Identification of multiple serine hydrolases involved in virulence and cell envelope integrity of Klebsiella pneumoniae

Klebsiella pneumoniae is a resident of the human gastro-intestinal tract and an opportunistic, critical priority pathogen that can cause severe systemic infections. To overcome emerging multi-drug resistance, discovery and validation of novel targets for developing new treatment options is essential. Here, we explored the highly druggable and functionally diverse enzyme family of serine hydrolases in K. pneumoniae . Using functionalized fluorophosphonate inhibitors as probes we identified 10 serine hydrolases by mass spectrometry-based activity-based protein profiling, 7 of which were previously uncharacterized. Functional validation using transposon mutants deficient in either the putative lysophospholipase PldB, esterase YjfP, or patatin-like phospholipase YchK revealed reduced virulence during Galleria mellonella infection and growth defects in co-culture with human colonic organoids. Mutants deficient in YjfP and the periplasmic protease DegP showed increased susceptibility for serum-killing and an antimicrobial peptide antibiotic, suggesting/confirming a role in maintaining cell envelope integrity. Biochemical characterization and structural analysis of YjfP suggest this protein is an acetyl esterase. This study gives important insights into the molecular mechanisms underlying virulence of K. pneumoniae and its cell physiology at the host interface. It positions several of these hydrolases as anti-virulence or antimicrobial target candidates, inhibition of which might synergize with antibiotics and human immune defenses.