A Tale of Two Toxins: Helicobacter Pylori CagA and VacA Modulate Host Pathways that Impact Disease

Helicobacter pylori is a pathogenic bacterium that colonizes more than 50% of the world's population, which leads to a tremendous medical burden. H. pylori infection is associated with such varied diseases as gastritis, peptic ulcers, and two forms of gastric cancer: gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. This association represents a novel paradigm for cancer development; H. pylori is currently the only bacterium to be recognized as a carcinogen. Therefore, a significant amount of research has been conducted to identify the bacterial factors and the deregulated host cell pathways that are responsible for the progression to more severe disease states. Two of the virulence factors that have been implicated in this process are cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA), which are cytotoxins that are injected and secreted by H. pylori, respectively. Both of these virulence factors are polymorphic and affect a multitude of host cellular pathways. These combined facts could easily contribute to differences in disease severity across the population as various CagA and VacA alleles differentially target some pathways. Herein we highlight the diverse types of cellular pathways and processes targeted by these important toxins

A Single Nucleotide Change Affects Fur-Dependent Regulation of sodB in H. pylori

Helicobacter pylori is a significant human pathogen that has adapted to survive the many stresses found within the gastric environment. Superoxide Dismutase (SodB) is an important factor that helps H. pylori combat oxidative stress. sodB was previously shown to be repressed by the Ferric Uptake Regulator (Fur) in the absence of iron (apo-Fur regulation) [1]. Herein, we show that apo regulation is not fully conserved among all strains of H. pylori. apo-Fur dependent changes in sodB expression are not observed under iron deplete conditions in H. pylori strains G27, HPAG1, or J99. However, Fur regulation of pfr and amiE occurs as expected. Comparative analysis of the Fur coding sequence between G27 and 26695 revealed a single amino acid difference, which was not responsible for the altered sodB regulation. Comparison of the sodB promoters from G27 and 26695 also revealed a single nucleotide difference within the predicted Fur binding site. Alteration of this nucleotide in G27 to that of 26695 restored apo-Fur dependent sodB regulation, indicating that a single base difference is at least partially responsible for the difference in sodB regulation observed among these H. pylori strains. Fur binding studies revealed that alteration of this single nucleotide in G27 increased the affinity of Fur for the sodB promoter. Additionally, the single base change in G27 enabled the sodB promoter to bind to apo-Fur with affinities similar to the 26695 sodB promoter. Taken together these data indicate that this nucleotide residue is important for direct apo-Fur binding to the sodB promoter

Helicobacter pylori Adapts to Chronic Infection and Gastric Disease via pH-Responsive BabA-Mediated Adherence

International audienceThe BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease

Expanding the Helicobacter pylori Genetic Toolbox: Modification of an Endogenous Plasmid for Use as a Transcriptional Reporter and Complementation Vector▿

Helicobacter pylori is an important human pathogen. However, the study of this organism is often limited by a relative shortage of genetic tools. In an effort to expand the methods available for genetic study, an endogenous H. pylori plasmid was modified for use as a transcriptional reporter and as a complementation vector. This was accomplished by addition of an Escherichia coli origin of replication, a kanamycin resistance cassette, a promoterless gfpmut3 gene, and a functional multiple cloning site to form pTM117. The promoters of amiE and pfr, two well-characterized Fur-regulated promoters, were fused to the promoterless gfpmut3, and green fluorescent protein (GFP) expression of the fusions in wild-type and Δfur strains was analyzed by flow cytometry under iron-replete and iron-depleted conditions. GFP expression was altered as expected based on current knowledge of Fur regulation of these promoters. RNase protection assays were used to determine the ability of this plasmid to serve as a complementation vector by analyzing amiE, pfr, and fur expression in wild-type and Δfur strains carrying a wild-type copy of fur on the plasmid. Proper regulation of these genes was restored in the Δfur background under high- and low-iron conditions, signifying complementation of both iron-bound and apo Fur regulation. These studies show the potential of pTM117 as a molecular tool for genetic analysis of H. pylori

Role of the −5 bp in <i>sodB</i> regulation.

<p>WT G27, WT 26695, and the “−5 bp swap” strain were grown as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005369#s2" target="_blank">Materials and Methods</a>, and RNA was isolated under iron replete and iron-depletion shock conditions. RPAs were performed on RNA isolated from 4 biologically independent experiments using <i>sodB</i> and <i>pfr</i> riboprobes. Data from <i>sodB</i> RPAs are presented in Panel A, and data from <i>pfr</i> RPAs are presented in Panel B. Each square, diamond, triangle, and circle represent the average fold decrease calculated from three technical repeats with each independent set of RNA for each strain and growth condition combination. Median fold decrease is represented as a bar for each combination, and the dotted-dashed line represents the 2-fold significance cut-off. ^*p-value of 0.0001. ^#p-value of 0.006.</p

Flow Cytometry analysis of <i>sodB</i> and <i>pfr</i> GFP reporters.

<p>Strains bearing <i>sodB::gfpmut3</i> or <i>pfr::gfpmut3</i> promoter fusions were grown overnight in either iron replete or iron depleted media. Changes in fluorescence were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005369#s2" target="_blank">Materials and Methods</a>. Results for the <i>sodB</i> promoter fusions are displayed in Panel 1A, and results for the <i>pfr</i> promoter fusions are displayed in Panel 1B. For both A and B, solid lines indicate the plasmid in WT <i>H. pylori</i> G27 grown in iron replete conditions, dotted lines indicate the plasmid in WT bacteria grown in iron deplete conditions, and dashed lines indicate the plasmid in Δ<i>fur</i> bacteria grown in iron replete conditions. Fluorescence is measured in relative units, and the data are representative of multiple independent flow analyses.</p

Polymorphism in the CagA EPIYA Motif Impacts Development of Gastric Cancer▿ §

Helicobacter pylori causes diseases ranging from gastritis to peptic ulcer disease to gastric cancer. Geographically, areas with high incidences of H. pylori infection often overlap with areas with high incidences of gastric cancer, which remains one of the leading causes of cancer-related deaths worldwide. Strains of H. pylori that carry the virulence factor cytotoxin-associated gene A (cagA) are much more likely to be associated with the development of gastric cancer. Moreover, particular C-terminal polymorphisms in CagA vary by geography and have been suggested to influence disease development. We conducted a large-scale molecular epidemiologic analysis of South Korean strains and herein report a statistical link between the East Asian CagA EPIYA-ABD genotype and the development of gastric cancer. Characterization of a subset of the Korean isolates showed that all strains from cancer patients expressed and delivered phosphorylatable CagA to host cells, whereas the presence of the cagA gene did not strictly correlate to expression and delivery of CagA in all noncancer strains