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

金黄色葡萄球菌是一种引起广泛疾病的细菌病原体，并且因其对抗生素的耐药性而臭名昭著。耐甲氧西林沙门氏菌感染。金黄色葡萄球菌菌株（MRSA）经常与医院相关，通常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，这些突变不可避免地赋予对万古霉素的中间抗性（VISA表型）和对达托霉素的完全抗性。还已经证明，这些菌株也更好地保护免受构成先天免疫系统的天然抗生素的阳离子肽（例如，G.抗微生物肽LL-37）。本项目将对S.金黄色葡萄球菌VC 40，其显示对两种抗生素的完全抗性（万古霉素MIC：64 μg/ml和达托霉素MIC：4 μg/ml）。该菌株的全基因组测序显示编码组氨酸激酶VraS和WalK的基因发生突变。这两种蛋白质都位于细胞膜上，作为传感器蛋白发挥作用，响应迄今为止尚未识别的信号，控制细胞包膜相关过程。基因表达谱分析表明，在菌株VC 40中由这些激酶调节的基因的表达增加。将VraS激酶（VraS（VC 40））中存在的突变重建到易感的S.金黄色葡萄球菌NCTC 8325背景导致对万古霉素、替考拉宁和达托霉素的抗性显著增加，MIC超过这些抗生素的折点，从而产生VISA（万古霉素中间抗性S. aureus）菌株。S. aureus VC 40以及菌株NCTC 8325的vraS突变体的特征在于细胞壁增厚和自溶活性降低。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，该项目将分析菌株VC 40及其后代、临床分离株以及过表达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，将研究控制胞壁素的周转的自溶酶的活性及其在抗性中的作用，以及菌株的膜和细胞外蛋白质组成。该项目将试图提出一个全面的VISA细胞壁结构和代谢模型，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.