Identifying protease drug targets to combat ESKAPE pathogen infections

Bacterial proteases, although not always essential for growth, regulate virulence, stress responses, and biofilm formation, making them attractive anti-virulence drug targets. The aim of this thesis was to investigate the roles of LonA protease, the Clp proteolytic system (ClpA and ClpS), and C13 legumain-like peptidases in Acinetobacter baumannii (A. baumannii) and Pseudomonas aeruginosa (P. aeruginosa) to evaluate their roles in virulence, biofilm formation, stress response, and their potential as anti-virulence drug targets.<br><br>In the first experimental chapter, the C13 protease of P. aeruginosa (C13-PA) was cloned and expressed in E. coli.

Unlike the homologous enzyme in A. baumannii (C13-AB), which was confirmed to be active, C13-PA showed no detectable protease activity, suggesting divergence in substrate specificity, regulation, or function.<br><br>The second chapter assessed A. baumannii transposon mutants lacking LonA, ClpA, ClpS, or C13-AB. Growth was unaffected; however, LonA and ClpA mutants displayed significantly reduced biofilm formation and attenuated virulence in the Galleria mellonella model, confirming LonA and the Clp proteostasis system as important contributors to pathogenicity.<br>The third chapter focused on LonA as a drug target.