Progress 12/15/01 to 12/31/05
Outputs
Fusobacterium necrophorum, a gram-negative, rod-shaped and anaerobic bacterium, is a primary or secondary etiological agent in a variety of necrotic, purulent infections in animals and humans. The major virulence factor is leukotoxin. Screening of genomic DNA library with polyclonal antisera raised against native affinity-purified leukotoxin, and further extension of the sequence using inverse PCR, led to cloning of the entire leukotoxin gene. The leukotoxin gene (lktA) is part of three genes operon (lktBAC), consists of 9,726 bp, and encodes a protein of 3,241 amino acids with an overall molecular weight of 335,956. The leukotoxin does not share sequence similarity with any other bacterial leukotoxin. Because of the apparent instability of the full-length recombinant leukotoxin, five overlapping leukotoxin gene truncations were created, and the recombinant polypeptides expressed in E. coli. Polyclonal antisera raised against two of the five truncated polypeptides
(BSBSE and GAS) neutralized the toxicity of F. necrophorum leukotoxin against bovine neutrophils. The effectiveness of the purified truncated polypeptides to invoke protective immunity was determined by experimental infection following immunization in mice. Two proteins (BSBSE and SH) invoked significant protection against F. necrophorum experimental challenge. Leukotoxin, at moderately high concentrations, induced apoptosis in bovine mononuclear cells and at very high concentrations, caused necrotic cell death of bovine peripheral leukocytes. Southern and western blot analyses and flow cytometry analysis for biological activity of the culture supernatants were carried out to determine if the leukotoxin is present in other species of the genus Fusobacterium. Only the two subspecies of F. necrophorum were found to possess the leukotoxin locus and produce the toxin. Attempts were made to express the lktA and lktB genes in E. coli to determine if the presence of LktB protein facilitates
the export of leukotoxin. Growth profiles of cells expressing both the LktA and LktB proteins were compared to those of cells not producing the LktB protein. If lktB gene is involved in leukotoxin transport, than the bacteria will survive and grow better because the toxic LktA protein will be transported out of the cell. However, the presence of the lktB containing plasmids did not improve the growth of the leukotoxin expressing host cells. SDS-polyacrylamide gel electrophoresis followed by western blot analysis using antisera against the LktA or LktB protein was used to evaluate the level of expression in E. coli cultures. The purpose of these experiments were to determine if LktB exported leukotoxin. Results indicated that only low levels of leukotoxin were produced and there was no increased amount of leukotoxin in the culture supernatants. The promoter regions of the leukotoxin operons from the two subspecies were identified and their nucleotide sequence compared. The promoter
regions were found to differ in sequence, in length of the sequence between the upstream determinant (oppF) and the first gene of the leukotoxin operon (lktB), and in promoter strength.
Impacts
Liver abscesses are of significant economic concern in the feedlot industry. It is a major cause of liver condemnation in this country and cattle with liver abscesses have reduced feed efficiency. The cloning of leukotoxin gene and expression of the recombinant protein could lead to development of a vaccine for the prevention of liver abscesses in cattle. Such an approach would alleviate the use of antibiotics in the feed.
Publications
- Narayanan, Sanjeevkumar. 2001. Molecular analysis and functional characterization of Fusobacterium necrophorum leukotoxin. Ph. D. Dissertation. Kansas State University.
- Narayanan, S. K., T. G. Nagaraja, M. M. Chengappa, G. C. Stewart. 2001. Electrophoretic mobility anomalies associated with PCR amplification of the intergenic spacer region between 16S and 23S ribosomal RNA genes of Fusobacterium necrophorum. J. Microbiol. Meth., 46:165-169.
- Narayanan, S. K., T. G. Nagaraja, M. M. Chengappa, G. C. Stewart 2001. Cloning, sequencing, and expression of the leukotoxin gene from Fusobacterium necrophorum. Infect. Immun., 69: 5447-5455.
- Narayanan, S. K., M. M. Chengappa, G. C. Stewart, and T. G. Nagaraja. 2003. Immunogenicity. and protective effects of truncated recombinant leukotoxin proteins of Fusobacterium necrophorum in mice. Vet. Microbiol., 93: 335-347.
- Oelke, A. M., T. G. Nagaraja and G. C. Stewart. 2005. The leukotoxin operon of Fusobacterium necrophorum is not present in other species of Fusobacterium. Anaerobe, 11: 123-129.
- Nagaraja, T. G., S. K. Narayanan, G. C. Stewart, and M. M. Chengappa. 2005. Fusobacterium necrophorum infections in animals: Pathogenesis and pathogenic mechanisms. Anaerobe, 11: 235-246.
- Zhang F, T. G. Nagaraja, D. George, G. C. Stewart. 2006. The two major subspecies of Fusobacterium necrophorum have distinct leukotoxin operon promoter regions. Vet. Microbiol. 112:73-78.
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Progress 01/01/04 to 12/31/04
Outputs
Fusobacterium necrophorum, a gram-negative, rod-shaped and anaerobic bacterium, is a primary or secondary etiological agent in a variety of necrotic, purulent infections in animals and humans. The major virulence factor is leukotoxin, a secreted protein, toxic to ruminant leukocytes. The complete nucleotide sequence of the leukotoxin operon of F. necrophorum has been determined. The operon consists of three genes (lktBAC) of which the leukotoxin structural gene is the middle determinant. Southern and western blot analyses and flow cytometry analysis for biological activity of the culture supernatants were carried out to determine if the leukotoxin is present in other species of the genus Fusobacterium. Only the two subspecies of F. necrophorum were found to possess the leukotoxin locus and produce the toxin. The human periodontal pathogen, F. nucleatum does not produce detectable leukotoxin. The F. necrophorum leukotoxin was found to be active against human
neutrophils. Deletion plasmids were constructed by using upstream and downstream fragments immediately outside the lktA, lktB or both. Each deletion plasmid would use allelic exchange to precisely delete each gene and replace it with an ampicillin resistant expressing gene. Introduction of the deletion plasmids into F. necrophorum by conjugation or electroporation was attempted without success. Attempts were made to express the lktA and lktB genes in E. coli to determine if the presence of LktB protein facilitates the export of leukotoxin. Growth profiles of cells expressing both the LktA and LktB proteins were compared to those of cells not producing the LktB protein. If lktB gene is involved in leukotoxin transport, than the bacteria will survive and grow better because the toxic LktA protein will be transported out of the cell. However, the presence of the lktB containing plasmids did not improve the growth of the leukotoxin expressing host cells. SDS-polyacrylamide gel
electrophoresis followed by western blot analysis using antisera against the LktA or LktB protein was used to evaluate the level of expression in the uninduced and IPTG induced E. coli cultures. The purpose of these experiments was to ensure that IPTG induced the expression of the leukotoxin and to determine if LktB exported leukotoxin. Results indicated that only low levels of leukotoxin were produced and there was no increased amount of leukotoxin in the culture supernatants.
Impacts
Liver abscesses are of significant economic concern in the feedlot industry. It is a major cause of liver condemnation in this country and cattle with liver abscesses have reduced feed efficiency. The cloning of leukotoxin gene and expression of the recombinant protein could lead to development of a vaccine for the prevention of liver abscesses in cattle. Such an approach would alleviate the use of antibiotics in the feed.
Publications
- Zhang, Fengqui. 2004. Characterization of the leukotoxin promoter of Fusobacterium necrophorum. M. S., Thesis. Kansas State University.
- Smith, Erin, 2004. Development of a genetic system for Fusobacterium necrophorum. M. S., Thesis. Kansas State University
- Oelke, A. M., T. G. Nagaraja, Melinda J. Wilkerson, and George C. Stewart. 2004. The leukotoxin operon of Fusobacterium necrophorum. Anaerobe (In Press).
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Progress 01/01/03 to 12/31/03
Outputs
Fusobacterium necrophorum, a gram-negative, anaerobic and rod-shaped bacterium, is generally an opportunistic pathogen and causes a wide variety of necrotic infections in animals and humans. In cattle, the organism is the primary causative agent in liver abscesses and foot rot. Leukotoxin, a secreted protein, is a major virulence factor. The gene encoding the leukotoxin (lktA) in F. necrophorum has been cloned, sequenced and expressed in E. coli. Because of low expression levels, problems associated with purifying full-length recombinant protein, and of the physical instability of the protein, five overlapping leukotoxin gene truncations were constructed. The recombinant polypeptides (BSBSE, SX, GAS, SH, and FINAL) were expressed in E. coli and purified by nickel affinity chromatography. The objectives were to investigate the effectiveness of the purified truncated polypeptides to induce protective immunity in mice challenged with F. necrophorum. The polypeptides,
individually or in combination, and inactivated native leukotoxin or culture supernatant of F. necrophorum were homogenized with an adjuvant and injected into mice on days 0 and 21. Blood samples were collected to measure serum antileukotoxin antibody titers on days 0, 21 and 42 and on day 42, mice were experimentally challenged with F. necrophorum. All polypeptides were immunogenic, with GAS polypeptide eliciting the least antibody response. Two polypeptides (BSBSE and SH) induced significant protection in mice against F. necrophorum infection. Protection was better than the full-length native leukotoxin or inactivated supernatant. The study demonstrated that the leukotoxin of F. necrophorum carries epitopes that induce protective immunity against experimental fusobacterial infection, thus providing further evidence to the importance of leukotoxin as a major virulence factor.
Impacts
Identification of the protective region of the leukotoxin molecule could potentially lead to the development of a recombinant vaccine for the prevention of liver abscesses and foot rot in cattle.
Publications
- Narayanan, S. K., M. M. Chengappa, G. C. Stewart, and T. G. Nagaraja. 2003. Immunogenicity and protective effects of truncated recombinant leukotoxin proteins of Fusobacterium necrophorum in mice. Vet. Microbiol., 93: 335-347.
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Progress 01/01/02 to 12/31/02
Outputs
Fusobacterium necrophorum, a gram-negative, rod-shaped, anaerobic bacterium, is a primary or secondary etiological agent in a variety of necrotic, purulent infections in humans and animals. In cattle, the organism is the primary causative agent in liver abscesses and foot rot. The major virulence factor F. necrophorum is leukotoxin, a high molecular weight secreted protein, primarily toxic to ruminant leukocytes. The nucleotide and the deduced amino acid sequences of F. necrophorum leukotoxin suggested that it is a novel protein unrelated to any known leukotoxins or other bacterial exotoxins. The mode of action of leukotoxiicity of the toxin has not been investigated. We have characterized the biological effects of F. necrophorum leukotoxin on bovine peripheral leukocytes using flow-cytometric and electron microscopy techniques. Bovine peripheral blood leukocytes were exposed to various concentrations of immunoaffinity-purified leukotoxin. At very low toxin
concentrations, polymorphonuclear leukocytes showed activation, indicated by translocation of primary and secondary granules to the periphery of the cytoplasm. Furthermore, these cells showed changes characteristic of apoptosis, including decreased cell size, organelle condensation, cytoplasmic membrane blebbing (zeiosis), and chromatin condensation and margination, and decrease in cellular DNA content. At moderately high concentrations of leukotoxin, bovine mononuclear cells were also induced to undergo programmed cell death. At very high concentrations, leukotoxin caused necrotic cell death of bovine peripheral leukocytes. The ability of F. necrophorum leukotoxin to modulate the host immune system by its toxicity, including cellular activation of PMNs and apoptosis-mediated killing of phagocytes and immune effector cells, represents a potentially important mechanism of its pathogenesis.
Impacts
Our studies have shown that the leukotoxin of F. necrophorum is a novel toxin. Understanding the mode of action of the toxin in killing leukocytes is critical to our understanding of the pathogenesis of F. necrophorum infections in animals. The ultimate goal of the project is to develop an effective vaccine for the prevention of liver abscesses and foot rot in cattle.
Publications
- Narayanan, S. K., G. C. Stewart, M. M. Chengappa, L. Willard, W. Shuman, M. Wilkerson, and T. G. Nagaraja 2002. Fusobacterium necrophorum leukotoxin induces activation and apoptosis of bovine leukocytes. Infect. Immun., 70: 4609-4620.
- Narayanan, S., T. G. Nagaraja, M. M. Chengappa, and G. C. Stewart. 2002. Immunogenicity and protective effects of truncated recombinant leukotoxin polypeptides of Fusobacterium necrophorum. Anaerobe 8: 127-128 (Presented at the 6 th Biennial Congress of the Anaerobe Society of the Americas, Park City, Utah, June 29-July 2, 2002)
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