脂磷壁酸合成途径在万古霉素中介金黄色葡萄球菌向高耐药菌转化中的作用与机制

Vancomycin has traditionally been reserved as a drug of last resort to treat the infections caused by Staphylococcus aureus. Recently, the discovery and epidemic of heterogeneous vancomycin-intermediate S. aureus (hVISA), vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) have brought a great challenge to clinical treatment. Most studies have demonstrated that the mutations in some specific genes are associated with the vancomycin resistance in S. aureus, however, the mechanisms underling the transformation of VISA to VRSA remain to be elucidated. We previously isolated and characterized a VISA strain XN108 (MIC=12 μg/ml), and the transposon mutant library experiments revealed that the mutation of ypfp or ltaA gene both responsible for the biosynthesis of staphylococcal lipoteichoic acid (LTA) has resulted in an increasing of vancomycin resistant phenotype from the VISA to VRSA, XN24+ with a vancomycin MIC value of 24 μg/ml and XN36+ with an MIC of 36 μg/ml. We suppose that the disruption of LTA biosynthesis may affect the expression and recruitment of PBP4, an important enzyme for peptidoglycan biosynthesis, to the division septum and the subsequently decreasing of peptidoglycan cross-linking enhances the vancomycin resistant phenotype. To test this hypothesis, the present project aims to reveal the defects of LTA synthetic pathway in XN108, XN16+, XN24+ and XN36+ through whole genome sequencing and LTA detection. The gene knock-out, replacement and complement experiments will be performed to test the effects of ypfP and ltaA genes on the expression and site-specific position of PBP4. The diversities of peptidoglycan cross-linking in XN108 and its variants will be determined using mass chromatographic analysis and vancomycin binding experiment. The elucidation of roles and mechanisms of LTA biosynthesis pathway in the transformation of VISA to VRSA will lay down a great foundation in the development of new strategies to control infections caused by vancomycin resistant S. aureus.

万古霉素是临床治疗金葡菌感染的最后防线。近年来，异质性、中介及万古霉素全耐药金葡菌（hVISA,VISA,VRSA）的发现和流行给临床治疗带来了严峻挑战。研究表明，特定基因的突变与万古霉素耐药密切相关，但尚未见对VISA向VRSA转化的研究报道。我们前期分离并鉴定了一株VISA，转座子突变文库研究发现当该菌脂磷壁酸（LTA）合成功能酶YpfP或LtaA突变后，细菌万古耐药性明显增强，推测LTA合成异常，影响了胞壁合成功能酶PBP4的表达和定位，使肽聚糖交联度下降而呈现高耐药性。本项目拟通过基因组序列分析和LTA检测，明确LTA合成途径的缺损；利用基因敲除、基因替换和回补技术研究YpfP、LtaA等对金葡菌PBP4表达和定位的影响；再通过万古霉素结合实验和质谱分析探讨不同突变菌细胞壁交联度的变化，阐明LTA合成途径在VISA向VRSA转化中的作用与机制，为寻找控制金葡菌感染的新途径奠定基础。

研究表明，特定基因的突变与万古霉素中介耐药金葡菌（VISA）的形成密切相关。然而，VISA菌具有细胞壁增厚、自溶能力下降等共同表型，上游众多基因突变如何介导耐药菌共同表型的产生一直不清。本研究首先对XN108及其耐药性升高突变株XN16+、XN24+、XN36+进行了全基因组测序和比较基因组学分析，证实GtaB(G205D)、YpfPΔC117及LtaAΔC192突变的存在，还发现一个新WalK(S221P)突变可能与VISA表型形成有关。为证实脂磷壁酸（LTA）合成途径在VISA耐药性升高中的作用，我们构建了N315-ΔgtaB，N315-ΔypfP和N315-GtaB (G205D)等LTA合成相关基因的突变株，耐药性检测发现gtaB和ypfP突变不是引起菌株万古霉素耐药性升高的主要原因。接着对WalK(S221P)在金葡菌万古霉素耐药性升高中的作用和机制进行了研究。发现将该突变引入N315则可使菌株耐药性显著升高，而将XN108中的WalK(S221P进行回复，则菌株耐药性下降，同时伴随细胞壁厚度下降，自溶能力恢复等变化；WalK(S221P)突变使其自磷酸化能力降低，对WalR的激活能力下降，调控靶基因能力随之降低，出现VISA耐药性升高表型；进一步研究发现，WalKR可通过CcpA调控分子调控VISA的耐药性，WalK(S221P)突变使WalR结合CcpA基因启动子活性下降，CcpA表达上调，促进金葡菌细胞壁合成，导致细胞壁增厚，菌株出现VISA表型；抑制CcpA的活性可使VISA菌株恢复对万古霉素的耐药性，为VISA感染的控制提供了新的靶点。发表论文6篇，其中SCI收录期刊论著5篇，获科技进步奖1项，参加学术会议及受邀报告7次，培养博士生2名，硕士生1名。