Analysis of cell wall architecture and metabolism of a vancomycin resistant Staphylococcus aureus strain

金黄色葡萄球菌是一种细菌病原体，可导致广泛的疾病，并以其对抗生素的耐药性而臭名昭著。感染耐甲氧西林金黄色葡萄球菌(MRSA)通常与医院相关，通常使用糖肽抗生素万古霉素或替考拉宁或脂肽抗生素达托霉素治疗。不幸的是，MRSA菌株能够获得突变，同时对万古霉素产生中等抗药性(VISA表型)和对达托霉素产生完全抗药性。研究还表明，这些菌株对构成天然免疫系统抗生素的阳离子多肽(如抗菌肽LL-37)也有更好的保护作用。该项目将分析对两种抗生素(万古霉素MIC：微克/毫升和达托霉素MIC：4微克/毫升)完全耐药的金黄色葡萄球菌VC40的细胞壁结构。对该菌株的全基因组测序显示，编码组氨酸激酶VraS和Walk的基因发生了突变。这两种蛋白都位于细胞膜上，作为传感器蛋白，对迄今尚未识别的信号做出反应，控制与细胞包膜相关的过程。基因表达谱显示，在菌株VC40中，受这些激酶调控的基因表达增加。VraS激酶(VraS(VC40))突变被重组到敏感的金黄色葡萄球菌NCTC 8325背景中，导致对万古霉素、替考拉宁和达托霉素的耐药性显著增加，MIC超过了这些抗生素的临界点，从而产生了VISA(万古霉素中间耐药金黄色葡萄球菌)菌株。金黄色葡萄球菌VC40和菌株NCTC8325的vraS突变体具有细胞壁增厚和自溶活性降低的特点。为了进一步描述这种耐药机制，并深入了解VraSR和WalRK系统对细胞壁结构的影响，该项目将分析VC40菌株及其后代、临床分离株以及高表达VraSR和WalRK的菌株中细菌细胞壁聚合物肽聚糖、磷壁酸和脂磷壁酸的组成。此外，还将研究控制毛霉素酶翻转的自溶酶的活性及其在抗药性中的作用，以及菌株的膜和胞外蛋白组成。该项目将试图展示VISA细胞壁结构和新陈代谢的全面模型，由VraSR和WalRK介导。

Staphylococcus aureus is a bacterial pathogen that causes a broad range of diseases and that is notorious for its resistance to antibiotic agents. Infections with methicillin-resistant S. aureus strains (MRSA), which are frequently hospital-associated, are often treated with the glycopeptide antibiotics vancomycin or teicoplanin or the lipopeptide antibiotic daptomycin. Unfortunately, MRSA strains are able to acquire mutations that simultanteously confer intermediate resistance to vancomycin (VISA phenotype) and full resistance to daptomycin. It has also been demonstrated that these strains are also better protected against the cationic peptides which constitute the natural antibiotics of the innate immune system (e. g. the antimicrobial peptide LL-37). The project will analyse the cell wall structure of S. aureus VC40 which shows full resistance to both antibiotics (vancomycin MIC: 64 µg/ml and daptomycin MIC: 4 µg/ml). Full genome sequencing of this strain revealed mutations in genes encoding the histidine kinases VraS and WalK. Both proteins are located in the cell membrane, and function as sensor proteins which, in answer to signals that have not been identified so far, control cell envelope related processes. Gene expression profiling indicated an increased expression of the genes that are regulated by these kinases in strain VC40. Reconstitution of the mutation present in the VraS kinase (VraS(VC40)) into the susceptible S. aureus NCTC 8325 background resulted in a considerably increased resistance to vancomycin, teicoplanin and daptomycin with MICs surpassing the breakpoints for these antibiotics, thereby generating a VISA (vancomycin intermediary resistant S. aureus) strain. S. aureus VC40 as well as the vraS mutant of strain NCTC 8325 are characterized by a thickened cell wall and a reduced autolytic activity. In order to describe this resistance mechanism further and obtain insight into the influence of the VraSR and WalRK system on cell wall structure, the project will analyse the composition of the bacterial cell wall polymers peptidoglycan, teichoic acid and lipoteichoic acid in strain VC40 and its descendants, clinical isolates, as well as strains that overexpress VraSR and WalRK. Furthermore, the activity of the autolytic enzymes that control the turn-over of the murein and their role in resistance will be investigated as well as the membrane and extracellular protein composition of the strains. The project will attempt to present a comprehensive model of VISA cell wall architecture and metabolism as mediated by VraSR and WalRK.