Dissecting the mechanism and regulation of bacterial secreted peptidases and their role in biofilms
Lead Research Organisation:
University of St Andrews
Department Name: Biology
Abstract
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.
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.
Technical Summary
Pseudomonas aeruginosa is an opportunistic pathogen that causes serious illness, especially in immunocompromised individuals, including those with cystic fibrosis. P. aeruginosa extensively forms biofilms which significantly contribute to its growth and persistence in a wide range of environments. Its success can be attributed to several secreted proteases and other virulence factors.
This project will dissect the mechanism of the aminopeptidase PaAP from P. aeruginosa, which is one of the most abundant extracellular proteins in the biofilm matrix. Prior work revealed that bacteria lacking PaAP die in late stage biofilms. We designed inhibitors that specifically target PaAP, killing cells in biofilms. The precise role PaAP is playing in biofilms that leads to bacterial death, however, remains undetermined, as well as PaAP detailed mechanism and substrate preference.
Additionally, PaAP carries a conserved "protease associated domain", with a proposed role in substrate selection and enzyme activation. We will study other proteins that contain PA domains, and determine their impact on activity, inhibition with peptide analogues, and on biofilms.
Our work will be impactful, answering the outstanding questions below:
1- What makes a potent and stable inhibitor for an extracellular peptidase such as PaAP?
2- What defines the kinetics and specificity of peptide cleavage?
3- Is the PA domain in other proteins playing a similar role, and can this be used to inhibit peptidases from other bacteria?
4- What causes cell death in biofilms when PaAP is inhibited?
This project will dissect the mechanism of the aminopeptidase PaAP from P. aeruginosa, which is one of the most abundant extracellular proteins in the biofilm matrix. Prior work revealed that bacteria lacking PaAP die in late stage biofilms. We designed inhibitors that specifically target PaAP, killing cells in biofilms. The precise role PaAP is playing in biofilms that leads to bacterial death, however, remains undetermined, as well as PaAP detailed mechanism and substrate preference.
Additionally, PaAP carries a conserved "protease associated domain", with a proposed role in substrate selection and enzyme activation. We will study other proteins that contain PA domains, and determine their impact on activity, inhibition with peptide analogues, and on biofilms.
Our work will be impactful, answering the outstanding questions below:
1- What makes a potent and stable inhibitor for an extracellular peptidase such as PaAP?
2- What defines the kinetics and specificity of peptide cleavage?
3- Is the PA domain in other proteins playing a similar role, and can this be used to inhibit peptidases from other bacteria?
4- What causes cell death in biofilms when PaAP is inhibited?
