Abstract:
Discovery and characterization of an apicomplexan Fab I gene and encoded enzyme and discovery of the triclosan as a lead compound, provide means to rationally design novel inhibitory compositions useful for prevention and treatment of apicomplexan related diseases.

Description:
BACKGROUND  
         [0001]    Discovery and characterization of an apicomplexan Fab I gene and encoded enzyme and the discovery of triclosan as a lead compound, provide means to rationally design novel inhibitory compositions useful for prevention and treatment of apicomplexan and microbial related diseases.  
           [0002]    Fab I, enoyl acyl carrier protein reductase (ENR), is an enzyme used in fatty acid synthesis. It is a single chain polypeptide in plants, bacteria, and mycobacteria, but is part of a complex polypeptide in animals and fungi. Certain other enzymes in fatty acid synthesis in apicomplexan parasites appear to have multiple forms, homologous to either a plastid sequence, a plant-like single chain enzyme, or more like the animal complex polypeptide.  
           [0003]    Apicomplexan infections are among the most common and devastating infectious diseases. Malaria (Plasmodium) kills one child every eleven seconds and three million people every year. It is a cause of substantial morbidity in pregnant women and young children.  Toxoplasmosis gondii  results in a chronic central nervous system infection in more than a third of the world population, as well as acute life threatening disease in immunocomprised individuals. New medicines are greatly needed for the treatment of these diseases.  
           [0004]    Recently a number of plant-like biochemical pathways associated with the vestigial plastid organelle of  T. gondii  and Plasmodium species have been suggested as new targets for such medicines (Roberts et al, 1998; Waller et al., 1998; Zuther et al., 1999). A particularly attractive target in this respect is the fatty acid biosynthesis pathway because there are major differences between the structure of the plastid-associated enzymes found in plants and the cytosolic enzymes found in mammals (Roberts et al., 1998; Jomaa et al., 1999; Zuther et al., 1999; Waller et al., 2000). Importantly, enzymes of mammalian lipid synthesis form domains on a multi-functional protein, whereas those enzymes in plants and certain bacteria are found on discrete mono-functional polypeptides. These differences have already been exploited by a number of compounds which selectively inhibit bacterial or plant enzymes, but do not inhibit mammalian enzymes (Roberts et al., 1998; Waller et al., 1998; Zuther et al., 1999; Payne et al., 2000). Notably, both  T. gondii  and  P. falciparum  have been shown to possess mono-functional, plant- or bacterial-like fatty acid biosynthesis enzymes which are targeted to the plastid organelle via a bipartite, N-terminal transit sequence (Waller et al., 1998; Zuther et al., 1999; Roos et al., 1999; DeRocher et al., 2000). Compounds such as aryloxyphenoxypropionates (Zuther et al., 1999), cyclohexanedione (Zuther et al., 1999) herbicides and thiolactomycin (Waller et al., 1998) which inhibit acetyl-CoA carbozylase (ACC) and β-ketoacyl-ACP synthase (Fab H) respectively, have been demonstrated to restrict the growth of  T. gondii  in vitro.  
           [0005]    Enoyl acyl carrier protein reductase catalyses the NAD (P)-dependent reduction of a trans-2,3 enoyl moiety into a saturated acyl chain, the second reductive step in the fatty acid biosynthesis pathway. Recent studies on the inhibition of ENR by compounds such as the diazaborines (Turnowsky et al., 1989; Baldock et al., 1996) and triclosan (McMurray et al., 1998; Heath et al., 1998; Levy et al., 1999; Payne et al., 2000; and Jones et al., 2000) have validated this enzyme as a target for the development of new antibacterial agents. In particular, triclosan, which is found in many house-hold formulations including soaps, deodorants, hand lotion, toothpaste and impregnated into plastics as an anti-bacterial agent is an extremely potent ENR inhibitor (Ward et al., 1999). A question is whether Fab I is in apicomplexan parasites and, if so, whether inhibition of Fab I can inhibit parasite growth and/or survival.  
         SUMMARY OF THE INVENTION  
         [0006]    The present invention relates the first report of apicomplexan Fab I (enoyl acyl carrier protein reductase, ENR) and discloses the effects of triclosan, a potent and specific inhibitor of this enzyme, on the in vitro growth of  T. gondii  and  P. falciparum  chain. A plant-like Fab I in  P. falciparum  was identified by the inventors and the structure was modeled on the  Brassia napus  and  Escherichia coli  structures, alone and complexed to triclosan (5-chloro-2-[2,4 dichloropheoxyl] phenol), which confirmed all the requisite features of an enoyl acyl carrier protein reductase (ENR) and its interactions with triclosan. Like the remarkable effect of triclosan on a wide variety of bacteria, this compound markedly inhibits growth and survival of the apicomplexan parasites  P. falciparum  and  Toxoplasma gondii  at low concentrations (i.e., IC50≅150−2000 and 62 nanogram/ml respectively).  
           [0007]    Initially, a sequence for a putative Plasmodium falciparum Fab I was located on the aggregate  P. falciparum  chromosomes referred to as “blob” (GenBank Accession Number AF338731). The deduced amino acid sequence and a multisequence alignment with representative enoyl acyl carrier protein reductases are shown in FIG. 1 (GenBank Accession Number AF33781). GenBank web site is www.ncbi.nlm.nih.gov. The gene sequence of Plasmodium ENR was obtained with a BLAST search using the sequences from both the  B. napus  and  E. coli  enzymes within the  P. falciparum  database “PlasmoDB” (found at www.PlasmoDB.org). (See Materials and Methods). This sequence was then converted to an amino acid sequence at www.expasy.ch/tools/dna.html. The sequence was aligned using the “Multiple Sequence Alignment at http://searchlauncher.bcm.tmc.edu.  
           [0008]    Subsequently, the molecules were prepared and tested in a laboratory setting (see Example 1). Errors in the published sequence for the Plasmodium genome were found. FIG. 1 shows the correct amino acid sequence for Plasmodium.  
           [0009]    Analysis of the pattern of sequence conservation confirmed that this protein has all the residues that have been identified as essential for enzyme activity. Interestingly, there is much greater sequence similarity with the plant enzyme than with the ENRs of bacterial origin. The  P. falciparum  enoyl acyl carrier protein reductase appears to have a plastid targeting sequence (Waller et al., 2000) and has a number of internal insertions. In addition, the  P. falciparum  protein has an extremely polar additional internal insertion for which no counterpart exists in any of the previously described enoyl acyl carrier protein reductases. This is important to target sequences among that are unique species of Fab I targets that can be attacked with antisense.  
           [0010]    Because Fab I was located, the effects of triclosan on Plasmodium falciparum in vitro were investigated. For  P. falciparum , the in vitro assays (Milhous et al., 1985; Oduola et al., 1988) were conducted using a modification of the semiautomated microdilution technique for assessing antifolate antagonists. Instead of dialysed human plasma, 10% Albumax I (Gibco BRL), a serum-free substitute, was used to supplement the RPMI 1640 medium. All test compounds were dissolved in DMSO and diluted 400-fold into complete medium before serial dilution over 11 concentrations. Incubation was at 37° C. in 5% O2, 5% CO2 and 90% N2 for 48 h. [3H]-Hypoxanthine incorporation was measured as described previously (Milhous et al, 1985; Oduola et al, 1988).  P. falciparum  strain W2 is susceptible to mefloquine, but resistant to pyrimethamine, sulphadoxine and quinine and less susceptible to chloroquine than  P. falciparum  strain D6. Strain D6 is susceptible to pyrimethamine and sulphadoxine, but similar to  P. falciparum  strains TM90C2A and TM90C2B, and strain TM91C235 is less susceptible to mefloquine.  
           [0011]    The effect of triclosan on  P. falciparum  in vitro was studied with pyrimethamine sensitive and resistant organisms, and those with varying sensitivity to chloroquine and mefloquine, simultaneously with studies of effect of chloroquin or mefloquine on these parasites (Table 1). Triclosan was effective against pyrimethamine resistant  P. falciparum  (W2) at low concentrations (IC50s of 150 nanograms/ml [triclosan] and 160 ngm/ml [Chloroquin], respectively) (Table 1). Interestingly, the pattern of relative susceptibility of triclosan and mefloquine were identical. This similarity suggests that triclosan and mefloquine may share a common mechanism of influx or efflux, because such differences in transporters are believed to be the basis of the differences in susceptibility of malaria parasites to mefloquin although other mechanisms are possible.  
           [0012]    For  T. gondii , growth inhibition was assessed over a 4-day period as described previously (Mack et al., 1984; Roberts et al., 1998; Zuther et al., 1999) using human foreskin fibroblasts (HFF) infected with 105 tachyzoites of the RH strain of  T. gondii . The assays are based on microscopic visual inspection of infected and inhibitor treated cultures, and on quantitation of nucleic acid synthesis of the parasite by measuring uptake of 3H uracil into the parasite&#39;s nucleic acid. Uracil is not utilized by mammalian cells. Parasites present as tachyzoites (RH, Ptg., a clone derived from the Me49 strain), bradyzoites (Me49), and R5 mutants (mixed tachyzoites/bradyzoites of the Me49 strain that can be stage switched by culture conditions) (Bohne et al., 1993; Soete et al., 1994; Tomovo and Boothroyd, 1995; Weiss et al., 1992) are suitable for assay systems used to study effects of inhibitors. Only the RH strain tachyzoites, cultured for up to 72 hours, had been used in previously reported assays. The use of Me49, Ptg, and R5 mutants are unique aspects of the methods used in these assays in this invention.  
           [0013]    Results using the assay systems are shown in FIGS.  6 - 8 . In these assays toxicity of a candidate inhibitor was assessed by its ability to prevent growth of human foreskin fibroblasts (HFF) after 4 days and after 8 days as measured by tritiated thymidine uptake and microscopic evaluation. Confluent monolayers of HFF were infected with tachyzoites and bradyzoites. Inhibitor was added one hour later. Non-toxic doses were used in parasite growth inhibition assays. Parasite growth was measured by ability to incorporate tritiated uracil during the last 48 hours of culture.  
           [0014]    Triclosan also was effective against  T. gondii , in nanomolar amounts (FIG. 2). IC50 was 62 nanograms/ml. There was no toxicity to host cells at these concentrations.  
           [0015]    Analysis of the binding site for triclosan in  B. napus  and  E. coli  ENR shows that 11 residues have contacts less than 4 Å with one of more atoms of the triclosan (FIG. 3). Inspection of the sequence for  P. falciparum  ENR reveals that it shares sequence identity at each of these positions with either the sequence of the  B. napus  or  E. coli  enzymes providing a clear explanation for the inhibitory properties of this agent against  P. falciparum.    
           [0016]    The discovery and characterization of an apicomplexan Fab I and discovery of triclosan as a lead compound provide means to rationally design novel inhibitory compounds with considerable promise. The invention provides novel ways to counteract the increasing resistance of Plasmodium to the current armoury of antimalarial agents and provides a new approach to the great need for additional, less toxic antimicrobial agents effective against  T. gondii . The inventors (Zuther et al., 1999) and others (Waller et al., 1998) have also identified other novel inhibitors of sequential enzymatic steps in the apicomplexan lipid synthesis pathway, that are predicted to be synergistic with triclosan and other inhibitors of Fab I (Baldock, et al., 1996). This also raises the exciting possibility of a rational basis for discovery of synergistic inhibitors of this pathway effective against multiple different microorganisms (Payne et al., 2000). 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0017]    [0017]FIG. 1 shows a multiple structure-based sequence alignment of the enoyl reductases from  E. coli, H. pylori, B. subtilis, S. aureus, P. falciparum  and  B. napus . The secondary structures and sequence numbers of the  E. coli  and  B. napus  enzymes are shown above and below the alignment, respectively. The residues which are completely conserved are all black with white faced type and those involved in triclosan binding are indicated with a black, filled circle above. The N terminal sequence in the  P. falciparum  Fab I with no corresponding sequence in  E. coli  is a plastid target sequence which is a suitable separate target from the entire enzyme for inhibition.  
         [0018]    [0018]FIG. 2 demonstrates inhibition of  T. gondii  by triclosan. (a) no inhibitory effect of triclosan on the host cells uptake of thymidine; appearance of the monolayer also was unchanged. (b) effect of triclosan on  T. gondii  uracil uptake; triclosan reduces uracil uptake by intracellular  T. gondii  4 days after infection; IC50 was ≅62 nanograms per ml; effect increased between days 1 to 4, for example, in a separate experiment, for 125 nanograms per ml of triclosan on day 1, percentage inhibition was 20% and on day 4 was 72% and for pyrimethamine/sulfadiazine percentages of inhibition at these times were 63% and 100% respectively. Abbreviations: RH=RH strain of  T. gondii  within fibroblasts; No RH=control with fibroblasts alone; DMSO=fibroblasts with highest concentration of DMSO; P/S=fibroblasts,  T. gondii , pyrimethamine and sulfadiazine used as a positive control for the assay; CPM=counts per minute.  
         [0019]    [0019]FIG. 3 is a stereo view of the three dimensional arrangement of the atoms that form the binding pocket for triclosan, in  E. coli  enoyl reductase, with the 11 residues that have any atom within 4 Å of the inhibitor, labeled. This is important in assigning the relative contributions made to the interaction with triclosan by the critical amino acids that are also present in the  P. falciparum  enzyme.  
         [0020]    [0020]FIG. 4 shows FabIt fusion protein cut with Factor Xa protease (lane 2) was applied to a cation exchange column (SP Sepharose) separating the fusion protein (lane 3) from Fab It (lanes 4-10). Molecular weight markers (Sigma Wide) are shown in lane 1.  
         [0021]    [0021]FIG. 5 shows the expression and purification of recombinant Fab I. FabI-MBP fusion protein cut with Factor Xa protease (lane 2) was applied to a cation exchange column (SP Sepharose) separating MBP (lane 3) from Fab I (lanes 4-10). A small amount of uncut fusion protein (FabI-MBP) can be seen in the elution fractions as well as some lower molecular weight fragments resulting from overdigestion of Fab I.  
         [0022]    [0022]FIG. 6(A) is a schematic representation of the pathway for conversion of shikimate to chorismate in  T. gondii . The inhibitor of EPSP synthase is NMPG; (B) shows uptake of tritiated uracil by tachyzoites (RH strain) is inhibited by NPMG. Toxicity of NPMG was assessed by its ability to prevent growth of human foreskin fibroblasts (HFF) after 4 days, as measured by tritiated thymidine uptake and microscopic evaluation; (C) shows product rescue of NPMG&#39;s inhibitory effect of EPSP synthase on PABA. The effect of PABA on sulfadiazine is similar, but the effect of pyrimethamine, as predicted reduces the enzyme to the levels that were present when media alone was utilized, as measured by the uracil uptake. S=sulfdiazine; PYR=pyrimethamine; and PABA=para amino benzoic acid; (D) shows functional and enzymatic evidence for the shikimate pathway in  T. gondii  with inhibition of EPSP synthase enzyme activity by 1 mM glyosate. Squares, without glyphosate. Circles, with glyphosate; (E) shows evidence for the shikimate pathway in  P. falciparum  with functional evidence for the shikimate pathway in  P. falciparum . Glyphosate inhibition of in vitro growth of asexual erythrocytic forms and PABA and folate antagonism of growth inhibition. Effect of NPMG on  C. parvum  was not abrogated by PABA. This suggests that either uptake of PABA by  C. parvum  differs or effect of NPMG is on a different branch from the shikimate pathway in  C. parvum.    
         [0023]    [0023]FIG. 7 shows inhibitory effects of NPMG, gabaculine, SHAM 8-OH-quinoline and on Cryptosporidia. 3NPA also inhibited Cryptosporidia.  
         [0024]    [0024]FIG. 8 shows the effect of NPMG, pyrimethamine, and pyrimethamine plus NPMG on survival of mice following intraperitoneal infection with 500 tachyzoites of the RH strain of  T. gondii . Dosage of NPMG was 200 mg/kg/day and pyrimethamine was 12.5 mg/kg/day.  
         [0025]    [0025]FIG. 9 is an illustrative copy of a web page for the  Plasmodium falciparum  genomic sequence.  
         [0026]    [0026]FIG. 10 is an illustrative copy of a web page with a tool to translate nucleotides to protein sequences.  
         [0027]    [0027]FIG. 11 is an illustrative copy of a web page for various searches.  
         [0028]    [0028]FIG. 12 shows acetyl Co-A carboxylases of apicomplexan were identified.  T. gondii  was ionhibited by the herbicide, clodinafop, 1 micromolar. A and C control; B and D with clodinafop.  
         [0029]    [0029]FIG. 13 shows a model of triclosan binding to its target enzyme, ENR.  
         [0030]    [0030]FIG. 14 shows the fatty acid synthesis pathway.  
         [0031]    [0031]FIG. 15 is the molecular formula and model for triclosan. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0032]    A plant-like FAB I was identified in  Plasmodium falciparum . The nucleotide sequence and deduced amino acid sequence was prepared and correct sequences were confirmed. FAB I is a single chain, discrete enzyme. All requisite residues for FAB I enzyme activity were confirmed. The  P. falciparum  enayl acyl carrier protein reductase has a putative plastid targeting sequence and unique polar insertions. The FAB I structure is modeled on  E. coli  and  B. napus  FAB I structure alone and complexed to triclosan. Key amino acids were identified for 2° structure. Residues for binding triclosan were conserved providing explanation for inhibition by triclosan. Triclosan inhibits  P. falciparum, T. gondii  (nm) in a pattern similar to the action of mefloquine. Soluble protein can be overexpressed.  
         [0033]    Information obtained from  P. falciparum  because FAB I was purified include that the N terminal sequence is the same as  B. napus  FAB I, enzyme activity is NADH dependent and inhibited by triclosan. FAB I is involved in synthesis of 10, 12 C fatty acids. In a  P. berghei  murine model, Triclosan administered subcutaneously (3 or 38 mg/kg) was nontoxic, cleared parasitemia and prevented death. Synergy was demonstrated in vitro with cerulein, an inhibitor of Fab F, B, H.  
         [0034]    Materials and Methods  
         [0035]    [0035] T. gondii  in vitro. Growth inhibition was assessed over a 4-day period as described previously by Roberts et al., 1998; Zuther et al., 1999; and Mack et al., 1984, all incorporated by reference, using human foreskin fibroblasts (HFF) infected with 105 tachyzoites of the RH strain of  T. gondii . Uptake of 3H uracil was determined. Evaluation of slides of preparations containing HFF, Toxoplasma and inhibitors were made.  
         [0036]    [0036] P. falciparum . The in vitro assays (Oduala et al., 1988; Milhous et al., 1985) were conducted using a modification of the semiautomated microdilution technique for assessing antifolate antagonists. Instead of dialysed human plasma, 10% Albumaz I (Gibco BRL), a serum-free substitute, was used to supplement the RPMI 16-40 media. All test compounds were dissolved in DMSO and diluted 400-fold into complete media before serial dilution over 11 concentrations. Incubation was at 37° C. in 5% O2, 5% CO2 and 90% N2 for 48h, [3H]-Hypoxanthine incorporation was measured as described previously (13, 14). W2 is susceptible to mefloquine, but resistant to pyrimethamine, sulphadoxine, but similar to TM90C2A and TM90C2B, and TM91C235 is less susceptible to mefloquin.  
         [0037]    TABLE 1: IC 50 1  OF Triclosan, Chloroquine, AND Mefloquine When Cultured with  P Falciparum  (Nanograms/Ml)  
         [0038]    The activity of triclosan, mefloquine, and chloroquine were tested against a series of  P. falciparum  isolates and clones with differing susceptibilities to antimalarial drugs. D6, a clone from the African Sierra I/UNC isolate, is chloroquine and pyrimethamine susceptible; W2 is a clone of the Indochina I isolate and is chloroquine and pyrimethamine resistant. TM90C2A, TM90C2B, and TM91C235 are isolates from Thailand and all are chloroquine and mefloquine resistant. TM91C235 was isolated from a patient that failed mefloquine twice, whereas TM90-C2a and TM90-Cb are admission and recrudescent isolates, respectively, of the first patient who failed treatment with atovaquone (alone) in Thailand. Subsequent susceptibility testing demonstrated that the recrudescent isolate (2B) was approximately 2000 fold resistant to atovaquone, when compared with the admission isolate and other atovaquone—susceptible isolates from Thailand.  
                                                                                       Antimicrobial   Parasite Strain            Agents   D6   TM90C2A   W2   TM90C2B   TM91C235                    Triclosan   387.1   1891.4   154.4   1330.4   1800.5       Mefloquine   5.3   24.5   2.0   19.3   19.6       Chloroquine   3.8   57.3   162.4   82.7   46.1                  
 
         [0039]    Cloning of the FabI gene. The FabI gene from Plasmodium falciparum is located on chromosome 4 and codes for a 432 amino acid protein. The FabI gene from gDNA of the 3D7 strain of  P. falciparum  was amplified using Pfu Turbo polymerase (Stratagene) and two primers ( 5 ′-GGTGGTGAATTCATGAATAAAATATCACAACGG- 3 ′ and  5 ′-GGTGGTGTCGACTTATTCATTTTCATTGCGATATATATC- 3 ′). The resulting amplicon was digested with EcoRI and SaII endonucleases and gel purified using the QIAquick Gel Extraction Kit from Qiagen. The digested ampicon was ligated with T4 DNA ligase (Boehringer Mannheim) into the pMAL-c2x vector (New England Biolabs) which had previously been digested with the same endonucleases and treated with Shrimp Alkaline Phosphatase (USB). A second construct, lacking FabI residues 1-84, was prepared in the same way using the following two primers: 5′-GGTGGTGAATTCTCAAACATAAACAAAATTAAAGAAG-3′ and 5′-GGTGGTGTCGACTTATTCATTTTCATTGCGATATATATC-3′. This truncated construct was called FabIt.  
         [0040]    Overexpression of FabIt in bacterial culture. The pMAL-c2x vector containing the FabIt construct was transformed into BL21-CodonPlus(DE3) cells (Stratagene). Bacterial cultures were grown in shaker flasks at 37° to an OD600 of 0.6 and then induced with IPTG (Sigma) to a final concentration of 0.4 mM. Induced cultures were transferred to a 20° shaker and incubated for an additional 12 hours. After this period, the cells were harvested by centrifugation at 5,000×G for 15 minutes and the cell pellet was frozen at −20°.  
         [0041]    Purification of Recombinant Fab It fusion protein. Cell lysis buffer (20 mM Na/K phosphate pH 7.5, 1 mg/ml lysozyme (Sigma), 2.5 □g/ml DNAse I (Sigma), 200 mM NaCl) was added to the frozen cell pellets (20 mL per liter of original culture) and gently vortexed. Resuspended cells were incubated on ice for 10 minutes followed by 30 seconds of sonication. Cell lysate was clarified by centrifugation at  20 , 000 ×G for 15 minutes at 4° and applied to a 10 ml amylose column (New England Biolabs) equilibrated in 20 mM Na/K phosphate pH 7.5, 200 mM NaCl. The column was washed with 5 column volumes of 20 mM Na/K phosphate pH 7.5, 500 mM NaCl followed by elution with 20 mM Na/K phosphate pH 7.5, 200 mM NaCl, 100 mM Maltose.  
         [0042]    Cleavage of FabIt fusion protein and purification of FabIt. Purified FabIt fusion protein was digested with Factor Xa (New England Biolabs) at ratio of 1 mg Factor Xa per 500 mg of fusion protein. Calcium chloride was added to the reaction mixture at a final concentration of 1 mM and the mixture was incubated at 4° for 24 hours. The reaction mixture was desalted with a HiPrep 26/10 Desalting column (Pharmacia) equilibrated in 20 mM Na/K phosphate pH 8.0, IODM NAD+. Desalted protein was applied to a SP Sepharose cation exchange column (Phaimacia) equilibrated in 20 mM Na/K phosphate pH 8.0, 10□M NAD+ and washed for 10 column volumes with the same buffer. Adsorbed proteins were eluted from the column with a linear gradient to 20 mM Na/K phosphate pH 8.0, 10□M NAD+, 500 mM NaCl in 20 column volumes. Fractions containing pure Fablt protein were pooled for further analysis.  
         [0043]    Overexpression of Recombinant Fab I. The FabI gene was amplified from cDNA of the 3D7 strain of  P. falciparum  and inserted into the pMAL-c2x vector (New England Biolabs) for expression in  E. coli . Recombinant FabI fused the Maltose Binding Protein (FabI-MBP) was purified from clarified cell lysate using a 10 ml amylose column (New England Biolabs). The pure FabI-MBP fusion protein was cleaved with Factor Xa protease yielding FabI and MBP, which were the separated with a 5 ml SP Sepharose column (Pharmacia).  
       DOCUMENTS CITED  
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Met Gly Phe Leu Ser Gly Lys Arg Ile Leu Val Thr Gly Val Ala Ser 
  1               5                  10                  15 

Lys Leu Ser Ile Ala Tyr Gly Ile Ala Gln Ala Met His Arg Glu Gly 
             20                  25                  30 

Ala Glu Leu Ala Phe Thr Tyr Gln Asn Asp Lys Leu Lys Gly Arg Val 
         35                  40                  45 

Glu Glu Phe Ala Ala Gln Leu Gly Ser Asp Ile Val Leu Gln Cys Asp 
     50                  55                  60 

Val Ala Glu Asp Ala Ser Ile Asp Thr Met Phe Ala Glu Leu Gly Lys 
 65                  70                  75                  80 

Val Trp Pro Lys Phe Asp Gly Phe Val His Ser Ile Gly Phe Ala Pro 
                 85                  90                  95 

Gly Asp Gln Leu Asp Gly Asp Tyr Val Asn Ala Val Thr Arg Glu Gly 
            100                 105                 110 

Phe Lys Ile Ala His Asp Ile Ser Ser Tyr Ser Phe Val Ala Met Ala 
        115                 120                 125 

Lys Ala Cys Arg Ser Met Leu Asn Pro Gly Ser Ala Leu Leu Thr Leu 
    130                 135                 140 

Ser Tyr Leu Gly Ala Glu Arg Ala Ile Pro Asn Tyr Asn Val Met Gly 
145                 150                 155                 160 

Leu Ala Lys Ala Ser Leu Glu Ala Asn Val Arg Tyr Met Ala Asn Ala 
                165                 170                 175 

Met Gly Pro Glu Gly Val Arg Val Asn Ala Ile Ser Ala Gly Pro Ile 
            180                 185                 190 

Arg Thr Leu Ala Ala Ser Gly Ile Lys Asp Phe Arg Lys Met Leu Ala 
        195                 200                 205 

His Cys Glu Ala Val Thr Pro Ile Arg Arg Thr Val Thr Ile Glu Asp 
    210                 215                 220 

Val Gly Asn Ser Ala Ala Phe Leu Cys Ser Asp Leu Ser Ala Gly Ile 
225                 230                 235                 240 

Ser Gly Glu Val Val His Val Asp Gly Gly Phe Ser Ile Ala Ala Met 
                245                 250                 255 

Asn Glu Leu Glu Leu Lys 
            260 

 
           
             2  
             275  
             PRT  
             Helicobacter pylori  
           
            2 

Met Gly Phe Leu Lys Gly Lys Lys Gly Leu Ile Val Gly Val Ala Asn 
  1               5                  10                  15 

Asn Lys Ser Ile Ala Tyr Gly Ile Ala Gln Ser Cys Phe Asn Gln Gly 
             20                  25                  30 

Ala Thr Leu Ala Phe Thr Tyr Leu Asn Glu Ser Leu Glu Lys Arg Val 
         35                  40                  45 

Arg Pro Ile Ala Gln Glu Leu Asn Ser Pro Tyr Val Tyr Glu Leu Asp 
     50                  55                  60 

Val Ser Lys Glu Glu His Phe Lys Ser Leu Tyr Asn Ser Val Lys Lys 
 65                  70                  75                  80 

Asp Leu Gly Ser Leu Asp Phe Ile Val His Ser Val Ala Phe Ala Pro 
                 85                  90                  95 

Lys Glu Ala Leu Glu Gly Ser Leu Leu Glu Thr Ser Lys Ser Ala Phe 
            100                 105                 110 

Asn Thr Ala Met Glu Ile Ser Val Tyr Ser Leu Ile Glu Leu Thr Asn 
        115                 120                 125 

Thr Leu Lys Pro Leu Leu Asn Asn Gly Ala Ser Val Leu Thr Leu Ser 
    130                 135                 140 

Tyr Leu Gly Ser Thr Lys Tyr Met Ala His Tyr Asn Val Met Gly Leu 
145                 150                 155                 160 

Ala Lys Ala Ala Leu Glu Ser Ala Val Arg Tyr Leu Ala Val Asp Leu 
                165                 170                 175 

Gly Lys His His Ile Arg Val Asn Ala Leu Ser Ala Gly Pro Ile Arg 
            180                 185                 190 

Thr Leu Ala Ser Ser Gly Ile Ala Asp Phe Arg Met Ile Leu Lys Trp 
        195                 200                 205 

Asn Glu Ile Asn Ala Pro Leu Arg Lys Asn Val Ser Leu Glu Glu Val 
    210                 215                 220 

Gly Asn Ala Gly Met Tyr Leu Leu Ser Ser Leu Ser Ser Gly Val Ser 
225                 230                 235                 240 

Gly Glu Val His Phe Val Asp Ala Gly Tyr His Val Met Gly Met Gly 
                245                 250                 255 

Ala Val Glu Glu Lys Asp Asn Lys Ala Thr Leu Leu Trp Asp Leu His 
            260                 265                 270 

Lys Glu Gln 
        275 

 
           
             3  
             258  
             PRT  
             Bacillus subtilis  
           
            3 

Met Asn Phe Ser Leu Glu Gly Arg Asn Ile Val Val Met Gly Val Ala 
  1               5                  10                  15 

Asn Lys Arg Ser Ile Ala Trp Gly Ile Ala Arg Ser Leu His Glu Ala 
             20                  25                  30 

Gly Ala Arg Leu Ile Phe Thr Tyr Ala Gly Glu Arg Leu Glu Lys Ser 
         35                  40                  45 

Val His Glu Leu Ala Gly Thr Leu Asp Arg Asn Asp Ser Ile Ile Leu 
     50                  55                  60 

Pro Cys Asp Val Thr Asn Asp Ala Glu Ile Glu Thr Cys Phe Ala Ser 
 65                  70                  75                  80 

Ile Lys Glu Gln Val Gly Val Ile His Gly Ile Ala His Cys Ile Ala 
                 85                  90                  95 

Phe Ala Asn Lys Glu Glu Leu Val Gly Glu Tyr Leu Asn Thr Asn Arg 
            100                 105                 110 

Asp Gly Phe Leu Leu Ala His Asn Ile Ser Ser Tyr Ser Leu Thr Ala 
        115                 120                 125 

Val Val Lys Ala Ala Arg Pro Met Met Thr Glu Gly Gly Ser Ile Val 
    130                 135                 140 

Thr Leu Thr Tyr Leu Gly Gly Glu Leu Val Met Pro Asn Tyr Asn Val 
145                 150                 155                 160 

Met Gly Val Ala Lys Ala Ser Leu Asp Ala Ser Val Lys Tyr Leu Ala 
                165                 170                 175 

Ala Asp Leu Gly Lys Glu Asn Ile Arg Val Asn Ser Ile Ser Ala Gly 
            180                 185                 190 

Pro Ile Arg Thr Leu Ser Ala Lys Gly Ile Ser Asp Phe Asn Ser Ile 
        195                 200                 205 

Leu Lys Asp Ile Glu Glu Arg Ala Pro Leu Arg Arg Thr Thr Thr Pro 
    210                 215                 220 

Glu Glu Val Gly Asp Thr Ala Ala Phe Leu Phe Ser Asp Met Ser Arg 
225                 230                 235                 240 

Gly Ile Thr Gly Glu Asn Leu His Val Asp Ser Gly Phe His Ile Thr 
                245                 250                 255 

Ala Arg 

 
           
             4  
             264  
             PRT  
             Staphylococcus aureus  
           
            4 

Met Thr Thr Lys Ile Ser Met Leu Asn Leu Thr Gly Lys Asn Ala Leu 
  1               5                  10                  15 

Val Thr Gly Ile Ala Asn Asn Arg Ser Ile Ala Trp Gly Ile Ala Gln 
             20                  25                  30 

Gln Leu His Ala Ala Gly Ala Asn Leu Gly Ile Thr Tyr Leu Pro Asp 
         35                  40                  45 

Glu Arg Gly Lys Phe Glu Lys Lys Val Ser Glu Leu Val Glu Pro Leu 
     50                  55                  60 

Asn Pro Ser Leu Phe Leu Pro Cys Asn Val Gln Asn Asp Glu Gln Ile 
 65                  70                  75                  80 

Gln Ser Thr Phe Asp Thr Ile Arg Asp Lys Trp Gly Arg Leu Asp Ile 
                 85                  90                  95 

Leu Ile His Cys Leu Ala Phe Ala Asn Arg Asp Asp Leu Thr Gly Asp 
            100                 105                 110 

Phe Ser Gln Thr Ser Arg Ala Gly Phe Ala Thr Ala Leu Asp Ile Ser 
        115                 120                 125 

Thr Phe Ser Leu Val Gln Leu Ser Gly Ala Ala Lys Pro Leu Met Thr 
    130                 135                 140 

Glu Gly Gly Ser Ile Ile Thr Leu Ser Tyr Leu Gly Gly Val Arg Ala 
145                 150                 155                 160 

Val Pro Asn Tyr Asn Val Met Gly Val Ala Lys Ala Gly Leu Glu Ala 
                165                 170                 175 

Ser Val Arg Tyr Leu Ala Ser Glu Leu Gly Ser Gln Asn Ile Arg Val 
            180                 185                 190 

Asn Ala Ile Ser Ala Gly Pro Ile Arg Thr Leu Ala Ser Ser Ala Val 
        195                 200                 205 

Gly Gly Ile Leu Asp Met Ile His His Val Glu Gln Val Ala Pro Leu 
    210                 215                 220 

Arg Arg Thr Val Thr Gln Leu Glu Val Gly Asn Thr Ala Ala Phe Leu 
225                 230                 235                 240 

Ala Ser Asp Leu Ala Ser Gly Ile Thr Gly Gln Val Leu Tyr Val Asp 
                245                 250                 255 

Ala Gly Tyr Glu Ile Met Gly Met 
            260 

 
           
             5  
             420  
             PRT  
             Plasmodium falciparum  
           
            5 

Met Asn Lys Ile Ser Gln Arg Leu Leu Phe Leu Phe Leu His Phe Tyr 
  1               5                  10                  15 

Thr Ile Val Cys Phe Ile Gln Asn Asn Thr Gln Lys Thr Phe His Asn 
             20                  25                  30 

Val Leu His Asn Glu Gln Ile Arg Gly Lys Glu Lys Ala Phe Tyr Arg 
         35                  40                  45 

Lys Glu Lys Arg Glu Asn Ile Phe Ile Gly Asn Lys Met Lys His Leu 
     50                  55                  60 

Asn Asn Met Asn Asn Thr His Asn Asn Asn His Tyr Met Glu Lys Glu 
 65                  70                  75                  80 

Glu Gln Asp Ala Ser Asn Ile Tyr Lys Ile Lys Glu Glu Asn Lys Asn 
                 85                  90                  95 

Glu Asp Ile Cys Phe Ile Ala Ile Gly Asp Thr Asn Gly Tyr Gly Trp 
            100                 105                 110 

Gly Ile Lys Glu Leu Ser Lys Arg Asn Val Lys Ile Ile Phe Gly Ile 
        115                 120                 125 

Trp Pro Pro Val Tyr Asn Ile Phe Met Lys Asn Tyr Lys Asn Gly Lys 
    130                 135                 140 

Phe Asp Asn Asp Met Ile Ile Asp Lys Asp Lys Lys Met Asn Ile Leu 
145                 150                 155                 160 

Asp Met Leu Pro Phe Asp Ala Ser Phe Asp Thr Ala Asn Asp Ile Asp 
                165                 170                 175 

Glu Glu Thr Lys Asn Asn Lys Arg Tyr Asn Met Leu Gln Asn Tyr Thr 
            180                 185                 190 

Ile Glu Asp Val Ala Asn Leu Ile His Gln Lys Tyr Gly Lys Ile Asn 
        195                 200                 205 

Met Leu Val His Ser Leu Ala Asn Ala Lys Glu Val Gln Lys Lys Asp 
    210                 215                 220 

Leu Leu Asn Thr Ser Arg Lys Gly Tyr Leu Asp Leu Ser Lys Ser Tyr 
225                 230                 235                 240 

Leu Ile Ser Leu Cys Lys Tyr Phe Val Asn Ile Met Lys Pro Gln Ser 
                245                 250                 255 

Ser Ile Ile Ser Thr His Ala Ser Gln Lys Val Val Pro Gly Gly Gly 
            260                 265                 270 

Gly Ser Ser Ala Leu Glu Ser Asp Thr Arg Val Ala Tyr His Leu Gly 
        275                 280                 285 

Arg Asn Tyr Asn Ile Arg Ile Asn Thr Ile Ser Ala Gly Pro Leu Lys 
    290                 295                 300 

Ser Arg Ala Ala Thr Ala Ile Asn Lys Leu Asn Asn Thr Tyr Glu Asn 
305                 310                 315                 320 

Asn Thr Asn Gln Asn Lys Asn Arg Asn Ser His Asp Val His Asn Ile 
                325                 330                 335 

Met Asn Asn Ser Gly Glu Lys Glu Glu Lys Lys Asn Ser Ala Ser Gln 
            340                 345                 350 

Asn Tyr Thr Phe Ile Asp Tyr Ala Ile Glu Tyr Ser Glu Lys Tyr Ala 
        355                 360                 365 

Pro Leu Arg Gln Lys Leu Leu Ser Thr Asp Ile Gly Ser Val Ala Ser 
    370                 375                 380 

Phe Leu Leu Ser Arg Glu Ser Arg Ala Ile Thr Gly Gln Thr Ile Tyr 
385                 390                 395                 400 

Val Asp Asn Gly Leu Asn Ile Met Phe Leu Pro Asp Asp Ile Tyr Arg 
                405                 410                 415 

Asn Glu Asn Glu 
            420 

 
           
             6  
             374  
             PRT  
             Brassica napus  
           
            6 

Met Ala Ala Thr Ala Ala Ala Ser Ser Leu Gln Met Ala Thr Thr Arg 
  1               5                  10                  15 

Pro Ser Ile Ser Ala Ala Ser Ser Lys Ala Arg Thr Tyr Val Val Gly 
             20                  25                  30 

Ala Asn Pro Arg Asn Ala Tyr Lys Ile Ala Cys Thr Pro His Leu Ser 
         35                  40                  45 

Asn Leu Gly Cys Leu Arg Asn Asp Ser Ala Leu Pro Ala Ser Lys Lys 
     50                  55                  60 

Ser Phe Ser Phe Ser Thr Lys Ala Met Ser Glu Ser Ser Glu Ser Lys 
 65                  70                  75                  80 

Ala Ser Ser Gly Leu Pro Ile Asp Leu Arg Gly Lys Arg Ala Phe Ile 
                 85                  90                  95 

Ala Ile Ala Asp Asp Asn Gly Tyr Gly Trp Ala Val Lys Ser Leu Ala 
            100                 105                 110 

Ala Ala Gly Ala Glu Ile Leu Val Gly Thr Trp Val Pro Ala Leu Asn 
        115                 120                 125 

Ile Phe Glu Thr Ser Leu Arg Arg Gly Lys Phe Asp Gln Ser Arg Val 
    130                 135                 140 

Leu Pro Asp Gly Ser Leu Met Glu Ile Lys Lys Val Tyr Pro Leu Asp 
145                 150                 155                 160 

Ala Val Phe Asp Asn Pro Glu Asp Val Pro Glu Asp Val Lys Ala Asn 
                165                 170                 175 

Lys Arg Tyr Ala Gly Ser Ser Asn Trp Thr Val Gln Glu Ala Ala Glu 
            180                 185                 190 

Cys Val Arg Gln Asp Phe Gly Ser Ile Asp Ile Leu Val His Ser Leu 
        195                 200                 205 

Ala Asn Gly Pro Glu Val Ser Lys Lys Pro Leu Leu Glu Thr Ser Arg 
    210                 215                 220 

Lys Gly Tyr Leu Ala Ile Ser Ala Ser Tyr Phe Val Ser Leu Leu Ser 
225                 230                 235                 240 

His Phe Leu Pro Ile Met Asn Pro Gly Gly Ala Ser Ile Ser Thr Ile 
                245                 250                 255 

Ala Ser Glu Arg Ile Ile Pro Gly Gly Gly Gly Ser Ser Ala Leu Glu 
            260                 265                 270 

Ser Asp Thr Arg Val Leu Ala Phe Glu Ala Gly Arg Lys Gln Asn Ile 
        275                 280                 285 

Arg Val Asn Thr Ile Ser Ala Gly Pro Leu Gly Ser Arg Ala Ala Lys 
    290                 295                 300 

Ala Ile Gly Phe Ile Asp Thr Met Ile Glu Tyr Ser Tyr Asn Asn Ala 
305                 310                 315                 320 

Pro Ile Gln Lys Thr Leu Thr Ala Asp Glu Val Gly Asn Ala Ala Ala 
                325                 330                 335 

Phe Leu Val Ser Pro Leu Ala Ser Ala Ile Thr Gly Ala Thr Ile Tyr 
            340                 345                 350 

Val Asp Asn Gly Leu Asn Ser Met Gly Val Ala Leu Asp Ser Pro Val 
        355                 360                 365 

Phe Lys Asp Leu Asn Lys 
    370 

 
           
             7  
             33  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      primer  
             
           
            7 

ggtggtgaat tcatgaataa aatatcacaa cgg                                  33 

 
           
             8  
             39  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      primer  
             
           
            8 

ggtggtgtcg acttattcat tttcattgcg atatatatc                            39 

 
           
             9  
             37  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      primer  
             
           
            9 

ggtggtgaat tctcaaacat aaacaaaatt aaagaag                              37 

 
           
             10  
             39  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      primer  
             
           
            10 

ggtggtgtcg acttattcat tttcattgcg atatatatc                            39