Loss of membrane‐bound lytic transglycosylases increases outer membrane permeability and β‐lactam sensitivity in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial infections. Its relatively impermeable outer membrane (OM) limits antibiotic entry, and a chromosomally encoded AmpC β-lactamase inactivates β-lactam antibiotics. AmpC expression is linked to peptidoglycan (PG) recycling, and soluble (sLT) or membrane-bound (mLT) lytic transglycosylases are responsible for generating the anhydromuropeptides that induce AmpC expression. Thus, inhibition of LT activity could reduce AmpC-mediated β-lactam resistance in P. aeruginosa. Here, we characterized single and combination LT mutants. Strains lacking SltB1 or MltB had increased β-lactam minimum inhibitory concentrations (MICs) compared to wild type, while only loss of Slt decreased MICs. An sltB1 mltB double mutant had elevated β-lactam MICs compared to either the sltB1 or mltB single mutants (96 vs. 32 μg/mL cefotaxime), without changes to AmpC levels. Time-kill assays with β-lactams suggested that increased MIC correlated with a slower rate of autolysis in the sltB1 mltB mutant - an antisuicide phenotype. Strains lacking multiple mLTs were more sensitive to β-lactams and up to 16-fold more sensitive to vancomycin, normally incapable of crossing the OM. Multi-mLT mutants were also sensitive to bile salts and osmotic stress, and were hyperbiofilm formers, all phenotypes consistent with cell envelope compromise. Complementation with genes encoding inactive forms of the enzymes - or alternatively, overexpression of Braun's lipoprotein - reversed the mutants' cell envelope damage phenotypes, suggesting that mLTs help to stabilize the OM. We conclude that P. aeruginosa mLTs contribute physically to cell envelope stability, and that Slt is the preferred target for future development of LT inhibitors that could synergize with β-lactams.