抑制性消减杂交结合基因芯片研究淋球菌耐头孢曲松的分子机制

Identification of genes related to induced resistance to ceRriaxone in Neisseria gonorrhoeae using suppression subtractive hybridization and DNA microarray

目的探讨体外人工诱导的耐头孢曲松淋球菌的分子耐药机制。方法在成功诱导淋球菌标准菌株ATCC49226和临床菌株ZSSY00205对头孢曲松耐药的基础上，分别对标准菌株和临床菌株进行诱导后耐药株对诱导前敏感株的抑制性消减杂交，构建差异基因文库。从文库中随机挑取192个差异基因作为探针点样于基因芯片，用分别标记Cy3、Cy5的敏感株、耐药株基因组DNA的RsaI酶切片段同时与芯片杂交，根据芯片扫描图选取差异荧光探针对应的基因进行测序和Blast分析。结果分别构建淋球菌标准菌株ATCC49226和临床菌株ZSSY00205的诱导后耐药株DNA特异性的消减文库，并分别获得高分辨的基因芯片扫描图，发现两组菌株间有共同的耐药相关基因mtrR、mtrC、gyrB、rpsJ、PJD1。结论淋球菌对头孢曲松耐药与mtrR、mtrC、gyrB、rpsJ等基因有关，很可能通过外排泵活性增强的途径介导耐药。伴随淋球菌对头孢曲松产生耐药的同时，可能引起对青霉素、四环素、红霉素、喹诺酮等多种抗菌药物产生耐药，即多重耐药现象。

Objective To elucidate the molecular basis for induced resistance ofN. gonorrhoeae to ceftriaxone in vitro. Methods The reference strain ATCC49226 and clinical isolate ZSSY00205 of N. gonorrhoeae were exposed to subinhibitory concentration of ceftriaxone for the induction of resistance. Then, suppression subtractive hybridization was performed with the pre-induction parent strains as drivers and post-induction mutant strains as testers to create a subtractive cDNA library. Following that, a total of 192 clones were randomly selected from the library, and arrayed by spotting onto nylon membranes. Finally, differentially expressed genes were screened by hybridization with labeled-RsaI restriction fragments from the sensitive and resistant N.gonorrhoeae strains respectively, and analyzed by sequencing and homology research using Blast program. Results A subtractive library for these resistant N.gonotrhoeae strains was generated by SSH technique. Microarray analysis and homology research confirmed 5 genes related to ceftriaxone resistance, i.e. mtrR, mtrC, gyrB, rpsJ and PJD1. Conclusions The induced resistance ofN. gonorrhoeae to ceftriaxone may be associated with mtrR, mtrC, gyrB, rpsJ and PJD1 genes which probably mediate the resistance by enhancing the activity of efflux pump system.