Targeting the permeability barrier and peptidoglycan recycling pathways to disarm Pseudomonas aeruginosa against the innate immune system

Abstract

Antimicrobial resistance is a continuously increasing threat that severely compromises our antibiotic arsenal and causes thousands of deaths due to hospital-acquired infections by pathogens such as Pseudomonas aeruginosa, situation further aggravated by the limited development of new antibiotics. Thus, alternative strategies such as those targeting bacterial resistance mechanisms, virulence or potentiating the activity of our immune system resources are urgently needed. We have recently shown that mutations simultaneously causing the peptidoglycan recycling blockage and the β-lactamase AmpC overexpression impair the virulence of P.aeruginosa. These findings suggested that peptidoglycan metabolism might be a good target not only for fighting antibiotic resistance, but also for the attenuation of virulence and/or potentiation of our innate immune weapons. Here we analyzed the activity of the innate immune elements peptidoglycan recognition proteins (PGRPs) and lysozyme against P. aeruginosa. We show that while lysozyme and PGRPs have a very modest basal effect over P. aeruginosa, their bactericidal activity is dramatically increased in the presence of subinhibitory concentrations of the permeabilizing agent colistin. We also show that the P. aeruginosa lysozyme inhibitors seem to play a very residual protective role even in permeabilizing conditions. In contrast, we demonstrate that, once the permeability barrier is overpassed, the activity of lysozyme and PGRPs is dramatically enhanced when inhibiting key peptidoglycan recycling components (such as the 3 AmpDs, AmpG or NagZ), indicating a decisive protective role for cell-wall recycling and that direct peptidoglycan-binding supports, at least partially, the activity of these enzymes. Finally, we show that recycling blockade when occurring simultaneously with AmpC overexpression determines a further decrease in the resistance against PGRP2 and lysozyme, linked to quantitative changes in the cell-wall. Thus, our results help to delineate new strategies against P. aeruginosa infections, simultaneously targeting β–lactam resistance, cell-wall metabolism and virulence, ultimately enhancing the activity of our innate immune weapons.

Interplay between Peptidoglycan Biology and Virulence in Gram-Negative Pathogens

“…Further, the analysis of a set of P. aeruginosa mutants very similar to those used in the above-mentioned work by Pérez-Gallego and colleagues (223) has recently revealed that, besides fitness and virulence attenuation, PGN recycling impairment (together or not with AmpC derepression) determines a great increase in bacterial susceptibility to innate immune weapons targeting the PGN, such as lysozyme and the four human PGN recognition proteins (PGRPs) (203). Lysozyme (also known as muramidase or N-acetylmuramide glycanhydrolase) is especially abundant in secretions and in neutrophil granules and catalyzes the hydrolysis of 1,4-beta linkages between NAcMur and NAcGlc.…”

“…First, it is worth mentioning that some works have suggested that the muramidase activity of lysozyme is not necessary for its bactericidal power, since it displays membrane permeabilization action, therefore actually working as a cationic peptide (198,(200)(201)(202). Additionally, it has to be stated that many works have proposed that lysozyme generally has low activity per se against Gram-negative bacteria, unless the outer membrane is permeabilized by some other innate immune compound (198,203). However, different bacterial inhibitors of the three types of lysozyme that exist in the animal kingdom (chicken or conventional [c-type], goose [g-type], and invertebrate [i-type] lysozymes) have been described (204).…”

“…Indeed, it is tempting to speculate that the production of an intrinsic ␤-lactamase, at least at regular levels, should not entail any biological cost, but what about when the enzyme is overproduced? Very few works have tried to answer this question (203,223). Either way, the targets that we have identified in the contexts mentioned above are displayed in Table 7.…”

Constructing and deconstructing the bacterial cell wall

“…A credible strategy to create meaningful clinical adjuvants to preserve β‐lactam efficacy would be to suppress muropeptide recycling in its entirety. Indeed, preliminary data support fully this hypothesis . One focus of such an effort would be the understanding of the enigmatic functions of the LT family and the respective means to their regulation .…”

“…Indeed, preliminary data support fully this hypothesis. [280][281][282] One focus of such an effort would be the understanding of the enigmatic functions of the LT family and the respective means to their regulation. 283,284 Accordingly, we have undertaken extensive mechanistic and structural studies of this family.…”