RESUMO
Bacillus anthracis, B. thuringiensis and B. cereus are members of the B. cereus group. They share high genetic similarity. Whereas plcR (Phospholipase C regulator) usually encodes a functional pleiotropic activator protein in B. cereus and B. thuringiensis isolates, a characteristic nonsense mutation is found in all B. anthracis strains investigated, making the gene dysfunctional. To study the function of PlcR in B. anthracis, we used the B. cereus CMCC63301 genome as a template and constructed a recombinant expression plasmid pBE2A-plcR, and introduced it into the B. anthracis vaccine strain A16R, and then analyzed the activity of the hemolysin and sphingomyelinase. The results showed that transformation of B. anthracis with plasmid pBE2A-plcR carrying the native B. cereus plcR gene active the expression of sphingomyelinase gene, but did not activate expression of hemolysin genes of B. anthracis A16R.
Assuntos
Bacillus anthracis/genética , Bacillus cereus/genética , Proteínas de Bactérias/metabolismo , Vacinas Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Transativadores/metabolismo , Bacillus anthracis/enzimologia , Bacillus anthracis/crescimento & desenvolvimento , Bacillus anthracis/metabolismo , Bacillus cereus/metabolismo , Proteínas de Bactérias/genética , Vacinas Bacterianas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Transativadores/genéticaRESUMO
AIM: To construct and evaluate a polyvalent recombinant vaccine strain Shigella flexneri 2a T32 against enterotoxigenic E.coli (ETEC). METHODS: By using a host-plasmid balanced lethal system based on asd gene, a polyvalent recombinant strain was constructed to highly express CS3 and regularly express fusion enterotoxin of LTB subunit and mutant ST (LTB/STm) in a vaccine strain Shigella flexneri 2a T32 with specific deletion of asd gene. Fimbria CS3 was observed by immunofluorescence and electron microscopy assay. The security of LTB/STm was examined by ileal loop assay and suckling mouse assay. To evaluate this new candidate vaccine, it was compared with a previous vaccine strain in plasmid and protein level, growth assay and immunogenicity in Balb/c mice. RESULTS: The newly constructed vaccine expressed CS3 and grew better than the previously constructed vaccine except for the lower expression of LTB/STm. Serum IgG and mucosal IgA against CS3, LTB, ST, and host lipopolysaccharide (LPS) were produced after immunization of Balb/c mice by oral route with the new strain. The titers were not significantly different from the Balb/c mice with the previous strain. CONCLUSION: This novel candidate diarrheal vaccine can effectively induce serum and mucosal antibody responses against ETEC and Shigella.
Assuntos
Toxinas Bacterianas/genética , Disenteria Bacilar/prevenção & controle , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Proteínas de Fímbrias/genética , Vetores Genéticos/genética , Vacinas contra Shigella/genética , Shigella flexneri/genética , Animais , Feminino , Cobaias , Camundongos , Camundongos Endogâmicos BALB C , CoelhosRESUMO
Among the known colonization factors of enterotoxigenic Escherichia coli (ETEC), CFA/I and CS6 (the common antigen in the CFA/IV fimbrial antigens ) are two of the most prevalent fimbriae found in clinical isolates but are never expressed by the same wild-type strains. In this study, CFA/I and CS6 of ETEC were co-expressed in Shigella flexneri 2a T32 derivative strain FWL01 by using a host-plasmid lethal balancing system based on asd gene. The results indicate that the recombinant plasmid carrying CFA/I and CS6 could be stably integrated in FWL01. Expression of the two antigens did not interfere the host growth. The results of immunofluorescence analysis showed that CFA/I and CS6 were localized on the surface of the strain FWL01. In Balb/c mice orally immunized with the recombinant strain, the immune responses against CFA/I and CS6 were observed. Those observations show the feasibility of a multivalent vaccine expressing different fimbrial antigens in attenuated Shigella flexneri.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Escherichia coli/genética , Proteínas de Fímbrias/genética , Shigella flexneri/genética , Animais , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Western Blotting , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/imunologia , Proteínas de Fímbrias/metabolismo , Regulação Bacteriana da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Shigella flexneri/crescimento & desenvolvimento , Shigella flexneri/metabolismo , Vacinas Atenuadas/imunologiaRESUMO
Enterotoxigenic Escherichia coli (ETEC) causes watery dehydrating diarrhea in infants in developing countries, and is the most common cause of travelers diarrhea. It has been known that the colonazition factor antigens (CFAs) and enterotoxins are important virulence factors of ETEC, and these two kinds of proteins should be included in any effective vaccine against ETEC. In this study, a host-plasmid lethal balancing system was constructed based on asd gene in an avirulent strain of S.flexneri to express CS3 antigens and the fusion LT-B/ST enterotoxins of Escherichia coli. Both of these antigens were expressed steadily in the S. flexneri vector without any antibiotic markers. Antibodies against CS3, LT, ST and LPS of Shigella were detected in sera of mice that were immunized with recombinant bacteria either oragastrically (o.g.) or intranasally (i.n.). SIgA against CS3 and enterotoxins were detected simultaneously in feces of mice. This work is helpful for constructing multivalent recombinant vaccine for prevention of bacterial diarrhea.
