The Mechanism Research Of Bifunctional Enzyme Spot Mediates Clarithromycin Adaption In Helicobacter Pylori

OBJECTIVEHelicobacter pylori(Hp) is a helical or spiral-shaped gram-negative bacterium that colonizes in the gastric mucosa selectively. Most people in the world are infected with Hp in early childhood and carry it throughout their life. Hp infection is related to human gastritis and ulcers and the long-term chronic infection will increase the risk of gastric adenocarcinoma and mucosa-associated lymphoid-tissue lymphoma. Therefore, Hp was classified as a definite carcinogen by the World Health Organization’s International Agency for Research on Cancer in1994. So the treatment and eradication of Hp infection is a scientific issue to be solved urgently.The first-line clinical treatment of Hp infection is the use of the triple therapy that includes colloidal bismuth or proton pump inhibitors and two antibiotics at the present time. Clarithromycin (CLR) is the new generation of macrolide antibiotics with the characteristic of being better absorbed and stability to gastric acid, for the reason that, CLR-based triple therapy has been recommended as the first choice in children and adults by the latest Maastricht Consensus. But recent years, the antibiotics resistance of Hp becomes more serious at home and abroad due to the excessive and repeated use of CLR. The current research for CLR tolerance mainly focuses on the analysis of23S rRNA gene point mutation. Other studies about CLR resistance are reported rarely, and some studies found that23S RNA of many CLR resistance-strains did not mutated, suggesting that point mutation of23S rRNA is not the only reason for CLR-resistance, the mechanism needs to be further studied.The function of SpoT firstly discovered is to regulate the stringent response in Escherichia coli(E.coli). The stringent response is a bacterial adaptation which affects global gene expression under nutrient limitation and other pressure conditions. In E.coli, the stringent response is regulated by a small molecular-guanosine3’-diphosphate,5’-triphosphate and guanosine3’,5’-bispyrophosphate ((p)ppGpp).These small molecular effectors can be combined with the promoter of RNA polymerase, which results in the change of the specificity of promoter and transcription efficiency.(P)ppGpp is synthesized by the enzyme RelA and SpoT in E.coli, but the genome of Hp contains only spoT and lacks relA. SpoT is conserved and is a bifunctional enzyme with both synthetase and hydrolase activities. Previous researches have shown that the (p)ppGpp mediates Vancomycin Tolerance in Enterococcus faecalis which implied that the antibiotics stress may also activate stringent response. So we predict that (p)ppGpp may be the key regulator for antibiotics resistance in Hp.Multple studies have showed that the depression of membrane permeability and activation of efflux pump are the main mechanisms of membrane proteins contributing to antibioitics tolerance in gram-negative bacteria. In our work, a large amount of membrane proteins were found to alter in transcriptional level by microarray prediction under the pressure of CLR, which indicated that membrane protein may play a key role in CLR resistance of Hp, but the mechanism is not clear.Our research mainly discusses the mechanism of the bifunctional enzyme SpoT and its production-(p)ppGpp in mediating Hp adapted to CLR pressure and expression differentiation of genes regulated by SpoT. our study can provide a new mechanism for CLR resistance and lay a foundation for getting new drug targets and fairly use of antibiotics to treat Hp infection.METHODS1. Construction of spoT mutant strain(⊿spoT), kefB mutant strain (⊿kefB) and spoT complement strain (spoT*) by the method of homologous recombination.2. Determination of the minimal inhibitory concentration (MIC) of the CLR by agar plate dilution method using multipoint inoculator. Different doses of CLR were added to the quantitative MH agar and the medium prepared was inoculated with bacteria.The mininum dose of inhibiting the growth of the bacteria was the MIC.3. Detection of expresssion of SpoT and its production (p)ppGpp of Hp exposed to CLR.(1) Minimum inhibitory concentrations of CLR was used to treat the Hp26695, while a control group was not, after1h, RNA was extracted from these cells for QRT-PCR to detect the expression of the spoT.(2) Minimum inhibitory concentrations of CLR was used to treat the Hp26695,⊿spoT and spoT*tagged by32P for one generation and the control group was not, after lh, acid extracts were extracted and the (p)ppGpp was detected by spotted onto polyethyleneimine (PEI)-cellulose plates for separation by thin-layer chromatography (TLC).4. The survival ability and MIC change of Hp26695,△spoT and spoT*were compared through plate culture count, fluorescence staining and confocal microscopy and antibiotics sensitivity test by minimal inhibitory concentration (MIC).5. Analysis of gene differential expression between Hp26695and AspoT by mRNA microarray. The data of four groups which include the Hp26695and-⊿spoT treated with CLR and the others not treated was compared to forcast the downstream genes possibly regulated by SpoT.6. The gene differential expression from mRNA microarray were confirmed by the method of QRT-PCR. Minimum inhibitory concentrations of CLR was used to treat the Hp26695,⊿spoT and spoT*and the control group was not treated. After1h, RNA was extracted for QRT-PCR to detect the expression of the downstream genes forcasted by microarray.7. The survival ability and MIC change of mutant strain regulated by SpoT exposed to inhibitory concentrations of CLR was determined through plate culture count, fluorescence staining and confocal microscopy and antibiotics sensitivity test by minimal inhibitory concentration (MIC). RESULTS1. The spoT mutant strain(⊿spoT), kefB mutant strains(⊿kefB) and spoT complement strains(spoT*) were constructed successfully.2. SpoT and its production (p)ppGpp involved in the regulation of Hp stress reponse to CLR. Compared with control which was not treated with CLR, the expression level of gene spoT in Hp26695treated with inhibitory concentrations of CLR is much higher(P<0.05).Consistent with it, TLC analysis of nucleotide extracts clearly indicated that (p)ppGpp accumulated to significantly high levels in Hp26695and spoT*cells treated with inhibitory concentrations of CLR but no accumulation in⊿spoT.3. Hp was more sensitive when its spoT was mutated. After exposed to inhibitory concentrations of CLR for2hours, the survival ability of⊿spoT is pretty weak. Consistant with the result, the deletion of spoT resulted in the MIC of CLR reduced by50%compared with Hp26695.4. Membrane proteins may involved in CLR resistance. mRNA microarray analysis was performed according to the following plan:Hp26695no treated with CLR, Hp26695treated with CLR,⊿spoT no treated with CLR and⊿spoT treated with CLR which were analyzed by microarray. By the analysis of microarray’ data, about221genes were differention expression under the stress of CLR in Hp26695(Fold>3), and the largest proportion of these genes were transport proteins and membrane proteins. So our study focused on this part. In total,35genes coding transport proteins and membrane proteins changed tremendously under the stress of CLR in Hp26695, further analysis was done to find that13genes changed dramatically in Hp26695but not in⊿spoT strain under the same CLR stress condition.5. The efflux system protein KefB regulated by SpoT participated in the stress response of CLR. The expression level of all the13out membrane genes were confirmed by QRT-PCR, and the results showed that most of these genes may not be regulated by SpoT. and only glutathione-regulated potassium-efflux system protein KefB was regulated by SpoT directly.6. To explain the role of KefB in the process of CLR tolerance further, we constructed the kefB mutant(⊿kefB) and compared the difference of survival rate and MIC between Hp26695and⊿kefB by the methods of PCR absolute quantification, fluorescence staining and confocal microscopy and antibiotics sensitivity test by minimal inhibitory concentration (MIC). We found that the survival rate of⊿kefB strain dropped to1%or less after4h, but Hp26695still kept more than10%after8h. Consistent with the result, the deletion of kefB resulted in the reduction of MIC of CLR by25%compared with Hp26695.CONCLUSIONThe bifunctional enzyme SpoT and its production (p)ppGpp involved in the adaption of Hp to CLR pressure. The efflux system protein KefB was also proved to involve in the process of CLR resistance and its expression was regulated by SpoT. The high expression of efflux system protein KefB protected Hp from the damage of CLR, which laid the basis of time and material for further antibiotic resistance.