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1.
Mol Microbiol ; 110(1): 114-127, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30039535

RESUMO

Chronic infection with Helicobacter pylori can lead to the development of gastric ulcers and stomach cancers. The helical cell shape of H. pylori promotes stomach colonization. Screens for loss of helical shape have identified several periplasmic peptidoglycan (PG) hydrolases and non-enzymatic putative scaffolding proteins, including Csd5. Both over and under expression of the PG hydrolases perturb helical shape, but the mechanism used to coordinate and localize their enzymatic activities is not known. Using immunoprecipitation and mass spectrometry we identified Csd5 interactions with cytosolic proteins CcmA, a bactofilin required for helical shape, and MurF, a PG precursor synthase, as well as the inner membrane spanning ATP synthase. A combination of Csd5 domain deletions, point mutations, and transmembrane domain chimeras revealed that the N-terminal transmembrane domain promotes MurF, CcmA, and ATP synthase interactions, while the C-terminal SH3 domain mediates PG binding. We conclude that Csd5 promotes helical shape as part of a membrane associated, multi-protein shape complex that includes interactions with the periplasmic cell wall, a PG precursor synthesis enzyme, the bacterial cytoskeleton, and ATP synthase.


Assuntos
Parede Celular/metabolismo , Citoesqueleto/metabolismo , Helicobacter pylori/citologia , Helicobacter pylori/enzimologia , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptídeo Sintases/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Deleção de Genes , Helicobacter pylori/genética , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/genética , Peptídeo Sintases/química , Peptídeo Sintases/genética , Periplasma/metabolismo , Análise de Sequência de Proteína
2.
Int J Med Microbiol ; 305(3): 392-403, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25804332

RESUMO

Helicobacter pylori from different individuals exhibits substantial genetic diversity. However, the kinetics of bacterial diversification after infection with a single strain is poorly understood. We investigated evolution of H. pylori following long-term infection in the primate stomach; Rhesus macaques were infected with H. pylori strain USU101 and then followed for 10 years. H. pylori was regularly cultured from biopsies, and single colony isolates were analyzed. At 1-year, DNA fingerprinting showed that all output isolates were identical to the input strain; however, at 5-years, different H. pylori fingerprints were observed. Microarray-based comparative genomic hybridization revealed that long term persistence of USU101 in the macaque stomach was associated with specific whole gene changes. Further detailed investigation showed that levels of the BabA protein were dramatically reduced within weeks of infection. The molecular mechanisms behind this reduction were shown to include phase variation and gene loss via intragenomic rearrangement, suggesting strong selective pressure against BabA expression in the macaque model. Notably, although there is apparently strong selective pressure against babA, babA is required for establishment of infection in this model as a strain in which babA was deleted was unable to colonize experimentally infected macaques.


Assuntos
Variação Genética , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Adesinas Bacterianas/genética , Animais , Biópsia , Hibridização Genômica Comparativa , Impressões Digitais de DNA , DNA Bacteriano/genética , Modelos Animais de Doenças , Rearranjo Gênico , Estudos Longitudinais , Macaca mulatta , Análise em Microsséries , Seleção Genética , Estômago/microbiologia
3.
Infect Immun ; 81(1): 209-15, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23115044

RESUMO

Animal models are important tools for studies of human disease, but developing these models is a particular challenge with regard to organisms with restricted host ranges, such as the human stomach pathogen Helicobacter pylori. In most cases, H. pylori infects the stomach for many decades before symptoms appear, distinguishing it from many bacterial pathogens that cause acute infection. To model chronic infection in the mouse, a human clinical isolate was selected for its ability to survive for 2 months in the mouse stomach, and the resulting strain, MSD132, colonized the mouse stomach for at least 28 weeks. During selection, the cagY component of the Cag type IV secretion system was mutated, disrupting a key interaction with host cells. Increases in both bacterial persistence and bacterial burden occurred prior to this mutation, and a mixed population of cagY(+) and cagY mutant cells was isolated from a single mouse, suggesting that mutations accumulate during selection and that factors in addition to the Cag apparatus are important for murine adaptation. Diversity in both alleles and genes is common in H. pylori strains, and natural competence mediates a high rate of interstrain genetic exchange. Mutations of the Com apparatus, a membrane DNA transporter, and DprA, a cytosolic competence factor, resulted in reduced persistence, although initial colonization was normal. Thus, exchange of DNA between genetically heterogeneous H. pylori strains may improve chronic colonization. The strains and methods described here will be important tools for defining both the spectrum of mutations that promote murine adaptation and the genetic program of chronic infection.


Assuntos
Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Alelos , Animais , Proteínas de Bactérias/genética , Doença Crônica , Modelos Animais de Doenças , Feminino , Infecções por Helicobacter/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Estômago/microbiologia
4.
Mol Microbiol ; 84(6): 1050-61, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22519812

RESUMO

Genetic diversification of Helicobacter pylori adhesin genes may allow adaptation of adherence properties to facilitate persistence despite host defences. The sabA gene encodes an adhesin that binds sialyl-Lewis antigens on inflamed gastric tissue. We found variability in the copy number and locus of the sabA gene and the closely related sabB and omp27 genes due to gene conversion among 51 North American paediatric H. pylori strains. We determined that sabB to sabA gene conversion is predominantly the result of intra-genomic recombination and RecA, RecG and AddA influence the rate at which it occurs. Although all clinical strains had at least one sabA gene copy, sabA and sabB were lost due to gene conversion at similar rates in vitro, suggesting host selection to maintain the sabA gene. sabA gene duplication resulted in increased SabA protein production and increased adherence to sialyl-Lewis antigens and mouse gastric tissue. In conclusion, gene conversion is a mechanism for H. pylori to regulate sabA expression level and adherence.


Assuntos
Adesinas Bacterianas/genética , Aderência Bacteriana , Conversão Gênica , Helicobacter pylori/fisiologia , Adesinas Bacterianas/metabolismo , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Mucosa Gástrica/microbiologia , Dosagem de Genes , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Helicobacter pylori/patogenicidade , Humanos , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Camundongos , Seleção Genética
5.
PLoS Pathog ; 6(7): e1001026, 2010 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-20686662

RESUMO

Many organisms respond to DNA damage by inducing expression of DNA repair genes. We find that the human stomach pathogen Helicobacter pylori instead induces transcription and translation of natural competence genes, thus increasing transformation frequency. Transcription of a lysozyme-like protein that promotes DNA donation from intact cells is also induced. Exogenous DNA modulates the DNA damage response, as both recA and the ability to take up DNA are required for full induction of the response. This feedback loop is active during stomach colonization, indicating a role in the pathogenesis of the bacterium. As patients can be infected with multiple genetically distinct clones of H. pylori, DNA damage induced genetic exchange may facilitate spread of antibiotic resistance and selection of fitter variants through re-assortment of preexisting alleles in this important human pathogen.


Assuntos
Dano ao DNA , Helicobacter pylori/genética , Transformação Genética , Helicobacter pylori/patogenicidade , Humanos , Recombinases Rec A/genética , Transcrição Gênica
6.
J Biol Chem ; 284(25): 16759-16766, 2009 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-19395381

RESUMO

Helicobacter pylori infection of the human stomach is associated with disease-causing inflammation that elicits DNA damage in both bacterial and host cells. Bacteria must repair their DNA to persist. The H. pylori AddAB helicase-exonuclease is required for DNA repair and efficient stomach colonization. To dissect the role of each activity in DNA repair and infectivity, we altered the AddA and AddB nuclease (NUC) domains and the AddA helicase (HEL) domain by site-directed mutagenesis. Extracts of Escherichia coli expressing H. pylori addA(NUC)B or addAB(NUC) mutants unwound DNA but had approximately half of the exonuclease activity of wild-type AddAB; the addA(NUC)B(NUC) double mutant lacked detectable nuclease activity but retained helicase activity. Extracts with AddA(HEL)B lacked detectable helicase and nuclease activity. H. pylori with the single nuclease domain mutations were somewhat less sensitive to the DNA-damaging agent ciprofloxacin than the corresponding deletion mutant, suggesting that residual nuclease activity promotes limited DNA repair. The addA(NUC) and addA(HEL) mutants colonized the stomach less efficiently than the wild type; addB(NUC) showed partial attenuation. E. coli DeltarecBCD expressing H. pylori addAB was recombination-deficient unless H. pylori recA was also expressed, suggesting a species-specific interaction between AddAB and RecA and also that H. pylori AddAB participates in both DNA repair and recombination. These results support a role for both the AddAB nuclease and helicase in DNA repair and promoting infectivity.


Assuntos
DNA Helicases/metabolismo , Exodesoxirribonucleases/metabolismo , Helicobacter pylori/enzimologia , Helicobacter pylori/patogenicidade , Animais , Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , DNA Helicases/química , DNA Helicases/genética , Reparo do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Feminino , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética , Virulência/genética , Virulência/fisiologia
7.
Mol Microbiol ; 69(4): 994-1007, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18573180

RESUMO

Helicobacter pylori colonization of the human stomach is characterized by profound disease-causing inflammation. Bacterial proteins that detoxify reactive oxygen species or recognize damaged DNA adducts promote infection, suggesting that H. pylori requires DNA damage repair for successful in vivo colonization. The molecular mechanisms of repair remain unknown. We identified homologues of the AddAB class of helicase-nuclease enzymes, related to the Escherichia coli RecBCD enzyme, which, with RecA, is required for repair of DNA breaks and homologous recombination. H. pylori mutants lacking addA or addB genes lack detectable ATP-dependent nuclease activity, and the cloned H. pylori addAB genes restore both nuclease and helicase activities to an E. coli recBCD deletion mutant. H. pylori addAB and recA mutants have a reduced capacity for stomach colonization. These mutants are sensitive to DNA damaging agents and have reduced frequencies of apparent gene conversion between homologous genes encoding outer membrane proteins. Our results reveal requirements for double-strand break repair and recombination during both acute and chronic phases of H. pylori stomach infection.


Assuntos
Proteínas de Bactérias/fisiologia , DNA Helicases/fisiologia , Exodesoxirribonucleases/fisiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/fisiologia , Recombinases Rec A/fisiologia , Estômago/microbiologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , Reparo do DNA , Escherichia coli/enzimologia , Escherichia coli/genética , Exodesoxirribonucleases/genética , Feminino , Helicobacter pylori/enzimologia , Helicobacter pylori/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Recombinases Rec A/genética , Recombinação Genética
8.
J Clin Microbiol ; 47(6): 1680-8, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19386830

RESUMO

Helicobacter pylori strains display remarkable genetic diversity, and the presence of strains bearing the toxigenic vacA s1 allele, a complete cag pathogenicity island (PAI), cagA alleles containing multiple EPIYA phosphorylation sites, and expressing the BabA adhesin correlates with development of gastroduodenal disease in adults. To better understand the genetic variability present among pediatric strains and its relationship to disease, we characterized H. pylori strains infecting 47 pediatric North American patients. Prevalence of mixed infection was assessed by random amplified polymorphic DNA analysis of multiple H. pylori clones from each patient. Microarray-based comparative genomic hybridization was used to examine the genomic content of the pediatric strains. The cagA and vacA alleles were further characterized by allele-specific PCR. A range of EPIYA motif configurations were observed for the cagA gene, which was present in strains from 22 patients (47%), but only 19 (41%) patients contained a complete cag PAI. Thirty patients (64%) were infected with a strain having the vacA s1 allele, and 28 patients (60%) had the babA gene. The presence of a functional cag PAI was correlated with ulcer disease (P = 0.0095). In spite of declining rates of H. pylori infection in North America, at least 11% of patients had mixed infection. Pediatric strains differ in their spectrum of strain-variable genes and percentage of absent genes in comparison to adult strains. Most children were infected with H. pylori strains lacking the cag PAI, but the presence of a complete cag PAI, in contrast to other virulence markers, was associated with more severe gastroduodenal disease.


Assuntos
Proteínas de Bactérias/genética , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Fatores de Virulência/genética , Adolescente , Técnicas de Tipagem Bacteriana , Criança , Pré-Escolar , Hibridização Genômica Comparativa , Impressões Digitais de DNA , Feminino , Genótipo , Helicobacter pylori/classificação , Humanos , Masculino , América do Norte , Técnica de Amplificação ao Acaso de DNA Polimórfico , Adulto Jovem
9.
10.
PLoS One ; 11(8): e0161587, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27537185

RESUMO

The peptidoglycan (PG) cell wall is an essential component of the cell envelope of most bacteria. Biogenesis of PG involves a lipid-linked disaccharide-pentapeptide intermediate called lipid II, which must be translocated across the cytoplasmic membrane after it is synthesized in the inner leaflet of this bilayer. Accordingly, it has been demonstrated that MurJ, the proposed lipid II flippase in Escherichia coli, is required for PG biogenesis, and thereby viability. In contrast, MurJ is not essential in Bacillus subtilis because this bacterium produces AmJ, an unrelated protein that is functionally redundant with MurJ. In this study, we investigated why MurJ is not essential in the prominent gastric pathogen, Helicobacter pylori. We found that in this bacterium, Wzk, the ABC (ATP-binding cassette) transporter that flips the lipid-linked O- or Lewis- antigen precursors across the inner membrane, is redundant with MurJ for cell viability. Heterologous expression of wzk in E. coli also suppresses the lethality caused by the loss of murJ. Furthermore, we show that this cross-species complementation is abolished when Wzk is inactivated by mutations that target a domain predicted to be required for ATPase activity. Our results suggest that Wzk can flip lipid II, implying that Wzk is the flippase with the most relaxed specificity for lipid-linked saccharides ever identified.


Assuntos
Escherichia coli/metabolismo , Helicobacter pylori/metabolismo , Peptidoglicano/biossíntese , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antígenos de Bactérias/metabolismo , Membrana Celular/metabolismo , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Glicosilação , Helicobacter pylori/crescimento & desenvolvimento , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo
11.
PLoS One ; 10(6): e0129055, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26076489

RESUMO

BACKGROUND: The incidence of esophageal adenocarcinoma (EAC) has increased nearly five-fold over the last four decades in the United States. Barrett's esophagus, the replacement of the normal squamous epithelial lining with a mucus-secreting columnar epithelium, is the only known precursor to EAC. Like other parts of the gastrointestinal (GI) tract, the esophagus hosts a variety of bacteria and comparisons among published studies suggest bacterial communities in the stomach and esophagus differ. Chronic infection with Helicobacter pylori in the stomach has been inversely associated with development of EAC, but the mechanisms underlying this association remain unclear. METHODOLOGY: The bacterial composition in the upper GI tract was characterized in a subset of participants (n=12) of the Seattle Barrett's Esophagus Research cohort using broad-range 16S PCR and pyrosequencing of biopsy and brush samples collected from squamous esophagus, Barrett's esophagus, stomach corpus and stomach antrum. Three of the individuals were sampled at two separate time points. Prevalence of H. pylori infection and subsequent development of aneuploidy (n=339) and EAC (n=433) was examined in a larger subset of this cohort. RESULTS/SIGNIFICANCE: Within individuals, bacterial communities of the stomach and esophagus showed overlapping community membership. Despite closer proximity, the stomach antrum and corpus communities were less similar than the antrum and esophageal samples. Re-sampling of study participants revealed similar upper GI community membership in two of three cases. In this Barrett's esophagus cohort, Streptococcus and Prevotella species dominate the upper GI and the ratio of these two species is associated with waist-to-hip ratio and hiatal hernia length, two known EAC risk factors in Barrett's esophagus. H. pylori-positive individuals had a significantly decreased incidence of aneuploidy and a non-significant trend toward lower incidence of EAC.


Assuntos
Bactérias , Esôfago de Barrett/genética , Esôfago de Barrett/microbiologia , Instabilidade Genômica , Microbiota , Trato Gastrointestinal Superior/microbiologia , Adenocarcinoma/epidemiologia , Adenocarcinoma/etiologia , Idoso , Idoso de 80 Anos ou mais , Bactérias/classificação , Bactérias/genética , Esôfago de Barrett/complicações , Esôfago de Barrett/patologia , Biodiversidade , Suscetibilidade a Doenças , Neoplasias Esofágicas/epidemiologia , Neoplasias Esofágicas/etiologia , Feminino , Mucosa Gástrica/metabolismo , Humanos , Incidência , Masculino , Metagenoma , Microvilosidades/microbiologia , Pessoa de Meia-Idade , Mucosa/metabolismo , Mucosa/microbiologia , Mucosa/patologia , Filogenia , Característica Quantitativa Herdável , Fatores de Risco , Estômago/microbiologia , Relação Cintura-Quadril
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