Project description:The spread of antimicrobial resistance (AMR), coupled with the decline in antibiotic development, has become a major public health concern. Recent studies estimate that around 700,000 people die each year from infections caused by multidrug-resistant (MDR) bacteria. This led the WHO to publish the ESKAPEE list of high priority pathogens for AMR, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli. Among these, Gram-negative bacteria (K. pneumoniae, A. baumannii, P. aeruginosa, Enterobacter spp., and E. coli) are particularly overrepresented. This is mainly due to their high propensity to develop multiple resistance mechanisms, in addition to their intrinsic resistance to many antimicrobials, which is due to their membrane composition and the expression of broad-spectrum efflux pumps. One strategy to combat such AMR is the use of drug enhancers that are able to restore the antibacterial activity of poorly active antibiotics. In this context, we demonstrated that the polyamino-isoprenyl enhancer, NV716, efficiently potentiates the antibacterial activity of two families of multi-target Ser/Cys-based enzyme inhibitors, namely the oxadiazolone derivatives (OX) and the Cyclipostins and Cyclophostin analogs (CyC), against Enterobacter cloacae, while remaining inactive against other Gram-negative bacteria. We confirmed that NV716 potentiates some OX & CyC compounds by permeabilizing the outer membrane and thus by increasing the inhibitor accumulation as shown by fluorescence confocal microscopy. By using bio-orthogonal click-chemistry activity-based protein profiling (CC-ABPP) approach coupled to proteomic analysis, we also identified the target proteins of the best OX & CyC inhibitors from E. cloacae lysate, thereby confirming their multi-target nature. Interestingly, 6 of the latter proteins were also captured via CC-ABPP in P. aeruginosa lysate, and are highly conserved in all Gram-negative bacteria. These results provide proof of concept that both OX & CyC, if successfully potentiated, could be used against a wide range of ESKAPEE Gram-negative bacteria.

Project description:Pseudomonas aeruginosa is a major opportunistic pathogen causing a wide range of infections and one of the most problematic bacteria regarding antibiotic resistance, with an increasing incidence of multidrug and extensively-drug resistant strains, including resistance to last resource antibiotics such as carbapenems. Resistances are often due to complex interplays of naturally and acquired resistance mechanisms which are enhanced by its remarkably large regulatory network. Thus, the use of non-targeted shotgun methodologies such as mass spectrometry-based proteomics is crucial to understand these interplays and to reveal possible strain and species-specific novel mechanisms of antibiotic resistance. The aim of this study was to determine the proteomic response of two carbapenem-resistant and extensively-drug-resistant P. aeruginosa strains to subminimal inhibitory concentrations (sub-MICs) of meropenem. The strains belonged to high-risk clones ST235 and ST395, one carrying a class 1 integron-encoded VIM-4 metallo-β-lactamase and one carrying no acquired antibiotic resistance genes. Each strain was cultivated with three different sub-MICs of meropenem, and a quantitative shotgun proteomic approach was applied, using tandem mass tag (TMT) isobaric labeling followed by nano-liquid chromatography tandem-mass spectrometry, to determine significantly up- or down-regulated proteins and enriched groups of proteins and pathways. Cultivation of both strains with ½ and ¼ of the MIC, resulted in hundreds of differentially regulated proteins, including several β-lactamases, transport-related proteins (including multiple porins and efflux pumps), proteins associated with peptidoglycan metabolism and cell wall organization and dozens of regulatory proteins. Remarkably, all components of the H1 type VI secretion system were up-regulated in one of the strains. Enrichment analyses revealed that multiple metabolic pathways were affected. Additionally, numerous proteins of unknown function were also differentially-regulated in each strain. In conclusion, high subminimal-inhibitory concentrations of meropenem cause massive changes in the proteomes of carbapenem-resistant P. aeruginosa strains, involving a wide range of common and strain-specific mechanisms and proteins, many still uncharacterized which might potentially play a role in the susceptibility of P. aeruginosa to meropenem.

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Transcriptome and proteome reveal the effects of asmA on efflux and metabolic functions in Enterobacter cloacae ST2260

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