Pared celular, β-lactamasa AmpC, sistema inmune y virulencianuevas conexiones y hallazgos en Pseudomonas aeruginosa

Abstract

In the current scenario of progressive increase of antimicrobial resistance, our antibiotic arsenal is increasingly compromised. This is especially worrisome in pathogens as relevant as Pseudomonas aeruginosa, one of the first causes of acute nosocomial and chronic infections in patients with underlying respiratory diseases, and which in fact pose a serious clinical-epidemiological threat due to their high morbidity and mortality. Therefore, the search for alternative antipseudomonal options from different perspectives is of extreme urgency, such as those aimed at disabling its antibiotic resistance mechanisms, or those aimed at attenuating the virulence of the pathogen. In this sense, our group demonstrated in the past that the combination of peptidoglycan (PGN) recycling blockade and overexpression of chromosomal β-lactamase AmpC (the main resistance mechanism of P. aeruginosa) causes a drastic attenuation of virulence in this species. Furthermore, we also previously demonstrated the indispensable role of the aforementioned recycling to enable the hyper-production of AmpC and therefore the resistance based on this β-lactamase. In order to know the therapeutic implications of these antecedents, we decided to carry out a multidisciplinary study, in which we have described for the first time the usefulness of colistin at subinhibitory concentration to enhance the antipseudomonal effectiveness of the elements of our immune system targeting the PGN (lysozyme and Peptidoglycan Recognition Proteins), and we have managed to link the blockage of PGN recycling to a drastic increase in the susceptibility of P. aeruginosa to these immune weapons in vitro. Furthermore, we have shown that the combination of the blockade of recycling plus hyper-production of AmpC is the cause of a decrease in the amount of PGN/cell, most likely linked to the phenotype of extreme susceptibility to the aforementioned immune attacks. Additionally, the characterization of large collections of P. aeruginosa strains (from acute vs chronic infection) allowed us to: i) find new potentially therapeutic targets such as Mpl ligase (also a participant in PGN recycling), whose inactivation causes an additional increase in the antipseudomonal capacity of lysozyme; and ii) to appreciate that the PGN of P. aeruginosa is a very stable structure and little subject to adaptive changes from the biochemical point of view, in comparison with other species. In addition, we have validated these in vitro findings in a murine model, so that by using strains defective in key genes for PGN recycling (ampG or nagZ), we were able to corroborate a spectacular decrease in virulence on the animal (probably thanks to sensitization to immune weapons targeting the cell-wall), as well as a total deactivation of AmpC-dependent resistance. Thus, all these findings validate the PGN recycling of P. aeruginosa as an excellent target for the future development of both anti-virulence and anti-resistance therapies. On the other hand, due to the lack of knowledge about the fragments from PGN (muropeptides) that support the stable hyper-production of AmpC in P. aeruginosa, we decided to carry out a study [using UPLC-MS (Ultra High Performance Liquid Chromatography - Mass Spectrometry)] to identify and characterize these signals. This has allowed us to establish a pioneer model in P. aeruginosa that identifies the anhydro-muramil-pentapeptides and anhydro-muramil-tripeptides as the activating signals of the expression of ampC, although with interesting qualitative and quantitative differences depending on the mutational pathways involved and levels of AmpC production. This model, in which the previously identified repressor signals seem to have little impact, helps us to better understand the bases for AmpC-dependent resistance in P. aeruginosa, bringing us closer to new therapeutic conceptions aimed at interfering with PGN-derived signaling and consequently block the production of AmpC.