# Patent

## Title
Genes of an otitis media isolate of nontypeable Haemophilus influenzae

## Abstract

The invention relates to the polynucleotide sequence of a nontypeable stain of Haemophilus influenzae (NTHi) and polypeptides encoded by the polynucleotides and uses thereof. The invention also relates to NTHi genes which are upregulated during or in response to NTHi infection of the middle ear and/or the nasopharynx.

## Background
RELATED APPLICATIONS
The present application is a continuation of U.S. application Ser. No. 10/795,159, filed Mar. 5, 2004, now U.S. Pat. No. 7,241,867, which claims priority benefit from U.S. Provisional Application 60/453,134 filed Mar. 6, 2003 which is incorporated herein by reference in its entirety.


FIELD OF INVENTION
The invention relates to the polynucleotide sequence of a nontypeable strain of Haemophilus influenzae (NTHi) genome, NTHi genes contained within the genome and polypeptides encoded by the polynucleotides. The invention also relates to uses of these NTHi polynucleotides and NTHi polypeptides including vaccines and methods of treating and preventing NTHi related disorders. The invention also relates to NTHi genes which are upregulated during or in response to NTHi infection of the middle ear or nasopharynx.
BACKGROUND
Otitis media (OM) is a highly prevalent pediatric disease worldwide and is the primary cause for emergency room visits by children (Infante-Rivand and Fernandez, Epidemiol. Rev., 15: 444-465, 1993). Recent statistic indicate that 24.5 million physician office visits were made for OM in 1990, representing a greater than 200% increase over those reported in the 1980's. While rarely associated with mortality any longer, the morbidity associated with OM is significant. Hearing loss is a common problem associated with this disease, often times affecting a child's behavior, education and development of language skills (Baldwin, Am. J. Otol., 14: 601-604, 1993; Hunter et al., Ann. Otol. Rhinol. Laryngol. Suppl., 163: 59-61, 1994; Teele et al., J. Infect. Dis., 162: 685-694, 1990). The socioeconomic impact of OM is also great, with direct and indirect costs of diagnosing and managing OM exceeding $5 billion annually in the U.S. alone (Kaplan et al., Pediatr. Infect. Dis. J., 16: S9-11, 1997).
Whereas antibiotic therapy is common and the surgical placement of tympanostomy tubes has been successful in terms of draining effusions, clearing infection and relieving pain associated with the accumulation of fluids in the middle ear, the emergence of multiple antibiotic-resistant bacteria and the invasive nature associated with tube placement, has illuminated the need for more effective and accepted approaches to the management and preferably, the prevention of OM. Surgical management of chronic OM involves the insertion of tympanostomy tubes through the tympanic membrane while a child is under general anesthesia. While this procedure is commonplace (prevalence rates are  ̃13%; Bright et al., Am. J. Public Health, 83(7): 1026-8, 1993) and is highly effective in terms of relieving painful symptoms by draining the middle ear of accumulated fluids, it too has met with criticism due to the invasive nature of the procedure and its incumbent risks (Berman et al., Pediatrics, 93(3):353-63, 1994; Bright et al., supra.; Cimons, ASM News, 60: 527-528; Paap, Ann. Pharmacother., 30(11): 1291-7, 1996).
Progress in vaccine development is most advanced for Streptococcus pneumoniae, the primary causative agent of acute OM (AOM), as evidenced by the recent approval and release of a seven-valent capsular-conjugate vaccine, PREVNAR® (Eskola and Kilpi, Pedriatr. Infect. Dis. J. 16: S72-78, 2000). While PREVNAR® has been highly efficacious for invasive pneumococcal disease, coverage for OM has been disappointing (6-8%) with reports of an increased number of OM cases due to serotypes not included in the vaccine (Black et al., Pedriatr. Infect. Dis J., 19: 187-195; Eskola et al., Pedriatr. Infect. Dis. J., 19: S72-78, 2000; Eskola et al., N. Engl. J. Med. 344: 403-409, 2001; Snow et al., Otol. Neurotol., 23: 1-2, 2002). Less progress has been made for non-typeable Haemophilus influenzae (NTHi), the gram-negative pathogen that predominates in chronic OM with effusion (Klein, Pedriatr. Infect. Dis. J., 16: S5-8, 1997; Spinola et al., J. Infect. Dis., 154: 100-109, 1986). Hampering development of effective vaccines against NTHi, is the currently incomplete understanding of the pathogenesis of NTHi-induced middle ear disease. Contributing to this delay is a lack of understanding of the dynamic interplay between microbe-expressed virulence factors and the host's immune response as the disease progresses from one of host immunological tolerance of a benign nasopharyngeal commensal, to that of an active defensive reaction to an opportunistic invader of the normally sterile middle ear space.
Currently there is a poor understanding of how NTHi causes OM in children. The identification of putative virulence factors necessary for induction of OM will contribute significantly to the understanding of the host-pathogen interaction and ultimately, the identification of potential vaccine candidates and targets of chemotherapy. There is a tremendous need to develop more effective and accepted approaches to the management and preferably, the prevention of otitis media. Vaccine development is a very promising and cost effective method to accomplish this goal (Giebank, Pedriatr. Infect. Dis. J., 13(11): 1064-8, 1994: Karma et al., Int. J. Pediatr. Otorhinolaryngol., 32(Suppl.): S127-34, 1995).
SUMMARY OF INVENTION
The present invention provides for the identification and characterization of the genomic sequence of NTHi H. influenzae strain 86-028NP and the polypeptide sequences encoded thereby. The 3-fold analysis of the NTHi genomic sequence is set out in a series of contig sequences denoted as SEQ ID NO: 1-576, and the subsequent 8-fold analysis of the genomic sequence is set out in a series of 11 contig sequences denoted as SEQ ID NOS: 675-685. These contigs are raw data and one of skill in the art may assemble these contigs by comparing overlapping sequences to construct the complete genome of the NTHi stain 86-028NP using routine methods.
The present invention also provides for antibodies specific for the NTHi polypeptides of the invention. Methods of detecting NTHi bacteria in a human or in sample, such as serum, sputum, ear fluid, blood, urine, lymphatic fluid and cerebrospinal fluid are contemplated. These methods include detecting NTHi polynucleotides with specific polynucleotide probes or detecting NTHi polypeptides with specific antibodies. The invention also contemplates diagnostic kits which utilize these methods of detecting NTHi bacteria.
The present invention also contemplates methods of eliciting an immune response by administering a NTHi polypeptide of the invention or a NTHi peptide thereof. These methods include administering the NTHi polypeptide or NTHi peptide as a vaccine for treatment and/or prevention of diseases caused by NTHi infection, such as OM. The following NTHi genes are upregulated during or in response to middle ear and/or nasopharynx infections; and the polypeptides encoded by these genes and peptides thereof are contemplates as possible OM vaccine candidates and/or target of chemotherapy: hisB, lppB, sapA, lolA, rbsC, purE, ribB, arcB, uxuA, dsbB, ureH, licC, HI1647, ispZ, radC, mukF, glpR, ihfB, argR, cspD, HI0094, HI1163, HI1063, HI0665, HI1292, HI1064. NTHi hisB gene is set out as nucleotide sequence SEQ ID NO: 615 and encodes the amino acid sequence set out as SEQ ID NO: 616. NTHi sapA gene is set out as nucleotide sequence SEQ ID NO: 617 and encodes the amino acid sequence set out as SEQ ID NO: 618. NTHi rbsC gene is set out as nucleotide sequence SEQ ID NO: 619 and encodes the amino acid sequence set out as SEQ ID NO: 620. NTHi purE gene is set out as nucleotide sequence SEQ ID NO: 621 and encodes the amino acid sequence set out as SEQ ID NO: 622. NTHi ribB gene is set out as nucleotide sequence SEQ ID NO: 623 and encodes the amino acid sequence set out as SEQ ID NO: 624. NTHi arcB gene is set out as nucleotide sequence SEQ ID NO: 625 and encodes the amino acid sequence set out as SEQ ID NO: 626. NTHi uxuA gene is set out as nucleotide sequence SEQ ID NO: 627 and encodes the amino acid sequence set out as SEQ ID NO: 628. NTHi dsbB gene is set out as nucleotide sequence SEQ ID NO: 629 and encodes the amino acid sequence set out as SEQ ID NO: 630. NTHi ureH gene is set out as nucleotide sequence SEQ ID NO: 631 and encodes the amino acid sequence set out as SEQ ID NO: 632. NTHi licC gene is set out as nucleotide sequence SEQ ID NO: 633 and encodes the amino acid sequence set out as SEQ ID NO: 634. NTHi HI1647 gene is set out as nucleotide sequence SEQ ID NO: 635 and encodes the amino acid sequence set out as SEQ ID NO: 636. NTHi ispZ gene is set out as nucleotide sequence SEQ ID NO: 637 and encodes the amino acid sequence set out as SEQ ID NO: 638. NTHi radC gene is set out as nucleotide sequence SEQ ID NO: 639 and encodes the amino acid sequence set out as SEQ ID NO: 640. NTHi mukF gene is set out as nucleotide sequence SEQ ID NO: 641 and encodes the amino acid sequence set out as SEQ ID NO: 642. NTHi glpR gene is set out as nucleotide sequence SEQ ID NO: 643 and encodes the amino acid sequence set out as SEQ ID NO: 644. NTHi ihfB gene is set out as nucleotide sequence SEQ ID NO: 645 and encodes the amino acid sequence set out as SEQ ID NO: 646. NTHi argR gene is set out as nucleotide sequence SEQ ID NO: 647 and encodes the amino acid sequence set out as SEQ ID NO: 648. NTHi cspD gene is set out as nucleotide sequence SEQ ID NO: 649 and encodes the amino acid sequence set out as SEQ ID NO: 650. NTHi HI1163 gene is set out as nucleotide sequence SEQ ID NO: 651 and encodes the amino acid sequence set out as SEQ ID NO: 652. NTHi HI1063 gene is set out as nucleotide sequence SEQ ID NO: 653 and encodes the amino acid sequence set out as SEQ ID NO: 654. NTHi HI0665 gene is set out as nucleotide sequence SEQ ID NO: 655 and encodes the amino acid sequence set out as SEQ ID NO: 656. NTHi HI1292 gene is set out as nucleotide sequence SEQ ID NO: 657 and encodes the amino acid sequence set out as SEQ ID NO: 658.
The novel NTHi genes included in the polynucleotide sequences presented as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Tables 4 and 4B are also up-regulated during infection of the middle ear and/or the nasopharynx, and therefore are contemplated to encode OM vaccine candidates and/or targets of chemotherapy. In addition, the following NTHi genes are contemplated to be virulence-associated genes and therefore are contemplated to encode possible OM vaccine candidates and/or targets of chemotherapy: HI1386, HI1462, HI1369, lav, HI1598. NTHi HI1386 gene sequence is set out as SEQ ID NO: 659 and encodes the amino acid sequence set out as SEQ ID NO: 660. NTHi HI1462 gene sequence is set out as SEQ ID NO: 661 and encodes the amino acid sequence set out as SEQ ID NO: 662. NTHi HI1369 gene sequence is set out as SEQ ID NO: 665 and encodes the amino acid sequence set out as SEQ ID NO: 666. NTHi lav gene sequence is set out as SEQ ID NO: 663 and encodes the amino acid sequence set out as SEQ ID NO: 664. NTHi HI1598 gene sequence is set out as SEQ ID NO: 669 and SEQ ID NO: 671 and encodes the amino acid sequence set out as SEQ ID NO: 670 and SEQ ID NO: 672. Additional NTHi genes associated with virulence include the polynucleotide sequences presented as SEQ ID NO: 667 and SEQ ID NO: 673.
As a method of treating or preventing NTHi infection, the present invention contemplates administering a molecule that inhibits expression or the activity of the NTHi polypeptides, which are upregulated or active during infection. In particular, the invention contemplates methods of treating or preventing NTHi infection comprising modulating NTHi protein expression by administering an antisense oligonucleotide that specifically binds to NTHi genes that are upregulated during NTHi infections, such genes include hisB, lppB, sapA, lolA, rbsC, purE, ribB, arcB, uxuA, dsbB, ureH, licC, HI1647, ispZ, radC, mukF, glpR, ihfB, argR, cspD, HI0094, HI1163, HI1063, HI0665, HI1292, HI1064. The invention also contemplates methods of treating or preventing NTHi infection comprising administering antibodies or small molecules that modulate the activity of the proteins encoded by theses genes. The novel NTHi genes included in the polynucleotide sequences presented as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Tables 4 and 4B are also up-regulated during infection of the middle ear and/or the nasopharynx and therefore antisense oligonucleotides that specifically bind these polynucleotide sequences are also contemplated.
Polynucleotides and Polypeptides of the Invention
The present invention provides for the sequences of the NTHi strain 86-028NP genome. This genomic sequence is presented as a series of contig sequences denoted herein as “contigs 1-576”. Each contig is assigned a sequence identification number that correlates with its “contig number”. Therefore, the contigs of the present invention as set out as SEQ ID NOS: 1-576. These contig polynucleotide sequences may be assembled into the complete genome sequence of the NTHi strain 86-028NP using routine methods. Upon completion of 8-fold sequence analysis of the NTHi strain 82-028NP genome, the genomic sequence was assembled into 11 contigs which are denoted herein as SEQ ID NOS: 675-685.
The present invention provides for the NTHi polynucleotide sequences and open reading frames contained within the contigs of SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5. The present invention also provides for the polypeptide sequences encoded by the NTHi polynucleotides of the present invention such as the amino acid sequences set out in Table 3B, Table 4B and Table 5. The invention provides for polynucleotides that hybridize under stringent conditions to (a) the complement of the nucleotides sequence of SEQ ID NOS: 1-576; SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein (b) a polynucleotide which is an allelic variant of any polynucleotides recited above; (c) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (d) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the NTHi polypeptides of the present invention.
The NTHi polynucleotides of the invention also include nucleotide sequences that are substantially equivalent to the polynucleotides recited above. Polynucleotides according to the invention can have, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94% and even more typically at least 95%, 96%, 97%, 98% or 99% sequence identity to the NTHi polynucleotides recited above.
Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to the NTHi nucleotide sequences of SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein, or compliments thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides. Fragments of, e.g., 15, 17, or 20 nucleotides or more that are selective for (i.e., specifically hybridize to any one of the polynucleotides of the invention) are contemplated. Probes capable of specifically hybridizing to a polynucleotide can differentiate NTHi polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate NTHi genes from other bacterial genes, and are preferably based on unique nucleotide sequences.
The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent. Hybridization stringency is principally determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68° C. or 0.015 M sodium chloride, 0.0015M sodium citrate, and 50% formamide at 42° C. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, N.Y. 1989). More stringent conditions (such as higher temperature, lower ionic strength, higher formamide, or other denaturing agent) may also be used, however, the rate of hybridization will be affected. In instances wherein hybridization of deoxyoligonucleotides is concerned, additional exemplary stringent hybridization conditions include washing in 6×SSC 0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).
Other agents may be included in the hybridization and washing buffers for the purpose of reducing non-specific and/or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecylsulfate, NaDodSO4, (SDS), ficoll, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, although other suitable agents can also be used. The concentration and types of these additives can be changed without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually carried out at pH 6.8-7.4, however, at typical ionic strength conditions, the rate of hybridization is nearly independent of pH. See Anderson et al., Nucleic Acid Hybridisation: A Practical Approach, Ch. 4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by one skilled in the art in order to accommodate these variables and allow DNAs of different sequence relatedness to form hybrids.
The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof. Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, and nucleotide sequences out in Table 3B, Table 4B and Table 5 herein, preferably the open reading frames therein, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to the open reading frames within SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 with a sequence from another isolate of the same species. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res., 12: 387, 1984; Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215: 403-410, 1990). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well known Smith Waterman algorithm may also be used to determine identity.
Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific open reading frames (ORF) disclosed herein. In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.
The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences encoded by the nucleotide sequences included within the polynucleotide sequences set out as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5, or the corresponding full length or mature protein. The polypeptides of the invention include the amino acid sequences of SEQ ID NO: 616, SEQ ID NO: 618, SEQ ID NO: 620, SEQ ID NO: 622, SEQ ID NO: 624, SEQ ID NO: 626, SEQ ID NO: 628, SEQ ID NO: 628, SEQ ID NO: 630, SEQ ID NO: 632, SEQ ID NO: 634, SEQ ID NO: 636, SEQ ID NO: 638, SEQ ID NO: 640, SEQ ID NO: 642, SEQ ID NO: 644, SEQ ID NO: 646, SEQ ID NO: 648, SEQ ID NO: 650, SEQ ID NO: 652, SEQ ID NO: 654, SEQ ID NO: 656, SEQ ID NO: 658, SEQ ID NO: 660, SEQ ID NO: 662, SEQ ID NO: 664, SEQ ID NO: 666, SEQ ID NO: 668, SEQ ID NO: 670, SEQ ID NO: 672, SEQ ID NO: 674, SEQ ID NO: 687, SEQ ID NO: 689, SEQ ID NO: 691, SEQ ID NO: 693, SEQ ID NO: 695, SEQ ID NO: 697, SEQ ID NO: 699, SEQ ID NO: 701, SEQ ID NO: 703, SEQ ID NO: 705, SEQ ID NO: 707, SEQ ID NO: 709, SEQ ID NO: 711, SEQ ID NO: 713, SEQ ID NO:715, SEQ ID NO: 717, SEQ ID NO: 719, SEQ ID NO: 721, SEQ ID NO:723, SEQ ID NO:725, SEQ ID NO:727, SEQ ID NO:729, SEQ ID NO: 731, SEQ ID NO: 733, SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, SEQ ID NO: 749, SEQ ID NO: 751, SEQ ID NO: 753, SEQ ID NO: 755, SEQ ID NO: 757, SEQ ID NO: 759, SEQ ID NO: 761, 763, SEQ ID NO: 765, SEQ ID NO: 767, SEQ ID NO: 769 or SEQ ID NO: 771 which are set out in Table 3B, Table 4B and Table 5 herein.
Polypeptides of the invention also include polypeptides preferably with biological or immunogenic activity that are encoded by: (a) an open reading frame contained within the nucleotide sequences set forth as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5, or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.
The invention also provides biologically active or immunologically active variants of the amino acid sequences of the present invention; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological and/or immunogenic activity. Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides encoded by the polynucleotides included within the nucleotide sequences presented in SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein, and the polypeptides having an amino acid sequence set out in Table 3B, Table 4B and Table 5 herein
NTHi peptides refer to fragments of the NTHi polypeptides encoded by the nucleotide sequences presented in SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 or the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein, and the polypeptides having the amino acid sequences set out in Table 3B, Table 4B and Table 5 herein. The preferred NTHi peptides are biologically and/or immunologically active.
The present invention further provides isolated NTHi polypeptides or NTHi peptides encoded by the NTHi nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention. The term “degenerate variant” refers to nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical NTHi polypeptide sequence. Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.
The invention also provides for NTHi polypeptides with one or more conservative amino acid substitutions that do not affect the biological and/or immunogenic activity of the polypeptide. Alternatively, the NTHi polypeptides of the invention are contemplated to have conservative amino acids substitutions which may or may not alter biological activity. The term “conservative amino acid substitution” refers to a substitution of a native amino acid residue with a nonnative residue, including naturally occurring and nonnaturally occurring amino acids, such that there is little or no effect on the polarity or charge of the amino acid residue at that position. For example, a conservative substitution results from the replacement of a non-polar residue in a polypeptide with any other non-polar residue. Further, any native residue in the polypeptide may also be substituted with alanine, according to the methods of “alanine scanning mutagenesis”. Naturally occurring amino acids are characterized based on their side chains as follows: basic: arginine, lysine, histidine; acidic: glutamic acid, aspartic acid; uncharged polar: glutamine, asparagine, serine, threonine, tyrosine; and non-polar: phenylalanine, tryptophan, cysteine, glycine, alanine, valine, proline, methionine, leucine, norleucine, isoleucine. General rules for amino acid substitutions are set forth in Table 1 below.







TABLE 1







Amino Acid Substitutions









Original

Preferred


Residues
Exemplary Substitutions
Substitutions





Ala
Val, Leu, Ile
Val


Arg
Lys, Gln, Asn
Lys


Asn
Gln
Gln


Asp
Glu
Glu


Cys
Ser, Ala
Ser


Gln
Asn
Asn


Glu
Asp
Asn


Gly
Pro, Ala
Ala


His
Asn, Gln, Lys, Arg
Arg


Ile
Leu, Val, Met, Ala, Phe,
Leu


Leu
Norleucine, Ile, Val, Met,
Leu


Lys
Arg, 1,4 Diaminobutyric
Arg


Met
Leu, Phe, Ile
Leu


Phe
Leu, Val, Ile, Ala, Tyr
Arg


Pro
Ala
Gly


Ser
Thr, Ala, Cys
Thr


Thr
Ser
Ser


Trp
Tyr, Phe
Tyr


Tyr
Trp, Phe, Thr, Ser
Phe


Val
Ile, Met, Leu, Phe, Ala,
Leu









Antisense polynucleotides complementary to the polynucleotides encoding the NTHi polypeptides are also provided.
The invention contemplates that polynucleotides of the invention may be inserted in a vector for amplification or expression. For expression, the polynucleotides are operatively linked to appropriate expression control sequence such as a promoter and polyadenylation signal sequences. Further provided are cells comprising polynucleotides of the invention. Exemplary prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella and Serratia. 
The term “isolated” refers to a substance removed from, and essentially free of, the other components of the environment in which it naturally exists. For example, a polypeptide is separated from other cellular proteins or a DNA is separated from other DNA flanking it in a genome in which it naturally occurs.
Antibodies and Methods for Eliciting an Immune Response
The invention provides antibodies which bind to antigenic epitopes unique to (i.e., are specific for) NTHi polypeptides. Also provided are antibodies which bind to antigenic epitopes common among multiple H. influenzae subtypes but unique with respect to any other antigenic epitopes. The antibodies may be polyclonal antibodies, monoclonal antibodies, antibody fragments which retain their ability to bind their unique epitope (e.g., Fv, Fab and F(ab)2 fragments), single chain antibodies and human or humanized antibodies. Antibodies may be generated by techniques standard in the art.
It is known in the art that antibodies to the capsular polysaccharide of H. influenzae exhibit the ability to kill bacteria in vitro assays. These antibodies are also known to protect against challenge with H. influenzae in animal model systems. These studies indicate antibody to the capsular polysaccharides are likely to elicit a protective immune response in humans. The present invention provides for antibodies specific for the NTHi polypeptides of the present invention and fragments thereof, which exhibit the ability to kill both H. influenzae bacteria and to protect humans from NTHi infection. The present invention also provides for antibodies specific for the NTHi polypeptides of the invention which reduce the virulence, inhibit adherence, inhibit cell division, and/or inhibit penetration into the epithelium of H. influenzae bacteria or enhance phagocytosis of the H. influenzae bacteria.
In vitro complement mediated bactericidal assay systems (Musher et al., Infect. Immun. 39: 297-304, 1983; Anderson et al., J. Clin. Invest. 51: 31-38, 1972) may be used to measure the bactericidal activity of anti-NTHi antibodies. Further data on the ability of NTHi polypeptides and NTHi peptides to elicit a protective antibody response may be generated by using animal models of infection such as the chinchilla model system described herein.
It is also possible to confer short-term protection to a host by passive immunotherapy via the administration of pre-formed antibody against an epitope of NTHi, such as antibodies against NTHi OMP, LOS or noncapsular proteins. Thus, the contemplated vaccine formulations can be used to produce antibodies for use in passive immunotherapy. Human immunoglobulin is preferred in human medicine because a heterologous immunoglobulin may provoke an immune response to its foreign immunogenic components. Such passive immunization could be used on an emergency basis for immediate protection of unimmunized individuals exposed to special risks. Alternatively, these antibodies can be used in the production of anti-idiotypic antibody, which in turn can be used as an antigen to stimulate an immune response against NTHi epitopes.
The invention contemplates methods of eliciting an immune response to NTHi in an individual. These methods include immune responses which kill the NTHi bacteria and immune responses which block H. influenzae attachment to cells. In one embodiment, the methods comprise a step of administering an immunogenic dose of a composition comprising a NTHi protein or NTHi peptide of the invention. In another embodiment, the methods comprise administering an immunogenic dose of a composition comprising a cell expressing a NTHi protein or NTHi peptide of the invention. In yet another embodiment, the methods comprise administering an immunogenic dose of a composition comprising a polynucleotide encoding a NTHi protein or NTHi peptide of the invention. The polynucleotide may be a naked polynucleotide not associated with any other nucleic acid or may be in a vector such as a plasmid or viral vector (e.g., adeno-associated virus vector or adenovirus vector). Administration of the compositions may be by routes standard in the art, for example, parenteral, intravenous, oral, buccal, nasal, pulmonary, rectal, or vaginal. The methods may be used in combination in a single individual. The methods may be used prior or subsequent to NTHi infection of an individual.
An “immunological dose” is a dose which is adequate to produce antibody and/or T cell immune response to protect said individual from NTHi infection, particularly NTHi infection of the middle ear and/or the nasopharynx or lower airway. Also provided are methods whereby such immunological response slows bacterial replication. A further aspect of the invention relates to an immunological composition which, when introduced into an individual capable or having induced within it an immunological response. The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells. A NTHi protein or an antigenic peptide thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties. Thus fused recombinant protein, preferably further comprises an antigenic co-protein, such as Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-proteins which solubilize the protein and facilitate production and purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system. The co-protein may be attached to either the amino or carboxy terminus of the first protein. Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the NTHi polypeptides encoded by the polynucleotide of the invention or antigenic peptides thereof.
The invention correspondingly provides compositions suitable for eliciting an immune response to NTHi infection, wherein the antibodies elicited block binding of NTHi bacterium to the host's cells. The compositions comprise NTHi proteins or NTHi peptides of the invention, cells expressing the NTHi polypeptide, or polynucleotides encoding the polypeptides. The compositions may also comprise other ingredients such as carriers and adjuvants.
Genes that are up-regulated in NTHi infection of the middle ear and/or the nasopharynx and genes that are associated with NTHi virulence are described herein. The polypeptides and peptides thereof which are encoded by these NTHi genes are contemplated to be useful for eliciting an immune response for treating or preventing disorders associated with NTHi infection, such as OM. Some of the polypeptides encoded by these genes include: histidine biosynthesis protein, lipoprotein B, peptide ABC transporter, periplasmic SapA precursor, outer membrane lipoproteins carrier protein precursor, ribose transport system permease protein, phosphoribosylaminoimidazole carboxylase catalytic subunit, PurE, phosphoribosylaminoimidazole carboxylase catalytic subunit, ornithine carbamolytransferase, mannonate dehydratase, disulfide oxidoreductase, urease accessory protein, phosphocholine cytidylytransferase, putative pyridoxine biosynthesis protein, singlet oxygen resistance protein, intracellular septation protein, DNA repair protein, MukF protein, glycerol-3-phosphate regulon repressor, integration host factor beta subunit, arginine repressor, cold shock like protein, stress response protein, LicA, MukF, RadA and those hypothetical proteins encoded by HI0094, HI1163, HI0665, HI1292, HI1064, HI186, HI0352 genes. NTHi OMPs, LOS and noncapsular proteins are also contemplated to elicit an immune response for prevention and treatment of disorders associated with NTHi infection.
An “immunogenic dose” of a composition of the invention is one that generates, after administration, a detectable humoral and/or cellular immune response in comparison to the immune response detectable before administration or in comparison to a standard immune response before administration. The invention contemplates that the immune response resulting from the methods may be protective and/or therapeutic.
The invention includes methods of blocking binding of NTHi bacteria to host cells in an individual. The methods comprise administering antibodies or polypeptides of the invention that block binding of NTHi cellular attachment. Alternatively, administration of one or more small molecules that block binding of NTHi cell attachment is contemplated. In vitro assays may be used to demonstrate the ability of an antibody, polypeptide or small molecule of the invention to block NTHi cell attachment.
Pharmaceutical compositions comprising antibodies of the invention, polypeptides of the invention and/or small molecules of the invention that block NTHi cellular attachment are provided. The pharmaceutical compositions may consist of one of the foregoing active ingredients alone, may comprise combinations of the foregoing active ingredients or may comprise additional active ingredients used to treat bacterial infections. The pharmaceutical compositions may comprise one or more additional ingredients such as pharmaceutically effective carriers. Dosage and frequency of the administration of the pharmaceutical compositions are determined by standard techniques and depend, for example, on the weight and age of the individual, the route of administration, and the severity of symptoms. Administration of the pharmaceutical compositions may be by routes standard in the art, for example, parenteral, intravenous, oral, buccal, nasal, pulmonary, rectal, or vaginal.
Also provided by the invention are methods for detecting NTHi infection in an individual. In one embodiment, the methods comprise detecting NTHi polynucleotides of the invention in a sample using primers or probes that specifically bind to the polynucleotides. Detection of the polynucleotide may be accomplished by numerous techniques routine in the art involving, for example, hybridization and PCR.
The antibodies of the present invention may also be used to provide reagents for use in diagnostic assays for the detection of NTHi antigens (NTHi polypeptides and peptides thereof) in various body fluids of individuals suspected of H. influenzae infection. In another embodiment, the NTHi proteins and peptides of the present invention may be used as antigens in immunoassays for the detection of NTHi in various patient tissues and body fluids including, but not limited to: blood, serum, ear fluid, spinal fluid, sputum, urine, lymphatic fluid and cerebrospinal fluid. The antigens of the present invention may be used in any immunoassay system known in the art including, but not limited to: radioimmunoassays, ELISA assays, sandwich assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, fluorescent immunoassays, protein A immunoassays and immunoelectrophoresis assays.
Vaccines and Chemotherapeutic Targets
An aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with a NTHi antigen protein or an antigenic peptide thereof.
The present invention also provides for vaccine formulations which comprise an immunogenic recombinant NTHi protein or NTHi peptide of the invention together with a suitable carrier. The NTHi polypeptides and peptides thereof contemplated as vaccine candidates and/or targets of chemotherapy include, but are not limited to, histidine biosynthesis protein, lipoprotein B, peptide ABC transporter, periplasmic SapA precursor, outer membrane lipoproteins carrier protein precursor, ribose transport system permease protein, phosphoribosylaminoimidazole carboxylase catalytic subunit, PurE, 3,4-dihydroxt-2-butone 4-phosphate synthase, ornithine carbamolytransferase, mannonate dehydratase, disulfide oxidoreductase, urease accessory protein, phospshocholine cytidylytransferase, putative pyridoxine biosynthesis protein, singlet oxygen resistance protein, intracellular septation protein, DNA repair protein, MUKF protein, glycerol-3-phosphate regulon repressor, integration host factor beta subunit, arginine repressor, cold shock like protein, stress response protein, LicA, RadA and those hypothetical proteins encoded by HI0094, HI1163, HI0665, HI1292, HI1064, HI1386, HI10352 genes, NTHi OMPs, NTHi LOS and NTHi noncapsular proteins and polypeptides encoded by the novel NTHi polynucleotide sequences present in the nucleotide sequences set out as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein, and the polypeptides having the amino acid sequences set out in Table 3B, Table 4B and Table 5 herein.
Since the protein may be broken down in the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
A. Peptide Vaccines
Peptide therapeutic agents, such as peptide vaccines, are well known in the art and are of increasing use in the pharmaceutical arts. Consistent drawbacks to the parenteral administration of such peptide compounds have been the rapidity of breakdown or denaturation. Infusion pumps, as well as wax or oil implants, have been employed for chronic administration of therapeutic agents in an effort to both prolong the presence of peptide-like therapeutic agents and preserve the integrity of such agents. Furthermore, the peptide-like agent should (with particular reference to each epitope of the peptide-like agent) ideally maintain native state configuration for an extended period of time and additionally be presented in a fashion suitable for triggering an immunogenic response in the challenged animal or immunized human.
The NTHi antigenic peptides of the invention can be prepared in a number of conventional ways. The short peptides sequences can be prepared by chemical synthesis using standard means. Particularly convenient are solid phase techniques (see, e.g., Erikson et al., The Proteins (1976) v. 2, Academic Press, New York, p. 255). Automated solid phase synthesizers are commercially available. In addition, modifications in the sequence are easily made by substitution, addition or omission of appropriate residues. For example, a cysteine residue may be added at the carboxy terminus to provide a sulfhydryl group for convenient linkage to a carrier protein, or spacer elements, such as an additional glycine residue, may be incorporated into the sequence between the linking amino acid at the C-terminus and the remainder of the peptide. The short NTHi peptides can also be produced by recombinant techniques. The coding sequence for peptides of this length can easily be synthesized by chemical techniques, e.g., the phosphotriester method described in Matteucci et al., J Am Chem Soc., 103: 3185 (1981).
Some of the NTHi peptide sequences contemplated herein may be considered too small to be immunogenic, they may be linked to carrier substances in order to confer this property upon them. Any method of creating such linkages known in the art may be used. Linkages can be formed with heterobifunctional agents that generate a disulfide link at one functional group end and a peptide link at the other, such as a disulfide amide forming agent, e.g., N-succidimidyl-3-(2-pyridyldithio) proprionate (SPDP) (See, e.g., Jansen et al., Immun. Rev. 62:185, 1982) and bifunctional coupling agents that form a thioether rather than a disulfide linkage such as reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimido-methyl) cyclohexane-1-carboxylic acid and the like, and coupling agent which activate carboxyl groups by combining them with succinimide or 1-hydroxy-2-nitro-4-sulfonic acid, for sodium salt such as succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC).
B. Vaccine Compositions and Administration
A priming dose of the immunogen that is followed by one or more booster exposures to the immunogen may be necessary to be an effective vaccine (Kramp et al., Infect. Immun., 25: 771-773, 1979; Davis et al., Immunology Letters, 14: 341-8 1986 1987). Examples of proteins or polypeptides that could beneficially enhance the immune response if co-administered include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g. Leaf) or costimulatory molecules. Helper (HTL) epitopes could be joined to intracellular targeting signals and expressed separately from the CTL epitopes. This would allow direction of the HTL epitopes to a cell compartment different than the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the MHC class II pathway, thereby improving CTL induction. In contrast to CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases.
Ideally, an immunogen will exhibit two properties; the capacity to stimulate the formation of the corresponding antibodies and the propensity to react specifically with these antibodies. Immunogens bear one or more epitopes which are the smallest part of an immunogen recognizable by the combing site of an antibody. In particular instances, immunogen, fractions of immunogens or conditions under which the immunogen is presented are inadequate to precipitate the desired immunological response resulting in insufficient immunity. This is often the case with peptides or other small molecules used as immunogens. Other substances such as immunomodulators (e.g., cytokines such as the interleukins) may be combined in vaccines as well.
The vaccine art recognizes the use of certain substances called adjuvants to potentate an immune response when used in conjunction with an immunogen. Adjuvants are further used to elicit an immune response that is faster or greater than would be elicited without the use of the adjuvant. In addition, adjuvants may be used to create an immunological response using less immunogen than would be needed without the inclusion of adjuvant, to increase production of certain antibody subclasses that afford immunological protection or to enhance components of the immune response (e.g., humoral, cellular). Known adjuvants include emulsions such as Freund's Adjuvants and other oil emulsions, Bordetella pertussis, MF59, purified saponin from Quillaja saponaria (QS21), aluminum salts such as hydroxide, phosphate and alum, calcium phosphate, (and other metal salts), gels such as aluminum hydroxide salts, mycobacterial products including muramyl dipeptides, solid materials, particles such as liposomes and virosomes. Examples of natural and bacterial products known to be used as adjuvants include monophosphoryl lipid A (MPL), RC-529 (synthetic MPL-like acylated monosaccharide), OM-174 which is a lipid A derivative from E. coli, holotoxins such as cholera toxin (CT) or one of its derivatives, pertussis toxin (PT) and heat-labile toxin (LT) of E. coli or one of its derivatives, and CpG oligonucleotides. Adjuvant activity can be affected by a number of factors, such as carrier effect, depot formation, altered lymphocyte recirculation, stimulation of T-lymphocytes, direct stimulation of B-lymphocytes and stimulation of macrophages.
Vaccines are typically prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, e.g., water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants, which enhance the effectiveness of the vaccine. The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25-70%.
Vaccines may also be administered through transdermal routes utilizing jet injectors, microneedles, electroporation, sonoporation, microencapsulation, polymers or liposomes, transmucosal routes and intranasal routes using nebulizers, aerosols and nasal sprays. Microencapsulation using natural or synthetic polymers such as starch, alginate and chitosan, D-poly L-lactate (PLA), D-poly DL-lactic-coglycolic microspheres, polycaprolactones, polyorthoesters, polyanhydrides and polyphosphazenes polyphosphatazanes are useful for both transdermal and transmucosal administration. Polymeric complexes comprising synthetic poly-ornithate, poly-lysine and poly-arginine or amphipathic peptides are useful for transdermal delivery systems. In addition, due to their amphipathic nature, liposomes are contemplated for transdermal, transmucosal and intranasal vaccine delivery systems. Common lipids used for vaccine delivery include N-(1)2,3-(dioleyl-dihydroxypropyl)-N,N,N,-trimethylammonium-methyl sulfate (DOTAP), dioleyloxy-propyl-trimethylammonium chloride DOTMA, dimystyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE), dimethyldioctadecyl ammonium bromide (DDAB) and 9N(N′,N-dimethylaminoethane)carbamoyl) cholesterol (DC-Chol). The combination of helper lipids and liposomes will enhance up-take of the liposomes through the skin. These helper lipids include, diolcoyl phosplhatidylethanolamine (DOPE), dilauroylphosphatidylethanolamine (DLPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE). In addition, triterpenoid glycosides or saponins derived from the Chilean soap tree bark (Quillaja saponaria) and chitosan (deacetylated chitan) have been contemplated as useful adjuvants for intranasal and transmucosal vaccine delivery.
The proteins may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, e.g., hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, and procaine.
The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per individual. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed in one or three month intervals by a subsequent injection or other administration.
Upon immunization with a vaccine composition as described herein, the immune system of the host responds to the vaccine by producing large amounts of CTLs specific for the desired antigen, and the host becomes at least partially immune to later infection, or resistant to developing chronic infection. Vaccine compositions containing the NTHi polypeptide or NTHi peptides of the invention are administered to a patient susceptible to or otherwise at risk of bacterial infection to elicit an immune response against the antigen and thus enhance the patient's own immune response capabilities. Such an amount is defined to be an “immunogenically effective dose.” In this use, the precise amounts again depend on the patient's state of health and weight, the mode of administration, the nature of the formulation, etc., but generally range from about 1.0 μg to about 5000 per 70 kilogram patient, more commonly from about 10 to about 500 mg per 70 kg of body weight. For therapeutic or immunization purposes, the NTHi polypeptide or NTHi peptides of the invention can also be expressed by attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into an acutely or chronically infected host or into a noninfected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL response.
Humoral immune response may be measured by many well known methods, such as Single Radial Inmunodiffussion Assay (SRID), Enzyme Immunoassay (EIA) and Hemagglutination Inhibition Assay (HAI). In particular, SRID utilizes a layer of a gel, such as agarose, containing the immunogen being tested. A well is cut in the gel and the serum being tested is placed in the well.
Diffusion of the antibody out into the gel leads to the formation of a precipitation ring whose area is proportional to the concentration of the antibody in the serum being tested. EIA, also known as ELISA (Enzyme Linked Immunoassay), is used to determine total antibodies in the sample. The immunogen is adsorbed to the surface of a microtiter plate. The test serum is exposed to the plate followed by an enzyme linked immunoglobulin, such as IgG. The enzyme activity adherent to the plate is quantified by any convenient means such as spectrophotometry and is proportional to the concentration of antibody directed against the immunogen present in the test sample. HAI utilizes the capability of an immunogen such as viral proteins to agglutinate chicken red blood cells (or the like). The assay detects neutralizing antibodies, i.e., those antibodies able to inhibit hemagglutination. Dilution of the test serum are incubated with a standard concentration of immunogen, followed by the addition of the red blood cells. The presence of neutralizing antibodies will inhibit the agglutination of the red blood cells by the immunogen. Tests to measure cellular immune response include determination of delayed-type hypersensitivity or measuring the proliferative response of lymphocytes to target immunogen.
Nontypeable Haemophilus influenzae (NTHi) p H. influenzae is a small, nonmotile gram negative bacterium. Unlike other H. influenzae strains, the nontypeable H. influenzae (NTHi) strains lack a polysaccharide capsule and are sometimes denoted as “nonencapsulated.” NTHi strains are genetically distinct from encapsulated strains and are more heterogenous than the type b H. influenzae isolates. NTHi presents a complex array of antigens to the human host. Possible antigens that may elicit protection include OMPs, lipopolysaccharides, lipoproteins, adhesion proteins and noncapsular proteins.
Humans are the only host for H. influenzae. NTHi strains commonly reside in the upper respiratory tract including the nasopharynx and the posterior oropharynx, the lower respiratory tract and the female genital tract. NTHi causes a broad spectrum of diseases in humans, including but not limited to, otitis media, pneumonia, sinusitis, septicemia, endocarditis, epiglottitis, septic arthritis, meningitis, postpartum and neonatal infections, postpartum and neonatal sepsis, acute and chromic salpingitis, epiglottis, pericarditis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intraabdominal infections, urinary tract infection, mastoiditis, aortic graft infection, conjunctitivitis, Brazilian purpuric fever, occult bacteremia and exacerbation of underlying lung diseases such as chronic bronchitis, bronchietasis and cystic fibrosis.
Epidemiologic studies of NTHi have indicated that the strains are heterogeneous with respect to outer membrane protein profiles (Barenkamp et al., Infect. Immun., 36: 535-40, 1982), enzyme allotypes (Musser et al., Infect. Immun., 52: 183-191, 1986), and other commonly used epidemiologic tools. There have been several attempts to subtype NTHi, but none of the methodologies have been totally satisfactory. The outer-membrane protein composition of NTHi consists of approximately 20 proteins. All NTHi strains contains two common OMP's with molecular weights of 30,000 and 16,600 daltons. NTHi strains may be subtyped based on two OMP's within the 32,000-42,000 dalton range. The NTHi liposaccharide profile is fundamentally different than the enteric gram negative bacteria and separates into 1-4 distinct bands ranging from less than 20,000 daltons.
A prototype NTHi isolate is the low passage isolate 86-028NP which was recovered from a child with chronic otitis media. This strain has been well characterized in vitro (Bakaletz et al., Infect. Immun., 53: 331-5, 1988; Holmes et al., Microb. Pathog., 23: 157-66, 1997) as well as in the chinchilla OM model (described herein) (Bakaletz et al., Vaccine, 15: 955-61, 1997; Suzuki et al., Infect. Immun., 62: 1710-8, 1994; DeMaria et al., Infect. Immun., 64: 5187-92, 1996). The 86-028NP strain was used, as described herein, to identify genes that are up-regulated in expression in the chinchilla model of otitis media and genes that are necessary for NTHi survival in the chinchilla middle ear.
DFI Strategy
A differential fluorescence induction (DFI) strategy was used herein to identify NTHi genes induced during GM in a chinchilla animal model. Several methods have been developed to identify bacterial genes that contribute to the virulence of an organism during infection. Such methods include in vivo expression technology (IVET) in which bacterial promoters regulate the expression of gene(s) required for synthesis of essential nutrients required for survival in the host; signature-tagged mutagenesis (STM) enabling tag-specific identification of genes that alter the virulence properties of a microorganism when mutated; DNA microarray technology to globally screen for transcriptionally active genes, and DFI which uses Fluorescent Activated Cell Sorting (FACS) analysis to select for transcriptionally active promoters (Chiang et al., Annu. Rev. Microbiol., 53: 129-154, 1999). DFI is a high-throughput method that allows for the identification of differentially regulated genes regardless of the basal level of expression and does not exclude those that are essential for growth in vitro.
DFI has been successfully utilized in many microorganisms. For example, a Green Fluorescent Protein (GFP) reporter system and flow cytometry was used to study mycobacterial gene expression upon interaction with macrophages (Dhandayuthapani et al., Mol. Microbiol., 17: 901-912, 1995). A promoter trap system was used to identify genes whose transcription was increased when Salmonellae were subjected to environments simulating in vivo growth and when internalized by cultured macrophage-like cells (Valdivia and Falkow, Mol. Micro boil., 22: 367-378, 1996; Valdivia and Falkow, Science, 277: 2007-2011, 1997; Valdivia and Falkow, Curr. Opin. Microbiol., 1:359-363, 1998). In addition, DFI has been used to identify promoters expressed in S. pneumoniae and S. aureus when grown under varied in vitro conditions simulating infection (Marra et al., Infect. Immun., 70(3): 1422-1433, 2002; Schneider et al., Proc. Nati. Acad. Sci. U.S.A., 97: 1671-1676, 2000). In addition, DFI has been utilized to study gene regulation in Bacillus cereus in response to environmental stimuli (Dunn and Handelsman, Gene, 226: 297-305, 1999), in S. pneumoniae in response to a competence stimulatory peptide (Bartilson et al., Mol. Microbiol., 39: 126-135, 2001), and upon interaction with and invasion of host cells in Bartonella henselae Lee and Falkow, Infect. Immun., 66: 3964-3967, 1998), Listeria monocytogenes Wilson et al., Infect. Immun., 69: 5016-5024, 2001), Brucella abortus (Eskra et al., Infect. Immun., 69: 7736-7742, 2001), and Escherichia coli (Badger et al., Mol. Microbiol., 36: 174-182, 2000).
Whereas DFI has been successfully used to identify promoters active in cell culture models of infection or in vitro conditions designed to simulate an in vivo environment, few have applied DFI to identify promoters regulated in a specific biological niche within the whole animal. This is likely due to the numerous challenges associated with sorting from an in vivo environment. The host inflammatory response, dissemination and/or clearance of bacterial cells from the site of infection, as well as adherence of bacteria to epithelial cells, possibly via biofilm formation, can make bacteria inaccessible for retrieval from the living animal. These factors, among others, contribute to the complexity of the microenvironment and the heterogeneity of gene expression as the bacteria sense and respond to these changes. Recently, DFI has been used to identify promoters expressed in S. pneumoniae when the bacteria were screened in a mouse model of respiratory tract infection and a gerbil infection model of OM (Marra et al., Infect. Immun. 70: 1422-33, 2002; Marra et al., Microbiol., 148: 1483-91, 2002).
Animal Model
The chinchilla model is a widely accepted experimental model for OM. In particular, a chinchilla model of NTHi-induced OM has been well characterized (Bakaletz et al., J. Infect. Dis., 168: 865-872, 1993; Bakaletz and Holmes, Clin. Diagn. Lab. Immunol., 4: 223-225, 1997; Suzuki and Bakaletz, Infest. Immun., 62: 1710-1718, 1994), and has been used to determine the protective efficacy of several NTHi outer membrane proteins, combinations of outer membrane proteins, chimeric synthetic peptide vaccine components, and adjuvant formulations as vaccinogens against OM (Bakaletz et al., Vaccine, 15; 955-961, 1997; Bakaletz et al., Infect. Immun., 67: 2746-2762, 1999; Kennedy et al., Infect. Immun., 68: 2756-2765, 2000).
In particular, there is an unique in vivo model wherein adenovirus predisposes chinchillas to H. influenzae-induced otitis media, which allowed for the establishment of relevant cell, tissue and organ culture systems for the biological assessment of NTHi (Bakaletz et al., J. Infect. Dis., 168: 865-72, 1993; Suzuki et al., Infect. Immunity 62: 1710-8, 1994). Adenovirus infection alone has been used to assess for the transudation of induced serum antibodies into the tympanum (Bakaletz et al., Clin. Diagnostic Lab Immunol., 4(2): 223-5, 1997) and has been used as a co-pathogen with NTHi, to determine the protective efficacy of several active and passive immunization regimens targeting various NTHi outer membrane proteins, combinations of OMPs, chimeric synthetic peptide vaccine components, and adjuvant formulations as vaccinogens against otitis media (Bakaletz et al., Infect Immunity, 67(6): 2746-62, 1999; Kennedy et al., Infect Immun., 68(5): 2756-65, 2000; Novotny et al., Infect Immunity 68(4): 2119-28, 2000; Poolman et al., Vaccine 19 (Suppl. 1): S108-15, 2000).
Genes Upregulated In Vivo in Response to NTHi Infection of the Middle Ear
In order to identify differentially regulated promoters in response to NTHi infection of the middle ear, a promoter trap library was constructed and sorting parameters defined. A portion of the promoter trap library was inoculated directly into the chinchilla middle ear and OM development was monitored by video otoscopy and tympanometry at 24 and 48 hours. In addition, the middle ear fluids were recovered 24 and 48 hours after infection. Two-color FACS analysis was used to isolated bacteria that were expressing GFP from other cells and debris associated with the effusion. Following isolation, DNA sequence of the Haemophilus inserts 5′ of the gfpmut3 gene were determined and analyzed. In this manner, we identified genes that are up-regulated as NTHi sense and respond to the environment of the chinchilla middle ear during AOM. The following genes were identified and due to their up-regulation during NTHi infection, they may play a role in NTHi infection and virulence.
As described below in Example 7, following the DFI procedure described above and subsequent FACS analysis of gfp-expressing clones, 52 candidate clones containing potential in vivo-regulated promoters were isolated. The genes these clones control were categorized based upon general description and function within the cell and include general metabolic processes, environmental informational processing and membrane transport, membrane proteins and hypothetical proteins. Eight of these 52 clones contain sequences that are unique to NTHi strain 86-028NP. Importantly, 3 clones were isolated from independent screens in more than one animal thereby verifying the method of isolation.
In order to independently confirm the FACS data, we determined the relative expression of candidate genes by quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). The parent strain 86-028NP, was used for these studies. Thus, wild-type gene expression without the influence of plasmid copy number on gene regulation was analyzed, allowing for the indication of false-positive clone identification by FACS. Of the 44 candidate clones containing sequence similar to that identified in H. influenzae strain Rd, quantitative comparison of gene expression in vitro and in vivo confirmed up-regulated gene expression for twenty-six genes (60%) when NTHi respond to environmental cues present in the chinchilla middle ear. This analysis identified in vivo-regulated promoters which drive expression of genes involved in membrane transport, environmental informational processing, cellular metabolism, gene regulation, as well as hypothetical proteins with unknown function. (See Table 4 in Example 6).
Quantitative RT-PCR demonstrated a two-fold increase in lolA expression, enabling lipoprotein transport from the inner membrane to the outer membrane. Bacteria grow rapidly in the middle ear environment reaching 5.0×108 CFU NTHi ml middle ear fluid within 48 hours. The bacteria sense and respond to the environment, acquiring or synthesizing the necessary nutrients for growth and survival. The gene encoding the membrane component in ribose sugar transport, rbsC (SEQ ID NO: 619), showed a 5-fold increase in expression in vivo compared to cells growing in vitro. In addition, many genes involved in metabolic processes show a dramatic increase in gene expression in vivo compared to cells growing in vitro. These include a riboflavin synthesis gene, ribB (SEQ ID NO: 623), a purine nucleotide biosynthetic gene purE (SEQ ID NO: 621), ornithine carbamoyltransferase, arcB (SEQ ID NO: 625), involved in arginine degradation via the urea cycle and uxuA (SEQ ID NO: 627), encoding mannonate hydrolase, required for the uptake of D-glucuronate and transformation into glyceraldehyde 3-phosphate. In addition, but to a lesser degree, genes for histidine biosynthesis (hisB; SEQ ID NO: 615), DNA repair (radC; SEQ ID NO: 639) and a putative intracellular septation transmembrane protein (ispZ; SEQ ID NO: 637) were up-regulated.
Disulfide bond formation is important for folding and assembly of many secreted proteins in bacteria. In prokaryotes, DsbA and DsbB make up the oxidative pathway responsible for the formation of disulfides. DsbB reoxidizes DsbA, which donates disulfide bonds directly to unfolded polypeptides, and DsbB has been demonstrated to generate disulfides de novo from oxidized quinones (Collet and Bardwell, Mol. Microbiol., 44: 1-8, 2002). In H. influenzae strain Rd, DsbA is required for competence for transformation (Tomb, Proc. Natl. Acad. Sci. U.S.A., 89: 10252-10256, 1992). Herein, an approximate 3-fold increase in dsbB gene (SEQ ID NO: 629) transcription was demonstrated, illuminating an important role for disulfide interchange for NTHi growing in the middle ear environment.
Bacteria colonization of the middle ear, a normally sterile environment, results in a host inflammatory response and subsequent neutrophil infiltration. Bacteria have evolved numerous strategies to combat this host response. NTHi increase gene expression (4-fold) of ureH (SEQ ID NO:631), a homologue of a gene required for expression of active urease in Helicobacter, shown to be involved in acid tolerance (Young et al., J. Bacterol., 178: 6487-6495, 1996). Recently, it has been reported that urease activity may play a role in chronic Actinobacillus pleuropneumoniae infection by counteracting the decrease in pH occurring upon infection (Baltes et al., Infect. Immun., 69: 472-478, 2000; Baltes et al., Infect. Immun., 69: 472-478, 2001; Bosse and MacInnes, Can. J. Vet. Res., 64: 145-150). A biotype analysis on NTHi isolates from middle ear effusions demonstrated that 87% are urease positive (DeMaria et al., J. Clin. Microbiol., 20: 1102-1104, 1984). However, the role of urease in NTHi virulence is unknown. Similarly, an increase in expression of a gene whose product demonstrates 88% sequence identity to a pyridoxine biosynthesis protein in S. pneumoniae and 60% homology to a putative singlet oxygen resistance protein that may function as an antioxidant. Phosphorylcholine (ChoP) has been implicated in the pathogenesis of NTHi (Weiser et al., Infect. Immun., 65: 943-950, 1997). NTHi modulates ChoP expression by phase variation, decorating the LOS on the cell surface. ChoP may contribute to NTHi persistence in the respiratory tract via decreased susceptibility to antimicrobial peptides (Lysecko et al., Infect. Immun., 68: 1664-1671, 2000) and alter the sensitivity to serum killing mediated by C-reactive protein (CRP) (Weiser et al., J. Exp. Med., 187: 631-640, 1998). The microenvironment of the nasopharynx and middle ear cavity may select for the ChoP+ phenotype, as ChoP+ strains show greater colonization of the chinchilla nasopharynx (Tong et al., Infect. Immun., 68: 4593-4597, 2000). Expression of the licC gene (SEQ ID NO: 633) was also increased. The licC gene encodes a phosphorylcholine cytidylyltransferase that plays a role in the biosynthesis of phosphorylcholine-derivatized LOS (Rock et al., J. Bacterol., 183: 4927-4931, 2001).
Also included among the in vivo-induced genes is a set whose products subsequently regulate gene expression or DNA replication. These genes include transcriptional regulation of glycerol metabolism by the glp repressor, glpR (SEQ ID NO: 643), the arginine repressor gene, argR (SEQ ID NO: 647), and the integration host factor (IHF) beta subunit, ihfB (SEQ ID NO: 645). IHF is a histone-like protein that binds DNA at specific sequences, an accessory factor involved in replication, site-specific recombination and transcription, altering the activity of a large number of operons (Goosen and van de Putte, Mol. Microbiol. 16: 1-7, 1995). In addition, CspD inhibits DNA replication during stationary phase-induced stress response in E. coli (Yamanaka et al., Mol. Microbiol., 39: 1572-1584, 2001) and the mukF (SEQ ID NO: 641) gene protein homologue contributes to a remodeling of the nucleiod structure into a more compact form prior to cell segregation (Sawitzke and Austin, Proc. Natl. Acad. Sci. U.S.A., 62: 1710-1718, 2000). The DFI strategy described herein also identified promoters induced in vivo for genes of unknown function. The hypothetical protein, HI0094, demonstrated an 8-fold increase in gene expression during early OM but its role remains unknown. HI1163 (SEQ ID NO: 651) showed 58% amino acid identity with the hypothetical YdiJ proteins, a putative oxidase, of E. coli. 
A high-density transposon mutagenesis strategy was used to identify H. influenzae genes essential for growth on rich medium (Akerley et al., Proc. Natl. Acad. Sci. U.S.A., 99: 966-971, 2002). Six genes were identified in the screen described herein that are included in essential gene set described in Akerley' et al., supra. (hisB, lppB, lolA, ispZ, mukF and unknown HI0665). Recently genes of non-typeable H. influenzae that are expressed upon interaction with two human respiratory tract-derived epithelial cell lines have been identified. These genes included those involved in metabolic processes, stress responses, gene expression, cell envelope biosynthesis, DNA-related processes, cell division and ORF's encoding proteins of unknown function. (Ulsen et al., Mol. Microbiol., 45:485-500, 2,002). Similarly the stress response gene, cspD (SEQ ID NO: 649), genes involved in purine and riboflavin biosynthesis, and a protein of unknown function, vapA was identified in the screen described herein. Expression of vapA was detected in vitro, yet vapA gene expression increased two-fold in vivo. These unique approaches identified known genes that are upregulated in NTHi-induced OM and therefore are likely to play a role in NTHi infection and virulence; and may be potential candidates for vaccines and antisense therapies and other therapeutic methods of treatment of NTHi related disorders.
The DFI strategy resulted in the identification of promoters induced in vivo for genes of unknown function as well. The hypothetical protein, HI0094, demonstrated a 8-fold increase in gene expression during early OM but its role remains unknown. HI1163 (SEQ ID NO: 651) showed 58% amino acid identity with the hypothetical YdiJ proteins, a putative oxidase, of E. coli. Therefore, these hypothetical genes are likely to play a role in OM induced by NTHi infection.



BRIEF DESCRIPTION OF FIGURES
FIG. 1 depicts the LKP gene region in a panel of Haemophilus isolates. The strain 86-028NP sequence is identical in this region to the sequence in NTHi strain R3001. Both of these NTHi lack the hif gene cluster encoding the hemagglutinating pilus.
FIG. 2 depicts the rfaD region in a panel of Haemophilus isolates. The gene arrangement in the rfaD region of the strain 86-028NP genome is similar to that of the strain Rd genome but different than the arrangement of these genes seen in the genome of most NTHi examined.
FIGS. 3A-3M set out the nucleotide sequences (SEQ ID NOS: 589-614) described in Table 4, which were identified to be upregulated during OM infection (see Example 6). The nucleotides (nt.) which correspond to known genes and those nt. which correspond to the contig sequences set out as SEQ ID NO: 1-576 are also presented.



DETAILED DESCRIPTION
The following examples illustrate the invention wherein Example 1 describes the sequence of the NTHi genome, Example 2 describes the identified contigs and initial gene discovery, Example 3 describes construction of the NTHi promoter trap library, Example 4 describes the analyses of 86-028NP derivatives expressing GFP, Example 5 demonstrates direct labelling of bacteria from middle ear fluids, Example 6 describes identification of promoters induced in vivo in acute otitis media, Example 7 describes identification of virulence-associated genes, and Example 8 describes identification of unique NTHi gene sequences.
EXAMPLE 1
Sequence of a Non-Typeable Haemophilus influenzae Genome
NTHi strain 86-028NP is a minimally passaged clinical isolate obtained from a pediatric patient who underwent tympanostomy and tube insertion for chronic OM at Columbus Children's Hospital. (Bakaletz et al. Infection and Immunity, 56(2): 331-335, 1988) The 86-028NP strain was deposited with the American Type Tissue Collection (Manassas, Va. 20108 USA) on Oct. 16, 2002 and assigned accession no. PTA-4764.
In an effort to more broadly approach the identification of the virulence determinants in NTHi, the genome of the NTHi 86-028NP strain was sequenced to 3-fold coverage. Chromosomal DNA was prepared from strain 86-028NP using the Puregene protocol and sheared to 2-4 kb in size with a Hydroshear instrument (Gene Machines). The sheared DNA was ethanol-precipitated, end-repaired using a mixture of Klenow enzyme and T4 DNA polymerase, and size-selected by agarose gel electrophoresis to obtain 2-4 kb fragments as described in Chissoe et al. (Methods: a Companion to Methods of Enzymology 3: 55-65, 1991) and Sambrook et al. (Molecular Cloning: a Laboratory Manual, 2nd Ed. Cold Springs Harbor, N.Y., 1989). These fragments were cloned into vector pUC18 using the SmaI restriction site (phosphatase-treated) and transformed into E. coli XL-1 Blue, selecting for ampicillin resistance. Colonies that contain inserts were identified by blue/white screening on LB-Amp plates containing X-gal, and transferred into 96-deep well plates containing 1.5 ml of TB-Amp (TB=Terrific Broth) broth. The deep-well plate cultures were grown overnight (18-22 hours) at 37° C. Template preparation, sequencing and contig assembly were performed.
Automated template preparation was performed on the Beckman Biomek 2000 automated robotics workstation as described in Chissoe et al., (supra.) Briefly, each 96-deep well plate, containing the clones prepared above, was centrifuged to pellet the cells, the supernatant decanted, and the cells frozen (if necessary) at −20° C. Four 96-deep well blocks were placed on the Biomek table, and the liquid handling robot was used to prepare the template using an automated version of a typical SDS-NaOH lysis protocol as described in Chissoe et al., (supra.). The final ethanol-precipitated templates were each dissolved in 50 μl ddH2O, and used for DNA sequencing.
Sequencing reactions were run by re-arraying the templates (from 96-well plates) into 384-well plates, using the Robbins Hydra 96 robot. Cycle-sequencing reactions were run using PE Big-DyeTM terminators and universal primers (M13 forward and reverse), cleaned up over Sephadex G50 columns, and analyzed on a PE Biosystems 3700 capillary electrophoresis DNA sequencer according to the manufacturer's instructions. Sequencing reads (8219) were assembled into 576 contigs (SEQ ID NOS: 1-576 herein). The statistics for the 3-fold sequencing are shown in Table 2A. The total unique sequence in assembly 17 is 1.74 Mb.










TABLE 2A





Contig Size
Total Number
Total Length
% of Cumulative


















0-1 kb
65
55961
3.2%


1-2 kb
228
333665
19.2%


2-3 kb
101
243059
14.0%


3-4 kb
49
172385
9.9%


4-5 kb
45
196699
11.3%


5-10 kb 
74
515152
29.6%


10-20 kb 
11
144591
8.3%


20-30 kb 
3
77352
4.4%









Subsequently, 8-fold sequencing analysis of the NTHi genome was carried out. The 8-fold sequencing assembled the NTHi genome into 11 contigs. Contigs 5, 8, 9, 10, 12-18 are denoted as SEQ ID NOS: 675-685 herein. The statistics for the 8-fold sequencing are shown in Table 2B.










TABLE 2B





Contig Size
Total Number
Total Length
% of Cumulative



















0-1
kb
5
3950
0.2%


1-2
kb
3
4316
0.2%


2-3
kb
0
0
0.0%


3-4
kb
1
3964
0.2%


4-5
kb
0
0
0.0%


5-10
kb
0
0
0.0%


10-20
kb
1
15147
0.8%


20-30
kb
2
51888
2.7%


30-40
kb
0
0
0.0%


40-50
kb
0
0
0.0%


50-100
kb
1
85814
4.5%


>100
kb
5
1760339
91.4%









EXAMPLE 2
Contig Description and Initial Gene Discovery
Seventy-five of the 88 contigs with length≧5000bp, identified with the 3-fold sequence analysis, show significant similarity via BLASTN to genes in H. influenzae strain Rd. To visualize the potential relationship between the gene order in H. influenzae strain 86-028NP and H. influenzae strain Rd, the 86-028NP three-fold contig set and the Rd gene set were bidirectionally compared using BLASTN. The results were plotted in gene-order verses contig space by sorting the contigs based on gene coordinates of the Rd genes hit, anchoring each contig at the smallest coordinate found as described in Ray et al., (Bioinformatics 17: 1105-12, 2001). Compared in this fashion, an incomplete assembly of a genome with identical gene order to a completely known genome would display a monotonically increasing stair-stepped form.
BLASTX was used to identify hits to sequences with homology to genes in the strain Rd genome as well as genes not found in H. influenzae strain Rd. Hits to strain Rd sequences were removed from the data set and the other hits summarized in Table 3A. The data are presented as follows: contig # (=SEQ ID NO: #), column 1; E score for each hit, column 2; the name of the protein that had homology to a portion of the amino acid translation of the cited contig, column 3; the organism producing the homologue, column 4; and the Genbank protein identifier for each of the proteins cited in column 3, column 5; the corresponding nucleotides within the contig (referenced by SEQ ID NO:). In most instances, several homologues were identified but for clarity, the protein of greatest homology is cited in Table 3A.
The sequences for some of the genes listed in Table 3A were identified within the 8-fold sequencing of the NTHi genome. Table 3B lists the location of these genes within the 11 contigs, the full length open reading frame sequence (identified by SEQ ID NO:), the derived amino acid sequence encoded by the open reading frame and the gene with high homology identified by BLASTX (as listed in Table 3A).
To examine the relative short range gene arrangements in NTHi and the Rd strain, the gene order in two gene clusters that have been well-described were compared. First, the genes present in the hemagglutinating pilus (LKP) gene region were examined. (Mhlanga-Mutangadura et al., J. Bacteriol. 180(17): 4693-703, 1998). The pilus gene cluster is located between the purE and pepN genes, only fragments of which are depicted in FIG. 1. The serotype b strain, Eagan, contains the hifABCDE gene cluster and produces hemagglutinating pili. Strain Rd lacks the hicAB genes as well as the hifABCDE gene cluster. In general, the nontypeable strains previously examined contained the hicAB genes but not the hifgenes that encode the hemagglutinating pilus. The strain 86-028NP sequence (described herein) is identical in this region to the sequence in NTHi strain R3001 (FIG. 1). The rfaD gene encodes an enzyme involved in the biosynthesis of endotoxin. In addition, the rfaD gene from NTHi strain 2019 has been characterized by Nichols et al. (Infect Immunity 65(4): 1377-86, 1997). In strain 2019, the rfaD gene is immediately upstream of the rfaF gene that encodes another enzyme involved in endotoxin biosynthesis. The gene arrangement in strain Rd is different; the rfaD and rfaF genes are separated by approximately 11 kb of sequence. Most nontypeable strains examined contained the gene arrangement seen in strain 2019. In contrast, strain 86-028NP has a gene arrangement identical to that seen in strain Rd (FIG. 2).
A global analysis of the current assembly indicates that the gene content and order are similar to that in strain Rd. A more detailed analysis revealed that there are a substantial number of NTHi genes not previously seen in the Pasteurellaceae and some regions where the NTHi gene content and order is different than that seen in strain Rd. Thus, the current data suggest that the strain 86-028NP genome will contain a complex mosaic of Rd and non-Rd like features.
The DFI strategy also identified novel NTHi sequences that had increased gene expression. A list of these novel contig sequences that contain genes or gene fragments that have homology to ORFs in other organisms (primarily gram-negative bacteria) is set out in Table 3A. For example, the nucleotide sequence of contig 442 (SEQ ID NO: 442), nucleotides 1498-1845 are highly homologous to the sequences encoding amino acids 1-116 of H. influenzae strain Rd lipoprotein B (LppB). The gene is positioned between the stationary phase survival gene, surE, and a gene encoding a 43 kD antigenic outer membrane lipoprotein that is highly homologous to the recently identified bacterial lipoprotein, LppB/NlpD, which has been associated with virulence (Padmalayam et al., Infect. Immun., 68: 4972-4979, 2000). Recently, Zhang and coworkers demonstrated that nlpD and sure gene expression was induced during stationary phase of bacterial growth in Thermotoga maritima (Zhang et al., Structure (Camb), 9: 1095-1106, 2001). Therefore, under stress-induced conditions in the middle ear, this NTHi lipoprotein may be expressed.












TABLE 3A









Genbank



Contig
E score
Hit Identity
Organism
Protein
SEQ ID NO:




















104
4.00E−59
CpdB
Pasteurella
NP_246953.1
nt. 204-659 of





multocida

SEQ ID NO: 104


106
9.00E−10
hypothetical protein
Pyrococcus
G71244
nt. 40-309 of




PH0217-
horikoshii

SEQ ID NO: 106


106
1.00E−08
unknown
Pasteurella
NP_246871.1
nt. 605-694 of





multocida

SEQ ID NO: 106


106
200E−20
Orf122
Chlorobium
AAG12204.1
nt. 7-210 of





tepidum

SEQ ID NO: 106


110
3.00E−05
ArdC antirestriction
IncW plasmid pSa
AAD52160.1
compliment of




protein


nt. 959-1162 of







SEQ ID NO: 110


110
1.00E−33
hypothetical protein
Salmonella
NP_458676.1
compliment of





enterica subsp.

nt. nt. 181-825





enterica serovar

of SEQ ID NO:





Typhi

110


111
5.00E−12
putative membrane
Salmonella
NP_458664.1
compliment of




protein
enterica subsp.

nt. 45-287 of





enterica serovar

SEQ ID NO: 111





Typhi


111
6.00E−41
hypothetical protein
Salmonella
NP_458658.1
compliment of





enterica subsp.

nt. 1091-1480 of





enterica serovar

SEQ ID NO: 111





Typhi


114
7.00E−80
unknown
Pasteurella
NP_245828.1
compliment of





multocida

nt. 118-696 of







SEQ ID NO: 114


115
2.00E−09
A111R
Paramecium
NP_048459.1
nt. 555-869 of





bursaria Chlorella

SEQ ID NO: 115





virus 1


118
5.00E−45
DNA methylase
Vibrio cholerae
NP_231404.1
nt. 44-439 of




HsdM, putative


SEQ ID NO: 118


122
2.00E−18
unknown
Pasteurella
NP_245314.1
nt. 865-1302 of





multocida

SEQ ID NO: 122


123
4.00E−99
RNA
Proteus mirabilis
P50509
nt. 351-782 of




POLYMERASE


SEQ ID NO: 123




SIGMA-32




FACTOR


124
9.00E−58
ACETOLACTATE
Spirulina platensis
P27868
nt. 603-1025 of




SYNTHASE


SEQ ID NO: 124




(ACETOHYDROXY-




ACID SYNTHASE)




(ALS)


130
0
restriction
Neisseria
CAA09003.1
nt. 495-1559 of




modification
meningitidis

SEQ ID NO: 130




system-R protein


131
6.00E−91
uronate isomerase
Salmonella
NP_457532.1
compliment of




(glucuronate
enterica subsp.

nt. 661-1380 of




isomerase)
enterica serovar

SEQ ID NO: 131





Typhi


133
3.00E−30
GyrA
Pasteurella
NP_245778.1
compliment of





multocida

nt. 1447-1626 of







SEQ ID NO: 133


133
1.00E−27
DNA GYRASE
Pectobacterium
P41513
compliment of




SUBUNIT A
carotovorum

nt. 1302-1442 of







SEQ ID NO: 133


138
7.00E−06
KicA
Pasteurella
NP_245545.1
compliment of





multocida

nt. 92-157 of







SEQ ID NO: 138


138
1.00E−148
TYPE II
Haemophilus
O30869
compliment of




RESTRICTION
aegyptius

nt. 164-1045 of




ENZYME HAEII


SEQ ID NO: 138




(ENDONUCLEASE




HAEII) (R. HAEII)


143
4.00E−06
Gifsy-1 prophage
Salmonella
NP_461555.1
compliment of




protein
typhimurium LT2

nt. 228-632 of







SEQ ID NO: 143


143
1.00E−14
hypothetical protein
Bacteriophage
NP_050531.1
compliment of





VT2-Sa

nt. 778-1248 of







SEQ ID NO: 143


143
5.00E−09
hypothetical protein
Salmonella
CAD09979.1
compliment of





enterica subsp.

nt. 715-1026 of





enterica serovar

SEQ ID NO: 143





Typhi


143
6.00E−10
hypothetical 14.9 kd
Escherichia coli
NP_065324.1
nt. 3-173 of




protein


SEQ ID NO: 143


147
1.00E−38
GTP-binding
Escherichia coli
NP_289127.1
compliment of




elongation factor,
O157:H7 EDL933

nt. 172-342 of




may be inner


SEQ ID NO: 147




membrane protein


147
2.00E−14
GTP-binding
Borrelia
NP_212222.1
compliment of




membrane protein
burgdorferi

nt. 17-181 of




(IepA)


SEQ ID NO: 147


148
6.00E−17
galactokinase
Homo sapiens
AAC35849.1
compliment of







nt. 746-1246 of







SEQ ID NO: 148


148
7.00E−96
GALACTOKINASE
Actinobacillus
P94169
compliment of




(GALACTOSE
pleuropneumoniae

nt. 232-741 of




KINASE)


SEQ ID NO: 148


149
1.00E−92
GTP-binding
Buchnera sp.
NP_240245.1
compliment of




protein TypA/BipA
APS

nt. 265-1077 of







SEQ ID NO: 149


15
2.00E−21
ORF 1
Escherichia coli
CAA39631.1
nt. 665-850 of







SEQ ID NO: 15


150
6.00E−17
unknown
Pasteurella
NP_245919.1
nt. 171-665 of





multocida

SEQ ID NO: 150


153
7.00E−07
outer membrane
Rickettsia conorii
T30852
nt. 51-623 of




protein A


SEQ ID NO: 153


155
7.00E−40
cytochrome d
Vibrio cholerae
NP_233259.1
nt. 583-1002 of




ubiquinol oxidase,


SEQ ID NO: 155




subunit II


157
7.00E−13
unknown
Pasteurella
NP_245490.1
compliment of





multocida

nt. 1170-1367 of







SEQ ID NO: 157


157
2.00E−05
glycosyl
Neisseria
AAA68012.1
nt. 85-189 of




transferase
gonorrhoeae

SEQ ID NO: 157


158
1.00E−152
MltC
Pasteurella
NP_246259.1
compliment of





multocida

nt. 36-530 of







SEQ ID NO: 158


161
3.00E−25
lipoprotein, putative
Vibrio cholerae
NP_230232.1
nt. 870-1439 of







SEQ ID NO: 161


163
9.00E−53
chorismate
Caulobacter
NP_421948.1
nt. 1283-2029 of




synthase
crescentus

SEQ ID NO: 163


168
3.00E−13
COPPER-
Mus musculus
Q64430
nt. 66-995 of




TRANSPORTING


SEQ ID NO: 168




ATPASE 1




(COPPER PUMP




1)


168
2.00E−22
Cu transporting
Homo sapiens
2001422A
nt. 135-989 of




ATPase P


SEQ ID NO: 168


174
8.00E−48
magnesium/cobalt
Mesorhizobium
NP_103977.1
nt. 918-1205 of




transport protein
loti

SEQ ID NO: 174


175
5.00E−26
vacB protein
Buchnera sp.
NP_240369.1
compliment of





APS

nt. 1-1587 of







SEQ ID NO: 175


176
3.00E−21
putative ABC
Campylobacter
NP_282774.1
compliment of




transport system
jejuni

nt. 259-1089 of




permease protein [


SEQ ID NO: 176


183
5.00E−29
PROBABLE ATP
Ralstonia
NP_521442.1
compliment of




SYNTHASE A
solanacearum

nt. 42-677 of




CHAIN


SEQ ID NO: 183




TRANSMEMBRANE




PROTEIN


185
6.00E−85
putative exported
Salmonella
NP_458655.1
compliment of




protein
enterica subsp.

nt. 162-1529 of





enterica serovar

SEQ ID NO: 185





Typhi


187
2.00E−05
transketolase
Homo sapiens
AAA61222.1
nt. 709-819 of







SEQ ID NO: 187


188
1.00E−116
ribonuclease E
Xylella fastidiosa
NP_299884.1
compliment of





9a5c

nt. 280-1704 of







SEQ ID NO: 188


192
1.00E−38
ImpA
Pasteurella
NP_245829.1
nt. 35-448 of





multocida

SEQ ID NO: 192


193
3.00E−08
Orf80
Enterobacteria
NP_052285.1
nt. 1612-1818 of





phage 186

SEQ ID NO: 193


193
1.00E−06
holin
Haemophilus
AAC45168.1
nt. 370-576 of





somnus

SEQ ID NO: 193


193
0.007
unknown
Enterobacteria
NP_052260.1
nt. 1376-1609 of





phage 186

SEQ ID NO: 193


193
2.00E−48
lysozyme
Haemophilus
AAC45169.1
nt. 608-1093 of





somnus

SEQ ID NO: 193


199
4.00E−21
unknown protein
Escherichia coli
NP_288675.1
nt. 398-778 of





O157:H7

SEQ ID NO: 199





EDL933,





prophage CP-





933V


199
2.00E−49
hypothetical protein
Bacteriophage
NP_049495.1
compliment of





933W

nt. 1907-2392 of







SEQ ID NO: 199


20
1.00E−62
RpL14
Pasteurella
NP_246344.1
compliment of





multocida

nt. 233-601 of







SEQ ID NO: 20


200
2.00E−62
hypothetical protein
Salmonella
NP_458658.1
compliment of





enterica subsp.

nt. 431-997 of





enterica serovar

SEQ ID NO: 200





Typhi


200
3.00E−16
hypothetical protein
Salmonella
NP_458657.1
compliment of





enterica subsp.

nt. 1028-1264 of





enterica serovar

SEQ ID NO: 200





Typhi


201
2.00E−26
TsaA
Pasteurella
NP_245732.1
compliment of





multocida

nt. 1618-1809 of







SEQ ID NO: 201


209
6.00E−16
TsaA
Pasteurella
NP_245732.1
compliment of





multocida

nt. 2-136 of







SEQ ID NO: 209


211
2.00E−15
unknown
Pasteurella
NP_245535.1
compliment of





multocida

nt. 23-211 of







SEQ ID NO: 211


211
1.00E−70
PUTATIVE
Ralstonia
NP_520082.1
compliment of




ATPASE PROTEIN
solanacearum

nt. 475-915 of







SEQ ID NO: 211


212
3.00E−18
hypothetical protein
Escherichia coli
NP_309775.1
compliment of





O157:H7

nt. 895-1035 of







SEQ ID NO: 212


216
1.00E−173
unknown
Pasteurella
NP_245069.1
nt. 35-1543 of





multocida

SEQ ID NO: 216


217
9.00E−18
diacylglycerol
Vibrio cholerae
NP_233101.1
nt. 2083-2208 of




kinase


SEQ ID NO: 217


221
4.00E−34
Tail-Specific
Chlamydia
NP_219953.1
nt. 849-1421 of




Protease
trachomatis

SEQ ID NO: 221


222
4.00E−23
AGR_C_3689p
Agrobacterium
NP_355005.1
compliment of





tumefaciens str.

nt. 940-1305 of





C58 (Cereon)

SEQ ID NO: 222


224
9.00E−19
unknown
Pasteurella
NP_245536.1
nt. 15-308 of





multocida

SEQ ID NO: 224


225
1.00E−89
portal vector-like
Salmonella
NP_461651.1
nt. 31-750 of of




protein, in phage
typhimurium

SEQ ID NO: 225




P2 [Salmonella
LT2Fels-2




typhimurium LT2]
prophage


229
2.00E−25
anaerobic
Salmonella
CAB62266.1
nt. 1806-2108 of




ribonucleotide
typhimurium

SEQ ID NO: 229




reductase


234
3.00E−08
conserved
Xylella fastidiosa
NP_299850.1
nt. 1680-2048 of




hypothetical protein
9a5c

SEQ ID NO: 234


234
1.00E−42
Methionine
Clostridium
NP_348177.1
compliment of




sulfoxide reductase
acetobutylicum

nt. 415-654 of




C-terminal domain


SEQ ID NO: 234




related protein,




YPPQ ortholog


235
4.00E−16
phage-related tail
Wolbachia
AAK85310.1
compliment of




protein
endosymbiont of

nt. 931-1929 of





Drosophila

SEQ ID NO: 235





melanogaster


235
6.00E−56
similar to orfG
Salmonella
NP_461625.1
compliment of




protein in phage
typhimurium LT2,

nt. 313-1863 of




186, Salmonella
Fels-2 prophage

SEQ ID NO: 235




typhimurium LT2


236
6.00E−20
conserved
Pseudomonas
NP_252693.1
nt. 1572-1916




hypothetical protein
aeruginosa

of SEQ ID NO:







236


240
5.00E−27
MODIFICATION
Brevibacterium
P10283
compliment of




METHYLASE BEPI
epidermidis

nt. 922-1305 of







SEQ ID NO: 240


241
2.00E−15
phage-related
Xylella fastidiosa
NP_299573.1
compliment of




protein
9a5c

nt. 865-1305 of







SEQ ID NO: 241


241
4.00E−08
hypothetical protein
phage SPP1
T42296
nt. 73-636 of







SEQ ID NO: 241


241
4.00E−07
hypothetical protein
Salmonella
NP_458686.1
nt. 10-468 of





enterica subsp.

SEQ ID NO: 241





enterica serovar





Typhi


242
2.00E−29
translation
chloroplast-
S35701
compliment of




elongation factor
soybean

nt. 18-1085 of




EF-G


SEQ ID NO: 242


247
3.00E−23
GTP
Synechococcus
Q54769
compliment of




CYCLOHYDROLASE
sp. PCC 7942

nt. 1009-1257c




I (GTP-CH-I)


of SEQ ID NO:







247


248
6.00E−05
phospho-N-
Aquifex aeolicus
NP_213025.1
nt. 830-1747 of




acetylmuramoyl-


SEQ ID NO: 248




pentapeptide-




transferase


25
2.00E−86
PROBABLE
Ralstonia
NP_522358.1
compliment of




TRANSPORT
solanacearum

nt. 309-854 of




TRANSMEMBRANE


SEQ ID NO: 25




PROTEIN


25
7.00E−06
major facilitator
Caulobacter
NP_419155.1
compliment of




family transporter
crescentus

nt. 134-283 of







SEQ ID NO: 25


250
1.00E−150
CpdB
Pasteurella
NP_246953.1
compliment of





multocida

nt. 36-1016 of







SEQ ID NO: 250


252
3.00E−57
alanyl-tRNA
Vibrio cholerae
AAA99922.1
compliment of




synthetase


nt. 1418-1951 of







SEQ ID NO: 252


253
1.00E−108
similar to
Listeria
NP_464432.1
compliment of




glutathione
monocytogenes

nt. 411-1358 of




Reductase
EGD-e

of SEQ ID NO:







253


259
3.00E−39
hypothetical protein
Salmonella
NP_458654.1
compliment of





enterica subsp.

nt. 342-1037 of





enterica serovar

SEQ ID NO: 259





Typhi


259
3.00E−17
possible exported
Salmonella
NP_458653.1
compliment of




protein
enterica subsp.

nt. 1251-1607





enterica serovar

of SEQ ID NO:





Typhi

259


261
5.00E−74
hypothetical protein
Haemophilus
S27582
compliment of




6-Haemophilus
influenzae

nt. 3-422 of




influenzae


SEQ ID NO: 261


263
1.00E−94
putative
Haemophilus
AAD01406.1
nt. 2142-2672 of




transposase
paragallinarum

SEQ ID NO: 263


264
1.00E−126
unknown
Actinobacillus
NP_067554.1
nt. 40-714 of





actinomycetemco

SEQ ID NO: 264





mitans


264
1.00E−103
unknown
Actinobacillus
NP_067555.1
nt. 695-1309 of





actinomycetemco

SEQ ID NO: 264





mitans


264
2.00E−21
unknown
Actinobacillus
NP_067556.1
nt. 1302-1448 of





actinomycetemco

SEQ ID NO: 264





mitans


265
6.00E−27
Aminopeptidase 2
chloroplast
Q42876
nt. 556-1539 of







SEQ ID NO: 265


268
1.00E−116
MutY
Pasteurella
NP_246257.1
nt. 1003-1581 of





multocida

SEQ ID NO: 268


272
1.00E−07
hypothetical protein
Bacteriophage
NP_049495.1
compliment of





933W

nt. 77-232 of







SEQ ID NO: 272


274
3.00E−13
unknown
Pasteurella
NP_246952.1
compliment of





multocida

nt. 1658-1975 of







SEQ ID NO: 274


275
3.00E−20
CafA
Neisseria
AAG24267.1
nt. 1299-1571 of





gonorrhoeae

SEQ ID NO: 275


276
1.00E−45
mukE protein
Vibrio cholerae
NP_231351.1
compliment of







nt. 650-1390 of







SEQ ID NO: 276


276
1.00E−69
KicA
Pasteurella
NP_245545.1
compliment of





multocida

nt. 647-1321 of







SEQ ID NO: 276


278
2.00E−56
3-oxoacyl-[acyl-
Salmonella
NP_455686.1
nt. 1366-1944 of




carrier-protein]
enterica subsp.

SEQ ID NO: 278




synthase III
enterica serovar





Typhi


281
5.00E−56
unknown
Pasteurella
NP_246261.1
compliment of





multocida

nt. 31-678 of







SEQ ID NO: 281


282
3.00E−09
orf25; similar to T
bacteriophage phi
NP_490625.1
compliment of




gene of P2
CTX

nt. 511-1032 of







SEQ ID NO: 282


282
1.00E−08
orf11; similar to
Haemophilus
AAC45165.1
compliment of




phage P2 gene S-
somnus

nt. 1450-1584 of




like product, which


SEQ ID NO: 282




is involved in tail




synthesis,


282
9.00E−27
putative
Salmonella
NP_457167.1
compliment of




bacteriophage tail
enterica subsp.

nt. 3-509 of




protein
enterica serovar

SEQ ID NO: 282





Typhi


286
5.00E−18
plasmid-related
Listeria innocua
NP_471066.1
compliment of




protein
plasmid

nt. 887-1501 of







SEQ ID NO: 286


287
8.00E−20
GTP
Escherichia coli
NP_287920.1
nt. 2-145 of




cyclohydrolase II
O157:H7 EDL933

SEQ ID NO: 287


289
1.00E−168
MODIFICATION
Haemophilus
O30868
compliment of




METHYLASE
aegyptius

nt. 138-1091 of




HAEII


SEQ ID NO: 289


289
5.00E−11
TYPE II
Haemophilus
O30869
compliment of




RESTRICTION
aegyptius

nt. 22-132 of




ENZYME HAEII


SEQ ID NO: 289


289
6.00E−47
mukF homolog
Haemophilus
AAB70828.1
compliment of





influenzae biotype

nt. 1107-1385





aegyptius

of SEQ ID NO:







289


294
1.00E−171
LICA PROTEIN
Haemophilus
P14181
compliment of





influenzae

nt. 677-1564 of





RM7004

SEQ ID NO: 294


297
1.00E−158
DNA methylase
Vibrio cholerae
NP_231404.1
compliment of




HsdM, putative


nt. 12-1136 of







SEQ ID NO: 297


302
0
HEME-BINDING
Haemophilus
P33950
nt. 3-1316 of




PROTEIN A
influenzae DL42

SEQ ID NO: 302


304
6.00E−19
hypothetical protein 6
Haemophilus
S27582
nt. 121-267 of





influenzae

SEQ ID NO: 304


305
6.00E−40
putative
Streptococcus
NP_269557.1
nt. 65-805 of




recombinase-
pyogenes M1

SEQ ID NO: 305




phage associated
GAS


305
7.00E−22
single stranded
Shewanella sp.
AAB57886.1
nt. 1607-2014 of




DNA-binding
F1A

SEQ ID NO: 305




protein


305
1.00E−43
phage-related
Bacillus
NP_244410.1
nt. 92-751 of




protein
halodurans

SEQ ID NO: 305


312
1.00E−28
PUTATIVE
Ralstonia
NP_518994.1
nt. 1819-2673 of




BACTERIOPHAGE-
solanacearum

SEQ ID NO: 312




RELATED




TRANSMEMBRANE




PROTEIN


312
9.00E−25
similar to
Homo sapiens
XP_068727.1
nt. 27-1001 of




BASEMENT


SEQ ID NO: 312




MEMBRANE-




SPECIFIC




HEPARAN




SULFATE




PROTEOGLYCAN




CORE PROTEIN




PRECURSOR




(HSPG)


315
2.00E−45
uracil permease
Deinococcus
NP_296001.1
compliment of





radiodurans

nt. 525-1592 of







SEQ ID NO: 315


318
7.00E−15
CzcD
Pasteurella
NP_246276.1
compliment of





multocida

nt. 3-227 of







SEQ ID NO: 318


320
2.00E−60
orf3; similar to
Haemophilus
AAC45159.1
compliment of




endonuclease
somnus

nt. 606-1241 of




subunit of the


SEQ ID NO: 320




phage P2




terminase (gene M)


320
2.00E−09
orf4; similar to
Haemophilus
AAC45160.1
compliment of




head
somnus

nt. 52-285 of




completion/stabilization


SEQ ID NO: 320




protein (gene




L) of phage P2


320
3.00E−35
orf2; similar to
Haemophilus
AAC45158.1
compliment of




major capsid
somnus

nt. 1271-1624 of




protein precursor of


SEQ ID NO: 320




phage P2 (gene N)


323
4.00E−37
dedC protein
Escherichia coli
AAA23966.1
compliment of







nt. 74-463 of







SEQ ID NO: 323


324
1.00E−153
conserved
Neisseria
NP_274972.1
compliment of




hypothetical protein
meningitidis

nt. 930-1943 of





MC58

SEQ ID NO: 324


326
5.00E−52
selenophosphate
Eubacterium
CAB53511.1
compliment of




synthetase
acidaminophilum

nt. 1186-2292 of







SEQ ID NO: 326


328
1.00E−129
secretion protein
Pseudomonas
NP_252510.1
compliment of




SecD
aeruginosa

nt. 8-625 of







SEQ ID NO: 328


333
3.00E−08
unknown
Pasteurella
NP_245489.1
compliment of





multocida

nt. 5-418 of







SEQ ID NO: 333


336
6.00E−38
probable methyl
Pseudomonas
NP_253353.1
compliment of




transferase
aeruginosa

nt. 2547-2819 of







SEQ ID NO: 336


338
2.00E−98
Pmi
Pasteurella
NP_245766.1
nt. 144-842 of





multocida

SEQ ID NO: 338


339
2.00E−07
tRNA
Escherichia coli
QQECPE
nt. 2331-2540 of




nucleotidyltransferase


SEQ ID NO: 339


340
0
DNA gyrase,
Salmonella
NP_461214.1
compliment of




subunit A, type II
typhimurium LT2

nt. 93-1799 of




topoisomerase


SEQ ID NO: 340


342
4.00E−12
tolA protein
Haemophilus
JC5212
nt. 980-1318 of





influenzae

SEQ ID NO: 342


344
1.00E−07
MODIFICATION
Haemophilus
P50192
compliment of




METHYLASE
parahaemolyticus

nt. 849-1034 of




HPHIA


SEQ ID NO: 344


344
8.00E−05
ABC transporter
Leishmania major
AAF31030.1
compliment of




protein 1


nt. 17-205 of







SEQ ID NO: 344


349
3.00E−44
conserved
Neisseria
NP_273467.1
compliment of




hypothetical protein
meningitidis

nt. 1397-1903 of





MC58

SEQ ID NO: 349


349
8.00E−09
hypothetical protein
Pseudomonas
NP_252667.1
compliment of





aeruginosa

nt. 795-1121 of







SEQ ID NO: 349


349
9.00E−10
conserved
Helicobacter
NP_207009.1
compliment of




hypothetical
pylori 26695

nt. 1319-1816 of




secreted protein


SEQ ID NO: 349


349
2.00E−06
putative TPR
Salmonella
NP_463149.1
compliment of




repeat protein
typhimurium LT2

nt. 2244-2558 of







SEQ ID NO: 349


35
1.00E−23
type I restriction-
Xylella fastidiosa
NP_300003.1
compliment of




modification
9a5c

nt. 29-388 of




system specificity


SEQ ID NO: 35




determinant


352
1.00E−116
putative peptidase
Escherichia coli
NP_416827.1
compliment of





K12

nt. 951-1640 of







SEQ ID NO:







352


352
0
unknown
Pasteurella
NP_245275.1
compliment of





multocida

nt. 86-946 of







SEQ ID NO: 352


354
5.00E−86
putative uronate
Salmonella
NP_462052.1
compliment of




isomerase
typhimurium LT2

nt. 168-914 of







SEQ ID NO: 354


356
1.00E−07
isomerase-like
Escherichia coli
S57220
nt. 5-73 of




protein (DsbD)-


SEQ ID NO: 356


358
1.00E−07
USG protein
Pediococcus
CAC16793.1
nt. 534-1307 of





pentosaceus

SEQ ID NO: 358


358
0.005
HsdS protein
Escherichia coli
CAA10700.1
nt. 26-205 of







SEQ ID NO: 358


361
1.00E−152
maltodextrin
Escherichia coli
NP_289957.1
compliment of




phosphorylase
O157:H7 EDL933

nt. 77-922 of







SEQ ID NO: 361


363
6.00E−06
BH2505-unknown
Bacillus
NP_243371.1
nt. 554-844 of




conserved protein
halodurans

SEQ ID NO: 363


368
1.00E−12
H02F09.3.p
Caenorhabditis
NP_508295.1
compliment of





elegans

nt. 1069-1977 of







SEQ ID NO: 368


368
6.00E−27
hypothetical
Mesorhizobium
NP_102360.1
compliment of




glycine-rich protein
loti

nt. 1201-1986 of







SEQ ID NO: 368


37
6.00E−09
putative ATP-
Escherichia coli
NP_415469.1
compliment of




binding component
K12

nt. 455-691 of




of a transport


SEQ ID NO: 37




system


372
7.00E−18
conserved
Clostridium
BAB80319.1
compliment of




hypothetical protein
perfringens

nt. 1763-1924 of







SEQ ID NO: 372


376
7.00E−24
putative
Salmonella
NP_456379.1
compliment of




bacteriophage
enterica subsp.

nt. 158-808 of




protein
enterica serovar

SEQ ID NO: 376





Typhi


376
8.00E−10
hypothetical protein
Xylella fastidiosa
NP_298882.1
compliment of





9a5c

nt. 1129-1671







of SEQ ID







NO: 376


376
9.00E−06
lin1713
Listeria innocua
NP_471049.1
compliment of







nt 913-1557 of







SEQ ID NO: 376


377
6.00E−05
Vng1732c
Halobacterium sp.
NP_280487.1
nt. 2378-2587 of





NRC-1

SEQ ID NO: 377


377
1.00E−11
INVASIN
Yersinia
P31489
compliment of




PRECURSOR
enterocolitica

nt. 127-345 of




(OUTER


SEQ ID NO: 377




MEMBRANE




ADHESIN)


382
4.00E−16
unknown
Pasteurella
NP_246871.1
compliment of





multocida

nt. 967-1068 of







SEQ ID NO: 382


383
4.00E−36
putative
Streptomyces
BAB69302.1
nt. 488-1162 of




transposase
avermitilis

SEQ ID NO: 383


383
3.00E−58
recombinase
IncN plasmid R46
NP_511241.1
compliment of







nt. 1-393 of







SEQ ID NO: 383


383
4.00E−24
transposase
Escherichia coli
I69674
nt. 1294-1740 of







SEQ ID NO: 383


383
0
tnpA
Yersinia
CAA73750.1
nt. 1782-2834 of





enterocolitica

SEQ ID NO: 383


385
2.00E−31
unknown
Pasteurella
NP_246065.1
nt. 1515-1772 of





multocida

SEQ ID NO: 385


386
5.00E−65
cydC [
Escherichia coli
AAA66172.1
compliment of







nt. 3438-4115 of







SEQ ID NO: 386


386
4.00E−33
ABC transporter,
Mesorhizobium
NP_105463.1
compliment of




ATP-binding
loti

nt. 2569-3390 of




protein


SEQ ID NO: 386


388
3.00E−45
60 KDA INNER-
Coxiella burnetii
P45650
compliment of




MEMBRANE


nt. 3211-3759




PROTEIN


of SEQ ID NO:




HOMOLOG


388


390
4.00E−25
putative DNA-
Salmonella
NP_458175.1
nt. 1051-1416 of




binding protein
enterica subsp.

SEQ ID NO: 390





enterica serovar





Typhi


390
3.00E−13
transcriptional
Bacillus
NP_241773.1
compliment of




regulator
halodurans

nt. 84-578 of







SEQ ID NO: 390


390
3.00E−06
DNA translocase
Staphylococcus
NP_372265.1
compliment of




stage III sporulation
aureus subsp.

nt. 620-871 of




prot homolog
aureus Mu50

SEQ ID NO: 390


395
7.00E−31
ATPase, Cu++
Homo sapiens
NP_000044.1
compliment of




transporting, beta


nt. 615-1406 of




polypeptide


SEQ ID NO: 395


397
3.00E−23
terminase large
Bacteriophage
NP_112076.1
compliment of




subunit
HK620

nt. 2363-2725 of







SEQ ID NO: 397


397
3.00E−16
hypothetical protein
Xylella fastidiosa
NP_297824.1
compliment of





9a5c

nt. 1517-1744 of







SEQ ID NO: 397


398
4.00E−67
orf32
Haemophilus
NP_536839.1
compliment of





phage HP2

nt. 1288-1866 of







SEQ ID NO: 398


398
8.00E−24
putative
Salmonella
NP_463063.1
compliment of




cytoplasmic protein
typhimurium LT2

nt. 798-1220 of







SEQ ID NO: 398


398
2.00E−83
orf31
Haemophilus
NP_043502.1
compliment of





phage HP1

nt. 1881-2510 of







SEQ ID NO: 398


399
1.00E−94
HEME/HEMOPEXIN-
Haemophilus
P45355
nt. 88-774 of




BINDING
influenzae N182

SEQ ID NO: 399




PROTEIN


401
3.00E−63
Sty SBLI
Salmonella
CAA68058.1
nt. 1690-2742 of





enterica

SEQ ID NO: 401


401
3.00E−06
RESTRICTION-
Mycoplasma
NP_325912.1
nt. 79-489 of




MODIFICATION
pulmonis

SEQ ID NO: 401




ENZYME




SUBUNIT M3


402
2.00E−13
OPACITY
Neisseria
Q05033
compliment of




PROTEIN OPA66
gonorrhoeae

nt. 2634-2915 of




PRECURSOR


SEQ ID NO: 402


406
8.00E−13
type I restriction
Neisseria
NP_273876.1
nt. 281-520 of




enzyme EcoR124IIR
meningitidis

SEQ ID NO: 406





MC58


407
6.00E−65
unknown
Pasteurella
NP_246237.1
nt. 938-2450 of





multocida

SEQ ID NO: 407


407
5.00E−99
PepE
Pasteurella
NP_245391.1
nt. 1216-1917 of





multocida

SEQ ID NO: 407


407
1.00E−16
Hemoglobin-
Haemophilus
Q48153
nt. 1-141 of




haptoglobin binding
influenzae Tn106

SEQ ID NO: 407




protein A


409
1.00E−106
hypothetical protein 1
Haemophilus
S27577
compliment of





influenzae

nt. 2524-3159 of







SEQ ID NO: 409


411
4.00E−29
heme-repressible
Haemophilus
AAB46794.1
nt. 391-615 of




hemoglobin-binding
influenzae, type b,

SEQ ID NO: 411




protein
strain HI689


411
0
Hemoglobin-
Haemophilus
Q48153
nt. 651-3263 of




haptoglobin binding
influenzae Tn106

SEQ ID NO: 411




protein A


412
2.00E−07
REGULATORY
bacteriophage
P03036
compliment of




PROTEIN CRO
434

nt. 59-259 of




(ANTIREPRESSOR)


SEQ ID NO: 412


412
4.00E−06
hypothetical protein
Bacteriophage
CAC83535.1
nt. 1436-1654 of





P27

SEQ ID NO: 412


413
8.00E−07
hypothetical protein
Deinococcus
NP_294301.1
compliment of





radiodurans

nt. 791-1012 of







SEQ ID NO: 413


414
9.00E−65
conserved
Vibrio cholerae
NP_230092.1
nt. 1696-2103 of




hypothetical protein


SEQ ID NO: 414


414
3.00E−93
unknown
Pasteurella
NP_246834.1
nt. 1777-2109 of





multocida

SEQ ID NO: 414


416
2.00E−17
unknown
Pasteurella
NP_246629.1
compliment of





multocida

nt. 2565-2831 of







SEQ ID NO: 416


416
4.00E−26
hypothetical protein
Escherichia coli
S30728
compliment of




o154


nt. 1928-2254 of







SEQ ID NO:







416


416
3.00E−37
transport protein
Pseudomonas
NP_253757.1
compliment of




TatC
aeruginosa

nt. 1494-2018 of







of SEQ ID NO:







416


417
1.00E−66
weakly similar to
Listeria innocua
NP_471073.1
compliment of




methyltransferases


nt. 999-1928 of







SEQ ID NO: 417


417
5.00E−05
DNA-BINDING
Pectobacterium
Q47587
compliment of




PROTEIN RDGA
carotovorum

nt. 3526-4212 of







SEQ ID NO: 417


417
2.00E−29
putative phage-
Yersinia pestis
NP_407132.1
compliment of




related protein


nt. 2546-2938 of







SEQ ID NO: 417


417
3.00E−06
Adenine-specific
Thermoplasma
NP_393798.1
compliment of




DNA methylase
acidophilum

nt. 826-1020 of







SEQ ID NO: 417


43
9.00E−16
PcnB
Pasteurella
NP_245801.1
nt. 511-870 of





multocida

SEQ ID NO: 43


434
2.00E−97
beta' subunit of
Nephroselmis
NP_050840.1
compliment of




RNA polymerase
olivacea

nt. 32-1534 of







SEQ ID NO: 434


435
4.00E−52
MODIFICATION
Brevibacterium
P10283
compliment of




METHYLASE BEPI
epidermidis

nt. 11-565 of







SEQ ID NO: 435


435
4.00E−57
pentafunctional
Saccharomyces
NP_010412.1
compliment of




arom polypeptide
cerevisiae

nt. 757-2064 of




(contains: 3-


SEQ ID NO: 435




dehydroquinate




synthase, 3-




dehydroquinate




dehydratase (3-




dehydroquinase),




shikimate 5-




dehydrogenase,




shikimate kinase,




and epsp synthase


437
5.00E−70
dihydrofolate
Haemophilus
S52336
nt. 2393-2767 of




reductase
influenzae

SEQ ID NO: 437





(clinical isolate





R1042)


438
1.00E−106
polyA polymerase
Vibrio cholerae
NP_230244.1
nt. 3-1124 of







SEQ ID NO: 438


439
6.00E−60
Porphyrin
Salmonella
NP_457816.1
nt. 2343-2783 of




biosynthetic protein
enterica subsp.

SEQ ID NO: 439





enterica serovar





Typhi


441
5.00E−73
RimM
Pasteurella
NP_246234.1
compliment of





multocida

nt. 151-441 of







SEQ ID NO: 441


442
9.00E−31
LIPOPROTEIN
Salmonella
P40827
compliment of




NLPD
typhimurium

nt. 3362-3520 of







SEQ ID NO: 442


444
6.00E−24
glycine betaine
Staphylococcus
NP_371872.1
compliment of




transporter
aureus subsp.

nt. 2242-2514 of





aureus Mu50

SEQ ID NO: 444


452
6.00E−28
unknown
Pasteurella
NP_245616.1
compliment of





multocida

nt. 533-883 of







SEQ ID NO: 452


452
0
Type I restriction
Escherichia coli
Q47163
nt. 3291-4154 of




enzyme Ecoprrl M


SEQ ID NO: 452




protein


452
2.00E−75
type I restriction
Ureaplasma
NP_077929.1
nt. 4156-4562 of




enzyme M protein
urealyticum

SEQ ID NO: 452


455
9.00E−56
PROBABLE
Ralstonia
NP_520059.1
nt. 2028-2774 of




BACTERIOPHAGE
solanacearum

SEQ ID NO: 455




PROTEIN


455
2.00E−55
orf2; similar to
Haemophilus
AAC45158.1
nt. 2864-3490 of




major capsid
somnus

SEQ ID NO: 455




protein precursor of




phage P2 (gene N),


455
1.00E−175
gpP
Enterobacteria
NP_046758.1
compliment of





phage P2

nt. 127-1812 of







SEQ ID NO: 455


456
1.00E−38
hypothetical protein
Pseudomonas
NP_542872.1
compliment of





putida

nt. 1010-1282 of







SEQ ID NO: 456


456
1.00E−172
hypothetical protein
Pseudomonas
NP_542873.1
compliment of





putida

nt. 1443-2006 of







SEQ ID NO: 546


457
1.00E−116
hypothetical protein
Haemophilus
S15287
compliment of




(galE 5′ region)-
influenzae

nt. 62-961 of




Haemophilus


SEQ ID NO: 457




influenzae


457
1.00E−134
dTDPglucose 4,6-
Actinobacillus
T00102
nt. 2637-3656 of




dehydratase
actinomycetemco

SEQ ID NO: 457





mitans


459
2.00E−10
RNA polymerase
Synechocystis sp.
NP_441586.1
nt. 25-117 of




gamma-subunit
PCC 6803

SEQ ID NO: 459


461
9.00E−51
conserved
Staphylococcus
NP_370593.1
nt. 4124-4624 of




hypothetical protein
aureus subsp.

SEQ ID NO: 461





aureus Mu50


462
9.00E−06
NADH
Burkholderia
AAG01016.1
nt. 703-828 of




dehydrogenase
pseudomallei

SEQ ID NO: 462


465
3.00E−41
GTP-binding
Synechocystis sp.
NP_441951.1
compliment of




protein Era
PCC 6803

nt. 2470-2787 of







SEQ ID NO: 465


466
1.00E−15
putative
Salmonella
NP_455548.1
nt. 837-1478 of




bacteriophage
enterica subsp.

SEQ ID NO: 466




protein
enterica serovar





Typhi


466
1.00E−90
orf31
Haemophilus
NP_043502.1
nt. 2396-3199 of





phage HP1

SEQ ID NO: 466


469
0
Hemoglobin and
Haemophilus
Q9X442
compliment of




hemoglobin-
influenzae HI689

nt. 427-3459 of




haptoglobin binding


SEQ ID NO: 469




protein C precursor


471
8.00E−05
transposase,
Neisseria
NP_274608.1
nt. 2957-3217 of




putative
meningitidis

SEQ ID NO: 471





MC58


472
6.00E−08
hypothetical protein
Salmonella
NP_458660.1
compliment of





enterica subsp.

nt. 2881-3270 of





enterica serovar

SEQ ID NO: 472





Typhi


472
5.00E−23
antirestriction
Mesorhizobium
NP_106707.1
nt. 4908-5324 of




protein
loti

SEQ ID NO: 472


472
1.00E−75
hypothetical protein
Salmonella
NP_458661.1
compliment of





enterica subsp.

nt. 1931-2776 of





enterica serovar

SEQ ID NO: 472





Typhi


472
9.00E−72
hypothetical protein
Salmonella
NP_458662.1
compliment of





enterica subsp.

nt. 544-1689 of





enterica serovar

SEQ ID NO: 472





Typhi


475
3.00E−25
unknown
Pasteurella
NP_244952.1
nt. 3207-3626 of





multocida

SEQ ID NO: 475


476
8.00E−73
putative DNA-
Salmonella
NP_458175.1
compliment of




binding protein
enterica subsp.

nt. 3339-4310 of





enterica serovar

SEQ ID NO: 476





Typhi


476
6.00E−47
anticodon nuclease
Neisseria
NP_273873.1|
compliment of





meningitidis

nt. 4397-4885 of





MC58

SEQ ID NO: 476


478
3.00E−06
methionin
Arabidopsis
CAB38313.1
compliment of




synthase-like
thaliana

nt. 3554-3679 of




enzyme


SEQ ID NO: 478


478
3.00E−05
unknown
Pasteurella
NP_245444.1
compliment of





multocida

nt. 164-250 of







SEQ ID NO: 478


479
1.00E−18
conserved
Xylella fastidiosa
NP_298841.1
nt. 2302-2658 of




hypothetical protein
9a5c

SEQ ID NO: 479


48
3.00E−19
Dca
Neisseria
AAF12796.1
compliment of





gonorrhoeae

nt. 225-746 of







SEQ ID NO: 48


482
1.00E−06
hypothetical protein
Neisseria
NP_275122.1
nt. 2055-2189 of





meningitidis

SEQ ID NO: 482





MC58


482
9.00E−28
conserved
Neisseria
NP_274383.1
nt. 1689-1898 of




hypothetical protein
meningitidis

SEQ ID NO: 482





MC58


487
5.00E−75
conserved
Neisseria
NP_284304.1
nt. 2541-2978 of




hypothetical protein
meningitidis

SEQ ID NO: 487





Z2491


488
2.00E−64
unknown
Pasteurella
NP_246617.1
nt. 2983-3540 of





multocida

SEQ ID NO: 488


488
8.00E−93
1-deoxy-D-xylulose
Zymomonas
AAD29659.1
nt. 1344-1880 of




5-phosphate
mobilis

SEQ ID NO: 488




reductoisomerase


491
5.00E−51
rubredoxin
Clostridium
AAB50346.1
compliment of




oxidoreductase
acetobutylicum

nt. 1690-2439 of




homolog


SEQ ID NO: 491


492
1.00E−27
phosphotransferase
Staphylococcus
AAK83253.1
compliment of




system enzyme
aureus

nt. 755-970 of




IIA-like protein


SEQ ID NO: 492


493
2.00E−84
unknown
Actinobacillus
AAC70895.1
nt. 3333-3935 of





actinomycetemco

SEQ ID NO: 493





mitans


493
4.00E−49
unknown
Helicobacter
NP_223898.1
nt. 3345-4010 of





pylori J99

SEQ ID NO: 493


493
9.00E−31
transcriptional
Acinetobacter
AAF20290.1
nt. 1885-2793 of




factor MdcH
calcoaceticus

SEQ ID NO: 493


493
6.00E−30
HimA
Pasteurella
NP_245565.1
nt. 1129-1260 of





multocida

SEQ ID NO: 493


494
4.00E−85
putative prophage
Yersinia pestis
NP_404712.1
nt. 900-2099 of




integrase


SEQ ID NO: 494


494
4.00E−63
DNA
Xylella fastidiosa
NP_299063.1
compliment of




methyltransferase
9a5c

nt. 5544-6170 of







SEQ ID NO: 494


494
6.00E−19
MODIFICATION
Lactococcus lactis
P34877
compliment of




METHYLASE
subsp. cremoris

nt. 5019-6113 of




SCRFIA


SEQ ID NO: 494


497
0
transferrin-binding
Haemophilus
S70906
nt. 3251-4999 of




protein 1
influenzae (strain

SEQ ID NO: 497





PAK 12085)


50
5.00E−07
AcpP
Pasteurella
NP_246856.1
nt. 2-136 of





multocida

SEQ ID NO: 50


501
7.00E−50
conserved
Vibrio cholerae
NP_231403.1
compliment of




hypothetical protein


nt. 3649-4872 of







SEQ ID NO: 501


501
0
type I restriction
Vibrio cholerae
NP_231400.1
compliment of




enzyme HsdR,


nt. 1551-3440 of




putative


SEQ ID NO: 501


501
4.00E−13
ATP-dependent
Deinococcus
NP_295921.1
compliment of




DNA helicase
radiodurans

nt. 5317-5844 of




RecG-related


SEQ ID NO: 501




protein


501
5.00E−11
conserved
Ureaplasma
NP_077868.1
compliment of




hypothetical
urealyticum

nt. 5098-5769 of







SEQ ID NO: 501


504
2.00E−44
OUTER
Haemophilus
Q48218
compliment of




MEMBRANE
influenzae

nt. 4681-5019 of




PROTEIN P2
AG30010

SEQ ID NO: 504




PRECURSOR




(OMP P2)


507
0
SpoT
Pasteurella
NP_245857.1
compliment of





multocida

nt. 3685-5316 of







SEQ ID NO: 507


51
6.00E−87
glucosamine--
Vibrio cholerae
NP_230141.1
nt. 30-470 of




fructose-6-


SEQ ID NO: 51




phosphate




aminotransferase




(isomerizing)


512
2.00E−28
dipeptide transport
Yersinia pestis
NP_407439.1
compliment of




system permease


nt. 1095-1580 of




protein


SEQ ID NO: 512


512
3.00E−82
SapC
Pasteurella
NP_245850.1
compliment of





multocida

nt. 730-1095 of







SEQ ID NO: 512


514
9.00E−06
putative integral
Campylobacter
NP_281236.1
compliment of




membrane protein
jejuni

nt. 577-684 of







SEQ ID NO: 514


514
3.00E−11
orf, hypothetical
Escherichia coli
NP_286004.1
compliment of




protein
O157:H7 EDL933

nt. 449-568 of







SEQ ID NO: 514


518
0
putative inner
Neisseria
NP_284893.1
nt. 92-1927 of




membrane transacylase
meningitidis

SEQ ID NO: 518




protein
Z2491


519
4.00E−30
hypothetical protein
Mesorhizobium
NP_108196.1
compliment of





loti

nt. 2221-3159 of







SEQ ID NO: 519


519
2.00E−12
conserved
Listeria innocua
NP_471067.1
compliment of




hypothetical protein


nt. 3994-5241 of







SEQ ID NO: 519


519
6.00E−20
hypothetical protein
Mesorhizobium
NP_108198.1
compliment of





loti

nt. 707-1552 of







SEQ ID NO: 519


519
4.00E−26
putative
Salmonella
NP_455526.1
compliment of




bacteriophage
enterica subsp.

nt. 3982-5163 of




protein
enterica serovar

SEQ ID NO: 519





Typhi


52
3.00E−94
OUTER
Haemophilus
Q48218
nt. 45-788 of




MEMBRANE
influenzae

SEQ ID NO: 52




PROTEIN P2




PRECURSOR




(OMP P2)


520
0
excision nuclease
Escherichia coli
NP_418482.1
compliment of




subunit A
K12

nt. 6309-7745 of







SEQ ID NO: 520


521
5.00E−08
zinc/manganese
Rickettsia conorii
NP_359651.1
nt. 2236-2652 of




ABC transporter


SEQ ID NO: 521




substrate binding




protein


521
1.00E−140
unknown
Pasteurella
NP_245865.1|
nt. 338-1390 of





multocida

SEQ ID NO: 521


521
1.00E−86
ORF_f432
Escherichia coli
AAB40463.1
nt. 203-1390 of







SEQ ID NO: 521


522
3.00E−22
unknown
Pasteurella
NP_246093.1
nt. 670-885 of





multocida

SEQ ID NO: 522


526
5.00E−33
exodeoxyribonuclease
Yersinia pestis
NP_404635.1
nt. 5582-6202 of




V alpha chain


SEQ ID NO: 526


526
1.00E−62
exodeoxyribonuclease
Vibrio cholerae
NP_231950.1
nt. 5675-6193 of




V, 67 kDa


SEQ ID NO: 526




subunit


527
1.00E−147
unknown
Pasteurella
NP_245980.1
nt. 4283-5203 of





multocida

SEQ ID NO: 527


527
0
Mfd
Pasteurella
NP_245978.1
nt. 7545-8759 of





multocida

SEQ ID NO: 527


527
0
transcription-repair
Salmonella
NP_455708.1
nt. 7611-8762 of




coupling factor
enterica subsp.

SEQ ID NO: 527




(TrcF)
enterica serovar





Typhi


527
0
PROBABLE
Ralstonia
NP_519763.1
nt. 7611-8870 of




TRANSCRIPTION-
solanacearum

SEQ ID NO: 527




REPAIR




COUPLING




FACTOR




PROTEIN


528
1.00E−48
undecaprenyl
Chlamydia
NP_297109.1
nt. 2918-3712 of




pyrophosphate
muridarum

SEQ ID NO: 528




synthetase


528
0
leucyl-tRNA
Vibrio cholerae
NP_230603.1
compliment of




synthetase


nt. 180-2822 of







SEQ ID NO: 528


529
1.00E−104
DNA PRIMASE
Legionella
P71481
compliment of





pneumophila

nt. 3316-3960 of







SEQ ID NO: 529


534
9.00E−29
putative integrase
Salmonella
NP_461690.1
nt. 4668-5009 of





typhimurium LT2

SEQ ID NO: 534


534
6.00E−18
hypothetical protein
Neisseria
NP_283002.1
compliment of




NMA0153
meningitidis

nt. 5933-6337 of





Z2491

SEQ ID NO: 534


534
2.00E−23
hypothetical protein
Deinococcus
NP_294868.1
nt. 6908-7654 of





radiodurans

SEQ ID NO: 534


534
1.00E−88
prophage CP4-57
Escherichia coli
NP_417111.1
nt. 5057-5875 of




integrase
K12

SEQ ID NO: 534


535
1.00E−115
phosphate
Buchnera sp.
NP_240007.1
nt. 3385-4596 of




acetyltransferase
APS

SEQ ID NO: 535


536
3.00E−35
cobalt membrane
Actinobacillus
AAD49727.1
compliment of




transport protein
pleuropneumoniae

nt. 3531-4136 of




CbiQ


SEQ ID NO: 536


536
6.00E−37
unknown
Pasteurella
NP_245305.1
compliment of





multocida

nt. 6478-6921 of







SEQ ID NO: 536


539
2.00E−26
Orf122
Chlorobium
AAG12204.1
compliment of





tepidum

nt. 1778-2008 of







SEQ ID NO: 539


540
1.00E−77
heat shock protein
Neisseria
NP_273864.1
compliment of




HtpX
meningitidis

nt. 2567-3481 of





MC58

SEQ ID NO: 540


541
0
IleS
Pasteurella
NP_246601.1
nt. 3167-4549 of





multocida

SEQ ID NO: 541


545
2.00E−09
DNA-BINDING
Pectobacterium
Q47588
nt. 3816-3977 of




PROTEIN RDGB
carotovorum

SEQ ID NO: 545


545
2.00E−11
putative
Sinorhizobium
NP_437741.1
compliment of




transposase
meliloti

nt. 2786-3019 of







SEQ ID NO: 544


545
2.00E−07
Hypothetical 42.5 kd
Escherichia coli
BAA77933.1
compliment of




protein in thrW-


nt. 2614-2811 of




argF intergenic


SEQ ID NO: 545




region


545
4.00E−18
putative IS element
Salmonella
NP_454711.1
nt. 1955-2230 of




transposase
enterica subsp.

SEQ ID NO: 545





enterica serovar





Typhi


546
0
HEME/HEMOPEXIN-
Haemophilus
P45354
nt. 5551-7809 of




BINDING
influenzae

SEQ ID NO: 546




PROTEIN


546
0
HEME/HEMOPEXIN
Haemophilus
P45356
nt. 3842-5536 of




UTILIZATION
influenzae

SEQ ID NO: 546




PROTEIN B


546
0
HEME/HEMOPEXIN
Haemophilus
P45357
nt. 1638-3176 of




UTILIZATION
influenzae

SEQ ID NO: 546




PROTEIN C


546
2.00E−12
HasR
Pasteurella
NP_246561.1
nt. 3149-3763 of





multocida

SEQ ID NO: 546


549
0
unknown
Pasteurella
NP_246821.1
nt. 2526-3512 of





multocida

SEQ ID NO: 549


549
1.00E−121
putative membrane
Yersinia pestis
NP_404859.1
nt. 605-1108 of




protein


SEQ ID NO: 549


549
0
unknown
Pasteurella
NP_246822.1
nt. 1122-1664 of





multocida

SEQ ID NO: 549


551
1.00E−157
type I restriction-
Xylella fastidiosa
NP_300016.1
compliment of




modification
9a5c

nt. 7396-8322 of




system


SEQ ID NO: 551




endonuclease


552
1.00E−100
valyl-tRNA
Deinococcus
NP_293872.1
compliment of




synthetase
radiodurans

nt. 6691-8688 of







SEQ ID NO: 552


552
0
VALYL-TRNA
Haemophilus
P36432
compliment of




SYNTHETASE
parainfluenzae

nt. 5850-6647 of







SEQ ID NO: 552


553
0
DNA-directed RNA
Vibrio cholerae
NP_229982.1
nt. 2668-6699 of




polymerase, beta


SEQ ID NO: 553




subunit


554
0
iron utilization
Haemophilus
T10887
nt. 991-2508 of




protein B
influenzae

SEQ ID NO: 554


559
1.00E−100
PREPROTEIN
Bacillus firmus
P96313
nt. 3420-4472 of




TRANSLOCASE


SEQ ID NO: 559




SECA SUBUNIT


56
2.00E−23
RpL30
Pasteurella
NP_246336.1
compliment of





multocida

nt. 656-832 of







SEQ ID NO: 56


56
9.00E−13
RpS5
Pasteurella
NP_246337.1
compliment of





multocida

nt. 843-1064 of







SEQ ID NO: 56


560
1.00E−157
Na+/H+ antiporter
Vibrio cholerae
NP_231535.1
2 compliment of







nt. 279-2989 of







SEQ ID NO: 560


562
1.00E−72
putative biotin
Yersinia pestis
NP_404419.1
nt. 7862-8878 of




sulfoxide reductase 2


SEQ ID NO: 562


562
1.00E−125
restriction
Neisseria
CAA09003.1
nt. 2-985 of




modification
meningitidis

SEQ ID NO: 562




system-R protein


563
0
IMMUNOGLOBULIN
Haemophilus
P45384
compliment of




A1 PROTEASE
influenzae HK715

nt. 4127-9508 of







SEQ ID NO: 563


563
0
3-
Schizosaccharomyces
O14289
nt. 1980-3983 of




ISOPROPYLMALATE
pombe

SEQ ID NO: 563




DEHYDRATASE




(IPMI)


564
2.00E−79
orf32
Haemophilus
NP_536839.1
nt. 6241-6831 of





phage HP2

SEQ ID NO: 564


564
7.00E−33
probable variable
Salmonella
NP_457882.1
nt. 3707-4177 of




tail fibre protein
enterica subsp.

SEQ ID NO: 564





enterica serovar





Typhi


564
2.00E−14
M protein
Enterobacteria
NP_052264.1
nt. 1905-2213 of





phage 186

SEQ ID NO: 564


564
4.00E−44
similar to tail fiber
Salmonella
NP_461635.1
nt. 3171-3692 of




protein (gpH) in
typhimurium LT2,

SEQ ID NO: 564




phage P2
Fels-2 prophage


564
2.00E−85
gpJ
Enterobacteria
NP_046773.1
nt. 2267-3166 of





phage P2

SEQ ID NO: 564


564
1.00E−24
hypothetical protein
Neisseria
NP_284534.1
nt. 6852-7334 of





meningitidis

SEQ ID NO: 564





Z2491


564
4.00E−26
gpV
Enterobacteria
NP_046771.1
nt. 1337-1912 of





phage P2

SEQ ID NO: 564


564
2.00E−47
similar to
Escherichia coli
BAA16182.1
nt. 11383-11961




[SwissProt P44255


of SEQ ID NO:







564


564
2.00E−51
hypothetical protein
Neisseria
NP_284066.1
nt. 10452-11180




NMA1315
meningitidis

of SEQ ID NO:





Z2491

564


564
0
orf31
Haemophilus
NP_043502.1
nt. 4160-6226 of





phage HP1

SEQ ID NO: 564


564
2.00E−09
rep
Haemophilus
NP_536816.1
compliment of





phage HP2

nt. 9986-10234







of SEQ ID NO:







564


565
2.00E−57
resolvase/integrase-
Haemophilus
AAL47097.1
nt. 11885-12445




like protein
influenzae biotype

of SEQ ID NO:





aegyptius

565


565
1.00E−93
integrase
Actinobacillus
AAC70901.1
compliment of





actinomycetemco

nt. 4118-4900





mitans

of SEQ ID NO:







565


565
6.00E−35
probable phage
Salmonella
NP_458745.1
compliment of




integrase
enterica subsp.

nt. 4148-4990 of





enterica serovar

SEQ ID NO: 565





Typhi


565
1.00E−107
hypothetical protein
Xylella fastidiosa
NP_299042.1
compliment of





9a5c

nt. 5066-6817 of







SEQ ID NO: 565


566
1.00E−126
hypothetical protein
Haemophilus
S15287
compliment of




(galE 5′ region)-
influenzae

nt. 10726-11607







of SEQ ID NO:







566


567
0
unknown
Pasteurella
NP_246387.1
nt.5343-7688 of





multocida

SEQ ID NO: 567


568
1.00E−151
multidrug
Escherichia coli
NP_311575.1
nt. 6-1403 of




resistance
O157:H7

SEQ ID NO: 568




membrane




translocase


568
1.00E−141
YhbX/YhjW/YijP/YjdB
Neisseria
|NP_275002.1
compliment of




family protein
meningitidis

nt. 11213-12634





MC58

of SEQ ID NO:







568


570
1.00E−180
hypothetical protein
Haemophilus
S71024
compliment of




3 (ksgA-lic2B
influenzae (strain

nt. 12845-13720




intergenic region)
RM7004)

of SEQ ID NO:







570


571
0
glycerophosphodiester
Haemophilus
A43576
nt. 1656-2693 of




phosphodiesterase
influenzae (isolate

SEQ ID NO: 571





772)


571
1.00E−137
outer membrane
Haemophilus
A43604
nt. 6145-6909 of




protein P4
influenzae

SEQ ID NO: 571




precursor-




Haemophilus




influenzae


571
2.00E−72
CG8298 gene
Drosophila
AAF58597.1
nt. 3813-5339 of




product [alt 1]
melanogaster

SEQ ID NO: 571


572
1.00E−40
hypothetical protein
Chlamydia
G81737
nt. 3734-4099 of




TC0130
muridarum (strain

SEQ ID NO: 572





Nigg)


572
5.00E−10
hypothetical protein
Pyrococcus
NP_142215.1
nt. 4472-4888 of





horikoshii

SEQ ID NO: 572


572
3.00E−11
109aa long
Sulfolobus
NP_377117.1
nt. 7303-7470 of




hypothetical protein
tokodaii

SEQ ID NO: 572


572
8.00E−43
hypothetical protein
Chlamydophila
NP_445524.1
nt. 4289-4618 of





pneumoniae

SEQ ID NO: 572





AR39


572
9.00E−08
CDH1-D
Gallus gallus
AAL31950.1
nt. 7183-7521 of







SEQ ID NO: 572


575
1.00E−173
topoisomerase B
Salmonella
NP_458624.1
nt. 18980-20923





enterica subsp.

of SEQ ID NO:





enterica serovar

575





Typhi


575
1.00E−100
DNA helicase
Salmonella
NP_458617.1
nt. 10399-11706





enterica subsp.

of SEQ ID NO:





enterica serovar

575





Typhi


65
2.00E−53
Sufl
Pasteurella
NP_245041.1
nt. 3-821 of





multocida

SEQ ID NO: 65


67
4.00E−39
putative MFS
Salmonella
NP_462786.1
compliment of




family tranport
typhimurium LT2

nt. 125-1033 of




protein (1st mdule)


SEQ ID NO: 67


7
4.00E−29
putative membrane
Salmonella
NP_458664.1
compliment of




protein
enterica subsp.

nt. 2-559 of





enterica serovar

SEQ ID NO: 7





Typhi


72
2.00E−51
serine transporter
Vibrio cholerae
NP_230946.1
nt. 18-803 of







SEQ ID NO: 72


74
3.00E−90
hypothetical 21.8K
Haemophilus
JH0436
compliment of




protein (in locus
influenzae

nt. 248-766 of




involved in


SEQ ID NO: 74




transformation)-


77
2.00E−18
RecX protein
Legionella
CAC33485.1
nt. 480-920 of





pneumophila

SEQ ID NO: 77


82
4.00E−95
unknown
Pasteurella
NP_246414.1
nt. 128-955 of





multocida

SEQ ID NO: 82


83
2.00E−66
unknown
Pasteurella
NP_246777.1
nt. 5-556 of





multocida

SEQ ID NO: 83


83
6.00E−33
CTP SYNTHASE
Helicobacter
NP_223042.1
compliment of





pylori J99

nt. 1027-1338 of







SEQ ID NO: 83.


83
4.00E−34
CTP synthase
Campylobacter
NP_281249.1
compliment of





jejuni

nt. 1024-1275 of







SEQ ID NO: 83


84
1.00E−16
REPRESSOR
Bacteriophage
P14819
nt. 823-1233 of




PROTEIN CI
phi-80

SEQ ID NO: 84


84
2.00E−05
orf, hypothetical
Escherichia coli
NP_415875.1
compliment of




protein
K12

nt. 533-700 of







SEQ ID NO: 84


84
4.00E−11
orf33
bacteriophage phi
NP_490633.1
compliment of





CTX

nt. 32-466 of







SEQ ID NO: 84


85
3.00E−42
SpoT
Pasteurella
NP_245857.1
nt. 899-1261 of





multocida

SEQ ID NO: 85


90
1.00E−103
putative methylase
Bacteriophage
NP_108695.1
compliment of





Tuc2009

nt. 478-1206 of







SEQ ID NO: 90


90
4.00E−11
probable adenine
Thermoplasma
NP_394624.1
compliment of




specific DNA
acidophilum

nt. 397-1140 of




methyltransferase


SEQ ID NO: 90




















TABLE 3B






Full Length






Nucleotide
Amino Acid

Homology to Genbank


Hit Identity
Sequence
Sequence
Location in Contig
Protein







CpdB
SEQ ID NO:
SEQ ID NO:
nt. 38041-36068 of
NP_246953.1



686
687
SEQ ID NO: 681





(contig 14)


putative membrane
SEQ ID NO:
SEQ ID NO:
nt. 906601-908094
NP_458664.1


protein
688
689
of SEQ ID NO: 685





(contig 18)


GTP-binding
SEQ ID NO:
SEQ ID NO:
nt. 42557-40995 of
NP_240245.1


protein TypA/BipA
690
691
SEQ ID NO: 683





(contig 16)


outer membrane
SEQ ID NO:
SEQ ID NO:
nt. 7000420-704187
T30852


protein A
692
693
of SEQ ID





NO: 685 (contig 18)


vacB protein
SEQ ID NO:
SEQ ID NO:
nt. 39184-36836 of
NP_240369.1



694
695
SEQ ID NO: 683





(contig 16)


putative ABC
SEQ ID NO:
SEQ ID NO:
nt. 59155-58370 of
NP_282774.1


transport system
696
697
SEQ ID NO: 685


permease protein [


(contig 18)


putative exported
SEQ ID NO:
SEQ ID NO:
nt. 901142-902542
NP_458655.1


protein
698
699
of SEQ ID NO: 685





(contig 18)


ImpA
SEQ ID NO:
SEQ ID NO:
nt. 348187-347747
NP_245829.1



700
701
of SEQ ID NO: 685





(contig 18)


TsaA
SEQ ID NO:
SEQ ID NO:
nt. 74941-75548 of
NP_245732.1



702
703
SEQ ID NO: 684





(contig 17)


PROBABLE
SEQ ID NO:
SEQ ID NO:
nt. 74436-75176 of
NP_522358.1


TRANSPORT
704
705
SEQ ID NO: 685


TRANSMEMBRANE


(contig 18)


PROTEIN
SEQ ID NO:
SEQ ID NO.
nt. 75160-75660 of



706
707
SEQ ID NO: 685





(contig 18)


possible exported
SEQ ID NO:
SEQ ID NO:
nt. 899618-900262
NP_458653.1


protein
708
709
of SEQ ID NO: 685





(contig 18)


LICA PROTEIN
SEQ ID NO:
SEQ ID NO:
nt. 356917-355958
P14181



710
711
of SEQ ID NO: 685





(contig 18)


HEME-BINDING
SEQ ID NO:
SEQ ID NO:
NT. 26114-27739
P33950


PROTEIN A
712
713
of SEQ ID NO: 683





(contig 16)


similar to
SEQ ID NO:
SEQ ID NO:
nt. 311610-312683
XP_068727.1


BASEMENT
714
715
of SEQ ID NO: 685


MEMBRANE-


(contig 18)


SPECIFIC


HEPARAN


SULFATE


PROTEOGLYCAN


CORE PROTEIN


PRECURSOR


(HSPG)


CzcD
SEQ ID NO:
SEQ ID NO:
nt. 34865-35542 of
NP_246276.1



716
717
SEQ ID NO: 681





(contig 14)


conserved
SEQ ID NO:
SEQ ID NO:
nt. 194993-193977
NP_274972.1


hypothetical protein
718
719
of SEQ ID NO: 685





(contig 18)


secretion protein
SEQ ID NO:
SEQ ID NO:
nt. 203707-201857
NP_252510.1


SecD
720
721
of SEQ ID NO: 683





(contig 17)


ABC transporter
SEQ ID NO:
SEQ ID NO:
nt. 3943-5859 of
AAF31030.1


protein 1
722
723
SEQ ID NO: 681





(contig 14)


conserved
SEQ ID NO:
SEQ ID NO:
nt. 331090-331749
NP_273467.1


hypothetical protein
724
725
of SEQ ID NO: 685





(contig 18)



SEQ ID NO:
SEQ ID NO:
nt. 331938-332492



726
727
of SEQ ID NO: 685





(contig 18)



SEQ ID NO:
SEQ ID NO:
nt. 332681-33232



728
729
of SEQ ID NO: 685





(contig 18)


INVASIN
SEQ ID NO:
SEQ ID NO:
nt. 416757-417020
P31489


PRECURSOR
730
731
of SEQ ID NO: 685


(OUTER


(contig 18)


MEMBRANE


ADHESIN)


HEME/HEMOPEXIN-
SEQ ID NO:
SEQ ID NO:
nt. 229430-232195
P45355


BINDING
732
733
of SEQ ID NO: 384


PROTEIN


(contig 17)


OPACITY
SEQ ID NO:
SEQ ID NO:
nt. 375592-375879
Q05033


PROTEIN OPA66
734
735
of SEQ ID NO: 384


PRECURSOR


(contig 17)


Hemoglobin-
SEQ ID NO:
SEQ ID NO:
nt. 45709-42566 of
Q48153


haptoglobin binding
736
737
SEQ ID NO: 681


protein A


(contig 14)


transport protein
SEQ ID NO:
SEQ ID NO:
nt. 134452-135222
NP_253757.1


TatC
738
739
of SEQ ID NO: 384





(contig 17)


LIPOPROTEIN
SEQ ID NO:
SEQ ID NO:
nt. 18895-20112 of
P40827


NLPD
740
741
SEQ ID NO: 682





(contig 15)


Hemoglobin and
SEQ ID NO:
SEQ ID NO:
nt. 34181-31041 of
Q9X442


hemoglobin-
742
743
SEQ ID NO: 682


haptoglobin binding


(contig 15)


protein C precursor


HimA
SEQ ID NO:
SEQ ID NO:
nt. 382795-383085
NP_245565.1



744
745
of SEQ ID NO: 685





(contig 18)


transferrin-binding
SEQ ID NO:
SEQ ID NO:
nt. 178537-175799
S70906


protein 1
746
747
of SEQ ID NO: 683





(contig 16)


SapC
SEQ ID NO:
SEQ ID NO:
nt. 197754-196867
NP_245850.1



748
749
of SEQ ID NO: 685





(contig 18)


heat shock protein
SEQ ID NO:
SEQ ID NO:
nt. 40414-41265 of
NP_273864.1


HtpX
750
751
SEQ ID NO: 682





(contig 15)


HEME/HEMOPEXIN-
SEQ ID NO:
SEQ ID NO:
nt. 229430-232195
P45354


BINDING
752
753
of SEQ ID NO: 684


PROTEIN


(contig 17)


HEME/HEMOPEXIN
SEQ ID NO:
SEQ ID NO:
nt. 227721-229418
P45356


UTILIZATION
754
755
of SEQ ID NO: 684


PROTEIN B


(contig 17)


HEME/HEMOPEXIN
SEQ ID NO:
SEQ ID NO:
nt. 225516-227645
P45357


UTILIZATION
756
757
of SEQ ID NO: 684
NP_246561.1


PROTEIN C


(contig 17)


iron utilization
SEQ ID NO:
SEQ ID NO:
nt. 32076-33611 of
T10887


protein B
758
759
SEQ ID NO: 684





(contig 17)


PREPROTEIN
SEQ ID NO:
SEQ ID NO:
nt. 82314-84785 of
P96313


TRANSLOCASE
760
761
SEQ ID NO: 683


SECA SUBUNIT


(contig 16)


IMMUNOGLOBULIN
SEQ ID NO:
SEQ ID NO:
nt. 171647-166263
P45384


A1 PROTEASE
762
763
of SEQ ID NO: 683





(contig 16)


multidrug
SEQ ID NO:
SEQ ID NO:
nt. 74524-72992 of
NP_311575.1


resistance
764
765
SEQ ID NO: 683


membrane


(contig 16)


translocase


YhbX/YhjW/YijP/YjdB
SEQ ID NO:
SEQ ID NO:
nt. 61734-63200 of
NP_275002.1


family protein
766
767
SEQ ID NO: 683





(contig 16)


putative membrane
SEQ ID NO:
SEQ ID NO:
nt. 906601-908094
NP_458664.1


protein
768
769
of SEQ ID NO: 685





(contig 18)


putative membrane
SEQ ID NO:
SEQ ID NO:
nt. 16185-17942 of
NP_404859.1


protein
770
771
SEQ ID NO: 683





(contig)









EXAMPLE 3
Construction of the NTHi Promoter Trap Library
To identify potential virulence determinants of NTHi, bacterial gene expression was monitored by differential fluorescence induction (DFI) during early disease progression in one specific anatomical niche of a chinchilla model of NTHi-induced otitis media (OM). Genomic DNA fragments from NTHi strain 86-028NP were cloned upstream of the promoterless gfpmut3 gene using a promoter trap library. Plasmid pGZRS39A, a derivative of pGZRS-1 isolated from Actinobacillus pleuropneumoniae, is an A. pleuropneumoniae-Escherichia coli shuttle vector. This plasmid contains the origin of replication from A. pleuropneumoniae, the lacZa gene from pUC19 and the kanamycin resistance gene from Tn903. (West et al., Genes, 160: 81-86, 1995).
The promoter trap vector was constructed by cloning the GTP mutant gfpmut3 gene, as a BamHI to EcoRI fragment into pGZRS-39A to form pRSM2167. This mutant GTP gene contains two amino acid changes, S65G and S72A, that enhance fluorescence emission when excited at 488 nm. This mutant also has high solubility and fast kinetics of chromophore formation (Cormack et al., Gene, 173: 33-38, 1996). This plasmid was transformed by electroporation into NTHi strain 86-028NP, generating the parent-plasmid strain 86-028NP/pRSM2169.
Random genomic DNA fragments (described in Example 1) were prepared for ligation into the promoter probe vector. Genomic DNA was isolated from strain 86-028NP using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, Minn.) according to the manufacturer's protocol. Due to restriction barriers, it was necessary to isolate the plasmid DNA and use this for the library generation. The isolated DNA was partially digested with Sau3AI (NEB, Beverly, Mass.; 0.25 units/μg DNA) for 1 hour at 37° C., separated by gel electrophoresis and DNA fragments 0.5-1.5 kb in size were recovered using the Qiagen gel extraction kit. For vector preparation, pRSM2167 was isolated from an overnight culture using the Wizard Plus Maxiprep DNA purification system (Promega, Madison Wis.) according to the manufacturer's protocol.
Plasmid DNA was linearized by BamHI digestion and 5′ phosphate groups removed by treatment with calf intestinal alkaline phosphatase (CIAP; GibcoBRL Life Technologies). Genomic DNA fragments were ligated with the linearized, phosphatase-treated vector and electroporated into competent NTHi strain 86-028NP prepared for electroporation according to a modified protocol (Mitchell et al., Nucleic Acids Res., 19: 3625-3628, 1991). When plasmid DNA was electroporated back into NTHi strain 86-028NP, transformation efficiency was improved by one-thousand fold. Briefly, cells were grown to an OD600=0.3 in sBHI (brain heart infusion) broth at 37° C., 220 rpm. Cells were chilled on ice for 30 minutes and subsequently washed with an equal volume of 0.5×SG (1×SG: 15% glycerol, 272 mM sucrose) at 4° C. Washes were repeated a total of three times. Subsequently, the cells were diluted in 1×SG to a 100× concentrated volume. The cells were electroporated using the BioRad Gene Pulser II set at 200 ohms, 2.5 kV and 25 μF and then diluted in 1 ml prewarmed sBHI, incubated for 2 hours at 37° C., 5% CO2 and plated on chocolate agar for overnight growth of transformants.
Transformants were selected and frozen in pools of 1000 clones in skim milk containing 20% glycerol (vol/vol). A 68,000 member gfp promoter probe library was generated. Using the probability calculation of Clarke and Carbon (Cell, 9: 91-99, 1976), to achieve a 99% probability of having a given DNA sequence represented in a library of 300 bp fragments of strain 86-028NP DNA (1.8×106 bp/genome), a library of 27,629 clones was needed. Therefore the present library represents 2.5 fold coverage of the 86-028NP genome.
In order to assess the quality of the library, fifty clones were selected at random, grown overnight on chocolate agar and the plasmids were isolated and insert DNA sequenced. A majority (64%) of the selected clones had insert sizes ranging between 200 and 500 bp while 32% exceeded 500 bp. The majority of inserts showed homology to unique H. influenzae strain Rd open reading frames (ORFs), and 15 clones had sequence unique to strain 86-028NP DNA. Of those clones with homology to strain Rd, 60% were in the correct orientation, 36% of which contained sequence upstream an ORF. Although a majority of clones had an insert size less than 500 bp, no correlation was found between small insert size and increased GFP expression. In fact four clones exhibited slight to moderate fluorescence in vitro, 3 of which had insert sizes between 200-500 base pairs and one had an insert that was greater than 700 base pairs.
A fraction of the library (approximately 1000 clones) was grown on chocolate agar, harvested in PBS and analyzed by flow cytometry for GFP fluorescence. Compared to strain 86-028NP/pRSM2169 that contains the promoter trap vector without insert DNA, the pool of library clones displays an increased fluorescence intensity. Thus, the library contains clones with promoters at varying levels of activity.
EXAMPLE 4
Analysis of 86-028NP Derivatives Expressing GFP
In order to establish the FACS parameters necessary to identify and sort gfp-expressing bacteria, a panel of isolates demonstrating varying levels of gfp expression was utilized. Background fluorescence was assessed using strain 86-028NP/pRSM2169 (negative control), therefore any observed fluorescence would be due to the lacZ promoter driving gfp expression. However, this strain does not produce detectable levels of GFP and in fact, does not demonstrate increased fluorescence when compared to the parent strain 86-028NP. A high-level gfp-expressing isolate was generated by cloning a 500 bp fragment containing the strong promoter for outer membrane protein P2 expression into SalI-BamHI digested pRSM2167. This plasmid was transformed into 86-028NP by electroporation, generating the high-level gfp expressing strain 86-028NP/pRSM2211 (highly fluorescent control). This strain demonstrated an approximate 100 fold increase in GFP fluorescence compared to strain 86-028NP/pRSM2169. An intermediate fluorescent derivative clone, 86-028NP/pKMM4B5 (intermediate fluorescent control), was isolated by FACS analysis and used both in preliminary experiments and as a control for cell sorting. The DNA fragment containing a promoter driving gfp expression in vitro is unique to strain 86-028NP, having no known homology to DNA of other organisms. This clone exhibits an approximate 10 fold increase in fluorescence compared to strain 86-028NP/pRSM2169.
The control strains were resuspended from growth on chocolate agar and labeled with cross-reactive Phycoprobe R-PE anti-human IgG (H=L) antibody (10μg/ml in 100 μl PBS; Biomeda Corp) for 30 minutes at 40° C. Following three successive washes to remove unbound antibody, bacteria were resuspended in 300 μl Dulbecco's Phosphate Buffered Saline (DPBS) for FACS analysis. These control preparations were used to set the appropriate size and fluorescence gates using a Coulter Epics Elite flow cytometer (Coulter Corp.) equipped with an argon laser emitting at 488 nm. Bacteria were gated for size based on log forward angle and side scatter detection and for sorting by FITC/PE labeling of bacteria. Sorted cells were collected into cold sBHI and plated on chocolate agar. After overnight growth, cells were collected for a secondary round of infection or were individually selected and grown overnight, screened by individual clone for fluorescence when grown in vitro, and frozen in skim milk containing 20% (vol/vol) glycerol prior to plasmid isolation and sequencing of insert DNA. Sorting efficiency of control strains was confirmed using a Coulter EPICS flow cytometer (Coulter Corp.).
Many plasmids were segregated rapidly in vitro in the absence of antibiotic selection. Thus, in order to assess whether the promoter trap vector used here was prone to this event, a single colony of strain 86-028NP/pRSM2211 (highly fluorescent control) was isolated on chocolate agar and passaged 20 times in the absence of antibiotic selection. No significant decrease in fluorescence intensity was observed when compared to bacteria grown in the presence of antibiotic. In addition, the plasmid is maintained in the absence of antibiotic selection in vivo. Similar bacterial counts were observed when bacteria-containing middle ear fluids collected from a chinchilla were plated on chocolate agar with or without kanamycin. These data demonstrate that the promoter trap vector was stably maintained in the absence of antibiotic selection.
In addition to problems with plasmid stability, early studies on the use of GFP as a reporter to study host-pathogen interactions demonstrated that GFP could be continuously synthesized as a cytoplasmic protein with low toxicity, having minimal effects on the bacterial cell-surface dynamics (Chalfie et al., Science, 263: 802-805, 1994). The construction of a high level gfp-expressing derivative allowed the assessment of the GFP toxicity on NTHi. Growth curves of both the wild-type strain (86-028NP) and the high GFP producing strain 86-028NP/pRSM2211 were compared when grown under similar conditions. The growth rates were similar, indicating that GFP expression was not toxic to the cells.
The 86-028NP gfp-expressing derivatives were used to define the parameters for efficient cell sorting. Strain 86-028NP/pRSM2169 was mixed with the intermediate gfp-expressing derivative, strain 86-028NP/pKMM4B5, at a 100:1 ratio, simulating the in vivo environment that is expected to contain a small percentage of gfp-expressing clones relative to the total bacterial population. This mixture was subjected to FACS analysis, collecting the 1.8% most fluorescent population and the 52% least fluorescent population. Flow cytometric analysis of the sorted populations revealed an enrichment of strain 86-028NP/pKMM4B5 to 65% of the bacterial population, a phenomenon that was not observed when sorting on the negative population. Subsequent rounds of sorting would be expected to further enrich for this intermediate fluorescent population. The inability to decrease the amount of fluorescent bacteria in the negative sort was attributed to the size of the gate set for negative sorting. GFP-negative cells were enriched by gating on the 10% least fluorescent population.
EXAMPLE 5
Direct Labeling of Bacteria from Middle Ear Fluids
A similar strategy (as described in Example 5) was applied to sort fluorescent clones from effusions obtained from the chinchilla middle ear during AOM. Our ability to use differential fluorescence induction (DFI) in vivo was dependent upon our ability to sort gfp-expressing bacteria from non-fluorescent bacteria, fluorescent and non-fluorescent cellular debris, and eukaryotic cells.
Healthy adult chinchillas (Chinchilla lanigera) with no evidence of middle ear infection by either otoscopy or tympanometry were used to screen the library for promoter activity in vivo. Two pools of the NTHi/pRSM2169 library (1000 clones each) were grown overnight on chocolate agar containing kanamycin. The library was combined and diluted in cold 10 mM sterile PBS to 3.3×106 CFU/ml and 300 μl (1.0×106 CFU; 500 CFU/clone) was used to inoculate the left and the right chinchilla transbullar cavity (2000 clones/ear). OM development was monitored by video otoscopy and tympanometry at 24 and 48 hours. The bacteria multiplied in the middle ear cavity, reaching a concentration 500 times the inoculum dose by 48 hours as expected (Bakaletz et al., Infect. Immunity 7: 2746-62, 1999). This bacterial adaptation to the host environment results in an inflammatory response, indicated by erythema, vessel dilation and bulging of the tympanic membrane, infiltration of polymorphonuclear cells (PMN's), and accumulation of fluid in the middle ear cavity as observed by otoscopy and microscopic examination of recovered effusions. Twenty-four and 48 hours later, middle ear fluids were retrieved by epitympanic tap, and prepared for FACS.
It is important to note that this analysis was limited to those bacteria recoverable in the middle ear fluid. In some cases it was necessary to lavage the middle ear cavity to collect the bacteria for FACS analysis. Thus, this analysis includes genes up-regulated when NTHi are loosely adherent to mucosae. NTHi has been observed to form a biofilm in the middle ear cavity in a chinchilla model of OM (Erhlich et al., JAMA, 287: 1710-5, 2002). Since the protocols described herein select for clones recovered from the planktonic population, it is not expected to recover those clones in which genes are up-regulated when the bacteria are associated with mucosal biofilms. Homogenization of middle ear mucosae and subsequent bacterial cell isolation however, would enable us to recover these clones. It is also possible that some GFP-expressing clones were recovered in the effusion, yet were adherent to eukaryotic cells present in the effusion as exfoliated cells, or in aggregates. These bacteria are difficult to recover from the effusion without compromising the sorting efficiency. Therefore the middle ear fluids were treated with a mucolytic agent, then centrifuged to remove large aggregates and eukaryotic cells and prior to labeling.
Chinchilla middle ear fluids were diluted, if necessary, to 250 μl with sterile saline. An equal volume of N-acetyl-L-cysteine (0.5%; w/v) in DPBS (pH 7.4) was added for 5 minutes at room temperature as a mucolytic agent (Miyamoto and Bakaletz, Microb. Pathog., 21: 343-356 1996). Fluids were centrifuged (300×g, 5 min) to remove cellular debris, red blood cells and inflammatory cells, and supernatants containing bacteria were transferred to a fresh tube. Bacteria were incubated with chinchilla antiserum (1:50 dilution) directed against a whole OMP preparation, derived from NTHi strain 86-028NP, for 45 minutes at 4° C., pelleted by centrifugation (2000×g, 5 min) and washed twice with cold DPBS containing 0.05% bovine serum albumin. Bacteria were subsequently labeled with cross-reactive phycoprobe R-PE anti-human IgG (H+L) antibody (10 μ/ml in 100 μl PBS; Biomeda Corp) for 30 minutes at 4° C. Following three successive washes to remove unbound antibody, cells were resuspended in 300 μl DPBS for FACS analysis.
EXAMPLE 6
Identification of Promoters Induced in Vivo in Acute Otitis Media
H. influenzae 86-028NP transformed with the promoter trap library was grown overnight on chocolate agar. To select against those clones containing promoters that expressed gfp in vitro, the library was subjected to one round of FACS analysis (as described in Example 6), collecting only those clones expressing low-level amounts of GFP. These clones were pooled and used to inoculate the chinchilla middle ear transbullarly. Following 24 and 48 hours of infection, bacteria-containing effusions were removed by epitympanic tap. Bacteria were indirectly labeled with R-PE-labeled antibody and subjected to FACS analysis by gating on fluorescently tagged bacteria but sorting for those that were also expressing. These clones were used to reinfect animals for further enrichment. Following the final round of sorting, single colony isolates were screened in vitro for lack of fluorescence.
Those clones isolated by FACS analysis (positive for GFP fluorescence in vivo), which did not emit fluorescence in vitro were prepared for plasmid isolation and identification of insert DNA sequence. These clones were grown overnight on chocolate agar plates containing kanamycin and prepared for plasmid isolation using the Qiaprep Miniprep Kit (Qiagen) according to the manufacturer's protocol. Plasmid insert DNA was sequenced using the primer 5′-TGCCCATTAACATCACCATCTA-3′ (SEQ ID NO: 588) that is complementary to the gfpmut3 gene and downstream of the insert DNA. Sequencing reactions were performed using the ABI prism BigDye® terminator cycle sequencing ready reaction kit (Applied Biosystems) according to manufacturer's protocol using a GeneAmp PCR System 9700 (Applied Biosystems). The sequences were then purified by passage through sephadex G-50 in a 96-well multiscreen HV plate (Millipore) and subsequently analyzed on an ABI Prism 3100 DNA analyzer (Applied Biosystems).
Insert sequences were compared to the complete annotated sequence of H. influenzae strain Rd. Those inserts with no nucleotide homology to strain Rd were subsequently analyzed using the BLASTN and BLASTX algorithms. Further sequence analysis was performed with DNASTAR (Madison, Wis.). Inserts in the correct orientation and containing sequence 5′ to a predicted ORF contained a putative promoter that was preferentially active when the NTHi bacteria were in the chinchilla middle ear.
Fifty-two clones with putative promoters that were regulated in vivo were isolated. Of the 44 candidate clones containing sequence similar to that identified in H. influenzae strain Rd, quantitative comparison of gene expression in vitro and in vivo confirmed up-regulated gene expression for twenty-six genes (60%) when NTHi respond to environmental cues present in the chinchilla middle ear and these genes are summarized in Table 4A below. The in vivo-regulated promoters driving expression of genes are predicted to be involved in membrane transport, environmental informational processing, cellular metabolism, gene regulation, as well as hypothetical proteins with unknown function.
In order to confirm the induction of putative promoter candidates in vivo, the relative amount of messenger RNA expression was compared when NTHi strain 86-028NP was grown in vitro to mid-log phase or in vivo for 48 hours. The RNA was isolated using TRIzol LS reagent (Gibco Life Technologies) according to the manufacturer's protocol. DNA was removed from the RNA preparation using DNA-free kit (Ambion) according to the manufacturer's protocol. DNase I treated RNA samples were purified by passage through a Qiagen RNeasy column. RNA purity and integrity was assessed by 260/280 nm spectrophotometer readings and on the Agilent 2100 Bioanalyzer (Agilent Technologies), respectively.
In order to independently confirm the FACS data, we determined the relative expression of candidate genes by quantitative RT-PCR. The parent strain 86-028NP, was used for these studies. Real-time quantitative RT-PCR using the one-step QuantiTect SYBR Green RT-PCR kit (Qiagen) assessed transcription levels according to the manufacture's instructions. Briefly, using primers generated to an open reading frame downstream of the putative in vivo-induced promoters identified by FACS analysis, gene-specific mRNA was reverse transcribed and amplified by RT-PCR on the ABI Prism 7700 sequence detection system (Applied Biosystems). The amount of product was calculated using a standard curve generated to known amounts of bacterial genomic DNA (102-107 genomic copies DNA) by amplifying a fragment of the gyrase (gyr) gene. Controls were analyzed in parallel to verify the absence of DNA in the RNA preparation (−RT control) as well as the absence of primer dimers in control samples lacking template RNA. In addition, RT-PCR products were analyzed by gel electrophoresis and, in all cases, a single product was observed at the appropriate base pair size. Amounts of bacterial RNA between samples were normalized relative to gyr expression, shown to be constitutively expressed under various growth conditions that we tested in vitro. Known amounts of bacterial genomic DNA (102-107 genomic copies DNA) were used to generate a standard curve for RT-PCR quantitation by amplifying a fragment of the gyrase (gyr) gene. Gyrase is constitutively expressed in vitro under various growth conditions and was therefore used to normalize total bacterial RNA levels between samples. Relative gene expression in vivo was compared to that of gene expression in vitro and data expressed as fold-increase are summarized in Table 4A.
The 8-fold sequencing of the NTHi genome identified the full length open reading frames for the majority of genes listed in Table 4A. Table 4B provides the full length nucleotide sequence within the NTHi genome and the corresponding amino acid sequence. The fold induction of the gene due to environmental cues present in the chinchilla middle ear and the product or function of the gene are repeated in Table 4B for convenience.












TABLE 4A







SEQ






Gene or
ID
GenBank
Fold


Category
ORF
NO:
Protein ID
Induction
Product or Function




















Amino acid
hisB
589
NP_438632
2.9
Histidine biosynthesis


metabolism




bifunctional protein


Lipoprotein
lppB
590
NP_438862.1
2.6
Lipoprotein B homologue


Membrane transport
sapA
591
NP_439780.1
2.8
Peptide ABC transporter;







periplasmic SapA precursor



lolA
592
NP_439736.1
2.4
Outer membrane lipoproteins







carrier protein precursor



rbsC
593
NP_438661.1
5.1
Ribose transport system







permease protein


Purine synthesis
purE
594
NP_439757.1
51.7
Phosphoribosylaminoimidazole







carboxylase catalytic







subunit; PurE


Biosynthetic and
ribB
595
NP_438923.1
8.3
3,4-dihydroxy-2-butanone 4-


metabolic functions




phosphate synthase;







riboflavin biosynthesis



arcB
596
NP_438753.1
10
Ornithine







carbamolytransferase;







arginine degradation



uxuA
597
NP_438228.1
3.1
Mannonate dehydratase;







production of glyceraldehyde







3-phosphate



dsbB
598
NP_438589.1
2.6
Disulfide oxidoreductase;







disulfide bond formation







protein B



ureH
599
NP_438693.1
3.9
Urease accessory protein



licC
600
NP_439688.1
2.3
Phosphocholine (ChoP)







cytidylyltransferase



HI1647
601
NP_439789.1
2.0
Putative pyridoxin







biosynthesis protein; singlet







oxygen resistance protein


DNA replication,
ispZ
602
P43810
2.5
Probable intracellular


repair




septation protein



radC
603
NP_439113.1
2.1
DNA repair protein



mukF
604
P45185
2.0
MukF protein homologue;







remodeling of nucleiod







structure


Gene regulation
glpR
605
NP_438777.1,
2.8
Glycerol-3-phosphate regulon





NP_439170.1

repressor



ihfB
606
P43724
2.5
Integration host factor beta







subunit



argR
607
NP_439365.1
2.7
Arginine repressor



cspD
608
NP_439584.1
2.1
Cold shock like protein;







stress response protein


Hypothetical or
HI0094
609
NP_438267.1
8.3
Hypothetical protein


unknown
HI1163
610
NP_439321.1
2.3
Conserved hypothetical


proteins




protein; putative oxidase



HI1063
611
NP_439221.1
2.7
Hypothetical protein



HI0665
612
NP_438824.1
2.8
Hypothetical protein



HI1292
613
NP_439444.1
2.6
Hypothetical protein



HI1064
614
NP_439222.1
2.6
Hypothetical protein






















TABLE 4B







Full







Gene
Length



or
Nucleotide
Amino Acid

Fold
Product or


Category
ORF
Sequence
Sequence
Location in Contig
Induction
Function





















Amino
hisB
SEQ ID NO:
SEQ ID NO:
nt. 68378-67290
2.9
Histidine


acid

615
616
of SEQ ID NO:

biosynthesis


metabolism



680 (contig 13)

bifunctional protein


Membrane
sapA
SEQ ID NO:
SEQ ID NO:
nt. 200403-198709
2.8
Peptide ABC


transport

617
618
of SEQ

transporter;






ID NO: 685

periplasmic SapA






(contig 18)

precursor



rbsC
SEQ ID NO:
SEQ ID NO:
nt. 42773-41802
5.1
Ribose transport




619
620
of SEQ ID NO:

system permease






680 (contig 13)

protein


Purine
purE
SEQ ID NO:
SEQ ID NO:
nt. 219625-219131
51.7
Phosphoribosylaminoimidazole


synthesis

621
622
of SEQ

carboxylase catalytic






ID NO: 685

subunit; PurE






(contig 18)


Biosynthetic
ribB
SEQ ID NO:
SEQ ID NO:
nt. 131537-132184
8.3
3,4-dihydroxy-2-


and

623
624
of SEQ

butanone 4-


metabolic



ID NO: 682

phosphate synthase;


functions



(contig 15)

riboflavin








biosynthesis



arcB
SEQ ID NO.
SEQ ID NO:
nt. 49710-48706
10
Ornithine




625
626
of SEQ ID NO:

carbamolytransferase;






681 (contig 14)

arginine








degradation



uxuA
SEQ ID NO:
SEQ ID NO:
nt. 840671-841855
3.1
Mannonate




627
628
of SEQ

dehydratase;






ID NO: 685

production of






(contig 18)

glyceraldehyde 3-








phosphate



dsbB
SEQ ID NO:
SEQ ID NO:
nt. 388050-388583
2.6
Disulfide




629
630
of SEQ

oxidoreductase;






ID NO: 384

disulfide bond






(contig 17)

formation protein B



ureH
SEQ ID NO:
SEQ ID NO:
nt. 4452-5267 of
3.9
Urease accessory




631
632
SEQ ID NO: 680

protein






(contig 13)



licC
SEQ ID NO:
SEQ ID NO:
nt. 355083-354382
2.3
Phosphocholine




633
634
of SEQ

(ChoP)






ID NO: 385

cytidylyltransferase






(contig 18)



HI1647
SEQ ID NO:
SEQ ID NO:
nt. 664017-664892
2.0
Putative pyridoxin




635
636
of SEQ

biosynthesis protein;






ID NO: 685

singlet oxygen






(contig 18)

resistance protein


DNA
ispZ
SEQ ID NO:
SEQ ID NO:
nt. 4512-5069 of
2.5
Probable


replication,

637
638
SEQ ID NO: 683

intracellular


repair



(contig 16)

septation protein



radC
SEQ ID NO:
SEQ ID NO:
nt. 132695-132030
2.1
DNA repair protein




639
640
of SEQ






ID NO: 683






(contig 16)



mukF
SEQ ID NO:
SEO ID NO:
nt. 504549-503215
2.0
MukF protein




641
642
of SEQ

homologue;






ID NO: 685

remodeling of






(contig 18)

nucleiod structure


Gene
glpR
SEQ ID NO:
SEQ ID NO:
nt. 72716-73483
2.8
Glycerol-3-


regulation

643
644
of SEQ ID NO:

phosphate regulon






682 (contig 15)

repressor



ihfB
SEQ ID NO:
SEQ ID NO:
nt. 661004-660720
2.5
Integration host




645
646
of SEQ

factor beta subunit






ID NO: 685






(contig 18)



argR
SEQ ID NO:
SEQ ID NO:
nt. 178540-178085
2.7
Arginine repressor




647
648
of SEQ






ID NO: 685






(contig 18)



cspD
SEQ ID NO:
SEQ ID NO:
nt. 435310-435528
2.1
Cold shock like




649
650
of SEQ

protein; stress






ID NO: 685

response protein






(contig 18)


Hypothetical
HI1163
SEQ ID NO:
SEQ ID NO:
nt. 137202-134119
2.3
Conserved


or

651
652
of SEQ

hypothetical protein;


unknown



ID NO: 685

putative oxidase


proteins



(contig 18)



HI1063
SEQ ID NO:
SEQ ID NO:
nt. 35158-34937
2.7
Hypothetical protein




653
654
of SEQ ID NO:






685 (contig 18)



HI0665
SEQ ID NO:
SEQ ID NO:
nt. 17949-18980
2.8
Hypothetical protein




655
656
of SEQ ID NO:






679 (contig 12)



HI1292
SEQ ID NO:
SEQ ID NO:
nt. 555002-555799
2.6
Hypothetical protein




657
658
of SEQ






ID NO: 685






(contig 18)









EXAMPLE 7
Identification of Virulence-Associated Genes
In many bacterial species, a subset of virulence-associated genes is regulated by errors in replication of short repeats. These repeats may be 5′ to a gene or in the coding sequence, and their presence is an indication of controlled expression of the gene, which indicates association with virulence. Addition or deletion of a repeat results in the expression or of lack of expression of the particular virulence determinant.
The NTHi H. influenzae strain 86-028NP contig set was queried for short oligonucleotide repeats. The region surrounding the repeats was analyzed to identify the gene(s) associated with the repeat. Table 5 lists the identified repeats and the ORF (identified by BLAST) associated with each repeat.
Further sequence analysis has identified the full length nucleotide sequence of the virulence-associated genes and the corresponding amino acid sequences encoded by the ORF. The derived amino acid sequences are highly homologous to the listed Genbank sequence.












TABLE 5






Location in
Location in
Full Length
Amino




3-fold
8-fold
Nucleotide
Acid
Genebank


Repeat
Contigs
Contigs
Sequence
Sequence
Accession No.







SEQ ID
115
nt. 484533-483643
SEQ ID
SEQ ID
NP_439538.1


NO: 581
nt. 473-540
of
NO: 659
NO: 660



of
SEQ ID



SEQ ID
NO: 685



NO: 115
(contig 18)


SEQ ID
377
nt. 416274-414910
SEQ ID
SEQ ID
P45217


NO: 582
nt. 546-597
of
NO: 661
NO: 662



of
SEQ ID NO:



SEQ ID
685 (contig



NO: 337
18)


SEQ ID
505
nt. 414500-416614
SEQ ID
SEQ ID
AAK76425


NO: 583
nt. 310-393
of
NO: 663
NO: 664



of
SEQ ID NO:



SEQ ID
684 (contig



NO: 505
17)


SEQ ID
508
nt. 506516-507913
SEQ ID
SEQ ID
NP_439520


NO: 584
nt. 2079-2120
of
NO: 665
NO: 666



of
SEQ ID NO:



SEQ ID
685 (contig



NO: 508
18)


SEQ ID
518
nt. 354274-352406
SEQ ID
SEQ ID
NP_284893


NO: 585
nt. 758-789
of
NO: 667
NO: 668



of
SEQ ID NO:



SEQ ID
684 (contig



NO: 518
17)


SEQ ID
543
nt. 347864-243236
SEQ ID
SEQ ID
AAA20524


NO: 586
nt. 1814-196
of
NO: 669
NO: 670



of
SEQ ID NO:



SEQ ID
685 (contig



NO: 543
18)


SEQ ID
543
nt. 699709-704187
SEQ ID
SEQ ID
AAD56660


NO: 586
nt. 1814-196
of
NO: 671
NO: 672



of
SEQ ID NO:



SEQ ID
685 (contig



NO: 543
18)


SEQ ID
567
nt. 85546-84689
SEQ ID
SEQ ID
ZP_00053190


NO: 587
nt. 13309-13320
of
NO: 673
NO: 674



of
SEQ ID NO:



SEQ ID
681 (contig



NO: 567
14)









EXAMPLE 8
Identification of Unique NTHi Gene Sequences
Genes associated with NTHi virulence were also identified by comparing the level of expression of the gene when the NTHi bacterium was infecting a tissue verses the level of expression of the same gene when the NTHi was grown on artificial laboratory media. These novel genes were identified using the promoter trap techniques described above in Examples 4-6, and subsequently comparisons with the known Rd genome demonstrated these genes are unique to NTHi strain 86-028NP.
The DNA sequence identified using this screening procedure are set forth as SEQ ID NOS: 577-580. These sequences did not contain genes or gene fragments that have homologues in the H. influenzae Rd. genome sequence. Even though these are completely novel sequences, due to their expression level during NTHi infection in the chinchilla middle ear, it is likely that expression of these genes are involved in NTHi virulence.

## Claims

1. An isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 663.

2. A method for detecting NTHi bacteria in a biological sample comprising
(a) contacting a polynucleotide of claim 1 with polynucleotides from a biological sample under stringent hybridization conditions, wherein the hybridization conditions comprise washing with 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68°C., and
(b) detecting hybridization of the polynucleotide within the sample, wherein hybridization indicates the presence of NTHi bacteria in the biological sample.

3. The method of claim 2 wherein the biological sample is selected from the group consisting of serum, sputum, ear fluid, blood, urine, lymphatic fluid, and cerebrospinal fluid.

4. A vector comprising the polynucleotide of claim 1.

5. The vector of claim 4, wherein the polynucleotide is operatively linked to an expression control sequence.

6. A host cell comprising the vector of claim 4.

7. A cell comprising to vector of claim 5.

8. A host cell comprising the polynucleotide of claim 1.