RESUMO
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.
Assuntos
Proteínas de Bactérias , Vacinas Bacterianas/imunologia , Exotoxinas/imunologia , Infecções por Fusobacterium/imunologia , Fusobacterium necrophorum/imunologia , Proteínas Hemolisinas/imunologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/genética , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Exotoxinas/genética , Citometria de Fluxo , Infecções por Fusobacterium/prevenção & controle , Proteínas Hemolisinas/genética , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinação , Fatores de VirulênciaRESUMO
Leukotoxins are a group of exotoxins that produce their primary toxic effects against leukocytes, especially polymorphonuclear cells (PMNs). Leukotoxins include a variety of chemicals ranging from 9,10-epoxy 12-octadecenoate, a fatty acid derivative secreted by leukocytes themselves, to proteins such as RTX (repeats in toxin). This review focuses on leukotoxins of three species of gram-negative bacteria, Mannheimia (Pasteurella) haemolytica, Actinobacillus actinomycetemcomitans, and Fusobacterium necrophorum.
