CagA Protein Research Articles

Amino acid deletions at positions 893 and 894 of cytotoxin-associated gene A protein affect Helicobacter pylori gastric epithelial cell interactions

BACKGROUND Helicobacter pylori (H. pylori ) persistently colonizes the human gastric mucosa in more than 50% of the global population, leading to various gastroduodenal diseases ranging from chronic gastritis to gastric carcinoma. Cytotoxin-associated gene A (CagA) protein, an important oncoprotein, has highly polymorphic Glu-Pro-Ile-Tyr-Ala segments at the carboxyl terminus, which play crucial roles in pathogenesis. Our previous study revealed a significant association between amino acid deletions at positions 893 and 894 and gastric cancer. AIM To investigate the impact of amino acid deletions at positions 893 and 894 on CagA function. METHODS We selected a representative HZT strain from a gastric cancer patient with amino acid deletions at positions 893 and 894. The cagA gene was amplified and mutated into cagA -NT and cagA -NE (sequence characteristics of strains from nongastric cancer patients), cloned and inserted into pAdtrack-CMV, and then transfected into AGS cells. The expression of cagA and its mutants was examined using real-time polymerase chain reaction and Western blotting, cell elongation via cell counting, F-actin cytoskeleton visualization using fluorescence staining, and interleukin-8 (IL-8) secretion via enzyme-linked immunosorbent assay. RESULTS The results revealed that pAdtrack/cagA induced a more pronounced hummingbird phenotype than pAdtrack/cagA -NT and pAdtrack/cagA -NE (40.88 ± 3.10 vs 32.50 ± 3.17, P < 0.001 and 40.88 ± 3.10 vs 32.17 ± 3.00, P < 0.001) at 12 hours after transfection. At 24 hours, pAdtrack/cagA- NE induced significantly fewer hummingbird phenotypes than pAdtrack/cagA and pAdtrack/cagA- NT (46.02 ± 2.12 vs 53.90 ± 2.10, P < 0.001 and 46.02 ± 2.12 vs 51.15 ± 3.74, P < 0.001). The total amount of F-actin caused by pAdtrack/cagA was significantly lower than that caused by pAdtrack/cagA- NT and pAdtrack/cagA- NE (27.54 ± 17.37 vs 41.51 ± 11.90, P < 0.001 and 27.54 ± 17.37 vs 41.39 ± 14.22, P < 0.001) at 12 hours after transfection. Additionally, pAdtrack/cagA induced higher IL-8 secretion than pAdtrack/cagA- NT and pAdtrack/cagA- NE at different times after transfection. CONCLUSION Amino acid deletions at positions 893 and 894 enhance CagA pathogenicity, which is crucial for revealing the pathogenic mechanism of CagA and identifying biomarkers of highly pathogenic H. pylori .

MARK2/MARK3 kinases are catalytic co-dependencies of YAP/TAZ in human cancer.

The Hippo signaling pathway is commonly dysregulated in human cancer, which leads to a powerful tumor dependency on the YAP/TAZ transcriptional coactivators. Here, we used paralog co-targeting CRISPR screens to identify the kinases MARK2/3 as absolute catalytic requirements for YAP/TAZ function in diverse carcinoma and sarcoma contexts. Underlying this observation is direct MARK2/3-dependent phosphorylation of NF2 and YAP/TAZ, which effectively reverses the tumor suppressive activity of the Hippo module kinases LATS1/2. To simulate targeting of MARK2/3, we adapted the CagA protein from H. pylori as a catalytic inhibitor of MARK2/3, which we show can regress established tumors in vivo. Together, these findings reveal MARK2/3 as powerful co-dependencies of YAP/TAZ in human cancer; targets that may allow for pharmacology that restores Hippo pathway-mediated tumor suppression.

Virulence gene polymorphisms in Shandong Helicobacter pylori strains and their relevance to gastric cancer.

Helicobacter pylori (H. pylori) virulence factors, particularly the cagA and vacA genotypes, play important roles in the pathogenic process of gastrointestinal disease. The cagA and vacA genotypes of 87 H. pylori strains were determined by PCR and sequencing. The EPIYA and CM motif patterns were analyzed and related to clinical outcomes. We examined the associations between the virulence genes of H. pylori and gastrointestinal diseases in Shandong, and the results were analyzed via the chi-square test and logistic regression model. Overall, 76 (87.36%) of the strains carried the East Asian-type CagA, with the ABD types being the most prevalent (90.79%). However, no significant differences were observed among the different clinical outcomes. The analysis of CagA sequence types revealed 8 distinct types, encompassing 250 EPIYA motifs, including 4 types of EPIYA or EPIYA-like sequences. Additionally, 28 CM motifs were identified, with the most prevalent patterns being E (66.67%), D (16.09%), and W-W (5.75%). Notably, a significant association was discovered between strains with GC and the CM motif pattern D (P < 0.01). With respect to the vacA genotypes, the strains were identified as s1, s2, m1, m2, i1, i2, d1, d2, c1, and c2 in 87 (100%), 0 (0), 26 (29.89%), 61 (70.11%), 73 (83.91%), 14 (16.09%), 76 (87.36%), 11 (12.64%), 18 (20.69%), and 69 (79.31%), respectively. Specifically, the vacA m1 and c1 genotypes presented a significantly greater prevalence in strains from GC compared to CG (P < 0.05). Following adjustment for age and sex, the vacA c1 genotype demonstrated a notable association with GC (OR = 5.174; 95% CI, 1.402-20.810; P = 0.012). This association was both independent of and more pronounced than the correlations between vacA m1 and GC. CagA proteins possessing CM motif pattern D were more frequently observed in patients with GC (P < 0.01), implying a potentially higher virulence of CM motif pattern D than the other CM motif patterns. Moreover, a strong positive association was identified between the vacA c1 genotype and GC, indicating that the vacA c1 genotype is a robust risk indicator for GC among male patients aged ≥55 years in Shandong.

Hereditary Gastric Cancer Is Linked With Hereditary Breast and Ovarian Cancer.

Helicobacter pylori (H. pylori), a bacterium which chronically infects the stomach of approximately half the world's population, is a risk factor for the development of gastric cancer (GC). However, the underlying mechanism whereby H. pylori infection induces GC development remains unclear. Intermittent injection of the H. pylori cytotoxin-associated gene A antigen (CagA) protein into its host cell inhibits nuclear translocation of BRCA1/BRCA2, DNA repair proteins involved in the development of breast cancer/ovarian cancer. Interestingly, hereditary breast and ovarian cancer (HBOC) syndrome is associated with GC development. Here, we aimed to clarify the molecular link between H. pylori infection, BRCA1/2 pathogenic variants (PVs), GC and higher GC incidence in HBOC families. We retrospectively reviewed data from Japanese patients undergoing precision treatment using cancer genomic medicine. We found a higher GC incidence in HBOC families having germline pathogenic variants (GPVs) of BRCA1/2 (2.95% vs. 0.78% in non-HBOC families). Next, we found that 96.1% of H. pylori-infected patients received cancer genomic medicine for advanced GC, and > 16% advanced GC patients had gBRCA2 PVs. Furthermore, expressing wild-type BRCA1/2 in Gan mice (a mouse model of human GC) inhibited GC development. Thus, gBRAC1/2 PVs and H. pylori infection synergistically increase the risk of GC development. Our study highlights the need to investigate the potential of therapeutic agents against BRCA1/2 PVs to avoid the development of GC in HBOC families. In addition, our results suggest that poly (ADP-ribose) polymerase (PARP) inhibitors could potentially inhibit GC development and progression with gBRCA1/2 PVs.

AUF1-mediated inhibition of autophagic lysosomal degradation contributes to CagA stability and Helicobacter pylori-induced inflammation

ABSTRACT CagA, a virulence factor of Helicobacter pylori (H. pylori), is known to drive inflammation in gastric epithelial cells and is typically degraded through autophagy. However, the molecular mechanism by which CagA evades autophagy-mediated degradation remains elusive. This study found that H. pylori inhibits autophagic flux by upregulating the expression of AU-rich element RNA-binding factor 1 (AUF1). We confirmed that AUF1 does not affect autophagy initiation but instead hampers lysosomal clearance, as evidenced by treatments with 3-MA, CQ and BafA1. Upregulated AUF1 stabilizes CagA protein levels by inhibiting the autolysosomal degradation of intracellular CagA in H. pylori-infected gastric epithelial cells. Knocking down AUF1 promotes CagA degradation, an effect that can be reversed by the lysosome inhibitor BafA1 and CQ. Transcriptome analysis of AUF1-knockdown gastric epithelial cells infected with H. pylori indicated that AUF1 regulates the expression of lysosomal-associated hydrolase genes, specifically CTSD, to inhibit autolysosomal degradation. Moreover, we observed that knockdown of AUF1 enhanced the stability of CTSD mRNA and identified AUF1 binding to the 3’UTR region of CTSD mRNA. AUF1-mediated downregulation of CTSD expression contributes to CagA stability, and AUF1 overexpression leads to an increase in CagA levels in exosomes, thus promoting extracellular inflammation. In clinical gastric mucosa, the expression of AUF1 and its cytoplasmic translocation are associated with H. pylori-associated gastritis, with CagA being necessary for the translocation of AUF1 into the cytoplasm. Our findings suggest that AUF1 is a novel host-positive regulator of CagA, and dysregulation of AUF1 expression increases the risk of H. pylori-associated gastritis.

Assessment of Helicobacter pylori cytotoxin-associated Gene A (Cag A) protein and its association with ferritin and vitamin B12 deficiencies among adult healthy asymptomatic residents in Sharjah, United Arab Emirates

IntroductionThe United Arab Emirates (UAE) serves as an effective epidemiological site for assessing Helicobacter pylori (H. pylori) infection due to its diverse population. However, comprehensive studies on the prevalence of H. pylori in the UAE are notably scarce. In depth prevalence studies are needed as a preventive measure against gastric cancer and other emerging extra gastric diseases associated with H. pylori infection. Aim: This study aimed to assess H. pylori infection and its virulent oncoprotein, the Cytotoxin-Associated Gene (Cag A) and its association with ferritin and vitamin B12 deficiencies. Methods: The study was conducted on 1094 healthy asymptomatic volunteers residents in the Sharjah Emirate, UAE. Enzyme-linked immunosorbent assay (ELISA) was performed to assess H. pylori infection using H. pylori antibodies (IgG), and detection of CagA protein using Cag A antibody (IgG) in the human serum. Ferritin and vitamin B12 serum levels were assessed and correlated to H. pylori infection. Results: This study focuses mainly on the assessment of H. pylori and its virulent factor CagA, in relation to vitamin B12 and ferritin deficiencies. Remarkably, 49.6 % of the participants were detected positive for H. pylori, with over half of these cases involving CagA positive strains. Notably, among Emirati participants, 76.11 % of those with H. pylori infection were CagA positive. Statistical analysis revealed a significant correlation between H. pylori, CagA level, and ferritin/vitamin B12 deficiencies. Conclusion: These findings emphasize the importance of timely detection and eradication of H. pylori not only as a preventive strategy against gastric cancer but also as an effective strategy to rescue the adverse effects from ferritin and vitamin B12 deficiencies, thereby improving the overall health outcomes of individuals affected by H. pylori infection.

Gastric Cancer in the Era of Epigenetics.

Gastric cancer (GC) remains a significant contributor to cancer-related mortality. Novel high-throughput techniques have enlightened the epigenetic mechanisms governing gene-expression regulation. Epigenetic characteristics contribute to molecular taxonomy and give rise to cancer-specific epigenetic patterns. Helicobacter pylori (Hp) infection has an impact on aberrant DNA methylation either through its pathogenic CagA protein or by inducing chronic inflammation. The hypomethylation of specific repetitive elements generates an epigenetic field effect early in tumorigenesis. Epstein-Barr virus (EBV) infection triggers DNA methylation by dysregulating DNA methyltransferases (DNMT) enzyme activity, while persistent Hp-EBV co-infection leads to aggressive tumor behavior. Distinct histone modifications are also responsible for oncogene upregulation and tumor-suppressor gene silencing in gastric carcinomas. While histone methylation and acetylation processes have been extensively studied, other less prevalent alterations contribute to the development and migration of gastric cancer via a complex network of interactions. Enzymes, such as Nicotinamide N-methyltransferase (NNMT), which is involved in tumor's metabolic reprogramming, interact with methyltransferases and modify gene expression. Non-coding RNA molecules, including long non-coding RNAs, circular RNAs, and miRNAs serve as epigenetic regulators contributing to GC development, metastasis, poor outcomes and therapy resistance. Serum RNA molecules hold the potential to serve as non-invasive biomarkers for diagnostic, prognostic or therapeutic applications. Gastric fluids represent a valuable source to identify potential biomarkers with diagnostic use in terms of liquid biopsy. Ongoing clinical trials are currently evaluating the efficacy of next-generation epigenetic drugs, displaying promising outcomes. Various approaches including multiple miRNA inhibitors or targeted nanoparticles carrying epigenetic drugs are being designed to enhance existing treatment efficacy and overcome treatment resistance.

In vitro anti-Helicobacter pylori activity and the underlining mechanism of an empirical herbal formula - Hezi Qingyou.

Helicobacter pylori (H. pylori) is thought to primarily colonize the human stomach and lead to various gastrointestinal disorders, such as gastritis and gastric cancer. Currently, main eradication treatment is triple or quadruple therapy centered on antibiotics. Due to antibiotic resistance, the eradication rate of H. pylori is decreasing gradually. Therefore, searching for anti-H. pylori drugs from herbal sources has become a strategy for the treatment. Our team proposed a Hezi Qingyou Formula (HZQYF), composed of Chebulae Fructus, Ficus hirta Vahl and Cloves, and studied its anti-H. pylori activity and mechanism. Chemical components of HZQYF were studied using UHPLC-MS/MS and HPLC. Broth microdilution method and agar dilution method were used to evaluate HZQYF's antibacterial activity. The effects of HZQYF on expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF), and flagellar genes (flaA, flaB) were explored using Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) technology. Effects on morphology and permeability of the extracellular membrane were studied using scanning electron microscopy (SEM) and N-phenylnaphthalen-1-amine (NPN) uptake. Effect on urease activity was studied using a urease kinetics analysis in vitro. Immunofluorescence staining method was used to examine the effect on adhesion. Western blot was used to examine the effect on cagA protein. Minimum inhibitory concentration (MIC) values of the formula against H. pylori clinical strains and standard strains were 80-160 μg/mL, and minimum bactericidal concentration (MBC) values were 160-320 μg/mL. The formula could down-regulate the expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF) and flagellar genes (flaA, flaB), change the morphology of H. pylori, increase its extracellular membrane permeability, and decrease its urease activity. Present studies confirmed that HZQYF had promising in vitro anti-H. pylori activities and demonstrated its possible mechanism of action by down-regulating the bacterial adhesion, urease, and flagellar gene expression, which provided scientific bases for further clinical investigations.

Inhibition of the type IV secretion system from antibiotic-resistant Helicobacter pylori clinical isolates supports the potential of Cagα as an anti-virulence target.

Helicobacter pylori resistance to antibiotics is a growing problem and it increasingly leads to treatment failure. While the bacterium is present worldwide, the severity of clinical outcomes is highly dependent on the geographical origin and genetic characteristics of the strains. One of the major virulence factors identified in H. pylori is the cag pathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS) used to translocate effectors into human cells. Here, we investigated the genetic variability of the cagPAI among 13 antibiotic-resistant H. pylori strains that were isolated from patient biopsies in Québec. Seven of the clinical strains carried the cagPAI, but only four could be readily cultivated under laboratory conditions. We observed variability of the sequences of CagA and CagL proteins that are encoded by the cagPAI. All clinical isolates induce interleukin-8 secretion and morphological changes upon co-incubation with gastric cancer cells and two of them produce extracellular T4SS pili. Finally, we demonstrate that molecule 1G2, a small molecule inhibitor of the Cagα protein from the model strain H. pylori 26695, reduces interleukin-8 secretion in one of the clinical isolates. Co-incubation with 1G2 also inhibits the assembly of T4SS pili, suggesting a mechanism for its action on T4SS function.

Exploring Helicobacter pylori Infection Rates and Associated Risk Factors in Dyspeptic Individuals from Brack Al-Shati, Southern Libya"

Helicobacter pylori, sometimes known as H. pylori, is the most prevalent bacterium worldwide; it colonizes the stomachs of people on the planet, and the incidence of the disease is 85–90% in underdeveloped nations. The pathogenicity of bacteria varies according to the type of strain and the virulence factors it carries, such as the cytotoxin-related gene cagA, in addition to risk factors related to the host and environment. Due to the absence of data about the seroprevalence of antibodies to H. pylori in patients with dyspepsia in Libya in general and in the south in particular, the study aimed to assess the prevalence of H. pylori infection and its risk factors in Brack Al-Shati which is located in the Southwest of Libya. The study included 100 serum samples from dyspeptic patients whose ages varied in age from 18 to 72, with an average of 37.27 ± 1.515. All samples were examined using enzyme-linked immunosorbent assays (ELISA) to detect the presence of IgG antibodies against the CagA protein and H. pylori of the bacteria. The findings indicated that the rate of infection with H.pylori was 73% of the total patient samples, and 68.2% of the samples were positive for the CagA protein. Taking into account the frequency of CagA-positive strains and the seroprevalence of H. pylori in dyspeptic patients, we recommend paying attention to eradicating the bacteria through treatment, especially when more than one risk factor is present.

Seroprevalence of Helicobacter pylori infections in children and adults in Poland in the years 2020-2023.

Helicobacter pylori is a common cause of gastrointestinal infections in people around the world. Various microbiological methods are used in the laboratory diagnosis of infections, including determining the presence of specific antibodies in the serum. Serological tests can also be used in epidemiological studies aimed at determining the incidence of H. pylori infections. The aim of the study was to obtain insight into the incidence of antibodies to H. pylori in subjects of different ages living in Poland in the years 2020-2023. The research used serum samples obtained between January 2020 and September 2023 from 600 subjects living in Poland. The Anti-Helicobacter pylori ELISA IgG enzyme immunoassay from Euroimmun was used to test the level of IgG antibodies to H. pylori antigens. Additionally, selected serum samples were tested for the presence of antibodies to the most important protein virulence factors of H. pylori by Western Blot. IgG antibodies to H. pylori, at a diagnostically significant level, were detected in 28.3% of the examined persons. Antibodies to H. pylori were least frequently detected in children under 10 years of age (12.1%) and teenagers (13.2%). In adults aged 20 to 50, these antibodies were more common (23.9% to 29.5%). Statistically, H. pylori antibodies were most often detected in subjects over 50 years of age (52.3%). There were no statistically significant differences in the frequency of antibodies to H. pylori depending on the gender of the examined persons. In most serum samples tested by Western Blot, the presence of antibodies to the CagA protein was detected (66.7%). The conducted research and analysis of literature data showed a similar percentage of serum samples with a diagnostically significant level of antibodies to H. pylori in people living in Poland as in people living in other European countries. The epidemiology of infections is also very similar, characterized by low morbidity in children and adolescents and an increase in the incidence of infections with the age of the examined persons. Importantly, compared to research conducted in our country several years ago, the percentage of positive results is much lower, which may be due to the improvement of social and living conditions and hygiene habits.

Helicobacter pylori CagA promotes immune evasion of gastric cancer by upregulating PD-L1 level in exosomes

Cytotoxin-associated gene A (CagA) of Helicobacter pylori (Hp) may promote immune evasion of Hp-infected gastric cancer (GC), but potential mechanisms are still under explored. In this study, the positive rates of CagA and PD-L1 protein in tumor tissues and the high level of exosomal PD-L1 protein in plasma exosomes were significantly associated with the elevated stages of tumor node metastasis (TNM) in Hp-infected GC. Moreover, the positive rate of CagA was positively correlated with the positive rate of PD-L1 in tumor tissues and the level of PD-L1 protein in plasma exosomes, and high level of exosomal PD-L1 might indicate poor prognosis of Hp-infected GC. Mechanically, CagA increased PD-L1 level in exosomes derived from GC cells by inhibiting p53 and miRNA-34a, suppressing proliferation and anticancer effect of CD8+ Tcells. This study provides sights for understanding immune evasion mediated by PD-L1. Targeting CagA and exosomal PD-L1 may improve immunotherapy efficacy of Hp-infected GC.

Role of curcumin's phenolic –OH group in the inhibition of Helicobacter pylori CagA phosphorylation

Helicobacter pylori is a pathogenic bacterium that causes various gastric diseases in humans. It secretes virulence factors such as the CagA protein, which plays a crucial role in its pathogenesis. The C-terminal domain (CTD) of the CagA protein plays a crucial role in facilitating its attachment to host cell membranes and interacting with various intracellular proteins. These interactions are essential for the growth and infection of H. pylori. Strain-specific sequence polymorphism of H. pylori, specifically at the C-terminal of the CagA protein, is responsible for its differential oncogenic potential.Curcumin is a natural compound with pharmacological properties like anti-oxidant, anti-bacterial, anti-inflammatory, and anti-cancer effects. Additionally, several studies have reported the anti-H. pylori effects of curcumin. In this study, we investigated mechanisms of binding of curcumin and its non-oxidizable derivatives (acetalcurcumin and diacetalcurcumin) with CTD of CagA protein. We used computational strategies such as docking and molecular dynamics simulation for the analysis. Curcumin was found to have more binding affinity with the CagA protein than its acetal and diacetal analogs. This suggests that the OH-group present in the structure of curcumin, which gets modified in its acetal and diacetal analogs, may have an essential role in the interaction with the CagA protein.Results showed that curcumin binds to the residues within the active pocket formed by the EPIYA-C motif and CM motif located in the C-terminal domain (CTD) of the CagA protein. Additionally, curcumin was observed to interact with the TYR970 residue within the EPIYA-C motif of the CTD of the CagA protein. Notably, TYR970 phosphorylation is a known event during H. pylori-mediated pathogenesis. Therefore, this study suggests that curcumin's anti-H. pylori activity may be triggered by inhibiting the phosphorylation of the CagA protein.

A novel role for Helicobacter pylori cytotoxin-associated gene A in negative regulation of autophagy in human gastric cells

BackgroundAutophagy plays an important role in carcinogenesis and tumor progression in many cancers, including gastric cancer. Cytotoxin-associated gene A (CagA) is a well-known virulent factor in Helicobacter pylori (H. pylori) infection that plays a critical role in gastric inflammation and gastric cancer development. However, its role in autophagy during these processes remains unclear. Therefore, we aimed to clarify the role of CagA in autophagy in CagA-related inflammation.MethodsWe evaluated the autophagic index of AGS cells infected with wild-type cagA-positive H. pylori (Hp-WT) and cagA-knockout H. pylori (Hp-ΔcagA) and rat gastric mucosal (RGM1) cells transfected with CagA genes. To identify the mechanisms underlying the down regulation of autophagy in AGS cells infected with H. pylori, we evaluated protein and mRNA expression levels of autophagy core proteins using western blotting and quantitative reverse transcription-polymerase chain reaction (RT-PCR). To determine whether autophagy induced the expression of the pro-inflammatory mediator, cyclooxygenase-2 (COX-2), we evaluated COX-2 expression in AGS cells treated with an autophagy inducer and inhibitor and infected with H. pylori. In addition, we evaluated whether COX-2 protein expression in AGS cells influenced beclin-1 (BECN1) expression with si-RNA transfection when infected with H. pylori.ResultsAutophagic flux assay using chloroquine showed that autophagy in AGS cells was significantly suppressed after H. pylori infection. The autophagic index of AGS cells infected with Hp-WT was decreased significantly when compared with that in AGS cells infected with Hp-ΔcagA. The autophagic index of RGM1 cells transfected with CagA was lower, suggesting that CagA inhibits autophagy. In addition, BECN1 expression levels in AGS cells infected with Hp-WT were reduced compared to those in AGS cells infected with Hp-ΔcagA. Furthermore, COX-2 expression in AGS cells infected with H. pylori was controlled in an autophagy-dependent manner. When AGS cells were transfected with small interfering RNA specific for BECN1 and infected with Hp-WT and Hp-ΔcagA, COX-2 was upregulated significantly in cells infected with Hp-ΔcagA.ConclusionsIn conclusion, the H. pylori CagA protein negatively regulated autophagy by downregulating BECN1. CagA-induced autophagy inhibition may be a causative factor in promoting pro-inflammatory mediator production in human gastric epithelial cells.

Role of the CagY antenna projection in Helicobacter pylori Cag type IV secretion system activity.

Helicobacter pylori strains containing the cag pathogenicity island (PAI) are associated with the development of gastric adenocarcinoma and peptic ulcer disease. The cag PAI encodes a secreted effector protein (CagA) and a type IV secretion system (Cag T4SS). Cag T4SS activity is required for the delivery of CagA and non-protein substrates into host cells. The Cag T4SS outer membrane core complex (OMCC) contains a channel-like domain formed by helix-loop-helix elements (antenna projections, AP) from 14 copies of the CagY protein (a VirB10 ortholog). Similar VirB10 antenna regions are present in T4SS OMCCs from multiple bacterial species and are predicted to span the outer membrane. In this study, we investigated the role of the CagY antenna region in Cag T4SS OMCC assembly and Cag T4SS function. An H. pylori mutant strain with deletion of the entire CagY AP (∆AP) retained the capacity to produce CagY and assemble an OMCC, but it lacked T4SS activity (CagA translocation and IL-8 induction in AGS gastric epithelial cells). In contrast, a mutant strain with Gly-Ser substitutions in the unstructured CagY AP loop retained Cag T4SS activity. Mutants containing CagY AP loops with shortened lengths were defective in CagA translocation and exhibited reduced IL-8-inducing activity compared to control strains. These data indicate that the CagY AP region is required for Cag T4SS activity and that Cag T4SS activity can be modulated by altering the length of the CagY AP unstructured loop.

Hp0521 inhibited the virulence of H. pylori 26,695 strain via regulating CagA expression

Hp0521 is the number of cag pathogenicity island (cagPAI) family in Helicobacter pylori (H. pylori, Hp), which encoded Cag2 protein. The aim of this study was to investigate the role of hp0521 on the H. pylori 26,695 strain. We constructed the recombinant prokaryotic expression plasmid pET-32a-hp0521 and pET-32a-hpc0521. Then, we co-cultured the H. pylori wild strain 26,695 and Δhp0521 strain with GES-1 cells to detect CagA protein transport and IL-8 secretion. We found that Δhp0521 mutation increased the expression of cagA, rpoB and promoted the transportation of CagA protein in GES-1 cells. In addition, we also observed that Δhp0521 mutation had no effect on other cagPAI protein stability and the expression of IL-8. Our findings suggested that hp0521 may down-regulated the expression of cagA, rpoB and inhibited the transportation of CagA protein in GES-1 cells and had no effect on growth.

Predictive profiling of gram-negative antibiotics in CagA oncoprotein inactivation: a molecular dynamics simulation approach

ABSTRACT Gastric cancer (GC) is the fifth most prevalent form of cancer worldwide. CagA - positive Helicobacter pylori infects more than 60% of the human population. Moreover, chronic infection of CagA-positive H. pylori can directly affect GC incidence. In the current study, we have repurposed FDA-approved antibiotics that are viable alternatives to current regimens and can potentially be used as combination therapy against the CagA of H. pylori. The 100 FDA-approved gram negative antibiotics were screened against CagA protein using the AutoDock 4.2 tool. Further, top nine compounds were selected based on higher binding affinity with CagA. The trajectory analysis of MD simulations reflected that binding of these drugs with CagA stabilizes the system. Nonetheless, atomic density map and principal component analysis also support the notion of stable binding of antibiotics to the protein. The residues ASP96, GLN100, PRO184, and THR185 of compound cefpiramide, doxycycline, delafloxacin, metacycline, oxytetracycline, and ertapenem were involved in the binding with CagA protein. These residues are crucial for the CagA that aids in entry or pathogenesis of the bacterium. The screened FDA-approved antibiotics have a potential druggability to inhibit CagA and reduce the progression of H. pylori borne diseases.

A review of signal pathway induced by virulent protein CagA of Helicobacter pylori.

Gastric cancer (GC), a common and high-mortality disease, still occupies an important position in current cancer research, and Helicobacter pylori (H. pylori) infection as its important risk factor has been a hot and challenging research area. Among the numerous pathogenic factors of H. pylori, the virulence protein CagA has been widely studied as the only bacterial-derived oncoprotein. It was found that CagA entering into gastric epithelial cells (GECs) can induce the dysregulation of multiple cellular pathways such as MAPK signaling pathway, PI3K/Akt signaling pathway, NF-κB signaling pathway, Wnt/β-catenin signaling pathway, JAK-STAT signaling pathway, Hippo signaling pathway through phosphorylation and non-phosphorylation. These disordered pathways cause pathological changes in morphology, adhesion, polarity, proliferation, movement, and other processes of GECs, which eventually promotes the occurrence of GC. With the deepening of H. pylori-related research, the research on CagA-induced abnormal signaling pathway has been updated and deepened to some extent, so the key signaling pathways activated by CagA are used as the main stem to sort out the pathogenesis of CagA in this paper, aiming to provide new strategies for the H. pylori infection and treatment of GC in the future.

Hematological manifestations of Helicobacter Pylori (literature review)

It is known that Helicobacter pylori to be a key factor in the etiology of various gastrointestinal diseases, ranging from chronic gastritis without clinical symptoms to peptic ulcer, autoimmune gastritis, adenocarcinoma and gastric MALT lymphoma. However, current research suggests that Helicobacter pylori may be associated with numerous extra-gastric diseases that lead to chronic local or systemic inflammation and the initiation of autoimmune reactions, including hematological ones. The article describes the role of Helicobacter pylori CagA in the pathogenesis of iron deficiency anemia, immune thrombocytopenic purpura and MALT lymphoma. Studies of the iron-deficiency anemia pathogenesis in infected H. pylori patients have shown a connection between the CagA oncoprotein and iron homeostasis. It was established that transferrin endocytosis is mediated and iron absorption increases. In the development of immune thrombocytopenic purpura, CagA leads to a systemic host immune response through mechanisms of molecular mimicry. In pathogenesis of MALT lymphoma, it is considered significant that after the transfer of CagA to B-cell lymphocytes, through the type 4 secretory system (T4SS), a phosphorylated CagA-SHP2 complex is formed by affecting endoplasmic reticulum kinases 1 and 2 (ERK1, ERK2), p38MAPK, BCL2 and NF -κB, as well as through inhibition of p53 accumulation or inhibition of the JAK-STAT signaling pathway, ultimately promoting lymphogenesis and immortalization of B-cell lymphocytes. So, it was established that the presence of CagA protein in the Helicobacter pylori strain is key to the development of inflammation and tumor transformation. The disclosure of these mechanisms is necessary for a more accurate understanding of some pathological processes caused by this bacterium, both in the stomach and outside it. This will help improve diagnosis, guide treatment and predict clinical prognosis. Keywords: hypomagnesaemia, blood pressure, diabetes mellitus.

Use of sodium polyprenylphosphate to correct changes in the immune response caused by Helicobacter pylori CagA proteins in the experiment

Helicobacter pylori (HP) bacteria have a wide range of pathogenicity factors. The HP genome contains genes of the CagA group (cytotoxin-associated genes). Infection with CagA-positive HP strains is associated with the production of pro-inflammatory cytokines, which are involved in the maintenance and development of destructive and inflammatory changes in the organism. Here we studied the role of CagA proteins in the regulation of the immune response in DBA mice and evaluated the corrective effect of polyprenyl phosphate (PP) on this process. Genetically modified strains of E. coli were used, differing by the presence of an island of genes encoding the synthesis of HP CagA proteins. The drug Phosprenyl was used as a source of PP. The subpopulation composition of spleen cells was evaluated by flow cytofluorimetry using monoclonal antibodies. The level of IL 10, TGF-β in blood serum was determined by the enzyme immunoassay. The proliferative activity of splenocytes was measured by the standard procedure based on the inclusion of 3 H-thymidine in the DNA. CagA HP proteins caused the polarization of the immune response by the Th1-dominant type, which was expressed by an increase in the population of CD4 + cells, CD25 + and CD25 + Foxp3 + T cells and increase proliferation T- limphocytes. Inoculation of CagA + bacteria was accompanied by a quantitative increase in TGF-β produced by activated Treg cells. PP inoculation led to the normalization of the CD4 and CD8 T cells, a decrease in the populations of CD25 + , Foxp3 + , CD25 + Foxp3 + cells, and further increase in the IL-10 levels. Thus, PP corrected cellular immune response, prevented the pronounced polarization of the Th1 immune response, and reduced the activation of Treg population. The results obtained indicate a possible decrease in the pro-inflammatory immune response under the influence of PP.