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Disulfide bond formation

The formation of disulfide bonds in proteins is an oxidative process that generates a covalent bond linking the sulfur atoms of two cysteine residues. Disulfide bond formation has a central role in protein folding of both eukaryotes and prokaryotes.

Disulfide-bonded proteins are located in more oxidizing environments such as the bacterial cell envelope. In Escherichia coli the enzymes involved in disulfide bond formation are the periplasmic thioredoxin homologue DsbA, which introduces disulfide bonds into substrate proteins, and the membrane protein DsbB, which maintains DsbA active by oxidizing it.

three pathways of disulfide bond formation

Most Proteobacteria (like Pseudomonas aeruginosa) use DsbA- and DsbB-like proteins to form disulfide bonds in exported proteins. However, Actinobacteria (like Mycobacterium tuberculosis), Cyanobacteria and aerobic δ-proteobacteria maintain DsbA oxidized not by DsbB, but by the protein VKOR, a homologue of human Vitamin K epoxide reductase.

In bacteria, disulfide bonds play a role in the folding and stability of proteins involved in important cellular processes such as cell division, assembly of the outer membrane and virulence of gram-negative bacteria. Therefore, the pathways that introduce disulfide bonds into proteins represent good targets for the development of new antibacterial therapies.

The goal of our research is to use E. coli as a tool to characterize the pathways that introduce disulfide bonds in pathogens as well as to find inhibitors of this process that is crucial for growth and virulence of pathogenic bacteria for which new antibacterial agents are most desperately needed.

We have developed a method to look for molecules that could be used as antibiotics, antivirulence therapeutics or anticoagulants using E. coli cells. After several rounds of screening of several synthetic small molecule libraries we have found few candidate inhibitors of the DsbB and VKOR enzymes of pathogenic bacteria. We will continue searching for more effective inhibitors of DsbB and VKOR as well as to ellucidate their mechanisms of inhibition. We are interested in expanding our inhibitor search using natural products libraries and molecules derived from the microbiome given their chemical diversity.

Here are the steps we took in our previous high throughput screenings (HTS) to find inhibitors of the disulfide bond forming enzymes of Pseudomonas aeruginosa and Mycobacterium tuberculosis and the steps to characterize them. We will apply similar strategies using more diverse libraries.

screening steps

The goal of this project is to enable the systematic identification of substrates of the disulfide bond formation pathway of two human pathogens, Pseudomonas aeruginosa and Mycobacterium tuberculosis. We will identify the proteins that interact with DsbA using two model organisms, P. aeruginosa and M. smegmatis. Our focus will be the study of substrates that are required for virulence and growth of these two microorganisms.

Research

Disulfide bond formation

Antimicrobial screenings

Identification of DsbA substrates

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