RESUMO
Helicobacter pylori CagA is translocated into gastric epithelial cells by a type IV secretion system and interacts with the Src homology 2 phosphatase, altering cell morphology. Multiple EPIYA motifs in CagA are associated with increased activity in cells and with gastric cancer. The aim of this work was to study the heterogeneity in activity in cells of multiple H. pylori single colonies isolated from a single patient and its association with polymorphism in cagA. The presence of cagA, cagE, cagT, and cag10 was studied with 318 H. pylori isolates from the antra and corpora of 18 patients. AGS gastric epithelial cells were infected with 75 isolates, and interleukin-8 (IL-8) secretion, cytoskeletal changes, CagA translocation, and tyrosine phosphorylation were measured. The cagA 3'-variable region was sequenced for 30 isolates to determine the number and types of EPIYA motifs. Isolates from an individual stomach were usually genetically related and had quantitatively similar phenotypic effects on cells (IL-8 induction and cytoskeletal changes). However, strains from different patients with similar CagA EPIYA motif patterns varied widely in these phenotypes. Among isolates with an EPIYA-ABC pattern, the phenotype was variable: IL-8 induction ranged from 200 to 1,200 pg/ml, and morphological changes occurred in 20 to 70% of cells. In several cases, cagA sequence diversity appeared to explain the lack of CagA activity, as isolates with an EPIYA-ACC pattern or a modified B motif had reduced cell activity. cag pathogenicity island-positive H. pylori isolates displayed a high level of heterogeneity in the capacity to induce IL-8 secretion and morphological changes; an absent or modified B motif was associated with low activity.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Células Epiteliais/microbiologia , Variação Genética , Helicobacter pylori/classificação , Helicobacter pylori/patogenicidade , Estômago/citologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Linhagem Celular , Criança , Células Epiteliais/imunologia , Feminino , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Humanos , Interleucina-8/biossíntese , Masculino , México , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fenótipo , Análise de Sequência de DNA , Estômago/imunologiaRESUMO
Expression of bfpA, the gene coding for the structural subunit of the bundle-forming pili (BFP) in enteropathogenic Escherichia coli (EPEC), requires the product of bfpT (also called perA), a member of the AraC family of transcriptional regulators. Here, we show that bfpT-cat fusions were not expressed in a bfpT - or in a non-EPEC strain, unless a functional bfpT was present, indicating that an autoregulatory mechanism is involved in expression. Further experiments with bfpT-cat fusions and primer extension analysis showed that bfpT is transcribed from a conventional sigma-70 promoter and that it is expressed throughout the growth curve. It is regulated in response to the ammonium concentration, temperature and growth media, in the same proportions as those described previously for bfpA. In addition, bfpT and bfpA expression was also modulated by osmolarity, but was not affected by pH, iron excess or limitation. Deletion analysis of the bfpT upstream region revealed that a DNA segment of 81 bp, extending upstream from the transcriptional start site, contained all the sequence elements required for maximal expression of bfpT. Furthermore, it shares significant homology with a bfpA upstream AT-rich region, which has been shown to be involved in the BfpT-dependent regulation of bfpA. Interestingly, ammonium repression was observed only when bfpT-cat or bfpA-cat expression was complemented in an EPEC background, whereas low-temperature regulation was observed in both EPEC and non-EPEC strains. This suggests that specific regulatory elements are present in EPEC, while others are shared with non-pathogenic E. coli.
Assuntos
Proteínas de Bactérias , Meios de Cultura/farmacologia , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Fímbrias , Regulação Bacteriana da Expressão Gênica , Compostos de Amônio Quaternário/farmacologia , Proteínas Repressoras/genética , Fatores de Transcrição , Transcrição Gênica/genética , Sequência de Aminoácidos , Fator de Transcrição AraC , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/genética , Sequência de Bases , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/patogenicidade , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Genes Reporter , Concentração de Íons de Hidrogênio , Ferro/farmacologia , Dados de Sequência Molecular , Família Multigênica , Concentração Osmolar , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Repressoras/biossíntese , Proteínas Repressoras/fisiologia , Deleção de Sequência , Homologia de Sequência do Ácido Nucleico , Fator sigma/metabolismo , Temperatura , Virulência/genéticaRESUMO
The Salmonella typhi ompS1 gene codes for an outer membrane protein of the OmpC/OmpF porin family. It is expressed at very low levels, relative to the major porins. However, deletion analysis of the 5' regulatory region showed that the gradual removal of nucleotides -310 to -88, upstream from the P1 major transcriptional start-point, resulted in a stepwise increase in expression, reaching levels 10-fold above those for the ompC major porin gene. Hence, this 222 bp segment contains cis-acting regulatory elements involved in negative control. Primer extension analysis revealed the presence of three promoters: P1 activity was OmpR dependent; P2 was expressed at a lower level in the absence of OmpR; and P3 had a minor constitutive activity. OmpR bound preferentially to box II, an 18 bp F1/C1 canonical site, the removal (-88 to -66) of which resulted in a decrease in expression thus supporting its role in positive control. Expression of ompS1 was not induced by a set of stress conditions, including a shift in osmolarity, nor was the IHF regulator involved in negative control. An ompS1 homologue was found in E. coli K-12, which contains a nonsense codon and a shift in the reading frame, whereas Salmonella typhimurium contains an open reading frame in this region. Thus, S. typhi ompS1 provides novel features in OmpR regulation.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Salmonella typhi/crescimento & desenvolvimento , Salmonella typhi/genética , Transativadores/metabolismo , Sequência de Bases , Pegada de DNA , Eletroforese em Gel de Poliacrilamida , Deleção de Genes , Genes Bacterianos , Genes Reporter , Dados de Sequência Molecular , Porinas , Regiões Promotoras Genéticas , Salmonella typhi/metabolismo , Análise de Sequência de DNA , Transativadores/genética , Transcrição Gênica , Equilíbrio HidroeletrolíticoRESUMO
bfpA expression in enteropathogenic Escherichia coli is regulated by growth medium, temperature, and ammonium concentration and requires the BfpT protein (also called PerA), a member of the AraC family of transcriptional activators. Site-directed and PCR random mutagenesis, as well as deletion analysis of the bfpA upstream regulatory region, supported assignment of the promoter elements and demonstrated that the cis-acting elements that mediate BfpT-dependent regulation of bfpA are located between positions -85 and -46. Interestingly, this region shares 73% identity with a 40-bp-long AT-rich tract located upstream of the bfpT gene, which is essential for bfpT autoregulation.