RESUMO
Infections from Campylobacter jejuni pose a serious public health problem and are now considered the leading cause of foodborne bacterial gastroenteritis throughout the world. Sequencing of C. jejuni genomes has previously allowed a number of loci to be identified, which encode virulence factors that aid survival and pathogenicity. Recently, a Type VI secretion system (T6SS) consisting of 13 conserved genes was described in C. jejuni strains and recognised to promote pathogenicity and adaptation to the environment. In this study, we determined the presence of this T6SS in 63 Spanish C. jejuni isolates from the food chain and urban effluents using whole-genome sequencing. Our findings demonstrated that nine (14%) strains harboured the 13 ORFs found in prototype strain C. jejuni 108. Further studies will be necessary to determine the prevalence and importance of T6SS-positive C. jejuni strains.
Assuntos
Infecções por Campylobacter/genética , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/genética , Doenças Transmitidas por Alimentos/genética , Doenças Transmitidas por Alimentos/microbiologia , Sistemas de Secreção Tipo VI/genética , Animais , Campylobacter jejuni/isolamento & purificação , Galinhas/microbiologia , Microbiologia de Alimentos , Humanos , Prevalência , Análise de Sequência , Pele/microbiologia , EspanhaRESUMO
The host cell environment can alter bacterial pathogenicity. We employed a combination of cellular and molecular techniques to study the expression of Campylobacter jejuni polysaccharides cocultured with HCT-8 epithelial cells. After two passages, the amount of membrane-bound high-molecular-weight polysaccharide was considerably reduced. Microarray profiling confirmed significant downregulation of capsular polysaccharide (CPS) locus genes. Experiments using conditioned media showed that sugar depletion occurred only when the bacterial and epithelial cells were cocultured. CPS depletion occurred when C. jejuni organisms were exposed to conditioned media from a different C. jejuni strain but not when exposed to conditioned media from other bacterial species. Proteinase K or heat treatment of conditioned media under coculture conditions abrogated the effect on the sugars, as did formaldehyde fixation and cycloheximide treatment of host cells or chloramphenicol treatment of the bacteria. However, sugar depletion was not affected in flagellar export (fliQ) and quorum-sensing (luxS) gene mutants. Passaged C. jejuni showed reduced invasiveness and increased serum sensitivity in vitro. C. jejuni alters its surface polysaccharides when cocultured with epithelial cells, suggesting the existence of a cross talk mechanism that modulates CPS expression during infection.
Assuntos
Campylobacter jejuni/fisiologia , Células Epiteliais/microbiologia , Polissacarídeos Bacterianos/biossíntese , Linhagem Celular , Técnicas de Cocultura , Meios de Cultivo Condicionados , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Transcriptional profiling of Campylobacter jejuni during colonization of the chick cecum identified 59 genes that were differentially expressed in vivo compared with the genes in vitro. The data suggest that C. jejuni regulates electron transport and central metabolic pathways to alter its physiological state during establishment in the chick cecum.
Assuntos
Adaptação Fisiológica , Infecções por Campylobacter/metabolismo , Campylobacter jejuni/genética , Oxigênio/metabolismo , Animais , Campylobacter jejuni/metabolismo , Ceco/microbiologia , Galinhas/microbiologia , Transporte de Elétrons , Expressão Gênica/fisiologia , Perfilação da Expressão Gênica , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Campylobacter jejuni is the leading cause of bacterial food-borne diarrhoeal disease throughout the world, and yet is still a poorly understood pathogen. Whole genome microarray comparisons of 11 C. jejuni strains of diverse origin identified genes in up to 30 NCTC 11168 loci ranging from 0.7 to 18.7 kb that are either absent or highly divergent in these isolates. Many of these regions are associated with the biosynthesis of surface structures including flagella, lipo-oligosaccharide, and the newly identified capsule. Other strain-variable genes of known function include those responsible for iron acquisition, DNA restriction/modification, and sialylation. In fact, at least 21% of genes in the sequenced strain appear dispensable as they are absent or highly divergent in one or more of the isolates tested, thus defining 1300 C. jejuni core genes. Such core genes contribute mainly to metabolic, biosynthetic, cellular, and regulatory processes, but many virulence determinants are also conserved. Comparison of the capsule biosynthesis locus revealed conservation of all the genes in this region in strains with the same Penner serotype as strain NCTC 11168. By contrast, between 5 and 17 NCTC 11168 genes in this region are either absent or highly divergent in strains of a different serotype from the sequenced strain, providing further evidence that the capsule accounts for Penner serotype specificity. These studies reveal extensive genetic diversity among C. jejuni strains and pave the way toward identifying correlates of pathogenicity and developing improved epidemiological tools for this problematic pathogen.
Assuntos
Campylobacter jejuni/genética , Campylobacter jejuni/isolamento & purificação , Genoma Bacteriano , Análise de Sequência com Séries de Oligonucleotídeos/métodos , DNA Bacteriano/química , Variação Genética , Humanos , Hibridização de Ácido Nucleico , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/genética , Reprodutibilidade dos Testes , Especificidade da EspécieAssuntos
Campylobacter jejuni/genética , Genoma Bacteriano , Cápsulas Bacterianas/química , Cápsulas Bacterianas/genética , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/patogenicidade , Perfilação da Expressão Gênica , Variação Genética , Humanos , Ácido N-Acetilneuramínico/biossíntese , Ácido N-Acetilneuramínico/genética , Análise de Sequência com Séries de Oligonucleotídeos , Polissacarídeos/química , Proteoma/análiseRESUMO
Hemolysins have been found to possess a variety of functions in bacteria, including a role in virulence. Helicobacter pylori demonstrates hemolytic activity when cultured on unlysed blood agar plates which is increased under iron-limiting conditions. However, the role of an H. pylori hemolysin in virulence is unclear. Scrutiny of the H. pylori 26695 genome sequence suggests the presence of at least two distinct hemolysins, HP1086 and HP1490, in this strain. Previous studies have shown that the in vitro hemolytic activity of H. pylori is reduced when it is coincubated with dextran 5000, suggesting the presence of a pore-forming cytolysin. HP1086 has homology to pore-forming cytolysins (TlyA) from other bacterial species, and the introduction of the cloned H. pylori tlyA gene into a nonhemolytic Escherichia coli strain conferred hemolytic activity. An H. pylori tlyA defined mutant showed reduced in vitro hemolytic activity, which appears to be due to pore formation, as the hemolytic activity of the wild-type strain is reduced to the same level as the tlyA mutant by the addition of dextran 5000. The mutant also showed reduced adhesion to human gastric adenocarcinoma cells and failed to colonize the gastric mucosa of mice. These data clearly suggest a role in virulence for H. pylori TlyA, contrary to the suggestion that hemolytic activity is an in vitro phenomenon for this pathogen.
Assuntos
Proteínas de Bactérias/toxicidade , Citotoxinas/toxicidade , Mucosa Gástrica/microbiologia , Helicobacter pylori/patogenicidade , Proteínas Hemolisinas/toxicidade , Fatores de Virulência/toxicidade , Animais , Aderência Bacteriana , Proteínas de Bactérias/genética , Citotoxinas/genética , Escherichia coli/genética , Infecções por Helicobacter/etiologia , Helicobacter pylori/genética , Proteínas Hemolisinas/genética , Humanos , Masculino , Camundongos , Mutação , Proteínas Recombinantes/toxicidade , Células Tumorais Cultivadas , Fatores de Virulência/genéticaRESUMO
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 two-component regulatory system PhoPQ has been identified in many bacterial species. However, the role of PhoPQ in regulating virulence gene expression in pathogenic bacteria has been characterized only in Salmonella species. We have identified, cloned, and sequenced PhoP orthologues from Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica. To investigate the role of PhoP in the pathogenicity of Y. pestis, an isogenic phoP mutant was constructed by using a reverse-genetics PCR-based strategy. The protein profiles of the wild-type and phoP mutant strains, grown at either 28 or 37 degrees C, revealed more than 20 differences, indicating that PhoP has pleiotrophic effects on gene expression in Y. pestis. The mutant showed a reduced ability to survive in J774 macrophage cell cultures and under conditions of low pH and oxidative stress in vitro. The mean lethal dose of the phoP mutant in mice was increased 75-fold in comparison with that of the wild-type strain, indicating that the PhoPQ system plays a key role in regulating the virulence of Y. pestis.
Assuntos
Proteínas de Bactérias/genética , Macrófagos/microbiologia , Fatores de Transcrição/genética , Yersinia pestis/genética , Yersinia pestis/patogenicidade , Animais , Células Cultivadas , Clonagem Molecular , Feminino , Perfilação da Expressão Gênica , Genes Bacterianos , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutagênese , Reação em Cadeia da Polimerase , Transdução de Sinais , VirulênciaRESUMO
The HtrA stress response protein has been shown to play a role in the virulence of a number of pathogens. For some organisms, htrA mutants are attenuated in the animal model and can be used as live vaccines. A Yersinia pestis htrA orthologue was identified, cloned and sequenced, showing 86% and 87% similarity to Escherichia coli and Salmonella typhimurium HtrAs. An isogenic Y. pestis htrA mutant was constructed using a reverse genetics approach. In contrast to the wild-type strain, the mutant failed to grow at an elevated temperature of 39 degrees C, but showed only a small increase in sensitivity to oxidative stress and was only partially attenuated in the animal model. However, the mutant exhibited a different protein expression profile to that of the wild-type strain when grown at 28 degrees C to simulate growth in the flea.
Assuntos
Proteínas de Choque Térmico , Proteínas Periplásmicas , Serina Endopeptidases/metabolismo , Yersinia pestis/patogenicidade , Animais , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Vacinas Bacterianas , Camundongos , Camundongos Endogâmicos BALB C , Peste/imunologia , Peste/fisiopatologia , Reação em Cadeia da Polimerase , Serina Endopeptidases/genética , Virulência , Yersinia pestis/genética , Yersinia pestis/imunologiaRESUMO
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.
Assuntos
Proteínas de Bactérias , Quimiotaxia , Mucosa Gástrica/microbiologia , Helicobacter pylori/enzimologia , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Proteínas de Membrana/fisiologia , Animais , Sangue/microbiologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Feminino , Flagelos/genética , Histidina Quinase , Proteínas de Membrana/genética , Proteínas Quimiotáticas Aceptoras de Metil , Camundongos , Movimento , Mutagênese Sítio-Dirigida , Suínos , VirulênciaRESUMO
BACKGROUND & AIMS: Phospholipase activity may play a role in the pathogenicity of Helicobacter pylori. Furthermore, some drugs that are effective against H. pylori infection are phospholipase inhibitors. Scrutiny of the H. pylori 26695 genome sequence revealed the presence of a putative protein with homology to Esherichia coli outer membrane phospholipase A (PldA). The aim of this study was to investigate the role of this putative PldA in the pathogenicity of H. pylori. METHODS: An isogenic pldA mutant was constructed and analyzed for in vitro phospholipase A(2) and hemolytic activity. Adherence of the mutant to human gastric adenocarcinoma cells and the ability to colonize mice were also investigated. RESULTS: The pldA mutant showed a marked reduction in phospholipase A(2) and hemolytic activity compared with the wild-type strain. The mutant was unable to colonize mice at 2 and 8 weeks, but it did induce a significant immune response. In contrast, the ability of the mutant to adhere to human gastric adenocarcinoma cells was unaffected. CONCLUSIONS: The results suggest a role for PldA in colonization of the gastric mucosa and possibly tissue damage after colonization.
Assuntos
Mucosa Gástrica/enzimologia , Helicobacter pylori/enzimologia , Helicobacter pylori/crescimento & desenvolvimento , Fosfolipases A/fisiologia , Animais , Anticorpos Antibacterianos/sangue , Contagem de Colônia Microbiana , Feminino , Helicobacter pylori/imunologia , Cavalos/sangue , Humanos , Camundongos , Mutação/fisiologia , Fosfolipases A/genética , Células Tumorais CultivadasRESUMO
During infection, Brucella species have to adapt to a range of different environments. Environmental sensing in bacteria often involves the concerted action of two-component regulatory systems consisting of sensor and response regulator components. In this study, we identified, cloned and sequenced four independent response regulator gene fragments from Brucella melitensis. One amplified gene fragment showed nearly 90% identity to the response regulator subfamily of NtrC transcriptional activators, and further analysis revealed the presence of an adjacent gene encoding the sensor protein NtrB. The NtrBC two-component regulatory system has been shown to play varying roles in nitrogen metabolism and potentially in virulence in other bacterial species. A B. suis ntrC isogenic mutant was constructed which showed no significant differences in growth rates compared to the wild-type strain when grown at different temperatures in vitro. However, the mutant exhibited a reduction in metabolic activity in the presence of many amino acids. The mutation did not affect survival or multiplication of B. suis in macrophages, but during the initial stages of infection in the murine brucellosis model, the ntrC mutant showed a reduced ability to multiply rapidly in splenic tissue.
Assuntos
Proteínas de Bactérias , Brucella/genética , Brucelose/microbiologia , Proteínas de Ligação a DNA/fisiologia , Transativadores , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Sequência de Bases , Brucella/metabolismo , Brucella/patogenicidade , Brucella melitensis/genética , Brucella melitensis/metabolismo , Brucella melitensis/patogenicidade , Carboidratos/química , Clonagem Molecular , Primers do DNA/química , DNA Bacteriano/química , Proteínas de Ligação a DNA/genética , Formazans/química , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutagênese , Proteínas PII Reguladoras de Nitrogênio , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sais de Tetrazólio/química , Fatores de Transcrição/fisiologia , VirulênciaRESUMO
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 , MutaçãoAssuntos
Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Gastropatias/microbiologia , Adesinas Bacterianas , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa , Genes Bacterianos , Glicoesfingolipídeos , Infecções por Helicobacter/etiologia , Gastropatias/etiologiaRESUMO
The ompR-envZ two-component regulatory system has been shown to contribute to virulence in a number of enteric bacterial pathogens. A Yersinia enterocolitica O:8 ompR homologue was amplified, cloned and sequenced, showing 99.2% homology to the Escherichia coli OmpR. An isogenic ompR mutant was constructed by reverse genetics-based methodology. The mutant was shown to have increased sensitivity to high osmolarity, high temperature and low pH stresses in vitro. In the murine yersiniosis model, the mutant was attenuated and offered partial protection against wild-type challenge.
Assuntos
Proteínas de Bactérias , Transativadores/genética , Yersinia enterocolitica/genética , Animais , Sequência de Bases , Clonagem Molecular , Contagem de Colônia Microbiana , Genes Reguladores , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutação , Concentração Osmolar , Reação em Cadeia da Polimerase , Temperatura , Transativadores/isolamento & purificação , Virulência , Yersinia enterocolitica/patogenicidadeRESUMO
To cause disease, Brucella species have to adapt to a range of different environments. Environmental sensing and adaptive responses in bacteria often involve the concerted action of a two-component regulatory system, consisting of sensor and response regulator components. Amplification and sequence analysis of response regulators from Brucella species identified a response regulator sequence with 96% similarity to Rhizobium leguminosarum FeuP. In R. leguminosarum, the FeuPQ two-component system is involved in the regulation of iron uptake. A Brucella suis feuP isogenic mutant was constructed but was not attenuated in the murine brucellosis model. The survival and multiplication of the mutant in macrophages was also unaffected. The FeuPQ regulon represents a newly characterised sub-family of response regulators.
Assuntos
Proteínas de Bactérias , Brucella/patogenicidade , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Brucella/genética , Brucella/crescimento & desenvolvimento , Brucella/metabolismo , Brucella melitensis/genética , Brucelose/microbiologia , Linhagem Celular , Clonagem Molecular , DNA Bacteriano/análise , Desferroxamina/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Monócitos/microbiologia , Mutação , Regulon/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Baço/microbiologia , Fatores de Transcrição/fisiologiaRESUMO
The high-temperature requirement (HtrA) family of stress response proteins are induced by different environmental stress conditions in a variety of bacteria and have been shown to contribute to the pathogenicity of some of these species. In this study, the htrA gene from Yersinia enterocolitica O:8 was amplified, cloned, and sequenced. Analysis of the deduced amino acid sequence predicted that the putative HtrA homolog contains a serine protease active site and a catalytic triad characteristic of trypsin-like serine proteases, structural features characteristic of previously described HtrA proteins. In order to evaluate the biological functions of Y. enterocolitica HtrA, an isogenic mutant was constructed by a reverse-genetics PCR-based approach. Characterization of the mutant provided evidence supporting a stress response function for the Y. enterocolitica htrA gene product. In contrast to the parent strain, the mutant showed increased sensitivity to killing by H2O2, O2- and temperature stress (50 degrees C). The mutant was avirulent in the murine yersiniosis injection model and offered partial protection to mice challenged with the parent strain. Further studies with the Y. enterocolitica htrA mutant should increase our knowledge of the host-pathogen interactions which occur during Yersinia infections.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Choque Térmico , Proteínas Periplásmicas , Serina Endopeptidases/genética , Yersinia enterocolitica/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutação , Serina Endopeptidases/fisiologiaRESUMO
The role of the phrA gene in the genetic control of photoreactivation in Escherichia coli has been a matter of some controversy. It has been proposed that the gene has no significant physiological role in photoreactivation. However, we have previously sequenced a restriction fragment thought to contain the phrA gene and shown it to contain a putative gene. When this gene, termed the putative phrA gene, was transformed into a phrAphrB mutant, a photoreactivable response above that of the phrAphrB mutant was observed. It has been suggested that the photorepair seen in phrB mutants is due to Type III photoreactivation, which is independent of temperature and fluence rate effects. Here we have shown that the photorecovery associated with the phrA gene is dependent on both temperature and fluence rate. This suggests that the photorecovery is not due to Type III photoreactivation but to an enzymatic reaction caused by an unknown photoactive protein, the phrA gene product, which acts on lesions other than pyrimidine dimers, possibly pyrimidine (6-4) pyrimidone photoproducts. We therefore propose that the phrA gene be reaccepted and its role in photoreactivation in Escherichia coli acknowledged.