Analysis The Mechanism Of Helicobacter Pylori OMP18(Hp1125) In Immune Escape

Helicobacter pylori (H. pylori) is a pathogenic microorganisms to colonize human stomach successly, its long-term engraftment can cause a variety of stomach disease, such as chronic gastritis, peptic ulcer, intestinal metaplasia, gastric carcinoma and mucosa-associated with lymphoid tissue (MALT) lymphomas. The infection rate of H.pylori is very high, about50%of the world’s population infected with the bacterium, in our country H.pylori infection rate is about88.5%, and most of them are positive cagA, which is a highly pathogenic strain, so the pathogenicity is more serious in our country.The host has a series of defense mechanism against H.pylori infection, such as gastric peristalsis, gastric acid, and the complex environment of stomach and host’s immune response. However, H.pylori with its special physiological structure, for example the urease, furthermore, its stress reaction induced by host immunity response can induce the expression of the antioxygen stress molecules, and promote phagosomes fuse to become megasomes protect itself can not be cleared by host. The past studies are focused on how H. pylori to escape innate immune of host, long term colonization of the H.pylori can activate the host adaptive immune response. Some studies have confirmed that H.pylori infection can affect dendritic cells (DCs) mature and function and induce Th1immune response, and the IFN-γ of Th1cells secreted has an important role for H.pylori colonization, H.pylori colonization rate in normal mice was significantly lower than in IFN-γ-/-mice. However, how H.pylori to escape the host adaptive immunity, especially how to escape IFN-y immune clearance has still not clear. Although some study results suggest between IFN-y and H.pylori can interact directly, but how do them do function is not clear.H.pylori has abundant outer membrane proteins (OMPs), about32species, they are important for the organism due to their participation in ion transport, bacterial adherence, osmotic and structural stabilities and inducing signal transduction. In addition, some membrane protein has unique antigenicity, which can be used as the vaccine. According to the research of Pseudomonas aeruginosa and Francisella novicida outer membrane proteins, we focused on the outer membrane protein Hp1125of H.pylori; it has higher homology with OprF(pseudomonas aeruginosa). In their study, IFN-y could combine with the outer membrane protein. Whether Hp1125can bind IFN-y is still not known. Because of the host immune response is a challenge for H.pylori live and colonization, so it can induce the stringent response of H.pylori, especially the expression of the spoT, and how stringent response regulate H.pylori adapt to IFN-γ to escape being cleared is not clear.Our research Mainly discusses the mechanism of outer membrane protein Hp1125of H.pylori adapt to host IFN-γ, and the role of stringent response of H.pylori in the process, explored the mechanism of H.pylori escape the host immune clearance, to provide some clues for clinical prophylaxis and treatment.METHOD1. In vitro studies confirmed IFN-y stimulation could induce H.pylori stringent response. In vitro cultured H. pylori to OD=0.8, add IFN-γ in a bottle, another bottle add the same amount of sterile water. After co-culture1h,2h,4h and8h, extraction RNA and reverse transcription for cDNA, Real-time PCR detection the expression of spoT.2. Search for the homologous sequence of pseudomonas aeruginosa outer membrane protein OprF in H.pylori through the method of bioinformatics. We analyzed and searched the protein sequence in BLAST and BioCyc database.3. Through the method of homologous recombination to construct Hp1125mutant in HP26695strains. With pILL570plasmid, pUC18K2plasmid and polymerase chain reaction (PCR) method to construct knockout plasmid, with correct plasmid to electrotransformation wild strains, screening monoclonal which successed insert the resistance gene (kanamycin).4. To analysis the role of outer membrane protein Hp1125in H.pylori adapt to IFN-γ. Use IFN-γ processing HP26695and Hpl125mutants respectively1h,2h,4h and8h, extraction RNA for RT-PCR, then the Real-Time PCR to detect the expression of the spoT; Respectively extract HP26695and Hp1125mutant outer membrane protein, Proteins were separated by non-degeneration polyacrylamide gel electrophoresis (PAGE), transferred onto polyvinylidene difluoride (PVDF), the membrane were blocked with5%BSA, then membranes were incubated with IFN-y at4℃, biotin labeled anti-human IFN-γ was then added to membranes. After extensively washing, HRP labeled streptavidin was incubated with membranes. Membranes were developed using ECL reagent.5. Testing related gene expression. After treating with IFN-y, through Western Blot to test the expression of CagA and NapA changes in HP26695and Hpl125mutant; Then detected the gene expression in RNA levels in8h point, including toxic related gene:cagA, vacA, napA, ureA and ureB; Protein degradation genes:clpX, clpP; Bacterial adhesion genes:babA.RESULT1. After incubating with IFN-y in vitro, H.pylori wild strain, compared with the contrast, its spoT expression is gradually raised, and the most significant change at8h. It demonstrated that IFN-γ could induce stringent response of H.pylori.2. Bioinformatics analysis found that H.pylori outer membrane protein Hp1125has high homology with oprF.3. Constructed Hpl125mutant in HP26695successfully.4. Compared with the expression of spoT in Hp1125mutant (stimulate use IFN-γ) and wild strain (not stimulate use IFN-γ), there is no significant changes. Respectively extraction the outer membrane proteins of HP26695and Hp1125mutant, western blot examination revealed that the IFN-γ protein only was seen in wild strains..5. Use IFN-γ stimulate, detected the expression of CagA and NapA in HP26695 and Hp1125mutant at different time points, found that the wild strain after incubating with IFN-y, the expression of CagA and NapA are decreased, but which there is no significant changes after the same treatment in Hp1125mutant,. mRNA expression level of which was also confirmed these results. The downregulation of vac A, ureA, ureB, clpX, clpP and babA in Hp1125mutant suggest that Hp1125affect on H.pylori pathogenesis, adhesion and the degradation of mis-folded protein.CONCLUSIONIFN-y incubated with H.pylori wild strain, the expression of spoT is higher, at same time the toxic genes expression are reduced. After knocking out Hp1125of H.pylori, incubated with IFN-y, the expression of spoT was not been induced, which as same as the change in wild strain without IFN-γ, at the same time the related toxicity gene expression is increased. Combining the results that toxic gene expression in wild strain significantly lower than ΔspoT mutations. We can assume that H. pylori outer membrane protein Hp1125could recognize IFN-γ, and induce stringent response gene spoT, then adjusting related molecular expression to adapt to the change of external environment and escape immune clearance.