VacA is a channel-forming toxin unrelated to other known bacterial toxins. Most
strains contain a
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
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.
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
gene expression. In this study, we utilized transcriptome sequencing (RNA-seq) methodology to compare the transcriptional profiles of
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
,
, and
are increased under high-salt conditions, whereas transcript levels of
and
are decreased under high-salt conditions. Transcription of
,
,
, and
is derepressed in an
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
transcriptional alterations that occur in response to high-salt environmental conditions.
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.
CagA is a secreted effector protein that contributes to gastric carcinogenesis. Previous studies showed that there is variation among
strains in the steady-state levels of CagA and that a ...strain-specific motif downstream of the
transcriptional start site (the +59 motif) is associated with both high levels of CagA and premalignant gastric histology. The
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
transcript levels and
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
transcript and the CagA protein. Additionally, these mutations resulted in a decreased
mRNA half-life. Mutagenesis of the +59 motif without altering the stem-loop structure resulted in reduced steady-state
transcript and CagA protein levels but did not affect
transcript stability.
transcript stability was not affected by increased sodium chloride concentrations, an environmental factor known to augment
transcript levels and CagA protein levels. These results indicate that both a predicted stem-loop structure and a strain-specific +59 motif in the
5' untranslated region influence the levels of
expression.
The
Cag type IV secretion system (T4SS) translocates the effector protein CagA and nonprotein bacterial constituents into host cells. In this study, we infected Mongolian gerbils with an
strain in ...which expression of the
operon (required for Cag T4SS activity) is controlled by a TetR/
system. Transcript levels of
were significantly higher in gastric tissue from
-infected animals receiving doxycycline-containing chow (to derepress Cag T4SS activity) than in tissue from infected control animals receiving drug-free chow. At 3 months postinfection, infected animals receiving doxycycline had significantly increased gastric inflammation compared to infected control animals. Dysplasia (a premalignant histologic lesion) and/or invasive gastric adenocarcinoma were detected only in infected gerbils receiving doxycycline, not in infected control animals. We then conducted experiments in which Cag T4SS activity was derepressed during defined stages of infection. Continuous Cag T4SS activity throughout a 3-month time period resulted in higher rates of dysplasia and/or gastric cancer than observed when Cag T4SS activity was limited to early or late stages of infection. Cag T4SS activity for the initial 6 weeks of infection was sufficient for the development of gastric inflammation at the 3-month time point, with gastric cancer detected in a small proportion of animals. These experimental results, together with previous studies of
mutant strains, provide strong evidence that Cag T4SS activity contributes to gastric carcinogenesis and help to define the stages of
infection during which Cag T4SS activity causes gastric alterations relevant for cancer pathogenesis.
The "hit-and-run model" of carcinogenesis proposes that an infectious agent triggers carcinogenesis during initial stages of infection and that the ongoing presence of the infectious agent is not required for development of cancer.
infection and actions of CagA (an effector protein designated a bacterial oncoprotein, secreted by the Cag T4SS) are proposed to constitute a paradigm for hit-and-run carcinogenesis. In this study, we report the development of methods for controlling
Cag T4SS activity
and demonstrate that Cag T4SS activity contributes to gastric carcinogenesis. We also show that Cag T4SS activity during an early stage of infection is sufficient to initiate a cascade of cellular alterations leading to gastric inflammation and gastric cancer at later time points.
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.DesignWhole 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.ResultsA 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).ConclusionThese results indicate that specific genetic variation arises within H. pylori strains during in vivo adaptation to conditions conducive for gastric carcinogenesis.
Stagnation in antimicrobial development has led to a serious threat to public health because some
infections have become untreatable. New therapeutics with alternative mechanisms of action to combat
...are therefore necessary to treat these infections. To this end, the virulence of
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
virulence associated with hemolysis, cytolysis of A549 human alveolar epithelial cells, and increased mortality in the
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
strains. Additionally, scanning electron microscopy of polarized A549 cells infected with bacteria of the
ATCC 19606
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
infections, wherein miltefosine treatment of animals infected with ATCC 19606
significantly decreased mortality. These data demonstrate that inhibition of phospholipase C activity results in the overall reduction of
virulence in both
and
models, making miltefosine a viable option for the treatment of
infections, particularly those caused by multidrug-resistant isolates.
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.
Helicobacter pylori colonization of the stomach is a strong risk factor for the development of stomach cancer and peptic ulcer disease. In this study, we tested the hypothesis that H. pylori ...infection triggers alterations in gastric lipid composition. Mongolian gerbils were experimentally infected with H. pylori for 3 months. Conventional histologic staining revealed mucosal inflammation in stomachs from the H. pylori-infected animals but not in stomachs from uninfected control animals. Atrophic gastritis (a premalignant condition characterized by loss of corpus-specific parietal and chief cells), gastric mucosal hyperplasia, dysplasia, and/or gastric cancer were detected in stomachs from several infected animals. We then used imaging mass spectrometry to analyze the relative abundance and spatial distribution of gastric lipids. We detected ions corresponding to 36 distinct lipids that were differentially abundant when comparing gastric tissues from H. pylori-infected animals with tissues from uninfected animals. Liquid chromatography-tandem mass spectrometry analysis of lipid extracts from homogenized gastric tissues provided additional supportive evidence for the identification of several differentially abundant lipids. Sixteen of the differentially abundant lipids were localized mainly to the gastric corpus in stomachs from uninfected animals and were markedly reduced in abundance in stomachs from H. pylori-infected animals with severe disease (atrophic gastritis and dysplasia or gastric cancer). These findings indicate that H. pylori infection can lead to alterations in gastric lipid composition and constitute a new approach for identifying biomarkers of gastric atrophy and premalignant changes.
H. pylori colonization of the stomach triggers a cascade of gastric alterations that can potentially culminate in stomach cancer. The molecular alterations that occur in gastric tissue prior to development of stomach cancer are not well understood. We demonstrate here that H. pylori-induced premalignant changes in the stomach are accompanied by extensive alterations in gastric lipid composition. These alterations are predicted to have important functional consequences relevant to H. pylori-host interactions and the pathogenesis of gastric cancer.
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