title: Immunogenicity and protective effects of truncated recombinant leukotoxin proteins of Fusobacterium necrophorum in mice.
abstract: 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. 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 Escherichia 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 anti-leukotoxin 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.
authors.affiliation: Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, 305 Coles Hall, Manhattan, KS 66506-5606, USA.
authors.forename: Sanjeev Kumar
authors.initials: SK
authors.lastname: Narayanan
authors.forename: M M
authors.initials: MM
authors.lastname: Chengappa
authors.forename: George C
authors.initials: GC
authors.lastname: Stewart
authors.forename: T G
authors.initials: TG
authors.lastname: Nagaraja
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001426:Bacterial Proteins; D001428:Bacterial Vaccines / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000235:genetics / Q000276:immunology*; D005434:Flow Cytometry; D005674:Fusobacterium Infections / Q000276:immunology* / Q000517:prevention & control; D005675:Fusobacterium necrophorum / Q000276:immunology*; D006460:Hemolysin Proteins / Q000235:genetics / Q000276:immunology*; D008099:Liver / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014611:Vaccination; D037521:Virulence Factors
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D004269:DNA, Bacterial; D005098:Exotoxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; D037521:Virulence Factors; C102677:lktA protein, bacteria