Cortactin Promotes Effective AGS Cell Scattering by Helicobacter pylori CagA, but Not Cellular Vacuolization and Apoptosis Induced by the Vacuolating Cytotoxin VacA

Cortactin is an actin-binding protein and actin-nucleation promoting factor regulating cytoskeletal rearrangements in eukaryotes. Helicobacter pylori is a gastric pathogen that exploits cortactin to its own benefit. During infection of gastric epithelial cells, H. pylori hijacks multiple cellular signaling pathways, leading to the disruption of key cell functions. Two bacterial virulence factors play important roles in this scenario, the vacuolating cytotoxin VacA and the translocated effector protein CagA of the cag type IV secretion system (T4SS). Specifically, by overruling the phosphorylation status of cortactin, H. pylori alternates the activity of molecular interaction partners of this important protein, thereby manipulating the performance of cytoskeletal rearrangements, endosomal trafficking and cell movement. Based on shRNA knockdown and other studies, it was previously reported that VacA utilizes cortactin for its cellular uptake, intracellular travel and induction of apoptosis by a mitochondria-dependent mechanism, while CagA induces cell scattering, motility and elongation. To investigate the role of cortactin in these phenotypes in more detail, we produced a complete knockout mutant of cortactin in the gastric adenocarcinoma cell line AGS by CRISPR-Cas9. These cells were infected with H. pylori wild-type or various isogenic mutant strains. Unexpectedly, cortactin deficiency did not prevent the uptake and formation of VacA-dependent vacuoles, nor the induction of apoptosis by internalized VacA, while the induction of T4SS- and CagA-dependent AGS cell movement and elongation were strongly reduced. Thus, we provide evidence that cortactin is required for the function of internalized CagA, but not VacA.

Statins’ Regulation of the Virulence Factors of Helicobacter pylori and the Production of ROS May Inhibit the Development of Gastric Cancer

Conventionally, statins are used to treat high cholesterol levels. They exhibit pleiotropic effects, such as the prevention of cardiovascular disease and decreased cancer mortality. Gastric cancer (GC) is one of the most common cancers, ranking as the third leading global cause of cancer-related deaths, and is mainly attributed to chronic Helicobacter pylori infection. During their co-evolution with hosts, H. pylori has developed the ability to use the cellular components of the host to evade the immune system and multiply in intracellular niches. Certain H. pylori virulence factors, including cytotoxin-associated gene A (CagA), vacuolating cytotoxin A (VacA), and cholesterol-α-glucosyltransferase (CGT), have been shown to exploit host cholesterol during pathogenesis. Therefore, using statins to antagonize cholesterol synthesis might prove to be an ideal strategy for reducing the occurrence of H. pylori-related GC. This review discusses the current understanding of the interplay of H. pylori virulence factors with cholesterol and reactive oxygen species (ROS) production, which may prove to be novel therapeutic targets for the development of effective treatment strategies against H. pylori-associated GC. We also summarize the findings of several clinical studies on the association between statin therapy and the development of GC, especially in terms of cancer risk and mortality.

VARIABILITY OF NEUTROPHIL-ACTIVATING PROTEIN AMONG HELICOBACTER PYLORI STRAINS

The Helicobacter pylori neutrophil activating protein (NAP) presents relevant inflammatory and immunomodulatory activity and has consequently been explored as a diagnosis and therapeutic target. In the present work, nap gene sequences, retrieved from H. pylori isolated world-wide, were analyzed, a high genetic diversity (with 88% of alleles) being observed in accordance with other virulence factors. The phylogenetic analysis did not reveal the separation of strains per geographical region according to a bacterial panmictic population. When compared to other genes of virulence factors of H. pylori, such as the vacuolating cytotoxin A (vacA), nap presents slightly lower genetic variability, concerning the number of alleles and polymorphic sites, pointing to a possible lower pressure of the host immune system. The nap genetic diversity is associated to a high proportion of synonymous substitutions in relation to non-synonymous substitutions, pointing to equilibrium between the need for antigenic diversity as a mechanism to escape the host immune system and the maintenance of the proteins function. All this information could be put to good use when planning the NAP application as a therapeutic or diagnostic target.

Campylobacter jejuni Cytolethal Distending Toxin C Exploits Lipid Rafts to Mitigate Helicobacter pylori-Induced Pathogenesis

Helicobacter pylori infection is associated with several gastrointestinal diseases, including gastritis, peptic ulcer, and gastrointestinal adenocarcinoma. Two major cytotoxins, vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA), interact closely with lipid rafts, contributing to H. pylori-associated disease progression. The Campylobacter jejuni cytolethal distending toxin consists of three subunits: CdtA, CdtB, and CdtC. Among them, CdtA and CdtC bind to membrane lipid rafts, which is crucial for CdtB entry into cells. In this study, we employed recombinant CdtC (rCdtC) to antagonize the functions of H. pylori cytotoxin in cells. Our results showed that rCdtC alleviates cell vacuolation induced by H. pylori VacA. Furthermore, rCdtC reduces H. pylori CagA translocation, which decreases nuclear factor kappa-B activation and interleukin-8 production, resulting in the mitigation of gastric epithelial cell inflammation. These results reveal that CdtC hijacks cholesterol to compete for H. pylori cytotoxin actions via lipid rafts, ameliorating H. pylori-induced pathogenesis.

Significance of pathogenicity factors in initiation of immune response in Helicobacter pylori infection

Helicobacter pylori is a unique microorganism capable of long-term colonization of the gastric mucosa, induction of the inflammatory process, antigenic mimicry and immune evasia. Flagella proteins, adhesins, invasive and aggressive enzymes, cytotoxin-associated protein, vacuolating cytotoxin can have a damaging effect on stomach epithelial cells. Recognition of molecular patterns of Helicobacter pylori by stomach cell receptors initiates activation of adapter proteins, protein kinases and transcription factors, leading to the production of proinflammatory cytokines, infiltration by neutrophilic granulocytes, absorption and killing of microorganisms by phagocytes with presentation of antigens to lymphocytes, while the activity and completeness of phagocytosis remain at a low level. Activation of CD8+-, CD16+- lymphocytes is accompanied by cytotoxic effect on both Helicobacter pylori and epithelial cells of the gastric mucosa. Weak immunogenicity of Helicobacter pylori antigens limits the production of anti-Helicobacter antibodies. Thus, activation of immune factors, in most cases, does not lead to complete elimination of the pathogen, but can aggravate the pathomorphological changes of the gastric epithelium.

Association of Combined Sero-Positivity to Helicobacter pylori and Streptococcus gallolyticus with Risk of Colorectal Cancer

Previously, we found that risk of colorectal cancer (CRC) is increased in individuals with serum antibody response to both Helicobacter pylori (HP) Vacuolating Cytotoxin (VacA) toxin or Streptococcus gallolyticus (SGG) pilus protein Gallo2178. In the present analysis, we tested the hypothesis that combined seropositivity to both antigens is a better indicator of CRC risk than seropositivity to single antigens. We used multiplex serologic assays to analyze pre-diagnostic serum for antibody responses from 4063 incident CRC cases and 4063 matched controls from 10 US cohorts. To examine whether combined SGG Gallo2178 and HP VacA sero-status was associated with CRC risk, we used conditional logistic regression models to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Compared to dual sero-negative individuals, there was no increased risk for individuals sero-positive to SGG Gallo2178 only (OR: 0.93; 95% CI: 0.66–1.31) or to HP VacA only (OR: 1.08; 95% CI: 0.98–1.19). However, dual sero-positive individuals had a >50% increased odds of developing CRC (OR: 1.54; 95% CI: 1.16–2.04), suggesting an interaction between antibody responses to these two pathogens and CRC risk (pinteraction = 0.06). In conclusion, this study suggests that dual sero-positivity to HP VacA and SGG Gallo2178 is an indicator of increased risk of CRC.