RESUMO
The enterohepatic Helicobacter species Helicobacter hepaticus colonizes the murine intestinal and hepatobiliary tract and is associated with chronic intestinal inflammation, gall stone formation, hepatitis, and hepatocellular carcinoma. Thus far, the role of H. hepaticus motility and flagella in intestinal colonization is unknown. In other, closely related bacteria, late flagellar genes are mainly regulated by the sigma factor FliA (sigma(28)). We investigated the function of the H. hepaticus FliA in gene regulation, flagellar biosynthesis, motility, and murine colonization. Competitive microarray analysis of the wild type versus an isogenic fliA mutant revealed that 11 genes were significantly more highly expressed in wild-type bacteria and 2 genes were significantly more highly expressed in the fliA mutant. Most of these were flagellar genes, but four novel FliA-regulated genes of unknown function were identified. H. hepaticus possesses two identical copies of the gene encoding the FliA-dependent major flagellin subunit FlaA (open reading frames HH1364 and HH1653). We characterized the phenotypes of mutants in which fliA or one or both copies of the flaA gene were knocked out. flaA_1 flaA_2 double mutants and fliA mutants did not synthesize detectable amounts of FlaA and possessed severely truncated flagella. Also, both mutants were nonmotile and unable to colonize mice. Mutants with either flaA gene knocked out produced flagella morphologically similar to those of wild-type bacteria and expressed FlaA and FlaB. flaA_1 mutants which had flagella but displayed reduced motility did not colonize mice, indicating that motility is required for intestinal colonization by H. hepaticus and that the presence of flagella alone is not sufficient.
Assuntos
Proteínas de Bactérias/fisiologia , Infecções por Helicobacter/microbiologia , Helicobacter hepaticus/fisiologia , Fator sigma/fisiologia , Animais , Proteínas de Bactérias/genética , Sequência de Bases , Western Blotting , Flagelina/genética , Helicobacter hepaticus/genética , Helicobacter hepaticus/ultraestrutura , Interações Hospedeiro-Patógeno , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Fator sigma/genéticaRESUMO
Helicobacter hepaticus is nongastric helicobacter that can reside in the hepatobiliary and intestinal systems of many animal hosts, leading to proliferative hepatitis, hepatocellular carcinoma, typhlitis, and colonitis. In this study, the intestinal mucosa was isolated from BALB/c mice to prepare tissue homogenate and spread onto selective C jejuni blood agar plates for incubation in the presence of trimethoprim, vancomycin, and polymyxin at 37 degrees Celsius; under microaerobic conditions in vented jars containing 5% O2, 10%CO2, and 85% N2. The bacteria were identified morphologically and biochemically. Gene sequence analysis of the 16s rRNA confirmed the presence of Helicobacter hepaticus, and the success in isolating this bacteria may have significant implications for studies of nongastric helicobacter.