The Research Of Efflux PUMP System Joining In Clarithromycin Adaption And Resistance In Helicobacter Pylori

OBJECTIVEHelicobacter pylori(H.pylori) is a helical or spiral-shaped gram-negative bacterium that selectively colonizes the gastric mucosa. Most of people in the world are infected in early childhood and carry it throughout their life. At present, H.pylori infection has been linked to human gastritis and ulcers, gastric adenocarcinoma and mucosa-associated lymphoid-tissue lymphoma(MALT). H.pylori was classified as a definite carcinogen by the World Health Organization’s International Agency for Research on Cancer in 1994. Therefore, treatment and eradication of the H.pylori infection is a scientific issue to be solved urgently. Today, the first-line treatment of H. pylori infection in clinical is triple therapy.Clarithromycin (CLR) is the new gengeration of macrolide antibiotics with the characteristic of being better absorbed and stable to gastric acid, for the reason that, clarithromycin-based triple therapy has been recommended in children and adults by the latest Maastricht Consensus. In recent years,however, this type of therapy isn’t as effective as before, most people consider that H.pylori obtaining antibiotics resistance is the main reason which leads to eradication failure.Nevertherless, the mechanism of resistance acquirement is only partly understood.Recent years, due to the excessive and repeated use of CLR, the rate of H. Pylori resistance shows an upward trend at home and abroad. The current research for CLR tolerance mainly focuses on the analysis 23 S rRNA gene point mutation. Many studies found that 23 S rRNA of some CLR resistance-strains had not mutated, suggesting that point mutation of 23 S rRNA is not the only reason causing CLR-resistance,the mechanism needs to be further studied.Multidrug transporters are made of a series proteins, which can efflux noxious compounds out of cells and protect cells from environmental stresses.Based on sequences imilarity,multidrug transporters are classified into five families.For a cell,there are many ways to develop drug resistance, including decrease of membrane permeability,altered target proteins,drug metabolism,and so on. In contrast,to efflux drugs out of the cell is the principal mechanism.In H. pylori, four RND families have been identified (Hp0605 to Hp0607; HefABC, Hp0971 to Hp0969; HefDEF, Hp1327 to Hp1329; HefGHI and Hp1489 to Hp1487) and the participation in multidrug resistance of these proteins has been reported in H. pylori.However, many efflux pump genes in Helicobacter pylori are not reported.So we wonder wether these unreported ones join in clarithromycin resistance,even take important roles in clarithromycin resistance.It remains to be researched.METHODS1.Construction of Hp0939 mutant strain(△yckJ) by the method of homologous recombination.2.Introduction of Hp26695 resistance strains with CLR in vitro by the method of consecutive batches on resistant plate.3.Determination of MIC by trace the broth dilution method.After diluting CLR for different concentrations with MH broth,Hp was inoculated together.The minimum dose of inhibiting the growth of the bacteria was the MIC.4.Analysis of gene differential expression between Hp26695 and △yckJ by mRNA microarray.The data of two groups which include the Hp26695 treated with CLR or not was compared to forcast expression of the efflux pump system genes.5.The gene differential expression from mRNA microarray and confirming the importance of Hp0939 and Hp1017 further were confirmed by the method of QRT-PCR.After stimulating Hp26695 wild strains with CLR for 30min,RNA was extracted for QRT-PCR to detect the expression of the efflux system genes forcasted by microarray.And then,we stimulated Hp26695 wild strains with different concentration of CLR for different time,in addition,we stimulated Hp26695 resistance strains with different concentration of CLR.After all,we extrated RNA for QRT-PCR to detect expression of Hp.939、Hp1017.6.The survival ability and MIC change of △yckJ exposed to CLR was determined through plate culture count,flourescence staining and confocal microscopy and antibiotics sensitivity test by minimal inhibitory concentration(MIC).RESULTS1.Hp26695 resistance strains with CLR was induced successfully in vitro.2.Efflux pump system proteins were involved in the regulation of Hp stress response to CLR.By the analysis of microarray data, we found about 47 genes of efflux pumps were changed under the stress of CLR in H.pylori 26695,and 24 genes changed tremendously(Fold≥3).3.Protein Hp0939 may be involved in CLR resistance.The result of mRNA microarray analysis was confirmed by QRT-PCR,we found the expression of Hp0939 is over 100 times higher than control,Hp1017 is over 50 times higher than control. So,we confirmed its expression level in resistance strains,and then,we stimulated Hp26695 wild strains with different concentration of CLR for different time,in addition,we stimulated Hp26695 resistance strains with different concentration of CLR.These results showed that expression of Hp0939 was increased in various degree.4.Hp0939 mutant strain was constructed successfully(△yckJ).5.To explain the role of Hp0939 in the process of CLR tolerance further,we constructed the Hp0939 mutant strain(△yckJ),as well as compared the difference of survival rate and MIC between △yckJ and Hp26695 by the methods of PCR absolute quantification,florescence staining and confocal microscopy and antibiotics sensitiviy test by MIC.We found that the survival rate of △yckJ dropped to 1% or less after 4h,but Hp26695 still kept more than 10% after 8h.Consistent with the results,the delection of Hp0939 resulted in the reduction of MIC of CLR by 87.5% compared with Hp26695.6.We stimulated Hp26695 wild strains with different concentration of CLR for different time,in addition,we stimulated Hp26695 resistance strains with different concentration of CLR.These results showed that expression of Hp1017 was increased in various degree.CONCLUSIONThe efflux pump system proteins are involved in the adaption of Hp to CLR pressure,especially yckJ and rocE which plays the most significant role.The high expression of efflux pump system protein yckJ protected Hp from the damage of, CLR,which laid the basis of time and material for further antibiotics resistance.