WalKR负调节耐甲氧西林金黄色葡萄球菌对阿米卡星适应性耐药的机制研究

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen and superbug. Antibiotic resistance of MRSA can be achieved by gene mutation or horizontal acquisition of resistance genes, meanwhile MRSA can adaptively resist to antibiotics by regulating its metabolism. We have previously reported that the cell wall thickening is an important cause for MRSA adaptively resist to amikacin, using two MRSA isolates isolated from one patient. However, the exactly molecular mechanism of the MRSA adaptive resistance to amikacin through cell wall thickening is unclear. The two-component system, WalKR, is essential for the viability of S. aureus, which plays a central role in controlling cell wall metabolism. In our previous study, we found that WalKR activity was decreased in amikacin resistant MRSA isolate, and we suppose that MRSA strains respond to amikacin stress by downregulating the expression of walKR genes, which leaded to the switch of bacterial central metabolism from anabolism of amino acids to purine/pyrimidine biosynthesis, and resulted in the increase of pools of UDP N-acetylglucosamine (UDP-GlcNAc), the important material for bacterial cell wall synthesis, to cause the cell wall thickeness in MRSA with amikacin resistance. Therefore, in the present study, we plan to use the gene expression profiles to analyze the differential expression genes between the two MRSA strains.Then we will construct walKR mutant strains and the gene complementstrains to test the expression of genes among amino acids and purine/ pyrimidine metabolisms by quantitative reverse transcription-PCR. The results of this project will elucidate the molecular mechanism of MRSA adaptive resistance to amikacin by cell wall thickening, and will provide a new way to combat MRSA infections.

耐甲氧西林金黄色葡萄球菌(MRSA)是重要的超级耐药病原菌。MRSA除发生基因突变和获得外源耐药基因形成获得性耐药菌外，也可通过调节自身的代谢产生适应性耐药。申请者前期对分离自同一患者的两株MRSA菌研究发现细胞壁增厚与MRSA对阿米卡星的适应性耐药有关，但机制不清。WalKR是调节细菌中心代谢的二元调控元件，前期发现阿米卡星适应性耐药菌walKR的表达降低，推测MRSA对阿米卡星的应急反应导致walKR表达下调，使中心代谢由氨基酸合成向核酸合成偏移，肽聚糖合成前体UDP-GlcNAC增加，细胞壁增厚。为此，本项目拟采用表达谱分析法探讨阿米卡星敏感MRSA和适应性耐药菌的基因表达差异；再通过构建walKR基因突变菌及回补菌，检测氨基酸代谢和核酸代谢关键酶的表达变化及对细胞壁合成的影响，阐明耐甲氧西林金黄色葡萄球菌对阿米卡星适应性耐药的分子机制，为寻找控制MRSA感染的新途径奠定基础。

耐甲氧西林金黄色葡萄球菌(MRSA)是引起医院和社区获得性感染的重要病原菌，其耐药性的快速增长使得MRSA感染面临无药可治的困境，MRSA耐药性可以分为获得性耐药和适应性耐药。本项目对乌鲁木齐地区金黄色葡萄球菌临床分离株的分子特征及其对临床常用抗生素的耐药情况进行了研究，同时对MRSA菌株通过细胞壁增厚从而对阿米卡星产生适应性耐药的具体分子机制进行了探讨。结果显示：（1）乌鲁木齐地区主要的MRSA流行克隆为ST239-t030-SCCmec III，共鉴定出197株，占75.48%(197/261)，以及ST59-t437-SCCmec IV型克隆，共鉴定出30株，占11.49%(30/261)；主要流行的MSSA克隆为ST22-t309(25.33%,77/304),该克隆主要在新疆地区流行。（2）表达谱测序结果分析显示，在阿米卡星存在下，MRSA菌株的嘧啶嘌呤代谢加强，金葡菌的中心代谢由氨基酸合成向核酸合成偏移，细胞壁肽聚糖合成的前体UDP-GlcNAC增加，细胞壁合成相关基因mraY,murF,murD基因表达上调，细胞壁合成加速，出现细胞壁增厚；可抑制细胞壁水解酶的活性的LrgA和LrgB蛋白表达增强，降低细菌自溶素的表达，有利于细胞壁增厚。还发现VraSR二元调控系统表达增高，提示VraSR二元调控系统在金黄色葡萄球菌通过细胞壁增厚对抗生素适应性耐药发挥重要的作用。

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