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

Abstract

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.

Pseudomonas Aeruginosa : Targeting Cell-Wall Metabolism for New Antibacterial Discovery and Development

“…However, the accumulation of multiple mutations -each of which confers small increases in resistance -can lead to high-level resistance in a stepwise manner [2,[45][46][47][48]. While most studies focus on single mutations that confer high-level resistance, recent work from our laboratory [46,47] and others [44,49], confirm that high-level resistance is achievable by the accumulation of multiple mutations that alone confer low level resistance. This phenomenon has been referred to as 'creeping baselines' [48]; however, the frequency of such a phenomenon is difficult to assess in clinical isolates because creeping baselines are likely to be missed by most clinical microbiology procedures that focus on defined break points [27].…”

“…Studies in E. coli showed that loss of LT activity prevented AmpC induction; however, a recent study from our laboratory showed that P. aeruginosa strains lacking up to five of the ten known LTs Pseudomonas aeruginosa: targeting cell-wall metabolism for new antibacterial discovery & development Review continued to exhibit robust induction [46]. That study also unexpectedly revealed that the membrane-bound LTs (mLTs) were required for maintaining the structural integrity of the OM in P. aeruginosa.…”

“…For example, the impermeability of P. aeruginosa and other Gramnegative pathogens is a formidable obstacle for many antibiotics; however, OM permeabilizers are promising for the treatment of such infections. Such a strategy has been used recently to sensitize Gram-negative pathogens to multiple classes of antibiotics, including the bulky antibiotics daptomycin, vancomycin, teicoplanin and telavancin, which are normally incapable of entering the cell [46,[75][76][77][78]. Thus, combination therapies hold great promise for rescuing the activity of common antibiotic classes -or previously abandoned scaffolds -and may be useful for extending the lifespan of current antibiotics while advances are made in other areas of antibacterial discovery and development.…”

“…In most cases, mutational resistance occurs upon treatment with sublethal concentrations of antibiotics, and a single mutation can confer high-level resistance [14,44]. However, the accumulation of multiple mutations -each of which confers small increases in resistance -can lead to high-level resistance in a stepwise manner [2,[45][46][47][48]. While most studies focus on single mutations that confer high-level resistance, recent work from our laboratory [46,47] and others [44,49], confirm that high-level resistance is achievable by the accumulation of multiple mutations that alone confer low level resistance.…”

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

“…In P. aeruginosa, the loss of Slt (PA3020) and MltF (PA3764) is associated with a decrease in b-lactam resistance [98]. However, the loss of SltB1 (PA4001) and MltB1 (PA4444) led to an AmpC-independent increase in resistance to b-lactams, piperacillin and cefotaxime [98,213]. The loss of the other P. aeruginosa LTs' MltA (PA1222), MltD (PA1812), MltF2 (PA2865), SltG (PA1171) and SltH (PA3992) did not change the resistance profile [98] ( Table 3).…”