Disulfide bond formation in Pseudomonas species

Disulfide bonds are ubiquitous covalent linkages, essential for the stability of hundreds of proteins in the harsh extracytoplasmic environment. Despite their simplicity, they are formed by dedicated protein systems in all organisms. In Gram-negative bacteria, the formation of these bonds is catalysed by the Disulfide bond (DSB) formation system which is also responsible for folding most factors that enable bacteria to be efficient pathogens. More importantly, the DSB systems of pathogens are divergent compared to non-pathogenic bacteria as they contain multiple copies of key DSB players. We propose that this understudied adaptation of the DSB system allows bacteria to efficiently handle protein substrates related to pathogenesis and in-host survival.

Pseudomonas aeruginosa is an opportunistic pathogen which is resistant to most available antibiotics, causing financial stress to healthcare systems worldwide. Preliminary bioinformatic analysis of genomes from Pseudomonas species clearly revealed the presence of divergent DSB systems. We are interested in understanding the role of the DSB proteins in the mechanisms of virulence and antibiotic resistance of Pseudomonas species, with special interest in the model pathogen P. aeruginosa.

The assessment of the role of the DSB proteins in Pseudomonas species will be based on a three-pronged approach. Firstly, we will use bioinformatics analysis to screen and characterise the DSB system in all Pseudomonas species. There is a breadth of sequenced genomes for Pseudomonas, including longitudinal samples obtained from cystic-fibrosis patients. We will take advantage of this to determine the composition of the DSB system in this bacterium, but also to determine if this composition if affected by chronic infections in the human host. Secondly, we will investigate the contribution of the DSB proteins in the folding and stability of protein substrates involved in antimicrobial resistance. Several protein pathways that endow bacteria with the ability to break down commonly used antibiotics are localised in the periplasm of Gram-negative bacteria, which is the cellular compartment the DSB system acts in. We will investigate if the folding of these proteins is dependent on DsbA, the DSB protein responsible for disulfide bond formation. Positive results on this front could lead to new ways of re-sensitisation of clinical strains to existing antibiotics. Finally, we will use gene reporter fusions and appropriate deletion strains to characterise the role of the divergent protein members of the DSB system in P. aeruginosa. We will characterise the effects of gene deletion on bacterial virulence and pathogenesis by phenotypic assays and by using the Galleria mellonella model. In this way we will understand the role of these proteins for Pseudomonas virulence and evaluate whether they are promising targets for the development of novel antibacterial strategies.

In addition to standard biochemical and microbiological techniques, this project will require specialised cysteine-chemistry assays as well as phenotypic assays on motility, biofilm formation, type VI secretion and overall virulence. This breadth of techniques will allow for an interdisciplinary approach where basic protein biochemistry, structure-to-function studies and microbiology experiments could lead to the development of potential organism-specific antibacterial strategies.