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

艰难梭菌是一种院内致病菌，可引起腹泻，发病率/死亡率高，医疗费用高。抗生素治疗引起的肠道微生物失衡是导致孢子萌发和毒素产生的关键因素。艰难梭菌感染的基础是这种细菌对许多抗生素的内在抗药性，包括以合成肽聚糖为目标的β-内酰胺类药物(如头孢菌素)，肽聚糖是细菌细胞膜的基本成分。在艰难梭菌中，肽聚糖的合成基本上依赖于一组名为L的酶，即D-转肽酶(LDT)，在其他病原体中与β-内酰胺耐药有关。因此，我们假设艰难梭菌对β-内酰胺类抗生素的广泛耐药性在很大程度上可以归因于这组酶。我们的目的是用多学科的方法探索L，D转肽通路在艰难梭菌中对细菌生长和耐药性的贡献。我们建议通过基因置换的方法构建突变体，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。为了进一步了解艰难梭菌LDT的个体作用，我们将在体外研究它们的酶活性，并通过X射线结晶学来探索它们的结构特性。最后，在我们对艰难梭菌模型菌株LDT功能的详细分析的基础上，我们将探索循环临床菌株中PG结构的自然变异，并确定这与一系列β-内酰胺类抗生素的耐药性谱有何关系。这个项目将进一步加深我们对艰难梭菌感染机制的理解，并产生知识来开发针对这种关键医院病原体的新疗法。

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.