TbpA or TbpB, or TbpA or TbpB related protems, may also be identified usmg the methods of the invention by comparmg the pattern and level of expression of TbpA or TbpB, or TbpA or TbpB related protems of the mvention, in cells in the presence, and in the absence of the substance. The substances identified usmg the methods of the invention may be used m the treatment of animals, particularly ruminants infected with P haemolytica and accordmgly they may be formulated into pharmaceutical compositions for adminstration to ruminants, such as cattle, sheep and goats suffermg from infection with P haemolytica or exposed to lnfechor by P haemolytica The present inventors have demonstrated that the TbpA or TbpB, or TbpA or TbpB related proteins of the mvention, are lmmunogemc Therefore, the mvention also relates to antibodies agamst the TbpA or TbpB, or TbpA or TbpB related protems of the mvention In an embodiment, the antibodies are cross reactive agamst TbpA or TbpB or TbpA, or TbpB related protems, from a wide range of serotypes of P haemolytica The antibodies may be used m the diagnosis and treatment of P haemloytica mfection and may be used, for example, m passive immunization to treat or prevent diseases in rummants caused by P haemolytica The mvention further mcludes vaccme compositions comprismg the TbpA or TbpB, or TbpA or TbpB related protems of the invention, either alone, or m combmation The inventior still further includes methods of immunizing a host, preferably a rummant host agamst mfection by P haemolytica by administering therapeutically effective amounts of such vaccmes The present inventors ha\ e demonstrated that different strains of P haemolytica, from a range of rummants, are able to bmd and utilize a range of rummant transferrms Thus it is contemplatec that the vaccme compositions of the mvention will be useful as broad spectrum vaccmes suitable for immunizing a range of ruminants, such as sheep, cows and goats agamst infection with a wide range of P haemolytica biotypes and serotypes The invention also contemplates the use of nucleic acid molecules of the invention encodmg TbpA or TbpB, or TbpA or TbpB related protems, m a recombmant viral vector vaccme for augmenting the immune response of a ruminant to P haemolytica or for treating P haemolytica mfection Recombmant viral vectors may be constructed usmg techniques known in the art Other ob j ects, features and advantages of the present mvention will become apparen from the followmg detailed description It should be understood, howev er, that the detailed description and the specific examples while mdicatmg preferred embodiments of the mvention are given by way of illustration only, smce various changes and modifications with the spirr and scope of the mvention will become apparent to those skilled m the art from this detailed description BRIEF DESCRIPTION OF THE DRAWINGS The mvention will now be described m relation to the drawmgs m which Figure 1 is a schematic diagram of the PCR procedure (a), and the 0 8 kb PCR product amplified by Tbpl primer and primer left (b), Figure 2 is a restriction endonuclease map of tbp plasmids 9, 10, and 482, Figure 3 is a preliminary nucleotide sequence of P haemolytica tbpA and tpbB, Figure 4 is the promoter region of P haemolytica tbpB (PHTBPB), Figure 5 is a blot of Southern hybridization of P haemolytica genomic DNA digested with Clαl and probed with tbpA gene, Figure 6 is a blot of Southern hybridization of P haemolytica genomic DNA digested with Hind fll and Bam l and probed with the tpbA gene, Figure 7 is a blot of Southern hybridization of A suis 37114, A pleuropneumoniae CM5 and shope 4074 genomic DNA digested with various restriction endonucieases and probed with P haemolytica tbpA, Figure 8 are restriction maps of the tbpA, tbpB regions m P haemolytica Al, A pleuropneumoniae CM5, Shope 4074, and A suis 3714, Figure 9 shows an alignment of the ammo acid of Tbpl of P haemolytica Λl, (PHTBP) and the Tbpl of N gonorrhoeae (NGTBP1) and N memngitidis (NM1), Figure 10 shows an alignment of the amino acid of Tbpl of P haemolytica Al, (PHTBP) and the A pleuropneumoniae serotype 1 and 7 TfbA protems (APL, APL7), Figure 11 is a dendogram illustrating the genetic relatedness among P haemolytica Λ ϊ , (PHTBP) and the Tbpl of N gonorrhoeae (NGTBP1), N memngitidis (NM1) and A pleuropneumoniae serotype 1 and 7 TfbA protems (APL, APL7), Figure 12 is a peptide alignment between P haemolytica Al Tbpl and TonB- dependent outer membrane receptors of E coli, Figure 13 is a blot showing T7 analysis of the P haemolytica Tbpl protem, Figure 14 is a Western immunoblot of inner and outer membranes from P haemolytica Al and E coli HBlOl, Figure 15 is a Western immunoblot of inner and outer membranes from P haemolytica Al and £ coli HBlOl usmg sera raised in calves to soluble antigens by vaccmation with Presponse®, Figure 16 is a blot showmg the bindmg of labelled transferrms by iron-deficient bacterial membranes, Figure 17 is an immunoblot showmg isolation of receptor proteins with transferrin affmity columns, Figure 18 are immunoblots showmg lmmunological analysis of receptor protems from different serotypes of P haemolytica from bovme, sheep, and goats, where Panel A is with anti- TbpB serum and Panel B is with anh-TbpA serum, Figure 19 are blots showmg the bindmg of labelled transferrin and anti-receptor antibody by intact cells, Figure 20 is a map of the P haemolytica tbp operon (Top) and P haemolytica tbp operon (Top) and regulatory sequences (Bottom), tbp A and tbpB are the genes encodmg for TbpA andTbpB, respectively, p, is the putative promoter region precedmg tbpB and denoted as -35 and -10 sites at the bottom, Figure 21 and SEQ ID NO 1 show the DNA sequence of the tbpA gene from P haemolytica strain hl96, Figure 22 and SEQ ID NO 2 show the predicted amino acid sequence of the TbpA protem from P haemolytica strain hl96, Figure 23 and SEQ ID NO 3 show the DNA sequence of the tbpB gene from P haemolytica strain hl96, Figure 24 and SEQ ID NO 4 show the predicted amino acid sequence of the TbpB protem from P haemolytica strain hl96, Figure 25 is a blot showmg the results of a solid-phase HRP-Tf bmdmg assay, Figure 26 are blots showmg silver stam (Panel A), and western blot (Panel B) studies with anti-TbpA and anti-TbpB antisera from P haemolytica serotype Al, Figure 27 is a blot showmg the results of cross-reactivity studies with monospecific anti-TbpA and anti-TbpB antisera from P haemolytica serotype Al against mtact cells, Figure 28 shows a gel with restriction endonuclease digestion patterns of PCR-amplified tbpA (Panel A) and tbpB (Panel B) genes from P haemolytica and P trehalosi strams, and Figure 29 is a gel showmg PCR amplification of variable segments of the tbpA (Panel A) andtbpB (Panel B) genes DETAILED DESCRIPTION OF THE INVENTION The following standard abbreviations for the amino acid residues are used throughout the specification A, Ala - alanine, C, Cys - cysteme, D, Asp- aspartic acid, E, Glu - glutamic acid, F, Phe - phenylalanme, G, Gly - glycine, H, His - histidme, I, He - isoleucme, K, Lys - lysrne, L, Leu - leucme, M, Met - methionine, N, Asn - asparagme, P, Pro - prolme, Q, Gin - glutamine, R, Arg - arginine, S, Ser - serme, T, Thr threonme, V, Val - va ne, W, Trp- tryptophan, Y, Tyr - tyrosme, and p Y , P Tyr - phosphotyrosme I. Nucleic Acid Molecules of the Invention As herembefore mentioned, the present invention provides a purified and isolated nucleic acid molecule comprismg a sequence encodmg a TbpA protem, or a purified and isolated nucleic acid molecule comprismg a sequence encodmg a TbpB protein The term ' isolated and purified" refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombmant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized An ' isolated and purified' nucleic acid is also free of sequences which naturally flank the nucleic acid (l e , sequences located at the 5 and 3 ends of the nucleic acid) from which the nucleic acid is derived The term "nucleic acid is intended to mclude DNA and RNA and can be either double stranded or s gle stranded In an embodiment of the mvention, a nucleic acid molecule is provided which encodes TbpA havmg the amino acid sequence as shown m Figure 22 or SEQ ID NO 2 In another embodiment, a nucleic acid molecule is provided which encodes TbpB havmg the amino acid sequence as shown in Figure 24 or SEQ ID NO 4 In preferred embodiments of the mvention, the nucleic acid molecule is a DNA comprising the nucleotide sequence as shown in Figure 21 or SEQ ID NO 1, or the nucleotide sequence as shown in Figure 23 or SEQ ID NO 3 The mvention mcludes nucleic acid sequences complementary to the nucleic acid (a) encodmg TbpA havmg the ammo acid sequence as shown m Figure 22 or SEQ ID NO 2, (b) encodmg TbpB havmg the ammo acid sequence as shown in Figure 24 or SEQ ID NO 4, (c) havmg the sequence as shown in Figure 21 or SEQ ID NO 1, or m Figure 23 or SEQ ID NO 3 Preferably, the sequences are complementary to the full length nucleic acid sequences sequence shown m Figure 21 or SEQ ID NO 1, or m Figure 23 or SEQ ID NO 3 The invention also includes nucleic acid molecules having substantial sequence identity or homology to the nucleic acid sequence as shown m Figure 21 or SEQ ID NO 1 or Figure 23 or SEQ ID NO 3, or encodmg TbpA or TbpB protems havmg substantial homology to the ammo acid sequences shown m Figure 22 or SEQ ID NO 2, or in Figure 24 or SEQ ID NO 4, respectively Homology refers to sequence similarity between sequences and can be determined by comparing a position m each sequence which may be aligned for purposes of comparison When a position m the compared sequence is occupied by the same nucleotide base or ammo acid, then the molecules are matchmg or have identical positions shared by the sequences Nucleic acid sequences having substantial homology include (a) nucleic acid sequences havmg at least 40-60%, preferably 60-80% most preferably 80-90% identity with the nucleic acid sequence as shown in Figure 21 or SEQ ID NO 1, and (b) nucleic acid sequences havmg at least 40-60%, preferably 60-80% most preferably 80-90% identity with the nucleic acid sequence as shown m Figure 23 or SEQ ID NO 3 Another aspect of the invention provides a nucleic acid molecule, and fragments thereof havmg at least 15 nucleotide bases, which hybridizes to the nucleic acid molecules of the mvention under hybridization conditions, preferably strmgent hybridization conditions Appropriate stringency conditions which promote DNA hybridization are known to those skilled m the art, or may be found m Current Protocols m Molecular Biology, John Wiley & Sons, N Y (1989), 6 3 1-6 3 6 For example, 6 0 x sodium chloride /sodium citrate (SSC) at about 45 a C, followed by a wash of 2 0 x SSC at 50°C may be employed The strmgency may be selected based on the conditions used m the wash step By way of example, the salt concentration m the wash step can be selected from a high strmgency of about 0 2 x SSC at 50°C In addition, the temperature m the wash step can be at high strmgency conditions, at about 65°C Isolated and purified nucleic acid molecules encodmg a protein havmg the activity of TbpA or TbpB, and havmg a sequence which differs from the nucleic acid sequence shown m Figure 21 or SEQ ID NO 1, or m Figure 23 or SEQ ID NO 3, respectively, due to degeneracy in the genetic code are also within the scope of the mvention Such nucleic acids should encode functionally equivalent TbpA or TbpB protems but differ in sequence from the sequence in Figure 21 or SEQ ID NO 1, or m Figure 23 or SEQ ID NO 3, respectively, due to degeneracy m the genetic code An isolated and purified nucleic acid molecule of the invention which comprises DNA can be isolated by preparmg a labelled nucleic acid probe based on all or part of the nucleic acid sequence shown m Figure 21 or SEQ ID NO 1, or in Figure 23 or SEQ ID NO 3, and usmg the labelled nucleic acid probe to screen an appropriate DNA library (e g a cDNA or genomic DNA library) Nucleic acids isolated by screenmg of a cDNA or genomic DNA library can be sequenced by standard techniques An isolated and purified nucleic acid molecule of the invention which is DNA can also be isolated by selectively amplifying a nucleic acid encodmg TbpA or TbpB usmg the polymerase chain reaction (PCR) methods and cDNA or genomic DNA It is possible to design synthetic oligonucleotide primers from the nucleotide sequence shown in Figure 21 or SEQ ID NO 1, or m Figure 23 or SEQ ID NO 3, for use m PCR A nucleic acid can be amplified from cDNA or genomic DNA using these oligonucleotide primers and standard PCR amplification techniques The nucleic acid so amplified can be cloned mto an appropriate vector and characterized by DNA sequence analysis It will be appreciated that cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by usmg the guanidinium-thiocyanate extraction procedure of Chirgwm et al , Biochemistry, 18, 5294-5299 (1979) cDNA is then synthesized from the mRNA using reverse transcπptase (for example, Moloney MLV reverse transcπptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcπptase available from Seikagaku America, Inc , St Petersburg, FL) An isolated and purified nucleic acid molecule of the mvention which is RNA can be isolated by cloning a cDNA encodmg TbpA or TbpB mto an appropriate vector which allows for transcription of the cDNA to produce an RNA molecule which encodes a protem which exhibits TbpA or TbpB activity A nucleic acid molecule of the invention may also be chemically synthesized usmg standard techniques Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated m commercially available DNA synthesizers (See e g , Itakura et al U S Patent No 4,598,049, Caruthers et al U S Patent No 4,458,066, and Itakura U S Patent Nos 4,401,796 and 4,373,071) Determination of whether a particular nucleic acid molecule encodes a protem havmg TbpA or TbpB activity can be accomplished by expressmg the DNA in an appropriate host cell by standard techniques, and testing the ability of the expressed protein to bmd rummant transferrms and /or mediate iron uptake A cDNA havmg such activity can be sequenced by standard techniques, such as dideoxvnucleotide cham termination or Maxam- Gilbert chemical sequencmg, to determme the nucleic acid sequence and the predicted ammo acid sequence of the encoded protem Regulatory elements of tbpA or tbpB can be identified usmg conventional techniques The function of the elements can be confirmed by usmg these elements to express a reporter gene which is operatively lmked to the elements These constructs may be introduced mto cultured cells usmg standard procedures The sequence of a nucleic acid molecule of the mvention may be mverted relative to its normal presentation for transcription to produce an antisense nucleic acid molecule An antisense nucleic acid molecule may be constructed usmg chemical synthesis and enzymatic hgation reactions usmg procedures known in the art II. Recombmant TbpA and TbpB The invention also contemplates a purified and isolated TbpA or TbpB protem from Pasteurella haemolytica Al which exhibits transferrin bindmg activity. In an embodiment of the invention, a purified TbpA protem is provided which has the ammo acid sequence as shown in Figure 22 or m SEQ ID NO 2 In another embodiment of the invention, a purified TbpB protem is provided which has the ammo acid sequence as shown m Figure 24 or m SEQ ID NO 4 Recombmant TbpB, unlike the native receptor complex, recognizes bmdmg determinants on the N-lobe and C-lobe of transferrin In addition to the full length TbpA or TbpB ammo acid sequences, the protems of the present mvention mclude truncations of TbpA or TbpB, and analogs, and homologs of TbpA or TbpB and truncations thereof as described herein Truncated protems may comprise peptides with at least 3 ammo acid residues The truncated protems may have an ammo group (-NH2), a hydrophobic group (for example, carbobenzoxyl, dansyl, or T-butyloxycarbonyl), an acetyl group, a 9-fluorenylmethoxy-carbonyl (PMOC) group, or a macromolecuie mcludmg but not limited to hpid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the ammo termmal end The truncated proteins may hav e a carboxyl group, an amido group, a T- butyloxycarbonyl group, or a macromolecuie including but not limited to hpid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the carboxy termmal end The protems of the mvention may also mclude analogs of TbpA or TbpB as shown in Figure 22 or m SEQ ID NO 2, or in Figure 24 or m SEQ ID NO 4, respectively, and /or truncations thereof as described here , which may mclude, but are not limited to TbpA or TbpB (Figure 22 or m SEQ ID NO 2, or in Figure 24 or m SEQ ID NO 4), contammg one or more ammo acid substitutions, insertions, and /or deletions Ammo acid substitutions may be of a conserved or non-conserved nature Conserved ammo acid substitutions mvolve replacmg one or more ammo acids of the TbpA or TbpB ammo acid sequence with ammo acids of simdar charge, size, and/or hydrophobicity characteristics When only conserved substitutions are made the resulting analog should be functionally equivalent to TbpA or TbpB Non-conserved substitutions mvolve replacmg one or more ammo acids of the TbpA or TbpB ammo acid sequence with one or more ammo acids which possess dissimilar charge, size, and/or hydrophobicity characteristics One or more ammo acid msertions may be introduced mto TbpA or TbpB (Figure 22 or in SEQ ID NO 2, or m Figure 24 or m SEQ ID NO 4) Ammo acid msertions may consist of smgle ammo acid residues or sequential ammo acids ranging from 2 to 15 ammo acids m length Deletions may consist of the removal of one or more amino acids, or discrete portions from the TbpA or TbpB (Figure 22 or in SEQ ID NO 2, or in Figure 24 or m SEQ ID NO 4), sequence The deleted ammo acids may or may not be contiguous The lower limit length of the resulting analog with a deletion mutation is about 10 ammo acids, preferably 100 amino acids The protems of the mvention also mclude homologs of TbpA or TbpB (Figure 22 or m SEQ ID NO 2, or m Figure 24 or m SEQ ID NO 4), and /or truncations thereof as described herein Such TbpA or TbpB, homlogs are protems whose ammo acid sequences are comprised of the amino acid sequences of TbpA or TbpB regions from other species that hybridize under strmgent hybridization conditions (see discussion of strmgent hybridization conditions herem) with a probe used to obtam TbpA or TbpB Protem sequences having substantial homology mclude protein sequences havmg at least 40-60%, preferably 60-80%, most preferably 80-90% identity with the ammo acid sequence as shown m Figure 22 (or SEQ ID NO 2) or Figure 24 (SEQ ID NO 4) The mvention also contemplates isoforms of the protems of the mvention An isoform contams the same number and kmds of amino acids as the protem of the invention, but the isoform has a different molecular structure The isoforms contemplated by the present invention are those havmg the same properties as the protem of the mvention as described herem The present invention also mcludes a TbpA, TbpB or aTbpA or TbpB related protems con j ugated with a selected protem, or a selectable marker protem (see below ) to produce fusion protems Additionally, lmmunogenic portions of TbpA, TbpB or a TbpA or TbpB related protems are withm the scope of the mvention TbpA, TbpB or a TbpA or TbpB related protein of the invention are prepared usmg recombmant DNA methods Accordmgly, the nucleic acid molecules of the present mvention havmg a sequence which encodes TbpA, TbpB, or a TbpA or TbpB related protem of the mvention may be mcorporated m a known manner mto an appropriate expression vector which ensures good expression of the protem The expression vectors are suitable for transformation of a host cell' , means that the expression vectors contam a nucleic acid molecule of the invention and regulatory sequences selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid molecule Operatively lmked is intended to mean that the nucleic acid is lmked to regulatory sequences in a manner which allows expression of the nucleic acid The invention therefore contemplates a recombmant expression vector of the mvention contammg a nucleic acid molecule of the invention, or a fragment thereof, and the necessary regulatory sequences for the transcription and translation of the inserted protem-sequence Suitable regulatory sequences may be derived from a v aπety of sources, including bacterial, fungal, viral, mammalian, or insect genes Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art The recombmant expression vectors of the mvention may also contain a selectable marker gene which facilitates the selection of host cells transformed or transfected with a recombmant molecule of the mvention Examples of selectable marker genes are genes encodmg a protem which confers resistance to certain drugs, or β-galactosidase The recombmant expression vectors may also contam genes which encode a fusion moiety which provides increased expression of the recombmant protem, mcreased solubility of the recombmant protem, and aid m the purification of the target recombmant protem by actmg as a ligand in affmity purification Recombmant expression vectors can be mtroduced into host cells to produce a transformant host cell The term ' transformant host cell' is mtended to mclude prokaryohc and eukaryotic cells which have been transformed or transfected with a recombmant expression vector of the invention The terms transformed with , transfected with , transformation and transfection are mtended to encompass mtroduction of nucleic acid (e g a vector) mto a cell by one of many possible techniques known m the art Suitable methods for transforming and transfecting host cells can be found m Sambrook et al (Molecular Cloning A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks The proteins of the mvention may also be prepared by chemical synthesis usmg techniques well known in the chemistry of proteins such as solid phase synthesis (Merπfield, 1964, J Am Chem Assoc 85 2149-2154) or synthesis m homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed E Wansch, Vol 15 I and II, Thieme, Stuttgart) N-terminal or C-terminal fusion protems comprismg TbpA, TbpB, or a TbpA or TbpB related protem of the mvention conjugated with other molecules, such as proteins, may be prepared by fusmg, through recombmant techniques, the N-terminal or C-terminal of TbpA, TbpB or a TbpA or TbpB related protein, and the sequence of a selected protem or selectable marker protem with a desired biological function The resultant fusion protems contam TbpA, TbpB or aTbpA or TbpB related protem, fused to the selected protem or marker protem III. Applications of the Invention The nucleic acid molecules of the invention allow those skilled in the art to construct nucleotide probes for use in the detection of nucleic acid sequences m samples Suitable probes mclude nucleic acid molecules based on nucleic acid sequences encodmg at least 6 sequential ammo acids from regions of the TbpA or TbpB protem as shown m Figure 22 and SEQ ID NO 2, or m Figure 24 and SEQ ID NO 4, respectively For example, a suitable probe may mclude nucleic acid molecules of TbpA selected from nucleotide nos 1741 to 2784 of the sequence of TbpA shown m Figure 21 and SEQ ID NO 1 A nucleotide probe may be labelled with a detectable substance such as a radioactive label which provides for an adequate signal and has sufficient half-life such as 32 P, 3 H, 14 C or the like Other detectable substances which may be used mclude antigens that are recognized by a specific labelled antibody fluorescent compounds, enzymes, antibodies specific for a labelled antigen, and lummescent compounds An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleotide to be detected and the amount of nucleotide available for hybridization Labelled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described m Sambrook et al, 1989, Molecular Clonmg, A Laboratory Manual (2nd ed ) The nucleic acid probes may be used to detect genes, preferably m human cells, that encode TbpA, TbpB, or TbpA or TbpB related protems TbpA, TbpB, or TbpA or TbpB related protems of the mvention can be used to prepare antibodies specific for the protems Conventional methods can be used to prepare the antibodies To produce polyclonal antibodies a mammal (such as a rabbit, mouse or rat) may be immunized with TbpA,TbpB, fragments of the protems or a mixture of the two The immunogenicity of the protem(s) may be enhanced by addmg an adjuvant to the protem mixture or by couplmg the protein to an lmmunogemc carrier Examples of carriers include keyhole limpet hemocyanιn(KLH) and bovme serum albumm(BSA) To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an animal (immunized as described above) and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybπdoma cells Such techniques are well known m the art, [e g , the hybπdoma technique originally developed by Kohler and Mύstein (Nature 256, 495-497 (1975)) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al , Immunol Today 4, 72 (1983)), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al Monoclonal Antibodies in Cancer Therapy (1985) Allen R Bliss, Inc , pages 77-96), and screening of combinatorial antibody libraries (Huse et al , Science 246, 1275 (1989)] Hybridoma cells can be screened lmmunochemically for production of antibodies specifically reactive with the peptide and the monoclonal antibodies can be isolated Therefore, the invention also contemplates hybridoma cells secretmg monoclonal antibodies with specificity for TbpA or TbpB, or TbpA or TbpB related protems, as described herein The term "antibody" as used herem is mtended to mclude fragments thereof which also specifically react with a protem, or peptide thereof, having the activity of TbpA or TbpB Antibodies can be fragmented using conventional techniques and the fragments screened for utility m the same manner as described above For example, F(ab ) 2 fragments can be generated by treating antibody with pepsin The resultmg F(ab ) 2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments Multivalent antibodies may be prepared by fusmg two or more F(ab ) 2 or Fab' fragments For example, a multivalent antibody may contam one F(ab') 2 fragment specific for TbpA and one F(ab )2 fragment specific for TbpB Chimeπc antibody derivatives, I e , antibody molecules that combine a non- rummant animal variable region and a rummant constant region are also contemplated within the scope of the invention Chimeπc antibody molecules can include, for example, the antigen binding domam from an antibody of a mouse, rat, or other species, with bovine constant regions Conventional methods may be used to make chimeπc antibodies contammg the immunoglobulm variable region which recognizes the gene product of novel Tbp genes of the mvention (See, for example, Morrison et al., Proc. Natl Acad Sci U S A 81,6851 (1985), Takeda et al , Nature 314, 452 (1985), Cabilly et al , U.S Patent No 4,816,567, Boss et al , U S Patent No. 4,816,397, Tanaguchi et al , European Patent Publication EP171496, European Patent Publication 0173494 Antibodies specifically reactive with TbpA, TbpB, or TbpA or TbpB related protems, or derivatives thereof, such as enzyme conjugates or labeled derivatives, may be used as probes to detect TbpA, TbpB, or TbpA or TbpB related protems m samples such as tissues and cells, for example they may be used in any known immunoassays which rely on the bindmg mteraction between an antigenic determinant of TbpA, TbpB, or TbpA or TbpB related proteins and the antibodies Examples of such assays are radioimmunoassays, enzyme immunoassays (e g ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination, and histochemical tests Thus, the antibodies may be used to detect and quantify TbpA, TbpB, or TbpA or TbpB related proteins in a sample In an embodiment, the antibodies are cross reactive agamst TbpA or TbpB or TbpA, or TbpB related protems, from a wide range of serotypes of P haemolytica When used as probes the antibodies are usually labelled by techniques known m the art The antibodies of the present mvention may also be used in the diagnosis and treatment of P haemloytica infection In one embodiment, the antibodies are used m passive irnmunization to treat or prevent diseases m rummants caused by P haemolytica In such a case, a mixture of antibodies or a multivalent antibody may be used The mvention still further provides a method for identifying a substance which is capable of bmdmg to TbpA or TbpB, or TbpA or TbpB related protems, or an activated form thereof, comprising reactmg TbpA or TbpB, or TbpA or TbpB related protems, or an activated form thereof, with at least one substance which potentially can bmd with TbpA or TbpB, or TbpA or TbpB related protems, or an activated form thereof, under conditions which permit the formation of complexes between the substance and TbpA or TbpB, or TbpA or TbpB related proteins, or an activated form thereof, and assaymg for complexes, for free substance, for non-complexed TbpA or TbpB or a TbpA or TbpB related protems or an activated form thereof Substances which potentially can bmd TbpA or TbpB, or TbpA or TbpB related proteins, mclude transferrms, particularly ruminant transferrms, analogs and derivatives of transferrms and antibodies agamst TbpA and TbpB, or TbpA or TbpB related protems Still further, the invention provides a method for assaying a medium tor the presence of an agonist or antagonist of the mteraction of TbpA or TbpB, or TbpA or TbpB related protems, and a substance which bmds to TbpA or TbpB, or TbpA or TbpB related protems or an activated form thereof In an embodiment, the method comprises providmg a known concentration of TbpA or TbpB, or TbpA or TbpB related protems, with a substance which is capable of bmdmg to TbpA or TbpB, or TbpA or TbpB related protems and a suspected agonist or antagonist substance under conditions which permit the formation of complexes between the substance and TbpA or TbpB, or TbpA or TbpB related protems, and assaymg for complexes, for free substance, for non-complexed TbpA or TbpB, or TbpA or TbpB related protems In a preferred embodiment of the invention, the substance is a rummant transferrin, analog, derivative or part thereof ,or an antibody against TbpA or TbpB, or TbpA or TbpB related prote s Substances which affect expression of TbpA or TbpB, or TbpA or TbpB related protems, may also be identified usmg the methods of the invention by comparmg the pattern and level of expression of TbpA or TbpB, or TbpA or TbpB related protems of the mvention, m cells m the presence, and m the absence of the substance The substances identified usmg the methods of the mvention may be used in the treatment of animals, particularly ruminants infected with P haemolytica and accordmgly they may be formulated mto pharmaceutical compositions for adminstrahon to rummants, such as cattle, sheep and goats suffermg from infection with P haemolytica, or exposed to infection by P haemolytica The substances may be formulated into pharmaceutical compositions for adminstration to subjects m a biologically compatible form suitable for administration in vivo By ' biologically compatible form suitable for administration in vivo' is meant a form of the substance to be administered m which any toxic effects are outweighed by the therapeutic effects The substances may be admmistered to living organisms mcludmg humans, and animals Administration of a therapeutically active amount of the pharmaceutical compositions of the present mvention is defmed as an amount effective, at dosages and for periods of time necessary to achieve the desired result For example, a therapeutically active amount of a substance may vary accordmg to factors such as the disease state, age, sex, and weight of the mdividual, and the ability of antibody to elicit a desired response m the individual Dosage regima may be adjusted to provide the optimum therapeutic response The active substance may be admmistered m a convenient manner such as by in j ection (subcutaneous, intravenous, etc ), oral administration, inhalation, transdermal application, or rectal administration Depending on the route of administration, the active substance may be coated m a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound The compositions described herem can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to sub j ects, such that an effective quantity of the active substance is combined m a mixture with a pharmaceutically acceptable vehicle Suitable vehicles are described, for example, in Remington s Pharmaceutical Sciences (Remmgton s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa , USA 1985) On this basis, the compositions include, albeit not exclusively, solutions of the substances m association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids TbpA or TbpB, and /or TbpA or TbpB related protems may be used as vaccmes for the prophylaxis and treatment of various infectious diseases in animals Infectious diseases contemplated by the mvention mclude infections caused by P haemolytica such as bovme pneumoniae m cattle and systemic disease and pneumonia in sheep In addition, vaccines according to the present mvention may be used for the prophylaxis and treatment of mfections caused by other Pasteurella spp One example is the prophylaxis and treatment of Pasteurella multocida in cattle, swine and poultry mcludmg respiratory and systemic mfections such as Hemorrhagic sephcemia and bovme mastisis The vaccmes can be mtended for administration to various animals, preferably rummants, mcludmg cattle, sheep, and goats The present mventors have demonstrated that different strams of P haemolytica, from a range of rummants, are able to bmd and utilize a range of rummant transferrms Thus it is contemplated that the vaccine compositions of the mvention will be useful as broad spectrum vaccmes suitable for immunizing a range of rummants, such as sheep, cows and goats agamst mfection with a wide range of P haemolytica biotypes and serotypes The vaccme compositions comprise the TbpA ,TbpB, and /or TbpA or TbpB related protems, either alone, or in combmation The vaccme compositions may contam any combination of the described protems or immunogenic fragments thereof Further, the composition may contam Tbp protems isolated from one or more biotypes or serotypes of P haemolytica or other microorganisms Recombmant protems comprismg TbpA, TbpB, and /or TbpA or TbpB related proteins are preferably employed in the vaccme compositions of the mvention In a preferred embodiment, one or more of recombmant TbpA or TbpB, and TbpA or TbpB related protems of the mvention, are used m the vaccme compositions In another embodiment of the mvention, the vaccme composition consists of purified and isolated TbpA and TbpB, preferably recombmant TbpA and TbpB The vaccme of the mvention contams an lmmunologically effective amount of one or more of TpbA, TbpB, TbpA related protem, and TbpB related protem The optimum amounts of the protems depends on the nature of the infection agamst which protection is required, the characteristics of the animals to be protected, and other factors known to persons skilled m the art In addition to the TpbA, TbpB, TbpA related protem, and/or TbpB related protem, the vaccine may comprise an immunologically acceptable earner such as aqueous diluents, suspendmg aids, buffers, excipients, and one or more adjuvants known in the art Suitable adjuvants mclude alummum hydroxide, Freund s adjuvant (complete or incomplete), bacteria such as Bordetella pertussis or E coli or bacterium derived matter, immune stimulating complex (iscom), oil, sapronin, oligopeptide, emulsified paraffin-Emulsigen™ (MVP Labs, Ralston, Nebraska), L80 adjuvant containing AL(OH) 3 (Reheis, New Jersey), Quil A (Superphos), or other ad j uvants known to the skilled artisan Preferably, the adjuvant is L80 adjuvant containing AL(OH)^ (Reheis, New Jersey) and Quil A (Superphos) The vaccine can be mcorporated mto a hposome system which will allow the slow release of the TbpA and /or TbpB protem in the recipient The vaccme may also contam preservatives such as sodium azide, thimersol, gentamicin, neomycin, and polymyxin The vaccine may be a multivalent vaccine and additionally contain other lmmunogens of P haemolytica or immunogens related to other diseases m a prophylactically or therapeutically effective manner For example, the vaccme composition of the inventon may consist of P haemolytica leukotoxm and TbpB The vaccmes of the mvention may be admmistered m a convenient manner, such as intravenously, intramuscularly, subcutaneously, intraperitoneally, intranatally or orally Preferably the vaccme is admmistered intramuscularly or subcutaneously The dosage will depend on the nature of the mfection, on the desired effect and on the chosen route of administration, and other factors known to persons skilled m the art The mvention also contemplates the use of recombmant viral vector vaccines and recombmant bacterial vector vaccines contammg nucleic acid molecules of the mvention encodmg TbpA, TbpB, TbpArelated protein, and/or TbpB related protein, for the treatment and/or prophylaxis of P haemolytica mfection In such systems, the TbpA or TbpB protems are synthesized in vivo m the recipient from the exogenous nucleic acid molecules m the vaccme The recombmant viral or bacterial vectors may be constructed usmg techniques known m the art, and as described herem Examples of bacterial systems include E coli and Salmonella spp The followmg non- mitmg examples are illustrative of the present mvention Examples EXAMPLE 1 The following materials and methods were used in the studies described in the example: MATERIALS AND METHODS BACTERIAL STRAINS AND CLONING VECTORS P haemolytica strains were provided by Dr P Shewen, Department of Vetermary Microbiology and Immunology (VMI), University of Guelph, and were originally obtained from Dr E Biberstein, University of California, Davis, Dr G Frank, USDA, Ames, Iowa, and Dr W Donachie, Moredun Research Institute, Edinburgh, U K Actinobacύlus suis stra 3714, A pleuropneumoniae strams CM5 and Shope 4074 were provided by Dr S Rosendal, VMI E coli strains HBlOl and TG-1 were provided by Dr R Lo, Department of Microbiology, University of Guelph, and were used as recipient strams for clonmg experiments E coli stram JM109 (DE3) was provided by Dr C Whitfield, Department of Microbiology, University of Guelph Pasteurella and Actinobactllw, strains were maintained on sheep's blood agar and cultured in bram heart mfusion broth (BHIB), (Difco Labs, Detroit, Michigan) E coh HBlOl was grown on Luπa-Bertaini plus thymidme (LT), supplemented with ampicillm (Sigma Chemical Co , St Louis, Missouri) at 100 mg/L for selection of recombmant plasmids Similarly, E coli TG- and JM109 (DE3) were grown on Davis minimal medium with ampicillm Iron- depleted conditions were prepared by adding the iron chelator ethylenedιamιne-dι(o- hydroxyphenylacetic) acid (EDDA) (Sigma) to a fmal concentration of lOOμM Iron-depleted conditions were prepared by adding FeCl 3 to 1 mM The plasmid pBR322, bacteriophage vectors M13/mpl8 and M13/mpl9 were used as previously described (Lo and Cameron, 1986, Lo et al , 1985, and Lo et al , 1987) The pBluescπpt vector was obtamed from Stratagene (La Jolla, California) The recombmant Clone 482 was provided by Dr A Schryvers, Department of Microbiology, University of Calgary ENZYMES, CHEMICALS AND ANTISERA Restriction endonucieases and DNA modifying enzymes were purchased from Bethesda Research Laboratories (BRL) (Burlington, Ontario) or Pharmacia Chemicals Incorporated (Dorval, Quebec) and were used as described by the manufacturers Radioisotopes were purchased from ICN Biomedical (Montreal, Quebec) or Amersham Laboratories (Oakville, Ontario) Goat anti rabbit immunoglobulin G-alkalme phosphatase conjugate and lmmunodectection reagents were purchased from Bio-Rad Laboratories (Mississauga, Ontario) Goat anti-bovme immunoglobulin G-Alkalme phosphatase conjugate was purchased from Jackson Immunoresearch (West Grove, PA) Rabbit anti-autologous antiserum and bovine anti- Presponse antisera were obtamed from Dr P Shewen, Department of Vetermary Microbiology and immunology, University of Guelph The rabbit "anti-autologous" antiserum was raised agamst the soluble antigens of P haemolytica Al cultured m RPMI 1640 supplemented with that rabbit's own serum It is important to note that RMPI 1640 is an iron poor medium DNA METHODS a ) Chromosomal DNA isolation Chromosomal DNA was isolated from bacterial cells accordmg to the method of Marmer (1961) Bacteria were moculated mto 250 ml of the appropriate medium and grown overnight at 37°C with 150 rpm shaking The followmg day, the cells were pelleted by centrifugation at 4,000 x g in a GSA rotor m a Sorvall RC5-B refrigerated centrifuge (Dupont Instruments, Mississauga, Ontario) for 10 min The pellet was suspended in 8 ml of a 0 6 M sorbitol, 0 05 mM Tπs-HCI (pH 8 0), 0 05 M EDTA solution Lysozyme (Sigma) was added to a f al concentration of 3 mg/ml and the sample was mcubated for 30 mm on ice Two ml of lytic solution (0 5% SDS, 0 05 M EDTA, 0 05 M Tπs-Cl [pH 8 0]) and 3 mg/ml of protemase K (Sigma) solution were added to the sample, which was then incubated for 4 h in a 37°C water bath, followed by mcubation at 56°C The suspension was extracted with an equal volume of phenol (Gibco/BRL) saturated with TE buffer (0 05 M Tris-HCl [pH 7 5], 0 001 M EDTA) and shaken at 30-50 rpm for 45 mm The phenol and aqueous phases were separated by centrifugation at 12,000 x g at 5°C for 10 mm in a SS34 rotor The supernatant was collected by a cut off wide mouth pasteur pipette and the DNA was precipitated with 2-3 volumes of ice-cold 95% ethanol The strands of DNA were spooled onto a glass rod and dissolved m a small volume of 0 1 X SSC (IX SSC contams 0 15 M NAC1, 0 015 M sodium citrate) The DNA was then treated with RNase to a final concentration of lOμg/ml and incubated at 37°C for 30 mm The DNA was again precipitated by 2-3 volumes of cold 95% ethanol, spooled onto a glass rod and dissolved m IX SSC Samples were stored at 4°C b) Restriction endonuclease digestion and ligation Plasmid and bacteriophage vectors were digested with the appropriate restriction endonucieases accordmg to manufacturer's instructions Vector and msert DNA were mixed to a fmal volume of 5μl and were ligated with 0 5 units of T4 DNA gase Ligation mixtures were either mcubated for 3-4 hours at room temperature, or overnight at 14°C prior to tranformation mto E coli cells c) Preparation of competent E. coli cells Transformation was used to introduce plasmid and bacteriophage DNA mto competent E coli cells (Mandel and Higa, 1970, Lederberg and Cohen et al , 1972) £ coli strams to be transformed were grown overnight in LT broth at 37°C with 150 rpm shakmg The following day, a 1 /40 subculture in 20 ml of the same medium was prepared and grown for an additional 60 mm at 37°C with 75 rpm shakmg The cells were collected by centrifugation at 3,000 x g m an SS34 rotor and resuspended m 10 ml sterile ice-cold 50 mM CaCl 2 The suspension was mcubated for 30 mm on ice, then the cells were collected by centrifugation and resuspended in 2 ml sterile ice-cold mM CaCl 2 The competent cells could then be stored at 4°C and used for up to 3 days For transformation, 0 2 ml of the competent cells were mixed with the DNA sample and mcubated for 30 mm on ice The cells were heat-shocked for 2 mm at 42°C and then 0 2 ml of LT broth was added The cells were mcubated at 37°C for 15 mm then plated onto LT plates contammg appropriate antibiotics and incubated overnight at 37°C d) Large-scale plasmid isolation Large-scale plasmid isolation was performed accordmg to the procedure of Clewell and Helinski (1969) with modifications E coli carrying the plasmid was inoculated mto 250 ml LT broth contammg ampicillm and grown overnight at 37°C with 150 rpm shakmg The following day, chloramphenicol (Sigma) was added to a fmal concentration of 25 mg/1 and the culture was grown for a further 4-6 h The cells were collected by centrifugation at 4,000 x g for IC 1 mm m a GSA rotor The cell pellet was resuspended m 4 ml of an ice-cold solution contammg 25°/ sucrose and 0 05 M Tris HC1 (pH 8 0), then 1 ml of a fresh 10 mg/ml lysozyme (Sigma) solution was added The mixture was mcubated m a 37°C waterbath for 30 min, placed on ice for 5 mm and then 2 ml of 0 25 M EDTA (pH 8 0) was then added After a further 5 mm incubation on ice, 5 ml of a lytic solution (0 05 M Tris-HCl [pH 8 0], 0 0625 M EDTA and 2% Triton X-100) were added The mixture was returned to the 37°C waterbath for 5-15 mm until cell lysis was complete The mixture was then centπfuged for 30 mm at 27,000 x g and the clear lysate was transferred to a clean test tube, the lysate was mixed with solid CsCl (Boehπnger Mannheim, Laval, Quebec) at 1 g/ml to a total of 4 5 ml One hundred μl of ethidium bromide (10 mg/ml) were then added in 4 5 ml of the sample The centrifuge tube was heat sealed and the sample was centnfuged at 240,000 x g for a minimum of 9 h at 15°C m a Beckman VTι65 vertical rotor Plasmid DNA was recovered by puncturmg the top and bottom of the centrifuge tube and collecting the lower of two bands in the tube To extract the ethidium bromide from the sample, the plasmid DNA solution was mixed with an equal volume of Cs/Cl satured n-butanol After allowing the phases to separate, the upper layer contammg n-butanol and ethidium bromide was removed This process was repeated three times Followmg ethidium bromide extraction, the lower aqueous phase was dialyzed to remove the CsCl Dialysis tubmg (Fisher) with a molecular cutoff of 10 kDa was prepared by boiling 2 x 15 mm in 0 1 M Na bicarbonate and 1 x 15 m 0 25 M EDTA (pH 75) and was stored at 4°C in 50% ethanol and ImM EDTA Prior to dialysis, the tubmg was rmsed m dH 2 0 and then filled with the plasmid DNA solution The DNA was dialyzed for 24 h m 4 x IL of dialysis buffer (0 01 M Tris-HCl [pH 7 5 at 4°C], and 0 001 M EDTA) at 4°C The sample was stored at -20°C Alternatively, the Flexi-prep kit from Pharmacia (Quebec City, Quebec) was used for small-scale plasmid preparation This method involved a standard alkaline cell lysis, including RNase treatment and isopropanol precipitation (Birnboim and Doly, 1979, Isch- Horowicz and Burke, 1981) The plasmid DNA was purified and concentrated usmg a silica matrix (Sephaglas FP™) m guanidine hydrochloride e) Radiolabelling of DNA probes by random priming DNA fragments were labelled with [α- 32 P] dATP (3,000 Ci/mmol, ICN) usmg the random primer DNA labelling system of GIBCO/BRL This labelling system is based on the method of Femberg and Vogelstetn (1983), with modifications (Feinberg and Vogelstem, 1984) The sample (25 ng of DNA in 10 μl of H 2 0) was denatured by boiling for 5 mm, then immediately cooled on ice While still on ice, the followmg reagents were added 2μl of each of dCTP, dGTP and dTTP, 15 μl of random primer buffer, 4 μl [otAPJdATP and H 2 0 to 49μl The sample was mixed briefly and 3 units of Klenow Fragment was added The reaction mixture was mcubated for 1 h at 25°C and terminated by the addition of 5 μl of stop buffer The radiolabelled DNA was separated from unincorporated radionucleotides by gel filtration through a mmi Sephadex G-50 column The column was prepared m a Pasteur pipette plugged with glass wool and was equilibrated with TE buffer prior to addition of the radiolabelled sample The migration of DNA through the column was monitored usmg a Geiger counter (Mini-Instruments Ltd , Essex, England) The first peak of radioactπ ltv corresponded to the labelled DNA, while the second peak corresponded to the unincorporated [ 32 P]-dATP The DNA probe was denatured by boiling for 5 mm before bemg added to the hybridization solution f ) Agarose gel electrophoresis and Southern hybridization Agarose gels were prepared by adding TAE buffer (40 mM Tris [pH 79], ImM EDTA) to electrophoresis grade agarose powder (regular or low-melting point, Sigma) to a fmal concentration of 0 7% to 1% The agarose gel was electrophoresed m a horizontal flatbed gel apparatus (Tyler Research, Edmonton, Alberta) DNA samples were mixed with 1 /2 volume of tracking dye (50% glycerol, 0 1% ladder (Gibco/BRI), or lambda DNA (Pharmacia) digested with Hindllϊ was used as a molecular standard A runnmg buffer of TAE supplemented with 1 μg/ml of ethidium bromide was used Samples were initially electrophoresed at 100V for 5 mm, then the voltage was reduced to 10-12V for overnight electrophoresis After electrophoresis, the samples were viewed with a medium range ultraviolet transilluminator and photographed usmg Polaroid type 57 black and white film (Sharp et al , 1973, Hayward, 1972) For Southern hybridization, the agarose gel was immersed m 0 25 M HC1 for 15 mm to depurinate the DNA The gel was transferred to an alkaline solution consisting of 0 5 M NaOH and 1 5 M NaCl for 15 mm, then neutralized a solution of 0 5 M Tris-HCl (pH 7 5), 1 5 M NaCl for 30 mm The DNA was transferred to a nitrocellulose membrane (Schleicher and Shuell, Willowdale, Ontario) by electrophoretic transfer in a semi-dry blotting apparatus (Tyler Research) m 20X SSPF buffer (3 6 M NaCl, 0 2 M Na 2 P0 4 [pH 7 0], 0 02 M Na 2 EDTA, 0 16 M NaOH) at a constant current of 150 mA for 30 mmutes (Wahl et al , 1979, Southern, 1975) After electrophorectic transfer, the nitrocellulose membrane was washed m 2X SSPE buffer for 10 min, and the DNA was cross-linked by a UV Cross-lmker (Stratagene) The membrane was prehybπdized in a sealed plastic bag contammg a solution of 25% (low strmgency) or 50% (high strmgency) formamide (Gibco/BRL) m 0 1% glycine, 5X BFP (100X BFP contams 2% w/v bovme serum albumin, Ficoll and polyvinyl pyrrolιdιne-40), 5X SSPE buffer and 0 1 mg/ml sonicated, boiled salmon sperm carrier DNA The sealed bags were placed m a 42°C shakmg waterbath where the membranes were allowed to prehybπdize for at least an hour The prehybπdization buffer was then discarded and replaced with hybridization buffer (10% dextran sulphate, 5X SSPE, 5X BFP, 0 1% SDS, 0 1 mg/ml carrier DNA and 25% or 50% formamide) contammg the boiled, radiolabelled DNA probe The bags were placed m a 42°C shakmg waterbath where the membranes were hybridized overnight After hybridization, the nitrocellulose membrane was removed from the plastic bag and washed 4 x 10 min in either high stringency (5X SSPE, 0 1% SDS) or low strmgency (2X SSPE, 0 1% SDS) wash buffer in a 42 C C shakmg waterbath The membrane was air-dried, placed on Whatman filter paper, covered with plastic wrap and exposed to X-ray film (Cronex, Willingmington, Delaware) at -20°C for 104 days until the desired exposure was obtamed The exposure time was determined by measurmg the intensity of the radioactive signal usmg a Geiger counter Autoradiographs were developed in Kodak GBX rapid developer (Eastman Kodak, Rochester, New York) for 2 mm The developing reaction was stopped by immersing the film m 2 5% > acetic acid for 1 mm and fixed for 2 mm in Kodak GBX fixer (Eastman Kodak) g) Southern colony blot A master template of bacterial colonies grown on LT plus ampicillm was grown overnight at 37°C The colonies on the master plate were duplicated onto a nitrocellulose membrane overlaid on an LT plus ampicillm plate and grown for 2-3 h at 37°C The membrane was then overlaid on Whatman filter paper soaked m a 0 5 M NaOH, 1 5 M NaCl solution and mcubated at room temperature (RT) for 5 mm to lyse the cells The nitrocellulose membrane was then transferred to Whatman filter paper soaked in a 0 5 M Tπs=HCl (pH 7 5), 1 5 M NaCl solution and incubated for 5 mm at RT to neutralize the membrane The membrane was transferred to Whatman filter paper soaked m 95% ethanol and sprayed with 95% ethanol to precipitate the DNA The DNA on the membrane was cross-lmked in a UV cross-lmker (Stratagene), then prehybπdized and hybridized as described above h ) Polymerase chain reaction (PCR) PCR reactions were carried out m thin-walled 500 μl GeneAmp microfuge tubes in a Perkm-Elmer Cetus 480 DNA Thermal Cycler, usmg the Perkm-Elmer Cetus PCR core reagent kit which mcluded deoxynucleotides tπphosphates, MgCl , reaction buffer and Amph-Taq DNA polymerase (Perkm-Elmer Cetus) Amplification reactions were performed accordmg to the method of Saiki et al (1988), with modifications by Perkm-Elmer Cetus PCR reactions were performed m 100 μl mixtures contammg IX reaction buffer (0 5 M KC1, 0 1 M Tπs-HCl [pH 9 0]), 0 2 mM of each of dNTP, 0 4 μM primer, 5 μg of template, 15 mM MgCl 2 and 2 5 units of Amph- Taq enzyme The reaction mixture was heated at 95°C for 2 minutes to denature the template DNA Then 30 cycles of denaturation, annealing and extension followed with temperatures and times of 95°C (1 mm) 52°C (1 mm) and 72°C (2 min) respectively The fastest available transitions between temperatures (ramp time of 0 01s) were used A negative control which did not contam template DNA was mcluded in each PCR run After amplification, the PCR products were examined by agarose gel electrophoresis PCR products were purified by electrophoretic separation through a low- meltmg pomt agarose gel followed by excision of required DNA fragments The DNA products were purified from the agarose usmg a glass-bead matrix purification kit (GENECLEAN) i ) Purification of DNA fragments from agarose gels DNA fragments were purified from agarose gels using the GENECLEAN kit from Bio/Can Scientific (Mississauga, Ontario) The GENECLEAN purification process is based on the procedure by Vogelstein and Gillespie (1979) The gel slice contammg the fragment was excised from the gel usmg a razor blade and placed m a 1 5 ml Eppendorf centrifuge tube An equal volume of stock Nal solution was added and the sample was mcubated for 5 mm m a 55°C waterbath until the agarose had completely melted GLASSMILK (Bio/Can Scientific) was added to the sample at a volume of 5 μl foi 5μg or less of DNA and the mixture was mcubated on ice for 5 mm The silica matrix was then collected by centrifugation at 16,000 x g for 10 sec and resuspended in 600 μl of NEW wash buffer (Bio/Can Scientific) The pellet was washed with NEW buffer a total of three times After the fmal wash, the silica matrix was resuspended m lOμl of TE buffer and mcubated for 5 mm at 55°C The sample was then centπfuged and the TE recovered, avoidmg the silica matrix pellet Samples were stored at -20°C j) DNA dideoxy sequencing DNA fragments were sequenced either by cloning mto M13 mpl8/mpl9 bacteriophage vectors (single-stranded sequencmg) or directly from recombmant plasmids (double-stranded sequencmg) using the Pharmacia T7-sequencιng kit as described by the manufacturer The Pharmacia T7-sequencrng kit procedure is based on the method outlined by Sanger et al (1977) For single stranded sequencmg, DNA fragements were cloned mto the M13 mpl8/mpl9 bacteriophage vector and transformed into competent E coh TG-1 cells Recombmant phage "plaques", which appeared white due to the loss of β-galactosidase production, were selected Each plaque was moculated mto 10 ml of LT broth seeded with 0 1 ml overnight culture of E coh TG-1 grown in Davis minimal medium and mcubated 4-5 h at 37°C with 75 rpm shakmg The sample was centπfuged at 12,000 x g for 10 mm to remove the E coh cells The phage were precipitated from the culture supernatant by the addition of 1 /4 volume of 20% polyethylene glycol (8,000 MW, Sigma), 2 5 M NaCl and mcubated for 30 mm on ice Precipitated phage were recovered by centrifugation m a microfuge at 12,000 x g for 10 mm The pellet was then resuspended in 0 6 ml of phage buffer (0 1 M Tris-HCl [pH 8 0], 0 001 M EDTA, 0 3 M NaCl) The phage DNA was extrac ted with 0 5 ml of phenol (Gibco/BRL) saturated with TE buffer The phenol and aqueous phases were separated by centrifugation at 14,000 x g for 10 mm The aqueous phase was extracted with 1 1 phenol chloroform and fmally with chloroform The phage DNA was then precipitated with 1 /10 volume 3 M sodium acetate (pH 7 0) and 2 volumes of cold 95% ethanol and mcubated at -20°C overnight Precipitated DNA was collected by centrifugation at 14,000 x g for 10 mm The aqueous phase was extracted with 1 1 phenol chloroform and fmally with chloroform The phage DNA was then precipitated with 1/10 volme 3 M sodium acetate (pH 7 0) and 2 volumes of cold 95% ethanol and mcubated at - 20°C overnight Precipitated DNA was collected by centrifugation at 14, 000 x g for 10 mm in a 4°C microfuge The pellet was air-dried and resuspended m 50 μl of TE buffer and was used foi sequencmg The DNA was annealed to either the universal M13 primer or specific primers m the presence of annealing buffer (Pharmacia T7 sequencmg kit) by mcubation at 65°C for 10 mm, then room temperature for 10 mm prior to the sequencmg reactions For double-stranded sequencing, the plasmid template was prepared usmg the procedure outlined m the Pharmacia 17 sequencmg kit protocol, with modifications Plasmid DNA was ad j usted to 1 5-2 0 μg/32 μl and denatured by the addition of 12 μl of 2M NaOH for 1 mm Denaturation was terminated by the addition of 11 μl of 3 M sodium acetate (pH 5 0) The DNA was precipitated with 7 μl of dH 2 0 and 120 μl of ice-cold absolute ethanol and mcubated at -20°C overnight The DNA was collected by centrifugation at 14, 000 x g in a 4°C microfuge for 10 mm The pellet was washed with 100 μl of ice-cold 70% ethanol, centπfuged and dried under vacuum The sample was resuspended in 5 μl of dH 2 0 and mixed with 5 μl of pπmer and 2 μl of annealing buffer The mixture was mcubated at 65 G C for 5 mm, 37°C for 10 min and 5 mm at RT prior to sequencing Either [ 32 P]dATP or [ 35 S]dATP (specific activity of 3000 Ci/mmol) were used m the sequencmg reactions For short autoradiography exposure time, [ 32 P]dATP was used For superior resolution, [ 35 S]dATP was used Oligonucleotide primers were synthesized on an Applied Biosystems International 391 PCR-Mate DNA synthesizer and purified accordmg to the manufacturer's instructions The primers were quantitated by measuring the optical density at 260 nm pπor to use For [ 32 P]dATP sequencmg, the sequencmg gel consisted of 18 g of urea (ICN, Montreal, Quebec), 3 75 ml of 10X TBE buffer (1 M Tris [pH 8 3], 0 02 M EDTA, and 0 865 M boric acid) and 7 5 ml of 40% acrylamide (19 1 ratio of acrylamide bisacrylamide, Bio-Rad) made up to 38 ml with dH 2 0 The solution was stirred until the urea was dissolved and then 0 23 ml of 10% ammonium persulfate (Sigma) and 10 μl of TEMED (N, N, N'N'-Tetramethylethylenediamine, Sigma) (0 01% fmal concentration) were added for polymerization For [ 35 S]dATP sequencmg the gel consisted of 16 8 g of urea (ICN), 4 8 ml of 10X TBE buffer and 4 ml of a modified acrylamide solution ("Long Ranger", J T Baker, Phillipsburg, New Jersey) The solution was made up to 40 ml with dH 2 0 and polymerized with 200 μl of ammonium persulfate (Sigma) and 20 μl of TEMED (Sigma) The runnmg buffer consisted of IX TBE The sequencmg gel was run at 40 W/gel constant power for 2-6 hr [ 5 SJdATP sequencmg gels were transferred onto a sheet of Whatman filter paper and dried under vacuum at 80°C for 45 mm Both types of sequencing gels were exposed to Cronex 4 X-ray film (Cronex for 18-48 h at -20°C IV. PROTEIN METHODS a ) Isolation of inner and outer membranes Inner and outer membrane preparations were prepared from E coh and P haemolytica Al by the procedure of Hancock and Carey (1979), with modifications (Lo et al , 1991) Bacteria were grown m 250 ml of the appropriate medium at 37°C overnight The cells were collected by centrifugation at 4,000 x g, washed twice in 0 01 M Tris-HCl (pH 6 8) and resuspended m 7 5 ml of a cold Sucrose-Tπs solution contammg 20% sucrose, 0 01 M Tris-HCl (pH 6 8), lysozyme (lmg/ml), DNase (50μg/ml), and RNase (100 μg/ml) The cells were lvsed by French pressure cell three times at 16,000 - 18,000 psi at 4°C The sample was then centπfuged at 1,085 x g for 5 mm to remove unlysed cells The supernatant was layered onto a 70 52 sample 12% sucrose gradient which consisted of 14 ml of the 70 and 52% sucrose followed by 5 ml of the sample lysate and 4-5 ml of 12% sucrose The gradient was centπfuged in a swinging bucket rotor at 80,000 x g for 16-18 h at 4°C The inner and outer membrane fractions were collected by aspiration The inner membrane fraction was located between the 12% and 52% sucrose regions and had a yellowish-brown colour The outer membrane fraction, which was white, was located near the 70% sucrose region The collected fractions were loaded mto centrifuge tubes, topped up with dH 2 0 and centπfuged in a fixed-angle Tι80 rotor at 225,000 x g for 1 h at 4°C The pellet was then air-dried and resuspended in a Tris-HCl (pH 6 8), 0 001 M dithiothreitol buffer Samples were stored at 20°C b) Bradford determination of protein concentration The protein concentrations of the inner and outer membrane fractions were determmed usmg the method of Bradford (1976) Dilutions of each membrane fraction were prepared and dH 2 0 was added to a fmal sample volume of 0 8 ml The sample was then mixed with 0 2 ml of Bradford reagent (Biorad, Mississauga, Ontario) and incubated at room temperature for 5 mm to allow colour development to occur The optical density (OD) of each sample was meausred m a spectrophotometer at a wavelength of 595 nm A standard curve was plotted usmg bovme serum albumm (BSA, Sigma) in the 1-25 μg range The protem concentration of each sample was extrapolated from the BSA standard curve c) Sodium dodecyl sulfate polyacrylamide gel electrophoresis Proteins were analyzed using sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE) with 4% (w/v) stackmg and 75% (w/v) separating gels (Laemelh, 1970) The gels were polymerized by the addition of 0 1% ammonium persulfate and TEMED to 0 01% The samples were solubilized at 100°C for five minutes in an equal volume of 2X sample buffer A high molecular weight standard was also boiled and loaded onto the gel A discontinuous buffer system was used (0 192 M glycine, 0 02 M Tπs-HCl [pH 8 4], 0 1% SDS) The gels were run at 100V until the samples entered the separatmg gel, when the voltage was increased to 150V The samples were run until the dve front ran off the bottom of the gel The stacking gel was removed and the separating gel was either stained with Coomassie Brilliant Blue or electrophoretically transferred to nitrocellulose for Western lmmunoblotting Gels were stamed m Coomassie Brilliant Blue R250 (0 05 Vo m 40% methanol, 10% acetic acid) (Eastman Kodak) overnight and then destained in a methanol acetic acid solution d) Western immunoblot analysis The protems on the acrylamide gel were transferred to a nitrocellulose membrane according to the method of Burnette (1981) The gel was soaked m blottmg buffer (0 192 M glycme, 0 025 M Tπs-Cl [pH 8 4], 20% methanol) for 10 minutes to remove the SDS A piece of nitrocellulose membrane (Schleicher and Shuell, Willowdale, Ontario) cut to fit the gel was also soaked m blottmg buffer The protems were transferred to the nitrocellulose membrane m a Bio-Rad Transblot apparatus at 450 mA for 3 h A water-cooling system was used to prevent heatmg and breakdown of the blottmg buffer After electrophorectic transfer, the nitrocellulose membrane was soaked m 3% gelatm in TTBS buffer (0 02 M Tπs-Cl [pH 7 5], 0 5 M NaCl, 0 05% Tween-20) for 30 mm to block the membrane The nitrocellulose membrane was transferred to a 1 /500 dilution of the first antibody in 1% gelatm and mcubated overnight at room temperature with gentle shakmg The membrane was then washed twice m TTBS buffer (15 mm per wash) and placed in the second antibody solution (1/2000 dilution) for an hour The second antibody was goat anti-rabbit or goat anti-bovine IgG-alkaline phosphatase conjugate (Bio-Rad) in 1% gelatm The membrane was washed twice in TTBS buffer (15 mm per wash) and then twice (5 mm per wash) m NBT buffer (0 1 M Tπs-Cl [pH 9 5], 0 1 M NaCl, 50 mM MgCl 2 ) The membrane was then placed in the developmg solution of 100 μl of each of the reagents 5-bromo-4-chloro-3-rndolyl phosphate (BCIP, 25 mg/ml m dimethylformamide, Sigma) and nitro-blue-tetrazohum (NBT, 50 mg/ml m 70% dimethylformamide, Sigma) Colour development was allowed to proceed until the desired visibility of the bands was obtamed The colour reaction was stopped by rmsing the membrane m H 2 0 The membrane was air-dried e) T7 protein expression Protems encoded by a recombmant plasmid were analyzed usmg the method of Tabor and Richardson (1985) The tbpA gene was cloned mto the plasmid vector pBluescipt The recombmant plasmid was transformed mto E co/ιJM109 (DE3), which is a stram of E colt JM109 with the T7 RNA polymerase gene mtegrated mto the chromosome and T7 polymerase gene under the control of the lac promoter (Yanmsch-Perron et al , 1985) After transformation, the cells were grown overnight at 37°C in Davis mmimal medium contammg 1 0% casammo acids, 0 4% glucose and the appropriate antibiotics A 1/50 subculture mto 20 ml of the same medium was prepared and mcubated at 37°C for an additional 3-4 h until the OD 550 =0 6 The cells were collected by centrifugation at 14,000 x g for 5 mm and the pellet was resuspended in Davis mmimal medium with 0 4% glucose The sample was mcubated for 90 mm at 37°C, then 100 μl of 5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) were added After the cells were mcubated at 37°C for 20 mm, πfampicin was added (fmal concentration of 400 μg/ml) The sample was mcubated at 37°C for 30 mm and then labelled for 60 mm with 5 μCι[ 35 S]-methιonιne ("Trans-Label", ICN Biomedical, Quebec) The cells were washed twice in ice-cold PBS and collected by centrifugation at 14,000 x g for 5 mm in a microfuge The pellet was resuspended in 2X SDS-PAGE sample buffer The proteins were separated using SDS-PAGE and the gel was stained with Coomassie blue R250 The gel was then soaked in Amplify " (Amersham, Oakville, Ontario) for 30 mm and dried under vacuum Autoradiographs were exposed 18-48 h RESULTS I. Preliminary Cloning of PutativerbpA, tbpB genes The first stage m clonmg the tbpA gene was to screen a P haemolytica Al gene library by polymerase chain reaction An oligo primer specific for the N-termmal ammo acid sequence of the Tbpl protein was synthesized A P haemolytica Al codon table (Lo, 1992) was used to optimize the primer sequence The Tbpl primer was used in conjuction with primers based on the j unction sequences of the clonmg vector pBR322 (Table 1) A 0 8 kbp PCR product was obtamed (Figure 1) and cloned mto the M13 vector and sequenced Sequence analysis of this PCR product demonstrated that the first twenty ammo acids matched the sequence obtamed by N- terminal amino acid sequencmg of Tbpl Table 1. Oligonucleotide primers used in PCR. * - the underlined sequence is the HindlW site ** - the underlined sequence is the EcoRI site The 0 8 kb PCR product was then radiolabelled and used as a specific probe to screen the E coh clones contammg P haemolytica Al gene library by Southern hybridization Two recombmant clones, 9 and 10, hybridized strongly with the tbpA probe The plasmid DNA from each recombmant clone was analyzed by restriction endonuclease mapping (Figure 2) The insert of P haemolytica Al was determmed to be approximately 8 7 kb and 2 3 kb for plasmids 9 and 10, respectively Initial sequence analysis of the plasmids confirmed that the msert DNA from both plasmids share an overlapping region Plasmid 9 contained the entire tbpA gene but the region directly uptstream of tbp A was different than the upstream region in plasmid 10 It is possible that the msert DNA in plasmid 9 was formed from two DNA fragments from separate regions of the genome In plasmid 10, the region directly upstream of tbpA contamed an additional open read g frame which corresponded to the tbpB gene This plasmid, therefore, contamed not only the 5' region of tbp A but also part of the tbpB gene, directly upstream from the tbp A gene A third recombmant clone, 482, contained the entire tbpB gene This plasmid shares an overlapping region with plasmid 10 (Figure 2) The msert DNA m plasmid 482 is a PCR product obtamed from P haemolytica A 1 genomic DNA using primers specific for the ammo acid sequence of Tbp2 prote This PCR product was then cloned mto the vector PCRII The 3 0 kbp tbpA gene was sequenced from plasmid 9 (startmg from the Bgl site) The 2 1 kbp tbpB gene and the 91 bp sequence between tbp A and tbpB was sequenced from plasmids 482 and 10 II. Sequence Analysis The 5 2 kbp of DNA was sequenced and shown to contam two open readmg frames arranged in tandem, with tbpB upstream of tbp A (Figure 3) This genetic organization is consistent with other iron uptake systems in other bacteria where the genes often arranged in an operon (Payne, 1988) Upon sequence analysis of the deduced Tbpl protem, a putative 28 ammo acid leader peptide was observed A putative cleavage sequence for hpoprotems was observed m the deduced Tbp2 protem The close proximity of tbpA and tbpB and the absence of a promoter region m tbp A suggests that the two protems may be coordmately expressed A Fur consensus sequence in the promoter region of tbpB suggests that the protems may be regulated m a Fur-like manner The Fur consensus sequence in P haemolytica tbpB is similar to the consenses sequence found m N gonorrhoeae and N memngitidis tbpB (Figure 4) The lsoelectπc pomt of Tbpl and Tbp2 was calculated by PCGene (Chargpro) to be 9 16 and 9 71 respectively, making them basic protems III. Predicted Protein Topology The sequence analysis program Gene Runner (Hastmgs Software) was used to analyze the physical characteristics and to predict the secondary structure of the P haemolytica Tbpl and Tbp2 protems The hydropathy plot of Tbpl and Tbp2 were generated usmg the method of Kyte and Doohttle (1982) The first 28 amino acids of Tbpl form a hydrophobic region, which is characteristic of all signal sequences There are six other hydrophobic regions m the protem, which may be transmembrane domams of the protem Hydrophihc areas of the protem may be either exposed at the cell surface or m the peπplasm The hydropathy plot of N gonorrhoeae was also generated The N gonorrhoeae Tbpl protem seems to be less hydrophobic than P haemolytica Tbpl but the location of some of the hydrophobic regions are similar For example, both protems have hydrophobic regions around the 200, 400, and 780 ammo acid residues The similarity in hydrophobic regions suggests that the two protems share a significant degree of homology and may have a similar structure The Kyte-Doo ttle plot of P haemolytica Tbp2 reveals several large hydrophobic regions m the centre of the protein and two smaller hydrophihc regions at each end This is significantly different than the hydropathy plot of N gonorrhoeae Tbp suggestmg that the two protems may have different structures Surface exposed regions of Tbpl and Tbp2 were determmed usmg the Emmi surface probablility method (Emmi et al , 1985) The peaks on the graph correspond to the regions with the highest probability of being exposed Surface exposed regions of each protein may be involved in ligand bindmg and may be antigenic The Emmi plot of P haemolytica Tbpl suggests that the hydrophihc regions near ammo acids 330, 410, 460, 560, 610 and 820 of the protem may be exposed at the cell surface The Emmi plot of N gonorrhoeae Tbpl showed a few common exposed regions with P haemolytica Tbpl (regions at 330, 580, 810) The Emini plot of P haemolytica Tbp2 suggests that the hydrophihc regions at ammo acids 140, 160 and 620 have the greatest probability of bemg exposed The Emmi plot oi N gonorrhoeae Tbp2 shows that only the exposed regions at 160, 330 are similar to the surface regions of P haemolytica Tbp2 The secondary structure of both Tbpl and Tbp2 were predicted usmg the method ol Chou-Fasman (1978) The Chou-Fasman plot of P haemolytica Tbpl predicts that the protem is primarily a β-sheet and β-turn structure The Chou-Fasman plot of N gonorrhoeae Tbpl predicts a similar structure These predictions are consistent with other topology predictions of other iron-regulated outer membrane proteins, which are also composed of amphipathic β-sheets (Moeck et al , 1994) It is also mteresting to note that the location of the β-sheets correspond to the location of the hydrophobic domams in the two Tbpl Kyte-Doo ttle graphs The Chou-Fasman plot of P haemolytica Tbp2 indicates that it also consists primarily of β-Sheets and β-turns The predicted pattern of B-sheets is different than N gonorrhoeae Tbp2, which also has β-sheet regions These results add to the evidence that the Tbp2 protems have different structures IV. Distribution of tbpA in P. haemolytica and related species Southern hybridization analysis was carried out to determme whether or not all sixteen serotypes of P haemolytica carried the gene for the Tbpl protem Chromosomal DNA from each of the serotypes was disgested with restriction endonuclease and probed with the 5' end of the typA gene from P haemolytica Al Similar hybridization experiments were performed on digested chromosomal DNA from A pleuropneumoniae CM% and shope 4074 and High-stringency Southern hybridization (50% formamide) with the P haemolytica Al tbpA probe demonstrated the presence of tbpA homologous sequences m all sixteen serotypes of P haemolytica (Figures 5,6) In addition, there is a considerable difference m the size of fragments which hybridized with the probe between the A and T biotypes It is important to note that m Figure 5, there was a problem with the quality of the serotype 7 DNA, which did not give a reactive band with Southern hybridization The reaction of this serotype should be identical to that of serotype 1 A similar problem is seen m Figure 6, where serotypes 12 and 16 DNA was not properly digested The reaction of these serotypes should be identical to that of serotype 1 Low-stringency Southern hybridization (25% formamide) with the tbp A probe mdicated that A suis 3714, A pleuropneumoniae CM5 and Shope 4074 genomic DNA hybridized with the P haemolytica tbpA probe (Figure 7) The two strams of A pleuropneumoniae both belong to serotype 1 and hybridized m the same fashion A preliminary restriction map of the tbp A, tbpB regions in P haemolytica Al, A suis and A pleuropneumoniae is shown m Figure 8 V. Homology Studies The predicted ammo acid sequence of P haemolytica Tbpl was compared with the predicted sequences for the Neisseria spp and A pleuropneumoniae transferrin binding protems as well as for several E coli TonB-dependent receptor protems All of the comparisons were performed accordmg to the Higgms and Sharp algorithm (Higgins and Sharp, 1988) The predicted ammo acid sequence of P haemolytica Tbpl was found to have a high degree of homology with both the N gonorrhoeae and N memngitidis Tbpl proteins (Cornehssen et al , 1992, Legram et al , 1993) (Figure 9) The homology, mcludmg identical and conserved amino acids, was found to be 41% This result agrees with the protem topology studies which suggested that the P haemolytica and Neisseria spp Tbpl proteins share a similar structure A homology comparison between P haemolytica Tbpl and A pleuropneumoniae serotype 7 and serotype 1 TfbA protems (Gerlach et al , 1992a, Gerlach et al , 1992b) reveals only a low degree (22%) of homology (Figure 10) The degree of genetic relatedness among the Pasteurella, Neisseria, and Actinobacillus transferrin bmdmg protems is shown m the form of a dendrogram m Figure 11 It is mterestmg to note that P haemolytica Tbpl is more closely related to Neisseria Tbpl than to Actinobacillus transferrin bindmg protems P haemolytica Tbpl also has localized regions of homology with E coh TonB dependent outer membrance receptors (Figure 12) Homology with these protems implies the P haemolytica Tbpl is also a TonB dependent receptor protem The first homologous domam mcludes the TonB box, which has been implicated in the direct mteraction between TonB and the receptor protem (Bell et al , 1990) The significance of the other homologous domams is not known, however, it is possible that they are also mvolved in TonB mteraction VI. T7 expression of Tbpl T7 expression was performed m order to express the protein encoded by tbpA (Figure 13) An attempt to express tbpA by maxi-cell analysis of E co under iron-depleted and lron- repleted conditions was not successful (data not shown) The T7 expression did not produce any reactive band at 100 kDa A 30 dDa positive control is shown m lane 1 There was no difference between the plasmid carrymg tbpA and the pBluescπpt vector alone (Figure 13, lanes 2 and 3) VII. Western Immunoblot Analysis Inner and outer membrane fractions from P fωemoli/fico Al and £ coh HBlOl cells grown under iron-limiting and iron-sufficient conditions, were prepared and analyzed by Western lmmunoblottrng The purpose of these experiments was to determme whether or not the iron-regulated protems would react antigenically with antiserum prepared agamst the soluble antigens of P haemolytica The inner and outer membrane fractions were im unoblotted with rabbit "anti-autologous" antiserum which was raised against the soluble antigens of P haemolytica Al cultured m RPMI 1640 that had been supplemented with the rabbit's own serum (to avoid inclusion of antibodies to serum protems) The antiserum was preabsorbed with E coh HBlOl cells in order to minimize reactivity with E coh antigens Immunoreactive bands corresponding to the transferrm bmdmg proteins were not observed in the outer membrane fraction from P haemolytica Al cells grown under iron-limitmg conditions (Figure 14, lane 3) The mner and outer membrane fractions were also immunoblotted with the serum from a calf vaccinated with Presponse® as a first antibody (Figure 15) The antiserum was preabsorbed with E coh HBlOl cells to limit the number of E coh immunoreactive Bands of 71, 77 and 100 kDa were observed m the outer membranes of P haemolytica cells which were grown under iron-limiting conditions (Figure 15, lane 3) These protem bands correspond to the size of the P haemolytica transferrm bmdmg proteins If these antigenic bands are the transferπn- bmdmg protems then this result suggests that these peptides are antigenic and also immunogenic in cattle DISCUSSION I. Preliminary Sequence analysis The preliminary nucleotide sequences of P haemolytica tbpA and tbpB are shown m Figure 3 The promoter region of P haemolytica tpbB is shown in Figure 4 Upon preliminary sequence analysis of the cloned DNA, the two tbp genes were found m tandem with tbpB directly upstream of tbpA This genetic organization is consistent with iron uptake systems in other bacteria such as Neisseria spp where the genes where are arranged in an operon (Anderson at al 1994) It is likely that the genes mvolved m P haemolytica Al iron uptake are also arranged in an operon The tbpA gene has only a πbosomal bmdmg sequence whereas the tbpB gene is preceded by a πbosomal bmdmg site and has a Fur consensus sequence m its promoter region The presence of a putative Fur consensus sequence implies that the two genes could be coordmately regulated by iron concentrations and that a Fur homolog exists m P haemolytica Al Fur homologs have been cloned and sequenced m pathogenic Neisseria Spp (Bensh et al , 1993, Thomas and Sparling, 1994) If a P haemolytica Al Fur homolog exists, it may be mvolved m the regulation of other antigens such as the leukotoxin Strathdee and Lo (1989) reported that under iron-limiting conditions, there was a decrease m the amount of leukotoxm produced This is the opposite of the situation in the diptheπa tox (Boyd et al , 1990) where toxin production mcreases when the cells are grown under iron-limitmg conditions It is possible that Fur acts as a positive regulator m P haemolytica leukotoxin production This would be consistent with the earlier observation by Gentry et al (1986) of increased toxm production m iron contammg media N memngitidis also produces iron-regulated proteins which are related to the RTX family of exoprotems (Thompson et al , 1993) The first 28 predicted ammo acids in the tbp A sequence form a putative signal sequence A signal sequence is essential for insertmg the precursor protem mto the membrane during the process of translocation across the membrane The signal sequence also acts as a recognition site for the proteolytic cleavage of the precursor protem into its mature form (vor Hei j ne, 1983, Benson and Silhavy, 1983) The presence of a signal sequence confirms that Tbpl i' located beyond the cytoplasmic membrane, but it does not contam anv sortmg mformation The predicted ammo acid sequence of Tbpl has a termmal phenylalanme residue at the carboxyl- termmal of the protein Phenylelanine is a hydrophobic aromatic ammo acid which facilitates the partitioning of the hydrophobic environment m the membrane The presence of a termmal phenylalanme residue has been shown to be important for outer membrane localization (Struve et al , 1991) and suggests that P haemolytica Tbpl is located m the outer membrane Sequence analysis of tbpB revealed a putative cleavage sequence for hpoprotems Lipoprotems have a characteristic cleavage sequence of Leu-X-Y-Cys, where X and Y are small neutral ammo acids (Wu, 1987) This suggests that Tbp2 is processed and lipid modified It is mterestmg to note that Tbp2 lacks a termmal phenylalanme residue which is mvolved m outer membrane localization Analogous transferrin binding hpoprotems have been found m H influenzae, N gonorrohoeae and N memngitidis (Legain et al , 1993, Anderson et al , 1994) Griffiths et al (1993) has demonstrated common antigenic domams among the Tbp2 protems in N gonorrohoeae, N memngitidis and H influenzae type b The lsoelectπc point (pi) of a protem is defmed as the pH at which the peptide has a net charge of zero The pi calculation assumes that there are no three-dimensional structures which interfere with the lonization states Therefore, calculated pi values are only approximate values and may differ from experimental results The pi of P haemolytica Tbpl and Tbp2 has been calculated to be 9 16 and 9 71, respectively It has been suggested that cationic polypeptides enhance in vivo membrane interactions It is possible that the basic nature of Tbpl enhances interaction with the host transferrin protems The Neisserial Tbpl (Cornehssen et al , 1992) and Fbp (Beπsh et al , 1990) protems are also basic protems In Legionella pneumophiha , basic surface proteins act to inhibit phagolysosomal fusion (Cianociotto et al , 1989) II. Preliminary Predicted protein topology The hydropathy plots of Tbpl and Tbp2 were generated usmg the method of Kyte and Doohttle (1982) In the hydropathy plot of P haemolytica Tbpl, the first peak is located from the ammo acids 1 to 30 This represents the hydrophobic core which is common to all signal sequences (Hayashi and Wu, 1990) The other hydrophobic regions may be transmembrane domains Hydrophihc domams may be regions of the protem which are exposed to either the cell surface or to the peπplasm The location of the transmembrane regions m the P haemolytica Tbpl protem is similar to many transmembrane regions predicted m N gonorrhoeae Tbpl This suggests that the Tbpl protems may have a similar structure and that they share a certam degree of homology at the ammo acid level The P haemolytica Tbp2 protem possesses a hydrophobic leader sequence as well as several large hydrophobic regions m the centre of the protem and it is significantly different than the hydropathy plot predicted for N gonorrhoeae Tbp2 This implies that both protems are structurally different Surface exposed regions of Tbpl and Tbp2 were predicted usmg the Emmi surface probability method Regions of Tbpl and Tbp2 which are exposed to the cell surface may be mvolved m ligand mteraction and may be antigenic The Chou-Fasman method is commonly used m predictmg the secondary structure of a protem This method is based on the tendency each ammo acid has for being m an a-helix, a β- sheet or a -turn The Chou-Fasman method predicts that P haemolytica Tbpl consists of many /J-sheets and -turns It is possible that these β-sheets cross the outer membrane repeatedly and that the mtervenmg sequences constitute surface or peπplasm exposed loops The Chou-Fasman method also predicts that N gonorrhoeae Tbpl also consists of β-sheets This structure has already been proposed for E coh outer membrane protems such as FepA (Moeck et al , 1994) P haemolytica and N gonorrhoeae Tbpl protems share a common structure which implies that they may also have a similar mechanism of removmg iron from the host transferrm molecule The Chou-Fasman plot of P haemolytica Tbp2 also predicts a predominantly /3-sheet and /ϊ-tum structure which is significantly different than the prediction for N gonorrhoeae Tbp2 III. Distribution of tbpA in P. haemolytica and Related Species Southern hybridization of the genomic DNA of the sixteen P haemolytica serotypes with the tbpA probe demonstrated that a highly homologous gene is present withm the A biotype The results also suggest that the genetic organization or the tbpA gene is significantly different in the T biotypes This supports the observations by Murray el al (1992) who demonstrated that iron-regulated proteins from the A and T biotypes were antigenically distmct Previous work on P haemolytica antigenic determmants demonstrated that the sialoglycoprotease, the serotype specific antigen, three hpoprotems and a LPS biosynthetic gene were either missmg or had a different genetic organization m the T biotype (Burrows, PhD thesis, 1993) The A and T biotypes do share phenotypic and biochemical traits (Holt, 1977), but they are only distantly related based on DNA DNA hybridization (Bingham et al , 1990) Sneath and Stevens (1990) proposed that the biotype T serotypes be renamed as the species P trehalosi Diversity m the genetic organization of transferrin bmdmg protems has also been demonstrated m A pleuropneumoniae TfbA (Gonzalez et al , 1990, Gerlach et al , 1992b) and N memngitidis Tbp2 (Legram et al , 1993, Rokbi, 1993) In A pleuropnuemomae, serotype 1 and serotype 7 TfbA proteins share only 55% homology at the ammo acid level (Gerlach et al , 1992b) N memngitidis Tbpl proteins are divided mto two classes based on their molecular weight, sequence similarity and antigenic heterogeneity (Robki et al , 1993) The diversity m transferrin bindmg proteins within a species may facilitate the bmdmg of different serotypes to avoid the host immune response agamst heterologous strams (Gerlach et al , 1992b) Southern hybridization experiments demonstrated that chromosomal DNA from A pleuropneumoniae strams CM5 and Shope 4074 hybridized with the tbp A probe only under low- stringency conditions This suggests that P haemolytica and A pleuropneumoniae transferrm bmd g protems share only a low degree of homology This result was confirmed by homology studies on the ammo acid sequence from both P haemolytica Tbpl and A pleuropneumoniae TfbA protems The A suis genomic DNA also hybridized with the tbp A probe, which suggests that it mav have an analogous transferrin bmdmg protem The results also suggest that A suis transferrm bmdmg protems may be more closely related to the Tbpl and P haemolytica than to A pleuropneumoniae TfbA IV. Homology Studies All of the protem sequences were aligned by PCGene (Clustal), which compares sequences accordmg to the method of Higgms and Sharp (1988) The first step m this method is to calculate all pairwise sequences similarities A dendrogram is then generated from the similarity matrix generated m the first step The dendrogram in Figure 11 was generated by a Higgms and Sharp alignment of the Pasteurella, Actinobacillus, and Neisseria transferrin bmdmg protems a) Neisseria spp. The predicted ammo acid sequence of P haemolytica tbp A has regions of homology with the predicted ammo acid sequence of tbpA m JV gonorrhoeae This suggests that the transferrm bmdmg protems are structurally similar and agrees with the observations made m the protein topology studies Ogunnaπwo and Schryvers (1990) reported that P haemolytica Al Tbpl was similar to N gonorrhoeae Tbpl protems in size and properties Both species produce 100 kDa receptor proteins which cannot bind transferrm after SDS-PAGE, which suggests that the conformation of the native protein is important in transferrm bindmg However, the two protems differ in their bindmg specificities N gonorrhoeae Tbpl bound only human transferrm whereas P haemolytica Al Tbpl bound only bovme transferrm This suggests that differences between the two tbpA sequences may be regions which encode for specificity of iron source b) Λ. pleuropnuemomae The predicted amino acid sequence of P haemolytica Al Tbpl has a low degree of homology with the sequence of A pleuropneumoniae TfbA This result is confirmed by the Southern hybridization experiments, which demonstrated that chromosomal DNA from A pleuropneumoniae strams CM% and Shope 4074 hybridized with the tbpA probe only under low- stringency conditions This is interesting because both bacteria belong to the family Pasteur ellaceae and would therefore be expected to have a similar transferrin bmdmg protem Previous work has suggested that the two proteins are functionally similar but structurally different The TfbA protein in A pleuropneumoniae has been shown to be a hpoprotein (Gonzalez et al , 1990), whereas the 100 kDa P haemolytica Al Tbpl is not (Ogunnaπwo and Schryvers, 1990) A pleuropneumoniae is able to distinguish between iron-saturated and iron- depleted transferrm) (Gerlach et al , 1992a), whereas N memngitidis cannot (Tsai et al , 1988) It is mterestmg to note that A pleuropneumoniae TfbA has homology with N gonorrhoeae Tbpl, which is also a poprotem This suggests that the TfbA protem is analogous to Tbp2, and that Tbpl of A pleuropneumoniae has not yet been identified c) TonB dependent-receptor proteins The P haemolytica Tbpl sequence also has ammo acids which are common to a group of E coh TonB dependent receptor proteins This finding suggests that P haemolytica Al belongs to this family and that a TonB homolog exists in Pasteurella species The first homologous doma or "TonB box" has been implicated m direct mteraction between the receptor protein and TonB (Bell et al , 1990, Brewer et al , 1990) The significance of the other homologous regions is not known, but they may be required for TonB interaction or may be necessary for outer membrane localization P haemolytica Tbpl, like many other TonB- dependent proteins, is a transmembrane protem which is iron-regulated and mvolved m iron utilization (Mietzner and Morse, 1994) It is possible that P haemolytica Tbpl functions as a gated channel as has been proposed for £ coh FepA (Rutz et al , 1992) Tbpl from both N gonorrhoeae (Cornehssen et al , 1992), and H influenzae (Jarosik et al , 1994) also belong to the family of TonB-dependent receptor protems V. Proposed Model for P. haemolytica Iron Uptake The existence of many analogous proteins in Neisseria, Pasteurella and Haemophilus suggests that a common mechanism may be utilized for iron acquisition A hypothetical model of iron acquisition has been proposed for Neisseria (Chen et al , 1993) which may be used as a model for P haemolytica Al Iron deprivation activates transcription of the iron-regulated proteins by a Fur-like regulatory system Host transferrm bmds to the bacterial cell surface via a specific iron receptor complex composed of two or more protems The iron is removed from the transferrm and transported across the outer membrane of the bacterium with the energy provided by TonB In the peπplasm, the iron is transiently complexed to a peπplasmic component, Fbp, which transports it to a cytoplasmic membrane permease The iron is transported across the cytoplasmic membrane by a peπplasmic binding protein transport system In the cytoplasmic the iron is reduced to Fe 2" " " and assimilated by the cell One feature which may be unique to P haemolytica Al iron uptake is the presence of a third iron-regulated outer membrane protem (71 kDa) which may form part of the receptor complex (Ogunnaπwo and Schryvers, 1990) In addition, P hemolytica does not have a receptor protem which is capable of bmdmg transferrm after SDS-PAGE and electroblottrng, while N gonorrhoea does (Schryvers and Morris, 1988) This suggests that the bindmg mechanism of P haemolytica receptor complex may be slightly different than the receptor complex in N gonorrhoea Protems which are similar to N gonorrhoea Fbp have been identified m the family Pasteurellacease In H influenzae, a 40 kDa peπplasmic protem was identified and its N- terminal sequence was found to be 81% homologous to N gonorrhoea Fbp (Harkness et al , 1992) In P haemolytica A3, 35 kDa peπplasmic iron-regulated protem has been described but no function has been found (Lainson et al , 1990) In addition, a 37 kDa iron regulated protem has been isolated by affmity procedure from P haemolytica Al (Ogunnaπwo and Schryvers, 1990) Based on size and location similarities, it is possible that both of these protems are analogous to N gonorrhoea Fbp VI. T7 Protein Expression T7 RNA polymerase-dependent production of a Tbpl gene product m E coh JM109 (DE3) was not successful (Figure 13) One possible explanation may be that the πbosomal bmdmg site of tbpA was inefficient The gene could perhaps be cloned mto a vector that carries a functional πbosome-brnding site Alternatively, the Tbpl protem may be unstable and requires the presence of other protems or factors in order to be correctly produced Components of heterodimeπc protems are often unstable when they are synthesized singly VII. Western Immunoblot Analysis Western l munoblots were performed on the inner and outer membrane fractions from P haemolytica Al cells which were grown under iron-sufficient or iron-limiting conditions (Figures 14 and 15) The iron-limiting conditions were simulated by addmg the iron chelator EDDA, which is a common synthetic iron chelator used to limit the availability of iron in culture media EDDA was chosen for these studies because of its specificity for iron and its lack of toxic side effects to bacteria (Neilands, 1981) P haemolytica Al membrane fractions immunostained with rabbit antiserum to soluble antigens did not react with the iron-regulated protems (Figure 14) Neither the 100 kDa nor the 77 kDa iron-regulated proteins were observe m this immunoblot, possibly because the original P haemolytica Al culture (used m the hyperirnmunization of the rabbit) was not grown under iron-restricted conditions The medium used contamed 7% serum to avoid the mclusion of antibodies of serum protems In contrast, peptides which may be the iron-regulated protems reacted with antisea from calves vaccmated with the Presponse® (Figure 15) Presponse® is produced from P haemolytica A 1 cells which are grown to late log phase m serum-free RPMI medium 1640 (Shewen and Wilkie, 1987, Shewen et al , 1988) It is possible that the low iron concentration of this medium mduced production of the transferrm bmdmg protems It is also possible that the calf responded to transferrm bmdmg protems produced by P haemolytica which are commensal organisms m the nasopharynx The presence of antibodies to transferrm bmdmg protems suggests that these protems are immunogenic EXAMPLE 2 Bacterial strains The bacterial strams used in this study are listed in Table 2 P haemolytica strains hl73, hl74, hl75 and hl76 were field isolates from ruminants with pneumonic pasteurellosis and were provided by Dr Frank Milward, Rhone Meπeux, Lyon, France P haemolytica strains h44-h46 were bovme clinical type Al isolates from bovine pneumonia obtamed from S Lundberg ,Veteπnary Laboratory, Regional Agricultural Building, Airdπe, Alberta h44 has been described previously (26) P haemolytica strains h93-h97 were bovme clmical type Al isolates from bovine pneumonia obtained from by Dr A Potter of the Vetermary and Infectious Diseases Organization (VIDO), Saskatoon Strains h98-hl07 are ATCC P haemolytica strains (5) also obtained from Dr A Potter A ctin obacill us (Haemophilus) equuh strain h50 was obtained from Dr Jane Pπtchard, Vetermary Laboratory, Regional Agricultural Building, Airdπe, Alberta new species, P trehalosi (34) T-type strams are now considered as a new species, P trehalosi (34) Growth conditions. All bacterial strains were stored frozen at -70°C m 30% glycerol Isolates from the frozen stocks were streaked onto chocolate agar plates and mcubated at 37°C m a 5% C0 9 mcubator Iron-restricted growth was achieved by growmg the bacteria m Brain Heart Infusion broth (BH1, Difco Laboratories) or O'Reilly Niven broth (25) supplemented with 2 μg/ml thiamme monophosphate and 3 μg/ml nicotmamide aden e dinucleotide (NAD) and contammg the iron chelator ethylenediammedihydroxyphenylacetic acid (EDDHA, Sigma) at a fmal concentration of 100 μM Growth experiments for use of different transferrms as iron source was performed as previously descπbed (26) Preparation of transferrms and derivatives. Bovme transferrm was obtained from Sigma The preparahon of equme (horse), ovme (sheep) and caprme (goat) transferrms (2), the iron loadmg of transferrms to 30% or 100% saturation (22) and conjugation of horse-radish peroxidase (HRP) to trannsferπn (37) was essentially as described previously In the preparation of conjugates of bovme, ovme, caprme and equme transferrms (HRP-bTf, HRP-oTf, HRP-cTf and HRP-eTf), the mixture of HRP and transferrm were subjected to gel filtration after chemical conjugation The fractions demonstratmg maximal activity were pooled, dialyzed and aliquots frozen and stored at -70°C Solid-phase binding assays. The solid phase bindmg assay was essentially derived from methods described previously (32) Aliquots of mtact cell suspensions or crude total membrane preparations were spotted onto nitrocellulose/cellulose acetate membranes (HA paper, Millipore Corporation, Bedford, MA) and after drymg the HA paper was blocked with buffer contammg 0 5% skim milk (blockmg solution) For the transferrm bmdmg assay, the paper was exposed to blockmg solution contammg 450 ng/ml of the HRP-conjugated transferrm, washed and developed with HRP substrate mixture essentially as previously described (32) For assessment of bmdmg of anti-receptor antibody by mtact cells a similar procedure was utilized except that the first bmdmg solution contamed a 1/1,000 diluhon of the anti-TbpA and anti-TbpB antisera and, after washing, the membrane was exposed to a second bmdmg solution contammg a 1/3,000 dilution of a HRP-conjugated goat anti-rabbit antibody preparation Affinity isolation of transferrin bindmg proteins (TbpA and TbpB). Bovine, ovme, caprme and equme transferrms were individually coupled to CNBr-activated Sepharose 4B accordmg to the manufacturers instructions usmg solutions contammg 3 5 mg/ml of iron-saturated transferrm Activated groups were blocked by addition of ethanolamine Noncoupled transferrin was removed by washmg with 10 to 20 column volumes of a 50 mM TπsHCl, 1 M NaCl, pH 8 0 buffer contammg 6 0 M guanidine hydrochloride and after further washing the bound transferrm was reloaded with iron using a solution contammg 5 μg/ml FeCl 3 in 0 1 M sodium cιtrate/0 1 M NaHCO^ pH 8 6 buffer Iron-deficient total membrane (200 mg protem) from P haemolytica or A equuh prepared as previously described (32) was diluted to 2 mg/ml m 50 mM Tπs pH 8 0 contammg 1 0 M NaCl The diluted membrane was solubihzed by addition of EDTA and sarkosyl to a fmal concentration of 10 mM and 0 75%, respectively followed by mcubation of the mixture at room temperature for 15-30 mm with gentle rockmg The solution was centπfuged at 10,000 rpm for 10 mm to remove insoluble debris The supernatant containing the solubihzed membrane was applied to a 1 5 x 10 cm transferrm-affmity column and then washed extensively (at least 10 bed volumes) with 50 mM Tris pH 8 0 contammg 1 0 M NaCl, 10 mM EDTA, 0 75% Sarksosyl to remove non-specifically bound protem In experiments using low salt washing conditions the washmg buffer contamed 100 mM NaCl in lieu of 1M NaCl In some instances, additional washmg with 2-3 bed volumes of washmg buffer contammg 0 2 M guanidme hydrochloride was necessary to remove contaminating protems Coelution of both transferrm bmdmg protems (TbpA and TbpB) was achieved by application of 2-3 bed volumes of 2 0 M guanidme hydrochloride m 50 mM Tris pH 8 0, contammg 1 0 M NaCl, 1 mM EDTA, 0 01% sarkosyl The eluant was collected for immediate dialysis agamst 50 mM Tris pH 8 0 Further treatment with higher concentrations of guanidme hydrochloride usually did not result in any further yield of receptor protein Individual isolation of TbpA and TbpB was attamed by sequential elution with 2 bed-volumes of each buffer contammg 0 2, 0 5, 0 75, 1 0, 1 5, 2 0 and 3 0 guanidme hydrochloride, respectively The eluates were dialyzed agamst 3 changes of 3 litres 50 mM Tris pH 8 0 over an 18-hour period and concentrated by ultrafiltration After SDS-PAGE analysis the fractions from the 0 5 and 0 75 M guanidme HCl elution buffers were pooled for a preparation of TbpB and fractions from the 1 5 and 2 M guanidme HCl elution buffers were pooled for a preparation of TbpA Analytical methods. Protem samples were analyzed by SDS-PAGE followed by silver stam g as previously described (32) For Western blot analysis, about 1-2 m of purified receptor protems or 40 m of outermembrane protem from iron-poor cells were separated on 10% polyacrylamide gels Protems were electrophoretically transferred to nitrocellulose (Millipore, Bedford, MA) overnight at 15V m 20 mM Tris, pH 7 5, 150 mM glycme, 20% methanol and 0 1% SDS The filters were blocked with 0 5% skim milk m 20 mM Tris pH 7 5, 500 mM NaCl (TBS) for 30 mmutes at room temperature A 1/300 dilution of the appropriate antibody in the blockmg solution was applied to the paper for 1 hour at room temperature followed by two, 10-mιnute washes each with TBS A 1 /3000 dilution of secondary antibody (goat anti-rabbit IgG-horse- radish peroxidase conjugate from BioRad) was allowed to bind for 1 hour at room temperature The conjugate was removed by three, 10-mιnute washes m TBS and developed usmg the HRP- substrate mixture Comparison of receptor specificity: Prior studies had demonstrated differences in specificities towards different ruminant transferrms (I e cattle, sheep and goat) by transferrm receptors from various pathogenic bacterial species of rummants (38) This probably reflects differences in the regions of the receptor protems mvolved m ligand bmdmg and thus suggests that these regions could not serve as the basis of a broad-spectrum transferrm receptor-based vaccme for rummant pathogens However, it does not preclude the possibility that a group of related rummant pathogens, such as the various Pasteurella species, may have common ligand bmdmg domams that could provide the basis for generation of a cross-protective response Thus it was important to determme whether the transferrm receptors from a collection of representative Pasteurella isolates possessed the same specificity for rummant pathogens As a preliminary analysis of receptor specificity, a collection of representative isolates were assessed for their ability to utilize various rummant transferrms as a source of iron for growth (Table 2) A simple plate assay described in the methods section was utilized The growth of all the representative rummant isolates of Pasteurella haemolytica and P trehalosi was stimulated by Fe-saturated transferrms from rummant (bovme, caprme and ovme) but not from non-rummant (equme) hosts The stimulation of the growth of the equme pathogen, Actinobacillus equuh (stram h50), by equme transferrin mdicated that the inability of the P haemolytica strams to use equme transferrm as iron source was not due to deficiencies m the preparation Table 3: Growth on different transferrins. eTf As a further assessment of the receptor specificity, bmdmg of transferrin by mtact cells or isolated membranes was assessed by a simple binding assay utilizmg horse-radish peroxidase (HRP) conjugates of transferrm Conjugates were prepared from bovme, ovme and caprme transferrin and then tested for their ability to bind to total membranes isolated from iron-deficient cells of several representative strams of P haemolytica and P trehalosi The results illustrate that all the selected strains were capable of binding the three rummant transferrms (bovme, caprme and ovme) but not equme transferrm (Figure 16), which is consistent with the results of the growth studies (Table 3) To confirm that the observed bmdmg by all three rummant transferrms was due to the same receptor m the selected species, competitive bindmg assays were performed in which the ability of unlabelled rummant transferrms were tested for their ability to block binding of the labelled transferrins In these experiments reciprocal inhibition by the various rummant transferrms was equally effective, mdicatmg that they bound to the same receptor with similar affinities (data not shown) The results of the growth and bmding studies suggested that bovme, ovme and caprme transferrms were capable of mteractmg with the receptor components mvolved m iror acquisition in P haemolytica The affmity procedures described in the methods section were used to identify the proteins mteractmg with the rummant transferrms by employing bovme caprme or ovme transferrm-sepharose resms As illustrated m Figure 17, a predominant receptoi protein of approximately 100,000 molecular weight was isolated with membrane preparations from the bovine isolate (h44), the caprine isolate (hl73) or the ovme isolate (hl75) when eithei bovme (lanes A and B), ovme (lane C) or caprme (lane D) transferrm affmity columns were used This protein is analogous to receptor protems of similar size that are found m other bacteria] pathogens (18,27,30,31), which have conventionally been termed transferrm bmding protem 1 (Tbpl) An alternate name, TbpA has been recommended (21)to be consistent with existing conventions of nomenclature A second protem of approximately 60,000 molecular weight was also evident m the samples isolated by affmity chromatography with the rummant transferrms (lanes B, C and D using membranes from the bovine isolate (h44) This protem is comparable to the lower molecular weight receptor protein, transferrin binding prote 2 (Tbp2), isolated from other pathogenic bacterial species (18,27,30,31) For reasons alluded to above, the alternate name, TbpB, has been recommended (21) A protem of this molecular weight is also detectable m mosl samples obtamed with the caprine (hi 73) and ovine (hi 75) isolates but the presence and yield of this component was sensitive to the conditions of isolation The characteristically low yielc of TbpB (Tbp2) relative to TbpA (Tbpl) observed m these species is not a general property of the bacterial receptor protems and may even reflect common properties of TbpB from related species Neither of the protems were isolated when equme transferπn-Sepharose was usec m the affmity isolation procedure (lane E) indicating that their isolation was specifically due to the presence of ruminant transferrm When less strmgent washing conditions were used during the affinity isolation procedure, additional proteins of approximately 38,000 and 70,00C molecular weight were retained by the affinity column (lane A) when membranes from the bovine (h44), caprine (hl73) or ovine (hl75) isolate were used An additional protein o approximately 77,000 molecular weight was also evident in the sample obtained with membranes from the bovme isolate Comparison of the immunological properties of the receptor proteins. The observation that bovme, caprine and ovine transferrins compete for the same receptors suggested that there is conservation at least m the bmdmg domam of the receptors Ir order to determine whether there was also a similarity with respect to presence of common immunological epitopes, antisera were prepared against purified receptor protems from one strain to evaluate their crossreactivity with receptor proteins from other isolates Affinitv puπfied preparations of TbpA and TbpB were obtamed from stram h44 (see methods section) and used for generation of monospecific antisera m rabbits These antisera were then tested agamst receptor protems isolated from representative strams of different serotypes including isolates obtained from cattle, sheep and goats The results in Figure 18, Panel A demonstrate that the anti-TbpB antisera reacted strongly with a protein of approximately 60,000 molecular weight (TbpB) that was affmity isolated with bTf-Sepharose from all of the representative strams Similarly, the anti-TbpA antisera crossreacted with TbpA isolated from all seven representative strams (Figure 18, Panel B) Extension of this analysis to the additional serotypes of ruminant isolates (Table 2) contmued to show considerable cross-reactivity with both receptor protems (data not shown) These data suggest that both receptor proteins are conserved amongst the different serotypes of P haemolytica causmg pneumonic pasteurellosis in cattle, sheep and goats Although the immunological cross-reactivity illustrated m Figure 18 mdicates that there are conserved epitopes in receptor protems from different species, there is no indication whether any of these epitopes are exposed at the bacterial surface, where they could serve as effective targets for the host immune effector mechanisms In order to address this issue, a solid- phase bindmg assay was used to assess the bmdmg of antireceptor antibodies by intact cells This assay demonstrated that there was strong bmdmg by cells grown under iron-deficient, but not iron-sufficient conditions, when a selection of bovme type Al isolates were tested (data not shown) When a selection of sheep isolates of varymg serotypes were tested, there was a variable degree of reactivity (Figure 19) Other serotypes of type A P haemolytica strams (h98 and hi 05, Figure 19) showed considerable reactivity agamst the anti-TbpA and anti-TbpB antisera In contrast, the T-type strams (P trehalosi, h99, hlOO and hi 06) showed only very weak reactivity against both of the anti-receptor antisera However, the fact that there was also weak bmdmg by labelled bTf indicates that there was limited production of receptor protems under the iron-deficient growth conditions used in this experiment Thus the lack of reactivity of the anti-receptor antisera cannot be attributed to a lack of surface-exposed, cross- reactive epitopes m the receptor protems from these species EXAMPLE 3 Cloning of the transfemn receptor genes from a type Al strain. The followmg materials and methods were used in the studies described m the example MATERIALS AND METHODS Bacterial, plasmids, phages and culture conditions. P haemolytica and E coh strams were from the mventors' laboratory collections The plasmid clone bank of P haemolytica Al DNA in pBR322 has been described (Lo et al , 1985) The 1 clone bank contammg P haemolytica Al DNA was obtained from G Weinstock P haemolytica Al strain H196 was from the Vetermary Infectious Diseases Organization (VIDO, Saskatoon, Saskatchewan, Canada) All bacterial strams were stored frozen at -70°C m 30% glycerol Isolates from the frozen stocks were streaked onto chocolate (P haemolytica) or Luπa-Bertani plus antibiotic (E coh) agar plates ami mcubated at 37°C m a 5% C0 2 mcubator PCR amplification.The primers for PCR were synthesized m an Applied Biosystems Model 390 Ξ Synthesizer and purified according to manufacturer s mstructions PCR was carried out m thin- walled 500 ml tube in a Perkm-Elmer Cetus 480 Thermal Cycler usmg the PCR coreagents and Taq DNA polymerase as recommended The PCR conditions consist of 95° C for 2 mm , followed by 30 cycles of denaturation, annealing and extension at 95° C (1 mm ), 52° C (1 mm ) and 72° C (2mm ) respectively A negative control which did not contam template DNA was mcluded in each PCR run Mapping of tbp region in the genome. Genomic DNA from P haemolytica Al were digested with a number of restriction enzymes, separated by agarose gel electrophoresis, blotted onto nitrocellulose membrane and hybridized with DNA probes specific for the different regions of tbp A or tbpB as described The restriction maps were compared with that obtamed from the recombmant plasmids as well as the sequenced regions to verify the correct positions of the tbp genes Preparation of transferrins and derivatives. Bovme transferrm (bTf) was obtamed from Sigma The apo-form of bTf was produced followmg a procedure described elsewhere (Mazuπer and Spik, 1980) Briefly, bTf was dissolved to a concentration of 0 5 1 0 % in 0 1 M Na acetate, 0 1 M Na phosphate, 25 mM EDTA, and adjusted to (pH 5 5) by addmg drops of concentrated glacial acetic acid The solution was equilibrated overnight at 4°C, and the iron removed using an acrylamide gel column equilibrated with the Na acetate/Na phosphate low pH buffer The low pH buffer was exchanged usmg a acrylamide gel column equilibrated with 50 mM Tπs HCl (pH 7 5) Finally, the protein was concentrated usmg Amicon filter The N-and C-termmal derivatives of bTf were produced as described (Yu and Schryvers, 1994) Briefly, 80 mg of ConA purified bTf were digested with 2 mg protemase K in 40 ml 0 1 M Tris-HCl (pH 8 2), 25 mM CaCl 2 at room temperature for 20h To stop the reaction, phenylmethylsulfonyl fluoride (PMSF) was added to 0 1 mg/ml Five ml of concentrated preparation were applied to a Sephadex G-100 column equilibrated with 50 mM Tris-HCl (pH 8 0) , the N-lobe and C-lobe fractions wen 3 dialyzed agamst 50 mM Na acetate (pH 6 9), ImM CaCl 2 , 1 mM MgCl 2 , 1 mM MnCl 2 and applied to a ConA-Sepharose column (which bmds glycosvlated C-lobe but not N-lobe of bTf) Eluants from the column washed with the buffer just described were retamed as the N-lob containing fraction The C-lobe contammg fraction was eluted by using the same buffer containing 0 2 M methyl-a-D-mannopyranoside Both C-lobe and N-lobe fractions wer 3 dialyzed agamst 50 mM Tris-HCl (pH 8 0), concentrated bv ultrafiltration, and frozen as aliquots at 70°C Expression of recombinant receptor protein. E coh strams carrymg the appropriate recombmant plasmid (DH5αF/pCRIIPHtbpB for TbpB and DH5αF/pCRIIPHtbpA for TbpA) were used to moculate 50 ml LB-broth starter cultures contammg 0 2% maltose and 150 mg/ml ampicillm After growth at 37°C for several hours the cultures were used to moculate 1 liter of the same medium to a startmg OD 600 of 0 05 Once the OD 600 reached 0 4, glucose was added to 4 mg/ml and grown until OD 600 reached 0 7-0 8 At that time MgS0 4 to 10 mM and 100 ml of a 10 10 pfu/ml 5 suspension of CE6 1 phage were added The cell culture was mcubated for an additional 2 hrs at 37°C and then harvested b centrifugation The cell pellet was resuspended in 5 ml of ice cold 50 mM Tris-HCl pH 8 0, 1 M NaCl for affinity isolation, SDS-PAGE, and Western Immunoblot analysis Affinity isolation of transferrin binding proteins and analytical methods. Bovme transferrm 10 was coupled to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer s instructions usmg solutions contammg 3 5 mg/ml of iron-saturated bTf Activated groups were blocked by addition of ethanolamine Non-coupled transferrm was removed by washmg with 10 to 20 column volumes of a 50 mM TπsHCl, 1 M NaCl, pH 8 0 buffer contammg 6 0 M guanidme hydrochloride After further washmg with 50 M Tπs-HCl (pH 8 0) the bound transferrm was 15 reloaded with iron usmg a solution contammg 5 mg/ml FeCl 3 in 0 1 M sodium cιtrate/0 1 M NaHC0 3 (pH 8 6) After washmg aga with 50 mM Tπs-HCl (pH 8 0), the bTf-Sepharose resm was pre-equihbrated with 50 mM Tris-HCl (pH 8 0), 1 M NaCl before it was used m affmity experiments E co cells contammg recombmant plasmids resuspended m 50 mMTπs-HCl (pH 8 0)/ l M NaCl were solubihzed m 20 mM EDTA, 2 % Sarkosyl and mcubated for 2 h at room 20 temperature The mixture was centπfuged for 15 mm at 8,000 rpm (4°C) and the supernatant contammg the solubihzed receptor was carefully decanted The supernatant was diluted 4 times with 50 mM HCl (pH 8 0), 1 M NaCl buffer and pre-rncubated for 30 mm at room temperature with an excess (1 mg/ml) of each of the followmg transferrms diluted in the same buffer iron- loaded bTf, goat or caprine transferrm (cTf), sheep or ovine transferrm (oTf), and human 25 transferrm (hTf), apo-bTf, C-lobe bTf, and N-lobe bTf Only the buffer was added to the positive control experiment After pre-incubation the supernatants were applied to a bTf- Sepharose column previously equilibrated with 50 mM Tris-HCl (pH 8 0), 1 M NaCl and mcubated for 15 mm at room temperature Each column was washed extensively with at least 12 column volumes of 50 mM Tris-HCl, (pH 8 0),1 M NaCl, 10 mM EDTA, 0 5% sarkosyl followed by 30 10 column volumes contammg only 0 05% sarkosyl to remove non-specifically bound protem Fmal wash was done with 50 mM Tris-HCl (pH 8 0), 0 5 M NaCl Elution of the recombmant TbpB was achieved by application of 1 bed volume of 2 x SDS-PAGE sample buffer under non-reducing condition and no boiling Each eluant (supernatant) was collected after centπfuging the mixture contammg the resin for 5 min at 13,000 x g m a 35 microfuge An aliquot of each supernatant (eluant) was further submitted to SDS-PAGE and electroblotted to Immobilon PVDF (Millipore) membrane (overnight at 15V m 20 mM Tπs, pH 75, 150 mM glycme, 20% methanol and 0 1% SDS) The membrane was blocked with 0 5% skim milk m 20 mM Tris (pH 7 5), 500 mM NaCl (TBS) for 30 minutes at room temperature A 1/1,000 dilution of the anti-TbpB serum m the blockmg solution was applied to the membrane for 1 houi at 37°C followed by two, 10-mιnute washes each with TBS A 1 /3000 dilution of secondary antibody (goat anti-rabbit IgG-horseradish peroxidase conjugate) was allowed to bmd for 1 houi at 37°C The conjugate was removed by three, 10-mιnute washes m TBS and developed usmg ar HRP-substrate mixture (Chloro-Naphtol/H 2 0 2 ) N-terminal amino acid sequence analysis. Samples of affmity-puπfied and sequentially eluted TbpA and TbpB from H196 were subjected to SDS-PAGE, electroblotted onto PVDF (Immob lon-P Millipore IPVH 00010) membrane, briefly stamed with Coomassie Blue, and strips contammg the mdividual protein bands were cut from the membrane for N-terminal ammo acid sequence analysis Preparation of anti-TbpA and anti-TbpB monospecific rabbit sera. Approximately 500 mg of purified TbpA and TbpB from P haemolytica strain H44 obtamed from the appropriate fractions in the affmity procedure after dialysis and concentration was mixed with Freund s complete adjuvant and mjected mtramuscularly mto two white female New Zealand rabbits, respectively The rabbits were boosted twice at 3-week intervals with the same amount of antigens plus Freund's mcomplete adjuvant Two weeks after the fmal boost, blood was collected to determine the serum titre to the l espective antigens usmg the dot assay in a dot-blot apparatus The rabbits were either further boosted if titre was unsatisfactory or terminally bled, if the titre was satisfactory The specificity of the sera agamst TbpA and TbpB from H44 was exammed by SDS-PAGE and Western Immunoblot analysis using goat anti-rabbit IgG con j ugated to HRP as secondary antibody Expectedly, both TbpA and TbpB antisera crossreacted with TbpA and TbpB from stram H196, respectively Nucleotide sequence analysis. Two separate strategies for sequencing the tbp region were adopted One approach primarily mvolved subclonmg fragments from recombmant plasmids mto the M13 vectors and then sequencmg subsequently isolated smgle stranded DNA prepared by the dideoxy chain termination method using vector primers In a limited number of cases, oligonucleotide primers were synthesized on the basis of the sequence results from the cloned mserts and used to complete the sequence of the cloned insert In this analysis the nucleotide sequences were complied and analyzed by the Pustell programs (IBI) An alternate approach primarily mvolved sequence determmation of a succession of cloned mserts obtamed by PCR amplification from chromosomal DNA Oligonucleotide primers were synthesized on the basis of the preceeding sequence analysis The PCR amplified products were cloned into the pCRII clonmg vector (Invitrogen) Double stranded DNA sequencmg was performed using purified recombmant plasmids by the oligonucleotide primer-directed procedure using synthetic oligonucleotides, fluorescent dye-labelled dideoxynucleotide triphosphate termmators, and cycle sequencmg with Taq polymerase Sequence reaction products were analysed on a Applied Bιosystems(ABI) model 373A automated fluorescent sequencer The results from successive sequencing runs were compared and the composite sequence was determined by comparison of the chromatograms usmg the SeqEd program This sequence was subsequently compared to the sequence obtamed by single strand sequencmg usmg the Mac- DNASIS program In addition the sequence was analyzed by comparmg the predicted protem sequence in all three reading frames with aligned sequences for Tbps from several different species Any areas of uncertainty identified by this analysis were subjected to repeated runs of sequence analysis RESULTS Cloning the transferrin receptor genes. Anti-receptor antisera and N-terminal ammo acid sequences were obtamed in order to facilitate clonmg of the P haemolytica transferrm receptor genes Monospecific antisera was obtamed by immunizing rabbits with the affmity puπfed receptor protems, TbpA and TbpB from a serotype Al stram (H44) of P haemolytica Ammo acid sequence analysis of an electroblotted preparation of purified native H196 TbpA yielded a readable sequence of 20 amino acids (top of Figure 20) A similar analysis with the purified TbpB failed to provide any sequence mformation suggestmg the N-termmus of this protem may be blocked The sequence of the first eight ammo acids of the purified TbpA was used to design an oligonucleotide primer based on a P haemolytica preferred codon usage table (tbpA primer 023, Table 4) This primer was used in combmation with either of two vector primers (RL2 and RL3, Table 4) for polymerase cham reaction (PCR) amplification of a portion of the tbpA gene from a P haemolytica plasmid bank (Lo et al , 1985) An 800 bp PCR product was obtamed with vector primers RL2 and 023 and its authenticity was verified by sequence analysis smce the predicted ammo acid sequence contamed a sequence that was identical to the N-terminal ammo acid sequence and exhibited homology with other TbpA protems The cloned PCR product was used as a hybridization probe for Southern analysis of restriction endonuclease-digested H196 P haemolytica chromosomal DNA and for screenmg of the plasmid bank The Southern analysis provided a restriction map of the chromosomal DNA m the tbp region for comparison to cloned mserts obtamed from the plasmid bank Table 4: Oligonucleotide primers. Primer Description Direc tion * Sequence N o . (gene/region - location) 023 tbpA - 5'end, 1 st 8 N-terminal aa's 5' 3' GGAAGCTTACTGAAAATAAAAAAATC GAAGAA 088 tbpA - 5'end 3 -5' * CACTACTTTCCCCAAGCCAG RL2 pBR322 - upstream of BamH l site V-5'* GGAATTCCCTCCTGTGGATC 198 tbpA - V end, 3 5'* GCIGCII(G/C)IGCICGIAAσ/C)T(T/A)(T C) 190 tbpB 5' end, leader peptide region 5'-3' CAAAGCTTGC1TG(TC)TCIGGIGG 352 upstream of tbpB - 5' end 5' V AGATCTCGATTCTAAATCAGACCC TTTG TATTTTAG 192 tbpB - conserved aa sequence near 3' 5'-3' GTI(T/A)(A/G/C)IGGIGGITT(C r)TA(T/C)GG end 401 tbpB - 5 enύ 5 -V TAAATTAAAGGAGACATl'ATGTTTAAACT 350 tbpB 3 end, flanking Ncol site 3 -5' * CGACGCCCATGGTTATTTTTCTATTTGAC GTTTTCC 199 tbpB - V end, flanking Hindlll site 3'-5' * GCGCAAGCI'I'ITATTΓTT ATTTGACG 349 tbp A 5' end, BamH l/Bglll sites 5'-3' GGATTCAGATCTTAAAGGAGACCCTATC upstredtn of rbs TAATGATAATG 255 tbpA - 5 end Ndel site at start codon 5'-3' CCCTATCATATGATAATGAAATATCATC 256 tbpA - 3 end, Hindlll site after stop 3' 5'* TAGCGCAAGCTTCTAAAACTTCATTTCAAAT * Direction relative to orientation of codmg strand for the relevant gene Initially, two strongly hybridizing colonies were identified from the E coh clones Plasmid p**(clone 9) contamed a 9 kb insert which mcluded most of the tbpA gene with adjacent downstream regions but was fused with DNA from another chromosomal locus (fis m Figure 20) The second plasmid, p**(clone 10), only contamed a 1 2 kb msert that was primarily situated around the 5 end of the tbpA gene The artificial junction in plasmid pRYCL9 was reminiscent of similar artifacts observed while attemptmg to clone the menmgococcal tbpB gene and the ensumg difficulties that were encountered (23) prompted consideration of alternative strategies for clonmg the P haemolytica tbpB region One strategy was based on the observation that m other species the tbpB gene was located upstream of the tbpA gene (19, 20, 23) and that there were short stretches of amino acid identity m the predicted sequences of the respective TbpBs A conserved amino acid sequence near the carboxy terminus of TbpBs was used to design a degenerate oligonucleotide primer (primer 192, Table 4) to obtam the remainder of the tbpA gene, the mtergenic region and a portion of the 3 ' end of the tbpB gene This primer was used m combmation with a primer based on the sequence from the 5 end of the tbpA gene (primer 088, Table 4) to amplify a 700 bp fragment from H196 chromosomal DNA The sequence from this msert enabled the design of an oligonucleotide primer based on the authentic sequence of the 3' end of the tbpB gene (primer 199, Table 4) which was used m combmation with degenerate oligonucleotide based on a conserved ammo acid sequence present m the leader peptide region of known TbpBs (oligo 190, Table 4) for PCR amplification The resulting 2 4 kb PCR product obtamed when H196 chromosomal DNA was used as a template contamed the authentic 3' end of the tbpB gene When this PCR fragment was cloned m the pCRII vector and used m expression experiments utilizing the T7 promoter, an intact recombmant TbpB was produced indicating thai the πbosomal bmdmg site and start of the tbpB gene was contained withm the insert A second strategy utilized anchored PCR in which Pstl-digested pBluescπpl plasmid was hgated to Pst I-digested H196 chromosomal DNA and used as template for a PCP reaction utilizmg a primer from the 3 ' end of the tbpB gene (oligo 199, Table 4) and the M1Z reverse primer from the vector The resulting 3 5 kb product was subcloned mto the PCRII vector producmg a plasmid that contamed the entire tbpB gene and a considerable amount of adjacent upstream regions (ORF and RNase T in Figure 20) Further subclonmg the tbp genes into expression vectors mvolved PCR amplification with oligonucleotide primers complementary to the 5 and 3 ends of the genes and mclusion of appropriate restriction sites One set of primers for amplifying the tbpA gene (primer 349 and 256, Table 4) involved mtroduction of a BamHI and Bgl II site immediately upstream of the predicted πbosomal bmdmg site (rbs) so that provision of an exogenous promoter should result m expression due to the presence of the native rbs An alternate 5 primer (255, Table 4) mvolved the introduction of an Nde I site at the start codon so that cloning mto the pT7- 7 expression vector, which supplies a properly position πbosomal bmdmg site, was possible Since the expression experiments preceeded definitive identification of the start of the tbpB gene, the subcloned tbpB gene was obtained by PCR amplification with an upstream sequencmg primer (primer 352, Table 4) and a primer contammg the authentic 3 end flanked by an Nco I site (primer 350, Table 4) Characterization of the transferrin receptor genes. As illustrated m Figure 20, the tbp genes appear to be m an operon arrangement, with the tbpB gene located upstream of the tbpA gene Comparative sequence analysis revealed that the tbpB gene was preceded by an open readmg frame (ORF) encodmg a protem whose sequence was highly identical to RNaseT from E coh and H influenzae This ORF was m turn preceeded by another ORF encoding a protein with considerable identity to hypothetical proteins identified in influenzae and Vibrio parahaemolyticus Downstream of the tbpA gene is an ORF encoding a protem whose sequence was 70% identical to factor-for-mversion stimulation (FIS protein - recombmational enhancer) proteins from H influenzae and E coh This effectively demarcates the boundaries of the transferrin receptor protem gene operon and mdicates that there are no immediately adjacent genes related to this iron acquisition pathway There is a 420 bp region between the end of the ORF encodmg the RNaseT homologue and tire start of the tbpB gene with potential πbosomal bmdmg sites, promoter sites and regulatory sites all present within the last 62 base pairs (Figure 20) The remammg 358 bp mtervening region could presumably contam transcπptional termmation signals for the RNaseT gene and sequences potentially mvolved in regulation of the tbp operon The putative promoter region contams 5/6 and 6/6 of the consensus bases for the E coh s70 -35 and -10 promoter regions, respectively Previous studies have demonstrated that the expression of the P haemolytica transferrm receptor proteins is regulated by the level of available iron in the medium (26) The identification of a putative Fur box overlapping the -10 site of the tbpB promoter (Figure 20) suggests that regulation by iron may be at the transcπptional level via the action of a Fur homologue in P haemolytica The putative Fur box had 12/19 bases identical to the E coh Fur bmdmg site consensus sequence (Litwin and Calderwood, 1993) Between the tbpB and tbpA genes there is a 96 bp intergenic region which contains a putative ribosomal bmding site upstream of the tbpA gene but no evident promoter. In addition, there is no evident transcriptional terminators in this region. Downstream of the tbpA gene stop codon and the stop codon of the gene encoding the FIS homologue is a 98 bp region with no evident transcriptional terminators. Sequence analysis of the tbpA gene region from serotype Al P. haemolytica strain H196 revealed an ORF of 2,790 bp encoding a protein with a predicted molecular mass of 106,921 Da (Figure 22). The putative signal peptidase cleavage site at residue 28 was confirmed by comparison with the known N-terminal amino acid sequence of the mature protein (top of Figure 22). The predicted amino acid sequence of TbpA was compared to the sequences of TbpA from N. memngitidis (23), N. gonorrhoeae (8), H. influenzae (20) and Actinobacillus pleuropneumoniae (19). The localization of identical amino acids between these proteins (bold- underlined amino acids, Figure 22) was compared to the proposed topology of these amino acic segments based on the model predicted by Tommassen (28). It is evident that most of the identical amino acids are clustered in regions corresponding to the short transmembrane β-sheets or in the segments of the internal and external loops that are immediately adjacent to the transmembrane sections. It is interesting to note that there are conserved pairs of cysteines ir proposed external loops 4, 6 and 7 and a unique cysteine pair in loop 10 of the P. haemolyticc TbpA. These likely represent disulfide bridges that would provide structural stability to the external loops. Analysis of the sequence in the tbpB gene from serotype Al P. haemolyticc' strain H196 revealed an ORF of 1,752 bp encoding a protein with a predicted molecular mass o 63,419 Da (Figure 23). This predicted protein sequence of TbpB was compared to the publishec. sequences of TbpBs from N. meningitidis (23), N. gonorrhoeae (1 ), H. influenzae (20) and Actinobacillus pleuropneumoniae (14). This predicted amino acid sequence included a 18 amino acid leader peptide, a signal peptidase II recognition sequence with a cysteine as the predictec N-terminal amino acid of the mature protein. The presence of an N-terminal cysteine, which has been shown to be lipidated in other species (14, 24), may explain the inability to obtain an N-terminal amino acid sequence for this protein and may serve as the primary means of anchoring the protein to the outer membrane. Regions that aligned with the putative binding regions of the A. pleuropneumoniae TbpB (TfbA) recently identified by Gerlach et. al. (36) are indicated by a double underline. It is apparent that there are several regions of homology found throughou : the length of the amino acid sequence which include several short stretches of identical aminc acids (Figure 24). Upon closer inspection it is apparent that there is some homology between regions in the N-terminal and C-terminal portions of the protein suggesting there may be ar underlying bilobed structure to the protein, analogous to what is observed for transferrins. Thus the sequences YKGYW (aa 185-189) and YRGTW (aa 449-453), FTADFANK (aa 237-244) and FDVDFVNK (aa 480-487), GNRFSG (aa 276-281) and GNGFGG (aa 513-518), and LEGGFFG (aa 300-306) and FEGGFYG (aa 546-552) represent consecutive stretches of homologous amino acids m equivalent positions of the N-termrnal and C-termmal portions of the protem Expression and analysis of the recombmant receptor proteins. The mtact tbpB and tbpA genes were PCR amplified from H196 chromosomal DNA and subcloned mto expression vectors for production of recombmant protems For initial attempts at expression of the tbpB gene, the subcloned tbpB gene was obtamed by PCR amplification with an upstream primer (352, Table 4) and a primer contammg the authentic 3 end flanked by an Ncol site (350, Table 4) When the PCR amplified fragment was subcloned into the pCRII vector, all five of the resultmg clones were m the same orientation, downsheam of the T7 promoter and in the opposite direction of the lac promoter Smce the lac promoter would not be tightly regulated m a high copy number vector, this result suggests that expression of the insert m E coh may be selected agamst Once the sequence of this region was available it became apparent that primer 352 was immediately upstream of the RNaseT gene and thus expression of either this gene or the tbpB gene could have been responsible for the selective pressure Expression of TbpB from the T7 promoter was accomplished by mfection with CE6 λ phage, which encodes the T7 RNA polymerase Two hours after mfection a protem of the anticipated molecular weight for TbpB was evident and this protem reacted with anti-TbpB antiserum after electroblotting There was detectable binding of labelled bovme transferrin (bTf) by immobilized mtact cells expressmg the TbpB protem and this level of bindmg did not mcrease significantly with prior sonication of the cells (data not shown) Although this could be mterpreted as proper processmg and export of the TbpB to the cell surface m the heterologous E coh system, disturbance of the outer membrane integrity by overexpression of a foreign protem antigen is an equally plausible explanation The preliminary bmding studies suggested that a functional TbpB protein was bemg produced, mdicatmg that further analysis might enable evaluation of the functional properties of this protem and ascertain its contribution to the previously characterized properties of the native receptor complex Thus, crude membranes were prepared from cells expressmg TbpB and used m affmity isolation experiments designed to evaluate its bmdmg characteristics These experiments indicated that the recombmant TbpB is capable of bemg affmity isolated by immobilized bTf and this isolation could be inhibited by an excess of bovme, ovme or caprme Tf, demonstrating its ability to effectively bmd to all three of these rummant Tfs Human transferrin as well as apo-bTf did not inhibit the affmity isolation of recombmant TbpB by immobilized iron-loaded bTf This assay also revealed that both N-lobe and C-lobe of bTf effectively blocked bmdmg of recombmant TbpB to the immobilized bTf For expressmg the tbpA gene, PCR amplification was performed with a set of primers (ohgos 349 and 256, Table 4) that maintained the predicted πbosomal bmdmg site (Figure 21) After subcloning the PCR product into the pCRII vector, no recombmant TbpA was expressed after mfection with CE6 phage (encodmg T7 RNA polymerase) Sequence analysis of one of the clones revealed excision of several base pairs that eliminated πbosomal bmdmg site Other repeated attempts at subcloning the tbpA gene from PCR usmg 255 and 256 primers (see Table 4) into the Ndel site of the pT7-7 vector (which provides an optimally positioned πbosomal bmdmg site) were unsuccessful Discussion In the initial studies demonstratmg the presence of a transferrm receptor rn Pasteurella haemolytica (26) only a smgle receptor protem (TbpA) was isolated by an affmity method that yielded two receptor protems (TbpA and TbpB) in other species (32) Thus the ability to use iron from bovme transferrm for growth (26) and the specific bmdmg of rummanl transferrms (39) was initially presumed to be largely mediated by this receptor protem Ir subsequent studies that demonstrated that the bmdmg region of bovme transferrm was localized to the C-lobe (41), two receptor protems (TbpA and TbpB) were isolated by a modified affrnit ) method, although the yield of TbpA greatly exceeded that of TbpB Thus it was not possible to conclusively attribute the observed binding characteristics of the receptor to either receptoi protem, and particularly not to TbpB The clonmg of the tbp genes and expression of recombmant TbpB has enabled specific evaluation of its bmding characteristics These studies have demonstrated that TbpE has a similar host specificity as the native receptor complex (TbpA and TbpB) as it specifically bmds Tfs from several rummant species In contrast, unlike the native receptor complex, the recombmant TbpB was able to recognize bindmg determmants on the N-lobe as well as the C- lobe of bTf, suggestmg that in the previous study (Yu and Schryvers, 1994) an mteraction betweer TbpA and TbpB might have interfered in the ability of TbpB to also bmd to the N-lobe of bTf In competitive binding assays with immobilized membranes (TbpA and TbpB) from P haemolytica there was no evident preference for the iron-loaded or apo form oJ bTf (30) which is similar to what has been observed m most other bacterial species (Blanton el al , 1990, 32, Tsai et al , 1988, 32, 37), except Moraxella catarrhahs (Yu and Schryvers, 1993) In the present study recombmant TbpB clearly showed a strong preference for the iron-loaded form of bTf This preference of TbpB may have functional relevance in mcreasmg the efficiency of iron acquisition in vivo Example 4 Vaccine Potential of Recombinant Tbp2 and Authentic Tbpl The transferrm-bindmg protems Tbpl and Tbp2 are attractive targets for a number of reasons a) Since acquisition of iron from transferrin is likely essential for bacterial survival, an antibody response agamst these antigens should be protective b) The genes coding for Tbpl and Tbp2 appear to be conserved within various isolates of P haemolytica Al This study deals with test g the vaccme potential of recombmant Tbp2 and authentic Tbpl, alone and m combmation, m an experimental P haemolytica challenge model Methods The Tbpl and Tbp2 protems were affmity purified from P haemolytica and recombmant E coli outer membranes, respectively, by affmity chromatography usmg standard techniques as described herein Vaccmes were formulated using a proprietary mineral oil-based ad j uvant (VSA3) such that the volume of each dose was 2 cc contammg the followmg amount of each antigen Tbp2 - 45 mg, Tbpl - 85 mg when used by itself or 100 mg when combmed with Tbp2 In addition, a placebo vaccme was prepared containing sterile diluent m place of antigen Five groups were mcluded m the trial, mcludmg one which received a smgle immunization of Tbp2 ten days before challenge, and groups which received two immunizations with Tbp2, Tbpl + Tbp2, placebo, or Tbpl The mterval between primary and secondary immunization was three weeks and all vaccmations were carried out at a farm m Southern Saskatchewan Vaccmes were delivered via the subcutaneous route Approximately ten days prior to challenge, animals were transported to Saskatoon and housed at the VIDO research station All groups contamed ten animals with the exception of the group receivmg Tbpl m which there were six This group was not in the original proposal and was added in order to determme the protective capacity of Tbpl by itself In addition, one calf which received one immunization with the Tbp2 formulation developed clmical signs of disease unrelated to vaccmation and was therefore excluded from the trial The composition of the vaccme groups is summarized in Table 5 Table 5 Composition of vaccme groups Calves were challenged via the aerosol route by first exposmg them to a suspension of bovme herpesvιrus-1 strain 108 contammg approximately 2 5 x 10 6 PFU/ml followed four days later by an aerosol of P haemolytica contammg approximately 5 x IC? CFU/ml Animals were examined daily by a veterinarian and animal health technician and the followmg data was recorded weight, temperature, nasal scores, depression, strength, respiratory distress and sickness Each of these criteria with the exception of weight and temperature were scored on a scale of 0-4 The serological response to vaccination was measured usmg an enzyme-linked immunosorbent assay (ELISA) Serum samples were collected at the time of the first and second immunizations plus on the day of challenge with BHV-1 The titers are presented as the reciprocal of the serum dilution which resulted m an optical density equivalent to the background plus two standard deviations Responses agamst Tbpl, Tbp2 and the P haemolytica leukotoxm were measured The latter was mcluded as a diagnostic test to determme if animals had been naturally exposed to the organism Results a) Response to Vaccmation None of the animals showed any adverse reaction to vaccmation with any of the formulations used The serological response to vaccmation was determmed usmg an ELISA procedure which measured the serum antibody levels to Tbpl Tbp2 and the P haemolytica leukotoxin The latter antigen was mcluded in order to ensure that none of the animals had increased titers due to the natural exposure to the bacteria The titers agamst each antigen are shown in Table 6 and it can be seen that the titers against leukotoxm were comparable at the time of initial vaccmation (Bleed 1), the second vaccmation (Bleed 2), and at challenge (Bleed 3) Animals which have a titer below 3,000 were considered to be clean Interestingly, none of the animals seroconverted to a significant degree to the Tbpl antigen Based upon the inventors experience with Tbpl from other organisms, the expected titers should be low but it was unexpected that no significant mcrease in antibody levels would be detectable All groups which received Tbp2 responded well to vaccmation and although the group which received Tbpl + 2 had titers approximately 1/2 of that m the Tbp2 group, this difference is not significant Table 6 Serological response to vaccmation ELISA titers agamst leukotoxm, Tbpl and Tbp2 were determined usmg serum samples taken at the time of the first immunization (Bleed 1), the second immunization (Bleed 2), and the day of challenge with bovme herpesvιrus-1 (Bleed 3) The numbers are expressed as the reciprocal of the dilution equaling a negative control plus two standard deviations b) Mortality The experimental disease model has been calibrated to obtam 60-70% mortality under normal conditions However, mortality was higher than usual in this trial, probably because of the extremely cold temperatures to which the animals were exposed throughout the time period after challenge Daily low temperatures were m the -40°C range and all animals were housed outdoors durmg the trial The mortality by group is shown m Table 7 and the only group which showed significant protection was that which received both Tbpl and Tbp2 This is compared to 50% mortality for Tbp2 by itself and 100%> for Tbpl A smgle immunization with Tbpl was of no benefit Table 7 Group mortality observed durmg the trial Vaccme Group Mortality (%) Tbp2 (1 dose) 78 Tbp2 50 Tbpl & 2 10 Placebo 90 Tbpl 100 c) Clmical Signs of Disease The clmical results are summarized by group m Table 8 It should be noted that Table 8 contams the clmical results for all days of the trial, mcludmg those pπor to P haemolytica mfection on day four Therefore, only the results from days 4 through ten have been used to determme the protective capacity of the vaccine formulations The high rate of mortality observed durmg this trial had the effect of reducmg the size of each group to the pomt where differences observed in all of the clmical parameters measured are not statistically significant However, it is clear that the group which received both Tbpl and Tbp2 showed lower scores in most categories between days 5 and 7 The groups which received two immunizations with Tbp2 alone also showed reduced clinical signs of disease m survivors although they did not do as well as the combination group The contribution of Tbpl to protection is unclear at present smce there did not appear to be any antibody response to this antigen d) Postmortem Results Necropsies were performed on all animals which died durmg the trial In all cases, P haemolytica was cultured from the lungs and the pathology observed was consistent with fibrmous pneumonia caused by P haemolytica Conclusions Two vaccinations with a formulation containing P aemolytica Tbpl and Tbp2 providec significant protection against experimental bovine pneumonic pasteurellosis The exact contribution which Tbpl provides to this protection is unclear smce there did not appear to be any serological response to the protem The beneficial effect may have been due to a cell mediated immune response Immunization with two doses of Tbp2 provided some degree of protection and it may be possible to mcrease this by testmg vaccme formulations contammg greater quantities of antigen or a different adjuvant It is likely that the immunological response to Tbp2 provided the bulk of the protection seen with the combination vaccme Vaccination with one dose of Tbp2 or two doses of Tbpl had no beneficial effect after experimental challenge -57- Table 8 Mean Clinic*! Scorea by Group Animals were challenged on day 0 wim BHV 1 and day 4 wHh P haβmofyt t EXAMPLE 5 Comparison of the transferrin receptor from various ruminant serotypes A collection of Pasteurella haemolytica and P trehalosi strams of various serotypes isolated from cattle,sheep and goats were analyzed for bindmg of rummant transferrms and utilization of transferrin iron for growth Some of the goals of the study were to determme the prevalence of transferrm receptors from different host species, to evaluate their specificities for different rummant transferrins and to determme if there is antigenic relatedness amongst the surface receptors from the different strams causmg shippmg fever m cattle, pneumonia m sheep and goats and septiceamia in lambs Materials Bacterial strains. The bacterial strams used m this study are listed in Table 9 Clmical type Al isolates of P haemolytica (h93-h97)(9) and representative ATCC strams (h98-hl07) from bovme pneumonia were provided by Dr Andrew Potter, VIDO, Saskatoon P trehalosi stram hi 4, field isolate from goat with pneumonic pasteurellosis was provided by Dr Frank Milward, Rhone Meneux, Lyon, France P haemolytica stram h44 , a bovme clmical type Al isolate from bovme pneumonia, has been described previously (26) Strain hl96 was obtamed from Dr Lo,Unιversιty of Guelph , Ontario, Canada Table 9. Bacterial strains, serotypes and sources. Growth conditions. All bacterial strams were stored frozen at -70C m 30% glycerol Isolates from the frozen stocks were streaked onto chocolate agar plates and mcubated at 37C m a 5% C0 2 mcubator Iron-restricted growth was achieved by growmg the bacteria m Bram Heart Infusion broth (BH1, Difco Laboratories) or O Reilly-Niven broth (25) supplemented with 3 0 g/ml nicotinamide adenine dinucleotide (NAD) and containing the iron chelator ethylenediammedmydroxyphenylacetic acid (EDDA, Sigma) at a fmal concentration of 100 M Growth experiments for use of different transferrms as an iron source was performed as previously described (26) Preparation of transferrins and derivatives. Bov e transferrm was obtained from Sigma The preparation of ovme (sheep) and caprme (goat) transferrms (2), the iron loadmg of transferrms to 30% or 100% saturation (Herrrngton et al 1985) and conjugation of horse-radish peroxidase (HRP) to transferrin (37) was essentially as described previously In the preparation of con j ugates of bovme, ovine and caprine transferrms (HRP-bTf, HRP-oTf and HRP-gTf ), the mixture of HRP and transferrin were subjected to gel filtration after chemical conjugation The fractions demonstrating maximal activity were pooled, dialyzed and aliquots frozen and stored at -70°C Transferrin binding assay. The solid phase bindmg assay for transferrin was essentially as described previously (32) After the membrane or concentrated eluates were spotted unto HA paper (Millipore Corporation, Bedford, MA) and blocked m 0 5% skim milk, the paper was exposed to blocking solution contammg 450 ng/ml of the HRP-conjugated transferrin The mcubation, washmg and development with HRP substrate mixture were performed essentially as previously described (32) Affinity isolation of transferrin binding protems Bovme, ovme and caprme transferrms were individually coupled to CNBr-activated Sepharose 4B according to the manufacturers mstructions usmg solutions contammg 3 5 mg/ml of iron-saturated transferrm Activated groups were blocked by addition of ethanolamme Non coupled transferrin was removed by washing with 10 to 20 column volumes of a 50 mM TπsHCl, 1 M NaCl, pH 8 0 buffer contammg 6 0 M guanidme hydrochloride and after further washing the bound transferrm was reloaded with iron usmg a solution contammg 5 mg/ml FeCl 3 ui O l M sodium cιtrate/0 1 M NaHC0 pH 8 6 buffer Iron-deficient total membrane (200 mg protein) from P haemolytica or P trehalosi prepared as previously described (32) was diluted to 2 mg/ml m 50 mM Tris pH 8 0 containing 1 0 M NaCl The diluted membrane was solubihzed by addition of EDTA and sarkosyl to a fmal concentration of 10 mM and 0 75%, respectively followed by mcubation of the mixture at room temperature for 15-30 mm with gentle rocking The solution was centπfuged at 10,000 rpm for 10 mm to remove insoluble debris The supernatant contammg the solubihzed membrane was applied to a 1 5 x 10 cm transferrm-affmity column and then washed extensively (at least 10 bed volumes) with 50 mM Tris pH 8 0 contammg 1 0 M NaCl, 10 mM FDTA, 0 75°/. Sarksosyl to remove non-specifically bound protem In experiments usmg low salt washing conditions th^ washing buffer contamed 100 mM NaCl in lieu of 1M NaCl In some instances, additional washmg with 2-3 bed volumes of washmg buffer contammg 0 2 M guanidme hydrochloride was necessary to remove contaminating protems Coelution of both transferrin binding proteins (TbpA and TbpB) was achieved by application of 2-3 bed volumes of 2 0 M guanidme hydrochloride in 50 mM Tris pH 8 0 contammg 1 0 M NaCl, 1 mM EDTA, 0 01% sarkosyl The eluant was collected for immediate dialysis agamst 50 mM Tris pH 8 0 Further treatment with higher concentrations of guanidme hydrochloride usually did not result in any further yield of receptor protein Individual isolation of TbpA and TbpB was attained by sequential elution with 2 bed-volumes of each buffer contammg 0 2, 0 5, 0 75, 1 0, 1 5, 2 0 and 3 0 guanidme hydrochloride, respectively The eluates were dialyzed agamst 3 changes of 3 litres 50 mM Tris pH 8 0 over an 18-hour period and concentrated by ultrafiltration After SDS-PAGE analysis the fractions from the 0 5 and 0 75 M guanidme HCl elution buffers were found to contam TbpB only and were thus pooled for a preparation of TbpB and fractions from the 1 5 and 2 M guanidme HCl elution buffers were pooled for a preparation of TbpA Preparation of anti-TbpA and anti-TbpB monospectfic rabbit sera. Approximately 500 μg ot purified TbpA or TbpB from P haemolytica strain h44 prepared as described above, was mixed with Freund s complete adjuvant and injected intramuscularly into two white female New Zealand rabbits The rabbits were boosted twice at 3-week mtervals with the same amount of antigens m Freund's mcomplete adjuvant and the immune sera collected 2weeks after the fmal boost The specificity of the sera agamst TbpA and TbpB was tested after SDS-PAGE and immunoblotting of the receptor protems and using goat anti-rabbit IgG conjugated to HRP as secondary antibody Analytical methods. Protein samples were analyzed by SDS-PAGE followed by silver stammg as previously described (32) For Western blot analysis, about 1-2 μg of purified receptor proteins or 40 μg of outer membrane protein from iron-limited cells were separated on 10% polyacrylamide gels Protems were electrophoretically transferred to nitrocellulose (Millipore, Bedford, MA) overnight at 15V m 20 M Tris, pH 7 5, 150 mM glycme, 20% methanol and 0 1% SDS The filters were blocked with 0 5% skim milk m 20 mM Tris pH 7 5, 500 mM NaCl (TBS for 30 mmutes at room temperature The membrane was exposed to 1 / 1000 dilution of the appropriate antibody m blockmg solution for 1 hour at room temperature,washed twice with TBS, and then exposed to a 1 /3000 dilution of secondary antibody (goat anti-rabbit IgG-horse radish peroxidase conjugate from BioRad) The conjugate was removed, washed three tιme 5 with TBS and then developed usmg the HRP-substrate mixture For the whole cell assay, iron deficient or iron-sufficient (control) cells were directly spotted onto HA paper After drymg, the HA paper was treated with blocking solution and washed with TBS and then tested for reactivity with anti TbpA or anti TbpB antisera as described above A control set of spotted - 6A cells was treated with HRP-bovme transferrm for 1 hour followed by development with HRP- substrate after washmg m TBS PCR amplification of tbp genes and restriction endonuclease digest analysis Amplification of tbpA and tbpB from P haemolytica and P trehalosi strains was performed on intact cells by the method of Saris et al (1990) Amplification of tbpA was carried out with oligonucleotides tbpA 5 and tbpA 3' (#255 and #256, table2) Oligonucleotides #401 and # 199, (Table 10) were used to amplify tbpB Reaction conditions consisted of 30 cycles of 94 °C for 1mm, 45°C for 1mm, and 74°C for 2mm The PCR product was separated by 1% agarose gel electrophorsis in 0 5X TBE buffer (45mM Tπs-borate, ImM EDTA, pH8 3) and stamed with 0 5mg of ethidium bromide per ml m the same buffer For Sau3A restriction endonuclease (Gibco BRL) digestion, PCR products were sub j ected to phenol chloroform and ethanol precipitation and thereafter were digested with Sau3A The digests were analysed with a 7 5% acrylamide gel run m 0 5X TBE buffer and visualised m the same way as the agarose gels described above Results Specificity of receptor binding. In a prior study it was demonstrated that transferrm receptors in representative isolates from pathogenic bacterial species varied in their interaction with goat, sheep and cattle transferrin (Yu and Schryvers 1996) Thus a set of representative isolates of P haemolytica and P trehalosi from cattle, sheep and goats (Table 9) were evaluated for their interactions with the different rummant transferrins All of these strains were capable of utilizmg bovme, caprme or ovme transferrin as a source of iron for growth (data not shown) Immobilized, iron-deficient cells were positive for binding all three of the transferrins m a solid-phase bindmg assay (Figure 25) and the three transferrins were equally effective at blockmg bmdmg to the cells m reciprocal competition bmdmg assays (not shown) In addition, both TbpAand TbpB, molecular weights lOOKda and 60Kda respectively were effectively isolated by affmity resms contammg immobilized bovme (Figure 26, Panel A), caprme or ovme transferrm (not shown) These results indicate that the specificity of transferrm-bmdmg within this group of related strains is indistinguishable Immunological analysis of transferrin receptor proteins. The observation that bovine, caprme and ovme transferrms compete for the same receptors suggested that there is conservation at least m the binding domain of the receptors However, the extent of similarity between the mdividual receptor protems from different serotypes was not known To address this question, antibodies were raised agamst TbpA and TbpB individually and as a complex m rabbits usmg purified receptor protems (TbpA and TbpB from a bovme stram h44) These antisera were then tested agamst receptor protems from representative strams of different serotypes mcludmg isolates obtamed from cattle, sheep and goats The results m Figure 26, Panel B demonstrate that the antisera raised agamst purified TbpA and TbpB receptors from P haemolytica serotype Al, stram h44, reacted strongly with similar purified receptors from all of the representative strains Since there was variation m the yield of receptor protems obtamed from the various strams (Figure 26, Panel A), this likely accounts for the slight differences observed m the reactivity of the antisera for a few of the strams (Figures 26, Panel B) Thus, these results suggest that both receptor protems are conserved amongst the different serotypes of P haemolytica causmg pasteurellosis m cattle, sheep and goats Although this analysis indicates that there are crossreactive epitopes on the transferrm receptor protems it does not provide any information as to whether there are crossreactive epitopes at the cell surface where they would be accessible to host immune effector mechanisms As a first step m attempting to address this question the reactivity of mtact rron- deficient cells from the different species to monospecific anti-receptor antisera was tested (Figure 27) These experiments demonstrated that the monospecific antisera prepared agamst TbpA and TbpB from a type Al stram of P haemolytica reacted with type Al strams (h44 and hl96, Figure 26, Panel B), several other A serotypes ( h98, hl03 , hl05, and hl07), and reacted moderately with several P trehalosi strains (h99, hlOO, hl06) The monospecific antisera prepared agamst TbpA and TbpB from stram h44 reacted strongly with iron-limited whole cells from an extended collection of type Al strams (data not shown), two of which (h44, hi 96) are illustrated m Figure 27 There were varying degrees of reactivity with the other serotypes withm P haemolytica ( h98, hl03, hl05 and hl07) and P trehalosi ( h99, hlOO and hl06 ) The correlation between the signal obtamed with the labelled bTf (Figure 27) and with the anti-TbpA and anti-TbpB antisera (Figure 27) m the different strains suggests that the observed reactivity in intact cells is primarily due to the receptor protems The absence of reactivity observed when control antiserum is utilized (data not shown) and the reduced reactivity m iron deficient cells (Figure 26, Panel C) also supports this conclusion Genetic analysis of transferrm receptor protein genes. As a complement to the immunological studies the variability of the tbp genes from the various strains of P haemolytica and P trehalosi was evaluated Usmg the sequence information obtained for the tbpA gene from a serotype Al stram (28), specific primers were prepared for the 5 and 3 ends of the gene (primer #255 and #256, Table 10) These primers were capable of amplifying the intact tbpA genes from all the tested strains although only small yields were consistently obtained for stram hlOO The mtact genes (except from hlOO) were then subjected to digestion by the Sau3A restriction endonuclease and the resultmg fragments were analyzed by electrophoresis on polyacrylamide gels A specific pattern was observed m type Al strams (h44 and hl96, Figure 28, Panel A) and was identical m all the seven type Al strams that were tested (data not shown) This patterr was also present in most of the other serotypes within P haemolytica (hl05, hl04, and h98 Figure 28) Subtle changes in the pattern, involving one or more fragments, that is likely attributed to alteration of a single site, was observed for other type A strams (hl03 and hl07 and withm the T strams (hi 06 and h99) The restriction digestion analysis was unable to identify any differences between the tbpA genes in the Al isolates Thus, an alternative approach was adopted for rapidly and simply exammmg variation amongst the tbpA genes This approach is based on the observation that there are segments of the TbpA (and LbpA) protem, proposed to be surface loops, which show the greatest variation m ammo acid sequence among protems from different species (21 and Legram et al 1996) In particular, there is one large predicted loop, demonstrated to be at the surface by reactivity with a monoclonal antibody (21), in which the greatest variation m ammo acid sequence was observed when aligning TbpAs from two meningococcal strams , one gonococcal stram and four H influenzae strams (1, Loosemore et al 1996,Schryvers and Gonzalez 1996) An oligonucleotide primer was prepared (#450,Table 10) based on the known ammo acid sequence (VEDTCPTLD) in this region for P haemolytica typeAl and used it in colony PCR amplification reactions m combmation with a 5 specific primer ( #255 , Table 10) with the various strams As illustrated in Figure 29, Panel A, this oligonucleotide pair readily amplified the anticipated 800 bp PCR product under high strmgency conditions from all of the strams except strain hlOO where a comigratmg band was barely discernable These results suggest that there is considerable homology amongst the different serotypes of P liaemolytica and P trehalosi even in the non-conserved' ammo acid regions in TbpA In order to evaluate the variation in the tbpB genes, a first attempt was made to amplify the intact genes from the various strams usmg specific primers for the 5 and 3 ends of the gene (primer* 401 and #199, Table 10) These primers readilv amplified the mtact tbpB gene from all the strams tested (Table 11) Restriction enzyme digestion analysis revealed that an identical digestion pattern was observed for all the seven Al strams tested (see h44 and hl96 m Figure 28, Panel B) and some strams from the other groups (hl05, hl04 and h98) Only subtle differences m the pattern were detected m several of the other strams (hl03,hl07, h99, hl06, Figure 28, Panel B) Thus PCR-based approaches were used with oligonucleotide primers to variable regions to see if the variable domams found m other studies are subject to variation m P haemolyttca A reverse oligonucleotide primer (#397, Table 10) was tested outside the reported conserved regions m tbpB (26) m combmation with the 5 primer (#401, Table 10) Similarly, a forward primer (#400) from another variable region m combmation with the 3 termmal oligonucleotide primer (#199) was used The anticipated tbpB partial products were obtained for all the A and T strams tested except one T strain, h99 (Fig 29B and Table 11) mdicatmg that a considerable homology does occur not only at the 5 and 3 termmi of the gene but also m a number of regions known to be variable m other species Table 10: Oligonucleotide primers Table 11. PCR-amplification of tbpA and tbpB gene segments from different serotypes of Pasteurella haemolytica. Ke : + = Product of anticipated size, comparable to the control (hi 96) obtained +/ = Product of anticipated size but much weaker in intensity than the control - = No product obtained Discussion A collection of P haemolytica and P trehalosi isolates from other ruminants were found to be capable of acquirmg iron from bovme, ovme and caprme transferrms (data not shown) which is presumed to be mediated by surface receptors that specifically bmd rummant transferrins (Al-Sultan and Aitken 1984) The collection of P haemolytica (biotype A) and P trehalosi (biotype T) strains involved m this study mcludes different serotypes (Table 9), suggesting that the receptor-mediated iron acquistion mechanism is fairly widespread withm these species There was some variability m the expression of the transferrm bmdmg activity amongst the various strains tested under the standard conditions used for the bmdmg assay Similar variation m the ability to bind the conjugated host transferrin (HRP-hTf) has been observed amongst H influenzae strams (Robki et al 1993) This may be partly attributable to the varymg growth characteristics of the different strams However, the highly sensitive and specific nature of the bmdmg assay enabled definitive identification of the presence of receptor activity in the strains tested The competitive bindmg experiments and the affinity isolation experiments mdicate that mteraction with all three ruminant transferrms is mediated by the same receptor protems which show considerable similarity amongst the various strams analyzed This conclusion is further substantiated by the immunological analysis of receptor protems prepared from a variety of P haemolytica strams with monospecific sera agamst TbpA and TbpB (Figure 26, Panel B) Smce the immunological analysis included a variety of different serotypes withm biotype A and T, it would appear that the transferrm receptor proteins are fairly conserved among P haemolytica disease isolates from rummants Although there is considerable immunological cross-rectivity amongst the two receptor protems in a variety of different P haemolytica strains, this cross-reactivity should mclude epitopes present at the surface in vivo Prehmmary results with the whole cell/antibody analysis (Figure 27) suggest the presence of such surface epitopes The consistent pattern observed for whole cell/HRP-btf and whole cell/anti-Tbp reactivities for any given strain (Figure 27) coupled with the greatly reduced reactivity observed for both reactions in the iron replete cells suggest that the reactivity observed is agamst iron regulated protems An important consideration in the development of an effective vaccme agamst any bacterial species, is the spectrum of the vaccme agamst the different serotypes/biotypes of the infecting bacterial species In N memngitidis while TbpA protems are relatively homogenous, the identification of two families m the species based upon the differences m the molecular mases and antigenic properties of their TbpB protems (18) dictates the formulation of a heterogenous Tbp vaccme representative of the two families The lOOKda transferrin receptor protein (TbpA), from bovine strain of P haemolytica, serotype Al (26) was identified by the affinity purification procedure of Schryver and Morris (32) Modification of that procedure, as described in the method section, has enabled us to identify the 60Kda as the second transferrm bindmg protem m P haemolytica analogous to TbpB in the other bacterial species (32 and Robki et al 1993) The affmity purification procedure isolated TbpA and TbpB of similar molecular weights from all of the strams of P haemolytica and P trehalosi examined (Figure 26, Panel A) Our results m P haemolytica and P trehalosi showed that antisera raised against these two purified receptor protems from a serotype Al, strain h44, specifically recognised the receptor protems from other Al strams (hl96, Figure 26, Panel B) and other A serotypes These mcluded an All serotype (hl07) known to differ from the other types m its reactiv ity with convalescent sera agamst the 35Kda and 70Kda iron regulated outer membrane protems The amounts of purified receptors from the T strams, h99 and hlOO (Figure 26, Panel A) and their with the antisera (Figure 26, Panel B) appeared to be reduced compared with most of the A strams This reduction m the antibody reactivity with the T strains was also observed in whole cell assay (Figure 27) The reduction in the whole cell-antibody reactivity m these strains, however, was consistent with their reduced reactivity with HRP-bTf (Figure 27) This suggests that the observed lower reactivity with the anti-TbpA and anti-TbpB antisera is likeh. due to differences m the expression of receptor protems under the standard growth conditions used m these expenments The genes encodmg TbpA and TbpB proteins, tbpA and tbpB are fairly homogeneous within serotype Al (causmg pneumonic pasteurellosis in cattle) and to a large extent, amongst the A types (causmg pneumonia m sheep and goats) m general (Figure 28) By extendmg the restriction enzyme digestion analysis to the P trehalosi strams, we found only marginal differences (Figure 28) The results of PCR- amplification experiments with specific 5' and 3' oligonucleotide primers and primers derived from the hypervaπable regions (Figure 29, Table 11) further supports the conclusion that the tbp genes withm P haemolytica species and P trehalosi are relatively homogenous These results contrast with the differences which have been observed for H influenzae and N memngitidis (1 and Loosemore et al ) but will need to be confirmed by sequence analysis of genes isolated from representative strams from a number of serotypes The lack of evident genetic hetereogeneity in the tbp genes and the apparent immunological crossreactivity of the Tbp protems from a variety of P haemolytica and P trehalosi strams underscore their potential as broad-spectrum vaccme antigens for prevention of infection m rummants While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples To the contrary, the mvention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims All publications, patents and patent applications are herein mcorporated by reference m their entirety to the same extent as if each mdividual publication, patent or patent application was specifically and individually indicated to be mcorporated by reference m its entirety FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION Abdullah et al., J Bacteπol 73 5597-5603, 1991 Adams et al., J Am Vet Med Assoc 134 85-89, 1959 Aisen and Listowski, Annu Rev Biochem 49 357393, 1980 Al-sultan and Aitken. 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Result of PCR analysis a ) Schematic diagram of the PCR procedure - Each circle represents a recombmant pBR321 plasmid and a possible PCR reaction The Tbpl primer, primer left and primer right an 3 represented by the letters t, 1 and r respectively The EcoRI site on the pBR322 plasmid I , denoted by the letter, E In the upper plasmid, the Tbpl primer and primer left would amplify a PCR product correspondmg to the heavy lme Similarly in the lower plasmid, the PCR produc amplified by Tbpl primer and primer right is represented by the heavy lme (b) The 0 8 kb PCR product amplified by Tbpl primer and primer left Figure 2. Restriction endonuclease map of tbp plasmids 9, 10 and 482 Open box - pBR32.' presented linearly Crosshatched box PCRII presented linearly The positions and the orientations of tbpA, tbpB are as shown by the dark arrows Figure 3. Preliminary nucleotide sequence of P haemolytica tbpA and tbpB Putative signal sequence cleavage sites are mdicated by an arrow The start codon (ATG) is underlined Figure 4. The promoter region of P haemolytica tbpB (PHTBPB) The putative Fur consensus sequence is indicated by asterisks The Fur consensus sequences of N gonorrhoeae tbpE (NGTBPB) and N memngitidis tbpB (NMTBPB) are also indicated Figure 5. Southern hybridization of P haemolytica genomic DNA digested with Clal and probed with the tbpA gene Lanes 1-16 represent P haemolytica serotypes 1 to 16 Lane M represents lambda DNA digested with Hind III and hybridized with lambda DNA radiolablled separately as size markers Figure 6. Southern hybridization of P haemolytica genomic DNA digested with Hindlll anc βαwHI and probed with the tbpA gene Lanes 1-16 represent P haemolytica serotypes 1-16 The molecular sizes are as mdicated on the left Figure 7. Southern hybridization of A suis 3714, A pleuropneumoniae CM5 and shope 4074 genomic DNA digested with vaπou restriction endonucieases and probed with P haemolytica tbpA Lane M represents lambda DNA digested with Hind III and hybridized with lambda DNA radiolabelled separately as size markers Figure 8. Restriction maps of the tbpA, tbpB regions m P haemolytica Al, A pleuropneumoniae CM5, Shope 4074 and A suis 3714 Line 1 represents the tbpA probe used in Figure 15 Lme 1 represents the tbpA probe used m Figures 16 and 17 Figure 9. Alignment of the ammo acid of Tbpl of P haemolytica Al (PHTBP) and the Tbpl of N gonorrhoeae (NGTBP1) and N memngitidis (NM1) The numbers to the right mdicate ammo acid positions Asterisks mdicate positions of complete identity in alignment, dots mdicate similar ammo acid residues Gaps were introduced to maximize sequence alignment and are mdicated by dashes (-) Figure 10. Alignment between P haemolytica Al Tbpl (PHTBPI) and the A pleuropneumoniae serotype 1 and 7 TfbA protems (APL, APL7) Asterisks mdicate positions of complete identity in alignment, dots indicatesimilar amino acid residues Gaps were introduced to maximize sequence alignment and are mdicated by dashes (-) Figure 11. Dendrogram illustrating the genetic relatedness among P haemolytica Tbpl (PHTBP), N gonorrhoeae Tbpl (NGTBP1), N memngitidis Tbpl (NM1) and the TfbA protems from A pleuropneumoniae serotype 1 and 7 (APL1, APL7) Figure 12. Peptide alignment between P haemolytica Al Tbpl and TonB-dependent outer membrane receptors of £ coh Asterisks show ammo acids with complete identity m alignment, dots mdicate similar ammo acid residues Gaps were introduced to maximize sequence alignment and are midcated by dashes (-) Figure 13. T7 analysis of the P haemolytica Tbpl protem The molecular weight marks in kDa are as indicated on the left Lane 1 - positive control recombmant plasmid Lane 2 - the recombmant plasmid contammg tbpA Lane 3 - the vector plasmid pBluescπpt (SK) Figure 14. Western immunoblot of inner and outer membranes from P haemolytica Al and £ coh HBlOl The first antibody was a rabbit antiserum raised to the soluble antigens of P haemolytica Al and the second antibody was goat an -rabbit alkaline phosphatase conjugate Lane M represents the molecular weight markers m kDa Lanes 1 4 represent outer membrane fractions and lanes 5-8 are inner membrane fractions 6μg of protem was added to each lane Lanes 1 and 5 - £ coh protems from cells grown m LT Lanes 2 and 6 - protems from cells grown m BHIB Lanes 3 and 7 protems from cells grown m BHIB plus 100 μM EDDA Lanes 4 and 8 - protems from cells grown in BHIB plus 100 μM EDDA with 1 mM FeSo 4 added Figure 15. Western immunoblot of inner and outer membranes from P haemolytica Al and E co HBlOl using sera raised m calves to soluble antigens by vaccination with Presponse® The second antibody was goat anti-bovine alkalme phosphatase conjugate Lane M represents the molecular weight markers m kDa Lanes 1-4 are outer membrane fractions and lanes 5-8 are inner membrane fractions 6 μg of protem was added to each lane Lanes 1 and 5 - £ coh protems from cells grown m LT Lanes 2 and 6 - protems from cells grown in BHIB Lanes 3 and 7 - protems from cells grown m BHIB plus 100 μM EDDA Lanes 4 and 8 - protems from cells grown m BHIB plus 100 μM EDDA with 1 mM FeSo 4 added Figure 16. Bmdmg of labelled transferrms by iron-deficient bacterial membranes Aliquots of total membranes (4 μg protem) prepared from iron-deficient cells from the mdicated bacterial strains were spotted onto strips of nitrocellulose /cellulose acetate paper and, after blockmg, the papers wereexposed to mixtures containing 450 ng/ml of the indicated HRP-con j ugated transferrm The filters were subsequently washed and developed with HRP substrate mixture as described m the Methods section hl73, hl74, hl75, hl76 and h44 are representative strams of P haemolytica whose serotype and source are listed in Table 1 h50 - A equuh HRP-bTf, oTf,-cTf and -eTf - HRP conjugates of bovme, ovme, caprme and equme transferrms Figure 17. Isolation of receptor protems with transferrin affinity columns Affmity isolation experiments were performed with iron-deficient total membranes prepared from P haemolytica strain h44 (top panel), hl73 (middle panel) and hl75 (bottom panel) Experiments were performed with bovine transferπn-Sepharose (lanes A and B), ovme transferπn-Sepharose (lane C), caprme transferπn-Sepharose (lane D) or equme transferπn-Sepharose (lane E) usmg standard washmg conditions (lanes B-E) or low salt washmg conditions (lane A) as outlmed in the methods section The samples eluted with buffer contammg 2M guanidme HCl were dialyzed, concentrated and aliquots analyzed by SDS-PAGE and silver staining as described m the methods section Figure 18. Immunological analysis of receptor proteins from different serotypes of P haemolytica from bovine, sheep and goats Aliquots of purified receptor proteins from representative serotypes of P haemolytica were subjected to SDS-PAGE, electroblotted and then probed with specific anti-TbpB serum (Panel A) or with anti-TbpA serum (Panel B) as described m the methods section The followmg P haemolytica strains of the indicated serotype were mcluded in the analysis, Lane 1 - stram h44 (Al), Lane 2 - hl73 (untypable), Lane 3 - hl75 (A7), Lane 4 - hl76 (A9), Lane 5 - hlOO (T4), Lane 6 - hl06 (T10), and Lane 7 - hl07 (All) The numbers on the left represent the molecular weights (X 1000) of standard protems Figure 19. Binding of labelled transferrin and anti-receptor antibody by mtact cells The indicated bacterial strains were grown under iron-limiting conditions, harvested by centrifugation and resuspend to a A 600 of 1-2 m 50 mM TrisHCl, 150 mM NaCl, pH 75 buffer A 5 μl aliquot of the suspensions were applied to HA membrane, the membrane was dried, blocked and then exposed to blocking solution contamg labelled transferrin (HRP-bTf) or antireceptor antibody (anti-TbpA, anti-TbpB) The latter membranes were washed and subsequently exposed to labelled second antibody prior to development with substrate Figure 20. Map of the P haemolytica tbp operon (Top) and regulatory sequences (Bottom) tbpA and tbpB are the genes encodmg for TbpA and TbpB, respectively, p, is the putative promoter region precedmg tbpB and denoted as -35 and -10 sites at the bottom A putative Fur box is represented as two arrows m opposite directions m the sequence at the bottom, rnaseT and fis are two ORFs flanking the P haemolytica tbp operon encodmg for protems highly homologous to E coh and H influenzae RNase transferase and factor-for-invertion stimulation proteins, respectively Additionally, putatives πbosomal bmdmg site or Shine-Dalgarno (SD) consensus sequence, transcriptional start (Met), and stop codons (SC) are also bolded Figure 21 . The DNA sequence of the tbp A gene from P haemolytica stram H196 Figure 22. Predicted am o acid sequence of the TbpA protein from Pasteurella haemolytica stram H196 Italicized amino acids correspond to the experimentaly determmed N-termmal ammo acids of the mature protem Residues indicated by stπkethrough constitute the leadei peptide region Residues that are identical in TbpAs from Neisseria memngitidis, N gonorrhoeae, H mfluenzae and Actinobacillus pleuropneumoniae are bold and underlmed Regions proposed as internal segments (dark shadmg), intermembrane b-strands (light shadmg) or external segments (no shading) based on the proposed topology model by Tommassen (28) are indicated Figure 23. The DNA sequence ot the tbpB gene from P haemolytica stram H196 Figure 24. Predicted ammo acid sequence of the TbpB protem from P haemolytica stram H196 Italicized amino acids correspond to the predicted N-terminal ammo acids of the mature protem Residues indicated by stπkethrough constitute the leader peptide region Regions of homology are identified by shadmg and residues that are identical m TbpBs from Neisseria menrngitidis, N gonorrhoeae, H influenzae and Actinobacillus pleuropneumoniae are bold and underlined Figure 25. Solid-phase HRP-Tf bmdmg assay Total membrane preparations from the mdicated strams of P haemolytica and P trehalosi were spotted onto nitrocellulose-cellulose acetate paper and blocked with skim milk prior to mcubation with HRP-bTf, -oTf or -gTf Bmdmg was detected with chloronaphthol reagent as in the methods The letters on the left mdicate the strams while the letters on the top mdicate the different HRP-labbelled rummant transferrms Figure 26. Western blot cross-reactivity studies with anti-TbpA and anti-TbpB antisera from P haemolytica serotype Al Aliquots of affmity purified receptor protems from the mdicated strams of P haemolytica and P trehalosi were separated by SDS-PAGE and Silver stam (panel A) or subjected to Western blottιng(panel B) as in Methods Tbp protems were identified by mcubation with a mixture of antι-TbpB(l /1000) and antι-TbpA(l / 1000) rabbit antisera as described m the methods section The numbers on the left represent the molecular weights (X 1000) of standard protems in kilodaltons Figure 27. Cross-reactivity studies with monospecific anti-TbpA and anti-TbpB antisera from P haemolytica serotype Al against intact cells Aliquots of iron-limited mtact cells from the indicated strams of P haemolytica and P trehalosi were spotted onto HA nitrocellulse paper and, after blockmg, the membrane was either exposed to HRP - labelled bTf, anti-TbpA antiserum or anti-TbpB antiserum Bound antibodies were subsequently detected by labelled goat anti-rabbit antibody as described m the methods A preparation of mtact P haemolytica stram h44 (type Al) cells grown under iron-sufficient cells (mdicated by h44 - Fe + ) was spotted onto the membranes and used as a control Figure 28 Restriction endonuclease digestion pattern of PCR-amphfied tbpA (Panel A) and TbpB (Panel B) genes from P haemolytica and P trehalosi strams The tbp genes amplified by colony PCR from the mdicated strams were digested with Sau3Al restriction endonuclease The resultmg digests were electrophoresed on a 7 5% polyacrylamide gel as described in Methods The letters above the lanes indicate the source stram template DNA used in PCR while the letters on the left mdicate the molecular weight standard in kilobases Imaging was done with a Hewlett-Parkard ScanJet Up In fig 29B, primer #s 397 and 400 from non conserved regions of P.hamolytica tbpB gene, were used m combmation with opposing primers (#s 401( 5') and 199(3')} respectively. Figure 29. PCR amplification of variable segments of the tbp A andtbpB genes. For tbp A gene , oligonucleotide primer # 450 made from the deduced ammo acid sequence from a hypervariable region of tbpA was used in combination with the 5' specific primer (#255) to amplify the gene segment from the various P.haemolytica strams . The products were then analysed on 1% agarose gel followed by staining with ethidium bromide. 31 - SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANTS: (A NAME: LO, Reggie, Y.C. (B STREET: 4 Birch Street (C CITY: Guelph (D STATE: Ontario (E COUNTRY: Canada (F POSTAL CODE: NIG 2N3 (A NAME: SCHRYVERS, Anthony, B. (B STREET: 39 Edforth Road, N. . (C CITY: Calgary (D STATE: Alberta (E COUNTRY: Canada (F POSTAL CODE: T3A 3V8 (A NAME: POTTER, Andrew, A. (B STREET: 521 Dalhousie Cres . (C CITY: Saskatoon (D STATE: Saskatchewan (E COUNTRY: Canada (F POSTAL CODE: F7H 3S5 (ii) TITLE OF INVENTION: Transferrin Binding Proteins of Pasteurella Haemolytica and Vaccines Containing Same (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: BERESKIN & PARR (B) STREET: 40 King Street, West (C) CITY: Toronto (D) STATE: Ontario (E) COUNTRY: Canada (F) ZIP: M5H 3Y2 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 60/008,569 (B) FILING DATE: 01-DEC-1995 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: CA 2,164,274 (B) FILING DATE: 01-DEC-1995 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Kurdydyk, Linda M. (B) REGISTRATION NUMBER: 34,971 (C) REFERENCE/DOCKET NUMBER: 6580-82 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (416) 364-7311 (B) TELEFAX: (416) 361-1398 (2) INFORMATION FOR SEQ ID NO: 1 : (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2784 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Pasteurella haemolytica (tbpA gene) (B) STRAIN: H196 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATGATAATGA AATATCATCA TTTTCGCTAT TCACCTGTTG CCTTAACAGT GTTATTTGCT 60 CTTTCTCATT CATACGGTGC TGCGACTGAA AATAAAAAAA TCGAAGAAAA TAACGATCTA 120 GCTGTTCTGG ATGAAGTTAT TGTGACAGAG AGCCATTATG CTCACGAACG TCAAAACGAA 180 GTAACTGGCT TGGGGAAAGT AGTGAAAAAT TATCACGAAA TGAGTAAAAA TCAAATTCTT 240 GGTATTCGTG ATTTAACTCG CTATGACCCT GGTATTTCGG TGGTGGAACA AGGTCGCGGT 300 GCAAGTAGTG GCTATGCCAT TCGAGGTGTA GATAAAAACC GTGTCAGCTT ACTTGTTGAT 360 GGGCTACCAC AAGCGCACAG TTATCATACG CTAGGTTCAG ATGCTAATGG TGGTGCAATT 420 AATGAGATTG AGTATGAAAA CATTCGTTCA ATTGAGTTAA GCAAAGGAGC AAGTTCTGCG 480 GAATATGGCT CTGGTGCGCA TGGTGGTGCT ATTGGTTTTC GTACTAAAGA TGCGCAGGAT 540 ATTATTAAAG AGGGGCAGCA TTGGGGCTTA GATAGTAAGA CCTCTTATGC CAGCAAAAAT 600 AGCCATTTTT TACAGTCTAT CGCAGCGGCT GGTGAGGCGG GTGGTTTTGA AGCACTTGTT 660 ATTGCAACTC ACCGACACGG TAAAGAGACC AAAATTCATT CCGAGGCAAA TAAATTAAAA 720 CATAATATTC GGCGTATAAC CGGCTTTGAA AATCGCTACG ACTTTACCCA AATTCCGCAC 780 AGAATGCTCC TGGAGGATCT CCTTTTAATT GTGGAAGATA CTTGCCCAAC ATTAGATTGT 840 ACTCCTCGTG CAAGGGTTAA GTTGAACCGC GATAATTTCC CAGTGAGAAC ATTTCCGGAA 900 TATACGCCTG AAGAGCGCAA ACAGCTTGAG CAGATTCCTT ATCGCACTGA GCAGCTCTCA 960 GCCCAAGAAT ATACCGGTAA AGATCGCATT GCACCAAACC CTTTAGATTA CAAGAGTAAT 1020 TCTGTTTTTA TGAAGTTTGG CTATCACTTC AACTCGTCTC ATTATCTTGG CGCAATCTTA 1080 GAAGATACAA AAACACGCTA CGATATCCGT GATATGCAAA CGCCAGCTTA CTATACAAAA 1140 GACGATATTA ACTTATCACT TAGGAACTAT GTTTATGAAG GGGATAATAT TTTAGATGGC 1200 TTAGTGTTCA AGCCAAGGAT CCCTTATGGG TTGCGCTATA GCCATGTGAA GTTTTTTGAT 1260 GAACGTCACC ACAAACGTCG TTTAGGATTC ACCTATAAAT ATAAACCAGA GAATAATCGC 1320 TGGTTGGATA GCATTAAACT CAGTGCGGAT AAACAAGATA TTGAACTATA TAGCCGGCTA 1380 CATCGCTTGC ATTGTAGCGA TTATCCTGTG GTAGATAAAA ATTGCCGCCC GACTTTGGAT 1440 AAATCTTGGT CTATGTATCG AACTGAGCGT AATAATTACC AAGAAAAGCA TCGTGTCATT 1500 CATTTAGAAT TTGATAAAGC GCTAAATGCT GGTCAAGGCG TATTTAACCA AACCCACAAA 1560 CTGAATTTAG GGTTGGGCTT TGATCGATTT AATTCGCTTA TGGATCATGG GGATATGACT 1620 GCCCAATATA CCAAAGGCGG TTATACCAGC TACCGCGGTA GAGGGCGTTT AGATAATCCA 1680 TATATTTATC GCCGCGATCC ACGCAGTATT GAAACGGTAT CTTTGTGTAA TAATACACGC 1740 GGCGACATCT TAAACTGTGA ACCGCGTAAA ATTAAAGGCG ATAGCCATTT TGTTAGCTTC 1800 CGCGATCTAG TGATAAGCGA GTATGTGGAT TTGGGATTAG GGGTGCGTTT TGATCAACAT 1860 CGATTTAAAT CTGATGATCC GTGGACACTT AGCCGAACTT ATCGAAATTG GTCTTGGAAT 1920 GGTGGGATTA CGCTTAAACC AACAGAGTTT GTATCGCTTT CTTATCGCAT TTCAAACGGT 1980 TTTAGAGTGC CTGCATTCTA TGAACTTTAT GGTAAACGTG ATCATATTGG GCTTAAAGAT 2040 AACGAATATG TGCAACGCGC GCAACGTAGC CACCAGTTAG AGCCAGAAAA ATCGACTAAT 2100 CATGAGATTG GAGTTAGCTT TAAAGGTCAA TTTGGTTACC TTGAATTTCC GTAATAACTA 2160 TAAAAATATG ATTGCGACAG CATGTAAAAG AATAATACAA AAATCACACT GTTTCTATAA 2220 CTACCATAAT ATTCAAGATG TAGCACTAAA CGGGATAAAT TTAGTCGCTA AATTTGACTT 2280 ACACGGTATT TTATCTATGC TGCCAGATGG TTTTTATTCA TCAGTTGCTT ATAACCGTGT 2340 AAAAGTAAAA GAGCGGAAAC TAACCGACTC AAGACTCGAT AGCGTAAACG ATCCTATTCT 2400 AGATGCGATT CAGCCAGCAC GCTATGTGCT TGGATTCGGC TACGATCACC CAGAAGAAAA 2460 ATGGGGAATT GGCATTACTA CCACCTATTC TAAAGCCAAA AACGCCGATG AGGTGGCAGG 2520 CACACGTCAT CACGGATACA TCGCGTTGAT TTAGGTGGCA AACTGACCGG TTCTTGGTAC 2580 ACCCATGATA TTACCGGTTA CATCAATTAT AAAAACTACA CCTTACGTGG AGGAATTTAT 2640 AATGTGACTA ATCGTAAATA TTCCACTTGG GAATCAGTGC GCCAATCCGG TGTGAATGCA 2700 GTAAACCAAG ACCGGGGTAG CAATTACACT CGATTTGGCG CTCCGGGGAG AAATTTCAGT 2760 TTAGCATTTG AAATGAAGTT TTAG 2784 (2) INFORMATION FOR SEQ ID NO: 2 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 930 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Pasteurella haemolytica (TbpA protein) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 : Met lie Met Lys Tyr His His Phe Arg Tyr Ser Pro Val Ala Leu Thr 1 5 10 15 Val Leu Phe Ala Leu Ser His Ser Tyr Gly Ala Ala Thr Glu Asn Lys 20 25 30 Lys lie Glu Glu Asn Asn Asp Leu Ala Val Leu Asp Glu Val lie Val 35 40 45 Thr Glu Ser His Tyr Ala His Glu Arg Gin Asn Glu Val Thr Gly Leu 50 55 60 Gly Lys Val Val Lys Asn Tyr His Glu Met Ser Lys Asn Gin lie Leu 65 70 75 80 Gly lie Arg Asp Leu Thr Arg Tyr Asp Pro Gly lie Ser Val Val Glu 85 90 95 Gin Gly Arg Gly Ala Ser Ser Gly Tyr Ala lie Arg Gly Val Asp Lys 100 105 110 Asn Arg Val Ser Leu Leu Val Asp Gly Leu Pro Gin Ala His Ser Tyr 115 120 125 His Thr Leu Gly Ser Asp Ala Asn Gly Gly Ala lie Asn Glu lie Glu 130 135 140 Tyr Glu Asn lie Arg Ser lie Glu Leu Ser Lys Gly Ala Ser Ser Ala 145 150 155 160 Glu Tyr Gly Ser Gly Ala His Gly Gly Ala He Gly Phe Arg Thr Lys 165 170 175 Asp Ala Gin Asp He He Lys Glu Gly Gin His Trp Gly Leu Asp Ser 180 185 190 Lys Thr Ser Tyr Ala Ser Lys Asn Ser His Phe Leu Gin Ser He Ala 195 200 205 Ala Ala Gly Glu Ala Gly Gly Phe Glu Ala Leu Val He Ala Thr His 210 215 220 Arg His Gly Lys Glu Thr Lys He His Ser Glu Ala Asn Lys Leu Lys 225 230 235 240 His Asn He Arg Arg He Thr Gly Phe Glu Asn Arg Tyr Asp Phe Thr 245 250 255 Gin He Pro His Arg Met Leu Leu Glu Asp Leu Leu Leu He Val Glu 260 265 270 Asp Thr Cys Pro Thr Leu Asp Cys Thr Pro Arg Ala Arg Val Lys Leu 275 280 285 Asn Arg Asp Asn Phe Pro Val Arg Thr Phe Pro Glu Tyr Thr Pro Glu 290 295 300 Glu Arg Lys Gin Leu Glu Gin He Pro Tyr Arg Thr Glu Gin Leu Ser 305 310 315 320 Ala Gin Glu Tyr Thr Gly Lys Asp Arg He Ala Pro Asn Pro Leu Asp 325 330 335 Tyr Lys Ser Asn Ser Val Phe Met Lys Phe Gly Tyr His Phe Asn Ser 340 345 350 Ser His Tyr Leu Gly Ala He Leu Glu Asp Thr Lys Thr Arg Tyr Asp 355 360 365 He Arg Asp Met Gin Thr Pro Ala Tyr Tyr Thr Lys Asp Asp He Asn 370 375 380 Leu Ser Leu Arg Asn Tyr Val Tyr Glu Gly Asp Asn He Leu Asp Gly 385 390 395 400 Leu Val Phe Lys Pro Arg He Pro Tyr Gly Leu Arg Tyr Ser His Val 405 410 415 Lys Phe Phe Asp Glu Arg His His Lys Arg Arg Leu Gly Phe Thr Tyr 420 425 430 Lys Tyr Lys Pro Glu Asn Asn Arg Trp Leu Asp Ser He Lys Leu Ser 435 440 445 Ala Asp Lys Gin Asp He Glu Leu Tyr Ser Arg Leu His Arg Leu His 450 455 460 Cys Ser Asp Tyr Pro Val Val Asp Lys Asn Cys Arg Pro Thr Leu Asp 465 470 475 480 Lys Ser Trp Ser Met Tyr Arg Thr Glu Arg Asn Asn Tyr Gin Glu Lys 485 490 495 His Arg Val He His Leu Glu Phe Asp Lys Ala Leu Asn Ala Gly Gin 500 505 510 Gly Val Phe Asn Gin Thr His Lys Leu Asn Leu Gly Leu Gly Phe Asp 515 520 525 Arg Phe Asn Ser Leu Met Asp His Gly Asp Met Thr Ala Gin Tyr Thr 530 535 540 Lys Gly Gly Tyr Thr Ser Tyr Arg Gly Arg Gly Arg Leu Asp Asn Pro 545 550 555 560 Tyr He Tyr Arg Arg Asp Pro Arg Ser He Glu Thr Val Ser Leu Cys 565 570 575 Asn Asn Thr Arg Gly Asp He Leu Asn Cys Glu Pro Arg Lys He Lys 580 585 590 Gly Asp Ser His Phe Val Ser Phe Arg Asp Leu Val He Ser Glu Tyr 595 600 605 Val Asp Leu Gly Leu Gly Val Arg Phe Asp Gin His Arg Phe Lys Ser 610 615 620 Asp Asp Pro Trp Thr Leu Ser Arg Thr Tyr Arg Asn Trp Ser Trp Asn 625 630 635 640 Gly Gly He Thr Leu Lys Pro Thr Glu Phe Val Ser Leu Ser Tyr Arg 645 650 655 He Ser Asn Gly Phe Arg Val Pro Ala Phe Tyr Glu Leu Tyr Gly Lys 660 665 670 Arg Asp His He Gly Leu Lys Asp Asn Glu Tyr Val Gin Arg Ala Gin 675 680 685 Arg Ser His Gin Leu Glu Pro Glu Lys Ser Thr Asn His Glu He Gly 690 695 700 Val Ser Phe Lys Gly Gin Phe Gly Tyr Leu Asp Val Ser Tyr Phe Arg 705 710 715 720 Asn Asn Tyr Lys Asn Met He Ala Thr Ala Cys Lys Arg He He Gin 725 730 735 Lys Ser His Cys Phe Tyr Asn Tyr His Asn He Gin Asp Val Ala Leu 740 745 750 Asn Gly He Asn Leu Val Ala Lys Phe Asp Leu His Gly He Leu Ser 755 760 765 Met Leu Pro Asp Gly Phe Tyr Ser Ser Val Ala Tyr Asn Arg Val Lys 770 775 780 Val Lys Glu Arg Lys Leu Thr Asp Ser Arα Leu Asp Ser Val Asn Asp 785 790 795 800 Pro He Leu Asp Ala He Gin Pro Ala Arg Tyr Val Leu Gly Phe Gly 805 810 815 Tyr Asp His Pro Glu Glu Lys Trp Gly He Gly lie Thr Thr Thr Tyr 820 825 830 Ser Lys Ala Lys Asn Ala Asp Glu Val Ala Gly Thr Arg His His Gly 835 840 845 He His Arg Val Asp Leu Gly Gly Lys Leu Thr Gly Ser Trp Tyr Thr 850 855 860 His Asp He Thr Gly Tyr He Asn Tyr Lys Asn Tyr Thr Leu Arg Gly 865 870 875 880 Gly He Tyr Asn Val Thr Asn Arg Lys Tyr Ser Thr Trp Glu Ser Val 885 890 895 Arg Gin Ser Gly Val Asn Ala Val Asn Gin Asp Arg Gly Ser Asn Tyr 900 905 910 Thr Arg Phe Gly Ala Pro Gly Arg Asn Phe Ser Leu Ala Phe Glu Met 915 920 925 Lys Phe 930 (2) INFORMATION FOR SEQ ID NO:3 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1755 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (i ) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Pasteurella haemolytica (TbpB gene) (B) STRAIN: H196 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 : ATGTTTAAAC TTAAAAGTAG TTTTGTACTG CTTAATGCGG CGCTACTTGC TGCTTGTTCC 60 TCAAATGGTG GAAGCTTTGA TGTTCAATCT GCCAAAGTTG AATCTCAAAC GCAAACTACC 120 CCCAAAAAGC CAAGTTTACA AGATGATAAT AGTAACGCAA GACGTACAGT AAGCGCTTCT 180 GAAACTGAAG CTTTATTGCA GCCGGGGTTT GGTTTTTCAG CCAAAATTCC GCGTCGTAAT 240 CTCCTTCCGC AGGGGAAGGA AGATGTAGCC CCTATTGGTG ATATAAAAGA GATTACTGGA 300 GATCTGCCAA AAATTCCGTA TGAAGAAGAG GTTAAAGCGT GCGGTAGTAG TGCTGATGGA 360 TTTAGCCATA CTCATGATAG AAATCATAAG TTGTATACAA GAGATTTTAA TTTTGTTCGT 420 TCCGGCTATG TTGTGCATTC TGGTCCAAAA CCTGAAATAA AGCCTAAAGA AATTTTGAGA 480 ACAGGTGCAC ATGGGTATGT TTACTATTTA GGTATAGAGC CGCCCAAAGC AATACCTACC 540 CAAAAACTAA CTTATAAAGG ATATTGGGAT TTTACTACCT ATGCGGCTAA GGGGAGAGAT 600 AGTAATATTT TTCTAATTCC CGCAGGCATC AATAGTGGCG CCATACCGGA AAATAGTCAC 660 GATATTAATG TTGATGATTC TGAAAAACCA ATGGGGCATA CAGGAGAATT TACGGCTGAT 720 TTTGCTAATA AAACTTTAAC TGGAACATTG GTTCGTAATG GGTATGTTAG TCGTAGCAAA 780 GAGCAAAAAA TTACAACAAT TTACGATATT GATGCGAAAA TTAAAGGTAA TCGCTTTTCT 840 GGTAAAGCAA ACCCAAAAAA ACCGATGATC CTTATTTTTG GGAAAAGCTC CACGACACTT 900 GAAGGTGGAT TTTTTGGTGG GGAGGCTCAA GAACTTGCCG GTAAATTCTT AGCTGATGAT 960 AAGTCGGTAT TTGTTGTTTT TGCTGGCACA CGAGATGCTA AAAAAGATGA TAGTGAATCT 1020 GCCTTTGATG CTTTCCCAAT TAAACTTAAA GATTTAAATA AATCTGAGAT GGATACTTTC 1080 GGGAATGCGA CACATTTGAT TATTAACAAT AAGCAGATTC CACTTATTGC GGAAGCCACA 1140 AAAAGCTTTG CCGAGATGAA ATTTGATGAT TTGGTTACCC GTACTATTGA TGGAAAAACG 1200 TATCGAGTTT CAGTCTGCTG TAATAATTTA GATTATGTCA AATTTGGGAT TTATAGCGAG 1260 GGAAATAATA GTGATACTGC TCTCCAAGAA TATTTAGTAG GAGAACGTAC AGCTCTGGCA 1320 GATTTGCCAA CAGGGACAGT AAAATATCGA GGTACTTGGG ACGGGGTAAT GTACAGTAAA 1380 TCTGGCTCGG CAGGGGTTGA ATCGCCAAGT AACAGCGAAA GTGGTACTCG TTCACTATTC 1440 GATGTAGATT TTGTCAATAA AAAAATTAAT GGCAAGCTGA TTGCTAATGA TGGTGTTGAA 1500 GAACGCCCAA TGCTGACACT GGAAGGCAAT CTGAAAGGGA ATGGTTTTGG AGGCACAGCC 1560 AAAACGGGCA ATTCTGGTTT TAATCTTGAT CCCAAAAGTA CGAATGGTGG CACGGTAGGG 1620 CATATAAATA CTCAATTTGA AGGGGGCTTT TATGGCCCTA AGGCGACGGA ATTAGGTGGT 1680 ATTGTACAAA ATACAGAAAC GGATAAAGAT AGAGTCAGTA TTACATTCGG CGGAAAACGT 1740 CAAATAGAAA AATAA 1755 (2) INFORMATION FOR SEQ ID NO:4- (l) SEQUENCE CHARACTERISTICS (A) LENGTH: 584 ammo acids (C) STRANDEDNESS smgle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: prote ( vi ) ORIGINAL SOURCE : ( A ) ORGANISM : Pas teurel la haemolytica ( TbpB prote in ) ( xi ) SEQUENCE DESCRI PTION : SEQ ID NO : 4 : Met Phe Lys Leu Lys Ser Ser Phe Val Leu Leu Asn Ala Al a Leu Leu 1 5 10 15 Ala Ala Cys Ser Ser Asn Gly Gly Ser Phe Asp Val Gin Ser Ala Lys 20 25 30 Val Glu Ser Gin Thr Gin Thr Thr Pro Lys Lys Pro Ser Leu Gin Asp 35 40 45 Asp Asn Ser Asn Ala Arg Arg Thr Val Ser Ala Ser Glu Thr Glu Ala 50 55 60 Leu Leu Gin Pro Gly Phe Gly Phe Ser Ala Lys He Pro Arg Arg Asn 65 70 75 80 Leu Leu Pro Gin Gly Lys Glu Asp Val Ala Pro He Gly Asp He Lys 85 90 95 Glu He Thr Gly Asp Leu Pro Lys He Pro Tyr Glu Glu Glu Val Lys 100 105 110 Ala Cys Gly Ser Ser Ala Asp Gly Phe Ser His Thr His Asp Arg Asn 115 120 125 His Lys Leu Tyr Thr Arg Asp Phe Asn Phe Val Arg Ser Gly Tyr Val 130 135 140 Val His Ser Gly Pro Lys Pro Glu He Lys Pro Lys Glu He Leu Arg 145 150 155 160 Thr Gly Ala His Gly Tyr Val Tyr Tyr Leu Gly He Glu Pro Pro Lys 165 170 175 Ala He Pro Thr Gin Lys Leu Thr Tyr Lys Gly Tyr Trp Asp Phe Thr 180 185 190 Thr Tyr Ala Ala Lys Gly Arg Asp Ser Asn He Phe Leu He Pro Ala 195 200 205 Gly He Asn Ser Gly Ala He Pro Glu Asn Ser His Asp He Asn Val 210 215 220 Asp Asp Ser Glu Lys Pro Met Gly His Thr Gly Glu Phe Thr Ala Asp 225 230 235 240 Phe Ala Asn Lys Thr Leu Thr Gly Thr Leu Val Arg Asn Gly Tyr Val 245 250 255 Ser Arg Ser Lys Glu Gin Lys He Thr Thr He Tyr Asp He Asp Ala 260 265 270 Lys He Lys Gly Asn Arg Phe Ser Gly Lys Ala Asn Pro Lys Lys Pro 275 280 285 Met He Leu He Phe Gly Lys Ser Ser Thr Thr Leu Glu Gly Gly Phe 290 295 300 Phe Gly Gly Glu Ala Gin Glu Leu Ala Gly Lys Phe Leu Ala Asp Asp 305 310 315 320 Phe Ala Gly Thr Arg Asp Ala Lys Lys Asp 330 335 Asp Ala Phe Pro He Lys Leu Lys Asp Leu 345 350 Thr Phe Gly Asn Ala Thr His Leu He He 360 365 Leu He Ala Glu Ala Thr Lys Ser Phe Ala 375 380 Leu Val Thr Arg Thr He Asp Gly Lys Thr 395 400 Cys Asn Asn Leu Asp Tyr Val Lys Phe Gly 410 415 Asn Ser Asp Thr Ala Leu Gin Glu Tyr Leu 425 430 Leu Ala Asp Leu Pro Thr Gly Thr Val Lys 440 445 Gly Val Met Tyr Ser Lys Ser Gly Ser Ala 455 460 Asn Ser Glu Ser Gly Thr Arg Ser Leu Phe 475 480 Lys Lys He Asn Gly Lys Leu He Ala Asn 490 495 Pro Met Leu Thr Leu Glu Gly Asn Leu Lys 505 510 Thr Ala Lys Thr Gly Asn Ser Gly Phe Asn 520 525 Asn Gly Gly Thr Val Gly His He Asn Thr 535 540 Tyr Gly Pro Lys Ala Thr Glu Leu Gly Gly 555 560 Thr Asp Lys Asp Arg Val Ser He Thr Phe 570 575 Glu Lys

Claims: WE CLAIM 1 A purified and isolated nucleic acid molecule comprising a sequence encoding ε transferrin bmding protem of P haemolytica 1 A purified and isolated nucleic acid molecule according to claim 1 comprismg a sequence encodmg TbpA of P haemolytica 3 A purified and isolated nucleic acid molecule as claimed m claim 2, which encodes ε protem havmg the ammo acid sequence as shown m Figure 22 and SEQ ID NO 2 4 A purified and isolated nucleic acid molecule as claimed in claim 2, havmg a sequence which comprises (a) a nucleic acid sequence as shown in Figure 21 and SEQ ID NO 1 (b) nucleic acid sequences complementary to (a), (c) nucleic acid sequences which are at least 80% homologous to (a), or (d) a fragment of (a) or (b) that is at least 15 bases and which will hybridize to (a) or (b) under strmgent hybridization conditions 5 A purified and isolated nucleic acid molecule accordmg to claim 1 comprismg a sequence encoding TbpB of P haemolytica 6 A purified and isolated nucleic acid molecule as claimed m claim 5, which encodes a protem havmg the ammo acid sequence as shown m Figure 24 and SEQ ID NO 4 7 A purified and isolated nucleic acid molecule as claimed m claim 5, havmg a sequence which comprises (a) a nucleic acid sequence as shown m Figure 23 and SEQ ID NO 3, (b) nucleic acid sequences complementary to (a), (c) nucleic acid sequences which are at least 80% homologous to (a), or (d) a fragment of (a) or (b) that is at least 15 bases and which will hybridize to (a) or (b) under strmgent hybridization conditions 8 A naturally occurring nucleic acid molecule which is characterized by the ability to hybridize to the purified and isolated nucleic acid molecule as claimed in claim 2 under stringent hybridization conditions 9 A naturally occurring nucleic acid molecule which is characterized by the ability to hybridize to the purified and isolated nucleic acid molecule as claimed in claim 5 undei strmgent hybridization conditions 10 An oligonucleotide comprismg at least 15 contiguous bases of a nucleic acid molecule as claimed in claim 2 which is characterized by the ability to hybridize to the nucleic acid molecule under strmgent hybridization conditions 11 An oligonucleotide comprismg at least 15 contiguous bases of a nucleic acid molecule as 5 claimed in claim 5 which is characterized by the ability to hybridize to the nucleic acid molecule under strmgent hybridization conditions 12 A recombmant expression vector adapted for transformation of a host cell comprismg a nucleic acid molecule as claimed in clai 2 and one or more transcription and translation elements operatively lmked to the nucleic acid molecule 10 13 A recombmant expression vector adapted for transformation of a host cell comprismg a nucleic acid molecule as claimed in claim 5 and one or more transcription and translation elements operatively lmked to the nucleic acid molecule 14 A host cell contammg a recombmant expression vector as claimed m claim 12 15 A host cell contammg a recombmant expression vector as claimed m claim 13 15 16 A purified and isolated TbpA protem 17 A purified and isolated TbpA protein as claimed in claim 16, which has the ammo acid sequence as shown Figure 22 and SEQ ID NO 2, or fragments thereof capable of bmdmg transferrin 18 A purified and isolated TbpB protem 20 19 A purified and isolated TbpB protem as claimed in claim 18, which has the ammo acid sequence as shown m Figure 24 and SEQ ID NO 4, or fragments thereof capable of bmdmg transferrin 20 A method for preparing a TbpA protem comprising (a)transferπng the recombmant expression vector accordmg to claim 12 mto a host cell, (b) selecting transformed host cells from 25 untransformed host cells, (c) culturmg a transformed host cell under conditions which allow expression of TbpA , and (d)ιsolatιng the recombmant TbpA 21 A method for preparing a TbpB protem comprismg (a)transferrmg the recombmant expression vector accordmg to claim 13 mto a host cell, (b) selecting transformed host cells from untransformed host cells, (c) culturmg a transformed host cell under conditions which allow expression of TbpB , and (d)ιsolatιng the recombmant TbpB 5 22 A polyclonal or monoclonal antibody havmg specificity agamst an epitope of TbpA or TbpB 23 A vaccme for the prophylaxis and treatment of an mfection caused by a Pasteurella spp , wherem said vaccme comprises an immunologically effective amount of at least one of TbpA and TbpB of P haemolytica and a pharmaceutically acceptable carrier 10 24 The vaccine according to claim 23 wherein said infection is caused by Pasteurella haemolytica 25 A vaccme for the prophylaxis and treatment of an mfection caused by a Pasteurella spp , wherem said vaccme comprises an immunologically effective amount of TbpA and TbpB and a pharmaceutically acceptable carrier 15 26 A vaccme for the prophylaxis and treatment of an infection caused by a Pasteurell i spp , wherein said vaccine comprises an immunologically effective amount of TbpB and a pharmaceutically acceptable carrier 27 The vaccine according to claim 26 wherem said TbpB has the ammo acid sequence a ^ shown m Figure 24 and SEQ ID NO 4 20 28 A vaccme for the prophylaxis and treatment of an mfection caused by a Pasteurella spp , wherein said vaccine comprises an immunologically effective amount of a recombinar t expression vector accordmg to claim 12 and a pharmaceutically acceptable earner 29 A vaccme for the prophylaxis and treatment of an mfection caused by a Pasteurella spp , wherein said vaccine comprises an immunologically effective amount of a recombinar t 25 expression vector accordmg to claim 13 and a pharmaceutically acceptable carrier

Inventors: LO REGGIE Y C, POTTER ANDREW ALLAN, SCHRYVERS ANTHONY BERNARD, LO, REGGIE, Y., C., POTTER, ANDREW, ALLAN, SCHRYVERS, ANTHONY, BERNARD
Applicants: LO REGGIE Y C, POTTER ANDREW ALLAN, SCHRYVERS ANTHONY BERNARD, LO, REGGIE, Y., C., POTTER, ANDREW, ALLAN, SCHRYVERS, ANTHONY, BERNARD
Application year: 1996
Main classification: C12N15
IPC codes: A61K31, A61K39, A61P31, C07K14, C07K16, C12N1, C12N15, C12R1