Helicobacter Pylori Recognizes IFN—γ By OMP18to Involve In Persistent Colonization

OBJECTIVEHelicobacter pylori (H. pylori) is a spiral-shaped gram-negative bacterium with multiple flagella and colonizes in the stomach of almost half of the world’s population. H. pylori infection causes chronic gastritis, which is asymptomatic in the majority of carriers but is considered a major risk factor for the development of gastric and duodenal ulcers and the gastric malignancies mucosa associated lymphoid tissue lymphoma and gastric adenocarcinoma. Unless antibiotic treatment is applied, H. pylori can be persistent colonization in the stomach, which indicates that H. pylori has explored multiple mechanisms to evade host immune surveillance. Simultaneously, the drug resistance by H. pylori is higher and higher and most antibiotics become invalid. Therefor it is very important to explore the detailed mechanism used by H. pylori to evade host immune surveillance.H. pylori infection invariably induces a chronic and active inflammation of the antral mucosa, with influx of B cells, T cells, and neutrophils. Although all H. pylori strains induce gastritis, cag+strains augment the risk for severe gastritis, atrophic gastritis, and distal gastric cancer compared with that incurred by cag-strains. H. pylori strains carrying the cag-PAI induce a far stronger interleukin-8response than cag-negative strains. Gastric epithelial cells are supposed to play a major role in the host responses to acute and chronic H. pylori infection by activating numerous signal transduction cascades. One result of the activation of these pathways is the production of large amounts of IL-8by the gastric epithelial cells. Chronic active H. pylori gastritis is characterized by a strong infiltration of the mucosa by neutrophilic granulocytes which is thought to be a reaction to neutrophil-attracting molecules (urease, NapA) released by H. pylori.IFN-y, a cytokine that can active many parts of the immune system including phagocytosis and antigen presentation, appears to be involved in protection from bacterial infection and in induction of inflammation. IFN-y is thought to be important in immune responses, because it induces the expression of the class II major histocompatibility complex of antigen-presenting cells and activates macrophages and natural killer cells. STAT1(signal transducer and activator of transcription-1) is a critical molecule in the IFN-γ pathway and IFN-y is associated with increased phosphorylation and activation of STAT1. P-STAT1actives iNOS to promote the production of nitric oxide (NO) which inhibits growth of logarithmic-phase H. pylori.Bacterial pathogens have several surface structures (including pili, fimbriae, outer membrane proteins and various secretion systems) that have the potential to interact with host tissues to mediate attachment and invasion. The outer membrane of Gram-negative bacteria is a complex structure with a major role of adaptation of the bacterium to various external environments while passively and selectively controlling influx and efflux of important solutes, peptides or proteins, nucleic acids, and other organic compounds such as lipids and polysaccharides. H. pylori contains an outer membrane protein (OMP) family consisting of approximately33genes. Most OMPs are surface exposed and, therefore, are potentially important in interfacing bacteria with the mammalian host and uptaking nutrients.Despite the fact that H. pylori elicits a strong inflammatory response, the immune system fails to clear the infection, suggesting that immune evasion strategies are used. The mechanisms for immune evasion include the induction of a strongly polarized immune response, a modulation of phagocytosis and neutrophil function, and an inhibition of lymphocyte proliferation. Some research has showed that IFN-y can bind to the surface of H. pylori and downregulated the expression of CagA. However the concrete mechanism of IFN-y binding to H. pylori is still unclear. In our work, we find Ompl8of H. pylori by DNA microarray and prove that IFN-y binds to Omp18to assist the long term colonization of H. pylori. What’s more,Omp18invovlved in H. pylori survival of NO oxidative stress and anti-phagocytosis.METHODS1. The detection of gene changes of H. pylori26695under the treatment of IFN-y by gene chips. IFN-y was used to treat H. pylori26695for8hours, while a control group was not, and gene chips were used to detect the changes by comparison between the two groups.2. Results of gene chips were confirmed by Real-Time PCR. To determine the mRNA expression of omp18in H. pylori26695treated by IFN-y at different time, the liquid bacterial cultures were harvested at0,2,4and8h. RNA was extracted and transformed to cDNA. Real-Time PCR was used to check the expression of omp18.3. Detection of expression of virulent factors CagA and Nap A under the treatment of IFN-y. Wild type and△ompl8strains were cultured overnight and IFN-y was used to treat H. pylori for8hours, while a control group was not. The liquid bacterial cultures were harvested for RNA and protein extraction and then Real-Time PCR and western bolt were used to check the expression of CagA and NapA.4. Animal experiments. Wild type and△ompl8strains were used to infection mongolian gerbils and the mice were killed at2,4,6and8w. The antrum tissues were moved to detect H. pylori colonization and the inflammation condition of stomachs were assayed by HE stain. Inflammation factors were checked by Real-Time PCR and ELISA kits.5. Detection of the difference of survival ability between wild type and△omp18strains.(1) Overnight cultured H. pylori were added with SNP and the survival ability of wild type and△ompl8strains were compared by agar plate dilution method and fluorescent staining.(2) Overnight cultured wild type and Aompl8strains were used to stimulate macrophages respectively. At the time points of2,6and24h, macrophages were splited and the numbers of viable bacteria (CFU) in the macrophage lysates were determined by plating serial dilutions on solid plates.6. The ability of Omp18binding with IFN-y was determined by immunofluorescence.7. Alignment of Omp18, OprF and IFN-binding domain protein sequences using T-COFFEE align software.8. The expression of protein p-STAT1of macrophages was checked by western blot.9. Determination of NO. NO secreted by H. pylori infected macrophages and stomach tissues of gerbils was determined by griess reagent.RESULTS1.Omp18was up-regulated by IFN-γ. Compared to control group,Omp18of wild type strain was up-regulated by10ng/mL IFN-γ.2. T-COFFEE align software showed that the sequence of Omp18was similar to OprF and IFN-binding domain.3. Immunofluorescence showed that IFN-γ binds to H. pylori Omp18.4. IFN-y reduced H. pylori26695virulence factor expression. The down-regulated expression of CagA and NapA were confirmed by western blot and Real-Time PCR.5.△ompl8strains showed a defect in colonization in gastric system of Mongolia gerbils. Animal experiments showed that both wild-type and△ompl8strains could colonize in the stomach of gerbils. Compared with wild-type, the colonization rate of△ompl8strains gradually decreased from2to8weeks in Mongolia gerbils, especially in the8th week.6.△ompl8strains infection induces more severe inflammation response compared with wild-type strains. Compared with wild-type infected mice, mice infected with△omp18showed more neutrophils infiltration and severe gastric tissue damage. Real-Time PCR and ELISA demonstrated that△omp18strains infection induced more inflammatory cytokines production in gastric tissues compared with wild-type strains infection. More IL-8and MIP-2were induced respectively in AGS and macrophages infected by△omp18strains, especially in the cases in the presence of IFN-γ.7.△omp18strains infection induces more nitric oxide (NO) produce. Treated with IFN-y, p-STATl of macrophages was inhibited by wild-type strains, but not by△ompl8strains, in parallel,△ompl8strains infection induced more NO production from the macrophages and stomach tissues.8.Omp18is involved in H. pylori26695survival of oxidative stress and anti-phagocytosis. The survival rate of△ompl8strains decreased sharply when exposed to SNP compared with the wild-type strains, and most of them transformed from normal helical bacillary features to coccoid features.△ompl8strains also exhibited weaken survival ability in macrophages (Raw264.7cells).CONCLUSIONH. pylori actively senses alterations in IFN-△by Ompl8and responds by optimizing their virulence phenotype to avoid inducing stronger immune response for persistent colonization. And,Ompl8involved in H. pylori survival of NO oxidative stress and anti-phagocytosis, which is a new mechanism for long term colonization of H.pylori.