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1.
Artigo em Inglês | MEDLINE | ID: mdl-30373804

RESUMO

Stagnation in antimicrobial development has led to a serious threat to public health because some Acinetobacter baumannii infections have become untreatable. New therapeutics with alternative mechanisms of action to combat A. baumannii are therefore necessary to treat these infections. To this end, the virulence of A. baumannii isolates with various antimicrobial susceptibilities was assessed when the isolates were treated with miltefosine, a phospholipase C inhibitor. Phospholipase C activity is a contributor to A. baumannii virulence associated with hemolysis, cytolysis of A549 human alveolar epithelial cells, and increased mortality in the Galleria mellonella experimental infection model. While the effects on bacterial growth were variable among strains, miltefosine treatment significantly reduced both the hemolytic and cytolytic activity of all treated A. baumannii strains. Additionally, scanning electron microscopy of polarized A549 cells infected with bacteria of the A. baumannii ATCC 19606T strain or the AB5075 multidrug-resistant isolate showed a decrease in A549 cell damage with a concomitant increase in the presence of A549 surfactant upon administration of miltefosine. The therapeutic ability of miltefosine was further supported by the results of G. mellonella infections, wherein miltefosine treatment of animals infected with ATCC 19606T significantly decreased mortality. These data demonstrate that inhibition of phospholipase C activity results in the overall reduction of A. baumannii virulence in both in vitro and in vivo models, making miltefosine a viable option for the treatment of A. baumannii infections, particularly those caused by multidrug-resistant isolates.


Assuntos
Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/patogenicidade , Antibacterianos/uso terapêutico , Fosforilcolina/análogos & derivados , Células A549 , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Animais , Linhagem Celular , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Testes de Sensibilidade Microbiana , Mariposas/microbiologia , Fosforilcolina/uso terapêutico , Fosfolipases Tipo C/antagonistas & inibidores , Virulência/efeitos dos fármacos
2.
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
3.
PeerJ ; 6: e4803, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29796347

RESUMO

Helicobacter pylori requires genetic agility to infect new hosts and establish long-term colonization of changing gastric environments. In this study, we analyzed H. pylori genetic adaptation in the Mongolian gerbil model. This model is of particular interest because H. pylori-infected gerbils develop a high level of gastric inflammation and often develop gastric adenocarcinoma or gastric ulceration. We analyzed the whole genome sequences of H. pylori strains cultured from experimentally infected gerbils, in comparison to the genome sequence of the input strain. The mean annualized single nucleotide polymorphism (SNP) rate per site was 1.5e-5, which is similar to the rates detected previously in H. pylori-infected humans. Many of the mutations occurred within or upstream of genes associated with iron-related functions (fur, tonB1, fecA2, fecA3, and frpB3) or encoding outer membrane proteins (alpA, oipA, fecA2, fecA3, frpB3 and cagY). Most of the SNPs within coding regions (86%) were non-synonymous mutations. Several deletion or insertion mutations led to disruption of open reading frames, suggesting that the corresponding gene products are not required or are deleterious during chronic H. pylori colonization of the gerbil stomach. Five variants (three SNPs and two deletions) were detected in isolates from multiple animals, which suggests that these mutations conferred a selective advantage. One of the mutations (FurR88H) detected in isolates from multiple animals was previously shown to confer increased resistance to oxidative stress, and we now show that this SNP also confers a survival advantage when H. pylori is co-cultured with neutrophils. Collectively, these analyses allow the identification of mutations that are positively selected during H. pylori colonization of the gerbil model.

4.
Gut ; 67(10): 1793-1804, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-28924022

RESUMO

OBJECTIVE: Helicobacter pylori is the strongest risk factor for gastric cancer; however, the majority of infected individuals do not develop disease. Pathological outcomes are mediated by complex interactions among bacterial, host and environmental constituents, and two dietary factors linked with gastric cancer risk are iron deficiency and high salt. We hypothesised that prolonged adaptation of H. pylori to in vivo carcinogenic microenvironments results in genetic modification important for disease. DESIGN: Whole genome sequencing of genetically related H. pylori strains that differ in virulence and targeted H. pylori sequencing following prolonged exposure of bacteria to in vitro carcinogenic conditions were performed. RESULTS: A total of 180 unique single nucleotide polymorphisms (SNPs) were identified among the collective genomes when compared with a reference H. pylori genome. Importantly, common SNPs were identified in isolates harvested from iron-depleted and high salt carcinogenic microenvironments, including an SNP within fur (FurR88H). To investigate the direct role of low iron and/or high salt, H. pylori was continuously cultured in vitro under low iron or high salt conditions to assess fur genetic variation. Exposure to low iron or high salt selected for the FurR88H variant after only 5 days. To extend these results, fur was sequenced in 339 clinical H. pylori strains. Among the isolates examined, 17% (40/232) of strains isolated from patients with premalignant lesions harboured the FurR88H variant, compared with only 6% (6/107) of strains from patients with non-atrophic gastritis alone (p=0.0034). CONCLUSION: These results indicate that specific genetic variation arises within H. pylori strains during in vivo adaptation to conditions conducive for gastric carcinogenesis.


Assuntos
Carcinogênese , Infecções por Helicobacter , Helicobacter pylori , Neoplasias Gástricas , Proteínas de Bactérias/genética , Infecções por Helicobacter/patologia , Infecções por Helicobacter/fisiopatologia , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Humanos , Técnicas In Vitro/métodos , Polimorfismo de Nucleotídeo Único/fisiologia , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Neoplasias Gástricas/fisiopatologia
5.
Infect Immun ; 86(3)2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29229727

RESUMO

Helicobacter pylori infection and high dietary salt intake are risk factors for the development of gastric adenocarcinoma. One possible mechanism by which a high-salt diet could influence gastric cancer risk is by modulating H. pylori gene expression. In this study, we utilized transcriptome sequencing (RNA-seq) methodology to compare the transcriptional profiles of H. pylori grown in media containing different concentrations of sodium chloride. We identified 118 differentially expressed genes (65 upregulated and 53 downregulated in response to high-salt conditions), including multiple members of 14 operons. Twenty-nine of the differentially expressed genes encode proteins previously shown to undergo salt-responsive changes in abundance, based on proteomic analyses. Real-time reverse transcription (RT)-PCR analyses validated differential expression of multiple genes encoding outer membrane proteins, including adhesins (SabA and HopQ) and proteins involved in iron acquisition (FecA2 and FecA3). Transcript levels of sabA, hopA, and hopQ are increased under high-salt conditions, whereas transcript levels of fecA2 and fecA3 are decreased under high-salt conditions. Transcription of sabA, hopA, hopQ, and fecA3 is derepressed in an arsS mutant strain, but salt-responsive transcription of these genes is not mediated by the ArsRS two-component system, and the CrdRS and FlgRS two-component systems do not have any detectable effects on transcription of these genes. In summary, these data provide a comprehensive view of H. pylori transcriptional alterations that occur in response to high-salt environmental conditions.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Cloreto de Sódio/metabolismo , Transcrição Genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Regulação Bacteriana da Expressão Gênica , Infecções por Helicobacter/microbiologia , Humanos , Óperon , Regulação para Cima
6.
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
7.
PLoS One ; 11(11): e0167068, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27875572

RESUMO

Acinetobacter baumannii is an opportunistic Gram-negative pathogen that causes a wide range of infections including pneumonia, septicemia, necrotizing fasciitis and severe wound and urinary tract infections. Analysis of A. baumannii representative strains grown in Chelex 100-treated medium for hemolytic activity demonstrated that this pathogen is increasingly hemolytic to sheep, human and horse erythrocytes, which interestingly contain increasing amounts of phosphatidylcholine in their membranes. Bioinformatic, genetic and functional analyses of 19 A. baumannii isolates showed that the genomes of each strain contained two phosphatidylcholine-specific phospholipase C (PC-PLC) genes, which were named plc1 and plc2. Accordingly, all of these strains were significantly hemolytic to horse erythrocytes and their culture supernatants tested positive for PC-PLC activity. Further analyses showed that the transcriptional expression of plc1 and plc2 and the production of phospholipase and thus hemolytic activity increased when bacteria were cultured under iron-chelation as compared to iron-rich conditions. Testing of the A. baumannii ATCC 19606T plc1::aph-FRT and plc2::aph isogenic insertion derivatives showed that these mutants had a significantly reduced PC-PLC activity as compared to the parental strain, while testing of plc1::ermAM/plc2::aph demonstrated that this double PC-PLC isogenic mutant expressed significantly reduced cytolytic and hemolytic activity. Interestingly, only plc1 was shown to contribute significantly to A. baumannii virulence using the Galleria mellonella infection model. Taken together, our data demonstrate that both PLC1 and PLC2, which have diverged from a common ancestor, play a concerted role in hemolytic and cytolytic activities; although PLC1 seems to play a more critical role in the virulence of A. baumannii when tested in an invertebrate model. These activities would provide access to intracellular iron stores this pathogen could use during growth in the infected host.


Assuntos
Infecções por Acinetobacter/enzimologia , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/patogenicidade , Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Fosfolipases Tipo C/metabolismo , Células A549 , Infecções por Acinetobacter/genética , Acinetobacter baumannii/genética , Animais , Proteínas de Bactérias/genética , Bovinos , Modelos Animais de Doenças , Cavalos , Humanos , Ovinos , Fosfolipases Tipo C/genética
8.
Infect Immun ; 84(12): 3338-3349, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27620719

RESUMO

Epidemiologic studies have provided conflicting data regarding an association between Helicobacter pylori infection and iron deficiency anemia (IDA) in humans. Here, a Mongolian gerbil model was used to investigate a potential role of H. pylori infection, as well as a possible role of diet, in H. pylori-associated IDA. Mongolian gerbils (either H. pylori infected or uninfected) received a normal diet or one of three diets associated with increased H. pylori virulence: high-salt, low-iron, or a combination of a high-salt and low-iron diet. In an analysis of all infected animals compared to uninfected animals (independent of diet), H. pylori-infected gerbils had significantly lower hemoglobin values than their uninfected counterparts at 16 weeks postinfection (P < 0.0001). The mean corpuscular volume (MCV) and serum ferritin values were significantly lower in H. pylori-infected gerbils than in uninfected gerbils, consistent with IDA. Leukocytosis and thrombocytosis were also detected in infected gerbils, indicating the presence of a systemic inflammatory response. In comparison to uninfected gerbils, H. pylori-infected gerbils had a higher gastric pH, a higher incidence of gastric ulcers, and a higher incidence of fecal occult blood loss. Anemia was associated with the presence of gastric ulceration but not gastric cancer. Infected gerbils consuming diets with a high salt content developed gastric ulcers significantly more frequently than gerbils consuming a normal-salt diet, and the lowest hemoglobin levels were in infected gerbils consuming a high-salt/low-iron diet. These data indicate that H. pylori infection can cause IDA and that the composition of the diet influences the incidence and severity of H. pylori-induced IDA.


Assuntos
Anemia Ferropriva/etiologia , Ração Animal/análise , Infecções por Helicobacter/complicações , Helicobacter pylori/patogenicidade , Úlcera Gástrica/microbiologia , Anemia Ferropriva/prevenção & controle , Animais , Dieta , Gerbillinae , Infecções por Helicobacter/microbiologia , Inflamação/etiologia , Inflamação/patologia , Úlcera Gástrica/prevenção & controle
9.
Infect Immun ; 83(4): 1354-65, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25605767

RESUMO

Acinetobacter baumannii is a Gram-negative opportunistic nosocomial pathogen that causes pneumonia and soft tissue and systemic infections. Screening of a transposon insertion library of A. baumannii ATCC 19606T resulted in the identification of the 2010 derivative, which, although capable of growing well in iron-rich media, failed to prosper under iron chelation. Genetic, molecular, and functional assays showed that 2010's iron utilization-deficient phenotype is due to an insertion within the 3' end of secA, which results in the production of a C-terminally truncated derivative of SecA. SecA plays a critical role in protein translocation through the SecYEG membrane channel. Accordingly, the secA mutation resulted in undetectable amounts of the ferric acinetobactin outer membrane receptor protein BauA while not affecting the production of other acinetobactin membrane protein transport components, such as BauB and BauE, or the secretion of acinetobactin by 2010 cells cultured in the presence of subinhibitory concentrations of the synthetic iron chelator 2,2'-dipyridyl. Outer membrane proteins involved in nutrient transport, adherence, and biofilm formation were also reduced in 2010. The SecA truncation also increased production of 30 different proteins, including proteins involved in adaptation/tolerance responses. Although some of these protein changes could negatively affect the pathobiology of the 2010 derivative, its virulence defect is mainly due to its inability to acquire iron via the acinetobactin-mediated system. These results together indicate that although the C terminus of the A. baumannii ATCC 19606T SecA is not essential for viability, it plays a critical role in the production and translocation of different proteins and virulence.


Assuntos
Acinetobacter baumannii/patogenicidade , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Canais Iônicos/genética , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , 2,2'-Dipiridil/química , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Adenosina Trifosfatases/genética , Animais , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Imidazóis/metabolismo , Canais Iônicos/metabolismo , Ferro/química , Proteínas de Membrana Transportadoras/genética , Mariposas/microbiologia , Mutação , Oxazóis/metabolismo , Transporte Proteico/genética , Transporte Proteico/fisiologia , Canais de Translocação SEC , Fatores de Virulência/genética
10.
Infect Immun ; 81(9): 3382-94, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23817614

RESUMO

Acinetobacter baumannii is an opportunistic pathogen that causes severe nosocomial infections. Strain ATCC 19606(T) utilizes the siderophore acinetobactin to acquire iron under iron-limiting conditions encountered in the host. Accordingly, the genome of this strain has three tonB genes encoding proteins for energy transduction functions needed for the active transport of nutrients, including iron, through the outer membrane. Phylogenetic analysis indicates that these tonB genes, which are present in the genomes of all sequenced A. baumannii strains, were acquired from different sources. Two of these genes occur as components of tonB-exbB-exbD operons and one as a monocistronic copy; all are actively transcribed in ATCC 19606(T). The abilities of components of these TonB systems to complement the growth defect of Escherichia coli W3110 mutants KP1344 (tonB) and RA1051 (exbBD) under iron-chelated conditions further support the roles of these TonB systems in iron acquisition. Mutagenesis analysis of ATCC 19606(T) tonB1 (subscripted numbers represent different copies of genes or proteins) and tonB2 supports this hypothesis: their inactivation results in growth defects in iron-chelated media, without affecting acinetobactin biosynthesis or the production of the acinetobactin outer membrane receptor protein BauA. In vivo assays using Galleria mellonella show that each TonB protein is involved in, but not essential for, bacterial virulence in this infection model. Furthermore, we observed that TonB2 plays a role in the ability of bacteria to bind to fibronectin and to adhere to A549 cells by uncharacterized mechanisms. Taken together, these results indicate that A. baumannii ATCC 19606(T) produces three independent TonB proteins, which appear to provide the energy-transducing functions needed for iron acquisition and cellular processes that play a role in the virulence of this pathogen.


Assuntos
Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/patogenicidade , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Acinetobacter baumannii/genética , Sequência de Aminoácidos , Transporte Biológico Ativo , Linhagem Celular , Transferência de Energia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genes Bacterianos , Humanos , Ferro/metabolismo , Dados de Sequência Molecular , Óperon/genética , Filogenia , Alinhamento de Sequência , Virulência/genética
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