Identification and characterization of bacteriocins active against clinically important strains of Clostridium difficile

本项目的目的是从环境样品中鉴定梭菌和其他细菌产生的新型细菌素，通过对一系列临床相关的梭菌进行测试来确定其宿主范围。艰难梭菌分离株，包括超毒力PCR-核糖体型027菌株，并确定细菌素的输入机制和对敏感细胞的活性。该策略包括从环境样品，如污水，土壤材料和动物物质中分离孢子形成和非孢子形成细菌。然后通过生物活性测定法测试所有细菌分离株产生针对艰难梭菌菌株的活性细菌素的能力。生产微生物周围的抑制活性区域指示阳性结果，并且使用革兰氏染色、16 S RNA测序和全基因组测序进一步鉴定和表征生产微生物。通过基因组的计算机数据挖掘进一步研究细菌素在基因组中的存在，并将新的细菌素基因克隆到表达质粒中并表达重组细菌素。凝胶覆盖测定已经显示重组蛋白的活性。进一步的工作将涉及细菌素基因的工程突变，以确定生物活性的丧失，并确认与已知细菌素的活性相关性

The aim of this project is to identify novel bacteriocins produced by clostridia and other bacteria from environmental samples, establish their host-range by testing against a range of clinically relevant C. difficile isolates including the hypervirulent PCR-ribotype 027 strains and determine the mechanisms of bacteriocin import and activity against sensitive cells.The strategy has involved isolating spore-forming and non-spore forming bacteria form environmental samples such as sewage, soil material and animal matter. All bacterial isolates were then tested for the ability to produce active bacteriocins against strains of Clostridium difficile by a biological activity assay. Zones of inhibitory activity around the producing organisms were indicative of a positive result and the producing organisms were identified and characterised further using Gram stain, 16S RNA sequencing and whole genome sequencing. The presence of bacteriocins in the genome were investigated further by in silico data mining of the genome, and novel bacteriocins genes were cloned into expression plasmids and the recombinant bacteriocins were expressed. Gel overlay assays have shown activity of the recombinant proteins. Additional work will involve engineering mutations in the bacteriocin genes to determine loss of biological activity and confirm association of activity with the known bacteriocin