RESUMO
Although flagellar motility is essential for the colonisation of the stomach by Helicobacter pylori, little is known about the regulation of flagellar biosynthesis in this organism. We have identified a gene in H. pylori, designated fliI, whose deduced amino acid sequence revealed extensive homology with the FliI/LcrB/InvC family of proteins which energise the export of flagellar and other virulence factors in several bacterial species. An isogenic mutant of fliI was non-motile and synthesised reduced amounts of flagellin and hook protein subunits. The majority (> 99%) of mutant cells were completely aflagellate. These results suggest that FliI is a novel ATPase involved in flagellar export in H. pylori.
Assuntos
Adenosina Trifosfatases/fisiologia , Proteínas de Bactérias/fisiologia , Flagelos/metabolismo , Helicobacter pylori/enzimologia , Proteínas/fisiologia , ATPases Translocadoras de Prótons , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Flagelina/análise , Genes Bacterianos/genética , Helicobacter pylori/genética , Helicobacter pylori/ultraestrutura , Dados de Sequência Molecular , Mutação , Proteínas/genética , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Expression of the two Helicobacter pylori flagellin proteins FlaA and FlaB is required for full motility and persistent infection of the gastric mucosa. The mechanisms and regulation of the biosynthesis and export of flagella in H. pylori are still poorly understood. Scrutiny of the H. pylori 26695 genome sequence revealed homologues of FliQ and FlhB. The roles of the fliQ and flhB genes in H. pylori were investigated by the construction and characterisation of defined isogenic mutants. The results indicate that these genes are involved in the flagellar expression, adhesion to and colonisation of the gastric mucosa.
Assuntos
Proteínas de Bactérias/genética , Flagelos/genética , Helicobacter pylori/patogenicidade , Locomoção/genética , Proteínas de Membrana , Animais , Camundongos , MutaçãoRESUMO
We investigated the roles of fliF, fliS, flhB, fliQ, fliG, and fliI of Helicobacter pylori, predicted by homology to encode structural components of the flagellar basal body and export apparatus. Mutation of these genes resulted in nonmotile, nonflagellate strains. Western blot analysis showed that all the mutants had considerably reduced levels of both flagellin subunits and of FlgE, the flagellar hook protein. RNA slot blot hybridization showed reduced levels of flaA mRNA, indicating that transcription of the major flagellin gene is inhibited in the absence of the early components of the flagellar-assembly pathway. This is the first demonstration of a checkpoint in H. pylori flagellar assembly.
Assuntos
Proteínas de Escherichia coli , Flagelina/genética , Regulação Bacteriana da Expressão Gênica , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Proteínas de Membrana , ATPases Translocadoras de Prótons , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Grupo Borrelia Burgdorferi/genética , Grupo Borrelia Burgdorferi/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Flagelina/biossíntese , Mutagênese Sítio-Dirigida , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Salmonella enterica/genética , Salmonella enterica/metabolismo , Transcrição GênicaRESUMO
The gene encoding a 29-kDa flagellar sheath protein was cloned and found to be similar to hpaA, reported to encode an N-acetylneuraminyllactose-binding fibrillar hemagglutinin (D. G. Evans, T. K. Karjalainen, D. J. Evans, Jr., D. Y. Graham, and C. H. Lee, J. Bacteriol. 175:674-683, 1993). The transcriptional start was mapped by primer extension from Helicobacter pylori mRNA, indicating an active consensus promoter at a location different from that suggested by Evans et al. Immunogold labelling of the flagellar sheath with a monoclonal antibody to HpaA was demonstrated in four strains, contrary to previous reports of a surface (D. G. Evans, T. K. Karjalainen, D. J. Evans, Jr., D. Y. Graham, and C. H. Lee, J. Bacteriol. 175:674-683, 1993) or a cytoplasmic (P. W. O'Toole, L. Janzon, P. Doig, J. Huang, M. Kostrzynska, and T. J. Trust, J. Bacteriol. 177:6049-6057, 1995) locale. Agglutination of erythrocytes and adherence to AGS cells by a delta hpaA mutant were no different from those of the parent strain, confirming a recent finding of O'Toole et al.
Assuntos
Flagelos/química , Helicobacter pylori/química , Hemaglutininas/análise , Hemaglutininas/genética , Adesinas Bacterianas , Células Cultivadas , Clonagem Molecular , Epitélio/microbiologia , Eritrócitos/microbiologia , Deleção de Genes , Genes Bacterianos/genética , Helicobacter pylori/genética , Helicobacter pylori/fisiologia , Hemaglutininas/fisiologia , Humanos , Dados de Sequência Molecular , Análise de Sequência de DNA , Estômago/citologia , Estômago/microbiologia , Transcrição Gênica/genéticaRESUMO
Infection of the mucous layer of the human stomach by Helicobacter pylori requires the bacterium to be motile and presumably chemotactic. Previous studies have shown that fully functional flagella are essential for motility and colonization, but the role of chemotaxis remains unclear. The two-component regulatory system CheA/CheY has been shown to play a major role in chemotaxis in other enteric bacteria. Scrutiny of the 26695 genome sequence suggests that H. pylori has two CheY response regulators: one a separate protein (CheY1) and the other (CheY2) fused to the histidine kinase sensor CheA. Defined deletion mutations were introduced into cheY1, cheY2, and cheA in H. pylori strains N6 and SS1. Video tracking revealed that the wild-type H. pylori strain moves in short runs with frequent direction changes, in contrast to movement of cheY2, cheAY2, and cheAY2 cheY1 mutants, whose motion was more linear. The cheY1 mutant demonstrated a different motility phenotype of rapid tumbling. All mutants had impaired swarming and greatly reduced chemotactic responses to hog gastric mucin. Neither cheY1 nor cheAY2 mutants were able to colonize mice, but they generated a significant antibody response, suggesting that despite impaired chemotaxis, these mutants were able to survive in the stomach long enough to induce an immune response before being removed by gastric flow. Additionally, we demonstrated that cheY1 failed to colonize gnotobiotic piglets. This study demonstrates the importance of the roles of cheY1, cheY2, and cheA in motility and virulence of H. pylori.