Structure and catalytic function of Pseudomonas aeruginosa secreted proteins

Secreted virulence factors are ideal targets for novel antimicrobials: they are readily accessible to externally applied chemicals.

Bacteria secrete proteins that specifically target and kill other bacteria (Bacteriocins): potentially useful novel antimicrobials.

Both types of protein could serve as a chassis to export proteins for biotechnological uses thereby reducing downstream purification.

This project seeks to characterize the structure and function of Pseudomonas aeruginosa virulence factors. P. aeruginosa is a major opportunistic pathogen for CF, bronchiectasis, COPD and wound patients, is intrinsically resistant to antibiotics, and almost impossible to eradicate from hospitals in part due to its ability to grow as biofilm communities.

Pyocin S3 is a bacteriocin produced by P. aeruginosa that kills other species of P. aeruginosa. A cognate immunity protein binds to the pyocin in the producing cell to prevent suicide. We have shown that pyocin S3 is a DNase, and have solved the crystal structure of the DNase domain in complex with its cognate immunity protein, revealing its uncharacteristic primary sequence still maintains an active site beta-beta-alpha secondary structure motif without the typical DNase HNH arrangement. In addition, a novel octopus grasp of the DNAse by the cognate immunity protein was discovered.

This project aims to purify and crystallize:

1. Pyocin S3 DNase in its native form with the receptor binding and translocation domains attached
2. Pyocin S3 DNase domain without its cognate Immunity protein to reveal the presence of bound metal ion at the active site motif. The purified DNase will then be used to investigate the mode of action of DNA catalysis.
3. The Pyocin S3 cognate Immunity protein.

Site directed mutagenesis will be used to inactivate residues predicted to be implicated in the function to increase our understanding of their functional domains.