Analysis of the L,D transpeptidation pathway in Clostridium difficile: contribution to peptidoglycan synthesis and antibiotic resistance

艰难梭菌是一种引起腹泻的医院病原体，与高发病率/死亡率和高医疗保健成本相关。抗生素治疗引起的肠道微生物失衡是孢子萌发和产生毒素导致疾病的关键因素。C.因此，艰难梭菌感染的基础是该细菌对许多抗生素的内在抗性，所述抗生素包括靶向肽聚糖（细菌细胞包膜的基本组分）合成的β-内酰胺类（如头孢菌素）。In C.在艰难梭菌中，肽聚糖合成基本上依赖于称为L，D-转肽酶（Ldts）的酶家族，其与其他病原体中的β-内酰胺抗性相关。因此，我们假设，广泛的耐药性的C。对β-内酰胺类抗生素的抗性很大程度上可归因于这组酶。我们的目的是探讨L，D转肽途径对细菌细胞生长和耐药性的影响。使用多学科的方法来解决困难。我们建议通过基因置换构建突变体，以研究这些酶在细胞生长和分裂过程中的作用，并阐明Ldts活性对肽聚糖结构和β-内酰胺抗性的影响。为了进一步了解C. difficile Ldts，我们将在体外研究其酶活性，并通过X射线晶体学探索其结构特性。最后，基于我们对模型C中Ldt功能的详细分析。艰难梭菌菌株，我们将探索循环临床菌株中PG结构的自然变异，并确定这与对一系列β-内酰胺类抗生素的耐药性谱的关系。这一项目将进一步加深我们对C.艰难的感染，并产生知识，以开发新的治疗方法，对这一关键的医院病原体。

Clostridium difficile is a nosocomial pathogen causing diarrhoea associated with high morbidity/mortality and a high healthcare cost. The microbial imbalance in the gut caused by antibiotic treatment is a key factor for the germination of spores and the production of toxins causing the disease. C. difficile infections are therefore underpinned by the intrinsic resistance of this bacterium to many antibiotics including beta-lactams (such as cephalosporins) that target the synthesis of peptidoglycan, the essential component of the bacterial cell envelope. In C. difficile, peptidoglycan synthesis essentially relies on a family of enzymes called L,D-transpeptidases (Ldts) that have been associated with beta-lactam resistance in other pathogens. We therefore hypothesise that the widespread resistance of C. difficile to beta-lactams can, in large part, be attributed to this group of enzymes. Our aim is to explore the contribution of the L,D transpeptidation pathway to bacterial cell growth and antibiotic resistance in C. difficile using a multidisciplinary approach. We propose to construct mutants by gene replacement to investigate the role of these enzymes during cell growth and division and elucidate the impact of Ldts activities to peptidoglycan structure and beta-lactam resistance. To gain further insights into the individual role of C. difficile Ldts, we will study their enzymatic activity in vitro and explore their structural properties by X-ray crystallography. Finally, building upon our detailed analysis of Ldt functionality in a model C. difficile strain, we will explore the natural variation in PG structure in circulating clinical strains and determine how this relates to the spectrum of resistance to a range of beta-lactam antibiotics. This project will further our understanding of the mechanism underpinning C. difficile infections and generate knowledge to develop new treatments against this key hospital pathogen.