RESUMO
BACKGROUND: Corynebacterium pseudotuberculosis, a facultative intracellular bacterial pathogen, is the etiological agent of caseous lymphadenitis (CLA), an infectious disease that affects sheep and goats and it is responsible for significant economic losses. The disease is characterized mainly by bacteria-induced caseous necrosis in lymphatic glands. New vaccines are needed for reliable control and management of CLA. Thus, the putative virulence factors SpaC, SodC, NanH, and PknG from C. pseudotuberculosis FRC41 may represent new target proteins for vaccine development and pathogenicity studies. RESULTS: SpaC, PknG and NanH presented better vaccine potential than SodC after in silico analyses. A total of 136 B and T cell epitopes were predicted from the four putative virulence factors. A cluster analysis was performed to evaluate the redundancy degree among the sequences of the predicted epitopes; 57 clusters were formed, most of them (34) were single clusters. Two clusters from PknG and one from SpaC grouped epitopes for B and T-cell (MHC I and II). These epitopes can thus potentially stimulate a complete immune response (humoral and cellular) against C. pseudotuberculosis. Several other clusters, including two from NanH, grouped B-cell epitopes with either MHC I or II epitopes. The four target proteins were expressed in Escherichia coli. A purification protocol was developed for PknG expression. CONCLUSIONS: In silico analyses show that the putative virulence factors SpaC, PknG and NanH present good potential for CLA vaccine development. Target proteins were successfully expressed in E. coli. A protocol for PknG purification is described.
Assuntos
Vacinas Bacterianas/genética , Corynebacterium pseudotuberculosis/genética , Corynebacterium pseudotuberculosis/patogenicidade , Expressão Gênica , Fatores de Virulência/genética , Fatores de Virulência/imunologia , Sequência de Aminoácidos , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/metabolismo , Análise por Conglomerados , Corynebacterium pseudotuberculosis/imunologia , Corynebacterium pseudotuberculosis/metabolismo , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/metabolismo , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Escherichia coli/metabolismo , Dados de Sequência Molecular , Plasmídeos/genética , Plasmídeos/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Fatores de Virulência/metabolismoRESUMO
Genome annotation of the plant pathogen Xanthomonas axonopodis pv. citri (Xac), identified flagellar genes in a 15.7 kb gene cluster. However, FlgN, a secretion chaperone for hook-associated proteins FlgK and FlgL, was not identified. We performed extensive screening of the X. axonopodis pv. citri genome with the yeast two-hybrid system to identify a protein with the characteristics of the flagellar chaperone FlgN. We found a candidate (XAC1990) encoded by an operon for components of the flagellum apparatus that interacted with FlgK. In order to further support this finding, Xac FlgK and XAC1990 were cloned, expressed, and purified. The recombinant proteins were characterized by spectroscopic methods and their interaction in vitro confirmed by pull-down assays. We, therefore, conclude that XAC1990 and its homologs in other Xanthomonas species are, in fact, FlgN proteins. These observations extend the sequence diversity covered by this family of proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/fisiologia , Xanthomonas axonopodis/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Técnicas do Sistema de Duplo-Híbrido , Xanthomonas axonopodis/genética , Xanthomonas axonopodis/metabolismoRESUMO
In a previous study, the Schistosoma mansoni Rho1 protein was able to complement Rho1 null mutant Saccharomyces cerevisiae cells at restrictive temperatures and under osmotic stress (low calcium concentration) better than the human homologue (RhoA). It is known that under osmotic stress, the S. cerevisiae Rho1 triggers two distinct pathways: activation of the membrane 1,3-beta-glucan synthase enzymatic complex and activation of the protein kinase C1 signal transduction pathway, promoting the transcription of response genes. In the present work the SmRho1 protein and its mutants smrho1E97P, smrho1L101T, and smrho1E97P, L101T were used to try to clarify the basis for the differential complementation of Rho1 knockout yeast strain by the human and S. mansoni genes. Experiments of functional complementation in the presence of caffeine and in the presence of the osmotic regulator sorbitol were conducted. SmRho1 and its mutants showed a differential complementation of the yeast cells in the presence of caffeine, since smrho1E97P and smrho1E97P, L101T mutants showed a delay in the growth when compared to the yeast complemented with the wild type SmRho1. However, in the presence of sorbitol and caffeine the wild type SmRho1 and mutants showed a similar complementation phenotype, as they allowed yeast growth in all caffeine concentrations tested.
Assuntos
Animais , Humanos , Cafeína/farmacologia , Proteína Quinase C/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Schistosoma mansoni/genética , Proteínas rho de Ligação ao GTP/genética , Genes de Helmintos , Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Schistosoma mansoni/metabolismo , Transdução de Sinais/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
In a previous study, the Schistosoma mansoni Rho1 protein was able to complement Rho1 null mutant Saccharomyces cerevisiae cells at restrictive temperatures and under osmotic stress (low calcium concentration) better than the human homologue (RhoA). It is known that under osmotic stress, the S. cerevisiae Rho1 triggers two distinct pathways: activation of the membrane 1,3-beta-glucan synthase enzymatic complex and activation of the protein kinase C1 signal transduction pathway, promoting the transcription of response genes. In the present work the SmRho1 protein and its mutants smrho1E97P, smrho1L101T, and smrho1E97P, L101T were used to try to clarify the basis for the differential complementation of Rho1 knockout yeast strain by the human and S. mansoni genes. Experiments of functional complementation in the presence of caffeine and in the presence of the osmotic regulator sorbitol were conducted. SmRho1 and its mutants showed a differential complementation of the yeast cells in the presence of caffeine, since smrho1E97P and smrho1E97P, L101T mutants showed a delay in the growth when compared to the yeast complemented with the wild type SmRho1. However, in the presence of sorbitol and caffeine the wild type SmRho1 and mutants showed a similar complementation phenotype, as they allowed yeast growth in all caffeine concentrations tested.
Assuntos
Cafeína/farmacologia , Proteína Quinase C/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Schistosoma mansoni/genética , Proteínas rho de Ligação ao GTP/genética , Animais , Genes de Helmintos , Humanos , Mutação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Schistosoma mansoni/metabolismo , Transdução de Sinais/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The secretion of bacterial virulence factors and flagellar components requires the assistance of specific type III and flagellar chaperones. Standard computational annotation of the genome of Xanthomonas axonopodis pv citri, a plant pathogen that causes citrus canker, initially did not identify any genes belonging to these chaperone categories since the primary sequence homology between them was very low. However, in a search for hypothetical proteins with characteristics similar to these chaperones, we have now identified 30 chromosomal and 10 plasmidial potential genes encoding chaperones belonging to types III/IV, and flagellar secretion systems in this organism. The significance of these findings is discussed.
Assuntos
Animais , Proteínas de Bactérias , Chaperonas Moleculares , Xanthomonas axonopodis , Sistemas de Secreção Tipo III , Sistemas de Secreção Tipo IVRESUMO
Continuing the Schistosoma mansoni Genome Project 363 new templates were sequenced generating 205 more ESTs corresponding to 91 genes. Seventy four of theses genes (81 per cent) had not previously been descibed in S. mansoni. Among the newly discovered genes there are several of significant biological interest such as synaptophysin, NIFs-like and rho-GDP dissociation inhibitor.