RESUMO
The difference in host range between Salmonella enterica serovar Typhimurium (S. Typhimurium) and S. enterica serovar Typhi (S. Typhi) can be partially attributed to pseudogenes. Pseudogenes are genomic segments homologous to functional genes that do not encode functional products due to the presence of genetic defects. S. Typhi lacks several protein effectors implicated in invasion or other important processes necessary for full virulence of S. Typhimurium. SopA and SopE2, effectors that have been lost by pseudogenization in S. Typhi, correspond to an ubiquitin ligase involved in cytokine production by infected cells, and to a guanine exchange factor necessary for invasion of epithelial cells, respectively. We hypothesized that sopA and/or sopE pseudogenization contributed to the virulence of S. Typhi. In this work, we found that S. Typhi expressing S. Typhimurium sopE2 exhibited a decreased invasion in different epithelial cell lines compared with S. Typhi WT. S. Typhimurium sopA completely abolished the hypo-invasive phenotype observed in S. Typhi expressing S. Typhimurium sopE2, suggesting that functional SopA and SopE2 participate concertedly in the invasion process. Finally, the expression of S. Typhimurium sopA and/or sopE2 in S. Typhi, determined changes in the secretion of IL-8 and IL-18 in infected epithelial cells.
Assuntos
Proteínas de Bactérias/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Salmonella typhi/genética , Salmonella typhi/patogenicidade , Febre Tifoide/microbiologia , Virulência/genética , Proteínas de Bactérias/metabolismo , Citocinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Expressão Gênica , Genótipo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Mutação , PseudogenesRESUMO
A total of 162 clinical isolates of Shigella collected from children in a semi-rural community of Chile were examined for the presence of genetic determinants of resistance to ampicillin, chloramphenicol, tetracycline, and trimethoprim. Ampicillin resistance was most frequently associated with the presence of bla(OXA) in S. flexneri and with bla(TEM) in S. sonnei. The bla(OXA) gene but not bla(TEM) was located in class 1 integrons. The dhfrIa gene encoding for resistance to trimethoprim was associated to class 2 integrons and detected exclusively in S. flexneri, whereas dhfrIIIc was found in all S. sonnei strains and in 10% of the S. flexneri isolates. Cat, coding for choramphenicol resistance, and bla(OXA) genes were located in the chromosome in all cases, whereas tetA gene, coding for tetracycline resistance, and bla(TEM), dhfrIa and dhfrIIIc genes were found either in the chromosome or in conjugative plasmids. Our results show a heterogenous distribution of antibiotic-resistance determinants between S. flexneri and S. sonnei.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Disenteria Bacilar/epidemiologia , Disenteria Bacilar/microbiologia , Shigella flexneri/genética , Shigella sonnei/genética , Southern Blotting , Criança , Chile/epidemiologia , Feminino , Humanos , Masculino , Plasmídeos/genética , Reação em Cadeia da Polimerase , População Rural , Shigella flexneri/efeitos dos fármacos , Shigella flexneri/isolamento & purificação , Shigella sonnei/efeitos dos fármacos , Shigella sonnei/isolamento & purificaçãoRESUMO
Electrophoretic analysis of outer membrane proteins showed that Salmonella typhi OmpC expression is not reciprocally regulated relative to OmpF as described for Escherichia coli and S. typhimurium. When bacteria were grown in minimal media, both OmpC and OmpF were repressed as the osmolarity increased. However, in Luria broth, expression of OmpC was slightly induced by osmolarity up to 0.3 osmM. Plasmids bearing E. coli ompC-lacZ or ompF-lacZ gene fusions were studied for their expression in S. typhi and E. coli. Under anaerobic growth conditions, expression of ompC-lacZ in S. typhi was maximal at 0.16 osmM, while in E. coli expression was maximal at 0.7 osmM. ompF-lacZ expression was similarly repressed by medium osmolarity and anaerobiosis in both species. In contrast, a drastic difference in the regulation of OmpF by temperature was observed; at 37 degrees C ompF-lacZ expression was repressed in E. coli, while in S. typhi it was induced.
Assuntos
Escherichia coli/genética , Porinas/genética , Salmonella typhi/genética , Proteínas da Membrana Bacteriana Externa/genética , Eletroforese em Gel de Poliacrilamida , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Concentração Osmolar , Oxigênio/metabolismo , Proteínas Recombinantes/genética , TemperaturaRESUMO
This paper describes a method to achieve stable MudJ insertions in the Salmonella typhi Ty2 chromosome. The method is a modification of the genetic complementation system described previously for Salmonella typhimurium, which consists in placing the defective transposon (MudJ) near the transposase genes of a helper Mu phage on a single DNA fragment. This fragment is then introduced into a new bacterial host by means of P22 transduction. We constructed a S. typhi strain which carries MudJ and the Mu helper phage in the chromosome. This strain was induced to lytic growth and the lysate was used to infect S. typhi Ty2. The frequency of mutation was 2.0 x 10(-6) mutants per recipient bacterium. Superinfection with the Mu helper phage was about 1%. To determine the number of MudJ insertions, several mutants were subjected to Southern blot analysis. From a total of 25 mutants analyzed, only 4 contained more than one insertion. Our procedure compares well with the method described previously for the S. typhimurium-P22 system and can be applied to other Mu sensitive bacteria.
Assuntos
Bacteriófago mu , Salmonella typhi/genética , Transdução Genética , Bacteriófago mu/isolamento & purificação , ÓperonRESUMO
This paper describes a method to achieve stable MudJ insertions in the Salmonella typhi Ty2 chromosome. The method is a modification of the genetic complementation system described previously for Salmonella typhimurium, which consists in placing the defective transposon (MudJ) near the transposase genes of a helper Mu phage on a single DNA fragment. This fragment is then introduced into a new bacterial host by means of P22 transduction. We constructed a S. typhi strain which carries MudJ and the Mu helper phage in the chromosome. This strain was induced to lytic growth and the lysate was used to infect S. typhi Ty2. The frequency of mutation was 2.0 x 10(-6) mutants per recipient bacterium. Superinfection with the Mu helper phage was about 1 percent. To determine the number of MudJ insertions, several mutants were subjected to Southern blot analysis. From a total of 25 mutants analyzed, only 4 contained more than one insertion. Our procedure compares well with the method described previously for the S. typhimurium-P22 system and can be applied to other Mu sensitive bacteria