Dissecting the mechanism and regulation of bacterial secreted peptidases and their role in biofilms

点击翻译按钮获取中文摘要

Bacterial communities often organise in structures called biofilms. These communities are associated with a self-produced matrix containing extracellular DNA, RNA, proteins and complex sugars. Biofilms are resilient to environmental stressors including nutrient fluctuation, antibiotic treatment, and changes in temperature. Biofilm resilience is vital to the bacterial collective but presents a colossal economic and health challenge as bacteria in these populations are difficult to eradicate. Up to 4 out of 5 of all bacterial cells in the planet are associated in biofilms. A report by the NIH indicates that over 80% of microbial infections in the human body are caused by biofilms, many of which are resistant to standard antibiotics. Pseudomonas aeruginosa is a model organism to understand biofilm development and its role in survival and recalcitrance to antibiotic treatment. It can also lead to serious infections specially in immunocompromised patients. No drug currently in the market specifically targets bacterial biofilms. All drugs currently in the market target actively dividing bacteria, and therefore are not as efficient eradicating slow growing bacteria, which is often the state encountered in biofilms. Extracellular enzymes that can degrade proteins and peptides are important components of biofilms. These enzymes are crucial for nutrient scavenging and to shape the structure of biofilms, in a process called remodelling, which is essential for biofilm survival and propagation. Extracellular enzymes are very attractive targets as antimicrobials do not need to enter cells and can be embedded in dressings and topic gels. The Czekster lab have demonstrated proof of principle that targeting an important extracellular component of the bacterial matrix leads to bacterial cell death in biofilms formed by the Pseudomonas aeruginosa. This work demonstrated the feasibility of exploiting the mechanism by which the activity of extracellular peptidases is regulated to design specific inhibitors, which lead to cell death in a biofilm. It also highlighted many unanswered questions in biofilm biology, regulation and survival. It set the stage to the project proposed here. This project will address outstanding questions in biofilm biology and enzymology, dissecting the mechanism and role that extracellular peptidases play in protein and peptide turnover in biofilm communities. We will determine whether a conserved element of these extracellular peptidases, the protein associated domain, can be exploited to target peptidases from other microbes. We will also determine the precise mechanism by which Pseudomonas aeruginosa are dying due to the peptidase inhibition. We will employ enzymology, inhibitor design and characterisation, microbiology, proteomics and metabolomics, in a tailored interdisciplinary programme to unveil novel strategies and compounds to specifically target bacterial biofilms.