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1.
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
2.
J Proteomics ; 202: 103374, 2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31063819

RESUMO

Helicobacter pylori infection and a high salt diet are each risk factors for gastric cancer. In this study, we tested the hypothesis that environmental salt concentration influences the composition of the H. pylori exoproteome. H. pylori was cultured in media containing varying concentrations of sodium chloride, and aliquots were fractionated and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified proteins that were selectively released into the extracellular space, and we identified selectively released proteins that were differentially abundant in culture supernatants, depending on the environmental salt concentration. We also used RNA-seq analysis to identify genes that were differentially expressed in response to environmental salt concentration. The salt-responsive proteins identified by proteomic analysis and salt-responsive genes identified by RNA-seq analysis were mostly non-concordant, but the secreted toxin VacA was salt-responsive in both analyses. Western blot analysis confirmed that VacA levels in the culture supernatant were increased in response to high salt conditions, and quantitative RT-qPCR experiments confirmed that vacA transcription was upregulated in response to high salt conditions. These results indicate that environmental salt concentration influences the composition of the H. pylori exoproteome, which could contribute to the increased risk of gastric cancer associated with a high salt diet. SIGNIFICANCE: Helicobacter pylori-induced alterations in the gastric mucosa have been attributed, at least in part, to the actions of secreted H. pylori proteins. In this study, we show that H. pylori growth in high salt concentrations leads to increased levels of a secreted VacA toxin. Salt-induced alterations in the composition of the H. pylori exoproteome is relevant to the increased risk of gastric cancer associated with consumption of a high salt diet.

3.
Infect Immun ; 87(2)2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30510104

RESUMO

Helicobacter pylori CagA is a secreted effector protein that contributes to gastric carcinogenesis. Previous studies showed that there is variation among H. pylori strains in the steady-state levels of CagA and that a strain-specific motif downstream of the cagA transcriptional start site (the +59 motif) is associated with both high levels of CagA and premalignant gastric histology. The cagA 5' untranslated region contains a predicted stem-loop-forming structure adjacent to the +59 motif. In the current study, we investigated the effect of the +59 motif and the adjacent stem-loop on cagA transcript levels and cagA mRNA stability. Using site-directed mutagenesis, we found that mutations predicted to disrupt the stem-loop structure resulted in decreased steady-state levels of both the cagA transcript and the CagA protein. Additionally, these mutations resulted in a decreased cagA mRNA half-life. Mutagenesis of the +59 motif without altering the stem-loop structure resulted in reduced steady-state cagA transcript and CagA protein levels but did not affect cagA transcript stability. cagA transcript stability was not affected by increased sodium chloride concentrations, an environmental factor known to augment cagA transcript levels and CagA protein levels. These results indicate that both a predicted stem-loop structure and a strain-specific +59 motif in the cagA 5' untranslated region influence the levels of cagA expression.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , DNA Bacteriano/ultraestrutura , Infecções por Helicobacter/genética , Helicobacter pylori/genética , Estabilidade de RNA/genética , RNA Mensageiro/ultraestrutura , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Humanos , Mutagênese Sítio-Dirigida
4.
PLoS One ; 12(11): e0188804, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29186206

RESUMO

Helicobacter pylori is a genetically diverse bacterial species that colonizes the stomach in about half of the human population. Most persons colonized by H. pylori remain asymptomatic, but the presence of this organism is a risk factor for gastric cancer. Multiple populations and subpopulations of H. pylori with distinct geographic distributions are recognized. Genetic differences among these populations might be a factor underlying geographic variation in gastric cancer incidence. Relatively little is known about the genomic features of African H. pylori strains compared to other populations of strains. In this study, we first analyzed the genomes of H. pylori strains from seven globally distributed populations or subpopulations and identified encoded proteins that exhibited the highest levels of sequence divergence. These included secreted proteins, an LPS glycosyltransferase, fucosyltransferases, proteins involved in molybdopterin biosynthesis, and Clp protease adaptor (ClpS). Among proteins encoded by the cag pathogenicity island, CagA and CagQ exhibited the highest levels of sequence diversity. We then identified proteins in strains of Western African origin (classified as hspWAfrica by MLST analysis) with sequences that were highly divergent compared to those in other populations of strains. These included ATP-dependent Clp protease, ClpS, and proteins of unknown function. Three of the divergent proteins sequences identified in West African strains were characterized by distinct insertions or deletions up to 8 amino acids in length. These polymorphisms in rapidly evolving proteins represent robust genetic signatures for H. pylori strains of West African origin.


Assuntos
Helicobacter pylori/genética , África Ocidental , Sequência de Aminoácidos , Proteínas de Bactérias/química , Genes Bacterianos , Homologia de Sequência de Aminoácidos
5.
Toxins (Basel) ; 9(10)2017 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-29023421

RESUMO

Helicobacter pylori VacA is a channel-forming toxin unrelated to other known bacterial toxins. Most H. pylori strains contain a vacA gene, but there is marked variation among strains in VacA toxin activity. This variation is attributable to strain-specific variations in VacA amino acid sequences, as well as variations in the levels of VacA transcription and secretion. In this review, we discuss epidemiologic studies showing an association between specific vacA allelic types and gastric cancer, as well as studies that have used animal models to investigate VacA activities relevant to gastric cancer. We also discuss the mechanisms by which VacA-induced cellular alterations may contribute to the pathogenesis of gastric cancer.


Assuntos
Proteínas de Bactérias , Helicobacter pylori , Neoplasias Gástricas/microbiologia , Alelos , Animais , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Helicobacter pylori/genética , Helicobacter pylori/fisiologia , Humanos , Fatores de Risco , Estômago/microbiologia , Estômago/patologia , Neoplasias Gástricas/epidemiologia , Neoplasias Gástricas/patologia , Virulência
6.
ACS Appl Mater Interfaces ; 9(32): 26719-26730, 2017 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-28696672

RESUMO

Acinetobacter baumannii is a Gram-negative bacterium of increasing concern due to its virulence and persistence in combat and healthcare environments. The incidence of both community-acquired and nosocomial A. baumannii infections is on the rise in foreign and domestic healthcare facilities. Treatment options are limited due to the acquisition of multidrug resistance to the few effective antibiotics. Currently, the most effective pharmaceutically based treatment for multidrug-resistant A. baumannii infections is the antibiotic colistin (polymyxin E). To minimize side effects associated with administration of colistin or other toxic antimicrobial agents, we propose the development of a nanotechnology-mediated treatment strategy. In this design-based effort, colistin-functionalized multilayered, inorganic, magnetoplasmonic nanoconstructs were fabricated to bind to the surface of A. baumannii. This result, for the first time, demonstrates a robust, pharmaceutical-based motif for high affinity, composite nanoparticulates targeting the A. baumannii surface. The antibiotic-activated nanomaterials demonstrated cytocompatibility with human cells and no acute bacterial toxicity at nanoparticle to bacterial concentrations <10 000:1. The magnetomotive characteristics of the nanomaterial enabled magnetic extraction of the bacteria. In a macroscale environment, maximal separation efficiencies exceeding 38% were achieved. This result demonstrates the potential for implementation of this technology into micro- or mesofluidic-based separation environments to enhance extraction efficiencies. The future development of such a mesofluidic-based, nanotechnology-mediated platform is potentially suitable for adjuvant therapies to assist in the treatment of sepsis.


Assuntos
Acinetobacter baumannii , Infecções por Acinetobacter , Antibacterianos , Colistina , Farmacorresistência Bacteriana Múltipla , Compostos Férricos , Humanos , Testes de Sensibilidade Microbiana
7.
PLoS One ; 11(9): e0163167, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27657881

RESUMO

The rise of multi-drug resistance has decreased the effectiveness of antibiotics, which has led to increased mortality rates associated with symptomatic bacteremia, or bacterial sepsis. To combat decreasing antibiotic effectiveness, extracorporeal bacterial separation approaches have been proposed to capture and separate bacteria from blood. However, bacteremia is dynamic and involves host-pathogen interactions across various anatomical sites. We developed a mathematical model that quantitatively describes the kinetics of pathogenesis and progression of symptomatic bacteremia under various conditions, including bacterial separation therapy, to better understand disease mechanisms and quantitatively assess the biological impact of bacterial separation therapy. Model validity was tested against experimental data from published studies. This is the first multi-compartment model of symptomatic bacteremia in mammals that includes extracorporeal bacterial separation and antibiotic treatment, separately and in combination. The addition of an extracorporeal bacterial separation circuit reduced the predicted time of total bacteria clearance from the blood of an immunocompromised rodent by 49%, compared to antibiotic treatment alone. Implementation of bacterial separation therapy resulted in predicted multi-drug resistant bacterial clearance from the blood of a human in 97% less time than antibiotic treatment alone. The model also proposes a quantitative correlation between time-dependent bacterial load among tissues and bacteremia severity, analogous to the well-known 'area under the curve' for characterization of drug efficacy. The engineering-based mathematical model developed may be useful for informing the design of extracorporeal bacterial separation devices. This work enables the quantitative identification of the characteristics required of an extracorporeal bacteria separation device to provide biological benefit. These devices will potentially decrease the bacterial load in blood. Additionally, the devices may achieve bacterial separation rates that allow consequent acceleration of bacterial clearance in other tissues, inhibiting the progression of symptomatic bacteremia, including multi-drug resistant variations.

8.
Infect Immun ; 84(9): 2662-70, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27382020

RESUMO

Helicobacter pylori secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly ß-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted by H. pylori The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the ß-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the ß-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly.


Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Helicobacter pylori/genética , Mutação/genética , Domínios Proteicos/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Linhagem Celular Tumoral , Células HeLa , Helicobacter pylori/metabolismo , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Microscopia Eletrônica/métodos
9.
Toxins (Basel) ; 8(6)2016 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-27271669

RESUMO

The VacA toxin secreted by Helicobacter pylori enhances the ability of the bacteria to colonize the stomach and contributes to the pathogenesis of gastric adenocarcinoma and peptic ulcer disease. The amino acid sequence and structure of VacA are unrelated to corresponding features of other known bacterial toxins. VacA is classified as a pore-forming toxin, and many of its effects on host cells are attributed to formation of channels in intracellular sites. The most extensively studied VacA activity is its capacity to stimulate vacuole formation, but the toxin has many additional effects on host cells. Multiple cell types are susceptible to VacA, including gastric epithelial cells, parietal cells, T cells, and other types of immune cells. This review focuses on the wide range of VacA actions that are detectable in vitro, as well as actions of VacA in vivo that are relevant for H. pylori colonization of the stomach and development of gastric disease.


Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Humanos , Canais Iônicos/metabolismo , Conformação Proteica , Transcrição Genética
10.
J Biomed Nanotechnol ; 12(9): 1806-19, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29345892

RESUMO

Gold nanoparticles (AuNPs) were functionalized for rapid binding of Acinetobacter baumannii (A. baumannii), a Gram-negative bacterium. AuNPs were functionalized with colistin (Col), a polycationic antibiotic, using a two-step self-assembly process, in which heterobifunctional polyethylene glycol (PEG) was used as a linker. Colistin was successfully conjugated to the AuNPs (Col-PEG-AuNP), as validated by dynamic light scattering (DLS) and proton nuclear magnetic resonance (H1 NMR). High angle annular dark field scanning transmission electron microscopy (HAADF-STEM) images, acquired simultaneously with X-ray energy dispersive spectroscopy (EDS) data, confirmed binding of Col-PEG-AuNPs to the cell envelope of A. baumannii. Results generated from a binding assay indicated that Col-PEG-AuNP complexation with A. baumannii occurred rapidly and reached half-maximum saturation in approximately 7 minutes, on average, for all A. baumannii strains evaluated. Quantitative measurement of the kinetics of Col-PEG-AuNP binding to A. baumannii is essential to inform the design of colistin-functionalized magnetic nanoparticles for magnetic separation of nanoparticle-bound A. baumannii.


Assuntos
Acinetobacter baumannii/isolamento & purificação , Técnicas Bacteriológicas/métodos , Separação Celular/métodos , Colistina/metabolismo , Ouro/química , Nanopartículas Metálicas/química , Acinetobacter baumannii/química , Acinetobacter baumannii/metabolismo , Colistina/química
11.
PLoS Pathog ; 11(5): e1004896, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25993478

RESUMO

Clostridium perfringens ε-toxin (ETX) is a potent pore-forming toxin responsible for a central nervous system (CNS) disease in ruminant animals with characteristics of blood-brain barrier (BBB) dysfunction and white matter injury. ETX has been proposed as a potential causative agent for Multiple Sclerosis (MS), a human disease that begins with BBB breakdown and injury to myelin forming cells of the CNS. The receptor for ETX is unknown. Here we show that both binding of ETX to mammalian cells and cytotoxicity requires the tetraspan proteolipid Myelin and Lymphocyte protein (MAL). While native Chinese Hamster Ovary (CHO) cells are resistant to ETX, exogenous expression of MAL in CHO cells confers both ETX binding and susceptibility to ETX-mediated cell death. Cells expressing rat MAL are ~100 times more sensitive to ETX than cells expressing similar levels of human MAL. Insertion of the FLAG sequence into the second extracellular loop of MAL abolishes ETX binding and cytotoxicity. ETX is known to bind specifically and with high affinity to intestinal epithelium, renal tubules, brain endothelial cells and myelin. We identify specific binding of ETX to these structures and additionally show binding to retinal microvasculature and the squamous epithelial cells of the sclera in wild-type mice. In contrast, there is a complete absence of ETX binding to tissues from MAL knockout (MAL-/-) mice. Furthermore, MAL-/- mice exhibit complete resistance to ETX at doses in excess of 1000 times the symptomatic dose for wild-type mice. We conclude that MAL is required for both ETX binding and cytotoxicity.


Assuntos
Toxinas Bacterianas/toxicidade , Clostridium perfringens/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Sítios de Ligação , Células CHO , Morte Celular/efeitos dos fármacos , Clostridium perfringens/patogenicidade , Cricetulus , Humanos , Injeções Intravenosas , Ligantes , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Insercional , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/química , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Domínios e Motivos de Interação entre Proteínas , Precursores de Proteínas/administração & dosagem , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Precursores de Proteínas/toxicidade , Ratos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/toxicidade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Distribuição Tecidual , Toxicocinética
12.
Infect Immun ; 82(1): 423-32, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24191302

RESUMO

Helicobacter pylori colonizes the human stomach and confers an increased risk for the development of peptic ulceration, noncardia gastric adenocarcinoma, and gastric lymphoma. A secreted H. pylori toxin, VacA, can cause multiple alterations in gastric epithelial cells, including cell death. In this study, we sought to identify host cell factors that are required for VacA-induced cell death. To do this, we analyzed gene trap and short hairpin RNA (shRNA) libraries in AZ-521 human gastric epithelial cells and selected for VacA-resistant clones. Among the VacA-resistant clones, we identified multiple gene trap library clones and an shRNA library clone with disrupted expression of connexin 43 (Cx43) (also known as gap junction protein alpha 1 [GJA1]). Further experiments with Cx43-specific shRNAs confirmed that a reduction in Cx43 expression results in resistance to VacA-induced cell death. Immunofluorescence microscopy experiments indicated that VacA did not colocalize with Cx43. We detected production of the Cx43 protein in AZ-521 cells but not in AGS, HeLa, or RK-13 cells, and correspondingly, AZ-521 cells were the most susceptible to VacA-induced cell death. When Cx43 was expressed in HeLa cells, the cells became more susceptible to VacA. These results indicate that Cx43 is a host cell constituent that contributes to VacA-induced cell death and that variation among cell types in susceptibility to VacA-induced cell death is attributable at least in part to cell type-specific differences in Cx43 production.


Assuntos
Proteínas de Bactérias/fisiologia , Morte Celular/fisiologia , Conexina 43/metabolismo , Células Epiteliais/fisiologia , Helicobacter pylori/fisiologia , Sobrevivência Celular , Células Cultivadas , Mucosa Gástrica/citologia , Humanos , RNA Interferente Pequeno/análise
13.
J Microbiol Methods ; 95(3): 336-41, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24113399

RESUMO

The lack of a versatile system to control gene expression in Helicobacter pylori has hampered efforts to study H. pylori physiology and pathogenesis. To overcome these limitations, we evaluated the utility of an inducible system based on the well-characterized Tet repressor (TetR) and Tet operator (tetO). As validation of this system, we introduced three copies of tetO into the promoter region upstream of the cagUT operon (encoding two virulence factors required for function of the H. pylori Cag type IV secretion system) and expressed tetR by introducing a codon-optimized gene into the chromosomal ureA locus. Introduction of the tetO copies upstream of cagUT did not disrupt promoter activity, as determined by immunoblotting for CagT. The subsequent introduction of tetR, however, did repress CagT synthesis. Production of CagT was restored when strains were cultured in the presence of the inducer, anhydrotetracycline. To demonstrate one potential application of this new tool, we analyzed the function of the Cag type IV secretion system. When the modified H. pylori strains were co-cultured with AGS cells, activity of the Cag type IV secretion system was dependent on the presence of anhydrotetracycline as evidenced by inducer-dependent induction of IL-8 secretion, CagA translocation, and appearance of type IV secretion system pili at the bacteria-host interface. These studies demonstrate the effectiveness of the tetR-tetO system to control gene expression in H. pylori and provide an improved system for studying H. pylori physiology and pathogenesis.


Assuntos
Regulação Bacteriana da Expressão Gênica , Genética Microbiana/métodos , Helicobacter pylori/genética , Biologia Molecular/métodos , Regiões Operadoras Genéticas , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/biossíntese , Western Blotting , Helicobacter pylori/efeitos dos fármacos , Recombinação Genética , Proteínas Repressoras/genética , Tetraciclinas/metabolismo , Ativação Transcricional/efeitos dos fármacos
14.
Genome Announc ; 1(5)2013 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-24072860

RESUMO

We present the genome sequences of three hpAfrica2 strains of Helicobacter pylori, which are postulated to have evolved in isolation for many millennia in people of San ethnicity. Although previously considered to be ancestral to Helicobacter acinonychis, the hpAfrica2 strains differ markedly from H. acinonychis in their gene arrangement. These data provide new insights into Helicobacter evolution.

15.
PLoS One ; 8(1): e55120, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23383074

RESUMO

Helicobacter pylori infection is a risk factor for the development of gastric adenocarcinoma, a disease that has a high incidence in East Asia. Genes that are highly divergent in East Asian H. pylori strains compared to non-Asian strains are predicted to encode proteins that differ in functional activity and could represent novel determinants of virulence. To identify such proteins, we undertook a comparative analysis of sixteen H. pylori genomes, selected equally from strains classified as East Asian or non-Asian. As expected, the deduced sequences of two known virulence determinants (CagA and VacA) are highly divergent, with 77% and 87% mean amino acid sequence identities between East Asian and non-Asian groups, respectively. In total, we identified 57 protein sequences that are highly divergent between East Asian and non-Asian strains, but relatively conserved within East Asian strains. The most highly represented functional groups are hypothetical proteins, cell envelope proteins and proteins involved in DNA metabolism. Among the divergent genes with known or predicted functions, population genetic analyses indicate that 86% exhibit evidence of positive selection. McDonald-Kreitman tests further indicate that about one third of these highly divergent genes, including cagA and vacA, are under diversifying selection. We conclude that, similar to cagA and vacA, most of the divergent genes identified in this study evolved under positive selection, and represent candidate factors that may account for the disproportionately high incidence of gastric cancer associated with East Asian H. pylori strains. Moreover, these divergent genes represent robust biomarkers that can be used to differentiate East Asian and non-Asian H. pylori strains.


Assuntos
Evolução Molecular , Genes Bacterianos/genética , Genômica , Helicobacter pylori/classificação , Helicobacter pylori/genética , Alelos , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , DNA Bacteriano/metabolismo , Extremo Oriente , Marcadores Genéticos/genética , Variação Genética/genética , Helicobacter pylori/metabolismo , Helicobacter pylori/fisiologia , Tipagem de Sequências Multilocus , Filogenia , Seleção Genética , Especificidade da Espécie , Neoplasias Gástricas/microbiologia
16.
PLoS One ; 7(10): e46866, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23056496

RESUMO

Evidence from multiple studies suggests that Clostridium perfringens ε-toxin is a pore-forming toxin, assembling into oligomeric complexes in the plasma membrane of sensitive cells. In a previous study, we used gene-trap mutagenesis to identify mammalian factors contributing to toxin activity, including caveolin-2 (CAV2). In this study, we demonstrate the importance of caveolin-2 and its interaction partner, caveolin-1 (CAV1), in ε-toxin-induced cytotoxicity. Using CAV2-specific shRNA in a toxin-sensitive human kidney cell line, ACHN, we confirmed that cells deficient in CAV2 exhibit increased resistance to ε-toxin. Similarly, using CAV1-specific shRNA, we demonstrate that cells deficient in CAV1 also exhibit increased resistance to the toxin. Immunoprecipitation of CAV1 and CAV2 from ε-toxin-treated ACHN cells demonstrated interaction of both CAV1 and -2 with the toxin. Furthermore, blue-native PAGE indicated that the toxin and caveolins were components of a 670 kDa protein complex. Although ε-toxin binding was only slightly perturbed in caveolin-deficient cells, oligomerization of the toxin was dramatically reduced in both CAV1- and CAV2-deficient cells. These results indicate that CAV1 and -2 potentiate ε-toxin induced cytotoxicity by promoting toxin oligomerization - an event which is requisite for pore formation and, by extension, cell death.


Assuntos
Toxinas Bacterianas/química , Caveolina 1/metabolismo , Caveolina 2/metabolismo , Multimerização Proteica , Animais , Toxinas Bacterianas/toxicidade , Caveolina 1/deficiência , Caveolina 1/genética , Caveolina 2/deficiência , Caveolina 2/genética , Sobrevivência Celular/efeitos dos fármacos , Cães , Técnicas de Silenciamento de Genes , Humanos , Células Madin Darby de Rim Canino , Estrutura Quaternária de Proteína , RNA Interferente Pequeno/genética
17.
Biochemistry ; 51(38): 7588-95, 2012 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-22938730

RESUMO

Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1.


Assuntos
Aminoácidos/metabolismo , Toxinas Bacterianas/metabolismo , Vírus da Hepatite A/metabolismo , Receptores Virais/metabolismo , Animais , Toxinas Bacterianas/genética , Linhagem Celular , Cães , Mutação , Ligação Proteica
18.
Mol Biosyst ; 8(8): 2097-105, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22688384

RESUMO

Recent studies demonstrated that a variety of bacterial pore-forming toxins induce cell death through a process of programmed necrosis characterized by the rapid depletion of cellular ATP. However, events leading to the necrosis and depletion of ATP are not thoroughly understood. We demonstrate that ATP-depletion induced by two pore-forming toxins, the Clostridium perfringens epsilon-toxin and the Aeromonas hydrophila aerolysin toxin, is associated with decreased mitochondrial membrane potential and opening of the mitochondrial permeability transition pore. To gain further insight into the toxin-induced metabolic changes contributing to necrosis and depletion of ATP, we analyzed the biochemical profiles of 251 distinct compounds by GC/MS or LC/MS/MS following exposure of a human kidney cell line to the epsilon-toxin. As expected, numerous biochemicals were seen to increase or decrease in response to epsilon-toxin. However, the pattern of these changes was consistent with the toxin-induced disruption of major energy-producing pathways in the cell including disruptions to the beta-oxidation of lipids. In particular, treatment with epsilon-toxin led to decreased levels of key coenzymes required for energy production including carnitine, NAD (and NADH), and coenzyme A. Independent biochemical assays confirmed that epsilon-toxin and aerolysin induced the rapid decrease of these coenzymes or their synthetic precursors. Incubation of cells with NADH or carnitine-enriched medium helped protect cells from toxin-induced ATP depletion and cell death. Collectively, these results demonstrate that members of the aerolysin family of pore-forming toxins lead to decreased levels of essential coenzymes required for energy production. The resulting loss of energy substrates is expected to contribute to dissipation of the mitochondrial membrane potential, opening of the mitochondrial permeability transition pore, and ultimately cell death.


Assuntos
Toxinas Bacterianas/farmacologia , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Trifosfato de Adenosina/metabolismo , Aeromonas hydrophila/química , Carnitina/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular , Coenzima A/metabolismo , Humanos , NAD/metabolismo
19.
Infect Immun ; 80(8): 2578-88, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22585965

RESUMO

Colonization of the human stomach with Helicobacter pylori is a risk factor for peptic ulceration, noncardia gastric adenocarcinoma, and gastric lymphoma. The secreted VacA toxin is an important H. pylori virulence factor that causes multiple alterations in gastric epithelial cells and T cells. Several families of vacA alleles have been described, and H. pylori strains containing certain vacA types (s1, i1, and m1) are associated with an increased risk of gastric disease, compared to strains containing other vacA types (s2, i2, and m2). Thus far, there has been relatively little study of the role of the VacA intermediate region (i-region) in toxin activity. In this study, we compared the ability of i1 and i2 forms of VacA to cause functional alterations in Jurkat cells. To do this, we manipulated the chromosomal vacA gene in two H. pylori strains to introduce alterations in the region encoding the VacA i-region. We did not detect any differences in the capacity of i1 and i2 forms of VacA to cause vacuolation of RK13 cells. In comparison to i1 forms of VacA, i2 forms of VacA had a diminished capacity to inhibit the activation of nuclear factor of activated T cells (NFAT) and suppress interleukin-2 (IL-2) production. Correspondingly, i2 forms of VacA bound to Jurkat cells less avidly than did i1 forms of VacA. These results indicate that the VacA i-region is an important determinant of VacA effects on human T cell function.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Regulação Bacteriana da Expressão Gênica/fisiologia , Helicobacter pylori/metabolismo , Linfócitos T/metabolismo , Sequência de Aminoácidos , Antígenos CD18/metabolismo , Humanos , Interleucina-2/metabolismo , Células Jurkat , Dados de Sequência Molecular , Ligação Proteica , Isoformas de Proteínas , Linfócitos T/imunologia , Linfócitos T/microbiologia
20.
PLoS Pathog ; 7(9): e1002237, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21909278

RESUMO

Colonization of the human stomach by Helicobacter pylori is an important risk factor for development of gastric cancer. The H. pylori cag pathogenicity island (cag PAI) encodes components of a type IV secretion system (T4SS) that translocates the bacterial oncoprotein CagA into gastric epithelial cells, and CagL is a specialized component of the cag T4SS that binds the host receptor α5ß1 integrin. Here, we utilized a mass spectrometry-based approach to reveal co-purification of CagL, CagI (another integrin-binding protein), and CagH (a protein with weak sequence similarity to CagL). These three proteins are encoded by contiguous genes in the cag PAI, and are detectable on the bacterial surface. All three proteins are required for CagA translocation into host cells and H. pylori-induced IL-8 secretion by gastric epithelial cells; however, these proteins are not homologous to components of T4SSs in other bacterial species. Scanning electron microscopy analysis reveals that these proteins are involved in the formation of pili at the interface between H. pylori and gastric epithelial cells. ΔcagI and ΔcagL mutant strains fail to form pili, whereas a ΔcagH mutant strain exhibits a hyperpiliated phenotype and produces pili that are elongated and thickened compared to those of the wild-type strain. This suggests that pilus dimensions are regulated by CagH. A conserved C-terminal hexapeptide motif is present in CagH, CagI, and CagL. Deletion of these motifs results in abrogation of CagA translocation and IL-8 induction, and the C-terminal motifs of CagI and CagL are required for formation of pili. In summary, these results indicate that CagH, CagI, and CagL are components of a T4SS subassembly involved in pilus biogenesis, and highlight the important role played by unique constituents of the H. pylori cag T4SS.


Assuntos
Proteínas de Bactérias/fisiologia , Fímbrias Bacterianas/fisiologia , Ilhas Genômicas , Infecções por Helicobacter/fisiopatologia , Helicobacter pylori/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Sequência de Aminoácidos , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Helicobacter pylori/patogenicidade , Humanos , Estômago/microbiologia
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