Patent application title: RECOMBINANT FUSOBACTERIUM NECROPHORUM LEUKOTOXIN VACCINE AND PREPARATION THEREOF
Tiruvoor G. Nagaraja (Manhattan, KS, US)
George C. Stewart (Columbia, MO, US)
Sanjeev K. Narayanan (Manhattan, KS, US)
Muckatira M. Chengappa (Manhattan, KS, US)
Kansas State University Research Foundation
IPC8 Class: AA61K39114FI
Class name: Antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.) amino acid sequence disclosed in whole or in part; or conjugate, complex, or fusion protein or fusion polypeptide including the same disclosed amino acid sequence derived from bacterium (e.g., mycoplasma, anaplasma, etc.)
Publication date: 2009-05-07
Patent application number: 20090117142
Patent application title: RECOMBINANT FUSOBACTERIUM NECROPHORUM LEUKOTOXIN VACCINE AND PREPARATION THEREOF
Tiruvoor G. Nagaraja
George C. Stewart
Sanjeev K. Narayanan
Muckatira M. Chengappa
POLSINELLI SHUGART PC
KANSAS STATE UNIVERSITY RESEARCH FOUNDATION
Origin: KANSAS CITY, MO US
IPC8 Class: AA61K39114FI
The F. necrophorum gene expressing leukotoxin was sequenced and cloned.
The leukotoxin open reading frame (lktA) is part of a multi-gene operon
containing 9,726 bp, and encoding a protein containing 3,241 amino acids
with an overall molecular weight of 335,956 daltons. The protein encoded
by the gene was truncated into five polypeptides having overlapping
regions by truncating the full length gene into five different sections
and amplifying, expressing, and recovering the protein encoded by each of
these sections. Additionally, a region upstream of the gene was sequenced
and the polypeptide encoded by that nucleotide sequence was purified and
isolated. These polypeptides along with the full length protein are then
tested to determine their immunogenicity and protective immunity in
comparison to the efficacy of immunization conferred by inactivated
native leukotoxin in F. necrophorum culture supernatant.
1. An isolated nucleotide sequence having a nucleotide sequence having at
least about 50% sequence homology with a sequence that is a truncated
form of SEQ ID No. 8.
2. The sequence of claim 1, said sequence having at least about 60% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 8-14.
3. The sequence of claim 1, said sequence having at least about 75% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 8-14.
4. The sequence of claim 1, said sequence having at least about 87% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 8-14.
5. The sequence of claim 1, said sequence having at least about 95% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 8-14.
6. An expression vector containing a nucleotide sequence having at least about 50% sequence homology with a truncated sequence from SEQ ID No. 8.
7. The vector of claim 6, said nucleotide sequence having at least about 60% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 8-14.
8. The vector of claim 6, said nucleotide sequence having at least about 75% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 8-14.
9. The vector of claim 6, said nucleotide sequence having at least about 87% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 8-14.
10. The vector of claim 6, said nucleotide sequence having at least about 95% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 8-14.
11. An isolated nucleotide sequence which differs from that of claim 1 due to a mutation event selected from the group consisting of point mutations, deletions, insertions and rearrangements.
12. A vaccine effective for conferring protective immunity against F. necrophorum comprising the protein expressed by a portion of SEQ ID No. 8 and a suitable pharmacologically compatible carrier.
13. The vaccine of claim 12, said vaccine being prepared by a method comprising the steps of:a) providing the F. necrophorum gene which expresses leukotoxin;b) truncating said F. necrophorum gene into a plurality of discrete nucleotide sequences, each of said discrete nucleotide sequences encoding for a respective polypeptide sequence;c) expressing and recovering said encoded polypeptide sequence expressed by at least one of said discrete nucleotide sequences;d) inactivating said recovered polypeptide sequence; ande) combining said inactivated polypeptide sequence with said suitable pharmacologically compatible carrier to produce said vaccine.
14. The vaccine of claim 13, said discrete nucleotide sequences having a sequence having at least about 50% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 9-14.
15. The vaccine of claim 13, further comprising the step of expressing and recovering said respective polypeptides using said nucleotide.
16. A recombinantly derived nucleotide sequence than encodes a polypeptide effective in conferring protective immunity against F. necrophorum infections in mice, said sequence comprising a truncated form of SEQ ID No. 8.
17. The sequence of claim 16, said sequence having at least about 50% sequence homology with a sequence selected from SEQ ID Nos. 9-14.
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of application Ser. No. 09/841,786 filed Apr. 24, 2001 which was a continuation-in-part application to application Ser. No. 09/558,257, Filed Apr. 25, 2000. The content and teachings of each of these applications is hereby incorporated by reference herein.
A printed Sequence Listing accompanies this application, and also has been submitted with identical contents in the form of a computer-readable ASCII file on a floppy diskette with application Ser. No. 09/558,257, filed Apr. 25, 2000. Use of this previously filed CRF sequence listing is requested.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is concerned with methods of cloning and expressing the leukotoxin gene from Fusobacterium necrophorum (F. necrophorum), sequencing and characterizing the leukotoxin protein expressed by this gene, truncating the gene into a series of nucleotide sequences, amplifying these sequences, expressing and recovering the polypeptides encoded by the nucleotide sequences, and utilizing the protein and the polypeptides in recombinant vaccines in order to confer effective immunity against infection caused by the production of leukotoxin by F. necrophorum. More particularly, it is concerned with production of an inactivated recombinant leukotoxin vaccine generated by amplifying five leukotoxin gene fragments and one upstream region through PCR, digesting the nucleotide sequences encoded by the gene fragments with restriction enzymes, expressing the polypeptide sequences coded by the nucleotide sequences through an expression vector, recovering these proteins as five truncated leukotoxin proteins (or polypeptides), purifying these proteins (or polypeptides) to apparent homogeneity, with or without inactivation of the truncated and full length proteins, and combining the inactivated recombinant leukotoxins with adjuvants.
2. Description of the Prior Art
Liver abscesses in feed lot cattle are a serious economic problem, causing condemnation of over 3 million livers and an estimated loss of $15 million annually in the United States. This estimate is based primarily on condemnation of liver and other organs, and does not include economic losses stemming from reduced feed intake, reduced feed efficiencies, decreased carcass dressing percentage and lowered weight gains. A number of studies have confirmed that cattle with abscessed livers gain less (average 4-5%) and have reduced feed efficiencies (average 7%) compared with cattle having healthy livers. The average incidence of abscessed liver in grain-fed cattle approximates 25-30%. To a lesser extent, liver abscesses in sheep and goats are also an economic problem.
F. necrophorum is a gram-negative, rod-shaped, nonsporeforming, nonmotile, strictly anaerobic and pleomorphic organism. Morphologically, the organism varies from short rods to filamentous with pointed and rounded ends. Cell lengths range from coccoid bodies of 0.5-0.7 μm in diameter to filaments over 100 μm. Surface colonies are 1-2 mm in diameter, circular, transparent to opaque, and with some strains producing α or β hemolysis. The organism ferments glucose, fructose and maltose only weakly with final pH around 5.0-6.3. It ferments lactate to acetate, propionate, and butyrate. Butyrate is the major product from lactate fermentation. Indole is produced from peptone. F. necrophorum has been isolated from the normal flora in the oral cavity, gastrointestinal cavity, and genitourinary tract of humans and animals. The organism is also known to survive in the soil.
F. necrophorum is a normal inhabitant of the gastrointestinal tracts of animals and humans. Virulence factors and pathogenic mechanisms that contribute to the transition of this otherwise commensal organism to a pathogen are poorly understood. A leukotoxin, endotoxin, hemolysin, hemagglutinin, and several enzymes such as deoxyribonuclease and proteases have been suggested as possible virulence factors. However, several studies implicate leukotoxin, a protein cytotoxic to ruminant polymorphonuclear cells, as the major virulence factor. The importance of leukotoxin as a virulence factor in F. necrophorum infections is indicated by a correlation between toxin production and ability to induce abscesses in laboratory animals, an inability of nonleukotoxin-producing strains to induce foot abscesses in cattle following intradermal inoculation, and a relationship between antileukotoxin antibody titers and protection against infection in experimental challenge studies.
F. necrophorum is an opportunistic pathogen that is the primary etiologic agent of liver abscesses in ruminant animals. (Scanlan, et al., (1983) Bovine rumenitis-liver abscess complex: a bacteriological review. Cornell Vet. 73:288-297; Nagaraja, T. G. et al., (1998) Liver abscesses in feedlot cattle: A review. J. Anim. Sci., 76:287-298; and Tan, et al., (1996) Fusobacterium necrophorum infections: virulence factors pathogenic mechanism and control measures. Vet. Res. Comm., 20:113-140). The organism has been recognized as an animal and human pathogen since the late 1800s, and is associated as a primary or secondary etiologic agent with numerous necrotic disease conditions in domestic and wild animals. In addition to liver abscesses, the organism is also the primary etiologic agent of foot rot, foot abscesses, calf diphtheria, and is frequently isolated from cases of mastitis, metritis, and necrotic lesions of the oral cavity.
Liver abscesses in cattle are part of a disease complex where the abscessation is secondary to primary foci of infection in the rumen epithelium. The pathogenesis can be summarized as follows: (1) ruminal lesions are induced by acidosis that follows rapid change in diet from high-roughage to high grain, prolonged feeding of high grain diet, or occasionally by foreign body penetration of the rumen epithelium; (2) bacteria present in the rumen invade the epithelium and form focal abscesses in the rumen wall; and (3) bacteria enter the portal circulation, and are carried to the liver where they localize in the parenchyma with subsequent abscess formation.
The ability of F. necrophorum to establish in the liver is attributed to the production of a toxin which is a secreted protein of high molecular weight active against leukocytes from ruminants called leukotoxin (or leucocidin). The toxin is a soluble extracellular protein that is cytotoxic to neutrophils, macrophages, hepatocytes, and ruminal cells. The leukotoxin protects against phagocytosis and is believed to aid in the establishment of F. necrophorum in the liver by directly impairing the normal defense mechanism and indirectly by the damage caused by cytolytic products released from neutrophils and macrophages to the hepatic cells. Therefore, the leukotoxin elaborated from F. necrophorum plays a critical role in F. necrophorum infection of the liver and is believed to be the primary virulence factor in the pathogenesis of liver abscesses (Tan et al., 1996).
Four biotypes (A, B, AB and C) of F. necrophorum have been described. (Langworth, (1977) Fusobacterium necrophorum: its characteristics and role as an animal pathogen. Bacteriol. Rev. 41:373-390) Biotype A, most frequently isolated from liver abscesses, is more pathogenic than biotype B, which predominates in ruminal wall abscesses. Biotypes AB and C are rarely isolated in liver abcesses (Berg, et al., (1982) Studies of Fusobacterium necrophorum from bovine hepatic abscesses: Biotypes guantitation, virulence, and antibiotic susceptibility. Am. J. Vet. Res. 43:1580-1586), and biotype A has pathogenicity intermediate that of biotypes A and B while biotype C is non-pathogenic. (Shinjo, et al., (1990) Recognition of biovar C of Fusobacterium necrophorum (flugge) Moore and Holdeman as Fusobacterium pseudonecrophorum sp. nov.nom.rev. (ex prevot 1940) Int. J. Sys. Bacteriol. 41:395-397) Biotypes A and B, the most frequent types encountered in liver abscesses, have been assigned subspecies status: subsp. necrophorum and subsp. funduliforme, respectively (Shinjo et al., 1990). The subsp necrophorum is more virulent, produces more leukotoxin and hemagglutinin, and is more frequently isolated from cattle liver abscesses than the subsp. funduliforme. Virulence factors and pathogenic mechanisms contributing to the formation of liver abscesses by F. necrophorum are poorly understood (Tan et al., 1996). However, several studies implicate leukotoxin to be a major virulence factor (Emery, et al., (1986) Generation of immunity against Fusobacterium necrophorum in mice inoculated with extracts containing leukotoxin. Vet. Microbiol. 12:255-268; Tan et al., 1996). The importance of leukotoxin is evidenced by correlation between toxin production and ability to induce abscesses in laboratory animals (Coyle-Dennis, et al., (1979) Correlation between leukocidin production and virulence of two isolates of Fusobacterium necrophorum. Am. J. Vet. Res. 40:274-276; Emery and Vaughn, 1986), inability of nonleukotoxin-producing strains to induce foot abscesses in cattle following intradermal inoculation (Emery, et al., (1985) Culture characteristics and virulence of strains of Fusobacterium necrophorum isolated from feet of cattle and sheep. Australian Vet. J. 62:43-46) and relationship between antileukotoxin antibody titers and protection in experimental challenge studies (Saginala, et al., (1996a) The serum neutralizing antibody response in cattle to Fusobacterium necrophorum leukotoxoid and possible protection against experimentally induced hepatic abscesses. Vet. Res. Comm., 20:493-504; Saginala, et al., (1996b) The serum neutralizing antibody response and protection against experimentally induced liver abscesses in steers vaccinated with Fusobacterium necrophorum. Am. J. Vet Res., 57:483-488; and Shinjo, et al., (1991) Proposal of two subspecies of Fusobacterium necrophorum (Flugge) Moore and Holdeman: Fusobacterium necrophorum subsp. necrophorum subsp.nov.,nom.rev. (ex Flugge 1886), and Fusobacterium necrophorum subsp. funduliforme subsp.nov.nom.rev. (ex Hall 1898). Int. J. Sys. Bacteriol. 41:395-397).
Several investigators have attempted to induce protective immunity against F. necrophorum by using a variety of antigenic components. The results of such attempts have varied from ineffectual to significant protection. Clark et al. reported that cattle injected with F. necrophorum culture supernatant containing leukotoxin had a low incidence of foot rot caused by F. necrophorum. (Clark, et al. (1986), Studies into immunization of cattle against interdigital necrobacillosis. Aust. Vet. J. 63:107-110) Cell-free culture supernatant of a high leukotoxin producing strain of F. necrophorum (Tan et al., (1992) Factors affecting leukotoxin activity of F. necrophorum. Vet. Microbiol. 33: 15-28), mixed with an adjuvant, was shown to elicit a high antileukotoxin antibody titer when injected in steers and provided significant protection to experimentally induced liver abscesses (Saginala et al., 1996a, b; 1997). F. necrophorum bacterin was used as an agent for immunizing cattle and sheep against liver necrosis as shown in EPO Application No. 460-480 of Dec. 11, 1991 (the teachings of which are incorporated herein by reference). Specifically, virulent F.-necrophorum isolates are inactivated using β-propiolactone, followed by addition of adjuvants. In addition, Abe et al., Infection and Immunity, 13:1473-1478, 1976 grew F.-necrophorum for 48 hours. Cells were obtained by centrifuging, washing three times with saline, and were inactivated with formalin (0.4% in saline). The inactivated cells were then injected into mice to induce immunity. Two weeks after the last booster injection, each mouse was challenged with viable cells of F. necrophorum. The mice immunized with killed cells and challenged with live cells had no detectable bacteria in the liver, lung or spleen for up to 28 days. It was concluded that immunization of mice with formalin-killed F.-necrophorum conferred protection against infection. Garcia et al. (Canadian J. Comp. Med, 38:222-226, 1974), conducted field trials to evaluate the efficacy of alum-precipitated toxoids of F. necrophorum. The vaccine preparation consisted of washed cells (unlikely to contain leukotoxin) that were ruptured by sonication. The most promising result was achieved with the injection of 15.5 mg protein of cytoplasmic toxoid. In this group, the incidents of liver abscesses was reduced to 10% from an average 35% in the control group. Emery et al., Vet. Microbiol., 12:255-268, 1986, prepared material by gel filtration of 18-hour culture supernate of F. necrophorum. This elicited significant immunity against challenge by with viable F. necrophorum. The injected preparation contained endotoxin and the majority of the leukotoxic activity. U.S. Pat. No. 5,455,034 (the teachings of which are incorporated herein by reference) by Nagaraja et al. disclosed that prevention of leukotoxin production (or inhibition of its activity) in immunized animals prevents the establishment of F. necrophoruim infection. Thus, immunization of the animals against F.-necrophorum leukotoxin, so that the animals' white blood cells or tissue macrophages may phagocytize the bacteria, presented a way to prevent diseases associated with F. necrophorum infection, e.g., liver abscesses in cattle and sheep, and foot rot in cattle. In order to produce such a leukotoxoid vaccine, the F. necrophorum bacteria was cultured in a way to enhance the elaboration of leukotoxin in the supernate. Thereupon, bacterial growth and leukotoxin elaboration was terminated, and a vaccine prepared by inactivating at least the leukotoxin-containing supernate. In more detail, the leukotoxin elaboration method of the '034 patent involved first forming a culture of F. necrophorum bacteria in growth media, and thereafter causing the bacteria to grow in the culture and to simultaneously elaborate leukotoxin in the supernate. At the end of the culturing step, i.e., at the end of the selected culture time within the range of from about 4-10 hours, the bacterial growth and leukotoxin elaboration were terminated, and the leukotoxoid vaccine was prepared. This involved first separating the leukotoxin-containing supernate from the bacteria, followed by inactivation through use of formalin, 13-propiolactone, heat, radiation or any other known method of inactivation. Alternately, the entire culture could be inactivated to form the vaccine.
Presently, the control of liver abscesses is with the use of antimicrobial feed additives. Antimicrobial compounds reduce the incidence of liver abscesses but do not eliminate the problem (Nagaraja et al., 1998). Therefore, an effective vaccine would be highly desirable to the feedlot industry. The vaccine approach also would alleviate public health concerns associated with the use of subtherapeutic levels of antibiotics in the feed. Because studies have indicated that antileukotoxin immunity reduces the incidence of hepatic abscesses and interdigital necrobacillosis (Garcia et al., 1974; Clark et al., 1986; Saginala et al., 1996a, b; 1997), the development of a recombinant leukotoxin vaccine will be of great value in the control of hepatic and interdigital necrobacillosis in cattle.
SUMMARY OF THE INVENTION
In order to better define the molecular nature of the F. necrophorum leukotoxin, and as a first step toward determining its specific role in the virulence of this bacterium, the leukotoxin gene was isolated, its nucleotide sequence determined, and the recombinant leukotoxin was expressed in E. coli.
The leukotoxin open reading frame (lktA) is part of a multi-gene operon containing 9,726 bp, and encoding a protein containing 3,241 amino acids with an overall molecular weight of 335,956 daltons. F. necrophorum leukotoxin is highly unstable as evidenced by western blot analysis of native leukotoxin (culture supernatant, sephadex gel or affinity purified) (FIG. 1). In this Figure, lane 1 contains whole cell lysate of E. coli cells expressing full-length recombinant leukotoxin, lane 2 contains Immuno-affinity purified native leukotoxin, lane 3 contains Sephadex gel purified leukotoxin, and lane 4 contains culture supernatant from F. necrophorum concentrated 60 times. The blots were probed with polyclonal antiserum raised in rabbits against affinity purified native leukotoxin. Because of the apparent instability of the full-length recombinant leukotoxin protein, the protein encoded by the gene was truncated into five recombinant polypeptides (or protein fragments, BSBSE, SX, GAS, SH and FINAL) having overlapping regions by truncating the full length gene into five different sections and amplifying, expressing in E. coli, and recovering the protein or polypeptide encoded by each of these sections. These polypeptides along with the full length protein are then tested to determine their immunogenicity and protective immunity in comparison to the efficacy of immunization conferred by inactivated native leukotoxin in F. necrophorum culture supernatant.
Specifically, the chromosomal DNA was extracted from F. necrophorum and partially digested by restriction endonucleases prior to being size-fractionated by sucrose gradient centrifugation. The 10-12 kb fragments were then ligated into a BamHI digested, dephosphorylated λZAP expression vector. Recombinant phages were infected into Escherichia coli and plated onto agar plates. Plaque lifts were performed (with polyclonal antiserum raised in rabbits against affinity purified leukotoxin) using an immunoscreening kit. Six immunoreactive recombinant phages were identified and denominated as clones 816, 611, 513, 911, 101, and 103. These clones were plaque-purified three times to ensure purity, phagemids rescued, and anti-leukotoxin immunoreactivity of the encoded proteins was confirmed. This immunoreactivity verified that the clones represented native leukotoxin F. necrophorum.
Expression of a polypeptide encoded by the 3.5 kb from the 5' end of the lktA caused immediate cessation of the growth and lysis of E. coli host cells suggesting that regions of leukotoxin could be toxic to E. coli. Of course, the objective was to create overlapping gene truncations extending over the entire lktA ORF so that the resulting polypeptide products are small and relatively stable on expression, but are large enough to be immunogenic. Also, the effectiveness of various recombinant truncated leukotoxin polypeptides alone or in combinations as immunogens and evaluated protective immunity against challenge with F. necrophorum in mice was investigated. The use of mice as an experimental model for F. necrophorum infection in cattle is well established (Abe et al., 1976; Conion et al., 1977; Smith et al., 1989; Garcia and McKay, 1978; Emery and Vaughan, 1986). Extension of the patterns of immunity and infection to cattle has shown that mice can be a valuable model to evaluate the immunogenicity and protection provided by various F. necrophorum fractions (Garcia et al., 1975; Garcia and McKay, 1978). Studies have also indicated that strains of F. necrophorum that are pathogenic in domestic animals, frequently are pathogenic in mice suggesting necrobacillosis as a disease is similar among these species of animals (Smith and Thornton, 1993).
The nucleotide sequence of the full length version of the gene is designated as SEQ ID No. 8 and the nucleotide sequences of the five truncations of the full length gene are designated as BSBSE (SEQ ID No. 9), SX (SEQ ID No. 10), GAS (SEQ ID No. 11), SH (SEQ ID No. 12), and FINAL (SEQ ID No. 13). Additionally, the nucleotide sequence of the upstream region of the full length gene is designated UPS (SEQ ID No. 14). The amino acid sequence of the full length protein encoded by the F. necrophorum gene is designated as SEQ ID No. 1 and the amino acid sequences of the truncated protein fragments respectively encoded by BSBSE, SX, GAS, SH and FINAL are designated as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6. In the case of UPS, the polypeptide or truncated protein fragment encoded for by UPS is designated as SEQ ID No. 7. Finally, SEQ ID No. 15 is the full length gene sequence along with contiguous sequences.
Truncated recombinant polypeptides were purified by nickel affinity chromatography, and injected into rabbits to raise polyclonal antisera. Antibodies raised against two of the five polypeptides (BSBSE and GAS) neutralized the toxicity of F. necrophorum leukotoxin against bovine neutrophils. The effectiveness of the purified truncated polypeptides to induce a protective immunity was determined by injecting the polypeptides, individually or in mixtures, homogenized with Ribi adjuvant in mice, followed by experimental challenge with F. necrophorum. Two polypeptides (BSBSE and SH) induced significant protection in mice against F. necrophorum infection and the extent of protection was greater than the full-length native leukotoxin or inactivated culture supernatant. The study provided further credence to the importance of leukotoxin as the major virulence factor of F. necrophorum and the protein carries a domain (s) or epitope (s) that induces protective immunity against experimental infection.
The DNA and deduced amino acid sequences were compared with sequences in Genbank but no significant similarities (no sequences having greater than 22% sequence identity) were found. Thus, the F. necrophorum leukotoxin appears to be distinct from all known leukotoxins and RTX-type toxins. When the deduced amino acid sequence of the lktA region was subjected to the Kyte-Doolittle hydropathy analysis (FIG. 3), 14 sites of sufficient length and hydrophobic character to be potential membrane spanning regions, were found. Upstream to the leukotoxin ORF is an open reading frame of at least 1.4 kb in length, which is in the same orientation. It encodes a protein that has significant sequence similarity (21% or 62 out of 283 residues) to the heme-hemopexin utilization protein (UxuB) of Haemophilus infuenzae.
Bacterial leukotoxins and cytotoxins generally have molecular masses of less than 200 kDa. This includes characterized leukotoxins of Pasteurella hemolytica (104,000 kDa; 10), Staphylococcus aureus (38,000+32,000 kDa; 20), or Actinomyces actinomycetecomitans (114,000 kDa; 15) or other pore-forming toxins of gram-negative bacteria (103,000 to 198,000 kDa; 30). However, leukotoxin secreted by F. necrophorum was shown to be approximately 300 kDa in size based on sephadex column purification and SDS-PAGE analyses.
As used herein, the following definitions will apply: "Sequence Identity" as it is known in the art refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position-by-position basis, e.g., the sequences are "identical" at a particular position if at that position, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give % sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. et al., eds., M. Stockton Press, New York (1991); and Carillo, H., et al. Applied Math., 48:1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "sequence identity" to a reference nucleotide sequence, it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence having at least 95% identity relative to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having a given amino acid sequence having at least, for example, 95% sequence identity to a reference amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 5 amino acid alterations per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 95% sequence identity with a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or the carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in the one or more contiguous groups within the reference sequence. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as a match when determining sequence identity.
Similarly, "sequence homology", as used herein, also refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned as described above, and gaps are introduced if necessary. However, in contrast to "sequence identity", conservative amino acid substitutions are counted as a match when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology with a reference sequence, 95% of the amino acid residues or nucleotides in the reference sequence must match or comprise a conservative substitution with another amino acid or nucleotide, or a number of amino acids or nucleotides up to 5% of the total amino acid residues or nucleotides, not including conservative substitutions, in the reference sequence may be inserted into the reference sequence.
A "conservative substitution" refers to the substitution of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties including size, charge, hydrophobicity, etc., such that the overall functionality does not change significantly.
Isolated" means altered "by the hand of man" from its natural state., i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Finally, all references and teachings cited herein which have not been expressly incorporated by reference are hereby incorporated by reference.
Preferably, sequences having at least about 50% sequence homology or at least about 60% sequence identity with any of SEQ ID Nos. 1-15 are used for purposes of the present invention. More preferably, sequences having at least about 60% sequence homology or at least about 70% sequence identity are used for purposes of the present invention. Still more preferably, sequences having at least about 75% sequence homology or at least about 85% sequence identity are used for purposes of the present invention. Even more preferably, sequences having at least about 87% sequence homology or at least about 92% sequence identity are used for purposes of the present invention. Most preferably, sequences having at least about 95% sequence homology or at least about 98% sequence identity are used for purposes of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a Western blot assay of native and recombinant leukotoxins.
FIG. 2 is an illustration of the full length F. necrophorum gene and a map of the truncated regions of the genes and the expression clones encoded by the truncated regions;
FIG. 3 is a Kyte-Doolittle hydropathy plot of the leukotoxin from F. necrophorum;
FIG. 4 is an illustration of the Southern Hybridization pattern of the chromosomal DNA of F. necrophorum with inserts from clones 513, 611, 816, 911, and 101;
FIG. 5 is a Kyte-Doolittle hydropathy plots of deduced amino acid sequences from the F. necrophorum leukotoxin gene wherein the lines above the plot correspond to the regions of the five truncated LktA polypeptides (BSBSE, SX, GAS, SH, and FINAL);
FIG. 6 is an illustration of the leukotoxin locus of F. necrophorum;
FIG. 7a is a Western blot analysis of truncated forms of purified recombinant leukotoxin protein probed with polyclonal antileukotoxin antiserum;
FIG. 7b is a Western blot analysis of truncated forms of purified recombinant leukotoxin protein probed with monoclonal antibody F7B10;
FIG. 7c is a Western blot of whole-cell lysates from E. coli clones expressing full-length recombinant leukotoxin probed with the monoclonal anti-leukotoxin antibody;
FIG. 8 is a graph illustrating the evaluation of leukotoxic activity by flow cytometry;
FIG. 9 is graph illustrating the toxicity of the recombinant leukotoxin and the truncated polypeptides by flow cytometry;
FIG. 10 is an illustration of the hybridization patterns of radiolabeled lktA with Southern blotted HaeIII digested restriction fragments of genomic DNAs from F. necrophorum subsp. necrophorum isolates from liver abscesses;
FIG. 11 is an illustration of the expression clones for the truncated proteins designated UPS, BSBSE, SX, GAS, SH, and FINAL;
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The following examples set forth preferred embodiments of the present invention. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention.
Cloning of the Leukotoxin Encoding F. necrophorum Gene
Chromosomal DNA, extracted from Fusobacterium necrophorum subsp. necrophorum, strain A25 (Hull et al., 1981, Construction and expression of recombinant plasmids encoding type 1 or D-mannose-resistant pili from a urinary tract infection Escherichia coli isolate. Infect. Immun. 33:933-938.), was partially digested with the restriction endonuclease Sau3AI, and size-fractionated by sucrose gradient centrifugation (Baxter-Gabbard, 1972, A simple method for the large scale preparation of sucrose gradients. FEBS. Lett. 20117-119). The 10-12 kb DNA fragments were ligated in BamHI-digested, dephosphorylated λZAP Express vector, packaged into lambda phage head and tail protein components (Stratagene, La Jolla, Calif.), and recombinant phages were infected into Escherichia coli XL1-Blue MRF' and plated onto agar plates. Plaque lifts were performed (with polyclonal antiserum raised in rabbits against affinity purified leukotoxin) using the Pico-blue immunoscreening kit (Stratagene, La Jolla, Calif.). Six immunoreactive Recombinant phages were identified (816, 611, 513, 911, 101, and 103; FIG. 2). These clones were plaque-purified three times to ensure purity, and anti-leukotoxin immunoreactivity of the proteins was confirmed.
Characterization of the Leukotoxin Gene
Excision of the Cloned DNA Insert Into a Phagemid Vector
The λZAP Express vector is composed of a plasmid, designated pBK-CMV, which flanks the cloned insert DNA and which can be readily excised in order to obtain a phagemid that contains the cloned insert DNA. Therefore, a recombinant phagemid containing cloned F. necrophorum DNA insert was obtained by simultaneously infecting E. coli XLOLR with ExAssist helper phage and the recombinant phage (containing the cloned F. necrophorum DNA) according to the manufacturers instructions (Stratagene, La Jolla, Calif.). Once the recombinant plasmid was recovered, the presence of the DNA insert was confirmed by restriction endonuclease digestion and agarose gel electrophoresis.
Physical Mapping of the F. necrophorum DNA Inserts
Restriction enzyme digestion and mapping of the recombinant phagemid was performed (Sambrook et al., 1989, Molecular cloning: a laboratory manual. Cold spring harbor laboratory, Cold Spring Harbor, N.Y.). Combinations of the restriction enzyme SacI, SalI, SpeI, BamHI, EcoRI, HindIII, PstI, DraI, XbaI, HaeIII, BglII, SmaI, and KpnI were used for restriction enzyme mapping since single sites for these enzymes exist in the multiple cloning site of pBK-CMV. Insert DNA from all the six immunoreactive clones contained EcoRI, PsiI, HindIII, DraI, HaeIII and BglII sites but not sites for Sac I, SmaI, SalI, XbaI, KpnI or BamHI.
Hybridization of the Cloned DNA Fragments with F. necrophorum Chromosomal DNA
Southern hybridization (Southern, 1975, Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503) experiments were performed to confirm that the cloned DNA encoding the putative leukotoxin gene originated from F. necrophorum strain A25. Inserts from clones 513, 611, 816 and 911 were separated from the vector sequence by agarose gel electrophoresis of DNA digested with restriction enzymes SalI and XbaI. The insert DNA was used as a probe to hybridize to chromosomal DNA of F. necrophorum digested with EcoRI, EcoRV, HaeIII, and HindIII. A negative control, E. coli DH5αDNA, was digested with EcoRV. The Southern hybridization patterns included common DNA fragments indicating that the six clones carried overlapping inserts (FIG. 4). FIG. 2 illustrates the overlapping of each of the six immunoreactive clones designated 816, 611, 513, 911, 101, and 103. The expression clones for truncated peptides are designated UPS, BSBSE, SX, GAS, SH, and FINAL while the numbers in parentheses indicate the size in kilo-bases of each insert. The overlaps illustrated in FIG. 2 were further confirmed by sequence analysis.
DNA Sequence Analysis of the F. necrophorum DNA Inserts
Subclones of the cloned insert DNAs were constructed based on the restriction enzyme map of the cloned insert. Plasmid DNA was isolated from the resulting subclones (Birnboim and Doly, 1979, A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic acids Res. 7:1513) and subjected to DNA sequence analysis using the Sanger dideoxy chain termination method (Sanger et al., 1977, DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. 74:5463-5467) using vector based primers. Additional sequence data were obtained by creating deletion clones utilizing restriction endonuclease sites discovered in the preliminary sequencing or by sequencing using primers derived from the sequenced DNA.
A total of 9.3 kb of the leukotoxin chromosomal region was cloned and sequenced. A single large open reading frame (designated lktA) is common to each of the immunoreactive clones. The ORF is preceded by a ribosome binding site (RBS) sequence (AAGGGGGT). Eight base pairs following the RBS sequence is a start codon (the ninth base pair) for the open-reading frame, which is approximately 8 kb in length. The stop codon of lktA was not found in this region. Therefore, the downstream sequences were extended by inverse PCR amplification, followed by cloning and sequencing of the amplified region.
Extension of the lktA Open Reading Frame Using Inverse PCR
Chromosomal DNA from F. necrophorum strain A25 was digested with restriction endonucleases TaqI, EcoRI, DdeI, or Sau3AI individually. After complete digestion of the chromosomal DNA with any one of these enzymes, the products were extracted with phenol and chloroform, and ethanol precipitated. Under dilute conditions (100 μl final volume) 200 ng of digested DNA was self-ligated using T4 DNA ligase at 16° C. overnight (Ochman et al., 1990, Amplification of flanking sequences by inverse PCR. In: M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (eds); PCR protocols; A guide to methods and applications. Acad. Press, Inc. Harcourt Brace Jovanovich, publishers, Sandiego, 219-227). Ligated DNA was phenol and chloroform extracted, ethanol precipitated and reconstituted in 10 μl of nuclease free water. Two microliters of the ligated DNA were used as template for PCR reaction with forward and reverse primers designed based on the sequence already known to us from previous sequencing reactions. Amplified products were cloned in the pCR 2.1 plasmid vector (Invitrogen) and sequenced using vector specific sequences. Sequencing six consecutive inverse PCR products enabled us to identify the stop codon for leukotoxin gene and the presence of another ORF downstream of lktA.
The entire leukotoxin gene was amplified using heat-stable DNA polymerase (ExTaq) as two fragments using F. necrophorum strain A25 chromosomal DNA as the template. The 5'4.3 kb of the lktA open-reading frame encoding the N-terminal half of the leukotoxin, and the 3'5.4 kb representing the C-terminal half of the leukotoxin protein. Making use of the unique Nhe I site present at this location (4.3 kb from the start codon), the leukotoxin gene was joined together to give the giant 9.726 kb ORF. The entire leukotoxin gene was cloned into the modified variant (with coding sequence for six histidine residues in the N-terminus of the expressed protein) of the expression vector pET 14b (Novagen Corp. Madison, Wis.). This T7 polymerase based system should enhance expression of toxic proteins, without damage to the host cell E. coli.
Preparation of Polyclonal Antileukotoxin Antiserum
Leukotoxin from F. necrophorum subsp.necrophorum strain A25 was purified using an immunoaffinity column containing antileukotoxin monoclonal antibody, F7B10 (Tan, Z. L., T. G. Nagaraja, M. M. Chengappa, J. J. Staats. 1994. Purification and quantification of Fusobacterium necrophorum leukotoxin using monoclonal antibodies. Vet. Microbiol. 42:121-133.). Affinity-purified native leukotoxin (0.5 mg) in 100 μl of PBS was homogenized with an equal volume of Freund's complete adjuvant and injected intramuscularly in rabbits. A booster dose was given on day 21 with 0.5 mg of native toxin in 100 μl of PBS homogenized with an equal volume of Freund's incomplete adjuvant. Serum samples were collected on day 42. Naturally occurring rabbit antibodies that react to E. coli proteins were removed from the antisera as follows. Cell pellets of E. coli XL1-Blue MRF' host cells grown overnight in Luria broth were sonicated in PBS and centrifuged to remove cellular debris, and the supernatant was incubated with 100 mm diameter nitrocellulose membranes at 37° C. for 3 hours. The nitrocellulose membranes were then washed twice in PBS-T (0.05% Tween 20 in PBS [pH 7.2]), blocked in 2% BSA, and washed three times again in PBS-T. Two ml of rabbit antileukotoxin polyclonal antiserum were diluted 10-fold in PBS-T containing 0.2% BSA and exposed to 10 changes of E. coli lysate-treated nitrocellulose membranes for 30 minutes duration each at 37° C. The resultant polyclonal antisera had minimal reactivity against E. coli proteins. Neutralizing activity of the serum, as determined by the MTT dye neutralization test and the indirect ELISA titer, were measured as described previously (Tan, Z. L., T. G. Nagaraja, M. M. Chengappa. 1992. Factors affecting leukotoxin activity of Fusobacterium necrophorum. Vet. Microbiol 33:15-28; Tan, Z. L., T. G. Nagaraja, M. M. Chengappa, and J. S. Smith. 1994. Biological and biochemical characterization of Fusobacterium necrophorum leukotoxin. Am. J. Vet. Res. 55:515-519; Tan, Z. L., T. G. Nagaraja, M. M. Chengappa, J. J. Staats. 1994. Purification and quantification of Fusobacterium necrophorum leukotoxin using monoclonal antibodies. Vet. Microbiol. 42:121-133).
Extraction of Genomic Dna from F. Necrophorum and E. Coli
Chromosomal DNA was extracted from highly virulent F. necrophorum subsp. necrophorum, strain A25 (18) and E. coli DH5α (F-X-φ80 Δ[lacZYA-argF] endA1 recA1 hsdR17deoR thi-1 supE44 gyrA96 relA1), using a modification of the method described by Hull and coworkers (Hull, R. A., R. E. Gill, P. Hsu, B. H. Minshew, and S. Falkow. 1981. Construction and expression of recombinant plasmids encoding type 1 or D-mannose-resistant pili from a urinary tract infection Escherichia coli isolate. Infect. Immun. 33:933-938). E. coli was cultured in Luria broth with shaking under aerobic conditions at 37° C. and F. necrophorum was grown overnight in a prereduced anaerobically sterilized brain heart infusion broth in serum bottles under anaerobic conditions at 39° C. Cell pellets were resuspended in TES buffer (25% sucrose, 50 mM Tris-HCl [pH 7.5] and 1 mM EDTA); spheroplasted with lysozyme at room temperature for 30 min; and lysed using sarkosyl in the presence of proteinase K at 60° C. for 1 hour. The product was extracted with buffer-saturated phenol and chloroform, and the DNA was precipitated in 2.5 volumes of ice-cold ethanol. The DNA pellet was resuspended in TE buffer (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA) and subjected to ultracentrifugation in a cesium-chloride step-gradient (43.5% to 60%) containing ethidium bromide (0.4 mg/ml final volume). The chromosomal DNA band was extracted with TE buffer and CsCl saturated isopropanol to remove ethidium bromide and dialyzed against double-distilled water. The DNA concentration and purity were checked spectrophotometrically.
Genomic Library and Screening
Genomic DNA of F. necrophorum A25 was digested partially with restriction endonuclease Sau3AI, and the fragments were size-fractionated in a sucrose gradient. Ten to 12 kb fragments were cloned into BamHI digested and alkaline phosphatase-treated Lambda zap Express vector (Stratagene Corp. La Jolla, Calif.) as per the manufacturer's instructions. Recombinant lambda DNA was packaged (Gigapack gold; Stratagene) and used to infect XL I Blue MRF' host cells (Stratagene). Plaques were lifted onto nitrocelluose membrane and screened with antileukotoxin polyclonal antiserum using a Picoblue immuno-screening kit as per the manufacturer's protocol (Stratagene). Immunoreactive clones were plaque purified three times using the polyclonal antiserum. The recombinant DNA from immunoreactive clones was rescued as phagemid (pBKCMV) clones using Exassist helper phage in E. coli XLOLR strain as per the manufacturer's protocol (Stratagene).
DNA Sequencing Analysis
Phagemids from immunoreactive clones, purified PCR products, and plasmid subclones were sequenced using vector-specific or internal primers with a model 373A automated DNA sequencer (Applied Biosystems, Foster City, Calif.). The DNA sequences were aligned and analyzed using Sequencher (version 3.1.1, Gene Codes Corp., Ann Arbor, Mich.) and DNA Strider (Version 1.2).
Inverse Pcr and Sequence Extension
Chromosomal DNA from F. necrophorum strain A25 was digested singly with restriction endonucleases TaqI, EcoRI, DdeI, or Sau3AI. After complete digestion of the chromosomal DNA with any one of these enzymes, the products were extracted with phenol and chloroform, and precipitated with ethanol. Under dilute conditions (200 ng of digested DNA in 100 μml total volume), DNA was self-ligated using T4 DNA ligase at 16° C. overnight. Ligated DNA was extracted with phenol and chloroform, precipitated with ethanol and reconstituted in 10 ml of nuclease free water. Two microliters of the ligated DNA were used as templates for 100 ml PCR reactions with forward and reverse primers designed based on the sequence obtained from previous sequencing reactions. The products from inverse PCR were cloned in pCR TOPO cloning vectors (TA, Blunt2 or Blunt4) as per the manufacturer's instructions (Invitrogen Corp. San Diego, Calif.), and sequenced directly or after subcloning, using vector specific primers. Six successive inverse PCRs were carried out to reach the 3' end of the leukotoxin gene.
Creation of Gene Truncations
Polymerase chain reaction using thermostable polymerase (EXTaq; Takara Corporation, Madison, Wis.) was used to amplify five overlapping regions of the leukotoxin gene ranging in size from 1.1 kb to 2.8 kb. Chromosomal DNA from F. necrophorum strain A25 was used as the template. The forward primers were designed to contain a SacI site, and the reverse primers had an XmaI site, for in-frame insertion into the His-tag expression vector pQE30 (Qiagen Inc. Valencia, Calif.). Each truncated gene product overlapped with the adjacent product by at least 100 bp. One kb of DNA from the 3' end of the upstream open reading frame (ups) was amplified and cloned in pQE30 vector as described above. Recombinant plasmids were transformed into E. coli host strain M15 for inducible expression of proteins encoded by cloned genes under the control of the lac promoter. The five truncated leukotoxin polypeptides and the C-terminus of the upstream polypeptide were purified using nickel chelation chromatography under denaturing conditions to apparent homogeneity as indicated by silver-stained SDS-PAGE gels (data not shown).
Preparation of Polyclonal Antiserum Against the Truncated Leukotoxin Polypeptides
New-Zealand White rabbits were injected intramuscularly with the five truncated leukotoxin polypeptides or the upstream polypeptide (0.5 mg/animal) precipitated with aluminum hydroxide. A booster dose was given on day 21 (0.5 mg/animal). Serum samples were collected on days 21 and 42 and antileukotoxin titers were determined by indirect ELISA using affinity purified native leukotoxin (Tan, Z. L., T. G. Nagaraja, M. M. Chengappa, J. J. Staats. 1994. Purification and quantification of Fusobacterium necrophorum leukotoxin using monoclonal antibodies. Vet. Microbiol. 42:121-133.). Leukotoxin neutralizing activities of the 42 day serum samples were determined by the MTT dye neutralization assay using 200 units of toxin (id.).
Affinity-purified native leukotoxin, the truncated leukotoxin polypeptides and upstream polypeptide purified over nickel columns, whole cell lysates from bacterial clones carrying recombinant expression plasmids, and concentrated culture supernatants were resolved by SDS-PAGE (6 or 10% acrylamide) and electroblotted to nitrocellulose membranes (BioRad minigel II electrophoresis and transfer unit). Monoclonal antibody against native leukotoxin (F7B10) or polyclonal antisera raised against native leukotoxin, various truncated leukotoxin or upstream polypeptides were used to probe the western blotted proteins. Goat antimouse or antirabbit IgG conjugated to alkaline phosphatase (Sigma Chemical Company, St. Louis, Mo.) was used as the secondary antibody, and the immunoreactive proteins were detected using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate as substrates.
Cloning and Expression of Full-Length Leukotoxin ORF
A 4.3 kb DNA fragment containing the 5' end of the lkta open reading frame up to the internal NheI restriction endonuclease recognition site was amplified from A25 chromosomal DNA. This fragment was cloned into the kanamycin resistance encoding vector pCR Blut II TOPO. A 5.4 kb DNA fragment extending from the NheI site to the 3' end of the lktA open reading frame was PCR amplified and cloned into the low-copy, spectinomycin resistance plasmid pCL1921 (Lerner, C. G., and M. Inouye. 1990. Low copy number plasmids for regulated low level expression of cloned genes in Escherichia coli with blue/white insert screening capability. Nuc. Acid. Res. 18:4631-4633.). The two resulting plasmid clones were ligated together making use of the unique NheI site present in lktA ORF, and the transformants were selected on media containing spectinomycin (100 μg/ml) and kanamycin (21 μg/ml). The pCR Blunt II vector specific sequences were then removed by digesting the resultant plasmid with SacI followed by ligation under dilute conditions and selection on L-agar containing 100 μg/ml spectinomycin. Thus the entire 9,726 base pairs of the leukotoxin ORF were cloned in a low-copy number plasmid pCL1921 to produce pSN1999. Making use of the unique XmaI site introduced into at the 3' end of the open reading frame and the SacI site introduced into the 5' end of the reading frame, the entire lktA coding sequence was cloned in-frame into the expression plasmid pQE30 to give pSN2000.
Flow Cytometric Analysis of Leukotoxin Biological Activity
Bovine peripheral polymorphonuclear leukocytes were isolated as described previously (Tan, Z. L., T. G. Nagaraja, M. M. Chengappa. 1992. Factors affecting leukotoxin activity of Fusobacterium necrophorum. Vet. Microbiol. 33:15-28; Tan, Z. L., T. G. Nagaraja, M. M. Chengappa, and J. S. Smith. 1994. Biological and biochemical characterization of Fusobacterium necrophorum leukotoxin. Am. J. Vet. Res 55:515-519). Untreated cells (negative control) or those treated with either 200 units of native leukotoxin from F. necrophorum (positive control) or whole-cell lysates from clones expressing full-length recombinant leukotoxin were tested for viability by flow cytometry (Facstar, Becton Dickinson Immunocytometry Systems, San Jose, Calif.). Briefly, 1 ml of bovine peripheral PMNs (9×106 cells/ml) was incubated with various preparations of toxin for 45 min at 37° C. in a chamber containing 5% CO2. The cells were then washed twice in 2 ml of HBSS (pH 7.2) and resuspended in 300 μl of HBSS. These cells were treated for 10 min in the dark at room temperature with 10 μl of 5 mg/ml propidium iodide (PI). The red fluorescence (FL-2 [585/42]) is proportional to the number of cells which have lost membrane integrity and, therefore, do not exclude the propidium iodide. Leukocyte subpopulations were displayed in a dot plot and gated according to size based on forward scatter (FSC) and granularity or 90 degree light scatter (SSC). A region was placed around granulocytes, cells of larger size and granularity and thus excluding monocytes, and data were collected on 10,000 gated cells. The identity of the gated cells as granulocytes by was indicated by indirect immunofluorescence labelling with monoclonal antibody DH59B (VMRD Inc., Pullman, Washington) which reacts with the granulocyte-monocyte-1 receptor. Fluorescence signals displayed as a dot plot were used to determine the percent positive cells by quadrant statistics.
Southern Blot Analysis
Genomic DNA was extracted from several strains of F. necrophorum subsp. necrophorum and subsp. funduliforme isolated from ruminal contents or liver abscesses. Chromosomal DNA was digested to completion with HaeIII, which cleaves the leukotoxin ORF once. The digested DNA was electrophoresed in a 1% agarose gel and Southern blotted onto a nitrocellulose membrane. The full-length lktA ORF cloned in pQE30 (pSN2000) was released by digestion with SacI and XmaI, and the insert DNA was gel purified, radiolabelled with [α-35S]dATP, and hybridized.
Nucleotide Sequence Accession Number
The nucleotide sequence of F. necrophorum subsp. necrophorum strain A25 lktA has been assigned GenBank accession number AF312861.
Cloning and Nucleotide Sequence of the F. Necrophorum Leukotoxin Determinant
A Sau3A-generated genomic library of F. necrophorum strain A25 DNA was screened using rabbit polyclonal antisera raised against immunoaffinity-purified native leukotoxin and immunoreactive clones were identified. The clones carried inserts of approximately 4.6, 5.5, and 6.3 kb in length. The immunoreactive clones containing the leukotoxin open reading frame (designated lktA) are depicted in FIG. 1. Inverse PCR was used to extend the cloned region to allow completion of the sequence of the lktA open reading frame. The 11,130 bp sequence of F. necrophorum DNA contained one complete and two partial ORFs. The upstream (orjB) partial ORF comprises the first 1,018 bp. The lktA ORF initiates 16 bp downstream of the lktB ochre codon. A putative ribosome-binding site (RBS) with the sequence AAGGGGGT precedes the lktA ORF. The first two bases of the RBS were the last two bases of the lktB stop codon. The leukotoxin determinant is 9,726 bp and encodes a protein of 3,241 amino acids with an overall molecular weight of 335,956. The deduced protein sequence is unusual in that it lacks cysteine residues. The protein has substantial hydrophobic character (FIG. 5) and possesses 14 regions with sufficient hydrophobic character and length to be membrane spanning. However, this is a secreted toxin in F. necrophorum. The potential transmembrane domains may provide a clue as to the mode of action of the leukotoxin on the target neutrophils.
A BLAST search of the protein database with the deduced leukotoxin did not indicate significant sequence similarity to any bacterial cytotoxins. Some sequence similarity, generally 17-20% amino acid identity over a window of 1,500 to 2,000 residues, was found to certain high molecular weight cell surface proteins. These include the SrpA serine-rich protein from Streptococcus cristatus (accession number U96166), the hemagglutinin from Streptococcus gordonii (AB029393), a surface protein from Xylella fastidiosa (AE003982), the outer membrane protein A from Rickettsia australis (AF149108), the 190 kDa surface antigen precursor from R. rickettsii (A41477), and the high molecular weight antigen (HmwA) of Haemophilus influenzae (AF180944). Given the molecular size of the leukotoxin protein, which is larger than any known bacterial exotoxin, its lack of cysteine residues, and its lack of sequence similarity to other bacterial leukotoxins, the LktA protein from F. necrophorum appears to be a novel leukotoxin.
The deduced amino acid sequence of the carboxy terminus of the OrfB protein has some sequence identity to heme-hemopexin utilization protein (HxuB) of Haemophilus influenzae (21% amino acid identity over a 283 residue window). The putative open reading frame upstream of the leukotoxin determinant does encode a protein product. The 1 kb sequence encoding the carboxyl terminus of this ORF was cloned into pQE30, and the polypeptide was expressed with the six histidine tag at its N-terminus. The protein was purified by nickel chelation chromatography, and the antiserum was raised against this protein in rabbits. Western blot analysis revealed that this antiserum recognized a 60 kDa protein in whole-cell lysates of F. necrophorum (data not shown). This protein was not present in culture supernatants or in purified outer membranes of F. necrophorum.
Downstream of lktA is another apparent open reading frame, which extends to the end of the cloned sequences (375 bp). The putative ATG start codon overlaps the opal stop codon of lktA. The nucleotide and deduced amino acid sequences do not show significant sequence similarity to any sequences currently in GenBank.
Creation of Truncated Leukotoxin Polypeptides and Characteristics of Polyclonal Antisera Raised Against Them
A 3.5 kb sequence from the 5 end of lktA gene was amplified by PCR and cloned in-frame in the expression vector pQE30. Induced expression of this truncated version of the leukotoxin protein with IPTG resulted in the immediate cessation of growth and lysis of the host E. coli cells. In order to obtain better expression of recombinant protein and less toxicity to E. coli host cells, smaller truncations of the leukotoxin gene were constructed. The truncated polypeptides were named BSBSE, SX, GAS, SH, and FINAL starting from the N-terminus and ending at the C-terminus of the leukotoxin protein (FIG. 6). In this Figure, the boxes represent the leukotoxin open reading frame (lktA) and its flanking putative open reading frames. The lines above the boxes represent the phagemid clones (816, 101, and 611) obtained from the immunoreactive plaques in the cloning experiments. The region designated iPCR represents the sequence obtained from sequencing a series of inverse PCR clones. The plasmid pSN2000 contains the entire lktA open reading frame. Below the boxes are the clones expressing the truncated leukotoxin polypeptides. The numbers refer to the nucleotide positions of the boundaries of each truncation relative to the 11,130 bp sequence deposited in GenBank.
Each polypeptide had an overlap of at least 21 amino acids with its adjacent polypeptide. The C-terminal truncated polypeptide of the upstream protein and the polyclonal antiserum raised against it, served as a negative control in our toxicity and toxin-neutralization studies. Purified truncated leukotoxin and upstream polypeptides were then analyzed by western blots, for their reactivity against polyclonal and monoclonal antisera raised against affinity-purified native leukotoxin, using western blot analysis. Antileukotoxin polyclonal antisera reacted strongly with polypeptides BSBSE, SX, and FINAL and weakly with polypeptides GAS and SH (FIG. 7a). Monoclonal antileukotoxin antibody reacted with the N-terminal polypeptide, BSBSE, but not any other truncated leukotoxin polypeptides (FIG. 7b). As expected, the UPS polypeptide did not react with polyclonal or monoclonal antileukotoxin antibodies. Polyclonal antisera raised in rabbits against each of the truncated leukotoxin polypeptides reacted strongly with the corresponding polypeptide and also the native leukotoxin. These results are shown below in Table 1. Antibodies raised against individual truncations reacted weakly to their adjacent polypeptides because of the presence of the overlapping amino acid sequences between them (data not shown). Antiserum raised against UPS (from the upstream ORF) failed to recognize the leukotoxin.
TABLE-US-00001 TABLE 1 Neutralization of Leukotoxin from F. Necrophorum by Rabbit Polyclonal Antisera Raised Against the Recombinant Truncated Polypeptides. ELISA Titer Neutralization Immunogen Self polypeptide Native Leukotoxin Titer UPS 9,600 ± 1,693 19 ± 17 <5 BSBSE 10,420 ± 1,142 10,680 ± 1,653 1,460 ±71 SX 8,754 ± 983 7,480 ± 1,593 <5 GAS 8,748 ± 865 8,100 ± 1,297 1,280 ± 89 SH 10,180 ± 1,789 8,220 ± 1,301 <5 FINAL 9,750 ± 1,343 9,440 ± 1,262 <5 ELISA titers are presented as the mean of three determinations expressed as the reciprocal of the highest dilution giving a positive reaction (± standard deviation). The neutralization titer is the reciprocal of the greatest dilution of antiserum able to neutralize the activity of 200 units of native leukotoxin in an MTT assay.
Antisera raised against the individual polypeptides were tested for neutralization activity against the native leukotoxin from F. necrophorum. An ELISA assay was utilized to measure the reactivity of each antiserum against the leukotoxin. An MTT dye reduction assay was then utilized to determine if the antiserum could neutralize the toxic effects of the leukotoxin against bovine peripheral leukocytes. As shown in Table 1, two of the antisera could neutralize the leukotoxin. The active antisera were raised against the N terminal polypeptide (BSBSE) and the middle polypeptide (GAS). The other three antisera did not have neutralizing activity in this assay, although the ELISA data indicated that each antiserum recognized the F. necrophorum leukotoxin.
Creation of Full-length Recombinant Leukotoxin and its Toxicity to Bovine Peripheral Blood Polymorphonuclear Cells
The entire leukotoxin gene (9,726 bp) was cloned into the pQE30 expression vector. Unlike certain truncated versions of the leukotoxin protein, full-length recombinant leukotoxin upon expression was not toxic to E. coli host cells. When whole-cell lysates from clones expressing full-length leukotoxin were subjected to western blot assays, both polyclonal (not shown) and monoclonal antileukotoxin antibodies reacted to high-molecular weight (>220 kDa) protein species (FIG. 7c). In this Figure, MW is molecular weight markers; Lkt, is affinity-purified leukotoxin from F. necrophorum; FL-I and FL-UI are full-length clone induced or uninduced with IPTG; Super is concentrated F. necrophorum A25 culture supernatant. Additionally, the arrows denote the positions of the reactive BSBSE band in FIG. 7b and the full-length leukotoxin in FIG. 7c. The amount of full-length leukotoxin in the culture supernatant in panel C was insufficient to be visualized as a distinct band in this blot. The protein was extremely unstable, as evident by the presence of numerous smaller molecular weight species, which presumably represent breakdown products. This instability was also observed with native leukotoxin that was immunoaffinity-purified from F. necrophorum culture supernatants. Antisera raised against all the truncated leukotoxin polypeptides, including the C-terminal FINAL polypeptide, reacted to recombinant leukotoxin suggesting that the protein may be expressed in its full-length (data not shown). As expected, antibody raised against the upstream polypeptide failed to react to the full-length recombinant leukotoxin.
Bovine peripheral polymorphonuclear leukocytes exposed to whole-cell lysates of full-length or truncated recombinant clones (12 mg/ml protein) prior to or after induction with IPTG were tested for membrane integrity using propidium iodide exclusion and flow cytometry. Control cells untreated with leukotoxin gave a baseline value of 5.4% PI-staining cells (FIG. 8). In this Figure, membrane damage was assessed by staining of the cells with propidium iodide. Shown are the values obtained after counting 10,000 PMNs (stippled bars) or the lymphocyte fraction (hatched bars). Cells were untreated (control), treated with 200 units of affinity purified leukotoxin from F. necrophorum (Fn leukotoxin) or lysates of E. coli harboring expression plasmids bearing the upstream polypeptide (pSN100) or the full-length lktA open reading frame (pSN2000). The "U" and "I" designations refer to lysates from uninduced cultures and cultures induced with 1 mM IPTG, respectively. Induced lysates were also tested after 1:5, 1:25, and 1:125 dilutions in PBS. The results shown are the averages of three experiments and the standard deviation is indicated.
The addition of 200 MTT units of affinity-purified native leukotoxin resulted in 75.4% of the PMNs taking up the dye. An MTT unit of the toxin is defined as the reciprocal of the dilution causing a 10% decrease in MTT-dye reduction activity. The affinity-purified leukotoxin preparation used in this study had an activity of 2×105 units/ml. Lysates from the clone expressing the upstream polypeptide (SN 100) did not increase the percentage of PI-staining cells, indicating that the truncated form of this protein lacked membrane-damaging activity. Whole-cell lysates from E. coli carrying recombinant full-length leukotoxin gene (SN2000), uninduced with IPTG, gave rise to 9.6% PI-staining bovine PMNs, whereas lysates from induced clones gave 27.3% staining PMNs. The low percentage of damaged cells from the uninduced lysate resulted from leaky expression of the toxin with this vector, consistent with the results obtained by western blot analysis (not shown). The membrane damaging activity in the induced lysate was proportionately lost when the samples were diluted in phosphate-buffered saline. The data indicate that recombinant full-length leukotoxin is toxic to bovine neutrophils.
Preparations of PMNs had residual contaminating cells of smaller size and granularity, which were found to be predominantly lymphocytes by immunophenotyping with anti-CD3 and anti-IgM specific monoclonal antibody. These cells were gated, and the effects of various leukotoxin preparations on the viability of these cells were measured as described for PMNs. Untreated control lymphocytes gave a baseline value of 13.6% staining cells, whereas inclusion of 200 units of affinity-purified native leukotoxin resulted in 31.3% of the lymphocytes taking up the PI (FIG. 8). The apparently lower sensitivity of lymphocytes compared to PMNs is characteristic of F. necrophorum leukotoxin. Furthermore, the recombinant toxin displayed the same degree of activity against lymphocytes as did the native leukotoxin. Among lymphocytes treated with lysates from E. coli carrying uninduced recombinant full-length lktA, 12.8% were PI-positive compared to 19.2% obtained with lysates from induced clones. Thus the expressed recombinant leukotoxin had toxicological properties similar to those of the native leukotoxin purified from F. necrophorum culture supernatant. Lysates from E. coli with IPTG-induced expression of the leukotoxin truncated polypeptides or the upstream polypeptide did not display membrane-damaging activity against either bovine PMNs or the lymphocyte-containing population (FIG. 9). In this Figure, membrane damage was assessed by staining of the cells with propidium iodide. Shown are the values obtained after counting 10,000 PMNs (stippled bars) or the lymphocyte fraction (hatched bars). Cells were untreated (control), treated with 200 units of affinity purified leukotoxin from F. necrophorum (native toxin), lysates from IPTG-induced cultures of clones expressing the truncated polypeptides (ups, BSBSE, SX, GAS, SH, and Final) or the whole recombinant leukotoxin (whole toxin). The results shown are the averages of three experiments and the standard deviation is indicated.
Presence of the Leukotoxin Determinant in F. Necrophorum Isolates
The leukotoxin gene was cloned and sequenced from F. necrophorum subsp. necrophorum A25, a strain originally isolated from a bovine liver abscess. Southern blot hybridization of the chromosomal DNA extracted from various F. necrophorum strains of both subspecies isolated from ruminal contents or liver abscesses was carried out using the leukotoxin ORF as a probe (FIG. 10). In this Figure, F. necrophorum subsp. necrophorum from liver abscesses are in lane 1 which is strain A21; lane 2 which is A25; and lane 3 which is A39. F. necrophorum subsp. necrophorum from ruminal contents are in lane 7 which is RA13; lane 8 which is RA15; lane 9 which is RA16; lane 10 which is RA18; lane 11 which is RA26; lane 12 which is RA28; and lane 13 which is RA29. The F. necrophorum subsp. funduliforme isolates from liver abscesses are in lane 4 which is B17; lane 5 which is B29; lane 6 which is B35 or ruminal contents which are in lane 14 which is RB33; and lane 15 which is RB37. Strains are described in reference 24. M, DNA molecular weight markers. The restriction endonuclease HaeIII was used to digest the chromosomal DNA from F. necrophorum isolates. A single recognition site for this enzyme occurs 5,933 bp from the start codon in the lktA ORF. Thus, two hybridizing fragments should be present in strains carrying this gene. All strains of F. necrophorum subsp. funduliforme isolated from liver abscesses (B17, B29, and B35) or ruminal contents (RB33 and RB37) were identical in their hybridization patterns showing two bands at approximately 7 and 8 kb each. Also, all isolates of F. necrophorum subsp. necrophorum, except A39, isolated from liver abscesses (A21 and A25) and those isolated from ruminal contents (RA13, RA15, RA16, RA18, RA26, RA28, and RA29) had identical hybridization patterns showing two bands of approximately 10 and 11 kb each. A single band of approximately 10.5 kb, presumably a doublet, hybridized to the leukotoxin gene in chromosomal DNA of strain A39 (FIG. 10, lane 4). This suggests that some heterogeneity may be present in the leukotoxin locus sequences among strains of F. necrophorum subsp. necrophorum. However, the hybridization pattern does appear to be a good indicator for subspecies determination.
Construction of Truncated Forms of the Leukotoxin
A 3.5 kb sequence from the 5' end of lktA gene was amplified by PCR and cloned in-frame in the expression vector pQE 30 (Qiagen Corporation). Induced expression of this truncated version of the leukotoxin protein with IPTG resulted in the immediate cessation of growth and caused lysis of the host E. coli cells. In order to obtain better expression of recombinant protein, smaller truncations of the leukotoxin gene were constructed. Polymerase chain reaction using thermostable polymerase with proof reading ability (EXTaq; Takara Corp.) was used to amplify five overlapping regions of the leukotoxin gene. The forward primers were designed to contain a SacI site, and the reverse primers had a XmaI site. F. necrophorum A25 chromosomal DNA was used as the template, and the amplified products were digested with restriction enzymes SacI and XmaI, and cloned in-frame in the His-tag expression vector pQE 30. Five truncated leukotoxin proteins and the C-terminus of the upstream protein were purified using nickel chelation chromatography to apparent homogeneity as indicated by silver-stained SDS-PAGE gels. The proteins were then tested for their reactivity with polyclonal antisera raised in rabbits against affinity purified native leukotoxin using western blot analysis. Purified proteins were injected in rabbits to produce polyclonal antisera, which in turn were used to carry out western blot analysis and neutralization tests (Table 2). Antisera raised against each protein recognized native leukotoxin from F. necrophorum. Antisera directed against the BSBSE9 and GAS polypeptides were able to neutralize the activity of native leukotoxin. Thus the cloned ORF does indeed represent the F. necrophorum leukotoxin.
TABLE-US-00002 TABLE 2 Characterization of the Truncated Upstream and Leukotoxin Proteins Antisera Truncated Recognized by Antisera Raised Neutralizes Leukotoxin Number Anti-native Against Truncated Activity of Proteins (N to C of Amino Size (in Leukotoxin Proteins Recognized Leukotoxin terminal) Acids Daltons) Antibodies Native Leukotoxin Against PMNs UPS 9 339 38324 - - - BSBSE 9 377 40810 + + + SX7 926 97453 + + - GAS 15 713 71949 - + + SH 12 628 63457 - + - FINAL 2 774 80590 + + -
Production of an Inactivated Recombinant Leukotoxin Vaccine
The immunogenicity and protective immunity of the recombinant full length and truncated leukotoxin proteins is determined in mice and compared to the efficacy of immunization with inactivated native leukotoxin in F. necrophorum culture supernatant. The usefulness of the mouse model in studying experimental Fusobacterium infections has been well documented (Abe et al., 1986, Emery and Vaughn, 1986).
Purified recombinant leukotoxins (described above) including the full-length protein are inactivated by the addition of formalin (final concentration 0.3%) and homogenized with Ribi or other suitable adjuvant (10% vol/vol; Ribi Immunochem, Hamilton, Mont.). The native leukotoxoid vaccine is prepared with culture supernatant from F. necrophorum subsp. necrophorum, strain A25 grown in PRAS-BHI broth (Saginala et al., 1997). The leukotoxic activities of the recombinant leukotoxin and culture supernatant, before and after formalin inactivation, are then tested by MTT-dye reduction assay using bovine polymorphonuclear (PMN) leukocytes as target cells (Tan et al., 1992). The quantity of native leukotoxin is then assayed using a sandwich ELISA using purified monoclonal antibody (Tan et al., 1994b).
Immunogenicity of the Inactivated Recombinant Leukotoxin in Mice
Immunogenicity and protective effects of the inactivated recombinant full length, and truncated leukotoxins are evaluated in comparison with the native leukotoxin (culture supernatant of F. necrophorum, strain A25). Five overlapping truncations and the recombinant full-length leukotoxin are purified using the nickel-affinity columns. The treatment groups include control (0.2 ml PBS), native leukotoxin, recombinant full length, and truncated leukotoxins individually or in combination (all five truncations individually, and a mixture of all five truncated proteins in equimolar ratio). Additionally, a mixture of the two truncated proteins BSBSE and GAS in equimolar concentrations is tested for immunogenicity, because polyclonal antisera raised against these two proteins neutralize the activity of native leukotoxin against bovine neutrophils. Each leukotoxin preparation is tested at 10 and 50 μg doses (total protein concentration), administered subcutaneously on days 0 and 21. Six mice (7-8 wk old BALB/c) are used in each treatment group. Blood samples are collected on days 0, 14, 21, 35, and 42. Serum is stored at -70° C. until assayed for antileukotoxin antibody. After the last blood sampling (on day 42), mice are challenged intraperitoneally with 0.4 ml of late-log phase F. necrophorum strain A25 culture (6-7 hour culture in PRAS-BHI broth with an absorbance of 0.65 at 600 nm and with a cell concentration of approximately 1 to 5×108 CFU/ml). The number of bacteria used for inoculation is enumerated by viable counts on blood agar plates in an anaerobic glove Box (Form a Scientific, Marietta, Ohio). Mice are observed for 4 days after challenge to record mortality and clinical signs, and those that survive the challenge are euthanized. Mice are then necropsied and examined grossly for abscesses in the liver. Additionally, other organs and liver tissue will be cultured for anaerobic bacterial isolation.
Following this study, the efficacious dose and the recombinant leukotoxin preparation is selected and one more immunization and challenge study in mice to confirm the protective effect of recombinant leukotoxin is conducted. Groups of 7-8 week old BALB/c mice (10 per group) are used and each group receives one of the following leukotoxin preparations: most immunogenic recombinant leukotoxin protein, combination (two or more) of most immunogenic recombinant leukotoxin proteins, and native leukotoxin (F. necrophorurm culture supernatant). The leukotoxin proteins are inactivated with 0.3% formalin, mixed with Ribi or any other suitable adjuvant and emulsified with a homogenizer and administered subcutaneously on days 0 and 21. Blood samples are collected on days 0, 14, 21, 35 and 42. Serum samples are assayed for antileukotoxin antibody. After the last blood sampling (on day 42), mice are challenged as described above. Overlapping variants of effective polypeptides (the truncated protein fragments) are identified and are constructed in order to identify the polypeptide sequences that are most effective in conferring protection.
Determination of Antileukotoxin Antibody Induced by Immunization
Mouse serum is analyzed for antileukotoxin antibody by two methods. First, serum samples are assayed for leukotoxin neutralizing antibody by testing its ability to neutralize the toxin using the MTT dye reduction assay with mouse and bovine PMNs as the target cells (Saginala, et al., 1996b; Tan et al., 1994a). Second, serum samples are tested for anti-leukotoxin IgG antibodies by enzyme linked immunosorbent assay (ELISA) using affinity-purified leukotoxin as the coating antigen. Affinity purification of the leukotoxin is carried out using monoclonal antibody MAbF7B10 (Tan et al., 1994b).
DNA Extraction and Polymerase Chain Reaction
Chromosomal DNA was isolated from F. necrophorum subspecies necrophorum, strain A25. Briefly, F. necrophorum was grown overnight in a PRAS-BHI broth in serum bottles at 39° C. Cell pellets were resuspended in TES buffer (25% sucrose, 50 mM Tris-HCl [pH 7.5] and 1 mM EDTA), spheroplasted with lysozyme at room temperature for 30 min, and lysed using sarkosyl in the presence of proteinase K at 60° C. for 1 hour. The DNA was extracted with buffer-saturated phenol and chloroform and was precipitated in 2.5 volumes of ice-cold ethanol and 1/10 volume of sodium acetate (3 M, pH 5.2). The DNA pellet was resuspended in TE buffer (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA) and was run for 20 hours in a cesium-chloride gradient (60% to 43.5%) containing ethidium bromide (0.4 mg/ml final volume). The chromosomal DNA band was extracted with cesium-chloride saturated isopropanol to remove ethidium bromide and dialyzed against double distilled water. DNA concentration and purity were checked spectrophotometrically.
The primers were designed to amplify the leukotoxin gene as five overlapping truncations (Table 3). The sites for annealing of the primers were chosen, so that there is an overlap of approximately 100 bp with the adjacent truncated leukotoxin gene product. Each forward primer was designed to contain a SacI site and reverse primers carried a XmaI site (Table 3). PCR amplifications were carried out under following conditions using a thermostable DNA polymerase with a proof-reading function ExTaq (Takara Corp., Madison, Wis.): initial denaturation 94° C. for 3 min; 36 cycles of denaturation 94° C. for 1 min, 59° C. for 45 sec, 67° C. for 30 sec, and 72° C. for 1 to 3 min (at min per kb), and a final extension at 72° C. for 4 min.
TABLE-US-00003 TABLE 3 PCR primers used for amplifying truncated leukotoxin gene segments. Location Truncated in lktA Desig- segment gene (bp) nation Primer Sequencea bsbse 1-22 BS-START tccgagctcATGAGCGGCA TCAAAAATAACG 1130-1112 BS-END tcgccccgggATAGGAGAA ATAGAACCTG sx 919-940 SX-START tccgagctcGGGAGATTTA TAAAGAAAGAAG 3698-3679 SX-END tcgccccgggGATCCGCCC CATGCTCCAAC gas 3553-3572 GAS-START tccgagctcGGAGCTTCTG GAAGTGTTTC 5693-5674 GAS-END tcgccccgggGTACTATTT TTTATATGTGC sh 5623-5641 SH-START tccgagctcGCTGCAGTAG GAGCTGGAG 7510-7492 SH-END tcgccccgggCTGCAGTTC CCAAACCACC final 7405-7425 FIN-START tccgagctcGGAATTAAAG CCATTGTGAAG 9726-9706 FIN-END tcgccccgggTCATTTTTT CCCTTTTTCTCC aLower case letters in primer sequences represent extra bases added to incorporate restriction sites.
Directional Cloning in an Expression Vector
The amplified gene products which are overlapping truncations extending from 5' to 3' end of the leukotoxin gene (lktA), were named BSBSE, SX, GAS, SH, and FINAL (FIG. 11). In this Figure the numbers in parentheses indicate the size in kilobases of each insert. They were extracted with phenol and chloroform and precipitated with ethanol as described above. The amplified lktA gene products and expression vector pQE30 (Qiagen Corp., Valencia, Calif.) were digested with restriction endonucleases SacI and XmaI as per manufacturer's instructions (New England Biolabs, Beverly, Mass.). After digestion, the vector and insert DNA were phenol and chloroform extracted, ethanol precipitated, and ligated overnight at 16° C. using T4 DNA ligase (Promega Corp., Madison, Wis.). Ligated DNA was digested with restriction enzyme KpnI before transforming chemically competent E. coli M15 cells as per standard procedures. Restriction sites for KpnI is absent in the entire lktA gene and present in a single location between SacI and XmaI sites in pQE 30. The expression vector pQE 30 lacks blue/white selection, thus the above manipulation helped us to enrich clones that carry truncated leukotoxin gene products. The transformants were plated on Luria-agar plates containing ampicillin (100 ug/ml) and kanamycin (20 ug/ml) to select for clones containing plasmids pQE30 and pRep4.
Expression of Truncated Leukotoxin Polypeptides
Plasmid DNA from the transformants were purified using Wizard SV miniprep columns (Promega), and the orientation of the insert was checked by sequencing with a vector specific 5'QE primer which anneals upstream to the MCS using a Applied Biosystems 373A automated sequencer. Positive clones were induced for the expression of polypeptides with IPTG, the whole cell lysates from uninduced and induced were compared for immunoreactive polypeptides in a western-blot using polyclonal antisera raised in rabbits against affinity purified native leukotoxin (Tan et al, 1994d).
Due to the presence of its codons in the sequence upstream of the MCS in the vector pQE 30, six histidine residues are added in the N-terminus of the expressed polypeptides. The expressed polypeptides were purified using nickel-affinity columns under denaturing conditions using guanidium hydrochloride, as per the manufacturer's instructions (Qiagen). The column purified polypeptides were dialyzed for 48 hours at 4° C. against sterile phosphate buffered saline (0.1 M, pH 7.2) to remove any traces of urea, and concentrated in Ultrafree-Biomax 30 filters (Millipore Corp. Bedford, Mass.), which retains molecules of sizes over 30 kDa. The protein concentrations were analyzed using the BCA assay (Pierce, Rockfort, Ill.) and the purity checked with SDS-PAGE analysis followed by silver staining. Native leukotoxin from F. necrophorum culture supernatant was purified using immunoaffinity columns with anti-leukotoxin monoclonal antibody (F7B10) as described previously. Also, leukotoxoid vaccine (12 hours culture supernatant inactivated with 0.3% formaldehyde) was made as described previously (Saginala et al., 1997).
Preparation of Polyclonal Antiserum Against Truncated Polypeptides
Five New-Zealand White rabbits were injected intramuscularly with the five truncated leukotoxin polypeptides (0.5 mg/animal) precipitated with aluminum hydroxide. A booster dose was given on day 21 (0.5 mg/animal). Serum samples were collected on days 21 and 42 and antileukotoxin titers were determined by indirect ELISA using affinity purified native leukotoxin. Leukotoxin neutralizing activities of the 42 day serum samples were determined by the MTT dye neutralization assay. A neutralization ratio, which was the dilution of the antiserum that caused neutralization divided by its ELISA titer, was calculated for each truncated polypeptide.
Vaccine and Immunization
One hundred (100) 8 to 10 week old mice, identified by ear-markings, were randomly divided into 10 groups of 10 mice each. The groups received five truncated leukotoxin polypeptides (BSBSE, SX, GAS, SH, and FINAL) individually, a mixture of BSBSE and GAS, a mixture of all five truncated polypeptides, affinity purified native leukotoxin, inactivated culture supernatant, or PBS emulsified with Ribi adjuvant. Each mouse was injected subcutaneously (in two locations of 100 μl each between the shoulder blades) on day 0 and day 21 with 200 μl of one of the above preparations. The total amount of antigen in each injection (except with culture supernatant or PBS) was 10 μg per animal. Inactivated culture supernatant (12 mg/ml protein concentration) was used without dilution to reconstitute Ribi adjuvant (Ribi Immunochem, Hamilton, Mont.) and each mouse was injected with 200 μl (2.4 mg protein) of the emulsified preparation. Negative control group received 200 μl of PBS emulsified with the Ribi adjuvant.
Determination of Antileukotoxin Antibodies Induced by Immunization
Blood for serum separation was collected from the right saphenous vein of each mouse on days 0, 21 and 42, and directly from the heart after euthanasia. Antileukotoxin antibody titers were assayed by an indirect ELISA as described previously with slight modifications. Briefly, 96-well microtiter plates (Falcon Probind assay plates, Beckton Dickinson Labware, Lincoln Park, N.J.) were coated with 50 μl (2 μg/ml) per well of affinity purified native leukotoxin at 37° C. for 2 hours. The wells were blocked with 3% bovine serum albumin (Sigma Chemical Company, St. Louis, Mo.) in PBS at 37° C. for 2 hours. Fifty μl of a 1 in 25 dilution of serum samples in PBS-T (0.05% Tween 20 in PBS) were added in duplicate and the plates were incubated at 37° C. for 1 hour. Following 6 washes with PBS-T, 100 μl of biotinylated goat anti-mouse immunoglobulin (Accurate Chemicals and Scientific Corp., Westbury, N.Y.) was added to each well and incubated at 37° C. for 1 hour. The plates were washed 6 times with PBS-T and 50 μl of streptavidin conjugated with horseradish peroxidase was added to each well, and incubated at 37° C. for 1 hour. After washing the wells 6 times with PBS-T, 100 μl of ABTS substrate (2,2'-azino-di-[3-ethyl-benzthiazoline-6-sulfonic acid]; Sigma) and H2O2 in phosphate-citrate buffer (pH 4.0) was added to each well, and the plates were incubated for 30 min, or until color development, at room temperature. The absorbance was measured clorimetrically at 410 nm in a 96-well plate reader (Molecular Devices, California).
Experimental Challenge with Fusobacterium necrophorum
Fusobacterium necrophorum subsp. necrophorum, strain A25 was grown to an OD600 of 0.7 in PRAS-BHI broth and 0.4 ml of this late-log-phase culture was injected intraperitoneally in mice. The inoculum had a bacterial concentration of 4.7×108 CFU/ml as determined by spread-plating on blood agar plates (Remel, Lenexa, Kans.) incubated in an anaerobic glove box (Form a Scientific, Marietta, Ohio). Mice were observed for 4 days post-challenge to record clinical signs and mortality. Mice that survived for 4 days post-challenge were euthanized, necropsied and examined for the presence of abscesses in liver and other internal organs.
Enumeration of Fusobacterium necrophorum Load in the Liver
Livers from mice were collected at necropsy, weighed and homogenized in a tissue homogenizer for 1 min in PRAS-BHI broth. A 10-fold dilution of the homogenate was taken inside an anaerobic Glove box for further processing. Two hundred μl of modified lactate medium was dispensed into each well of the 96-well tissue culture plate (Falcon, Beckton Dickinson Labware, Lincoln Park, N.J.). Fifty μl of 1 in 10 dilution of homogenated liver was transferred to the wells on the first lane (8 wells) and serially diluted (five-fold) up to the eleventh well. The wells in the 12th lane were negative controls. The plates were incubated in a Glove box at 39° C. for 48 hours. Kovac's reagent (20 μls each) was added to each well to detect indole production, presumptive of F. necrophorum. The bacterial load of F. necrophorum in liver was enumerated by most probable number (MPN) analysis (Rowe, R., Todd, R., and Waide, J. 1977. Microtechnique for most-probable-number analysis. Appl. Environ. Microbiol. 33:675-680.). Homogenized liver tissue samples were also streaked on blood agar plates and colonies identified using Rapid ANAII system (Innovative Diagnostic Systems, Norcross, Ga.).
Serum ELISA measurements (absorbance values per ml of serum) were analyzed using Proc Mixed procedure of SAS (SAS systems, Cary, N.C.). The weights of liver and bacterial counts, log-transformed, were analyzed using PROC GLM program of SAS. P-values less than 0.01 were considered significant.
Cloning and Expression of Leukotoxin Gene Truncations
In-frame cloning of the PCR amplified truncations of the leukotoxin gene (lktA) in plasmid pQE 30 was carried out as described above by incorporating restriction sites for Sac and XmaI in the forward and reverse primers respectively. Inducing the clones carrying various truncations did not produce inclusion bodies in the E. coli host cells. However, purification of the expressed polypeptides under native conditions was unsuccessful. Therefore, polypeptides were purified using nickel affinity columns after denaturation with guanidium isothiocyanate. The denatured truncated polypeptides, after dialysis against PBS, lacked toxicity to PMNs.
Antileukotoxin Antibody Titers in Rabbits.
The anti-leukotoxin antibody titers in rabbits injected with truncated polypeptides are shown below in Table 4. Antisera raised against truncated leukotoxin polypeptides, BSBSE and GAS, neutralized the toxicity of affinity purified native leukotoxin against bovine peripheral PMNs. The neutralizing activities for polyclonal antisera raised against BSBSE and GAS were similar as evident from their identical neutralization ratios (0.146).
TABLE-US-00004 TABLE 4 Anti leukotoxin antibody titers in rabbits injected with truncated leukotoxin proteins Neutral- LISA Titer ization Neutral- Truncated Size (in LISA titer on day 42 titer on ization proteins daltons) on day 21 (b) day 42 (a) ratio (a/b) BSBSE 40810 1250 10000 1460 0.146 SX 97453 1000 8750 0 0 GAS 71949 1150 8750 1280 0.146 SH 63457 1000 10000 0 0 FINAL 80590 875 9750 0 0
Anti-Leukotoxin Antibody Response in Mice
The mean absorbances per ml of serum, determined by ELISA, from mice vaccinated with various leukotoxin polypeptides are shown in Table 5.
TABLE-US-00005 TABLE 5 Anti-leukotoxin antibody response in mice injected with various leukotoxin preparations. D 46 Vaccine Preparations D 0 D 21 D 42 (post-mortem) PBS 63.6a 65.3a 66.9a 126.3d BSBSE 52.9a 90.2b 179.4c* 129.1d SX 54.1a 77.6ab 186.4c* 144.5d GAS 61.0a 77.6ab 97.1bc* 109.6cd SH 60.95a 101b* 163.8c* 130.0d FINAL 63.9a 66.2ab 95.7bc* 121.7cd BSBSE + GAS 79.7a 82.5a 161.1c* 172.7cd* ALL FIVE 66.1a 98.9b* 189c* 219d* Native Leukotoxin 59.6a 101.3b* 235.5c* 205.2d* Culture Supernatant 76.4a 105.7b* 205.4c* 230.1cd* Numbers with same superscripts were not significantly different from the ELISA values from mice belonging to same group at a different sampling period. *Significantly different from negative control (PBS).
On day 21, mice vaccinated with affinity purified native leukotoxin, truncations BSBSE or SH, mixture of all five, or culture supernatant had higher antileukotoxin antibody levels compared to day 0, Serum collected on day 21 from groups vaccinated with truncated polypeptide SH, mixture of five truncations, native affinity purified leukotoxin or culture supernatant, had significantly higher anti-leukotoxin antibody levels compared to the control (PBS) group (p<0.01). There was no significant rise in the antibody levels on day 21 among mice vaccinated with truncated polypeptides SX, GAS, FINAL, a combination of BSBSE and GAS or PBS. Mice belonging to group that was vaccinated with culture supernatant, had significantly higher (P<0.01) antibody titers to leukotoxin than mice in other groups.
On day 42, there was a significant increase in antibody response compared to day 21 among mice vaccinated with all leukotoxin preparations except GAS (P<0.01). Anti-leukotoxin antibody levels in serum from mice vaccinated with different leukotoxin polypeptides (including GAS) were significantly higher compared to the control. The antibody response to a mixture of BSBSE+GAS was similar to BSBSE alone but higher than GAS polypeptide. The antibody response to mixture of all five was similar to BSBSE, SX, SH but higher than GAS or FINAL polypeptides. Mice vaccinated with affinity purified native leukotoxin had the highest anti-leukotoxin antibody levels on day 42, followed by those vaccinated with the culture supernatant and a mixture of all five overlapping truncations. The truncated polypeptide GAS failed to raise anti-leukotoxin antibody levels significantly after the second vaccination compared to the day 21.
On day 46, 4 days after challenge with F. necrophorum (post-mortem), serum samples from mice vaccinated with leukotoxin polypeptides, BSBSE, SX, and SH, and affinity purified native leukotoxin had lower anti-leukotoxin antibody titers compared to day 42. Anti-leukotoxin antibody levels in mice vaccinated with GAS, FINAL, mixture of truncated polypeptides or culture supernatant had higher antibody levels compared to day 42. Also, anti-leukotoxin antibody levels in mice in the control group (vaccinated with PBS) on day 46 showed a significant increase than serum collected before challenge (day 42). However, antibody levels in mice injected with BSBSE+GAS, mixture of all five, native leukotoxin and culture supernatant were higher than the control group.
Following the challenge with F. necrophorum, mice in all groups exhibited acute shock within 24 hours perhaps induced by LPS. Mice in the control or in the group vaccinated with inactivated culture supernatant seemed to be affected most. The mice were listless, recumbent and did not seem to consume food or water. Mice vaccinated with various leukotoxin preparations recovered after 2 days post-challenge. Mice in the control group did not recover completely from the symptoms of shock even by day 4 after challenge. Two mice in the control group and one mouse in the group vaccinated with GAS polypeptide died about 36 hours after challenge. Pure cultures of F. necrophorum subsp. necrophorum were isolated from the heart blood of all three mice.
Mice were euthanized 4 days after challenge and the internal organs were examined for abscesses. None of the mice vaccinated with leukotoxin truncation SH had any liver abscesses (Table 6).
TABLE-US-00006 TABLE 6 Mortality, liver abscess formation, weight of liver and bacterial load in liver in mice vaccinated with leukotoxin preparations after experimental challenge with Fusobacterium necrophorum. Number No. of mice Average MPN counts Leukotoxin of dead with liver weight of in the preparations mice abscess (%) liver(g) liver Control (PBS) 2/10 0/8 (0)a 1.86 5.3 × 106 BSBSE 0/10 1/10 (10) 1.29* 1.2 × 103* SX 0/10 5/10 (50) 1.39* 8.2 × 105* GAS 1/10 3/9 (33) 1.32* 1.5 × 106 SH 0/10 0/10 (0) 1.20* 5.3 × 102* FINAL 0/10 3/10 (30) 1.44* 6.8 × 105* BSBSE + GAS 0/10 3/10 (30) 1.27* 1.4 × 105* ALL FIVE 0/10 3/10 (30) 1.33* 5.5 × 105* Native leukotoxin 0/10 3/10 (30) 1.31* 5.9 × 104* Culture supernatant 0/10 1/10 (10) 1.51* 1.6 × 104* *Differs from the control group (P < 0.01) aLivers lacked abscesses, but were highly congested and icteric.
The eight mice that survived in the control group had highly congested and icteric livers, but had no abscesses. Thirty percent of mice vaccinated with affinity purified native leukotoxin, truncations GAS or FINAL, or mixtures (BSBSE and GAS, or all five truncations) had liver abscesses. Five out of ten mice vaccinated with leukotoxin truncated polypeptide SX developed liver abscesses. However, in the groups vaccinated with the truncated leukotoxin polypeptide B SB SE or inactivated culture supernatant, only one out of 10 had liver abscesses.
The mean weight of livers from the control group was significantly higher than mean weights of livers from other groups. Livers from the group that received inactivated culture supernatant had the next biggest liver size. This correlated with the clinical signs of acute shock displayed by these two groups.
Enumeration of F. necrophorum in Liver Tissue
Fusobacterium necrophorum subsp. necrophorum was isolated from homogenized liver tissue and abscesses from all mice. The counts of F. necrophorum from livers of mice injected with any leukotoxin preparation were lower (p<0.01) than the control (Table 6). Livers from mice vaccinated with leukotoxin truncations BSBSE or SH showed significantly lower bacterial counts (p<0.01) than mice vaccinated with other preparations. Among leukotoxin truncations, SX showed least protection followed by FINAL and GAS polypeptides as evidenced by the bacterial counts in the livers of mice vaccinated with these polypeptides. Bacterial counts were considerably lower among groups vaccinated with mixtures of leukotoxin truncations (BSBSE and GAS or all five truncations), or affinity purified native leukotoxin as compared to the control group but higher than SH, BSBSE or inactivated culture supernatant (Table 6).
The five overlapping truncated leukotoxin polypeptides created allowed expression of the entire leukotoxin gene without toxicity to the E. coli host cells. Primers for the amplification of various truncated leukotoxin gene products were designed in such a way that the expressed polypeptides were not toxic to E. coli host cells, but were big enough (at least 30 kDa) to be a good immunogen. The nickel affinity column purified polypeptides were tested for purity in terms of contaminating proteins or lipopolysaccharides by silver-staining the SDS-PAGE separated proteins. Because all truncated polypeptides were purified under denaturing conditions, they were not toxic as determined by the MTT assays. Fusobacterium necrophorum culture supernatant and affinity purified native leukotoxin were inactivated with 0.3% formalin before injection, thus were nontoxic.
Neutralization of toxicity of F. necrophorum leukotoxin against bovine peripheral PMNs by antiserum raised against BSBSE and GAS polypeptides suggested that biologically important domains, such as those responsible for toxicity or host cell receptor binding was located in these regions. Therefore, a mixture of these two polypeptides (BSBSE+GAS) was also used in a vaccine preparation in our challenge experiments with mice.
The significantly higher antibody levels noticed among groups vaccinated with preparations containing full-length leukotoxin proteins (native affinity purified leukotoxin, culture supernatant, or a mixture of recombinant leukotoxin polypeptides containing all five truncations) may be due to determinant spreading, or due to augmentation of anti-leukotoxin antibody response by the presence of multiple immunodominant epitopes on the leukotoxin protein. Truncated leukotoxin GAS produced a low antibody response. The high hydrophobicity of this polypeptide may be the reason for its reduced immunogenicity. Also, the wells in the ELISA plates were coated with native immunoaffinity purified leukotoxin, and the domains represented by the GAS polypeptide could possibly be hidden and not exposed for the antibodies against GAS polypeptide to bind.
Decrease in anti-leukotoxin antibody levels among various groups of mice on day 46 (4 days after experimental challenge with F. necrophorum) suggested neutralizing effect and clearance of toxin secreted by F. necrophorum used for experimental challenge by these antibodies. Pure cultures of F. necrophorum subsp. necrophorum were isolated from the heart blood of the three mice (two from negative control group and one from group injected with GAS polypeptide) that died on day 2 after challenge, suggesting that death was due to septicemia induced by F. necrophorum. The hepatic tissue from the negative control group showed inflammation, congestion and icterus characteristic of an acute phase response, but showed no abscesses.
Multiple responses including mortality, clinical signs, weights of liver, presence of abscesses, and the bacterial load in liver were considered to evaluate the effectiveness of various vaccine preparations in providing immunity and protection against experimental challenge with F. necrophorum. Leukotoxin truncation SH was a very effective immunogen as evidenced by a rise in anti-leukotoxin antibody levels in serum samples on day 21 or 42. Also, there were no mortality, hepatic inflammation or abscesses in mice vaccinated with this polypeptide after experimental challenge. The mean bacterial load in the livers of mice from this group was the lowest (5.3×102). Interestingly, leukotoxin truncated polypeptide SH did not induce neutralizing antibodies in rabbits. Production of high-affinity antibodies against certain immunodominant domains that brings about effective opsonization and clearance of leukotoxin in an experimental challenge model may render this truncated polypeptide (SH) a protective antigen.
Vaccination with N-terminal truncation BSBSE or culture supernatant followed by experimental challenge with F. necrophorum caused no mortality, but livers were abscessed in 10% of the mice. Mice vaccinated with BSBSE, however, had less clinical signs of LPS induced shock after vaccinations or challenge, lower liver weights and lower hepatic-bacterial counts compared to mice vaccinated with inactivated culture supernatant.
Native leukotoxin purified by immunoaffinity columns from F. necrophorum culture supernatant was the fourth best vaccine preparation (behind SH, BSBSE, and culture supernatant) in terms of serum antibody levels, protection against formation of liver abscess (30%), and number of bacteria in the liver tissue. The vaccine consisting of a mixture of all five recombinant truncated leukotoxin polypeptides also protected 70% of mice from abscess formation and the bacterial counts in their hepatic tissue were not significantly different from mice that were vaccinated with native leukotoxin.
Truncated polypeptide GAS, although it invoked neutralizing antibodies in rabbits, was a poorer immunogen and protected 67% of the mice in its group from formation of liver abscesses but one of the ten mice in this group died after challenge. As mentioned above, this region could contain domain(s) of toxicological importance such as, target cell binding, biological activities. However, multiple host-factors such as, availability of specific lymphocyte sub-population for clonal selection, type of helper T-cells stimulated, ability to invoke antibodies capable of opsonization, decide if an antibody response to a particular protein is protective in the species of animal tested.
The truncated leukotoxin polypeptide SX provided least protection from liver abscess formation. The number of bacteria in the hepatic tissue of mice vaccinated with GAS or SX were significantly higher (P<0.01) than in livers of mice vaccinated with SH, BSB SE, culture supernatant or full-length native or recombinant leukotoxin (mixture of five truncations), but was lower than the mice in the negative control group. A mixture of BSBSE and GAS or the FINAL polypeptides provided only amediocre protection against experimental challenge. Polyclonal antisera raised in rabbits against BSBSE or GAS neutralized the activity of native leukotoxin against PMNs used as target cells and were thus chosen to be used in combination.
Recombinant truncated leukotoxin polypeptides SH and BSBSE provided significant protection in mice when used as a vaccine individually. Dilution of immunodominant and protective epitopes present within these regions by including other truncated polypeptides as seen in vaccine preparations containing affinity purified leukotoxin or combinations of truncated leukotoxin polypeptides possibly caused a decrease in overall protection. Further studies to test the effectiveness of leukotoxin truncations BSBSE and SH individually or in combination providing protection against natural or experimental infections with F. necrophorum infections need to be carried out. This study provided further credence to the importance of leukotoxin as the major virulence factor of F. necrophorum and the protein carries a domain (s) or epitope (s) that induces protective immunity against experimental infection. The vaccine that produced best antileukotoxin titer did not always afford good protection against experimental infection. Therefore, certain epitopes may be more important in conferring protective immunity to infection. The results of this study suggest that some of these important epitopes reside on the BSBSE and SH polypeptides.
Fusobacterium necrophorum subsp. necrophorum is isolated more often than subsp. funduliforme from necrotic abscesses. The strains of subsp. necrophorum produces the high molecular weight leukotoxin in greater quantities than strains of subsp. funduliforme. In this study, we have cloned the leukotoxin gene from the highly virulent F. necrophorum subsp. necrophorum strain A25. The evidence that the lktA determinant encodes the leukotoxin is as follows: (1) the ORF encodes a 336 kDa protein, a size consistent with previous studies of the toxin; (2) the protein encoded by the recombinant lktA determinant is recognized by both polyclonal and monoclonal antibodies raised against purified leukotoxin from F. necrophorum; (3) antisera raised against polypeptides from the cloned lktA determinant recognized the native toxin in western blots; (4) antisera raised against two of the truncated polypeptides neutralized the toxic activity of the leukotoxin; and (5) the recombinant protein expressed in E. coli is relatively more toxic to bovine neutrophils as compared to bovine lymphocytes. These differing degrees of toxicity toward neutrophils relative to lymphocytes is also observed with leukotoxin that was affinity-purified from F. necrophorum culture supernatants.
The leukotoxin ORF is 9,726 base pairs long encoding a 3,241 amino acid protein with an overall molecular mass of 335,956 daltons. The DNA and deduced amino acid sequences were compared with sequences in Genbank but no significant (greater than 25% identity) similarities were found with other bacterial toxins. For example, the closest identity was found with HmwA from Haemophilus influenzae (22% or 356 out of 1,625 residues). Other similar homologies were found in SrpA from Streptococcus cristatus (17% or 388 out of 2,239 residues), OmpA from Ricketsia australis (21% or 321 out of 1,489 residues) and the 190 kDa surface antigen of Rickettsia ricketsii (21% or 379 out of 1,770 residues). Other Thus, the F. necrophorum leukotoxin appears to be distinct from all known leukotoxins and RTX-type toxins. When the deduced amino acid sequence of the lktA region was subjected to the Kyte-Doolittle hydropathy analysis (FIG. 3), 14 sites of sufficient length and hydrophobic character to be potential membrane spanning regions, were found. Upstream to the leukotoxin ORF is an open reading frame of at least 1.4 kb in length, which is in the same orientation. It encodes a protein that has some sequence identity to the heme-hemopexin utilization protein (UxuB) of Haemophilus infuenzae.
Additionally, the protein is larger than any bacterial exotoxins identified to date and shows no sequence similarity to other known leukotoxins. Thus, this protein may represent a new class of bacterial leukotoxins. The protein is unusual in that it is devoid of cysteine. This is not a characteristic of proteins from anaerobes, as evidenced by the normal content of cysteine residues in the clostridial toxins including Clostridium botulinum neurotoxin, Cl. difficile cytotoxin B, Cl. septicum alpha-toxin, and Cl. tetani tetanus toxin (Genbank accession numbers AB037166, AB217292, D17668, and X06214, respectively). The leukotoxin protein has a sequence at its N-terminus that has the properties of a signal sequence. This may indicate that the protein is exported across the cytoplasmic membrane in F. necrophorum in a Sec pathway-dependent manner.
The DNA sequences flanking lktA suggests that this toxin gene may be part of a multigene operon with at least one ORF upstream and another downstream of this gene. The activity of the LktA protein expressed in E. coli indicates that the other proteins encoded in the putative leukotoxin operon are not required to produce a biologically active toxin. Their role may be in secretion of the toxin across the cytoplasmic and outer membranes of F. necrophorum into the culture fluid.
If the lktA determinant is part of an operon, it would be greater than 12 kb in length. A dilemma with such a large operon might be to efficiently translate the messenger RNA species without premature dissociation of ribosome from the message. A peculiarity in the cloned region is an abundance of potential ribosome binding site sequences. Within the cloned region, there are 26 occurrences of GGAGG, which is a perfect match to the sequence at the 3' end of the 16S rRNA. The complementary sequence, CCTCC, which has the same G+C content but does not act as a ribosome binding site, is present only two times in the sequence. The abundance of the GGAGG sequence could provide translation reinforcement sequences to help ensure that a ribosome remains associated with the message and completes the translation of the ORFs. The abundance of the putative RBS sequence (GGAGG) is due to the presence of di-glycine repeats in the amino acid sequence. The GGA glycine codon occurs 263 times in the leukotoxin ORF and 24 of the 26 occurrences of GGAGG in the 11,130 bp sequenced to date correspond to tandem repeats of this codon. This feature of the amino acid sequence in the protein may provide the additional benefit of enabling more efficient translation of the message.
Expressing the 3.5 kb sequence from the 5' end of lktA caused immediate cessation of growth and lysis of E. coli carrying this recombinant expression vector. Creation of overlapping truncations allowed the expression of the entire leukotoxin gene without significant toxicity to the E. coli host cells. Polyclonal antileukotoxin antiserum reacted strongly to three truncated polypeptides (BSBSE, SX and FINAL) and more weakly to the other two truncated polypeptides (GAS and SH) in western blot analysis. This low reactivity was not due to poor immunogenicity of these relatively hydrophobic polypeptides, because both polypeptides (GAS and SH), produced high antibody titers in rabbits. Thus, it may been due to the tertiary folding pattern of leukotoxin under native conditions. The toxin being a secreted protein, would have its hydrophobic domains internalized when the protein was properly folded. The epitopes corresponding to these domains may not be as accessible to the immune system. Antibodies against these epitopes would thus be underrepresented when the whole un-denatured toxin is used as the immunogen. Interestingly, antibodies to one of these polypeptides, GAS, was neutralizing. Thus at least some of the critical epitopes are available in the active toxin.
The intact leukotoxin gene was introduced into E. coli under the control of the lac promoter. Inducible expression of full-length leukotoxin protein was achieved without any recognizable toxicity to E. coli host cells. Expression of the full-length leukotoxin instead of truncated polypeptides may allow correct folding of the toxin. This would result in internalization of the hydrophobic domains with a corresponding reduction of toxicity in E. coli host cells. Both polyclonal and monoclonal antibodies against native leukotoxin recognized a protein species with a size consistent with that of the intact leukotoxin in western blot analysis of cell lysates of E. coli harboring pSN2000. Antibodies raised against all five truncated leukotoxin polypeptides, but not the upstream polypeptide, recognized full-length recombinant leukotoxin as well.
In order to determine the prevalence and heterogeneity of leukotoxin gene in this species, 15 F. necrophorum strains belonging to subsp. necrophorum and subsp. funduliforme isolated from liver abscesses (opportunistic pathogen) or rumen contents (normal inhabitant) were screened for lktA by Southern blotting. Strains belonging to F. necrophorum subsp. necrophorum, irrespective of its location of isolation (liver abscess or ruminal contents) had similar hybridizing patterns. Similarly, all strains of F. necrophorum subsp. funduliforme, irrespective of the site from which it was isolated had identical hybridization patterns, but which differed from the subspecies necrophorum pattern. The difference in Southern blot hybridization patterns suggest that the disparity in levels of leukotoxin produced between the two subspecies may be due to differences in genetic organization of the leukotoxin locus.
1513241PRTFusobacterium necrophorum 1Met Ser Gly Ile Lys Asn Asn Val Gln Arg Thr Arg Lys Arg Ile Ser 1 5 10 15Asp Ser Lys Lys Val Leu Met Ile Leu Gly Leu Leu Ile Asn Thr Met 20 25 30Thr Val Arg Ala Asn Asp Thr Ile Thr Ala Thr Glu Asn Phe Gly Thr 35 40 45Lys Ile Glu Lys Lys Asp Asn Val Tyr Asp Ile Thr Thr Asn Lys Ile 50 55 60Gln Gly Glu Asn Ala Phe Asn Ser Phe Asn Arg Phe Ala Leu Thr Glu 65 70 75 80Asn Asn Ile Ala Asn Leu Tyr Phe Gly Glu Lys Asn Ser Thr Gly Val 85 90 95Asn Asn Leu Phe Asn Phe Val Asn Gly Lys Ile Glu Val Asp Gly Ile 100 105 110Ile Asn Gly Ile Arg Glu Asn Lys Ile Gly Gly Asn Leu Tyr Phe Leu 115 120 125Ser Ser Glu Gly Met Ala Val Gly Lys Asn Gly Val Ile Asn Ala Gly 130 135 140Ser Phe His Ser Ile Ile Pro Lys Gln Asp Asp Phe Lys Lys Ala Leu145 150 155 160Glu Glu Ala Lys His Gly Lys Val Phe Asn Gly Ile Ile Pro Val Asp 165 170 175Gly Lys Val Lys Ile Pro Leu Asn Pro Asn Gly Ser Ile Thr Val Glu 180 185 190Gly Lys Ile Asn Ala Val Glu Gly Ile Gly Leu Tyr Ala Ala Asp Ile 195 200 205Arg Leu Lys Asp Thr Ala Ile Leu Lys Thr Gly Ile Thr Asp Phe Lys 210 215 220Asn Leu Val Asn Ile Ser Asp Arg Ile Asn Ser Gly Leu Thr Gly Asp225 230 235 240Leu Lys Ala Thr Lys Thr Lys Ser Gly Asp Ile Ile Leu Ser Ala His 245 250 255Ile Asp Ser Pro Gln Lys Ala Met Gly Lys Asn Ser Thr Val Gly Lys 260 265 270Arg Ile Glu Glu Tyr Val Lys Gly Asn Thr Lys Ala Asn Ile Glu Ser 275 280 285Asp Ala Val Leu Glu Ala Asp Gly Asn Ile Lys Ile Ser Ala Lys Ala 290 295 300Thr Asn Gly Arg Phe Ile Lys Lys Glu Gly Glu Lys Glu Thr Tyr Asn305 310 315 320Thr Pro Leu Ser Leu Ser Asp Val Glu Ala Ser Val Arg Val Asn Lys 325 330 335Gly Lys Val Ile Gly Lys Asn Val Asp Ile Thr Ala Glu Ala Lys Asn 340 345 350Phe Tyr Asp Ala Thr Leu Val Thr Lys Leu Ala Lys His Ser Phe Ser 355 360 365Phe Val Thr Gly Ser Ile Ser Pro Ile Asn Leu Asn Gly Phe Leu Gly 370 375 380Leu Leu Thr Ser Lys Ser Ser Val Val Ile Gly Lys Asp Ala Lys Val385 390 395 400Glu Ala Thr Glu Gly Lys Ala Asn Ile His Ser Tyr Ser Gly Val Arg 405 410 415Ala Thr Met Gly Ala Ala Thr Ser Pro Leu Lys Ile Thr Asn Leu Tyr 420 425 430Leu Glu Lys Ala Asn Gly Lys Leu Leu Ser Ile Gly Ala Gly Tyr Ile 435 440 445Ser Ala Lys Ser Asn Ser Asn Val Thr Ile Glu Gly Glu Val Lys Ser 450 455 460Lys Gly Arg Ala Asp Ile Thr Ser Lys Ser Glu Asn Thr Ile Asp Ala465 470 475 480Ser Val Ser Val Gly Thr Met Arg Asp Ser Asn Lys Val Ala Leu Ser 485 490 495Val Leu Val Thr Glu Gly Glu Asn Lys Ser Ser Val Lys Ile Ala Lys 500 505 510Gly Ala Lys Val Glu Ser Glu Thr Asp Asp Val Asn Val Arg Ser Glu 515 520 525Ala Ile Asn Ser Ile Arg Ala Ala Val Lys Gly Gly Leu Gly Asp Ser 530 535 540Gly Asn Gly Val Val Ala Ala Asn Ile Ser Asn Tyr Asn Ala Ser Ser545 550 555 560Arg Ile Asp Val Asp Gly Tyr Leu His Ala Lys Lys Arg Leu Asn Val 565 570 575Glu Ala His Asn Ile Thr Lys Asn Ser Val Leu Gln Thr Gly Ser Asp 580 585 590Leu Gly Thr Ser Lys Phe Met Asn Asp His Val Tyr Glu Ser Gly His 595 600 605Leu Lys Ser Ile Leu Asp Ala Ile Lys Gln Arg Phe Gly Gly Asp Ser 610 615 620Val Asn Glu Glu Ile Lys Asn Lys Leu Thr Asn Leu Phe Ser Val Gly625 630 635 640Val Ser Ala Thr Ile Ala Asn His Asn Asn Ser Ala Ser Val Ala Ile 645 650 655Gly Glu Ser Gly Arg Leu Ser Ser Gly Val Glu Gly Ser Asn Val Arg 660 665 670Ala Leu Asn Glu Ala Gln Asn Leu Arg Ala Thr Thr Ser Ser Gly Ser 675 680 685Val Ala Val Arg Lys Glu Glu Lys Lys Lys Leu Ile Gly Asn Ala Ala 690 695 700Val Phe Tyr Gly Asn Tyr Lys Asn Asn Ala Ser Val Thr Ile Ala Asp705 710 715 720His Ala Glu Leu Val Ser Glu Gly Lys Ile Asp Ile Asn Ser Glu Asn 725 730 735Lys Ile Glu Tyr Lys Asn Pro Ser Lys Met Ala Lys Ser Val Ile Asp 740 745 750Lys Leu Glu Leu Leu Lys Arg Ala Phe Gly Lys Glu Thr Lys Thr Pro 755 760 765Glu Tyr Asp Pro Lys Asp Ile Glu Ser Ile Glu Lys Leu Leu Asn Ala 770 775 780Phe Ser Glu Lys Leu Asp Gly Lys Pro Glu Leu Leu Leu Asn Gly Glu785 790 795 800Arg Met Thr Ile Ile Leu Pro Asp Gly Thr Ser Lys Thr Gly Thr Ala 805 810 815Ile Glu Ile Ala Asn Tyr Val Gln Gly Glu Met Lys Lys Leu Glu Glu 820 825 830Lys Leu Pro Lys Gly Phe Lys Ala Phe Ser Glu Gly Leu Ser Gly Leu 835 840 845Ile Lys Glu Thr Leu Asn Phe Thr Gly Val Gly Asn Tyr Ala Asn Phe 850 855 860His Thr Phe Thr Ser Ser Gly Ala Asn Gly Glu Arg Asp Val Ser Ser865 870 875 880Val Gly Gly Ala Val Ser Trp Val Glu Gln Glu Asn Tyr Ser Lys Val 885 890 895Ser Val Gly Lys Gly Ala Lys Leu Ala Ala Lys Lys Asp Leu Asn Ile 900 905 910Lys Ala Ile Asn Lys Ala Glu Thr Val Asn Leu Val Gly Asn Ile Gly 915 920 925Leu Ala Arg Ser Ser Thr Ser Gly Ser Ala Val Gly Gly Arg Leu Asn 930 935 940Val Gln Arg Ser Lys Asn Ser Ala Ile Val Glu Ala Lys Glu Lys Ala945 950 955 960Glu Leu Ser Gly Glu Asn Ile Asn Ala Asp Ala Leu Asn Arg Leu Phe 965 970 975His Val Ala Gly Ser Phe Asn Gly Gly Ser Gly Gly Asn Ala Ile Asn 980 985 990Gly Met Gly Ser Tyr Ser Gly Gly Ile Ser Lys Ala Arg Val Ser Ile 995 1000 1005Asp Asp Glu Ala Tyr Leu Lys Ala Asn Lys Lys Ile Ala Leu Asn Ser 1010 1015 1020Lys Asn Asp Thr Ser Val Trp Asn Ala Ala Gly Ser Ala Gly Ile Gly1025 1030 1035 1040Thr Lys Asn Ala Ala Val Gly Val Ala Val Ala Val Asn Asp Tyr Asp 1045 1050 1055Ile Ser Asn Lys Ala Ser Ile Glu Asp Asn Asp Glu Gly Gln Ser Lys 1060 1065 1070Tyr Asp Lys Asn Lys Asp Asp Glu Val Thr Val Thr Ala Glu Ser Leu 1075 1080 1085Glu Val Asp Ala Lys Thr Thr Gly Thr Ile Asn Ser Ile Ser Val Ala 1090 1095 1100Gly Gly Ile Asn Lys Val Gly Ser Lys Pro Ser Glu Glu Lys Pro Lys1105 1110 1115 1120Ser Glu Glu Arg Pro Glu Gly Phe Phe Gly Lys Ile Gly Asn Lys Val 1125 1130 1135Asp Ser Val Lys Asn Lys Ile Thr Asp Ser Met Asp Ser Leu Thr Glu 1140 1145 1150Lys Ile Thr Asn Tyr Ile Ser Glu Gly Val Lys Lys Ala Gly Asn Leu 1155 1160 1165Pro Ser Asn Val Ser His Thr Pro Asp Lys Gly Pro Ser Phe Ser Leu 1170 1175 1180Gly Ala Ser Gly Ser Val Ser Phe Asn Asn Ile Lys Lys Glu Thr Ser1185 1190 1195 1200Ala Val Val Asp Gly Val Lys Ile Asn Leu Lys Gly Ala Asn Lys Lys 1205 1210 1215Val Glu Val Thr Ser Ser Asp Ser Thr Phe Val Gly Ala Trp Gly Gly 1220 1225 1230Ser Ala Ala Leu Gln Trp Asn His Ile Gly Ser Gly Asn Ser Asn Ile 1235 1240 1245Ser Ala Gly Leu Ala Gly Ala Ala Ala Val Asn Asn Ile Gln Ser Lys 1250 1255 1260Thr Ser Ala Leu Val Lys Asn Ser Asp Ile Arg Asn Ala Asn Lys Phe1265 1270 1275 1280Lys Val Asn Ala Leu Ser Gly Gly Thr Gln Val Ala Ala Gly Ala Gly 1285 1290 1295Leu Glu Ala Val Lys Glu Ser Gly Gly Gln Gly Lys Ser Tyr Leu Leu 1300 1305 1310Gly Thr Ser Ala Ser Ile Asn Leu Val Asn Asn Glu Val Ser Ala Lys 1315 1320 1325Ser Glu Asn Asn Thr Val Ala Gly Glu Ser Glu Ser Gln Lys Met Asp 1330 1335 1340Val Asp Val Thr Ala Tyr Gln Ala Asp Thr Gln Val Thr Gly Ala Leu1345 1350 1355 1360Asn Leu Gln Ala Gly Lys Ser Asn Gly Thr Val Gly Ala Thr Val Thr 1365 1370 1375Val Ala Lys Leu Asn Asn Lys Val Asn Ala Ser Ile Ser Gly Gly Arg 1380 1385 1390Tyr Thr Asn Val Asn Arg Ala Asp Ala Lys Ala Leu Leu Ala Thr Thr 1395 1400 1405Gln Val Thr Ala Ala Val Thr Thr Gly Gly Thr Ile Ser Ser Gly Ala 1410 1415 1420Gly Leu Gly Asn Tyr Gln Gly Ala Val Ser Val Asn Lys Ile Asp Asn1425 1430 1435 1440Asp Val Glu Ala Ser Val Asp Lys Ser Ser Ile Glu Gly Ala Asn Glu 1445 1450 1455Ile Asn Val Ile Ala Lys Asp Val Lys Gly Ser Ser Asp Leu Ala Lys 1460 1465 1470Glu Tyr Gln Ala Leu Leu Asn Gly Lys Asp Lys Lys Tyr Leu Glu Asp 1475 1480 1485Arg Gly Ile Asn Thr Thr Gly Asn Gly Tyr Tyr Thr Lys Glu Gln Leu 1490 1495 1500Glu Lys Ala Lys Lys Lys Glu Gly Ala Val Ile Val Asn Ala Ala Leu1505 1510 1515 1520Ser Val Ala Gly Thr Asp Lys Ser Ala Gly Gly Val Ala Ile Ala Val 1525 1530 1535Asn Thr Val Lys Asn Lys Phe Lys Ala Glu Leu Ser Gly Ser Asn Lys 1540 1545 1550Glu Ala Gly Glu Asp Lys Ile His Ala Lys His Val Asn Val Glu Ala 1555 1560 1565Lys Ser Ser Thr Val Val Val Asn Ala Ala Ser Gly Leu Ala Ile Ser 1570 1575 1580Lys Asp Ala Phe Ser Gly Met Gly Ser Gly Ala Trp Gln Asp Leu Ser1585 1590 1595 1600Asn Asp Thr Ile Ala Lys Val Asp Lys Gly Arg Ile Ser Ala Asp Ser 1605 1610 1615Leu Asn Val Asn Ala Asn Asn Ser Ile Leu Gly Val Asn Val Ala Gly 1620 1625 1630Thr Ile Ala Gly Ser Leu Ser Thr Ala Val Gly Ala Ala Phe Ala Asn 1635 1640 1645Asn Thr Leu His Asn Lys Thr Ser Ala Leu Ile Thr Gly Thr Lys Val 1650 1655 1660Asn Pro Phe Ser Gly Lys Asn Thr Lys Val Asn Val Gln Ala Leu Asn1665 1670 1675 1680Asp Ser His Ile Thr Asn Val Ser Ala Gly Gly Ala Ala Ser Ile Lys 1685 1690 1695Gln Ala Gly Ile Gly Gly Met Val Ser Val Asn Arg Gly Ser Asp Glu 1700 1705 1710Thr Glu Ala Leu Val Ser Asp Ser Glu Phe Glu Gly Val Ser Ser Phe 1715 1720 1725Asn Val Asp Ala Lys Asp Gln Lys Thr Ile Asn Thr Ile Ala Gly Asn 1730 1735 1740Ala Asn Gly Gly Lys Ala Ala Gly Val Gly Ala Thr Val Ala His Thr1745 1750 1755 1760Asn Ile Gly Lys Gln Ser Val Ile Ala Ile Val Lys Asn Ser Lys Ile 1765 1770 1775Thr Thr Ala Asn Asp Gln Asp Arg Lys Asn Ile Asn Val Thr Ala Lys 1780 1785 1790Asp Tyr Thr Met Thr Asn Thr Ile Ala Val Gly Val Gly Gly Ala Lys 1795 1800 1805Gly Ala Ser Val Gln Gly Ala Ser Ala Ser Thr Thr Leu Asn Lys Thr 1810 1815 1820Val Ser Ser His Val Asp Gln Thr Asp Ile Asp Lys Asp Leu Glu Glu1825 1830 1835 1840Glu Asn Asn Gly Asn Lys Glu Lys Ala Asn Val Asn Val Leu Ala Glu 1845 1850 1855Asn Thr Ser Gln Val Val Thr Asn Ala Thr Val Leu Ser Gly Ala Ser 1860 1865 1870Gly Gln Ala Ala Val Gly Ala Gly Val Ala Val Asn Lys Ile Thr Gln 1875 1880 1885Asn Thr Ser Ala His Ile Lys Asn Ser Thr Gln Asn Val Arg Asn Ala 1890 1895 1900Leu Val Lys Ser Lys Ser His Ser Ser Ile Lys Thr Ile Gly Ile Gly1905 1910 1915 1920Ala Gly Val Gly Ala Gly Gly Ala Gly Val Thr Gly Ser Val Ala Val 1925 1930 1935Asn Lys Ile Val Asn Asn Thr Ile Ala Glu Leu Asn His Ala Lys Ile 1940 1945 1950Thr Ala Lys Gly Asn Val Gly Val Ile Thr Glu Ser Asp Ala Val Ile 1955 1960 1965Ala Asn Tyr Ala Gly Thr Val Ser Gly Val Ala Arg Ala Ala Ile Gly 1970 1975 1980Ala Ser Thr Ser Val Asn Glu Ile Thr Gly Ser Thr Lys Ala Tyr Val1985 1990 1995 2000Lys Asp Ser Thr Val Ile Ala Lys Glu Glu Thr Asp Asp Tyr Ile Thr 2005 2010 2015Thr Gln Gly Gln Val Asp Lys Val Val Asp Lys Val Phe Lys Asn Leu 2020 2025 2030Asn Ile Asn Glu Asp Leu Ser Gln Lys Arg Lys Ile Ser Asn Lys Lys 2035 2040 2045Gly Phe Val Thr Asn Ser Ser Ala Thr His Thr Leu Lys Ser Leu Leu 2050 2055 2060Ala Asn Ala Ala Gly Ser Gly Gln Ala Gly Val Ala Gly Thr Val Asn2065 2070 2075 2080Ile Asn Lys Val Tyr Gly Glu Thr Glu Ala Leu Val Glu Asn Ser Ile 2085 2090 2095Leu Asn Ala Lys His Tyr Ser Val Lys Ser Gly Asp Tyr Thr Asn Ser 2100 2105 2110Ile Gly Val Val Gly Ser Val Gly Val Gly Gly Asn Val Gly Val Gly 2115 2120 2125Ala Ser Ser Asp Thr Asn Ile Ile Lys Arg Asn Thr Lys Thr Arg Val 2130 2135 2140Gly Lys Thr Thr Met Ser Asp Glu Gly Phe Gly Glu Glu Ala Glu Ile2145 2150 2155 2160Thr Ala Asp Ser Lys Gln Gly Ile Ser Ser Phe Gly Val Gly Val Ala 2165 2170 2175Ala Ala Gly Val Gly Ala Gly Val Ala Gly Thr Val Ser Val Asn Gln 2180 2185 2190Phe Ala Gly Lys Thr Glu Val Asp Val Glu Glu Ala Lys Ile Leu Val 2195 2200 2205Lys Lys Ala Glu Ile Thr Ala Lys Arg Tyr Ser Ser Val Ala Ile Gly 2210 2215 2220Asn Ala Ala Val Gly Val Ala Ala Lys Gly Ala Gly Ile Gly Ala Ala2225 2230 2235 2240Val Ala Val Thr Lys Asp Glu Ser Asn Thr Arg Ala Arg Val Lys Asn 2245 2250 2255Ser Lys Ile Met Thr Arg Asn Lys Leu Asp Val Ile Ala Glu Asn Glu 2260 2265 2270Ile Lys Ser Gly Thr Gly Ile Gly Ser Ala Gly Ala Gly Ile Leu Ala 2275 2280 2285Ala Gly Val Ser Gly Val Val Ser Val Asn Asn Ile Ala Asn Lys Val 2290 2295 2300Glu Thr Asp Ile Asp His Ser Thr Leu His Ser Ser Thr Asp Val Asn2305 2310 2315 2320Val Lys Ala Leu Asn Lys Ile Ser Asn Ser Leu Thr Ala Gly Gly Gly 2325 2330 2335Ala Ala Gly Leu Ala Ala Val Thr Gly Val Val Ser Val Asn Thr Ile 2340 2345 2350Asn Ser Ser Val Ile Ala Arg Val His Asn Asn Ser Asp Leu Thr Ser 2355 2360 2365Val Arg Glu Lys Val Asn Val Thr Ala Lys Glu Glu Lys Asn Ile Lys 2370 2375 2380Gln Thr Ala Ala Asn Ala Gly Ile Gly Gly Ala Ala Ile Gly Ala Asn2385 2390 2395 2400Val Leu Val Asn Asn Phe Gly Thr Ala Val Glu Asp Arg Lys Asn Ser 2405 2410 2415Glu Gly Lys Gly Thr Glu Val Leu Lys Thr Leu Asp Glu Val Asn Lys 2420 2425 2430Glu Gln Asp Lys Lys Val Asn Asp Ala Thr Lys Lys Ile Leu Gln Ser 2435 2440 2445Ala Gly Ile Ser Thr Glu Asp Thr Ser Val Lys Ala Asp Arg Gly Asp 2450 2455 2460Thr Gln Gly Glu Gly Ile Lys Ala Ile Val
Lys Thr Ser Asp Ile Ile2465 2470 2475 2480Gly Lys Asn Val Asp Ile Thr Thr Glu Asp Lys Asn Asn Ile Thr Ser 2485 2490 2495Thr Gly Gly Leu Gly Thr Ala Gly Leu Ala Ser Ala Ser Gly Thr Val 2500 2505 2510Ala Val Thr Asn Ile Lys Arg Asn Ser Gly Val Thr Val Glu Asn Ser 2515 2520 2525Phe Val Lys Ala Ala Glu Lys Val Asn Val Arg Ser Asp Ile Thr Gly 2530 2535 2540Asn Val Ala Leu Thr Ala Tyr Gln Gly Pro Val Gly Ala Leu Gly Ile2545 2550 2555 2560Gly Ala Ala Tyr Ala Glu Leu Asn Ser Asn Gly Arg Ser Asn Ile Ser 2565 2570 2575Ile Lys Asn Ser Lys Leu Leu Gly Lys Asn Ile Asp Val Ile Val Lys 2580 2585 2590Asp Lys Ser Glu Leu Arg Ala Glu Ala Lys Gly Leu Thr Val Gly Ala 2595 2600 2605Val Ala Ala Gly Ala Ile Ile Ser Lys Ala Lys Asn Glu Met Asn Ser 2610 2615 2620Glu Val Glu Ile Glu Lys Ser Ile Phe Asn Glu Glu Asn Arg Val Thr2625 2630 2635 2640Ser Pro Ser Lys Gly Ile Gly Arg Glu Ile Asn Val Lys Val Glu Lys 2645 2650 2655Glu Asn Arg Val Thr Ala Glu Ser Gln Gly Ala Ser Val Gly Ala Val 2660 2665 2670Ala Gly Ala Gly Ile Ile Ser Glu Ala Lys Asp Ala Gly Ser Ser Tyr 2675 2680 2685Leu Lys Val Ser Thr Lys Ser Gly Arg Ser Ile Phe His Ala Asp Asn 2690 2695 2700Val Asn Met Glu Ala Thr His Lys Met Lys Val Thr Ala Val Ser Lys2705 2710 2715 2720Ala Val Thr Gly Ser Val Leu Gly Gly Val Gly Val Thr Lys Ala Glu 2725 2730 2735Ala Thr Ala Ala Gly Lys Thr Met Val Glu Val Glu Glu Gly Asn Leu 2740 2745 2750Phe Arg Thr Asn Arg Leu Asn Ala Ile Ser Lys Val Glu Gly Leu Asp 2755 2760 2765Glu Asp Lys Val Thr Ala Lys Ser Ser Val Val Ser Gly Asn Gly Gly 2770 2775 2780Gly Ile Ala Gly Ala Gly Val Asn Thr Ser Thr Ala Gln Ser Asn Thr2785 2790 2795 2800Glu Ser Val Val Arg Leu Arg Lys Gln Asp Tyr Glu Asn Asn Asp Tyr 2805 2810 2815Thr Lys Lys Tyr Ile Ser Glu Val Asn Ala Leu Ala Leu Asn Asp Thr 2820 2825 2830Lys Asn Glu Ala Asn Ile Glu Ser Leu Ala Val Ala Gly Val His Ala 2835 2840 2845Gln Gly Thr Asn Lys Ala Phe Thr Arg Ser Asn Lys Leu Thr Ser Thr 2850 2855 2860Thr Val Asn Gly Gly Asn Val Ser Gln Leu Arg Ala Lys Ala Leu Ala2865 2870 2875 2880Lys Asn Glu Asn Tyr Gly Asn Val Lys Gly Thr Gly Gly Ala Leu Val 2885 2890 2895Gly Ala Glu Thr Ala Ala Val Glu Asn Tyr Thr Lys Ser Thr Thr Gly 2900 2905 2910Ala Leu Val Ala Gly Asn Trp Glu Ile Gly Asp Lys Leu Glu Thr Ile 2915 2920 2925Ala Arg Asp Asn Thr Ile Val Arg Val Asn Gly Asp Gly Thr Lys Gly 2930 2935 2940Gly Leu Val Gly Lys Asn Gly Ile Ser Val Lys Asn Thr Ile Ser Gly2945 2950 2955 2960Glu Thr Lys Ser Ser Ile Glu Asp Lys Ala Arg Ile Val Gly Thr Gly 2965 2970 2975Ser Val Asn Val Asp Ala Leu Asn Glu Leu Asp Val Asp Leu Gln Gly 2980 2985 2990Lys Ser Gly Gly Tyr Gly Gly Ile Gly Ile Gly Asn Val Asp Val Asn 2995 3000 3005Asn Val Ile Lys Lys Asn Val Glu Ala Lys Ile Gly Arg His Ala Ile 3010 3015 3020Val Glu Thr Thr Gly Lys Gln Glu Tyr Gln Ala Phe Thr Arg Ala Lys3025 3030 3035 3040Val Asn Ile Leu Gly Lys Gly Asp Ala Ala Ala Ala Ala Ala Ile Ser 3045 3050 3055Asn Val His Ile Ser Asn Glu Met Asp Ile Lys Asn Leu Ala Lys Gln 3060 3065 3070Tyr Ala Ser Ser Gln Leu Ile Thr Lys Asn Ser Lys Asn Asn Ile Thr 3075 3080 3085Leu Ala Ser Ser Ser Glu Ser Asn Val Asn Val His Gly Val Ala Glu 3090 3095 3100Ala Arg Gly Ala Gly Ala Lys Ala Thr Val Ser Val Lys Asn Gln Ile3105 3110 3115 3120Asn Arg Thr Asn Asn Val Asp Leu Ala Gly Lys Ile Lys Thr Glu Gly 3125 3130 3135Asn Ile Asn Val Tyr Ala Gly Tyr Asp Lys Asn Tyr Asn Ile Ser Lys 3140 3145 3150Thr Asn Ser Lys Ala Ile Ala Asp Ala Lys Ser His Ala Ala Ala Ala 3155 3160 3165Ser Ala Thr Ala Thr Ile Glu Lys Asn Glu Val Lys Phe Asn Asn Ala 3170 3175 3180Ile Arg Glu Phe Lys Asn Asn Leu Ala Arg Leu Glu Gly Lys Ala Asn3185 3190 3195 3200Lys Lys Thr Ser Val Gly Ser Asn Gln Val Asp Trp Tyr Thr Asp Lys 3205 3210 3215Tyr Thr Trp His Ser Ser Glu Lys Ala Tyr Lys Lys Leu Thr Tyr Gln 3220 3225 3230Ser Lys Arg Gly Glu Lys Gly Lys Lys 3235 32402369PRTFusobacterium necrophorum 2Met Ser Gly Ile Lys Asn Asn Val Gln Arg Thr Arg Lys Arg Ile Ser 1 5 10 15Asp Ser Lys Lys Val Leu Met Ile Leu Gly Leu Leu Ile Asn Thr Met 20 25 30Thr Val Arg Ala Asn Asp Thr Ile Thr Ala Thr Glu Asn Phe Gly Thr 35 40 45Lys Ile Glu Lys Lys Asp Asn Val Tyr Asp Ile Thr Thr Asn Lys Ile 50 55 60Gln Gly Glu Asn Ala Phe Asn Ser Phe Asn Arg Phe Ala Leu Thr Glu 65 70 75 80Asn Asn Ile Ala Asn Leu Tyr Phe Gly Glu Lys Asn Ser Thr Gly Val 85 90 95Asn Asn Leu Phe Asn Phe Val Asn Gly Lys Ile Glu Val Asp Gly Ile 100 105 110Ile Asn Gly Ile Arg Glu Asn Lys Ile Gly Gly Asn Leu Tyr Phe Leu 115 120 125Ser Ser Glu Gly Met Ala Val Gly Lys Asn Gly Val Ile Asn Ala Gly 130 135 140Ser Phe His Ser Ile Ile Pro Lys Gln Asp Asp Phe Lys Lys Ala Leu145 150 155 160Glu Glu Ala Lys His Gly Lys Val Phe Asn Gly Ile Ile Pro Val Asp 165 170 175Gly Lys Val Lys Ile Pro Leu Asn Pro Asn Gly Ser Ile Thr Val Glu 180 185 190Gly Lys Ile Asn Ala Val Glu Gly Ile Gly Leu Tyr Ala Ala Asp Ile 195 200 205Arg Leu Lys Asp Thr Ala Ile Leu Lys Thr Gly Ile Thr Asp Phe Lys 210 215 220Asn Leu Val Asn Ile Ser Asp Arg Ile Asn Ser Gly Leu Thr Gly Asp225 230 235 240Leu Lys Ala Thr Lys Thr Lys Ser Gly Asp Ile Ile Leu Ser Ala His 245 250 255Ile Asp Ser Pro Gln Lys Ala Met Gly Lys Asn Ser Thr Val Gly Lys 260 265 270Arg Ile Glu Glu Tyr Val Lys Gly Asn Thr Lys Ala Asn Ile Glu Ser 275 280 285Asp Ala Val Leu Glu Ala Asp Gly Asn Ile Lys Ile Ser Ala Lys Ala 290 295 300Thr Asn Gly Arg Phe Ile Lys Lys Glu Gly Glu Lys Glu Thr Tyr Asn305 310 315 320Thr Pro Leu Ser Leu Ser Asp Val Glu Ala Ser Val Arg Val Asn Lys 325 330 335Gly Lys Val Ile Gly Lys Asn Val Asp Ile Thr Ala Glu Ala Lys Asn 340 345 350Phe Tyr Asp Ala Thr Leu Val Thr Lys Leu Ala Lys His Ser Phe Ser 355 360 365Phe3927PRTFusobacterium necrophorum 3Gly Arg Phe Ile Lys Lys Glu Gly Glu Lys Glu Thr Tyr Asn Thr Pro 1 5 10 15Leu Ser Leu Ser Asp Val Glu Ala Ser Val Arg Val Asn Lys Gly Lys 20 25 30Val Ile Gly Lys Asn Val Asp Ile Thr Ala Glu Ala Lys Asn Phe Tyr 35 40 45Asp Ala Thr Leu Val Thr Lys Leu Ala Lys His Ser Phe Ser Phe Val 50 55 60Thr Gly Ser Ile Ser Pro Ile Asn Leu Asn Gly Phe Leu Gly Leu Leu 65 70 75 80Thr Ser Lys Ser Ser Val Val Ile Gly Lys Asp Ala Lys Val Glu Ala 85 90 95Thr Glu Gly Lys Ala Asn Ile His Ser Tyr Ser Gly Val Arg Ala Thr 100 105 110Met Gly Ala Ala Thr Ser Pro Leu Lys Ile Thr Asn Leu Tyr Leu Glu 115 120 125Lys Ala Asn Gly Lys Leu Leu Ser Ile Gly Ala Gly Tyr Ile Ser Ala 130 135 140Lys Ser Asn Ser Asn Val Thr Ile Glu Gly Glu Val Lys Ser Lys Gly145 150 155 160Arg Ala Asp Ile Thr Ser Lys Ser Glu Asn Thr Ile Asp Ala Ser Val 165 170 175Ser Val Gly Thr Met Arg Asp Ser Asn Lys Val Ala Leu Ser Val Leu 180 185 190Val Thr Glu Gly Glu Asn Lys Ser Ser Val Lys Ile Ala Lys Gly Ala 195 200 205Lys Val Glu Ser Glu Thr Asp Asp Val Asn Val Arg Ser Glu Ala Ile 210 215 220Asn Ser Ile Arg Ala Ala Val Lys Gly Gly Leu Gly Asp Ser Gly Asn225 230 235 240Gly Val Val Ala Ala Asn Ile Ser Asn Tyr Asn Ala Ser Ser Arg Ile 245 250 255Asp Val Asp Gly Tyr Leu His Ala Lys Lys Arg Leu Asn Val Glu Ala 260 265 270His Asn Ile Thr Lys Asn Ser Val Leu Gln Thr Gly Ser Asp Leu Gly 275 280 285Thr Ser Lys Phe Met Asn Asp His Val Tyr Glu Ser Gly His Leu Lys 290 295 300Ser Ile Leu Asp Ala Ile Lys Gln Arg Phe Gly Gly Asp Ser Val Asn305 310 315 320Glu Glu Ile Lys Asn Lys Leu Thr Asn Leu Phe Ser Val Gly Val Ser 325 330 335Ala Thr Ile Ala Asn His Asn Asn Ser Ala Ser Val Ala Ile Gly Glu 340 345 350Ser Gly Arg Leu Ser Ser Gly Val Glu Gly Ser Asn Val Arg Ala Leu 355 360 365Asn Glu Ala Gln Asn Leu Arg Ala Thr Thr Ser Ser Gly Ser Val Ala 370 375 380Val Arg Lys Glu Glu Lys Lys Lys Leu Ile Gly Asn Ala Ala Val Phe385 390 395 400Tyr Gly Asn Tyr Lys Asn Asn Ala Ser Val Thr Ile Ala Asp His Ala 405 410 415Glu Leu Val Ser Glu Gly Lys Ile Asp Ile Asn Ser Glu Asn Lys Ile 420 425 430Glu Tyr Lys Asn Pro Ser Lys Met Ala Lys Ser Val Ile Asp Lys Leu 435 440 445Glu Leu Leu Lys Arg Ala Phe Gly Lys Glu Thr Lys Thr Pro Glu Tyr 450 455 460Asp Pro Lys Asp Ile Glu Ser Ile Glu Lys Leu Leu Asn Ala Phe Ser465 470 475 480Glu Lys Leu Asp Gly Lys Pro Glu Leu Leu Leu Asn Gly Glu Arg Met 485 490 495Thr Ile Ile Leu Pro Asp Gly Thr Ser Lys Thr Gly Thr Ala Ile Glu 500 505 510Ile Ala Asn Tyr Val Gln Gly Glu Met Lys Lys Leu Glu Glu Lys Leu 515 520 525Pro Lys Gly Phe Lys Ala Phe Ser Glu Gly Leu Ser Gly Leu Ile Lys 530 535 540Glu Thr Leu Asn Phe Thr Gly Val Gly Asn Tyr Ala Asn Phe His Thr545 550 555 560Phe Thr Ser Ser Gly Ala Asn Gly Glu Arg Asp Val Ser Ser Val Gly 565 570 575Gly Ala Val Ser Trp Val Glu Gln Glu Asn Tyr Ser Lys Val Ser Val 580 585 590Gly Lys Gly Ala Lys Leu Ala Ala Lys Lys Asp Leu Asn Ile Lys Ala 595 600 605Ile Asn Lys Ala Glu Thr Val Asn Leu Val Gly Asn Ile Gly Leu Ala 610 615 620Arg Ser Ser Thr Ser Gly Ser Ala Val Gly Gly Arg Leu Asn Val Gln625 630 635 640Arg Ser Lys Asn Ser Ala Ile Val Glu Ala Lys Glu Lys Ala Glu Leu 645 650 655Ser Gly Glu Asn Ile Asn Ala Asp Ala Leu Asn Arg Leu Phe His Val 660 665 670Ala Gly Ser Phe Asn Gly Gly Ser Gly Gly Asn Ala Ile Asn Gly Met 675 680 685Gly Ser Tyr Ser Gly Gly Ile Ser Lys Ala Arg Val Ser Ile Asp Asp 690 695 700Glu Ala Tyr Leu Lys Ala Asn Lys Lys Ile Ala Leu Asn Ser Lys Asn705 710 715 720Asp Thr Ser Val Trp Asn Ala Ala Gly Ser Ala Gly Ile Gly Thr Lys 725 730 735Asn Ala Ala Val Gly Val Ala Val Ala Val Asn Asp Tyr Asp Ile Ser 740 745 750Asn Lys Ala Ser Ile Glu Asp Asn Asp Glu Gly Gln Ser Lys Tyr Asp 755 760 765Lys Asn Lys Asp Asp Glu Val Thr Val Thr Ala Glu Ser Leu Glu Val 770 775 780Asp Ala Lys Thr Thr Gly Thr Ile Asn Ser Ile Ser Val Ala Gly Gly785 790 795 800Ile Asn Lys Val Gly Ser Lys Pro Ser Glu Glu Lys Pro Lys Ser Glu 805 810 815Glu Arg Pro Glu Gly Phe Phe Gly Lys Ile Gly Asn Lys Val Asp Ser 820 825 830Val Lys Asn Lys Ile Thr Asp Ser Met Asp Ser Leu Thr Glu Lys Ile 835 840 845Thr Asn Tyr Ile Ser Glu Gly Val Lys Lys Ala Gly Asn Leu Pro Ser 850 855 860Asn Val Ser His Thr Pro Asp Lys Gly Pro Ser Phe Ser Leu Gly Ala865 870 875 880Ser Gly Ser Val Ser Phe Asn Asn Ile Lys Lys Glu Thr Ser Ala Val 885 890 895Val Asp Gly Val Lys Ile Asn Leu Lys Gly Ala Asn Lys Lys Val Glu 900 905 910Val Thr Ser Ser Asp Ser Thr Phe Val Gly Ala Trp Gly Gly Ser 915 920 9254714PRTFusobacterium necrophorum 4Gly Ala Ser Gly Ser Val Ser Phe Asn Asn Ile Lys Lys Glu Thr Ser 1 5 10 15Ala Val Val Asp Gly Val Lys Ile Asn Leu Lys Gly Ala Asn Lys Lys 20 25 30Val Glu Val Thr Ser Ser Asp Ser Thr Phe Val Gly Ala Trp Gly Gly 35 40 45Ser Ala Ala Leu Gln Trp Asn His Ile Gly Ser Gly Asn Ser Asn Ile 50 55 60Ser Ala Gly Leu Ala Gly Ala Ala Ala Val Asn Asn Ile Gln Ser Lys 65 70 75 80Thr Ser Ala Leu Val Lys Asn Ser Asp Ile Arg Asn Ala Asn Lys Phe 85 90 95Lys Val Asn Ala Leu Ser Gly Gly Thr Gln Val Ala Ala Gly Ala Gly 100 105 110Leu Glu Ala Val Lys Glu Ser Gly Gly Gln Gly Lys Ser Tyr Leu Leu 115 120 125Gly Thr Ser Ala Ser Ile Asn Leu Val Asn Asn Glu Val Ser Ala Lys 130 135 140Ser Glu Asn Asn Thr Val Ala Gly Glu Ser Glu Ser Gln Lys Met Asp145 150 155 160Val Asp Val Thr Ala Tyr Gln Ala Asp Thr Gln Val Thr Gly Ala Leu 165 170 175Asn Leu Gln Ala Gly Lys Ser Asn Gly Thr Val Gly Ala Thr Val Thr 180 185 190Val Ala Lys Leu Asn Asn Lys Val Asn Ala Ser Ile Ser Gly Gly Arg 195 200 205Tyr Thr Asn Val Asn Arg Ala Asp Ala Lys Ala Leu Leu Ala Thr Thr 210 215 220Gln Val Thr Ala Ala Val Thr Thr Gly Gly Thr Ile Ser Ser Gly Ala225 230 235 240Gly Leu Gly Asn Tyr Gln Gly Ala Val Ser Val Asn Lys Ile Asp Asn 245 250 255Asp Val Glu Ala Ser Val Asp Lys Ser Ser Ile Glu Gly Ala Asn Glu 260 265 270Ile Asn Val Ile Ala Lys Asp Val Lys Gly Ser Ser Asp Leu Ala Lys 275 280 285Glu Tyr Gln Ala Leu Leu Asn Gly Lys Asp Lys Lys Tyr Leu Glu Asp 290 295 300Arg Gly Ile Asn Thr Thr Gly Asn Gly Tyr Tyr Thr Lys Glu Gln Leu305 310 315 320Glu Lys Ala Lys Lys Lys Glu Gly Ala Val Ile Val Asn Ala Ala Leu 325 330 335Ser Val Ala Gly Thr Asp Lys Ser Ala Gly Gly Val Ala Ile Ala Val 340 345 350Asn Thr Val Lys Asn Lys Phe Lys Ala Glu Leu Ser Gly Ser Asn Lys 355 360 365Glu Ala Gly Glu Asp Lys Ile His Ala Lys His Val Asn Val Glu Ala 370 375 380Lys Ser Ser Thr Val Val Val Asn Ala Ala Ser Gly
Leu Ala Ile Ser385 390 395 400Lys Asp Ala Phe Ser Gly Met Gly Ser Gly Ala Trp Gln Asp Leu Ser 405 410 415Asn Asp Thr Ile Ala Lys Val Asp Lys Gly Arg Ile Ser Ala Asp Ser 420 425 430Leu Asn Val Asn Ala Asn Asn Ser Ile Leu Gly Val Asn Val Ala Gly 435 440 445Thr Ile Ala Gly Ser Leu Ser Thr Ala Val Gly Ala Ala Phe Ala Asn 450 455 460Asn Thr Leu His Asn Lys Thr Ser Ala Leu Ile Thr Gly Thr Lys Val465 470 475 480Asn Pro Phe Ser Gly Lys Asn Thr Lys Val Asn Val Gln Ala Leu Asn 485 490 495Asp Ser His Ile Thr Asn Val Ser Ala Gly Gly Ala Ala Ser Ile Lys 500 505 510Gln Ala Gly Ile Gly Gly Met Val Ser Val Asn Arg Gly Ser Asp Glu 515 520 525Thr Glu Ala Leu Val Ser Asp Ser Glu Phe Glu Gly Val Ser Ser Phe 530 535 540Asn Val Asp Ala Lys Asp Gln Lys Thr Ile Asn Thr Ile Ala Gly Asn545 550 555 560Ala Asn Gly Gly Lys Ala Ala Gly Val Gly Ala Thr Val Ala His Thr 565 570 575Asn Ile Gly Lys Gln Ser Val Ile Ala Ile Val Lys Asn Ser Lys Ile 580 585 590Thr Thr Ala Asn Asp Gln Asp Arg Lys Asn Ile Asn Val Thr Ala Lys 595 600 605Asp Tyr Thr Met Thr Asn Thr Ile Ala Val Gly Val Gly Gly Ala Lys 610 615 620Gly Ala Ser Val Gln Gly Ala Ser Ala Ser Thr Thr Leu Asn Lys Thr625 630 635 640Val Ser Ser His Val Asp Gln Thr Asp Ile Asp Lys Asp Leu Glu Glu 645 650 655Glu Asn Asn Gly Asn Lys Glu Lys Ala Asn Val Asn Val Leu Ala Glu 660 665 670Asn Thr Ser Gln Val Val Thr Asn Ala Thr Val Leu Ser Gly Ala Ser 675 680 685Gly Gln Ala Ala Val Gly Ala Gly Val Ala Val Asn Lys Ile Thr Gln 690 695 700Asn Thr Ser Ala His Ile Lys Asn Ser Thr705 7105628PRTFusobacterium necrophorum 5Ala Val Gly Ala Gly Val Ala Val Asn Lys Ile Thr Gln Asn Thr Ser 1 5 10 15Ala His Ile Lys Asn Ser Thr Gln Asn Val Arg Asn Ala Leu Val Lys 20 25 30Ser Lys Ser His Ser Ser Ile Lys Thr Ile Gly Ile Gly Ala Gly Val 35 40 45Gly Ala Gly Gly Ala Gly Val Thr Gly Ser Val Ala Val Asn Lys Ile 50 55 60Val Asn Asn Thr Ile Ala Glu Leu Asn His Ala Lys Ile Thr Ala Lys 65 70 75 80Gly Asn Val Gly Val Ile Thr Glu Ser Asp Ala Val Ile Ala Asn Tyr 85 90 95Ala Gly Thr Val Ser Gly Val Ala Arg Ala Ala Ile Gly Ala Ser Thr 100 105 110Ser Val Asn Glu Ile Thr Gly Ser Thr Lys Ala Tyr Val Lys Asp Ser 115 120 125Thr Val Ile Ala Lys Glu Glu Thr Asp Asp Tyr Ile Thr Thr Gln Gly 130 135 140Gln Val Asp Lys Val Val Asp Lys Val Phe Lys Asn Leu Asn Ile Asn145 150 155 160Glu Asp Leu Ser Gln Lys Arg Lys Ile Ser Asn Lys Lys Gly Phe Val 165 170 175Thr Asn Ser Ser Ala Thr His Thr Leu Lys Ser Leu Leu Ala Asn Ala 180 185 190Ala Gly Ser Gly Gln Ala Gly Val Ala Gly Thr Val Asn Ile Asn Lys 195 200 205Val Tyr Gly Glu Thr Glu Ala Leu Val Glu Asn Ser Ile Leu Asn Ala 210 215 220Lys His Tyr Ser Val Lys Ser Gly Asp Tyr Thr Asn Ser Ile Gly Val225 230 235 240Val Gly Ser Val Gly Val Gly Gly Asn Val Gly Val Gly Ala Ser Ser 245 250 255Asp Thr Asn Ile Ile Lys Arg Asn Thr Lys Thr Arg Val Gly Lys Thr 260 265 270Thr Met Ser Asp Glu Gly Phe Gly Glu Glu Ala Glu Ile Thr Ala Asp 275 280 285Ser Lys Gln Gly Ile Ser Ser Phe Gly Val Gly Val Ala Ala Ala Gly 290 295 300Val Gly Ala Gly Val Ala Gly Thr Val Ser Val Asn Gln Phe Ala Gly305 310 315 320Lys Thr Glu Val Asp Val Glu Glu Ala Lys Ile Leu Val Lys Lys Ala 325 330 335Glu Ile Thr Ala Lys Arg Tyr Ser Ser Val Ala Ile Gly Asn Ala Ala 340 345 350Val Gly Val Ala Ala Lys Gly Ala Gly Ile Gly Ala Ala Val Ala Val 355 360 365Thr Lys Asp Glu Ser Asn Thr Arg Ala Arg Val Lys Asn Ser Lys Ile 370 375 380Met Thr Arg Asn Lys Leu Asp Val Ile Ala Glu Asn Glu Ile Lys Ser385 390 395 400Gly Thr Gly Ile Gly Ser Ala Gly Ala Gly Ile Leu Ala Ala Gly Val 405 410 415Ser Gly Val Val Ser Val Asn Asn Ile Ala Asn Lys Val Glu Thr Asp 420 425 430Ile Asp His Ser Thr Leu His Ser Ser Thr Asp Val Asn Val Lys Ala 435 440 445Leu Asn Lys Ile Ser Asn Ser Leu Thr Ala Gly Gly Gly Ala Ala Gly 450 455 460Leu Ala Ala Val Thr Gly Val Val Ser Val Asn Thr Ile Asn Ser Ser465 470 475 480Val Ile Ala Arg Val His Asn Asn Ser Asp Leu Thr Ser Val Arg Glu 485 490 495Lys Val Asn Val Thr Ala Lys Glu Glu Lys Asn Ile Lys Gln Thr Ala 500 505 510Ala Asn Ala Gly Ile Gly Gly Ala Ala Ile Gly Ala Asn Val Leu Val 515 520 525Asn Asn Phe Gly Thr Ala Val Glu Asp Arg Lys Asn Ser Glu Gly Lys 530 535 540Gly Thr Glu Val Leu Lys Thr Leu Asp Glu Val Asn Lys Glu Gln Asp545 550 555 560Lys Lys Val Asn Asp Ala Thr Lys Lys Ile Leu Gln Ser Ala Gly Ile 565 570 575Ser Thr Glu Asp Thr Ser Val Lys Ala Asp Arg Gly Asp Thr Gln Gly 580 585 590Glu Gly Ile Lys Ala Ile Val Lys Thr Ser Asp Ile Ile Gly Lys Asn 595 600 605Val Asp Ile Thr Thr Glu Asp Lys Asn Asn Ile Thr Ser Thr Gly Gly 610 615 620Leu Gly Thr Ala6256773PRTFusobacterium necrophorum 6Gly Ile Lys Ala Ile Val Lys Thr Ser Asp Ile Ile Gly Lys Asn Val 1 5 10 15Asp Ile Thr Thr Glu Asp Lys Asn Asn Ile Thr Ser Thr Gly Gly Leu 20 25 30Gly Thr Ala Gly Leu Ala Ser Ala Ser Gly Thr Val Ala Val Thr Asn 35 40 45Ile Lys Arg Asn Ser Gly Val Thr Val Glu Asn Ser Phe Val Lys Ala 50 55 60Ala Glu Lys Val Asn Val Arg Ser Asp Ile Thr Gly Asn Val Ala Leu 65 70 75 80Thr Ala Tyr Gln Gly Pro Val Gly Ala Leu Gly Ile Gly Ala Ala Tyr 85 90 95Ala Glu Leu Asn Ser Asn Gly Arg Ser Asn Ile Ser Ile Lys Asn Ser 100 105 110Lys Leu Leu Gly Lys Asn Ile Asp Val Ile Val Lys Asp Lys Ser Glu 115 120 125Leu Arg Ala Glu Ala Lys Gly Leu Thr Val Gly Ala Val Ala Ala Gly 130 135 140Ala Ile Ile Ser Lys Ala Lys Asn Glu Met Asn Ser Glu Val Glu Ile145 150 155 160Glu Lys Ser Ile Phe Asn Glu Glu Asn Arg Val Thr Ser Pro Ser Lys 165 170 175Gly Ile Gly Arg Glu Ile Asn Val Lys Val Glu Lys Glu Asn Arg Val 180 185 190Thr Ala Glu Ser Gln Gly Ala Ser Val Gly Ala Val Ala Gly Ala Gly 195 200 205Ile Ile Ser Glu Ala Lys Asp Ala Gly Ser Ser Tyr Leu Lys Val Ser 210 215 220Thr Lys Ser Gly Arg Ser Ile Phe His Ala Asp Asn Val Asn Met Glu225 230 235 240Ala Thr His Lys Met Lys Val Thr Ala Val Ser Lys Ala Val Thr Gly 245 250 255Ser Val Leu Gly Gly Val Gly Val Thr Lys Ala Glu Ala Thr Ala Ala 260 265 270Gly Lys Thr Met Val Glu Val Glu Glu Gly Asn Leu Phe Arg Thr Asn 275 280 285Arg Leu Asn Ala Ile Ser Lys Val Glu Gly Leu Asp Glu Asp Lys Val 290 295 300Thr Ala Lys Ser Ser Val Val Ser Gly Asn Gly Gly Gly Ile Ala Gly305 310 315 320Ala Gly Val Asn Thr Ser Thr Ala Gln Ser Asn Thr Glu Ser Val Val 325 330 335Arg Leu Arg Lys Gln Asp Tyr Glu Asn Asn Asp Tyr Thr Lys Lys Tyr 340 345 350Ile Ser Glu Val Asn Ala Leu Ala Leu Asn Asp Thr Lys Asn Glu Ala 355 360 365Asn Ile Glu Ser Leu Ala Val Ala Gly Val His Ala Gln Gly Thr Asn 370 375 380Lys Ala Phe Thr Arg Ser Asn Lys Leu Thr Ser Thr Thr Val Asn Gly385 390 395 400Gly Asn Val Ser Gln Leu Arg Ala Lys Ala Leu Ala Lys Asn Glu Asn 405 410 415Tyr Gly Asn Val Lys Gly Thr Gly Gly Ala Leu Val Gly Ala Glu Thr 420 425 430Ala Ala Val Glu Asn Tyr Thr Lys Ser Thr Thr Gly Ala Leu Val Ala 435 440 445Gly Asn Trp Glu Ile Gly Asp Lys Leu Glu Thr Ile Ala Arg Asp Asn 450 455 460Thr Ile Val Arg Val Asn Gly Asp Gly Thr Lys Gly Gly Leu Val Gly465 470 475 480Lys Asn Gly Ile Ser Val Lys Asn Thr Ile Ser Gly Glu Thr Lys Ser 485 490 495Ser Ile Glu Asp Lys Ala Arg Ile Val Gly Thr Gly Ser Val Asn Val 500 505 510Asp Ala Leu Asn Glu Leu Asp Val Asp Leu Gln Gly Lys Ser Gly Gly 515 520 525Tyr Gly Gly Ile Gly Ile Gly Asn Val Asp Val Asn Asn Val Ile Lys 530 535 540Lys Asn Val Glu Ala Lys Ile Gly Arg His Ala Ile Val Glu Thr Thr545 550 555 560Gly Lys Gln Glu Tyr Gln Ala Phe Thr Arg Ala Lys Val Asn Ile Leu 565 570 575Gly Lys Gly Asp Ala Ala Ala Ala Ala Ala Ile Ser Asn Val His Ile 580 585 590Ser Asn Glu Met Asp Ile Lys Asn Leu Ala Lys Gln Tyr Ala Ser Ser 595 600 605Gln Leu Ile Thr Lys Asn Ser Lys Asn Asn Ile Thr Leu Ala Ser Ser 610 615 620Ser Glu Ser Asn Val Asn Val His Gly Val Ala Glu Ala Arg Gly Ala625 630 635 640Gly Ala Lys Ala Thr Val Ser Val Lys Asn Gln Ile Asn Arg Thr Asn 645 650 655Asn Val Asp Leu Ala Gly Lys Ile Lys Thr Glu Gly Asn Ile Asn Val 660 665 670Tyr Ala Gly Tyr Asp Lys Asn Tyr Asn Ile Ser Lys Thr Asn Ser Lys 675 680 685Ala Ile Ala Asp Ala Lys Ser His Ala Ala Ala Ala Ser Ala Thr Ala 690 695 700Thr Ile Glu Lys Asn Glu Val Lys Phe Asn Asn Ala Ile Arg Glu Phe705 710 715 720Lys Asn Asn Leu Ala Arg Leu Glu Gly Lys Ala Asn Lys Lys Thr Ser 725 730 735Val Gly Ser Asn Gln Val Asp Trp Tyr Thr Asp Lys Tyr Thr Trp His 740 745 750Ser Ser Glu Lys Ala Tyr Lys Lys Leu Thr Tyr Gln Ser Lys Arg Gly 755 760 765Glu Lys Gly Lys Lys 7707338PRTFusobacterium necrophorum 7Ile Asn Met Ala Ser Gly Lys Val Pro Gly Thr Thr Asp Tyr Phe Val 1 5 10 15Gln Ile Tyr Glu Pro Lys Arg Gln Gln Phe Phe Val Phe Ala Asp Asn 20 25 30Leu Gly Gln Lys Asn Thr Gly Glu Leu Arg Trp Gly Leu Asn Tyr Ile 35 40 45Asn Asn Ser Val Thr Gly Asn Arg Asp Gln Leu Ser Leu Thr Ser Leu 50 55 60Val Thr Glu Gly Thr Ala Ser Leu Ser Ser Phe Tyr Thr Phe Pro Val 65 70 75 80Ser Lys Lys Gly Thr Lys Ile Ser Leu Gln His Ser Val Gly Lys Leu 85 90 95Lys His Ile Gln Gly Ala Leu Lys His Lys Ile Thr Gly Asn Ser Tyr 100 105 110Ser Tyr Gly Val Gly Ile Val His Pro Ile Leu Val His Glu Lys Asn 115 120 125Lys Val Glu Leu Ser Leu Asp Trp Val Lys Gln Arg Thr Val Thr Asp 130 135 140Leu Leu Lys Leu Lys Trp Val Asn Asn Arg Leu Ser Lys Tyr Thr Ala145 150 155 160Gly Ile Gly Ile Ser His Tyr Glu Glu Asp Ser Val Phe Tyr Thr Lys 165 170 175Gln Asn Ile Thr Lys Gly Lys Phe Ile Pro Ile Ser Gly Asp Ala Arg 180 185 190Asn Tyr Thr Lys Tyr Asp Met Phe Leu Ile Tyr Gln Lys Asn Leu Lys 195 200 205Tyr Asn Thr Leu Val Thr Leu Lys Met Ala Gly Gln Tyr Ser Leu Ser 210 215 220Lys Lys Leu Pro Ser Val Glu Gln Ile Tyr Ala Gly Gly Ala Tyr Asn225 230 235 240Val Arg Gly Tyr Pro Glu Asn Phe Met Gly Ala Glu His Gly Val Phe 245 250 255Phe Asn Ala Glu Leu Ser Lys Leu Val Glu Asn Lys Gly Glu Phe Phe 260 265 270Val Phe Leu Asp Gly Ala Ser Leu His Gly Glu Ser Ala Trp Gln Glu 275 280 285Asn Arg Ile Phe Ser Ser Gly Phe Gly Tyr Lys Ile Arg Phe Leu Glu 290 295 300Lys Asn Asn Ile Ala Val Ser Met Ala Phe Pro Trp Lys Lys Lys Ile305 310 315 320Asn Ser Ile Ser Val Asp Ser Asn Arg Ile Tyr Ile Thr Ile Asn His 325 330 335Glu Phe89726DNAFusobacterium necrophorum 8atgagcggca tcaaaaataa cgttcagagg acaaggaaga ggatatcaga ttctaaaaaa 60gttttaatga ttttgggatt gttgattaac actatgacgg tgagggctaa tgatacaatc 120accgcgactg agaattttgg aacaaaaata gaaaaaaagg ataatgttta tgacattact 180acaaacaaga ttcaagggga gaacgctttt aacagtttta atagatttgc tttaacagaa 240aataatatag caaatctata ttttggggaa aagaatagta cgggggtaaa taatcttttt 300aactttgtca atggaaaaat tgaagtagat gggattatca acggaattcg agaaaataaa 360attggaggaa atttatattt cttaagctcg gaagggatgg cagtaggaaa aaatggagtt 420atcaatgctg gttcttttca ttctattatt ccaaaacaag atgattttaa gaaggctttg 480gaagaagcca aacatggtaa agtttttaat ggaatcattc cagtagatgg aaaagtaaaa 540attccattga atccgaatgg aagcattacg gtagaaggaa aaatcaatgc tgttgaaggc 600atcggtttat atgcggcgga tattagattg aaagatactg caatactaaa gacaggaatt 660acagatttta aaaatttagt caatattagt gatcgaataa attctggtct gaccggagat 720ttaaaagcta ccaagacaaa atctggagat attattcttt cagctcacat agattctcct 780caaaaagcta tgggaaaaaa ttcaactgtt ggaaagagaa tagaagaata tgtaaaagga 840aataccaaag caaatattga atctgatgct gtattggaag cagatggaaa tataaaaatt 900agtgcgaaag ctacaaatgg gagatttata aagaaagaag gggaaaaaga aacttataac 960actcctttaa gtttatcaga tgtggaagct tccgtaagag taaataaagg aaaagtcata 1020ggaaagaatg ttgacattac agctgaagca aagaatttct atgatgcaac tttagttact 1080aagcttgcaa agcactcttt tagctttgtt acaggttcta tttctcctat caatttaaat 1140ggatttttag gtttattgac aagtaagtcc agtgtcgtta ttggaaaaga tgccaaagtc 1200gaagcaacag aaggaaaggc aaatattcat tcttacagtg gagtaagagc aactatggga 1260gcagctactt ctccattaaa aattaccaat ttatatttgg agaaagccaa tggaaaactt 1320ctcagtatcg gagcgggata tatttctgca aaaagtaatt ccaatgtaac tattgaagga 1380gaagtaaaat cgaagggaag agcagatatt acttcaaaat ctgaaaatac tattgatgct 1440tctgtttctg ttggaacgat gagagattcc aataaagtag ctctttcagt attggtgacg 1500gaaggagaaa ataaatcttc cgtcaagatt gctaaaggag caaaagtaga atcagaaacg 1560gatgatgtaa atgtgagaag tgaagcgatt aattccattc gagctgctgt aaaaggtgga 1620ttgggggata gtggtaatgg ggttgtggct gcaaatattt ctaactataa tgcttcctcc 1680cgtatagatg tagatggata tctacatgcc aagaagcgac taaatgtgga ggctcataac 1740attactaaaa atagtgttct gcaaacagga tctgatttgg gaacttccaa gtttatgaat 1800gatcacgttt atgaatcagg tcatctaaaa tcaattttag atgcaataaa acagcggttt 1860ggaggagaca gtgtcaatga ggaaataaag aataagctaa cgaacttatt tagtgtcggt 1920gtgtctgcaa ccatagcaaa tcataataat tctgcttctg tggcaatagg agagagtgga 1980agactttctt caggagtgga agggagtaat gtaagggcat taaatgaagc tcaaaatctt 2040cgagcgacta cgtcaagtgg aagtgtggct gtacgaaagg aagaaaaaaa gaaacttatt 2100ggaaatgcag cagtttttta tggaaactat aaaaataatg cttctgtgac aattgccgat 2160catgctgaat tggtatcgga aggaaaaatt gatatcaaca gtgaaaataa aattgaatat 2220aaaaatcctt caaaaatggc aaagtctgtt attgataaat tagaactttt aaagagagct 2280tttggaaaag aaacgaaaac tccagaatat gatccgaaag atattgaatc tattgaaaaa 2340ttattgaatg cattttcaga aaaattggat ggaaaaccgg agcttttact aaatggtgaa 2400agaatgacaa ttattcttcc ggatggaact
tcaaaaacag gaactgctat agaaattgca 2460aactatgttc agggagaaat gaaaaaatta gaggaaaaat taccgaaagg atttaaagct 2520ttttcagaag gattgagtgg actgattaaa gaaactttga attttacagg agtaggaaat 2580tatgcaaatt ttcacacttt tacctcttcc ggagctaatg gagaaagaga tgtttcttct 2640gtgggaggag ctgtttcgtg ggtagaacag gagaattata gcaaggtatc cgttggaaaa 2700ggagctaaac ttgctgcaaa aaaagattta aatataaaag ctatcaataa agcagaaaca 2760gtgaatttag ttggaaatat tggacttgcg agaagcagta catccggaag tgcagtcgga 2820ggaagattaa atgttcaaag atcgaaaaat tcagctatcg tagaagctaa agaaaaagct 2880gaattatcag gagaaaatat taatgcagat gcattgaaca gactttttca tgtagcggga 2940tcttttaatg gtggctcagg tgggaatgca atcaatggaa tgggaagtta tagtggaggt 3000atcagtaagg caagagtttc cattgatgac gaagcatatt tgaaagctaa taaaaaaatt 3060gctttaaaca gtaagaatga tacttctgtt tggaatgctg ccggttcagc gggaatcgga 3120acgaaaaatg cggcggtcgg ggttgctgtt gcggtaaatg attatgatat ttcaaacaaa 3180gcttccattg aagataatga cgaaggacaa agtaaatatg ataagaataa agatgatgaa 3240gtaacagtaa ctgcggaatc tttagaagta gatgcaaaaa cgaccggaac aatcaacagt 3300atttctgttg ccggaggaat taataaggtt ggaagtaaac cgagtgaaga aaaaccgaaa 3360tcagaagaaa gaccagaggg attttttggc aaaatcggaa acaaagtgga ctctgtaaaa 3420aataaaatta cggatagtat ggattcatta acagaaaaaa ttacaaatta catttctgaa 3480ggagtaaaaa aagcggggaa tcttccttcg aacgtttctc atactcccga taaaggaccg 3540tctttcagtt tgggagcttc tggaagtgtt tctttcaata atattaaaaa ggaaacatct 3600gctgtcgtag atggagtaaa gataaatttg aagggagcaa ataaaaaggt agaggtgact 3660tcttctgatt ctacttttgt tggagcatgg ggcggatctg ctgcacttca gtggaatcat 3720attggaagtg gaaatagcaa catcagtgct ggtttagctg gagcggctgc tgtaaataat 3780attcaaagta aaacaagtgc tttggttaaa aatagtgata ttcgaaatgc caataaattt 3840aaagtaaatg ctttgagtgg aggaactcaa gtagcagcag gagcaggttt ggaagcagtt 3900aaagaaagtg gaggacaagg aaaaagttat ctattgggaa cttctgcttc tatcaactta 3960gtgaacaatg aagtttctgc aaaatcagaa aataatacag tagcaggaga atctgaaagc 4020caaaaaatgg atgttgatgt cactgcttat caagcggaca cccaagtgac aggagcttta 4080aatttacaag ctggaaagtc aaatggaact gtaggggcta ctgtgactgt tgccaaatta 4140aacaacaaag taaatgcttc tattagtggt gggagatata ctaacgttaa tcgagcggac 4200gcaaaagctc ttttagcaac cactcaagtg actgctgcag tgacgacggg agggacaatt 4260agttctggag cgggattagg aaattatcaa ggggctgttt ctgtcaataa gattgacaat 4320gacgtggaag ctagcgttga taaatcttcc atcgaaggag ctaatgaaat caatgtcatt 4380gccaaagatg tcaaaggaag ttctgatcta gcaaaagaat atcaggcttt actaaatgga 4440aaagataaaa aatatttaga agatcgtggt attaatacga ctggaaatgg ttattatacg 4500aaggaacaac tagaaaaagc aaagaaaaaa gaaggagcgg tcattgtaaa tgctgcttta 4560tcggttgctg gaacggataa atccgctgga ggagtagcta ttgcagtcaa tactgttaaa 4620aataaattta aagcagaatt gagtggaagc aataaggaag ccggagagga taaaattcat 4680gcgaaacatg taaatgtgga ggcaaaatca tctactgttg ttgtgaatgc ggcttctgga 4740cttgctatca gcaaagatgc tttttcagga atgggatctg gagcatggca agacttatca 4800aatgacacga ttgcaaaggt ggataaagga agaatttctg ctgattcctt aaatgtgaac 4860gcaaataatt ccattcttgg ggtgaatgtt gcgggaacca ttgccggttc tctttctacg 4920gcggtaggag ctgcttttgc gaataatact cttcataata aaacctctgc tttgattaca 4980ggaacgaagg taaatccttt tagtggaaag aatacaaaag tcaatgtaca agctttgaat 5040gattctcata ttacaaacgt ttctgctgga ggcgctgcaa gtattaagca ggctggaatc 5100ggaggaatgg tatctgtcaa tcgtggttct gatgaaacgg aagctttagt tagtgattct 5160gagtttgaag gagtaagttc tttcaatgta gatgcaaaag atcaaaaaac aataaataca 5220attgccggaa atgcaaatgg aggaaaagcg gctggagttg gagcaacagt tgctcataca 5280aatattggaa aacaatcagt tatagctatt gtaaaaaaca gtaaaattac aacggcgaat 5340gatcaagata gaaaaaatat caatgtgact gcaaaagatt atactatgac caatactata 5400gcagtcggag ttggaggagc aaaaggagcc tctgtgcaag gagcttctgc aagtactacc 5460ttgaataaga cagtttcttc tcatgttgat caaactgata ttgacaaaga tttagaggaa 5520gaaaataatg gaaataagga aaaggcaaat gttaatgttc tagctgaaaa tacgagtcaa 5580gtggtcacaa atgcgacagt gctttccgga gcaagtggac aagctgcagt aggagctgga 5640gtagcagtta ataaaattac acaaaatact tctgcacata taaaaaatag tactcaaaat 5700gtacgaaatg ctttggtaaa aagcaaatct cattcatcta ttaaaacaat tggaattgga 5760gctggagttg gagctggagg agctggagtg acaggttctg tagcagtgaa taagattgta 5820aataatacga tagcagaatt aaatcatgca aaaatcactg cgaagggaaa tgtcggagtt 5880attacagagt ctgatgcggt aattgctaat tatgcaggaa cagtgtctgg agtggcccgt 5940gcagcaatag gagcctcaac cagtgtgaat gaaattacag gatctacaaa agcatatgta 6000aaagattcta cagtgattgc taaagaagaa acagatgatt atattactac tcaagggcaa 6060gtagataaag tggtagataa agtattcaaa aatcttaata ttaacgaaga cttatcacaa 6120aaaagaaaaa taagtaataa aaaaggattt gttaccaata gttcagctac tcatacttta 6180aaatctttat tggcaaatgc cgctggttca ggacaagccg gagtggcagg aactgttaat 6240atcaacaagg tttatggaga aacagaagct cttgtagaaa attctatatt aaatgcaaaa 6300cattattctg taaaatcagg agattacacg aattcaatcg gagtagtagg ttctgttggt 6360gttggtggaa atgtaggagt aggagcttct tctgatacca atattataaa aagaaatacc 6420aagacaagag ttggaaaaac tacaatgtct gatgaaggtt tcggagaaga agctgaaatt 6480acagcagatt ctaagcaagg aatttcctct tttggagtcg gagtcgcagc agccggggta 6540ggagccggag tggcaggaac cgtttccgta aatcaatttg caggaaagac ggaagtagat 6600gtggaagaag caaagatttt ggtaaaaaaa gctgagatta cagcaaaacg ttatagttct 6660gttgcaattg gaaatgccgc agtcggagtg gctgcaaaag gagctggaat tggagcagca 6720gtggcagtta ccaaagatga atcaaacacg agagcaagag tgaaaaattc taaaattatg 6780actcgaaaca agttagatgt aatagcagaa aatgagataa aatcaggtac tggaatcggt 6840tcagccggag ctggaattct tgcagccgga gtatctggag tggtttctgt caataatatt 6900gcaaataagg tagaaacaga tatcgatcat agtactttac actcttctac tgatgtaaat 6960gtaaaagctc ttaataaaat ttcgaattcc ttgacagccg gtggaggagc cgcaggtctt 7020gcagcagtta ccggagtggt ttctgttaac actataaata gttctgtgat agctcgagtt 7080cacaataact ctgatttgac ttccgtacga gaaaaagtaa atgtaacggc aaaagaggaa 7140aaaaatatta agcaaacagc agcaaatgca ggaatcggag gagcagcaat cggagccaat 7200gtcttggtaa ataattttgg aacagctgta gaagatagaa aaaattctga aggaaaagga 7260acagaagttt taaaaacttt agacgaagtt aacaaagaac aagataaaaa agtaaatgat 7320gctacgaaaa aaatcttaca atcagcaggt atttctacag aagatacttc tgtaaaagcg 7380gatagaggag atactcaggg agaaggaatt aaagccattg tgaagacttc tgatattatt 7440ggaaaaaatg tagatattac aacagaggac aagaataata tcacttctac tggtggtttg 7500ggaactgcag gtcttgcttc cgcatcagga acagtggcag ttacaaatat taaaagaaat 7560tccggagtta ctgttgaaaa ttcttttgtg aaagcagctg aaaaagtaaa tgttagatcg 7620gatattacag gaaatgttgc tttaacagca tatcaaggtc ctgtaggagc attgggaata 7680ggagctgcct atgcagaatt aaattctaat ggaagatcaa atatcagtat taaaaattct 7740aagctattag gaaaaaatat tgatgttatt gtaaaagata aatcggaatt gagagcggaa 7800gcaaaaggat taaccgtagg agcggtagct gccggagcca ttatctcaaa agcaaagaat 7860gaaatgaatt cagaggttga aattgagaag agtattttca atgaagaaaa tagagtaact 7920agcccttcta aaggaattgg aagagaaatc aatgtcaaag tggaaaaaga aaacagagtg 7980actgctgaat ctcaaggagc ttctgtagga gcagtagcag gggcaggaat tatttccgaa 8040gcaaaagatg ccggaagctc ttatttgaaa gttagtacaa aatccggaag aagtattttt 8100catgcagata atgtgaatat ggaagcaaca cataaaatga aagtaacagc agtttctaaa 8160gcagtaacag gttctgtatt gggaggagtt ggagtcacca aggcagaagc tactgctgca 8220ggtaaaacta tggtagaagt tgaggaagga aatttgttca gaacaaatcg attgaatgca 8280atttctaaag tagaaggttt ggatgaagat aaagtaactg ctaaatcttc tgtagtatca 8340ggaaatggag gaggaattgc cggagcagga gtgaatactt ctacagcaca aagtaatact 8400gaatccgtag ttcgtttacg aaagcaagat tatgaaaata atgattacac aaaaaaatat 8460atttcagaag tcaatgctct tgctttaaat gatacaaaga atgaagcgaa tatagaatct 8520ttagcggtag ccggtgtgca tgcacaagga acaaacaaag catttacgag atcaaacaag 8580ttaacttcta caactgtaaa tggaggaaac gtatctcaac ttcgtgcaaa agctttggct 8640aaaaatgaaa attatggaaa tgtaaaagga actggaggag ccttagtcgg agcggaaaca 8700gcagccgttg aaaattatac aaagagtact acaggagcat tggttgcagg aaattgggaa 8760attggagata aattagaaac gattgcaaga gataatacga ttgtaagagt caacggagac 8820ggaaccaaag gaggtcttgt cggaaagaat ggtatttctg tgaaaaatac aatttcaggg 8880gaaacaaaat catccattga agataaagcc agaattgttg gaaccggaag tgtaaatgta 8940gatgctttga atgaacttga tgtagatcta caaggaaaaa gtggtggcta tggtggaatt 9000ggtattggaa atgttgatgt aaataatgtg attaagaaaa atgtagaagc caaaatcgga 9060agacatgcta ttgtagaaac tactggaaaa caagaatatc aagcatttac aagagcaaaa 9120gtaaatattc ttggaaaagg agacgctgca gctgcagctg caatatcgaa tgtacacatt 9180tccaatgaga tggatattaa aaatttggca aagcagtatg catcttctca attaataacc 9240aaaaattcaa aaaataatat tactttagca tcaagtagtg aatcgaatgt gaatgttcat 9300ggggtggctg aagcaagagg tgcaggagcc aaagcgacag ttagtgtaaa gaatcaaata 9360aatagaacta ataatgttga tttagcagga aaaattaaaa cagagggaaa catcaatgta 9420tatgccggat atgataaaaa ttataatata agtaagacaa attctaaggc tattgcggat 9480gccaaaagtc atgctgcagc tgcttcggca actgccacta ttgaaaaaaa tgaagtaaaa 9540tttaataatg cgatccgaga atttaaaaat aatctggcaa gattggaagg gaaagctaat 9600aaaaaaacgt cggtaggatc taatcaggta gactggtata cggataaata tacatggcat 9660tcttctgaaa aagcatacaa aaaattgaca tatcaatcaa agagaggaga aaaagggaaa 9720aaatga 972691130DNAFusobacterium necrophorum 9atgagcggca tcaaaaataa cgttcagagg acaaggaaga ggatatcaga ttctaaaaaa 60gttttaatga ttttgggatt gttgattaac actatgacgg tgagggctaa tgatacaatc 120accgcgactg agaattttgg aacaaaaata gaaaaaaagg ataatgttta tgacattact 180acaaacaaga ttcaagggga gaacgctttt aacagtttta atagatttgc tttaacagaa 240aataatatag caaatctata ttttggggaa aagaatagta cgggggtaaa taatcttttt 300aactttgtca atggaaaaat tgaagtagat gggattatca acggaattcg agaaaataaa 360attggaggaa atttatattt cttaagctcg gaagggatgg cagtaggaaa aaatggagtt 420atcaatgctg gttcttttca ttctattatt ccaaaacaag atgattttaa gaaggctttg 480gaagaagcca aacatggtaa agtttttaat ggaatcattc cagtagatgg aaaagtaaaa 540attccattga atccgaatgg aagcattacg gtagaaggaa aaatcaatgc tgttgaaggc 600atcggtttat atgcggcgga tattagattg aaagatactg caatactaaa gacaggaatt 660acagatttta aaaatttagt caatattagt gatcgaataa attctggtct gaccggagat 720ttaaaagcta ccaagacaaa atctggagat attattcttt cagctcacat agattctcct 780caaaaagcta tgggaaaaaa ttcaactgtt ggaaagagaa tagaagaata tgtaaaagga 840aataccaaag caaatattga atctgatgct gtattggaag cagatggaaa tataaaaatt 900agtgcgaaag ctacaaatgg gagatttata aagaaagaag gggaaaaaga aacttataac 960actcctttaa gtttatcaga tgtggaagct tccgtaagag taaataaagg aaaagtcata 1020ggaaagaatg ttgacattac agctgaagca aagaatttct atgatgcaac tttagttact 1080aagcttgcaa agcactcttt tagctttgtt acaggttcta tttctcctat 1130102780DNAFusobacterium necrophorum 10gggagattta taaagaaaga aggggaaaaa gaaacttata acactccttt aagtttatca 60gatgtggaag cttccgtaag agtaaataaa ggaaaagtca taggaaagaa tgttgacatt 120acagctgaag caaagaattt ctatgatgca actttagtta ctaagcttgc aaagcactct 180tttagctttg ttacaggttc tatttctcct atcaatttaa atggattttt aggtttattg 240acaagtaagt ccagtgtcgt tattggaaaa gatgccaaag tcgaagcaac agaaggaaag 300gcaaatattc attcttacag tggagtaaga gcaactatgg gagcagctac ttctccatta 360aaaattacca atttatattt ggagaaagcc aatggaaaac ttctcagtat cggagcggga 420tatatttctg caaaaagtaa ttccaatgta actattgaag gagaagtaaa atcgaaggga 480agagcagata ttacttcaaa atctgaaaat actattgatg cttctgtttc tgttggaacg 540atgagagatt ccaataaagt agctctttca gtattggtga cggaaggaga aaataaatct 600tccgtcaaga ttgctaaagg agcaaaagta gaatcagaaa cggatgatgt aaatgtgaga 660agtgaagcga ttaattccat tcgagctgct gtaaaaggtg gattggggga tagtggtaat 720ggggttgtgg ctgcaaatat ttctaactat aatgcttcct cccgtataga tgtagatgga 780tatctacatg ccaagaagcg actaaatgtg gaggctcata acattactaa aaatagtgtt 840ctgcaaacag gatctgattt gggaacttcc aagtttatga atgatcacgt ttatgaatca 900ggtcatctaa aatcaatttt agatgcaata aaacagcggt ttggaggaga cagtgtcaat 960gaggaaataa agaataagct aacgaactta tttagtgtcg gtgtgtctgc aaccatagca 1020aatcataata attctgcttc tgtggcaata ggagagagtg gaagactttc ttcaggagtg 1080gaagggagta atgtaagggc attaaatgaa gctcaaaatc ttcgagcgac tacgtcaagt 1140ggaagtgtgg ctgtacgaaa ggaagaaaaa aagaaactta ttggaaatgc agcagttttt 1200tatggaaact ataaaaataa tgcttctgtg acaattgccg atcatgctga attggtatcg 1260gaaggaaaaa ttgatatcaa cagtgaaaat aaaattgaat ataaaaatcc ttcaaaaatg 1320gcaaagtctg ttattgataa attagaactt ttaaagagag cttttggaaa agaaacgaaa 1380actccagaat atgatccgaa agatattgaa tctattgaaa aattattgaa tgcattttca 1440gaaaaattgg atggaaaacc ggagctttta ctaaatggtg aaagaatgac aattattctt 1500ccggatggaa cttcaaaaac aggaactgct atagaaattg caaactatgt tcagggagaa 1560atgaaaaaat tagaggaaaa attaccgaaa ggatttaaag ctttttcaga aggattgagt 1620ggactgatta aagaaacttt gaattttaca ggagtaggaa attatgcaaa ttttcacact 1680tttacctctt ccggagctaa tggagaaaga gatgtttctt ctgtgggagg agctgtttcg 1740tgggtagaac aggagaatta tagcaaggta tccgttggaa aaggagctaa acttgctgca 1800aaaaaagatt taaatataaa agctatcaat aaagcagaaa cagtgaattt agttggaaat 1860attggacttg cgagaagcag tacatccgga agtgcagtcg gaggaagatt aaatgttcaa 1920agatcgaaaa attcagctat cgtagaagct aaagaaaaag ctgaattatc aggagaaaat 1980attaatgcag atgcattgaa cagacttttt catgtagcgg gatcttttaa tggtggctca 2040ggtgggaatg caatcaatgg aatgggaagt tatagtggag gtatcagtaa ggcaagagtt 2100tccattgatg acgaagcata tttgaaagct aataaaaaaa ttgctttaaa cagtaagaat 2160gatacttctg tttggaatgc tgccggttca gcgggaatcg gaacgaaaaa tgcggcggtc 2220ggggttgctg ttgcggtaaa tgattatgat atttcaaaca aagcttccat tgaagataat 2280gacgaaggac aaagtaaata tgataagaat aaagatgatg aagtaacagt aactgcggaa 2340tctttagaag tagatgcaaa aacgaccgga acaatcaaca gtatttctgt tgccggagga 2400attaataagg ttggaagtaa accgagtgaa gaaaaaccga aatcagaaga aagaccagag 2460ggattttttg gcaaaatcgg aaacaaagtg gactctgtaa aaaataaaat tacggatagt 2520atggattcat taacagaaaa aattacaaat tacatttctg aaggagtaaa aaaagcgggg 2580aatcttcctt cgaacgtttc tcatactccc gataaaggac cgtctttcag tttgggagct 2640tctggaagtg tttctttcaa taatattaaa aaggaaacat ctgctgtcgt agatggagta 2700aagataaatt tgaagggagc aaataaaaag gtagaggtga cttcttctga ttctactttt 2760gttggagcat ggggcggatc 2780112141DNAFusobacterium necrophorum 11ggagcttctg gaagtgtttc tttcaataat attaaaaagg aaacatctgc tgtcgtagat 60ggagtaaaga taaatttgaa gggagcaaat aaaaaggtag aggtgacttc ttctgattct 120acttttgttg gagcatgggg cggatctgct gcacttcagt ggaatcatat tggaagtgga 180aatagcaaca tcagtgctgg tttagctgga gcggctgctg taaataatat tcaaagtaaa 240acaagtgctt tggttaaaaa tagtgatatt cgaaatgcca ataaatttaa agtaaatgct 300ttgagtggag gaactcaagt agcagcagga gcaggtttgg aagcagttaa agaaagtgga 360ggacaaggaa aaagttatct attgggaact tctgcttcta tcaacttagt gaacaatgaa 420gtttctgcaa aatcagaaaa taatacagta gcaggagaat ctgaaagcca aaaaatggat 480gttgatgtca ctgcttatca agcggacacc caagtgacag gagctttaaa tttacaagct 540ggaaagtcaa atggaactgt aggggctact gtgactgttg ccaaattaaa caacaaagta 600aatgcttcta ttagtggtgg gagatatact aacgttaatc gagcggacgc aaaagctctt 660ttagcaacca ctcaagtgac tgctgcagtg acgacgggag ggacaattag ttctggagcg 720ggattaggaa attatcaagg ggctgtttct gtcaataaga ttgacaatga cgtggaagct 780agcgttgata aatcttccat cgaaggagct aatgaaatca atgtcattgc caaagatgtc 840aaaggaagtt ctgatctagc aaaagaatat caggctttac taaatggaaa agataaaaaa 900tatttagaag atcgtggtat taatacgact ggaaatggtt attatacgaa ggaacaacta 960gaaaaagcaa agaaaaaaga aggagcggtc attgtaaatg ctgctttatc ggttgctgga 1020acggataaat ccgctggagg agtagctatt gcagtcaata ctgttaaaaa taaatttaaa 1080gcagaattga gtggaagcaa taaggaagcc ggagaggata aaattcatgc gaaacatgta 1140aatgtggagg caaaatcatc tactgttgtt gtgaatgcgg cttctggact tgctatcagc 1200aaagatgctt tttcaggaat gggatctgga gcatggcaag acttatcaaa tgacacgatt 1260gcaaaggtgg ataaaggaag aatttctgct gattccttaa atgtgaacgc aaataattcc 1320attcttgggg tgaatgttgc gggaaccatt gccggttctc tttctacggc ggtaggagct 1380gcttttgcga ataatactct tcataataaa acctctgctt tgattacagg aacgaaggta 1440aatcctttta gtggaaagaa tacaaaagtc aatgtacaag ctttgaatga ttctcatatt 1500acaaacgttt ctgctggagg cgctgcaagt attaagcagg ctggaatcgg aggaatggta 1560tctgtcaatc gtggttctga tgaaacggaa gctttagtta gtgattctga gtttgaagga 1620gtaagttctt tcaatgtaga tgcaaaagat caaaaaacaa taaatacaat tgccggaaat 1680gcaaatggag gaaaagcggc tggagttgga gcaacagttg ctcatacaaa tattggaaaa 1740caatcagtta tagctattgt aaaaaacagt aaaattacaa cggcgaatga tcaagataga 1800aaaaatatca atgtgactgc aaaagattat actatgacca atactatagc agtcggagtt 1860ggaggagcaa aaggagcctc tgtgcaagga gcttctgcaa gtactacctt gaataagaca 1920gtttcttctc atgttgatca aactgatatt gacaaagatt tagaggaaga aaataatgga 1980aataaggaaa aggcaaatgt taatgttcta gctgaaaata cgagtcaagt ggtcacaaat 2040gcgacagtgc tttccggagc aagtggacaa gctgcagtag gagctggagt agcagttaat 2100aaaattacac aaaatacttc tgcacatata aaaaatagta c 2141121887DNAFusobacterium necrophorum 12ctgcagtagg agctggagta gcagttaata aaattacaca aaatacttct gcacatataa 60aaaatagtac tcaaaatgta cgaaatgctt tggtaaaaag caaatctcat tcatctatta 120aaacaattgg aattggagct ggagttggag ctggaggagc tggagtgaca ggttctgtag 180cagtgaataa gattgtaaat aatacgatag cagaattaaa tcatgcaaaa atcactgcga 240agggaaatgt cggagttatt acagagtctg atgcggtaat tgctaattat gcaggaacag 300tgtctggagt ggcccgtgca gcaataggag cctcaaccag tgtgaatgaa attacaggat 360ctacaaaagc atatgtaaaa gattctacag tgattgctaa agaagaaaca gatgattata 420ttactactca agggcaagta gataaagtgg tagataaagt attcaaaaat cttaatatta 480acgaagactt atcacaaaaa agaaaaataa gtaataaaaa aggatttgtt accaatagtt 540cagctactca tactttaaaa tctttattgg caaatgccgc tggttcagga caagccggag 600tggcaggaac tgttaatatc aacaaggttt atggagaaac agaagctctt gtagaaaatt 660ctatattaaa tgcaaaacat tattctgtaa aatcaggaga ttacacgaat tcaatcggag 720tagtaggttc tgttggtgtt ggtggaaatg taggagtagg agcttcttct gataccaata 780ttataaaaag aaataccaag acaagagttg gaaaaactac aatgtctgat gaaggtttcg 840gagaagaagc tgaaattaca gcagattcta agcaaggaat ttcctctttt ggagtcggag 900tcgcagcagc cggggtagga gccggagtgg caggaaccgt ttccgtaaat caatttgcag 960gaaagacgga agtagatgtg gaagaagcaa agattttggt aaaaaaagct gagattacag 1020caaaacgtta tagttctgtt gcaattggaa atgccgcagt cggagtggct gcaaaaggag 1080ctggaattgg agcagcagtg gcagttacca aagatgaatc aaacacgaga gcaagagtga 1140aaaattctaa aattatgact cgaaacaagt tagatgtaat agcagaaaat gagataaaat 1200caggtactgg aatcggttca gccggagctg gaattcttgc agccggagta tctggagtgg 1260tttctgtcaa taatattgca aataaggtag aaacagatat cgatcatagt actttacact 1320cttctactga tgtaaatgta aaagctctta ataaaatttc gaattccttg acagccggtg 1380gaggagccgc aggtcttgca gcagttaccg gagtggtttc tgttaacact ataaatagtt 1440ctgtgatagc tcgagttcac aataactctg
atttgacttc cgtacgagaa aaagtaaatg 1500taacggcaaa agaggaaaaa aatattaagc aaacagcagc aaatgcagga atcggaggag 1560cagcaatcgg agccaatgtc ttggtaaata attttggaac agctgtagaa gatagaaaaa 1620attctgaagg aaaaggaaca gaagttttaa aaactttaga cgaagttaac aaagaacaag 1680ataaaaaagt aaatgatgct acgaaaaaaa tcttacaatc agcaggtatt tctacagaag 1740atacttctgt aaaagcggat agaggagata ctcagggaga aggaattaaa gccattgtga 1800agacttctga tattattgga aaaaatgtag atattacaac agaggacaag aataatatca 1860cttctactgg tggtttggga actgcag 1887132322DNAFusobacterium necrophorum 13ggaattaaag ccattgtgaa gacttctgat attattggaa aaaatgtaga tattacaaca 60gaggacaaga ataatatcac ttctactggt ggtttgggaa ctgcaggtct tgcttccgca 120tcaggaacag tggcagttac aaatattaaa agaaattccg gagttactgt tgaaaattct 180tttgtgaaag cagctgaaaa agtaaatgtt agatcggata ttacaggaaa tgttgcttta 240acagcatatc aaggtcctgt aggagcattg ggaataggag ctgcctatgc agaattaaat 300tctaatggaa gatcaaatat cagtattaaa aattctaagc tattaggaaa aaatattgat 360gttattgtaa aagataaatc ggaattgaga gcggaagcaa aaggattaac cgtaggagcg 420gtagctgccg gagccattat ctcaaaagca aagaatgaaa tgaattcaga ggttgaaatt 480gagaagagta ttttcaatga agaaaataga gtaactagcc cttctaaagg aattggaaga 540gaaatcaatg tcaaagtgga aaaagaaaac agagtgactg ctgaatctca aggagcttct 600gtaggagcag tagcaggggc aggaattatt tccgaagcaa aagatgccgg aagctcttat 660ttgaaagtta gtacaaaatc cggaagaagt atttttcatg cagataatgt gaatatggaa 720gcaacacata aaatgaaagt aacagcagtt tctaaagcag taacaggttc tgtattggga 780ggagttggag tcaccaaggc agaagctact gctgcaggta aaactatggt agaagttgag 840gaaggaaatt tgttcagaac aaatcgattg aatgcaattt ctaaagtaga aggtttggat 900gaagataaag taactgctaa atcttctgta gtatcaggaa atggaggagg aattgccgga 960gcaggagtga atacttctac agcacaaagt aatactgaat ccgtagttcg tttacgaaag 1020caagattatg aaaataatga ttacacaaaa aaatatattt cagaagtcaa tgctcttgct 1080ttaaatgata caaagaatga agcgaatata gaatctttag cggtagccgg tgtgcatgca 1140caaggaacaa acaaagcatt tacgagatca aacaagttaa cttctacaac tgtaaatgga 1200ggaaacgtat ctcaacttcg tgcaaaagct ttggctaaaa atgaaaatta tggaaatgta 1260aaaggaactg gaggagcctt agtcggagcg gaaacagcag ccgttgaaaa ttatacaaag 1320agtactacag gagcattggt tgcaggaaat tgggaaattg gagataaatt agaaacgatt 1380gcaagagata atacgattgt aagagtcaac ggagacggaa ccaaaggagg tcttgtcgga 1440aagaatggta tttctgtgaa aaatacaatt tcaggggaaa caaaatcatc cattgaagat 1500aaagccagaa ttgttggaac cggaagtgta aatgtagatg ctttgaatga acttgatgta 1560gatctacaag gaaaaagtgg tggctatggt ggaattggta ttggaaatgt tgatgtaaat 1620aatgtgatta agaaaaatgt agaagccaaa atcggaagac atgctattgt agaaactact 1680ggaaaacaag aatatcaagc atttacaaga gcaaaagtaa atattcttgg aaaaggagac 1740gctgcagctg cagctgcaat atcgaatgta cacatttcca atgagatgga tattaaaaat 1800ttggcaaagc agtatgcatc ttctcaatta ataaccaaaa attcaaaaaa taatattact 1860ttagcatcaa gtagtgaatc gaatgtgaat gttcatgggg tggctgaagc aagaggtgca 1920ggagccaaag cgacagttag tgtaaagaat caaataaata gaactaataa tgttgattta 1980gcaggaaaaa ttaaaacaga gggaaacatc aatgtatatg ccggatatga taaaaattat 2040aatataagta agacaaattc taaggctatt gcggatgcca aaagtcatgc tgcagctgct 2100tcggcaactg ccactattga aaaaaatgaa gtaaaattta ataatgcgat ccgagaattt 2160aaaaataatc tggcaagatt ggaagggaaa gctaataaaa aaacgtcggt aggatctaat 2220caggtagact ggtatacgga taaatataca tggcattctt ctgaaaaagc atacaaaaaa 2280ttgacatatc aatcaaagag aggagaaaaa gggaaaaaat ga 2322141017DNAFusobacterium necrophorum 14atcaatatgg cttccggaaa agttccggga acgaccgatt attttgtgca aatctatgaa 60ccaaaaagac agcagttttt tgtttttgca gataatttag gacaaaaaaa tacaggagaa 120ttacgatggg ggctaaatta tattaataat agtgttacag gaaacagaga tcaactgtct 180cttacctctt tagtaacaga aggaacggct tctctatctt ctttttatac ttttcctgtt 240tctaaaaaag gaaccaaaat atcactacaa cattctgtag gaaagttgaa acatatacaa 300ggggctttaa agcataaaat aactggaaac tcttatagtt atggggttgg aatagttcat 360cctattctgg ttcatgaaaa aaataaagta gaactttcct tggattgggt aaaacaaagg 420actgttacag atctattgaa attgaaatgg gtaaataata gactttctaa gtatacagcg 480ggaattggaa taagccatta tgaggaagat agtgttttct atacaaagca aaatattaca 540aagggaaaat ttattccaat ttcgggagat gcaagaaatt atacaaagta tgatatgttt 600ctaatatatc agaaaaactt gaaatataac actttagtaa cactaaagat ggcagggcaa 660tattctctga gtaaaaaatt accctctgtc gagcaaattt atgcaggagg agcctataat 720gttcgtggtt atccggaaaa ttttatggga gctgaacacg gagttttttt caatgctgaa 780ttatcaaaat tagtagagaa taaaggagaa ttttttgttt ttttagatgg ggcttctctt 840catggagaga gtgcttggca ggaaaataga atttttagct caggttttgg atataaaata 900aggtttttag aaaaaaataa tattgctgtt agcatggcat ttccatggaa gaaaaaaata 960aatagtattt cagtagattc taatcgaatc tatattacaa taaatcatga attttaa 10171511130DNAFusobacterium necrophorum 15gatcaatatg gcttccggaa aagttccggg aacgaccgat tattttgtgc aaatctatga 60accaaaaaga cagcagtttt ttgtttttgc agataattta ggacaaaaaa atacaggaga 120attacgatgg gggctaaatt atattaataa tagtgttaca ggaaacagag atcaactgtc 180tcttacctct ttagtaacag aaggaacggc ttctctatct tctttttata cttttcctgt 240ttctaaaaaa ggaaccaaaa tatcactaca acattctgta ggaaagttga aacatataca 300aggggcttta aagcataaaa taactggaaa ctcttatagt tatggggttg gaatagttca 360tcctattctg gttcatgaaa aaaataaagt agaactttcc ttggattggg taaaacaaag 420gactgttaca gatctattga aattgaaatg ggtaaataat agactttcta agtatacagc 480gggaattgga ataagccatt atgaggaaga tagtgttttc tatacaaagc aaaatattac 540aaagggaaaa tttattccaa tttcgggaga tgcaagaaat tatacaaagt atgatatgtt 600tctaatatat cagaaaaact tgaaatataa cactttagta acactaaaga tggcagggca 660atattctctg agtaaaaaat taccctctgt cgagcaaatt tatgcaggag gagcctataa 720tgttcgtggt tatccggaaa attttatggg agctgaacac ggagtttttt tcaatgctga 780attatcaaaa ttagtagaga ataaaggaga attttttgtt tttttagatg gggcttctct 840tcatggagag agtgcttggc aggaaaatag aatttttagc tcaggttttg gatataaaat 900aaggttttta gaaaaaaata atattgctgt tagcatggca tttccatgga agaaaaaaat 960aaatagtatt tcagtagatt ctaatcgaat ctatattaca ataaatcatg aattttaaag 1020ggggtaagac aaaatgagcg gcatcaaaaa taacgttcag aggacaagga agaggatatc 1080agattctaaa aaagttttaa tgattttggg attgttgatt aacactatga cggtgagggc 1140taatgataca atcaccgcga ctgagaattt tggaacaaaa atagaaaaaa aggataatgt 1200ttatgacatt actacaaaca agattcaagg ggagaacgct tttaacagtt ttaatagatt 1260tgctttaaca gaaaataata tagcaaatct atattttggg gaaaagaata gtacgggggt 1320aaataatctt tttaactttg tcaatggaaa aattgaagta gatgggatta tcaacggaat 1380tcgagaaaat aaaattggag gaaatttata tttcttaagc tcggaaggga tggcagtagg 1440aaaaaatgga gttatcaatg ctggttcttt tcattctatt attccaaaac aagatgattt 1500taagaaggct ttggaagaag ccaaacatgg taaagttttt aatggaatca ttccagtaga 1560tggaaaagta aaaattccat tgaatccgaa tggaagcatt acggtagaag gaaaaatcaa 1620tgctgttgaa ggcatcggtt tatatgcggc ggatattaga ttgaaagata ctgcaatact 1680aaagacagga attacagatt ttaaaaattt agtcaatatt agtgatcgaa taaattctgg 1740tctgaccgga gatttaaaag ctaccaagac aaaatctgga gatattattc tttcagctca 1800catagattct cctcaaaaag ctatgggaaa aaattcaact gttggaaaga gaatagaaga 1860atatgtaaaa ggaaatacca aagcaaatat tgaatctgat gctgtattgg aagcagatgg 1920aaatataaaa attagtgcga aagctacaaa tgggagattt ataaagaaag aaggggaaaa 1980agaaacttat aacactcctt taagtttatc agatgtggaa gcttccgtaa gagtaaataa 2040aggaaaagtc ataggaaaga atgttgacat tacagctgaa gcaaagaatt tctatgatgc 2100aactttagtt actaagcttg caaagcactc ttttagcttt gttacaggtt ctatttctcc 2160tatcaattta aatggatttt taggtttatt gacaagtaag tccagtgtcg ttattggaaa 2220agatgccaaa gtcgaagcaa cagaaggaaa ggcaaatatt cattcttaca gtggagtaag 2280agcaactatg ggagcagcta cttctccatt aaaaattacc aatttatatt tggagaaagc 2340caatggaaaa cttctcagta tcggagcggg atatatttct gcaaaaagta attccaatgt 2400aactattgaa ggagaagtaa aatcgaaggg aagagcagat attacttcaa aatctgaaaa 2460tactattgat gcttctgttt ctgttggaac gatgagagat tccaataaag tagctctttc 2520agtattggtg acggaaggag aaaataaatc ttccgtcaag attgctaaag gagcaaaagt 2580agaatcagaa acggatgatg taaatgtgag aagtgaagcg attaattcca ttcgagctgc 2640tgtaaaaggt ggattggggg atagtggtaa tggggttgtg gctgcaaata tttctaacta 2700taatgcttcc tcccgtatag atgtagatgg atatctacat gccaagaagc gactaaatgt 2760ggaggctcat aacattacta aaaatagtgt tctgcaaaca ggatctgatt tgggaacttc 2820caagtttatg aatgatcacg tttatgaatc aggtcatcta aaatcaattt tagatgcaat 2880aaaacagcgg tttggaggag acagtgtcaa tgaggaaata aagaataagc taacgaactt 2940atttagtgtc ggtgtgtctg caaccatagc aaatcataat aattctgctt ctgtggcaat 3000aggagagagt ggaagacttt cttcaggagt ggaagggagt aatgtaaggg cattaaatga 3060agctcaaaat cttcgagcga ctacgtcaag tggaagtgtg gctgtacgaa aggaagaaaa 3120aaagaaactt attggaaatg cagcagtttt ttatggaaac tataaaaata atgcttctgt 3180gacaattgcc gatcatgctg aattggtatc ggaaggaaaa attgatatca acagtgaaaa 3240taaaattgaa tataaaaatc cttcaaaaat ggcaaagtct gttattgata aattagaact 3300tttaaagaga gcttttggaa aagaaacgaa aactccagaa tatgatccga aagatattga 3360atctattgaa aaattattga atgcattttc agaaaaattg gatggaaaac cggagctttt 3420actaaatggt gaaagaatga caattattct tccggatgga acttcaaaaa caggaactgc 3480tatagaaatt gcaaactatg ttcagggaga aatgaaaaaa ttagaggaaa aattaccgaa 3540aggatttaaa gctttttcag aaggattgag tggactgatt aaagaaactt tgaattttac 3600aggagtagga aattatgcaa attttcacac ttttacctct tccggagcta atggagaaag 3660agatgtttct tctgtgggag gagctgtttc gtgggtagaa caggagaatt atagcaaggt 3720atccgttgga aaaggagcta aacttgctgc aaaaaaagat ttaaatataa aagctatcaa 3780taaagcagaa acagtgaatt tagttggaaa tattggactt gcgagaagca gtacatccgg 3840aagtgcagtc ggaggaagat taaatgttca aagatcgaaa aattcagcta tcgtagaagc 3900taaagaaaaa gctgaattat caggagaaaa tattaatgca gatgcattga acagactttt 3960tcatgtagcg ggatctttta atggtggctc aggtgggaat gcaatcaatg gaatgggaag 4020ttatagtgga ggtatcagta aggcaagagt ttccattgat gacgaagcat atttgaaagc 4080taataaaaaa attgctttaa acagtaagaa tgatacttct gtttggaatg ctgccggttc 4140agcgggaatc ggaacgaaaa atgcggcggt cggggttgct gttgcggtaa atgattatga 4200tatttcaaac aaagcttcca ttgaagataa tgacgaagga caaagtaaat atgataagaa 4260taaagatgat gaagtaacag taactgcgga atctttagaa gtagatgcaa aaacgaccgg 4320aacaatcaac agtatttctg ttgccggagg aattaataag gttggaagta aaccgagtga 4380agaaaaaccg aaatcagaag aaagaccaga gggatttttt ggcaaaatcg gaaacaaagt 4440ggactctgta aaaaataaaa ttacggatag tatggattca ttaacagaaa aaattacaaa 4500ttacatttct gaaggagtaa aaaaagcggg gaatcttcct tcgaacgttt ctcatactcc 4560cgataaagga ccgtctttca gtttgggagc ttctggaagt gtttctttca ataatattaa 4620aaaggaaaca tctgctgtcg tagatggagt aaagataaat ttgaagggag caaataaaaa 4680ggtagaggtg acttcttctg attctacttt tgttggagca tggggcggat ctgctgcact 4740tcagtggaat catattggaa gtggaaatag caacatcagt gctggtttag ctggagcggc 4800tgctgtaaat aatattcaaa gtaaaacaag tgctttggtt aaaaatagtg atattcgaaa 4860tgccaataaa tttaaagtaa atgctttgag tggaggaact caagtagcag caggagcagg 4920tttggaagca gttaaagaaa gtggaggaca aggaaaaagt tatctattgg gaacttctgc 4980ttctatcaac ttagtgaaca atgaagtttc tgcaaaatca gaaaataata cagtagcagg 5040agaatctgaa agccaaaaaa tggatgttga tgtcactgct tatcaagcgg acacccaagt 5100gacaggagct ttaaatttac aagctggaaa gtcaaatgga actgtagggg ctactgtgac 5160tgttgccaaa ttaaacaaca aagtaaatgc ttctattagt ggtgggagat atactaacgt 5220taatcgagcg gacgcaaaag ctcttttagc aaccactcaa gtgactgctg cagtgacgac 5280gggagggaca attagttctg gagcgggatt aggaaattat caaggggctg tttctgtcaa 5340taagattgac aatgacgtgg aagctagcgt tgataaatct tccatcgaag gagctaatga 5400aatcaatgtc attgccaaag atgtcaaagg aagttctgat ctagcaaaag aatatcaggc 5460tttactaaat ggaaaagata aaaaatattt agaagatcgt ggtattaata cgactggaaa 5520tggttattat acgaaggaac aactagaaaa agcaaagaaa aaagaaggag cggtcattgt 5580aaatgctgct ttatcggttg ctggaacgga taaatccgct ggaggagtag ctattgcagt 5640caatactgtt aaaaataaat ttaaagcaga attgagtgga agcaataagg aagccggaga 5700ggataaaatt catgcgaaac atgtaaatgt ggaggcaaaa tcatctactg ttgttgtgaa 5760tgcggcttct ggacttgcta tcagcaaaga tgctttttca ggaatgggat ctggagcatg 5820gcaagactta tcaaatgaca cgattgcaaa ggtggataaa ggaagaattt ctgctgattc 5880cttaaatgtg aacgcaaata attccattct tggggtgaat gttgcgggaa ccattgccgg 5940ttctctttct acggcggtag gagctgcttt tgcgaataat actcttcata ataaaacctc 6000tgctttgatt acaggaacga aggtaaatcc ttttagtgga aagaatacaa aagtcaatgt 6060acaagctttg aatgattctc atattacaaa cgtttctgct ggaggcgctg caagtattaa 6120gcaggctgga atcggaggaa tggtatctgt caatcgtggt tctgatgaaa cggaagcttt 6180agttagtgat tctgagtttg aaggagtaag ttctttcaat gtagatgcaa aagatcaaaa 6240aacaataaat acaattgccg gaaatgcaaa tggaggaaaa gcggctggag ttggagcaac 6300agttgctcat acaaatattg gaaaacaatc agttatagct attgtaaaaa acagtaaaat 6360tacaacggcg aatgatcaag atagaaaaaa tatcaatgtg actgcaaaag attatactat 6420gaccaatact atagcagtcg gagttggagg agcaaaagga gcctctgtgc aaggagcttc 6480tgcaagtact accttgaata agacagtttc ttctcatgtt gatcaaactg atattgacaa 6540agatttagag gaagaaaata atggaaataa ggaaaaggca aatgttaatg ttctagctga 6600aaatacgagt caagtggtca caaatgcgac agtgctttcc ggagcaagtg gacaagctgc 6660agtaggagct ggagtagcag ttaataaaat tacacaaaat acttctgcac atataaaaaa 6720tagtactcaa aatgtacgaa atgctttggt aaaaagcaaa tctcattcat ctattaaaac 6780aattggaatt ggagctggag ttggagctgg aggagctgga gtgacaggtt ctgtagcagt 6840gaataagatt gtaaataata cgatagcaga attaaatcat gcaaaaatca ctgcgaaggg 6900aaatgtcgga gttattacag agtctgatgc ggtaattgct aattatgcag gaacagtgtc 6960tggagtggcc cgtgcagcaa taggagcctc aaccagtgtg aatgaaatta caggatctac 7020aaaagcatat gtaaaagatt ctacagtgat tgctaaagaa gaaacagatg attatattac 7080tactcaaggg caagtagata aagtggtaga taaagtattc aaaaatctta atattaacga 7140agacttatca caaaaaagaa aaataagtaa taaaaaagga tttgttacca atagttcagc 7200tactcatact ttaaaatctt tattggcaaa tgccgctggt tcaggacaag ccggagtggc 7260aggaactgtt aatatcaaca aggtttatgg agaaacagaa gctcttgtag aaaattctat 7320attaaatgca aaacattatt ctgtaaaatc aggagattac acgaattcaa tcggagtagt 7380aggttctgtt ggtgttggtg gaaatgtagg agtaggagct tcttctgata ccaatattat 7440aaaaagaaat accaagacaa gagttggaaa aactacaatg tctgatgaag gtttcggaga 7500agaagctgaa attacagcag attctaagca aggaatttcc tcttttggag tcggagtcgc 7560agcagccggg gtaggagccg gagtggcagg aaccgtttcc gtaaatcaat ttgcaggaaa 7620gacggaagta gatgtggaag aagcaaagat tttggtaaaa aaagctgaga ttacagcaaa 7680acgttatagt tctgttgcaa ttggaaatgc cgcagtcgga gtggctgcaa aaggagctgg 7740aattggagca gcagtggcag ttaccaaaga tgaatcaaac acgagagcaa gagtgaaaaa 7800ttctaaaatt atgactcgaa acaagttaga tgtaatagca gaaaatgaga taaaatcagg 7860tactggaatc ggttcagccg gagctggaat tcttgcagcc ggagtatctg gagtggtttc 7920tgtcaataat attgcaaata aggtagaaac agatatcgat catagtactt tacactcttc 7980tactgatgta aatgtaaaag ctcttaataa aatttcgaat tccttgacag ccggtggagg 8040agccgcaggt cttgcagcag ttaccggagt ggtttctgtt aacactataa atagttctgt 8100gatagctcga gttcacaata actctgattt gacttccgta cgagaaaaag taaatgtaac 8160ggcaaaagag gaaaaaaata ttaagcaaac agcagcaaat gcaggaatcg gaggagcagc 8220aatcggagcc aatgtcttgg taaataattt tggaacagct gtagaagata gaaaaaattc 8280tgaaggaaaa ggaacagaag ttttaaaaac tttagacgaa gttaacaaag aacaagataa 8340aaaagtaaat gatgctacga aaaaaatctt acaatcagca ggtatttcta cagaagatac 8400ttctgtaaaa gcggatagag gagatactca gggagaagga attaaagcca ttgtgaagac 8460ttctgatatt attggaaaaa atgtagatat tacaacagag gacaagaata atatcacttc 8520tactggtggt ttgggaactg caggtcttgc ttccgcatca ggaacagtgg cagttacaaa 8580tattaaaaga aattccggag ttactgttga aaattctttt gtgaaagcag ctgaaaaagt 8640aaatgttaga tcggatatta caggaaatgt tgctttaaca gcatatcaag gtcctgtagg 8700agcattggga ataggagctg cctatgcaga attaaattct aatggaagat caaatatcag 8760tattaaaaat tctaagctat taggaaaaaa tattgatgtt attgtaaaag ataaatcgga 8820attgagagcg gaagcaaaag gattaaccgt aggagcggta gctgccggag ccattatctc 8880aaaagcaaag aatgaaatga attcagaggt tgaaattgag aagagtattt tcaatgaaga 8940aaatagagta actagccctt ctaaaggaat tggaagagaa atcaatgtca aagtggaaaa 9000agaaaacaga gtgactgctg aatctcaagg agcttctgta ggagcagtag caggggcagg 9060aattatttcc gaagcaaaag atgccggaag ctcttatttg aaagttagta caaaatccgg 9120aagaagtatt tttcatgcag ataatgtgaa tatggaagca acacataaaa tgaaagtaac 9180agcagtttct aaagcagtaa caggttctgt attgggagga gttggagtca ccaaggcaga 9240agctactgct gcaggtaaaa ctatggtaga agttgaggaa ggaaatttgt tcagaacaaa 9300tcgattgaat gcaatttcta aagtagaagg tttggatgaa gataaagtaa ctgctaaatc 9360ttctgtagta tcaggaaatg gaggaggaat tgccggagca ggagtgaata cttctacagc 9420acaaagtaat actgaatccg tagttcgttt acgaaagcaa gattatgaaa ataatgatta 9480cacaaaaaaa tatatttcag aagtcaatgc tcttgcttta aatgatacaa agaatgaagc 9540gaatatagaa tctttagcgg tagccggtgt gcatgcacaa ggaacaaaca aagcatttac 9600gagatcaaac aagttaactt ctacaactgt aaatggagga aacgtatctc aacttcgtgc 9660aaaagctttg gctaaaaatg aaaattatgg aaatgtaaaa ggaactggag gagccttagt 9720cggagcggaa acagcagccg ttgaaaatta tacaaagagt actacaggag cattggttgc 9780aggaaattgg gaaattggag ataaattaga aacgattgca agagataata cgattgtaag 9840agtcaacgga gacggaacca aaggaggtct tgtcggaaag aatggtattt ctgtgaaaaa 9900tacaatttca ggggaaacaa aatcatccat tgaagataaa gccagaattg ttggaaccgg 9960aagtgtaaat gtagatgctt tgaatgaact tgatgtagat ctacaaggaa aaagtggtgg 10020ctatggtgga attggtattg gaaatgttga tgtaaataat gtgattaaga aaaatgtaga 10080agccaaaatc ggaagacatg ctattgtaga aactactgga aaacaagaat atcaagcatt 10140tacaagagca aaagtaaata ttcttggaaa aggagacgct gcagctgcag ctgcaatatc 10200gaatgtacac atttccaatg agatggatat taaaaatttg gcaaagcagt atgcatcttc 10260tcaattaata accaaaaatt caaaaaataa tattacttta gcatcaagta gtgaatcgaa 10320tgtgaatgtt catggggtgg ctgaagcaag aggtgcagga gccaaagcga cagttagtgt 10380aaagaatcaa ataaatagaa ctaataatgt tgatttagca ggaaaaatta aaacagaggg 10440aaacatcaat gtatatgccg gatatgataa aaattataat ataagtaaga caaattctaa 10500ggctattgcg gatgccaaaa gtcatgctgc agctgcttcg gcaactgcca ctattgaaaa 10560aaatgaagta aaatttaata atgcgatccg agaatttaaa aataatctgg caagattgga 10620agggaaagct aataaaaaaa cgtcggtagg atctaatcag gtagactggt atacggataa 10680atatacatgg cattcttctg aaaaagcata caaaaaattg acatatcaat caaagagagg 10740agaaaaaggg aaaaaatgaa tttaagagag agtaaattta gtgagttttt aaaaaattca 10800aacataactt gttttgaaag agaagaagtg aaagatgagt tagaaacagt tgtatatcga 10860agttttatgg aagtagaggg acaaaattta cctatggtaa ttgtgatgga taacagtatt 10920tatacgaata tccgagtgca aattgctcca aaagtcataa aagatactaa taaagaagcg 10980gtactttcct atatcaatga attgaaccga gaatacaaag tatttaaata ttatgtgaca 11040gaggatgcag atgtttgttt agatagttgt gtaacctcca ttgcagaaga atttaatcca 11100gaaatggttt acactatttt aaatgtgatc
Patent applications by George C. Stewart, Columbia, MO US
Patent applications by Kansas State University Research Foundation
Patent applications in class Disclosed amino acid sequence derived from bacterium (e.g., Mycoplasma, Anaplasma, etc.)
Patent applications in all subclasses Disclosed amino acid sequence derived from bacterium (e.g., Mycoplasma, Anaplasma, etc.)