VacA Toxin Research Articles

Taurine modulates host cell responses to Helicobacter pylori VacA toxin.

Colonization of the human stomach with Helicobacter pylori strains producing active forms of the secreted toxin VacA is associated with an increased risk of peptic ulcer disease and gastric cancer, compared with colonization with strains producing hypoactive forms of VacA. Previous studies have shown that active s1m1 forms of VacA cause cell vacuolation and mitochondrial dysfunction. In this study, we sought to define the cellular metabolic consequences of VacA intoxication. Untargeted metabolomic analyses revealed that several hundred metabolites were significantly altered in VacA-treated gastroduodenal cells (AGS and AZ-521) compared with control cells. Pathway analysis suggested that VacA caused alterations in taurine and hypotaurine metabolism. Treatment of cells with the purified active s1m1 form of VacA, but not hypoactive s2m1 or Δ6-27 VacA-mutant proteins (defective in membrane channel formation), caused reductions in intracellular taurine and hypotaurine concentrations. Supplementation of the tissue culture medium with taurine or hypotaurine protected AZ-521 cells against VacA-induced cell death. Untargeted global metabolomics of VacA-treated AZ-521 cells or AGS cells in the presence or absence of extracellular taurine showed that taurine was the main intracellular metabolite significantly altered by extracellular taurine supplementation. These results indicate that VacA causes alterations in cellular taurine metabolism and that repletion of taurine is sufficient to attenuate VacA-induced cell death. We discuss these results in the context of previous literature showing the important role of taurine in cell physiology and the pathophysiology or treatment of multiple pathologic conditions, including gastric ulcers, cardiovascular disease, malignancy, inflammatory diseases, and other aging-related disorders.

An Immunoinformatic Approach to Designing a Multi-epitope Vaccine against Helicobacter pylori with the VacA Toxin and BabA Adhesion

Objective: Helicobacter pylori, as a carcinogen, has been related to the development of gastric cancer, particularly in developing countries. The main challenge with therapy is the recurrence of antibiotic-resistant bacteria, and vaccination is still a problem. Therefore, the objective of the current study was to rationally design a multi-epitope vaccine using two immunogenic proteins found in H. pylori. Methods: Promising epitopes for the Leb-binding adhesin A (BabA) and vacuolating cytotoxin (VacA) proteins were characterized through an immunoinformatics approach. Epitope-rich fragments were selected based on high-binding affinities with HLA classes I and II to be specifically presented to B and T lymphocytes and to selectively elicit both humoral and cellular immune responses. Results: Six constructs were planned by fusing these fragments in different arrangements with the help of GPGPG linkers. The most stable three-dimensional structure was found in Construct 6 during molecular dynamics. To improve immunogenicity and stability, an adjuvant called human β- defensin 2 (hBD-2) was joined to the N-terminus of Construct 6. Following molecular docking, the final vaccine reacted appropriately with each toll-like receptor 2 (TLR-2), TLR3, and TLR-4. The final DNA sequence was optimized for expression in E. coli K12 and in silico cloned into a pET-28a(+) plasmid. As a result of the vaccination in silico, substantial responses were developed against H. pylori. Conclusion: According to the immune response simulation, activated B and T lymphocytes and memory cell production increased. Macrophages and dendritic cells proliferated continuously, and IFN-γ and Cytokines, such as IL-2 were raised.

New strategies for Helicobacter pylori isolation and sequencing analysis for virulence genes contributing to its pathogenicity.

Helicobacter pylori is a fastidious pathogen that is required a complicated medium for growth. Invading epithelial cells of the stomach. H. pylori virulence factors are classified by function, acidic resistivity, adhesion, chemotaxis and motility, molecular mimicry, immunological invasion and modulation, and toxins formation such as cytotoxin-associated genes A (cagA) and vacuolating cytotoxin A (vacA). This study aims to determine a simple and innovative technique to isolate H. pylori from gastric biopsies and assess pathogenicity by virulence factor gene detection. A total of 200 patients who were suspected of having H. pylori infection had two antral gastric biopsies undertaken. A rapid urease test (RUT) was used for one, and Brain Heart Infusion broth (BHI) was used to cultivate the other. The molecular study included diagnostics utilizing the 16sRNA housekeeping gene along with the identification of the virulence factors genes (cagA, cagT, and vacA) and sequencing, RESULT: Of the 200 antral gastric biopsies collected, 135 were positive rapid urease tests, and 17 H. pylori isolates were successfully obtained from 135 biopsies. The 16SrRNA as a housekeeping gene is confirmed, and about 53%, 70.5%, and 82.3% of the 17 isolates show carrying cagA, cagT, and vacA genes, respectively. All peptic ulcer isolates have the cagA gene, while Gastroesophageal Reflux Disease (GERD) and non-peptic ulcer disease (NPUD) isolates show the lack of the cagA gene. All bacteria, which were isolated from peptic ulcer, nodular gastritis, and gastritis patients, have a vacA gene. The effective method for isolating H. pylori is centrifuging the transport broth after 24h of incubation. The cagA toxin causes peptic ulcer while vacA toxin induces several histopathological changes in the stomach. Three virulence genes were present in all peptic ulcer-causing bacteria, while only one or none were present in the GERD and NPUD biopsy isolates.

Structural Analysis of Membrane-associated Forms of Helicobacter pylori VacA Toxin

Helicobacter pylori colonizes the stomach in about half of the human population, leading to an increased risk of peptic ulcer disease and gastric cancer. H. pylori secretes an 88 kDa VacA toxin that contributes to pathogenesis. VacA assembles into oligomeric complexes in solution and forms anion-selective channels in cell membranes. Cryo-electron microscopy (cryo-EM) analyses of VacA oligomers in solution provided insights into VacA oligomerization but failed to reveal the structure of the hydrophobic N-terminal region predicted to be a pore-forming domain. In this study, we incubated VacA with liposomes and used single particle cryo-EM to analyze detergent-extracted VacA oligomers. A 3D structure of detergent-solubilized VacA hexamers revealed the presence of six α-helices extending from the center of the oligomers, a feature not observed in previous studies of water-soluble VacA oligomers. Cryo-electron tomography analysis and 2D averages of VacA associated with liposomes confirmed that central regions of the membrane-associated VacA oligomers can insert into the lipid bilayer. However, insertion is heterogenous, with some membrane-associated oligomers appearing only partially inserted and others sitting on top of the bilayer. These studies indicate that VacA undergoes a conformational change when contacting the membrane and reveal an α-helical region positioned to extend into the membrane. Although the reported VacA 3D structure does not represent a selective anion channel, our combined single particle 3D analysis, cryo-electron tomography, and modeling allow us to propose a model for the structural organization of the VacA N-terminus in the context of a hexamer as it inserts into the membrane.

There Are No Insurmountable Barriers: Passage of the Helicobacter pylori VacA Toxin from Bacterial Cytoplasm to Eukaryotic Cell Organelle.

The Gram-negative bacterium Helicobacter pylori is a very successful pathogen, one of the most commonly identified causes of bacterial infections in humans worldwide. H. pylori produces several virulence factors that contribute to its persistence in the hostile host habitat and to its pathogenicity. The most extensively studied are cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA). VacA is present in almost all H. pylori strains. As a secreted multifunctional toxin, it assists bacterial colonization, survival, and proliferation during long-lasting infections. To exert its effect on gastric epithelium and other cell types, VacA undergoes several modifications and crosses multiple membrane barriers. Once inside the gastric epithelial cell, VacA disrupts many cellular-signaling pathways and processes, leading mainly to changes in the efflux of various ions, the depolarization of membrane potential, and perturbations in endocytic trafficking and mitochondrial function. The most notable effect of VacA is the formation of vacuole-like structures, which may lead to apoptosis. This review focuses on the processes involved in VacA secretion, processing, and entry into host cells, with a particular emphasis on the interaction of the mature toxin with host membranes and the formation of transmembrane pores.

Computational insights into VacA toxin inhibition: harnessing FDA-approved antibiotics against Helicobacter pylori

Cancer is a condition in which a few of the body’s cells grow beyond its control and spread to other outward regions. Globally, gastric cancer (GC) is third most common cause of cancer-related mortality and the fourth most common kind of cancer. Persistent infection of VacA-positive Helicobacter pylori (H. pylori) modulates cellular physiology and leads to GC. About ∼70% of H. pylori are positive for vacuolating cytotoxin-A (VacA), and it infects ∼80–90% of world populations. Herein, for first time, we repurposed FDA-approved gram-negative antibiotics, which are feasible alternatives to existing regimens and may be used in combinatorial treatment against VacA-positive H. pylori. Out of 110 FDA-approved antibiotics, we retrieved 92 structures, which were screened against the VacA protein. Moreover, we determined that the top eight hit antibiotics viz; cefpiramide, cefiderocol, eravacycline, doxycycline, ceftriaxone, enoxacin, tedizolid, and cefamandole show binding free energies of −9.1, −8.9, −8.1, −8.0, −7.9, −7.8, −7.8 and −7.8 Kcal/mol, respectively, with VacA protein. Finally, we performed 100 ns duplicate MD simulations on the top eight selected antibiotics showing strong VacA binding. Subsequently, five antibiotics, including cefiderocol, cefpiramide, doxycycline, enoxacin, and tedizolid show stable ligand protein distance and good binding affinity revealed by the MM-PBSA scheme. Among the five antibiotics cefiderocol act as the most potent inhibitor (-28.33 kcal/mol). Furthermore, we also identified the hotspot residue like Asn-506, Tyr-529, and Phe-483 which control the interaction. Concisely, we identified antibiotics that can be repurposed against VacA of H. pylori and explored their molecular mechanism of interaction with VacA. Communicated by Ramaswamy H. Sarma

Helicobacter pylori strains and their relationship with vacuolating cytotoxin A gene in the increased risk of gastric cancer

ObjectivesThe main objective of this study is to analyze the relationship of H. pylori vacuolating gene in gastric cancer. MethodsH. pyroli strains were isolated from the biopsy specimens collected from 50 patients suspected of gastritis, gastric carcinoma and peptic ulcer disease. H. pylori JK was identified and the virulence gene was determined. The presence of vacuolating cytotoxin A gene was amplified using a polymerase chain reaction. The growth kinetics of the strain JK were evaluated up to 96 h at 37 °C and the culture was used for the determination of VacA gene expression. ResultsThe screened H. pylori JK was positive for vacA gene and showed heterogeneity among H. pylori. Bacterial growth increased up to 48 h incubation and declined after 48 h. The isolated strain showed maximum growth after 72 h incubation, whereas, the toxic protein reached maximum yield after 96 h incubation and was not associated with growth. It was resistant to antibiotics such as ceftriaxone, cefuroxime, cefalexin, and co-trimoxazole. The intracellular H. pylori culture extracts induced vacuolation in HeLa cell lines in vitro. The isolated H. pylori strain showed more than 43% vacuolating activity after 24 h incubation. Immunoblotting assay revealed the presence of an immuno-reactive protein band in the culture supernatant of H. pylori JK. These results indicated the pathogenic activity of the VacA gene and toxin production. ConclusionsGenerally, H. pylori persists for the lifetime of the individual, revealing the significance of eradication from the host.

Helicobacter pylori infection in Bamako (Mali): Polymorphism, diversity and prophage

Helicobacter pylori (Hp) is a bacteria associated with gastritis and gastric cancer (GC). Its prevalence is higher than 80% in Mali and affects the incidence of GC, which is a major cancer in the department of digestive surgery. VacA and CagA toxins are responsible of Hp strain polymorphism. With prophage, they are also associated with a region depending diversity. The aim of this study was to identify the variants of Hp strains and their prophage identity in Bamako (Mali). A total of 61 gastric biopsies were isolated in patients with gastric diseases. The extracted DNA was amplified by PCR targeting cagA, vacA (s, m) and phage integrase (int) genes. cagA and int amplicons were sequenced according to Sanger method. Hp was detected in 23 (37.7%) samples. A predominance of housewives, commercial workers, farmers and students was observed, confirming the importance of socio-economic conditions and the associated factors in Hp infection. Carcinogenic vacAs1m1 was the second dominant polymorph. This would explain the prevalence of Hp's GC in the country. The phylogenetic tree from cagA sequences has revealed the specific variants strain of Hp that causes gastritis in Mali (HpML). Their prophage integration protein shares more than 90% of its sequence with species of the Schmidvirus genus. Like the HpML probable strains, Malian’s Hp prophage (HpMLpΦ) is geographically clustered with hspWestAfrica1 (hpAfrica1). These results confirm the existence of probable Hp and bacteriophage specific strains to Mali and suggest that using the molecular diagnostics method for effective pathogen identification and rapid patient management.

A molecularly imprinted polymer-based electrochemical biosensor for detection of VacA virulence factor of H. pylori causing gastric cancer

A novel MIP-based electrochemical sensing platform was developed for the ultrasensitive detection of vacuolating cytotoxin A (VacA) toxin of H. pylori bacteria. VacA virulence factor is majorly involved in H. pylori-associated pathogenesis and plays a vital role in the establishment of bacteria inside host gastric cells. Herein, we have developed a SiO2 nanoparticle decorated imprinting polymer-based sensing platform on a screen printed electrode (SPE) that is attributed as a receptor of VacA protein. The molecularly imprinted polymer has been developed by a polymerization process directly over the screen-printed electrode using template VacA antigen and the performance of the fabricated electrode was studied electrochemically. Under the optimized experimental conditions the fabricated VacA-MIP/SiO2 @SPE sensor exhibits high sensitivity (0.304 mA ng−1 ml−1) and a very low limit of detection (0.01 ng ml−1) in a 0.01–100 ng ml−1 linear range. The influence of various other possible interferents in analytical response has also been observed with the successful determination of VacA antigen.

Assessment of the mixed origin of the gastric epithelial extracellular vesicles in acellular transfer of Helicobacter pylori toxins and a systematic review

BackgroundH. pylori are generally considered as extracellular organisms, with exclusive colonization of the gastric milieu. Yet, several extra gastric manifestations are associated with this infection. The aim of the present study was to investigate the feasibility of toxin transfer by extracellular vesicles, from bacterial and epithelial origins. MethodsTox-positive H. pylori and its two cagA and vacA mutant strains were used to produce bacterial vesicles (BVs) and to infect AGS cells. The produced BVs and the infected cell vesicles (ICVs) were collected by ultracentrifugation and evaluated by western blotting, DLS and electron microscopy. These two sets of vesicles were applied to a second set of recipient AGS cells, in which the acellular transfer of toxins, IL-8 production and downstream morphologic changes were assessed, by western blotting, ELISA and light microscopy, respectively. ResultsThe BVs were positive for H. pylori membrane markers (BabA and UreB), VacA and CagA toxins, except for from the corresponding mutant strains. The ICVs were larger in size and positive for bacterial markers, as well as epithelial markers of CD9, LGR5, but negative for nuclear (Ki76) or cytoplasmic (β-actin) markers. Bacteria-independent transfer of CagA and VacA into the recipient cells occurred upon treatment of cells with BVs and ICVs, followed by cellular vacuolation and elongation. IL-8 production was induced in recipient AGS cells, treated with BVs (1279.4 ± 19.79 pg/106 cells), early (8 h, 1171.4 ± 11.31 pg/106 cells) and late (48 h, 965.4 ± 36.77 pg/106 cells) ICVs (P < 0.0001). ConclusionOur data indicates that ICVs, with mixed bacterial and epithelial constituents, similar to BVs, are capable of transferring bacterial toxins into the recipient cells, inducing IL-8 production and subsequent morphologic changes, in an acellular manner.

Expression analysis of miRNA-155 level in Helicobacter pylori related inflammation and chronic gastritis.

Helicobacter pylori, is a major etiologic agent associated with gastritis. There is more evidence of noncoding microRNAs (miRs) dysregulation in gastrointestinal diseases, including inflammation caused by Helicobacter pylori. Also, the classification of gastrointestinal malignancies using the miRs profile is better than the protein profile. MiRNA-155(miRNA-155) among other miRs plays an important role in control of inflammation and gastric malignancy, so it can be remarkable prognosis marker of gastric cancer in the phase of chronic gastritis. The aim of this study was to compare the expression of miRNA-155 in gastric biopsy and serum samples of adult patients with chronic gastritis. Biopsy and blood samples were collected from endoscopy candidates at Taleghani hospital, Tehran, during 2019. H. pylori infection was detected using histology, culture and molecular PCR methods. Based on cagA and vacA genotyping, the toxicity of H. pylori isolates were determined. After RNA extraction, the expression rate of miRNA-155 was evaluated by real-time polymerase chain reaction (RT-PCR) in gastric tissue and serum of adults infected by H. pylori (n = 30) compared with control group without infection (n = 20). RNU6 housekeeping miRNA were used as endogenous control and statistical analyses were performed using SPSS, ANOVA and Student's t-test. miRNA-155 expression in H. pylori infected adult patients increased significantly by 5.61 and 10.11 fold in serum and tissue respectively, compared to that observed in the control group. Evaluation of miRNA-155 expression pattern in relation to bacterial virulence factors showed that the increase in miRNA-155 expression is independent of CagA and VacA toxins. According to the differential expression patterns of miRNA-155 in serum samples of the infected adult patients, miRNA-155 has the potential to evaluate as chronic gastritis marker.

Functional Properties of Oligomeric and Monomeric Forms of Helicobacter pylori VacA Toxin.

Helicobacter pylori VacA is a secreted toxin that assembles into water-soluble oligomeric structures and forms anion-selective membrane channels. Acidification of purified VacA enhances its activity in cell culture assays. Sites of protomer-protomer contact within VacA oligomers have been identified by cryoelectron microscopy, and in the current study, we validated several of these interactions by chemical cross-linking and mass spectrometry. We then mutated amino acids at these contact sites and analyzed the effects of the alterations on VacA oligomerization and activity. VacA proteins with amino acid charge reversals at interprotomer contact sites retained the capacity to assemble into water-soluble oligomers and retained cell-vacuolating activity. Introduction of paired cysteine substitutions at these sites resulted in formation of disulfide bonds between adjacent protomers. Negative-stain electron microscopy and single-particle two-dimensional class analysis revealed that wild-type VacA oligomers disassemble when exposed to acidic pH, whereas the mutant proteins with paired cysteine substitutions retain an oligomeric state at acidic pH. Acid-activated wild-type VacA caused vacuolation of cultured cells, whereas acid-activated mutant proteins with paired cysteine substitutions lacked cell-vacuolating activity. Treatment of these mutant proteins with both low pH and a reducing agent resulted in VacA binding to cells, VacA internalization, and cell vacuolation. Internalization of a nonoligomerizing mutant form of VacA by host cells was detected without a requirement for acid activation. Collectively, these results enhance our understanding of the molecular interactions required for VacA oligomerization and support a model in which toxin activity depends on interactions of monomeric VacA with host cells.

Construction and preservation of a stable and highly expressed recombinant Helicobacter pylori vacuolating cytotoxin A with apoptotic activity

BackgroundH. pylori is closely related to the occurrence and development of various digestive gastritis, peptic ulcer and mucosa-associated lymphoid tissue (MALT) lymphoma. H. pylori is also a class I carcinogen of gastric cancer. VacA is the only exocrine toxin of H. pylori, which plays a very important role in the pathogenesis of H. pylori. The production of VacA in natural circumstances is complex with heavy workload and low yield. Therefore, it is very important to obtain recombinant VacA protein which is stable and biologically active. This study therefore aims to explore the expression, purification and stable storage of VacA toxin of H. pylori in E.coli, and to provide experimental basis for further exploration of the role of VacA in H. pylori -induced inflammation of cancer.ResultsA 2502-bp fragment and VacA gene were identified. An 89.7-kDa VacA34–854 recombinant protein was expressed and purified from the recombinant engineering bacteria and was preserved stably in 50 mM acetic acid buffer (pH 2.9). The amount of the recombinant protein was larger in the inclusion bodies than in the supernatant. In addition, after a 24-h culture with VacA recombinant protein, GES-1 cells demonstrated evidence of apoptosis including early nuclear immobilization and clustering under inverted microscope and TEM. It was found that VacA recombinant protein induced apoptosis by TUNEL assay.ConclusionsA VacA recombinant protein that is stably and highly expressed and possesses pro-apoptotic activity is successfully constructed. The protein is stably preserved in 50 mM acetic acid buffer (pH 2.9).

N-Terminally Added Tag Selectively Enhances Heterologous Expression of VacA Cytotoxin Variants from Helicobacter pylori.

Gastric pathogen Helicobacter pylori secretes VacA cytotoxin displaying a high degree of polymorphic variations of which the highest VacA pathogenicity correlates with m1-type variant followed by VacA-m2. To comparatively evaluate expression in Escherichia coli of the mature VacA variants (m1- and m2-types) and their 33- and 55/59-kDa domains fused with His(6) tag at N- or C-terminus. All VacA clones expressed in E. coli TOP10™ were analyzed by SDS-PAGE and Western blotting. VacA inclusions were solubilized under native conditions (~150-rpm shaking at 37°C for 2 h in 20 mM HEPES (pH7.4) and 150 mM NaCl). Membrane-perturbing and cytotoxic activities of solubilized VacA proteins were assessed via liposome-entrapped dye leakage and resazurin- based cell viability assays, respectively. VacA binding to human gastric adenocarcinoma cells was assessed by immunofluorescence microscopy. Side-chain hydrophobicity of VacA was analyzed through modeled structures constructed by homology- and ab initio-based modeling. Both full-length VacA-m1 and 33-kDa domain were efficiently expressed only in the presence of N-terminal extension while its 55-kDa domain was capably expressed with either N- or Cterminal extension. Selectively enhanced expression was also observed for VacA-m2. Protein expression profiles revealed a critical period in IPTG-induced production of the 55-kDa domain with N-terminal extension unlike its C-terminal extension showing relatively stable expression. Both VacA- m1 isolated domains were able to independently bind to cultured gastric cells similar to the full- length toxin, albeit the 33-kDa domain exhibited significantly higher activity of membrane perturbation than others. Membrane-perturbing and cytotoxic activities observed for VacA-m1 appeared to be higher than those of VacA-m2. Homology-based modeling and sequence analysis suggested a potential structural impact of non-polar residues located at the N-terminus of the mature VacA toxin and its 33-kDa domain. Our data provide molecular insights into selective influence of the N-terminally added tag on efficient expression of recombinant VacA variants, signifying biochemical and biological implications of the hydrophobic stretch within the N-terminal domain.

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.

Is there a relationship between Helicobacter pylori vacA i1 or i2 alleles and development into peptic ulcer and gastric cancer? A meta-analysis study on an Iranian population.

Helicobacter pylori has several virulance factor i.e. VacA, CagA, BabA, SabA, AlpA, AlpB and etc. VacA has several polymorphic region in the nucleotide sequence such as s,m,i,d and, c. It has been suggested that each variation in these polymorphic region has been influenced the toxicity of VacA toxin. We performed a comprehensive meta-analysis to determine the main role of VacAi1/i2 in development into peptic ulcer and gastric cancer in an Iranian population.

Molecular evolution of the VacA p55 binding domain of Helicobacter pylori in mestizos from a high gastric cancer region of Colombia.

The stomach bacterium Helicobacter pylori is one of the most prevalent pathogens in humans, closely linked with serious diseases such as gastric cancer. The microbe has been associated with its host for more than 100,000 years and escorted modern humans out of Africa. H. pylori is predominantly transmitted within families and dispersed globally, resulting in distinct phylogeographic patterns, which can be utilized to investigate migrations and bioturbation events in human history. Latin America was affected by several human migratory waves due to the Spanish colonisation that drastically changed the genetic load and composition of the bacteria and its host. Genetic evidence indicates that independent evolutionary lines of H. pylori have evolved in mestizos from Colombia and other countries in the region during more than 500 years since colonisation. The vacuolating cytotoxin VacA represents a major virulence factor of the pathogen comprising two domains, p33 and p55, the latter of which is essential for binding to the host epithelial cell. The evolution of the VacA toxin in Colombia has been strongly biased due to the effects of Spanish colonization. However, the variation patterns and microevolution of the p55 domain have not yet been described for this population. In the present study, we determined the genetic polymorphisms and deviations in the neutral model of molecular evolution in the p55 domain of 101 clinical H. pylori isolates collected in Bogotá, a city located in Andean mountains characterized by its high gastric cancer risk and its dominant mestizo population. The microevolutionary patterns of the p55 domain were shaped by recombination, purifying and episodic diversifying positive selection. Furthermore, amino acid positions 261 and 321 in the p55 domain of VacA show a high variability among mestizos clinical subsets, suggesting that natural selection in H. pylori may operate differentially in patients with different gastric diseases.

Effect of environmental salt concentration on the Helicobacter pylori exoproteome

Helicobacter pylori infection and a high salt diet are each risk factors for gastric cancer. In this study, we tested the hypothesis that environmental salt concentration influences the composition of the H. pylori exoproteome. H. pylori was cultured in media containing varying concentrations of sodium chloride, and aliquots were fractionated and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified proteins that were selectively released into the extracellular space, and we identified selectively released proteins that were differentially abundant in culture supernatants, depending on the environmental salt concentration. We also used RNA-seq analysis to identify genes that were differentially expressed in response to environmental salt concentration. The salt-responsive proteins identified by proteomic analysis and salt-responsive genes identified by RNA-seq analysis were mostly non-concordant, but the secreted toxin VacA was salt-responsive in both analyses. Western blot analysis confirmed that VacA levels in the culture supernatant were increased in response to high salt conditions, and quantitative RT-qPCR experiments confirmed that vacA transcription was upregulated in response to high salt conditions. These results indicate that environmental salt concentration influences the composition of the H. pylori exoproteome, which could contribute to the increased risk of gastric cancer associated with a high salt diet. SignificanceHelicobacter pylori-induced alterations in the gastric mucosa have been attributed, at least in part, to the actions of secreted H. pylori proteins. In this study, we show that H. pylori growth in high salt concentrations leads to increased levels of a secreted VacA toxin. Salt-induced alterations in the composition of the H. pylori exoproteome is relevant to the increased risk of gastric cancer associated with consumption of a high salt diet.

Intracellular Degradation of Helicobacter pylori VacA Toxin as a Determinant of Gastric Epithelial Cell Viability.

Helicobacter pylori VacA is a secreted pore-forming toxin that induces cell vacuolation in vitro and contributes to the pathogenesis of gastric cancer and peptic ulcer disease. We observed that purified VacA has relatively little effect on the viability of AGS gastric epithelial cells, but the presence of exogenous weak bases such as ammonium chloride (NH4Cl) enhances the susceptibility of these cells to VacA-induced vacuolation and cell death. Therefore, we tested the hypothesis that NH4Cl augments VacA toxicity by altering the intracellular trafficking of VacA or inhibiting intracellular VacA degradation. We observed VacA colocalization with LAMP1- and LC3-positive vesicles in both the presence and absence of NH4Cl, indicating that NH4Cl does not alter VacA trafficking to lysosomes or autophagosomes. Conversely, we found that supplemental NH4Cl significantly increases the intracellular stability of VacA. By conducting experiments using chemical inhibitors, stable ATG5 knockdown cell lines, and ATG16L1 knockout cells (generated using CRISPR/Cas9), we show that VacA degradation is independent of autophagy and proteasome activity but dependent on lysosomal acidification. We conclude that weak bases like ammonia, potentially generated during H. pylori infection by urease and other enzymes, enhance VacA toxicity by inhibiting toxin degradation.

Current information on the association of Helicobacter pylori with autophagy and gastric cancer.

Helicobacter pylori (H. pylori) is a Gram-negative bacterium and causative agent of gastric cancer. H. pylori induce defective autophagy or inhibit it by means of CagA and vacuolating cytotoxin A (VacA) toxins leading to the gastric cancer induction. Impaired or defective autophagy leads to the accumulation of cytotoxic materials, such as ROS and P62 that lead to increased mutations in the DNA, genome instability, and risk of cancer formation. H. pylori CagA may inhibit autophagy through the c-Met-PI3k/Akt-mTOR signaling pathway. However, VacA induces autophagy by some signaling pathways. In the gastric epithelial cells, VacA is a necessary and sufficient factor for the creation of autophagy. While CagA is a negative regulator of this phenomenon, the elimination of this gene from H. pylori has increased autophagy and the production of inflammatory cytokines is reduced. In gastrointestinal cancers, some of the microRNAs (miRNAs) act as tumor suppressors and some other are oncogenes by regulating various genes expression. H. pylori can also modify autophagy through a mechanism that includes the function of miRNAs. In autophagy, oncogenic miRNAs inhibit activation of some tumor suppressor signaling pathways (e.g., ULK1 complex, Beclin-1 function, and Atg4 messaging), whereas tumor suppressor miRNAs can block the activation of oncogenic signaling pathways. For instance, Beclin-1 is negatively regulated by miRNA-376b (oncogenic miRNA) and miRNA-30a (tumor suppressor miRNA). Similarly, Atg4 by miRNA-376b (oncogenic miRNA) and miRNA-101 (tumor suppressor miRNA). So, this apparent paradox can be explained as that both Beclin-1 and Atg4 play different roles in a particular cell or tissue.