RESUMO
While marine natural products have been investigated for anticancer drug discovery, they are barely screened against rare cancers. Thus, in our effort to discover potential drug leads against the rare cancer pseudomyxoma peritonei (PMP), which currently lacks effective drug treatments, we screened extracts of marine actinomycete bacteria against the PMP cell line ABX023-1. This effort led to the isolation of nine rearranged angucyclines from Streptomyces sp. CNZ-748, including five new analogues, namely, grincamycins P-T (1-5). The chemical structures of these compounds were unambiguously established based on spectroscopic and chemical analyses. Particularly, grincamycin R (3) possesses an S-containing α-l-methylthio-aculose residue, which was discovered in nature for the first time. All of the isolated compounds were evaluated against four PMP cell lines and some exhibited low micromolar inhibitory activities. To identify a candidate biosynthetic gene cluster (BGC) encoding the grincamycins, we sequenced the genome of the producing strain, Streptomyces sp. CNZ-748, and compared the BGCs detected with those linked to the production of angucyclines with different aglycon structures.
Assuntos
Antraquinonas/farmacologia , Antineoplásicos/farmacologia , Pseudomixoma Peritoneal/tratamento farmacológico , Streptomyces/química , Antraquinonas/isolamento & purificação , Antineoplásicos/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , California , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Sedimentos Geológicos/microbiologia , Humanos , Estrutura Molecular , Família Multigênica , Streptomyces/genéticaRESUMO
BACKGROUND: Indole-3-carbinol (I3C) and other aryl hydrocarbon receptor agonists are known to modulate the immune system and ameliorate various inflammatory and autoimmune diseases in animal models, including colitis induced by dextran sulfate sodium (DSS). MicroRNAs (miRNAs) are also gaining traction as potential therapeutic agents or diagnostic elements. Enterohepatic Helicobacter (EHH) species are associated with an increased risk of inflammatory bowel disease, but little is known about how these species affect the immune system or response to treatment. AIM: To determine whether infection with an EHH species alters the response to I3C and how the immune and miRNA responses of an EHH species compare with responses to DSS and inflammatory bowel disease. METHODS: We infected C57BL/6 mice with Helicobacter muridarum (H. muridarum), with and without DSS and I3C treatment. Pathological responses were evaluated by histological examination, symptom scores, and cytokine responses. MiRNAs analysis was performed on mesenteric lymph nodes to further evaluate the regional immune response. RESULTS: H. muridarum infection alone caused colonic inflammation and upregulated proinflammatory, macrophage-associated cytokines in the colon similar to changes seen in DSS-treated mice. Further upregulation occurred upon treatment with DSS. H. muridarum infection caused broad changes in mesenteric lymph node miRNA expression, but colitis-associated miRNAs were regulated similarly in H. muridarum-infected and uninfected, DSS-treated mice. In spite of causing colitis exacerbation, H. muridarum infection did not prevent disease amelioration by I3C. I3C normalized both macrophage- and T cell-associated cytokines. CONCLUSION: Thus, I3C may be useful for inflammatory bowel disease patients regardless of EHH infection. The miRNA changes associated with I3C treatment are likely the result of, rather than the cause of immune response changes.
Assuntos
Colite , MicroRNAs , Animais , Colite/induzido quimicamente , Colite/genética , Colo , Citocinas , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Helicobacter , Humanos , Indóis , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genéticaRESUMO
A Gram-stain-negative, microaerophilic, non-motile, rod-shaped bacterium strain designated PMP191FT, was isolated from a human peritoneal tumour. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the organism formed a lineage within the family Chitinophagaceae that was distinct from members of the genus Pseudoflavitalea (95.1-95.2â% sequence similarity) and Pseudobacter ginsenosidimutans (94.4â% sequence similarity). The average nucleotide identity values between strain PMP191FT and Pseudoflavitalea rhizosphaerae T16R-265T and Pseudobacter ginsenosidimutans Gsoil 221T was 68.9 and 62.3% respectively. The only respiratory quinone of strain PMP191FT was MK-7 and the major fatty acids were iso-C15â:â0, iso-C15â:â1 G and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). The polar lipids consisted of phosphatidylethanolamine and some unidentified amino and glycolipids. The G+C content of strain PMP191FT calculated from the genome sequence was 43.4 mol%. Based on phylogenetic, phenotypic and chemotaxonomic evidence, strain PMP191FT represents a novel species and genus for which the name Parapseudoflavitalea muciniphila gen. nov., sp. nov. is proposed. The type strain is PMP191FT (=DSM 104999T=ATCC BAA-2857T = CCUG 72691T). The phylogenetic analyses also revealed that Pseudobacter ginsenosidimutans shared over 98â% sequence similarly to members of the genus Pseudoflavitalea. However, the average nucleotide identity value between Pseudoflavitalea rhizosphaerae T16R-265T, the type species of the genus and Pseudobacter ginsenosidimutans Gsoil 221T was 86.8â%. Therefore, we also propose that Pseudobacter ginsenosidimutans be reclassified as Pseudoflavitalea ginsenosidimutans comb. nov.
Assuntos
Bacteroidetes/classificação , Neoplasias Peritoneais/microbiologia , Filogenia , Técnicas de Tipagem Bacteriana , Bacteroidetes/isolamento & purificação , Baltimore , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Glicolipídeos/química , Humanos , Fosfatidiletanolaminas/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMO
The collection of microbes that live in and on the human body - the human microbiome - can impact on cancer initiation, progression, and response to therapy, including cancer immunotherapy. The mechanisms by which microbiomes impact on cancers can yield new diagnostics and treatments, but much remains unknown. The interactions between microbes, diet, host factors, drugs, and cell-cell interactions within the cancer itself likely involve intricate feedbacks, and no single component can explain all the behavior of the system. Understanding the role of host-associated microbial communities in cancer systems will require a multidisciplinary approach combining microbial ecology, immunology, cancer cell biology, and computational biology - a systems biology approach.
Assuntos
Microbiota , Neoplasias/microbiologia , Analgésicos Opioides/uso terapêutico , Animais , Bactérias/metabolismo , Sistema Nervoso Central/fisiologia , Sinergismo Farmacológico , Microbiologia Ambiental , Gastrite/microbiologia , Microbioma Gastrointestinal , Infecções por Helicobacter/complicações , Interações Hospedeiro-Patógeno , Humanos , Imunoterapia , Camundongos , Microbiota/efeitos dos fármacos , Microbiota/efeitos da radiação , Neoplasias/etiologia , Neoplasias/terapia , Neoplasias/virologia , Vírus Oncogênicos/patogenicidade , Probióticos , Neoplasias Gástricas/etiologia , Neoplasias Gástricas/microbiologia , Simbiose , Infecções Tumorais por VírusRESUMO
A number of viruses and bacterial species have been implicated as contributors to atherosclerosis, potentially providing novel pathways for prevention. Epidemiological studies examining the association between Helicobacter pylori and cardiovascular disease have yielded variable results and no studies have been conducted in nonhuman primates. In this investigation, we examined the relationship between H. pylori infection and atherosclerosis development in socially housed, pre- and postmenopausal cynomolgus macaques consuming human-like diets. Ninety-four premenopausal cynomolgus monkeys (Macaca fascicularis) were fed for 36 months an atherogenic diet deriving its protein from either casein lactalbumin(CL) or high isoflavone soy (SOY). Animals were then ovariectomized and fed either the same or the alternate diet for an additional 36 months. Iliac artery biopsies were obtained at the time of ovariectomy and iliac and coronary artery sections were examined at the end of the study. Evidence of H. pylori infection was found in 64% of the monkeys and 46% of animals had live H. pylori within coronary atheromas as determined by mRNA-specific in situ hybridization. There was a significant linear relationship between the densities of gastric and atheroma organisms. Helicobactor pylori infection correlated with increased intimal plaque area and thickness at both the premenopausal and postmenopausal time points and regardless of diet (p< 0.01), although animals consuming the SOY diet throughout had the least amount of atherosclerosis. Additionally, plasma lipid profiles, intimal collagen accumulation, ICAM-1, and plaque macrophage densities were adversely affected by H. pylori infection among animals consuming the CL diet, while the SOY diet had the opposite effect. Plaque measurements were more highly associated with the densities of cagA-positive H. pylori within coronary atheromas than with the densities of gastric organisms, whereas plasma lipid changes were associated with H. pylori infection, but not cagA status. This study provides strong evidence that live H. pylori infects atheromas, exacerbates atherosclerotic plaque development, and alters plasma lipid profiles independently of diet or hormonal status. Finally, socially subordinate animals relative to their dominant counterparts had a greater prevalence of H. pylori, suggesting a stress effect. The results indicate that early H. pylori eradication could prevent or delay development of cardiovascular disease.
Assuntos
Aterosclerose , Dieta , Infecções por Helicobacter , Helicobacter pylori , Pós-Menopausa , Pré-Menopausa , Animais , Feminino , Artérias/metabolismo , Artérias/patologia , Aterosclerose/epidemiologia , Aterosclerose/etiologia , Aterosclerose/metabolismo , Aterosclerose/microbiologia , Biomarcadores/metabolismo , Infecções por Helicobacter/complicações , Helicobacter pylori/fisiologia , Lipídeos/sangue , Macaca fascicularis , PrevalênciaRESUMO
Pseudomyxoma peritonei (PMP) is a subtype of peritoneal carcinomatosis that is traditionally treated by cytoreductive surgery (CRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC). A growing body of evidence suggests that microbes are associated with various tumor types and have been found in organs and cavities that were once considered sterile. Prior and ongoing research from our consortium of PMP researchers strongly suggests that bacteria are associated with PMP tumors. While the significance of this association is unclear, in our opinion, further research is warranted to understand whether these bacteria contribute to the development, maintenance and/or progression of PMP. Elucidation of a possible causal role for bacteria in PMP could suggest a benefit for supplementation of antibiotics to current treatment protocols.
Assuntos
Antibacterianos/uso terapêutico , Procedimentos Cirúrgicos de Citorredução , Hipertermia Induzida , Pseudomixoma Peritoneal/microbiologia , Pseudomixoma Peritoneal/terapia , Terapia Combinada , Feminino , Humanos , MasculinoRESUMO
Many of the symptoms of Gulf War Illness (GWI) that include neurological abnormalities, neuroinflammation, chronic fatigue and gastrointestinal disturbances have been traced to Gulf War chemical exposure. Though the association and subsequent evidences are strong, the mechanisms that connect exposure to intestinal and neurological abnormalities remain unclear. Using an established rodent model of Gulf War Illness, we show that chemical exposure caused significant dysbiosis in the gut that included increased abundance of phylum Firmicutes and Tenericutes, and decreased abundance of Bacteroidetes. Several gram negative bacterial genera were enriched in the GWI-model that included Allobaculum sp. Altered microbiome caused significant decrease in tight junction protein Occludin with a concomitant increase in Claudin-2, a signature of a leaky gut. Resultant leaching of gut caused portal endotoxemia that led to upregulation of toll like receptor 4 (TLR4) activation in the small intestine and the brain. TLR4 knock out mice and mice that had gut decontamination showed significant decrease in tyrosine nitration and inflammatory mediators IL1ß and MCP-1 in both the small intestine and frontal cortex. These events signified that gut dysbiosis with simultaneous leaky gut and systemic endotoxemia-induced TLR4 activation contributes to GW chemical-induced neuroinflammation and gastrointestinal disturbances.
Assuntos
Lobo Frontal/metabolismo , Microbioma Gastrointestinal/fisiologia , Inflamação/metabolismo , Enteropatias/metabolismo , Enteropatias/microbiologia , Síndrome do Golfo Pérsico/microbiologia , Receptor 4 Toll-Like/metabolismo , Animais , Claudina-2/metabolismo , Modelos Animais de Doenças , Disbiose/metabolismo , Endotoxemia/metabolismo , Guerra do Golfo , Inflamação/microbiologia , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Síndrome do Golfo Pérsico/metabolismoRESUMO
OBJECTIVE To develop a risk score to predict probability of bloodstream infections (BSIs) due to extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBLE). DESIGN Retrospective case-control study. SETTING Two large community hospitals. PATIENTS Hospitalized adults with Enterobacteriaceae BSI between January 1, 2010, and June 30, 2015. METHODS Multivariate logistic regression was used to identify independent risk factors for ESBLE BSI. Point allocation in extended-spectrum ß-lactamase prediction score (ESBL-PS) was based on regression coefficients. RESULTS Among 910 patients with Enterobacteriaceae BSI, 42 (4.6%) had ESBLE bloodstream isolates. Most ESBLE BSIs were community onset (33 of 42; 79%), and 25 (60%) were due to Escherichia coli. Independent risk factors for ESBLE BSI and point allocation in ESBL-PS included outpatient procedures within 1 month (adjusted odds ratio [aOR], 8.7; 95% confidence interval [CI], 3.1-22.9; 1 point), prior infections or colonization with ESBLE within 12 months (aOR, 26.8; 95% CI, 7.0-108.2; 4 points), and number of prior courses of ß-lactams and/or fluoroquinolones used within 3 months of BSI: 1 course (aOR, 6.3; 95% CI, 2.7-14.7; 1 point), ≥2 courses (aOR, 22.0; 95% CI, 8.6-57.1; 3 points). The area under the receiver operating characteristic curve for the ESBL-PS model was 0.86. Patients with ESBL-PSs of 0, 1, 3, and 4 had estimated probabilities of ESBLE BSI of 0.7%, 5%, 24%, and 44%, respectively. Using ESBL-PS ≥3 to indicate high risk provided a negative predictive value of 97%. CONCLUSIONS ESBL-PS estimated patient-specific risk of ESBLE BSI with high discrimination. Incorporation of ESBL-PS with acute severity of illness may improve adequacy of empirical antimicrobial therapy and reduce carbapenem utilization. Infect Control Hosp Epidemiol 2017;38:266-272.
Assuntos
Bacteriemia/tratamento farmacológico , Infecções por Enterobacteriaceae/tratamento farmacológico , Enterobacteriaceae/isolamento & purificação , Fluoroquinolonas/uso terapêutico , beta-Lactamas/uso terapêutico , Idoso , Antibacterianos/uso terapêutico , Bacteriemia/epidemiologia , Estudos de Casos e Controles , Enterobacteriaceae/enzimologia , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , South Carolina , beta-Lactamases/biossínteseRESUMO
Peritoneal tumors from a rare malignancy, pseudomyxoma peritonei, frequently contain bacteria. Evidence suggests that tumor-associated bacteria contribute to pseudomyxoma peritonei development and/or progression. One unique isolate (PMP191F) was characterized via whole-genome sequencing using the Illumina MiSeq platform. PMP191F shows similarities to the Chitinophaga, Niastella, and Flavitalea genera.
RESUMO
Helicobacter pylori (H. pylori) is an extremely common, yet underappreciated, pathogen that is able to alter host physiology and subvert the host immune response, allowing it to persist for the life of the host. H. pylori is the primary cause of peptic ulcers and gastric cancer. In the United States, the annual cost associated with peptic ulcer disease is estimated to be $6 billion and gastric cancer kills over 700000 people per year globally. The prevalence of H. pylori infection remains high (> 50%) in much of the world, although the infection rates are dropping in some developed nations. The drop in H. pylori prevalence could be a double-edged sword, reducing the incidence of gastric diseases while increasing the risk of allergies and esophageal diseases. The list of diseases potentially caused by H. pylori continues to grow; however, mechanistic explanations of how H. pylori could contribute to extragastric diseases lag far behind clinical studies. A number of host factors and H. pylori virulence factors act in concert to determine which individuals are at the highest risk of disease. These include bacterial cytotoxins and polymorphisms in host genes responsible for directing the immune response. This review discusses the latest advances in H. pylori pathogenesis, diagnosis, and treatment. Up-to-date information on correlations between H. pylori and extragastric diseases is also provided.
Assuntos
Antibacterianos/uso terapêutico , Infecções por Helicobacter/diagnóstico , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/efeitos dos fármacos , Animais , Farmacorresistência Viral , Predisposição Genética para Doença , Infecções por Helicobacter/epidemiologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Incidência , Valor Preditivo dos Testes , Prevalência , Fatores de Risco , Gastropatias/diagnóstico , Gastropatias/tratamento farmacológico , Gastropatias/microbiologia , Resultado do Tratamento , VirulênciaRESUMO
BACKGROUND: Pseudomyxoma peritonei (PMP) is a malignancy characterized by dissemination of mucus-secreting cells throughout the peritoneum. This disease is associated with significant morbidity and mortality and despite effective treatment options for early-stage disease, patients with PMP often relapse. Thus, there is a need for additional treatment options to reduce relapse rate and increase long-term survival. A previous study identified the presence of both typed and non-culturable bacteria associated with PMP tissue and determined that increased bacterial density was associated with more severe disease. These findings highlighted the possible role for bacteria in PMP disease. METHODS: To more clearly define the bacterial communities associated with PMP disease, we employed a sequenced-based analysis to profile the bacterial populations found in PMP tumor and mucin tissue in 11 patients. Sequencing data were confirmed by in situ hybridization at multiple taxonomic depths and by culturing. A pilot clinical study was initiated to determine whether the addition of antibiotic therapy affected PMP patient outcome. MAIN RESULTS: We determined that the types of bacteria present are highly conserved in all PMP patients; the dominant phyla are the Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. A core set of taxon-specific sequences were found in all 11 patients; many of these sequences were classified into taxonomic groups that also contain known human pathogens. In situ hybridization directly confirmed the presence of bacteria in PMP at multiple taxonomic depths and supported our sequence-based analysis. Furthermore, culturing of PMP tissue samples allowed us to isolate 11 different bacterial strains from eight independent patients, and in vitro analysis of subset of these isolates suggests that at least some of these strains may interact with the PMP-associated mucin MUC2. Finally, we provide evidence suggesting that targeting these bacteria with antibiotic treatment may increase the survival of PMP patients. CONCLUSIONS: Using 16S amplicon-based sequencing, direct in situ hybridization analysis and culturing methods, we have identified numerous bacterial taxa that are consistently present in all PMP patients tested. Combined with data from a pilot clinical study, these data support the hypothesis that adding antimicrobials to the standard PMP treatment could improve PMP patient survival.
Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas/complicações , Microbiota , Neoplasias Peritoneais/microbiologia , Pseudomixoma Peritoneal/microbiologia , Antibacterianos/uso terapêutico , Bactérias/classificação , Bactérias/efeitos dos fármacos , Bactérias/genética , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Meios de Cultura , Humanos , Hibridização In Situ , Mucina-2/metabolismo , Mucinas/metabolismo , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/mortalidade , Peritônio/metabolismo , Peritônio/microbiologia , Prognóstico , Pseudomixoma Peritoneal/tratamento farmacológico , Pseudomixoma Peritoneal/mortalidade , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Taxa de Sobrevida , Resultado do TratamentoRESUMO
PURPOSE: Pseudomyxoma peritonei is an understudied cancer in which an appendiceal neoplasm invades the peritoneum and forms tumor foci on abdominal organs. Previous studies have shown that bacteria reside within pseudomyxoma peritonei tumors and mucin. Thus, we sought to analyze the effect of antibiotics on bacterial density and ß-catenin expression within pseudomyxoma peritonei samples. EXPERIMENTAL DESIGN: The study included 48 patients: 19 with disseminated peritoneal adenomucinosis (DPAM) and 29 with peritoneal mucinous carcinomatosis (PMCA). Fourteen patients were given antibiotics (30 mg lansoprazole, 1 g amoxicillin, and 500 mg clarithromycin) twice a day for 14 days. One week after completion of therapy, surgery was conducted and specimens were harvested for pathology, bacterial culture, ISH, and immunohistochemistry. RESULTS: ISH showed the presence of bacteria in 83% of the patient samples, with a higher Helicobacter pylori density observed in PMCA versus DPAM. PMCA patients treated with antibiotics had a significantly lower bacterial density and decreased ß-catenin levels in the cytoplasm, the cell nuclei, and mucin-associated cells. Although not significant, similar trends were observed in DPAM patients. Cell membrane ß-catenin was significantly increased in both DPAM and PMCA patients receiving antibiotics. CONCLUSIONS: Bacteria play an important role in pseudomyxoma peritonei. Antibiotic treatment improved the histopathology of tissue, particularly in PMCA patients. In PMCA, antibiotics decreased bacterial density and were associated with a significant ß-catenin decrease in the cytoplasm, cell nuclei, and mucin along with a small membrane increase. These results suggest that antibiotics offer potential protection against cell detachment, cellular invasion, and metastasis.
Assuntos
Adenocarcinoma Mucinoso/microbiologia , Antibacterianos/uso terapêutico , Infecções por Helicobacter/tratamento farmacológico , Neoplasias Peritoneais/microbiologia , Pseudomixoma Peritoneal/microbiologia , beta Catenina/metabolismo , Adenocarcinoma Mucinoso/tratamento farmacológico , Adenocarcinoma Mucinoso/cirurgia , Amoxicilina/farmacologia , Amoxicilina/uso terapêutico , Antibacterianos/farmacologia , Carga Bacteriana/efeitos dos fármacos , Membrana Celular/metabolismo , Núcleo Celular , Claritromicina/farmacologia , Claritromicina/uso terapêutico , Terapia Combinada , Helicobacter pylori/genética , Humanos , Hibridização In Situ , Lansoprazol/farmacologia , Lansoprazol/uso terapêutico , Pessoa de Meia-Idade , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/cirurgia , Transporte Proteico , Pseudomixoma Peritoneal/tratamento farmacológico , Pseudomixoma Peritoneal/cirurgia , Resultado do TratamentoRESUMO
AIM: To investigate the role of host and bacterial arginases in the colonization of mice by Helicobacter pylori (H. pylori). METHODS: H. pylori produces a very powerful urease that hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid. Urease is absolutely essential to H. pylori pathogenesis; therefore, the urea substrate must be in ample supply for urease to work efficiently. The urea substrate is most likely provided by arginase activity, which hydrolyzes L-arginine to L-ornithine and urea. Previous work has demonstrated that H. pylori arginase is surprisingly not required for colonization of wild-type mice. Hence, another in vivo source of the critical urea substrate must exist. We hypothesized that the urea source was provided by host arginase II, since this enzyme is expressed in the stomach, and H. pylori has previously been shown to induce the expression of murine gastric arginase II. To test this hypothesis, wild-type and arginase (rocF) mutant H. pylori strain SS1 were inoculated into arginase II knockout mice. RESULTS: Surprisingly, both the wild-type and rocF mutant bacteria still colonized arginase II knockout mice. Moreover, feeding arginase II knockout mice the host arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC), while inhibiting > 50% of the host arginaseâ Iâ activity in several tissues, did not block the ability of the rocF mutant H. pylori to colonize. In contrast, BEC poorly inhibited H. pylori arginase activity. CONCLUSION: The in vivo source for the essential urea utilized by H. pylori urease is neither bacterial arginase nor host arginase II; instead, either residual host arginaseâ Iâ or agmatinase is probably responsible.
Assuntos
Arginase/genética , Proteínas de Bactérias/genética , Helicobacter pylori/enzimologia , Helicobacter pylori/genética , Mutação , Animais , Arginase/antagonistas & inibidores , Arginase/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Ácidos Borônicos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Ratos , Ureia/metabolismo , Ureo-Hidrolases/metabolismoRESUMO
Helicobacter pylori persistently colonizes humans, causing gastritis, ulcers, and gastric cancer. Adherence to the gastric epithelium has been shown to enhance inflammation, yet only a few H. pylori adhesins have been paired with targets in host tissue. The alpAB locus has been reported to encode adhesins involved in adherence to human gastric tissue. We report that abrogation of H. pylori AlpA and AlpB reduces binding of H. pylori to laminin while expression of plasmid-borne alpA or alpB confers laminin-binding ability to Escherichia coli. An H. pylori strain lacking only AlpB is also deficient in laminin binding. Thus, we conclude that both AlpA and AlpB contribute to H. pylori laminin binding. Contrary to expectations, the H. pylori SS1 mutant deficient in AlpA and AlpB causes more severe inflammation than the isogenic wild-type strain in gerbils. Identification of laminin as the target of AlpA and AlpB will facilitate future investigations of host-pathogen interactions occurring during H. pylori infection.
Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/metabolismo , Infecções por Helicobacter/patologia , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno , Laminina/metabolismo , Animais , Escherichia coli/genética , Feminino , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Expressão Gênica , Gerbillinae , Infecções por Helicobacter/microbiologia , Inflamação/patologia , Masculino , PlasmídeosRESUMO
The human gastric pathogen Helicobacter pylori steals host cholesterol, modifies it by glycosylation, and incorporates the glycosylated cholesterol onto its surface via a cholesterol glucosyltransferase, encoded by cgt. The impact of cholesterol on H. pylori antimicrobial resistance is unknown. H. pylori strain 26695 was cultured in Ham's F12 chemically defined medium in the presence or absence of cholesterol. The two cultures were subjected to overnight incubations with serial 2-fold dilutions of 12 antibiotics, six antifungals, and seven antimicrobial peptides (including LL-37 cathelicidin and human alpha and beta defensins). Of 25 agents tested, cholesterol-grown H. pylori cells were substantially more resistant (over 100-fold) to nine agents than were H. pylori cells grown without cholesterol. These nine agents included eight antibiotics and LL-37. H. pylori was susceptible to the antifungal drug pimaricin regardless of cholesterol presence in the culture medium. A cgt mutant retained cholesterol-dependent resistance to most antimicrobials but displayed increased susceptibility to colistin, suggesting an involvement of lipid A. Mutation of lpxE, encoding lipid A1-phosphatase, led to loss of cholesterol-dependent resistance to polymyxin B and colistin but not other antimicrobials tested. The cgt mutant was severely attenuated in gerbils, indicating that glycosylation is essential in vivo. These findings suggest that cholesterol plays a vital role in virulence and contributes to the intrinsic antibiotic resistance of H. pylori.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Colesterol/farmacologia , Helicobacter pylori/efeitos dos fármacos , Antifúngicos/farmacologia , Proteínas de Bactérias/fisiologia , Bismuto/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Replicação do DNA/efeitos dos fármacos , Farmacorresistência Bacteriana , Ácido Fólico/metabolismo , Helicobacter pylori/crescimento & desenvolvimento , Helicobacter pylori/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lipídeo A/metabolismo , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Compostos Organometálicos/farmacologia , Monoéster Fosfórico Hidrolases/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , Salicilatos/farmacologia , CatelicidinasRESUMO
The human gastric pathogen Helicobacter pylori modifies host cholesterol via glycosylation and incorporates the glycosylated cholesterol into its membrane; however, the benefits of cholesterol to H. pylori are largely unknown. We speculated that cholesterol in the H. pylori membrane might alter the susceptibility of these organisms to membrane-disrupting antibacterial compounds. To test this hypothesis, H. pylori strains were cultured in Ham's F-12 chemically defined medium in the presence or absence of cholesterol. The two cultures were subjected to overnight incubations with serial 2-fold dilutions of 10 bile salts and four ceragenins, which are novel bile salt derivatives that mimic membrane-disrupting activity of antimicrobial peptides. H. pylori cultured with cholesterol was substantially more resistant to seven of the bile salts and three ceragenins than H. pylori cultured without cholesterol. In most cases, these cholesterol-dependent differences ranged from 2 to 7 orders of magnitude; this magnitude depended on concentration of the agent. Cholesterol is modified by glycosylation using Cgt, a cholesteryl glycosyltransferase. Surprisingly, a cgt knockout strain still maintained cholesterol-dependent resistance to bile salts and ceragenins, indicating that cholesterol modification was not involved in resistance. We then tested whether three putative, paralogous inner membrane efflux pumps, HefC, HefF, or HefI, played a role. While HefF and HefI appeared unimportant, HefC was shown to play a critical role in the resistance to bile salts and ceragenins by multiple methods in multiple strain backgrounds. Thus, both cholesterol and the putative bile salt efflux pump HefC play important roles in H. pylori resistance to bile salts and ceragenins.
Assuntos
Proteínas de Bactérias/metabolismo , Ácidos e Sais Biliares/farmacologia , Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Helicobacter pylori/metabolismo , Esteroides/farmacologia , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Colesterol/química , Farmacorresistência Bacteriana/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Estrutura Molecular , MutaçãoRESUMO
Helicobacter pylori chronically infects the gastric mucosa, where it can be found free in mucus, attached to cells, and intracellularly. H. pylori requires iron for growth, but the sources of iron used in vivo are unclear. In previous studies, the inability to culture H. pylori without serum made it difficult to determine which host iron sources might be used by H. pylori. Using iron-deficient, chemically defined medium, we determined that H. pylori can bind and extract iron from hemoglobin, transferrin, and lactoferrin. H. pylori can use both bovine and human versions of both lactoferrin and transferrin, contrary to previous reports. Unlike other pathogens, H. pylori preferentially binds the iron-free forms of transferrin and lactoferrin, which limits its ability to extract iron from normal serum, which is not iron saturated. This novel strategy may have evolved to permit limited growth in host tissue during persistent colonization while excessive injury or iron depletion is prevented.
Assuntos
Meios de Cultura/química , Helicobacter pylori/metabolismo , Ferro/metabolismo , Animais , Bovinos , Hemoglobinas/metabolismo , Humanos , Lactoferrina/metabolismo , Transferrina/metabolismoRESUMO
Helicobacter pylori bacteria cultured in a chemically defined medium without serum readily adhere to a variety of abiotic surfaces. Growth produces microcolonies that spread to cover the entire surface, along with a planktonic subpopulation. Serum inhibits adherence. Initial attachment is protein mediated, but other molecules are responsible for more permanent attachment.
Assuntos
Aderência Bacteriana/fisiologia , Biofilmes , Meios de Cultura/química , Helicobacter pylori/fisiologia , Soro/fisiologia , Helicobacter pylori/ultraestrutura , Microscopia Eletrônica de Varredura , TripsinaRESUMO
BACKGROUND: Gene complementation strategies are important in validating the roles of genes in specific phenotypes. Complementation systems in Helicobacter pylori include shuttle vectors, which transform H. pylori at relatively low frequencies, and chromosomally based approaches. Chromosomal complementation strategies are susceptible to polar effects and disruption of other H. pylori genes, leading to unwanted pleiotropic effects. MATERIALS AND METHODS: A new complementation strategy was developed for H. pylori by utilizing a suicide plasmid vector that contains fragments of an H. pylori intergenic region (hp0203-hp0204), a chloramphenicol acetyltransferase cassette (cat), and a multiple-cloning site. Genes of interest could be cloned into the intergenic plasmid and the genes integrated into H. pylori by homologous recombination into the intergenic chromosomal region without disrupting any annotated H. pylori gene. The complementation system was validated using the gene encoding arginase (rocF). RESULTS: A rocF mutant unable to hydrolyze or consume l-arginine regained these functions by complementation with the wild-type rocF gene. Complemented strains also had restored arginase protein as determined by Western blot analysis. The complementation system could be successfully applied to multiple H. pylori strains. The intergenic region varied in length and sequence across 17 H. pylori strains, but the flanking-3' ends of the hp0203 and hp0204 coding regions were highly conserved. Inserting a cat cassette and wild-type rocF into the intergenic region did not alter the ability of strain SS1 to colonize mice. CONCLUSIONS: This complementation strategy should greatly facilitate genetic experiments in H. pylori.