RESUMO
Shiga-toxin-producing Escherichia coli (STEC) is an important food-borne pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. Since no vaccines are available and antibiotic treatment is not recommended because promotes the appearance of HUS symptoms, the control of STEC intestinal colonization in cows, which is an important environmental reservoir, is crucial to control this zoonosis. Here, we evaluated the adaptation of an attenuated strain of Salmonella enterica serovar Typhimurium (ΔaroA mutant) as a vaccine platform for preventing STEC intestinal colonization that was studied in a mouse model. A chimeric antigen formed by the combination of the STEC peptides EspA36-192, Intimin653-935, Tir 258-361, and H7 flagellin352-374 (EITH7) was constructed and fused to the ß-lactamase signal sequence (bla SS) that drives the secretion of the chimeric antigen to the bacterial periplasmic space. Oral administration of ΔaroA-ST(EITH7) in a regime of three doses of immunization elicited both mucosal and humoral immune responses that protect mice against a STEC oral experimental infection. Remarkably, serum antibodies not only were able to bind the chimeric antigen EITH7 but also to block actin pedestal formation triggered by the type three secretion system (T3SS) in Enteropathogenic Escherichia coli (EPEC). Furthermore, a single-dose protocol was evaluated, and mice were orally immunized with ΔaroA-ST(EITH7). Interestingly, although with this protocol of immunization only fecal α-EITH7 IgA antibodies were induced and no α-EITH7 in sera were detected, mice were able to efficiently control an oral experimental infection with 1010 STEC (strain Escherichia coli O157:H7), suggesting that mucosal immune response was necessary and sufficient to control STEC intestinal colonization.
Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Vacinas contra Escherichia coli , Vacinas contra Salmonella , Escherichia coli Shiga Toxigênica , Animais , Anticorpos Antibacterianos , Bovinos , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/genética , Feminino , Camundongos , Salmonella typhimuriumRESUMO
Zoonoses that affect human and animal health have an important economic impact. In the study now presented, a bivalent vaccine has been developed that has the potential for preventing the transmission from cattle to humans of two bacterial pathogens: Brucella abortus and Shiga toxin-producing Escherichia coli (STEC). A 66kDa chimeric antigen, composed by EspA, Intimin, Tir, and H7 flagellin (EITH7) from STEC, was constructed and expressed in B. abortus Δpgm vaccine strain (BabΔpgm). Mice orally immunized with BabΔpgm(EITH7) elicited an immune response with the induction of anti-EITH7 antibodies (IgA) that clears an intestinal infection of E. coli O157:H7 three times faster (t=4 days) than mice immunized with BabΔpgm carrier strain (t=12 days). As expected, mice immunized with BabΔpgm(EITH7) strain also elicited a protective immune response against B. abortus infection. A Brucella-based vaccine platform is described capable of eliciting a combined protective immune response against two bacterial pathogens with diverse lifestyles-the intracellular pathogen B. abortus and the intestinal extracellular pathogen STEC.
Assuntos
Vacinas Bacterianas/imunologia , Vacinas Bacterianas/isolamento & purificação , Brucella abortus/imunologia , Brucelose Bovina/prevenção & controle , Infecções por Escherichia coli/prevenção & controle , Escherichia coli O157/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Brucella abortus/genética , Brucelose Bovina/imunologia , Brucelose Bovina/microbiologia , Bovinos , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Expressão Gênica , Imunoglobulina A/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
Numerous chromosomal virulence genes (chv) have been shown to play an important role in the ability of Agrobacterium tumefaciens to transform plants. The A. tumefaciens chvH gene encodes a protein similar in sequence to the Escherichia coli elongation factor P (EF-P). In A. tumefaciens this factor is required for tumor formation and for full expression of the vir genes, exerting its activity at a post-transcriptional level. Cross-complementation assays suggest that the chvH gene and the efp gene of E. coli are functionally homologous. We have cloned and characterized the efp homolog gene in Brucella abortus which has 45% identity to A. tumefaciens chvH and 35% identity to E. coli efp. The gene complemented detergent sensitivity and virulence in the chvH A. tumefaciens mutant, suggesting that both genes are functionally homologous; the growth rate in complex medium also increased to wild type levels. An efp mutant in B. abortus 2308 grew slower in complex media and showed more sensitivity to detergents. Infection assays in J774 macrophage like cells revealed no significant differences between the wild type and the efp mutant strains. The recovery of this mutant from spleens of inoculated mice was equivalent compared to that of the parental strain suggesting that B. abortus efp is not required for virulence in an animal model. However the efp mutant revealed significant differences at 1 h-4 h post-infection in HeLa infection assays compared to the wild type strain, indicating that cellular internalization was affected in non-professional phagocytes. Double immunofluorescence assays for detecting extracellular and intracellular bacteria, demonstrated that the mutant attaches to HeLa cells as the wild type but is deficient in the internalization process, thus indicating that efp is involved in the penetration of Brucella in non-professional phagocytes.
Assuntos
Proteínas de Bactérias/metabolismo , Brucella abortus/metabolismo , Brucelose/microbiologia , Fatores de Alongamento de Peptídeos/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Brucella abortus/química , Brucella abortus/genética , Brucella abortus/patogenicidade , Feminino , Regulação Bacteriana da Expressão Gênica , Células HeLa , Humanos , Kalanchoe/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/genética , Alinhamento de Sequência , VirulênciaRESUMO
Type III secretion systems (T3SS) have been found in several species of rhizobia. Proteins (termed effectors) secreted by this system are involved in host-range determination and influence nodulation efficiency. Mesorhizobium loti MAFF303099 possesses a functional T3SS in its symbiotic island whose expression is induced by flavonoids. As in other rhizobia, conserved cis-elements (tts box) were found in the promoter regions of genes or operons encoding T3SS components. Using a bioinformatics approach, we searched for other tts-box-controlled genes, and confirmed this transcriptional regulation for some of them using lacZ fusions to the predicted promoter regions. Translational fusions to a reporter peptide were created to demonstrate T3SS-mediated secretion of two new MAFF303099 effectors. Finally, we showed that mutation of the M. loti MAFF303099 T3SS affects its competitiveness on Lotus glaber and investigated, at the molecular level, responses of the model legume L. japonicus to the T3SS.
Assuntos
Alphaproteobacteria/genética , Proteínas de Bactérias/genética , Nódulos Radiculares de Plantas/genética , Simbiose/genética , Alphaproteobacteria/metabolismo , Alphaproteobacteria/fisiologia , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Biologia Computacional/métodos , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Lotus/genética , Lotus/crescimento & desenvolvimento , Lotus/microbiologia , Espectrometria de Massas , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/microbiologiaRESUMO
The intracellular pathogen Brucella abortus has an alternative sigma factor sigma54 (RpoN) highly similar to Sinorhizobium meliloti NtrA. RpoN was described to be required for the transcription of a wide range of genes involved in diverse physiological functions including the regulation of virulence-related factors in both plants and animal pathogens. B. abortus rpoN gene restored the normal growth of an S. meliloti ntrA mutant in minimal media with succinic acid as a sole carbon source as well as the formation of functional nodules in alfalfa, thus revealing that the gene is functional. B. abortus rpoN mutant and B. abortus wild-type strain harboring a multicopy plasmid coding for a wild-type rpoN gene displayed reduced survival under stationary-phase conditions suggesting that expression of RpoN must be tightly regulated. Real-time PCR analysis revealed that B. abortus rpoN expression is downregulated during the stationary phase of growth. This regulation is absent in the rpoN mutant background, indicating that RpoN regulates its own expression. Intracellular multiplication in HeLa or J774 cells, and survival in BALB/c mice of the rpoN mutant, are not affected. However 2weeks postinfection survival of rpoN mutant complemented with a multicopy plasmid containing a wild-type rpoN gene is reduced, thus suggesting that overexpression of rpoN may misregulate the expression of genes involved in this stage of infection.