Helicobacter Pylori Variants with ABC-Type Tyrosine Phosphorylation Motif in Gastric Biopsies of Ghanaian Patients

Background. Helicobacter pylori pathogenicity and disease severity are determined by the tyrosine phosphorylation motifs of CagA protein. This study is aimed at detecting the presence of H. pylori and identifying the CagA tyrosine phosphorylation motifs in Ghanaian patients. Material and Methods. A total of 94 archival genomic DNA samples from gastric biopsies were used for the study, and H. pylori was detected by amplifying the 16S rRNA gene. The 3 ′ -end variable region of the cagA gene was amplified, and the entire 3 ′ -end was sequenced and translated into amino acids. Results. H. pylori was detected in 53.2% (50/94) of the samples, and all the detected bacteria harboured the cagA gene. Two variants of the bacteria were identified based on the size of the amplified cagA gene: 207 bp and 285 bp. The 207 bp and 285 bp variants accounted for 74% and 22%, respectively, and 4% showed both fragments. Translated amino acid sequence of the cagA gene showed EPIYA-A, EPIYA-B, and EPIYA-C (ABC type) motifs, indicating the Western variant. The CagA protein C-terminal showed insertion of amino acids in the sequence flanking the EPIYA-A motif at the N-terminal and a complete deletion of the EPIYA-CC and EPIYA-CCC motifs together with the flanking sequences. Conclusions. H. pylori identified were Western variant (ABC type) with unique amino acid insertions, suggesting unique variants in Ghanaian patients. Further investigation is however required to understand the role of the molecular diversity of the variant in gastric disease outcome.

Antibodies crossreacting with human TNF-alfa receptor induced in the patients with coronary heart disease by Helicobacter pylori CagA positive strains

Introduction Coronary heart disease (CHD) is associated with inflammation and atherosclerosis. The hypothesis of autoimmune origin of CHD has been suggested. Chronic infection with gastric pathogen Helicobacter pylori may induce antibodies, potentially autoreactive, due to molecular mimicry between H. pylori cytotoxin associated gene A (CagA) protein and tumor necrosis factor (TNF)-α receptor (TNFR). Purpose To evaluate presence of antibodies against synthetic c peptide (P1), which mimics the common sequence of TNFR and H. pylori CagA, in the sera of CHD patients and healthy donors (HD), uninfected or infected with H. pylori. Furthermore, guinea pigs (Caviae porcellus), sensitive to H. pylori infection, were used to confirm the induction of antibodies crossreacting with TNFR by this pathogen. Material and methods Serum samples from CHD patients infected with H. pylori CagA(+) strain (43) or H.pylori CagA(−) strain (12), and uninfected HD (16) without symptoms of gastritis or CHD were selected. Moreover, the serum samples from control (10) or H. pylori infected guinea pigs, 7, 28 and 60 days from inoculation were used (10, 20, 10, respectively). The H. pylori status in humans was confirmed by 13C urea breath testing and anti-H. pylori antibody production whereas in Cavia porcellus by histological examination of gastric tissue, detection of H. pylori antigens in stool as well as serum anti-H. pylori antibodies. The ELISA assay with the surface H. pylori antigens – glycine acid extract (GE), recombinant CagA (IRIS, Siena, Italy), synthetic P1 or P2 peptides (Lipopharm, Gdansk, Poland), with or without common CagA/TNFR sequence, respectively, were used for detection of antibodies. Complement activation by P1-anti-P1 IgG complexes was determined by complement fixation assay with sheep erythrocytes (SRBC) and anti-SRBC antibodies. Results :Anti-P1 IgG were detected only in CHD patients infected with H. pylori CagA(+) but not CagA(−) strain. Such antibodies were not detected in H. pylori uninfected HD. Anti-P1 IgG were found in the sera of Cavia porcellus infected with H. pylori, 7 and 28 but not 60 days from inoculation. This may suggest the clearance of such antibodies during the course of infection or their early deposition. The role of H. pylori in anti-P1 IgG induction was confirmed by deletion of such antibodies by absorption of sera, which were positive for anti-P1 IgG, with heat inactivated H. pylori. Anti-P1 IgG present in human and animal sera were biologically active. They formed the immune complexes (IC) with P1 in vitro, which were able to activate complement, suggesting a pro-inflammatory potential of such IC in vivo. Conclusions During H. pylori infection in CHD patients the CagA protein of this pathogen may induce antibodies cross reacting with TNFR. They can potentially increase the inflammatory response by IC dependent activation of complement or due to modulation of TNFR-driven cell signaling pathways. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Statutory Fund of University of Lodz, Poland

Recombinant Helicobacter pylori CagA protein induces endoplasmic reticulum stress and autophagy in human cells

Helicobacter pylori (Hp) CagA protein has a key role in the development of gastric cancer by the intruding in many intracellular processes of host human cell. Endoplasmic reticulum (ER) stress is an essential process for cellular homeostasis that modulates survival and death and is linked to several complex diseases including cancer. CagA protein is found in the serum of Hp-positive individuals and also in the supernatant of Hp culture. Limited studies report that recombinant CagA can alter gene expression and signaling pathways and induce the death of human cells. In this study, we investigated the effect of exogenous recombinant CagA protein treatment on ER stress and autophagy of human cell. AGS, MKN45, and HEK293 cells were treated with 1 µg/ml of recombinant CagA protein and then ER stress was studied by quantitative-PCR of spliced XBP-1 mRNA, immunofluorescence staining of ATF6 protein nuclear localization and real-time quantitative-PCR and/or western blot expression of GRP78, GRP94, ATF4 and CHOP genes. Autophagy was studied by western blot assessment of the conversion of LC3-I to LC3-II and LC3 aggregation. Cell proliferation and death were investigated by MTT assay and trypan blue staining respectively. As result, treatment with recombinant CagA enhanced XBP-1mRNA splicing, nuclear localization of ATF6, and the expression of ER stress signaling target genes in the cells. Recombinant CagA also induced LC3 protein conversion and aggregation in the cells. Reduced cell proliferation and increased cell death were determined in the cells treated with recombinant CagA. These results show that exogenous recombinant CagA protein causes cell death by inducing ER stress and autophagy in human cells. We conclude that CagA protein exogenously localizes in/on human cells and induces ER stress via disturbing protein machinery leading the human cell to death, however, the mechanism of CagA-host cell interaction is to be investigated.

Analysis of the Carcinogenic Potential of Helicobacter Pylori Based on Determination of the Degree of Phosphorylation of the Caga Protein Bacteria

The developed molecular and genetic method for analysis of the carcinogenic potential of Helicobacter pylori based on determining the degree of phosphorylation of the cytotoxin-associated protein of the bacterium (CagA) has been presented. The degree of phosphorylation of CagA protein of Helicobacter pylori is estimated by determining the number and type of so-called EPIYA (Glu-Pro-Ile-Tyr-Ala) motifs at the carboxyl end of the CagA protein region using PCR (polymerase chain reaction) and sequencing of the CagA gene locus. The proposed method can be used to form groups of patients who need additional examination and follow-up observation for the purpose of early prediction of pathological conditions associated with the infectious process caused by Helicobacter pylori strains dominating in the Republic of Belarus. The determination of the number and type of EPIYA motifs can be used as an additional criterion for detection of risk groups for gastrointestinal diseases.