Transporter HP0497 Is Involved In The Clarithromycin Tolerance Of Helicobacter Pylori

OBJECTIVEHelicobacter pylori(H.pylori)is a spiral-shaped,flagellated and microaerophilic gram-negative bacteria colonized in the gastric epithelium of humans.It is currently the most significant pathogen that colonized in the human stomach.H.pylori infection is confirmed to be the main cause of chronic active gastritis and peptic ulcer,and it is closely related to gastric cancer and gastric mucosa-associated lymphoid tissue(MALT)lymphoma.The World Health Organization has classified H.pylori as a first class carcinogenic factor,making it a risk factor for gastric cancer.About half of the world's population has been infected with H.pylori so far.Therefore,the eradication of H.pylori and infection treatment has become an important issue in terms of maintaining the health of human stomach.The first-line treatment present for H.pylori infection called Triple therapy,which includes Colloidal bismuth or a proton pump inhibitor in combination with two kinds of antibiotics like amoxicillin or metronidazole(MTZ)and clarithromycin.Clarithromycin is the new generation of macrolide antibiotics.It being stable in acid environment,and easy to absorb make it one of the most commonly used antibiotics in treatment of H.pylori infection.However,due to years of the antibiotic abuse,the acquired resistance to antibiotics has become the main cause for the failure of H.pylori eradication.Increasing resistance to clarithromycin worldwide hampers the treatment of H.pylori infections.Clarithromycin is acting on the 23S rRNA of the ribosomal large subunit,thereby inhibiting protein synthesis in bacteria.The present studies of mechanisms of resistance to clarithromycin mostly focused on point mutations of 23S rRNA sites.Nevertheless,some researches have shown that point mutations in 23S rRNA is not the only mechanism responsible for clarithromycin resistance.Possible mechanisms of intrinsic drug resistance also involve decreased drug uptake or increased drug efflux.Nowadays three RND efflux systems have been identified already in H.pylori,namely hefABC,hefDEF,and hefGHI.L Transporters include multiple transporter systems besides efflux pumps and uptake pumps,which have different functions in antibiotic tolerance in bacteria.Whether they participate in clarithromycin tolerance remains unclear and needs to be determined.Transporter HP0497,which expressed differently in both wild type strain Hp26695 treated with clarithromycin and clarithromycin-resistant strain when comparing to wild type strain Hp26695,was identified by qPCR and further studied using variable scientific methods.This research is to identify the role of the transporter HP0497 in adaption to clarithromycin even to multidrug and its inner mechanism.This study gives light on solving the multidrug resistance problem in H.pylori and in many other pathogens,and also helps to identify novel drug targets,providing effective guidance for the rational use of antibiotics in clinical at the same time.METHODS1.The total mRNA of WT strain Hp26695 was extracted after treated with CLA,and was then reversely transcribed into cDNA.Transporters genes expressed differently were then detected by qPCR.Those expressed differently were tested in the CLA-resistant strain by qPCR,and finally select the ones expressed differently in both CLA-resistant strain and WT strain treated with CLA.2.Gene knockout mutant strain ?HP0497 was constructed using WT strain Hp26695 as parent strain.Gene knockout mutant strain ?HP0497-a was constructed using CLA-resistant strain as parent strain.3.To determine the involvement of transporter HP0497 in growth when WT strain and ?HP0497 strain grow in the same conditions,the growth curve of mutant strain was compared with that of WT strain.4.Time-kill assays were performed to determine the susceptibility to CLA of both WT strain and ?HP0497.5.The MICs(Minimum Inhibitory Concentration)for CLA of WT??HP0497 and?HP0497-a strains were determined by E-test,as well as by the agar dilution method.6.The viability changes of WT strain and ?HP0497 strain when exposed to CLA were determined by confocal microscopy.7.H33342 accumulation assays were performed to compare the difference in efflux ability between WT strain and ?HP0497 strain.8.The expressions of transporter HP0497 gene were detected in clinical CLA-sensitive strains and clinical CLA-resistant strains by qPCR,and compared with that of WT strain.9.The expressions of efflux pump genes(hefABC,hefGH1)were detected in WT strain and ?HP0497 strain by qPCR.RESULTS1.Transporter HP0497 gene showed a higher expression in WT strain Hp26695 treated with CLA and CLA-resistant strain compared with WT strain Hp26695,indicating that HP0497 might be responsible for the CLA tolerance of Helicobacter pylori.2.Gene knockout mutant strain ?HP0497 was constructed using WT strain Hp26695 as parent strain.Gene knockout mutant strain ?HP0497-a was constructed using CLA-resistant strain as parent strain.3.The growth curve of ?HP0497 showed no significant difference compared with that of WT strain.4.The time-kill assays,MICs and photos by confocal microscopy showed that the susceptibility of ?HP0497 was higher than that of WT strain.5.H33342 accumulated more in the ?HP0497 compared with WT strain.6.Transporter HP0497 showed higher expression in clinical CLA-resistant strain compared with clinical CLA-sensitive strain.7.Efflux pump genes(hefABC,hefGHl)were examined by the qPCR in both?HP0497 and WT strain,and the result showed that two of which expressed much less in the ?HP0497.CONCLUSIONTransporter HP0497 plays an important role in clarithromycin tolerance of Helicobacter pylori,and it could help pump out clarithromycin and various antibiotics by influencing the expression of efflux pump genes hefABC and hefGHl,protecting H.pylori from clarithromycin damage and providing time and material basis for further antibiotic resistance in H.pylori.