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2.
Infect Immun ; 88(2)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31712269

RESUMO

Helicobacter pylori colonizes the stomach in about half of the world's population. H. pylori strains containing the cag pathogenicity island (cag PAI) are associated with a higher risk of gastric adenocarcinoma or peptic ulcer disease than cag PAI-negative strains. The cag PAI encodes a type IV secretion system (T4SS) that mediates delivery of the CagA effector protein as well as nonprotein bacterial constituents into gastric epithelial cells. H. pylori-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and interleukin-8 (IL-8) secretion are attributed to T4SS-dependent delivery of lipopolysaccharide metabolites and peptidoglycan into host cells, and Toll-like receptor 9 (TLR9) activation is attributed to delivery of bacterial DNA. In this study, we analyzed the bacterial energetic requirements associated with these cellular alterations. Mutant strains lacking Cagα, Cagß, or CagE (putative ATPases corresponding to VirB11, VirD4, and VirB4 in prototypical T4SSs) were capable of T4SS core complex assembly but defective in CagA translocation into host cells. Thus, the three Cag ATPases are not functionally redundant. Cagα and CagE were required for H. pylori-induced NF-κB activation, IL-8 secretion, and TLR9 activation, but Cagß was dispensable for these responses. We identified putative ATP-binding motifs (Walker-A and Walker-B) in each of the ATPases and generated mutant strains in which these motifs were altered. Each of the Walker box mutant strains exhibited properties identical to those of the corresponding deletion mutant strains. These data suggest that Cag T4SS-dependent delivery of nonprotein bacterial constituents into host cells occurs through mechanisms different from those used for recruitment and delivery of CagA into host cells.


Assuntos
Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Epiteliais/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo , Transporte Biológico , DNA Bacteriano/metabolismo , Humanos , Interleucina-8/metabolismo , Lipopolissacarídeos/metabolismo , NF-kappa B/metabolismo , Peptidoglicano/metabolismo , Receptor Toll-Like 9/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
3.
Proc Natl Acad Sci U S A ; 116(11): 5077-5085, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30804204

RESUMO

Infection by Helicobacter pylori is the primary cause of gastric adenocarcinoma. The most potent H. pylori virulence factor is cytotoxin-associated gene A (CagA), which is translocated by a type 4 secretion system (T4SS) into gastric epithelial cells and activates oncogenic signaling pathways. The gene cagY encodes for a key component of the T4SS and can undergo gene rearrangements. We have shown that the cancer chemopreventive agent α-difluoromethylornithine (DFMO), known to inhibit the enzyme ornithine decarboxylase, reduces H. pylori-mediated gastric cancer incidence in Mongolian gerbils. In the present study, we questioned whether DFMO might directly affect H. pylori pathogenicity. We show that H. pylori output strains isolated from gerbils treated with DFMO exhibit reduced ability to translocate CagA in gastric epithelial cells. Further, we frequently detected genomic modifications in the middle repeat region of the cagY gene of output strains from DFMO-treated animals, which were associated with alterations in the CagY protein. Gerbils did not develop carcinoma when infected with a DFMO output strain containing rearranged cagY or the parental strain in which the wild-type cagY was replaced by cagY with DFMO-induced rearrangements. Lastly, we demonstrate that in vitro treatment of H. pylori by DFMO induces oxidative DNA damage, expression of the DNA repair enzyme MutS2, and mutations in cagY, demonstrating that DFMO directly affects genomic stability. Deletion of mutS2 abrogated the ability of DFMO to induce cagY rearrangements directly. In conclusion, DFMO-induced oxidative stress in H. pylori leads to genomic alterations and attenuates virulence.


Assuntos
Proteínas de Bactérias/genética , Carcinogênese/genética , Carcinogênese/patologia , Eflornitina/farmacologia , Helicobacter pylori/genética , Mutação/genética , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Animais , Dano ao DNA , Deleção de Genes , Rearranjo Gênico , Gerbillinae , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/patogenicidade , Masculino , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Virulência
4.
Cancer Res ; 79(7): 1600-1611, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30696658

RESUMO

Helicobacter pylori (H. pylori) is the strongest known risk for gastric cancer. The H. pylori cag type IV secretion system is an oncogenic locus that translocates peptidoglycan into host cells, where it is recognized by NOD1, an innate immune receptor. Beyond this, the role of NOD1 in H. pylori-induced cancer remains undefined. To address this knowledge gap, we infected two genetic models of Nod1 deficiency with the H. pylori cag + strain PMSS1: C57BL/6 mice, which rarely develop cancer, and INS-GAS FVB/N mice, which commonly develop cancer. Infected C57BL/6 Nod1-/- and INS-GAS Nod1-/- mice acutely developed more severe gastritis, and INS-GAS Nod1-/- mice developed gastric dysplasia more frequently compared with Nod1+/+ mice. Because Nod1 genotype status did not alter microbial phenotypes of in vivo-adapted H. pylori, we investigated host immunologic responses. H. pylori infection of Nod1-/- mice led to significantly increased gastric mucosal levels of Th1, Th17, and Th2 cytokines compared with Nod1 wild-type (WT) mice. To define the role of specific innate immune cells, we quantified cytokine secretion from H. pylori-infected primary gastric organoids generated from WT or Nod1-/- mice that were cocultured with or without WT or Nod1-/- macrophages. Infection increased cytokine production from gastric epithelial cells and macrophages and elevations were significantly increased with Nod1 deficiency. Furthermore, H. pylori infection altered the polarization status of Nod1-/- macrophages compared with Nod1+/+ macrophages. Collectively, these studies demonstrate that loss of Nod1 augments inflammatory and injury responses to H. pylori. Nod1 may exert its restrictive role by altering macrophage polarization, leading to immune evasion and microbial persistence. SIGNIFICANCE: These findings suggest that manipulation of NOD1 may represent a novel strategy to prevent or treat pathologic outcomes induced by H. pylori infection.


Assuntos
Helicobacter pylori/patogenicidade , Proteína Adaptadora de Sinalização NOD1/fisiologia , Neoplasias Gástricas/microbiologia , Animais , Carcinogênese , Citocinas/biossíntese , Mucosa Gástrica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Adaptadora de Sinalização NOD1/genética , Neoplasias Gástricas/imunologia
5.
Gut ; 67(7): 1247-1260, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-28473630

RESUMO

OBJECTIVE: Gastric cancer is the third leading cause of cancer death worldwide and infection by Helicobacter pylori is the strongest risk factor. We have reported increased epidermal growth factor receptor (EGFR) phosphorylation in the H. pylori-induced human carcinogenesis cascade, and association with DNA damage. Our goal was to determine the role of EGFR activation in gastric carcinogenesis. DESIGN: We evaluated gefitinib, a specific EGFR inhibitor, in chemoprevention of H. pylori-induced gastric inflammation and cancer development. Mice with genetically targeted epithelial cell-specific deletion of Egfr (EfgrΔepi mice) were also used. RESULTS: In C57BL/6 mice, gefitinib decreased Cxcl1 and Cxcl2 expression by gastric epithelial cells, myeloperoxidase-positive inflammatory cells in the mucosa and epithelial DNA damage induced by H. pylori infection. Similar reductions in chemokines, inflammatory cells and DNA damage occurred in infected EgfrΔepi versus Egfrfl/fl control mice. In H. pylori-infected transgenic insulin-gastrin (INS-GAS) mice and gerbils, gefitinib treatment markedly reduced dysplasia and carcinoma. Gefitinib blocked H. pylori-induced activation of mitogen-activated protein kinase 1/3 (MAPK1/3) and activator protein 1 in gastric epithelial cells, resulting in inhibition of chemokine synthesis. MAPK1/3 phosphorylation and JUN activation was reduced in gastric tissues from infected wild-type and INS-GAS mice treated with gefitinib and in primary epithelial cells from EfgrΔepi versus Egfrfl/fl mice. Epithelial EGFR activation persisted in humans and mice after H. pylori eradication, and gefitinib reduced gastric carcinoma in INS-GAS mice treated with antibiotics. CONCLUSIONS: These findings suggest that epithelial EGFR inhibition represents a potential strategy to prevent development of gastric carcinoma in H. pylori-infected individuals.


Assuntos
Antineoplásicos/uso terapêutico , Receptores ErbB/antagonistas & inibidores , Gastrite/patologia , Infecções por Helicobacter/patologia , Quinazolinas/uso terapêutico , Neoplasias Gástricas/prevenção & controle , Animais , Técnicas de Cultura de Células , Células Epiteliais , Gastrite/microbiologia , Gefitinibe , Gerbillinae , Helicobacter pylori , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia
6.
Cancer Res ; 77(9): 2401-2412, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28209611

RESUMO

Helicobacter pylori is the strongest risk factor for gastric adenocarcinoma, yet only a minority of infected persons ever develop this malignancy. One cancer-linked locus is the cag type 4 secretion system (cagT4SS), which translocates an oncoprotein into host cells. A structural component of the cagT4SS is CagY, which becomes rapidly altered during in vivo adaptation in mice and rhesus monkeys, rendering the cagT4SS nonfunctional; however, these models rarely develop gastric cancer. We previously demonstrated that the H. pylori cag+ strain 7.13 rapidly induces gastric cancer in Mongolian gerbils. We now use this model, in conjunction with samples from patients with premalignant lesions, to define the effects of a carcinogenic host environment on the virulence phenotype of H. pylori to understand how only a subset of infected individuals develop cancer. H. pylori cagY sequence differences and cagT4SS function were directly related to the severity of inflammation in human gastric mucosa in either a synchronous or metachronous manner. Serial infections of Mongolian gerbils with H. pylori strain 7.13 identified an oscillating pattern of cagT4SS function. The development of dysplasia or cancer selected for attenuated virulence phenotypes, but robust cagT4SS function could be restored upon infection of new hosts. Changes in the genetic composition of cagY mirrored cagT4SS function, although the mechanisms of cagY alterations differed in human isolates (mutations) versus gerbil isolates (addition/deletion of motifs). These results indicate that host carcinogenic phenotypes modify cagT4SS function via altering cagY, allowing the bacteria to persist and induce carcinogenic consequences in the gastric niche. Cancer Res; 77(9); 2401-12. ©2017 AACR.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Carcinogênese/genética , Helicobacter pylori/genética , Neoplasias Gástricas/genética , Animais , Modelos Animais de Doenças , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Gerbillinae/microbiologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Helicobacter pylori/patogenicidade , Humanos , Fatores de Risco , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia
7.
Am J Physiol Gastrointest Liver Physiol ; 311(5): G852-G858, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27758771

RESUMO

Helicobacter pylori (H. pylori) induces chronic gastritis in humans, and infection can persist for decades. One H. pylori strain-specific constituent that augments disease risk is the cag pathogenicity island. The cag island encodes a type IV secretion system (T4SS) that translocates DNA into host cells. Toll-like receptor 9 (TLR9) is an innate immune receptor that detects hypo-methylated CpG DNA motifs. In this study, we sought to define the role of the H. pylori cag T4SS on TLR9-mediated responses in vivo. H. pylori strain PMSS1 or its cagE- mutant, which fails to assemble a T4SS, were used to infect wild-type or Tlr9-/- C57BL/6 mice. PMSS1-infected Tlr9-/- mice developed significantly higher levels of inflammation, despite similar levels of colonization density, compared with PMSS1-infected wild-type mice. These changes were cag dependent, as both mouse genotypes infected with the cagE- mutant only developed minimal inflammation. Tlr9-/- genotypes did not alter the microbial phenotypes of in vivo-adapted H. pylori strains; therefore, we examined host immunological responses. There were no differences in levels of TH1 or TH2 cytokines in infected mice when stratified by host genotype. However, gastric mucosal levels of IL-17 were significantly increased in infected Tlr9-/- mice compared with infected wild-type mice, and H. pylori infection of IL-17A-/- mice concordantly led to significantly decreased levels of gastritis. Thus loss of Tlr9 selectively augments the intensity of IL-17-driven immune responses to H. pylori in a cag T4SS-dependent manner. These results suggest that H. pylori utilizes the cag T4SS to manipulate the intensity of the host immune response.


Assuntos
Infecções por Helicobacter/metabolismo , Inflamação/metabolismo , Receptor Toll-Like 9/metabolismo , Animais , Mucosa Gástrica/metabolismo , Helicobacter pylori , Interleucina-17/genética , Interleucina-17/metabolismo , Camundongos , Camundongos Knockout , Receptor Toll-Like 9/genética
8.
J Infect Dis ; 214(4): 644-8, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27190191

RESUMO

Helicobacter pylori is the strongest risk factor for gastric adenocarcinoma, which develops within a hypochlorhydric environment. We sequentially isolated H. pylori (strain J99) from a patient who developed corpus-predominant gastritis and hypochlorhydia over a 6-year interval. Archival J99 survived significantly better under acidic conditions than recent J99 strains. H. pylori arsRS encodes a 2-component system critical for stress responses; recent J99 isolates harbored 2 nonsynonymous arsS mutations, and arsS inactivation abolished acid survival. In vivo, acid-resistant archival, but not recent J99, successfully colonized high-acid-secreting rodents. Thus, genetic evolution of arsS may influence progression to hypochlorhydia and gastric cancer.


Assuntos
Acloridria/microbiologia , Evolução Molecular , Gastrite/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/enzimologia , Helicobacter pylori/genética , Histidina Quinase/genética , Ácidos/toxicidade , Animais , Proteínas de Bactérias/genética , Gastrite/complicações , Gerbillinae , Helicobacter pylori/isolamento & purificação , Helicobacter pylori/fisiologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Viabilidade Microbiana/efeitos dos fármacos , Mutação de Sentido Incorreto
9.
Am J Physiol Gastrointest Liver Physiol ; 309(4): G270-8, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26113297

RESUMO

Nonalcoholic fatty liver disease occurs frequently in the setting of metabolic syndrome, but the factors leading to nonalcoholic steatohepatitis (NASH) are not fully understood. This study investigated Toll-like receptor 4 (TLR4) signaling in human liver with the goal of delineating whether activation of this pathway segregates those with nonalcoholic fatty liver from those with NASH. Experiments were performed using liver biopsy tissue obtained from class III obese subjects undergoing bariatric surgery, and extended to an immortalized human hepatocyte HepaRG cell line and primary human hepatocytes. The bacterial endotoxin lipopolysaccharide (LPS) and total free fatty acid levels were significantly increased in plasma of NASH patients. TLR4 mRNA levels were significantly increased in subjects with NASH compared with NAFL as was interferon regulatory factor (IRF) 3 in the myeloid differentiation factor 88-independent signaling pathway. In HepaRG cells, nuclear factor-κB (NF-κB) nuclear translocation and functional activity increased following treatment with the fatty acid, palmitate, and following exposure to LPS compared with hepatocytes stimulated with a lipogenic treatment that induced de novo lipogenesis. Palmitate and LPS induction of NF-κB activity was partially attenuated by chemical- or small-interfering RNA-mediated inhibition of TLR4. Expression of TLR4 and its downstream mediators was upregulated with palmitate and LPS. Similar results were observed using primary human hepatocytes from a lean donor. Interestingly, NF-κB activity assays showed obese donor hepatocytes were resistant to chemical TLR4 inhibition. In conclusion, TLR4 expression is upregulated in a large cohort of NASH patients, compared with those with NAFL, and this occurs within the setting of increased LPS and fatty acids.


Assuntos
Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Adulto , Linhagem Celular , Células Cultivadas , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fator Regulador 3 de Interferon/metabolismo , Lipopolissacarídeos/sangue , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Hepatopatia Gordurosa não Alcoólica/complicações , Obesidade/complicações , Ácido Palmítico/sangue , Ácido Palmítico/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor 4 Toll-Like/genética
10.
Am J Physiol Gastrointest Liver Physiol ; 309(3): G193-201, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26045613

RESUMO

Acute Helicobacter pylori infection of gastric epithelial cells and human gastric biopsies represses H,K-ATPase α subunit (HKα) gene expression and inhibits acid secretion, causing transient hypochlorhydria and supporting gastric H. pylori colonization. Infection by H. pylori strains deficient in the cag pathogenicity island (cag PAI) genes cagL, cagE, or cagM, which do not transfer CagA into host cells or induce interleukin-8 secretion, does not inhibit HKα expression, nor does a cagA-deficient strain that induces IL-8. To test the hypothesis that virulence factors other than those mediating CagA translocation or IL-8 induction participate in HKα repression by activating NF-κB, AGS cells transfected with HKα promoter-Luc reporter constructs containing an intact or mutated NF-κB binding site were infected with wild-type H. pylori strain 7.13, isogenic mutants lacking cag PAI genes responsible for CagA translocation and/or IL-8 induction (cagA, cagζ, cagε, cagZ, and cagß), or deficient in genes encoding two peptidoglycan hydrolases (slt and cagγ). H. pylori-induced AGS cell HKα promoter activities, translocated CagA, and IL-8 secretion were measured by luminometry, immunoblotting, and ELISA, respectively. Human gastric biopsy acid secretion was measured by microphysiometry. Taken together, the data showed that HKα repression is independent of IL-8 expression, and that CagA translocation together with H. pylori transglycosylases encoded by slt and cagγ participate in NF-κB-dependent HKα repression and acid inhibition. The findings are significant because H. pylori factors other than CagA and IL-8 secretion are now implicated in transient hypochlorhydria which facilitates gastric colonization and potential triggering of epithelial progression to neoplasia.


Assuntos
Mucosa Gástrica/metabolismo , Helicobacter pylori , NF-kappa B/metabolismo , Bombas de Próton/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Acloridria/etiologia , Acloridria/metabolismo , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Ácido Gástrico/metabolismo , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/complicações , Infecções por Helicobacter/metabolismo , Helicobacter pylori/patogenicidade , Helicobacter pylori/fisiologia , Humanos , Interleucina-8/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Fatores de Virulência/metabolismo
11.
Cancer Res ; 75(8): 1749-59, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25732381

RESUMO

Helicobacter pylori (H. pylori) is the strongest known risk factor for gastric carcinogenesis. One cancer-linked locus is the cag pathogenicity island, which translocates components of peptidoglycan into host cells. NOD1 is an intracellular immune receptor that senses peptidoglycan from Gram-negative bacteria and responds by inducing autophagy and activating NF-κB, leading to inflammation-mediated bacterial clearance; however chronic pathogens can evade NOD1-mediated clearance by altering peptidoglycan structure. We previously demonstrated that the H. pylori cag(+) strain 7.13 rapidly induces gastric cancer in Mongolian gerbils. Using 2D-DIGE and mass spectrometry, we identified a novel mutation within the gene encoding the peptidoglycan deacetylase PgdA; therefore, we sought to define the role of H. pylori PgdA in NOD1-dependent activation of NF-κB, inflammation, and cancer. Coculture of H. pylori strain 7.13 or its pgdA(-) isogenic mutant with AGS gastric epithelial cells or HEK293 epithelial cells expressing a NF-κB reporter revealed that pgdA inactivation significantly decreased NOD1-dependent NF-κB activation and autophagy. Infection of Mongolian gerbils with an H. pylori pgdA(-) mutant strain led to significantly decreased levels of inflammation and malignant lesions in the stomach; however, preactivation of NOD1 before bacterial challenge reciprocally suppressed inflammation and cancer in response to wild-type H. pylori. Expression of NOD1 differs in human gastric cancer specimens compared with noncancer samples harvested from the same patients. These results indicate that peptidoglycan deacetylation plays an important role in modulating host inflammatory responses to H. pylori, allowing the bacteria to persist and induce carcinogenic consequences in the gastric niche.


Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/microbiologia , Amidoidrolases/genética , Proteínas de Bactérias/genética , Helicobacter pylori/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Peptidoglicano/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/microbiologia , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Adenocarcinoma/genética , Idoso , Amidoidrolases/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Feminino , Gastrite/genética , Gastrite/metabolismo , Gastrite/microbiologia , Inativação Gênica , Gerbillinae , Células HEK293 , Helicobacter pylori/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Organismos Geneticamente Modificados , Neoplasias Gástricas/genética
12.
Mol Microbiol ; 91(5): 858-61, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24471732

RESUMO

Helicobacter pylori is one of the most common bacterial infections worldwide, and virtually all infected persons develop coexisting gastritis, a signature feature of which is the capacity to persist for decades. In support of its lifestyle, H. pylori has evolved to express an array of diverse phenotypes, including enzyme functional diversity, that help to subvert obstacles presented by the human host, which permits long-term microbial colonization. The versatility of the newly discovered enzyme LpxJ may allow H. pylori to quickly adapt to dynamic and hostile conditions present within its cognate gastric niche.


Assuntos
Aciltransferases/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Lipídeo A/metabolismo , Família Multigênica
13.
PLoS Pathog ; 9(2): e1003189, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23468628

RESUMO

Helicobacter pylori causes clinical disease primarily in those individuals infected with a strain that carries the cytotoxin associated gene pathogenicity island (cagPAI). The cagPAI encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into epithelial cells and is required for induction of the pro-inflammatory cytokine, interleukin-8 (IL-8). CagY is an essential component of the H. pylori T4SS that has an unusual sequence structure, in which an extraordinary number of direct DNA repeats is predicted to cause rearrangements that invariably yield in-frame insertions or deletions. Here we demonstrate in murine and non-human primate models that immune-driven host selection of rearrangements in CagY is sufficient to cause gain or loss of function in the H. pylori T4SS. We propose that CagY functions as a sort of molecular switch or perhaps a rheostat that alters the function of the T4SS and "tunes" the host inflammatory response so as to maximize persistent infection.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/fisiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/metabolismo , Animais , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , DNA Bacteriano , Feminino , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/metabolismo , Helicobacter pylori/imunologia , Helicobacter pylori/ultraestrutura , Interações Hospedeiro-Patógeno , Interleucina-8/metabolismo , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Varredura , Recombinação Genética , Organismos Livres de Patógenos Específicos , Fatores de Virulência
14.
PLoS One ; 8(1): e54344, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23372710

RESUMO

Helicobacter pylori is the strongest known risk factor for the development of gastric adenocarcinoma. H. pylori expresses a repertoire of virulence factors that increase gastric cancer risk, including the cag pathogenicity island and the vacuolating cytotoxin (VacA). One host element that promotes carcinogenesis within the gastrointestinal tract is Krüppel-like factor 5 (KLF5), a transcription factor that mediates key cellular functions. To define the role of KLF5 within the context of H. pylori-induced inflammation and injury, human gastric epithelial cells were co-cultured with the wild-type cag(+) H. pylori strain 60190. KLF5 expression was significantly upregulated following co-culture with H. pylori, but increased expression was independent of the cag island or VacA. To translate these findings into an in vivo model, C57BL/6 mice were challenged with the wild-type rodent-adapted cag(+) H. pylori strain PMSS1 or a PMSS1 cagE(-) isogenic mutant. Similar to findings in vitro, KLF5 staining was significantly enhanced in gastric epithelium of H. pylori-infected compared to uninfected mice and this was independent of the cag island. Flow cytometry revealed that the majority of KLF5(+) cells also stained positively for the stem cell marker, Lrig1, and KLF5(+)/Lrig1(+) cells were significantly increased in H. pylori-infected versus uninfected tissue. To extend these results into the natural niche of this pathogen, levels of KLF5 expression were assessed in human gastric biopsies isolated from patients with or without premalignant lesions. Levels of KLF5 expression increased in parallel with advancing stages of neoplastic progression, being significantly elevated in gastritis, intestinal metaplasia, and dysplasia compared to normal gastric tissue. These results indicate that H. pylori induces expression of KLF5 in gastric epithelial cells in vitro and in vivo, and that the degree of KLF5 expression parallels the severity of premalignant lesions in human gastric carcinogenesis.


Assuntos
Adenocarcinoma/genética , Transformação Celular Neoplásica , Gastrite/genética , Infecções por Helicobacter/genética , Helicobacter pylori/genética , Fatores de Transcrição Kruppel-Like/genética , Neoplasias Gástricas/genética , Adenocarcinoma/etiologia , Adenocarcinoma/microbiologia , Adenocarcinoma/patologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cocultura , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Gastrite/etiologia , Gastrite/microbiologia , Gastrite/patologia , Expressão Gênica , Ilhas Genômicas , Infecções por Helicobacter/complicações , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/metabolismo , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Índice de Gravidade de Doença , Neoplasias Gástricas/etiologia , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia
15.
J Clin Invest ; 123(1): 479-92, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23257361

RESUMO

Gastric adenocarcinoma is strongly associated with Helicobacter pylori infection; however, most infected persons never develop this malignancy. H. pylori strains harboring the cag pathogenicity island (cag+), which encodes CagA and a type IV secretion system (T4SS), induce more severe disease outcomes. H. pylori infection is also associated with iron deficiency, which similarly augments gastric cancer risk. To define the influence of iron deficiency on microbial virulence in gastric carcinogenesis, Mongolian gerbils were maintained on iron-depleted diets and infected with an oncogenic H. pylori cag+ strain. Iron depletion accelerated the development of H. pylori-induced premalignant and malignant lesions in a cagA-dependent manner. H. pylori strains harvested from iron-depleted gerbils or grown under iron-limiting conditions exhibited enhanced virulence and induction of inflammatory factors. Further, in a human population at high risk for gastric cancer, H. pylori strains isolated from patients with the lowest ferritin levels induced more robust proinflammatory responses compared with strains isolated from patients with the highest ferritin levels, irrespective of histologic status. These data demonstrate that iron deficiency enhances H. pylori virulence and represents a measurable biomarker to identify populations of infected persons at high risk for gastric cancer.


Assuntos
Transformação Celular Neoplásica , Ferritinas/sangue , Infecções por Helicobacter , Helicobacter pylori , Ferro/deficiência , Neoplasias Gástricas , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Feminino , Ferritinas/genética , Ilhas Genômicas/genética , Gerbillinae , Infecções por Helicobacter/sangue , Infecções por Helicobacter/complicações , Infecções por Helicobacter/genética , Infecções por Helicobacter/patologia , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Helicobacter pylori/patogenicidade , Humanos , Masculino , Fatores de Risco , Neoplasias Gástricas/sangue , Neoplasias Gástricas/etiologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia
16.
Clin Vaccine Immunol ; 20(2): 227-38, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23239803

RESUMO

Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1(-) strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1(-) mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1(-) CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Imunização Passiva , Vacina contra a Peste/imunologia , Proteínas Recombinantes/imunologia , Yersinia pestis/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Soros Imunes , Imunização , Levofloxacino , Camundongos , Ofloxacino/uso terapêutico , Peste/imunologia , Peste/microbiologia , Peste/prevenção & controle , Vacina contra a Peste/uso terapêutico , Ativadores de Plasminogênio/genética , Ativadores de Plasminogênio/imunologia , Ratos , Proteínas Recombinantes/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/uso terapêutico , Fatores de Virulência/genética , Fatores de Virulência/imunologia , Yersinia pestis/genética
17.
J Biol Chem ; 287(44): 37030-41, 2012 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-22969084

RESUMO

The mono-ADP-ribosyltransferase (mART) toxins are contributing factors to a number of human diseases, including cholera, diphtheria, traveler's diarrhea, and whooping cough. VahC is a cytotoxic, actin-targeting mART from Aeromonas hydrophila PPD134/91. This bacterium is implicated primarily in diseases among freshwater fish species but also contributes to gastrointestinal and extraintestinal infections in humans. VahC was shown to ADP-ribosylate Arg-177 of actin, and the kinetic parameters were K(m)(NAD(+)) = 6 µM, K(m)(actin) = 24 µM, and k(cat) = 22 s(-1). VahC activity caused depolymerization of actin filaments, which induced caspase-mediated apoptosis in HeLa Tet-Off cells. Alanine-scanning mutagenesis of predicted catalytic residues showed the predicted loss of in vitro mART activity and cytotoxicity. Bioinformatic and kinetic analysis also identified three residues in the active site loop that were critical for the catalytic mechanism. A 1.9 Å crystal structure supported the proposed roles of these residues and their conserved nature among toxin homologues. Several small molecules were characterized as inhibitors of in vitro VahC mART activity and suramin was the best inhibitor (IC(50) = 20 µM). Inhibitor activity was also characterized against two other actin-targeting mART toxins. Notably, these inhibitors represent the first report of broad spectrum inhibition of actin-targeting mART toxins.


Assuntos
ADP Ribose Transferases/química , Actinas/química , Aeromonas hydrophila/enzimologia , Proteínas de Bactérias/química , Toxinas Bacterianas/química , ADP Ribose Transferases/antagonistas & inibidores , ADP Ribose Transferases/biossíntese , ADP Ribose Transferases/genética , Citoesqueleto de Actina/metabolismo , Motivos de Aminoácidos , Apoptose , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Domínio Catalítico , Forma Celular , Cristalografia por Raios X , Inibidores Enzimáticos/química , Células HeLa , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Saccharomyces cerevisiae , Propriedades de Superfície
18.
Gene ; 506(2): 369-76, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22814176

RESUMO

The repeat in toxin (Rtx) of an environmental isolate ATCC 7966 of Aeromonas hydrophila consists of six genes (rtxACHBDE) organized in an operon similar to the gene organization found for the Rtx of the Vibrio species. The first gene in this operon (rtxA) encodes an exotoxin in vibrios, while other genes code for proteins needed for proper activation of RtxA and in secretion of this toxin from Vibrio cholerae. However, the RtxA of ATCC 7966, as well as from the clinical isolate SSU of A. hydrophila, was exclusively expressed and produced during co-infection of this pathogen with the host, e.g., HeLa cells, indicating that rtxA gene expression required host cell contact. Within the RtxA, an actin cross-linking domain (ACD) exists and to investigate the functionality of this domain, several truncated versions of ACD were generated to discern its minimal biological active region. Such genetically modified genes encoding ACD, which were truncated on either the NH(2) or the COOH terminal, as well as on both ends, were expressed from a bidirectional promoter of the pBI-enhanced green fluorescent protein (EGFP) vector in a HeLa-Tet-Off cell system. We demonstrated that only the full-length ACD of RtxA from A. hydrophila catalyzed the covalent cross-linking of the host cellular actin, whereas the ACD truncated on the NH(2), COOH or both ends did not exhibit such actin cross-linking characteristics. Further, we showed that the full-length ACD of A. hydrophila RtxA disrupted the actin cytoskeleton of HeLa cells, resulting in their rounding phenotype. Finally, our data provided evidence that the full-length ACD of RtxA induced host cell apoptosis. Our study is the first to report that A. hydrophila possesses a functional RtxA having an ACD that contributes to the host cell apoptosis, and hence could represent a potential virulence factor of this emerging human pathogen.


Assuntos
Actinas/química , Aeromonas hydrophila/metabolismo , Apoptose , Toxinas Bacterianas/metabolismo , Animais , Toxinas Bacterianas/química , Sequência de Bases , Reagentes para Ligações Cruzadas/farmacologia , Escherichia coli/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Óperon , Proteínas Recombinantes/metabolismo
19.
Microbiology ; 156(Pt 12): 3678-3688, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20798163

RESUMO

Recently, we reported that the type 6 secretion system (T6SS) of Aeromonas hydrophila SSU plays an important role in bacterial virulence in a mouse model, and immunization of animals with the T6SS effector haemolysin co-regulated protein (Hcp) protected them against lethal infections with wild-type bacteria. Additionally, we showed that the mutant bacteria deleted for the vasH gene within the T6SS gene cluster did not express the hcp gene, while the vasK mutant could express and translocate Hcp, but was unable to secrete it into the extracellular milieu. Both of these A. hydrophila SSU mutants were readily phagocytosed by murine macrophages, pointing to the possible role of the secreted form of Hcp in the evasion of the host innate immunity. By using the ΔvasH mutant of A. hydrophila, our in vitro data showed that the addition of exogenous recombinant Hcp (rHcp) reduced bacterial uptake by macrophages. These results were substantiated by increased bacterial virulence when rHcp was added along with the ΔvasH mutant in a septicaemic mouse model of infection. Analysis of the cytokine profiling in the intraperitoneal lavage as well as activation of host cells after 4 h of infection with the ΔvasH mutant supplemented with rHcp indicated that this T6SS effector inhibited production of pro-inflammatory cytokines and induced immunosuppressive cytokines, such as interleukin-10 and transforming growth factor-ß, which could circumvent macrophage activation and maturation. This mechanism of innate immune evasion by Hcp possibly inhibited the recruitment of cellular immune components, which allowed bacterial multiplication and dissemination in animals, thereby leading to their mortality.


Assuntos
Aeromonas hydrophila/imunologia , Proteínas de Bactérias/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Aeromonas hydrophila/genética , Aeromonas hydrophila/patogenicidade , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Feminino , Humanos , Camundongos , Fagocitose , Transporte Proteico , Virulência
20.
Microb Pathog ; 49(3): 122-34, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20553837

RESUMO

We recently characterized a T3SS effector, AexU, from a diarrheal isolate SSU of Aeromonas hydrophila, which exhibited ADP-ribosyltransferase (ADPRT) activity. Here we provided evidence that AexU also possessed GTPase-activating protein (GAP) activity, which was mainly responsible for host cell apoptosis and disruption of actin filaments. Earlier, we showed that the DeltaaexU null mutant was attenuated in a mouse model, and we now demonstrated that while the parental A. hydrophila strain could be detected in the lung, liver, and spleen of infected mice, the DeltaaexU mutant was rapidly cleared from these organs resulting in increased survivability of animals. Further, AexU prevented phosphorylation of c-Jun, JNK and IkappaBalpha and inhibited IL-6 and IL-8 secretion from HeLa cells. Our data indicated that AexU operated by inhibiting NF-kappaB and inactivating Rho GTPases. Importantly, however, when the DeltaaexU null mutant was complemented with the mutated aexU gene devoid of ADPRT and GAP activities, a higher mortality rate in mice with concomitant increase in the production of pro-inflammatory cytokines/chemokines was noted. These data indicated that either such a mutated AexU is a potent inducer of them or that AexU possesses yet another unknown activity that is modulated by ADPRT and GAP activities and results in this aberrant cytokine/chemokine production responsible for increased animal death.


Assuntos
ADP Ribose Transferases/metabolismo , Aeromonas hydrophila/enzimologia , Aeromonas hydrophila/patogenicidade , Proteínas de Bactérias/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Virulência/metabolismo , ADP Ribose Transferases/genética , Animais , Proteínas de Bactérias/genética , Citocinas/metabolismo , Proteínas Ativadoras de GTPase/genética , Deleção de Genes , Teste de Complementação Genética , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/patologia , Células HeLa , Humanos , Fígado/microbiologia , Pulmão/microbiologia , Camundongos , Baço/microbiologia , Análise de Sobrevida , Fatores de Virulência/genética
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