Stéphane Canaan

Most antimicrobial peptides (AMPs) are cationic, with positively charged amino acid residues (i.e., arginine, lysine, and/or histidine) being responsible for their selective interaction with bacteria. In the present study, we investigated the influence of the nature of cationic residues taking the Lynronnes peptides as models. Derivatives were designed and synthetized with substitutions of the type of cationic residues present. All derivatives and parental peptides were studied in terms of secondary structure, antimicrobial activity, anti‐biofilm activity, mechanism of action, and in vitro toxicity. All‐His peptides were found inactive, whereas all‐Lys variants showed the highest potency against Gram‐negative strains, particularly against E. coli . All‐Arg analogues were more active against Gram‐positive strains with enhanced stability under salt or serum conditions but were also more hemolytic. Mechanistic studies revealed that the peptides act by membrane permeabilization. Finally, Lynronne‐3 and its all‐Lys derivative proved the most effective in both the prevention and eradication of biofilms. These findings confirm that the type of cationic residue present in AMPs affects their activity, toxicity, and sensitivity to host factors, with different effects depending on the rest of the amino acid sequence, Lys‐substitution emerging as a promising strategy in the case of Lynronnes for targeting E. coli .

Staphylococcus aureus is a Gram-positive opportunistic pathogen and a top priority bacterium in the fight against antimicrobial resistance. Its high propensity to develop resistance, its high virulence, and its ability to form biofilms and persist intracellularly result in difficult-to-treat infections against, which new chemical classes are urgently needed. Here, we investigated the antibacterial activity of oxadiazolone-core derivatives (OX) against planktonic, intracellular, and biofilm-associated S. aureus. Among the tested compounds, MpPPOX exhibited a bactericidal effect on extracellular bacteria with an MIC similar to that of vancomycin; iBPOX mainly inhibited intracellular replication, while HPOX strongly impaired initial biofilm formation. These results prompted us to identify the potential target enzymes of the three OXs via activity-based protein profiling, combined with mass spectrometry. The antibiofilm HPOX compound was indeed found to primarily react with enzymes involved in biofilm formation and associated virulence, while iBPOX and the most active MpPPOX inhibitor targeted multiple (Ser/Cys)-based enzymes. Among these, the FabH protein has been confirmed as a vulnerable target of MpPPOX. Overall, this study underscores the multitarget nature of the OXs, which covalently bind to several (Ser/Cys)-based enzymes of interest. Such property makes them highly versatile chemotypes that could be used as broad-spectrum antimicrobial agents, notably by improving the antibiofilm activity of ineffective or poorly active drugs.

Medical devices are critical for patient survival in hospitals, yet they represent a leading cause of infections.

The genetic basis underlying nontuberculous mycobacteria (NTM) pathogenesis remains poorly understood. This gap in knowledge has been partially filled over the years through the generation of novel and efficient genetic tools, including the recently developed CRISPR interference (CRISPRi) technology. Our group recently capitalized on the well‐established mycobacteria‐optimized dCas9 _Sth1 ‐mediated gene knockdown system to develop a new subset of fluorescence‐based CRISPRi vectors that enable simultaneous controlled genetic repression and fluorescence imaging. In this Research Protocol, we use Mycobacterium smegmatis ( M. smeg ) and Mycobacterium abscessus ( M. abs ) as NTM model species and provide simple procedures to assess CRISPRi effectiveness. We describe how to evaluate the efficacy of gene silencing when targeting essential genes but also genes involved in smooth‐to‐rough envelope transition, a critical feature in NTM pathogenesis. This protocol will have a broad utility for mycobacterial functional genomics and phenotypic assays in NTM species.

Tuberculosis (TB) is still a significant public health threat, with rising drug resistance and high incidence in multiple areas worldwide. In the search for novel antitubercular agents, this study explores the application of a bioactivity-guided molecular networking approach to identify bioactive compounds from seven plant species (Curatella americana, Davilla nitida, Dipteryx punctata, Indigofera suffruticosa, Quassia amara, Tetradenia riparia, and Zingiber zerumbet) collected in French Guiana. Using ultrasound-assisted extraction followed by liquid–liquid partitioning and UHPLC-HRMS/MS analysis, a library of 72 samples was tested against Mycobacterium tuberculosis. The non-polar fractions from Indigofera suffruticosa, Tetradenia riparia, and Zingiber zerumbet showed the highest activity. The integration of metabolomic and bioassay data on molecular networks allowed the prioritization and annotation of active compounds, revealing flavonoids as contributors to the antitubercular activity of the active samples. In addition, the use of computational tools such as GNPS, SIRIUS, and TIMA-R enabled dereplication and increased the confidence in the structural prediction of active metabolites. This approach demonstrated its potential in accelerating the identification of both known and novel bioactive compounds without requiring exhaustive isolation, offering a robust strategy for natural product-based drug development against TB.

Pyrazinamide (PZA) is a cornerstone drug in tuberculosis (TB) treatment with a strong bactericidal activity in vivo on both actively and non-replicating bacterial subpopulations. Yet the precise mode of action of its active form, pyrazinoic acid (HPOA), remains unclear. In this study, we comprehensively explore and challenge the two major and conflicted models of PZA mode of action. The pH-dependent model, where the drug is mostly effective at acidic pH by acidifying Mycobacterium tuberculosis ( Mtb ) cytosol, and the PanD-dependent model where PZA’s active form targets the aspartate decarboxylase PanD, therefore depleting pantothenate and subsequently coenzyme A (CoA) levels regardless of the surrounding pH. By combining standard antimicrobial susceptibility testing at various pH with fluorescence-based live recording of Mtb intrabacterial pH, we demonstrate that PZA kills Mtb by decreasing intrabacterial pH, independently of pantothenate levels. Comparative studies between a prototrophic Mtb strain and a pantothenate auxotrophic mutant lacking the panCD locus confirmed that PZA bactericidal activity is primarily driven by pH and its ability to acidify Mtb cytosol, independently of the aspartate decarboxylase PanD. Bio-electrophysiology experiments revealed that acidic pH promotes the conversion of the pyrazinoate anion POA⁻ into HPOA which in turn acts as conventional weak acid that facilitates membrane permeation and cytosolic acidification. Finally, using custom-based culture media, we demonstrate that PZA displays heterogeneous efficacy according to the media composition, therefore proposing a revisited biological model that might explain the discrepancies around PZA’s unique mode of action. Overall, this work constitutes the first comprehensive side-by-side investigation of the two models and univocally supports a pH-dependent mechanism of action underlying PZA sterilizing activity, providing new insights for the development of more effective PZA-like drugs.

Gram‐negative bacteria are particularly prone to developing antimicrobial resistance (AMR), as evidenced by the WHO's ESKAPEE list of high‐priority pathogens. One strategy that has increased is the use of antibiotic enhancers, which can re‐empower abandoned or poorly active antibiotics against the resistant strain of interest. In this study, the polyamino‐isoprenyl antibiotic enhancer, NV716, was tested in combination with two families of multi‐target Ser/Cys‐based enzyme inhibitors, the oxadiazolone derivatives (OX) and the Cyclipostins and Cyclophostin analogs (CyC), which are inactive against Gram‐negative ESKAPEE bacteria, to potentiate their antibacterial activity and thus make them active against these bacteria. We demonstrated that NV716 potentiates some OX and CyC compounds by permeabilizing the outer membrane and thus by increasing the inhibitor accumulation, as shown by fluorescence microscopy. By using the click‐chemistry activity‐based protein profiling (ABPP) approach coupled with proteomic analysis, we also confirmed the multi‐target nature of the best OX and CyC inhibitors by identifying their target proteins on a bacterial culture of Enterobacter cloacae. Remarkably, a large set of these identified proteins had already been captured in previous ABPP experiments conducted on Mycobacterium tuberculosis and/or Mycobacterium abscessus culture. Furthermore, we showed that five of the identified target proteins were present in a total lysate of Pseudomonas aeruginosa. Importantly, these latter enzymes are highly conserved among Gram‐negative bacteria, with two of them annotated as essential for bacterial survival. These results provide proof of concept that both OX and CyC, if successfully potentiated, could be used against ESKAPEE Gram‐negative bacteria.

Lipids play a critical role in the physiology, life cycle, and pathogenicity of mycobacteria. They largely participate in host-pathogen interactions and fulfill important functions ranging from cell wall biosynthesis/maintenance, bacterial growth dynamics, and long-term persistence. In that context, triacylglycerol, a specific subtype of neutral lipid, is stored as intrabacterial lipid inclusions (ILI), which have been described as important structures for long-term survival and persistence within the host. However, the enzymes involved in ILI formation and degradation remain largely undefined. Using bio-orthogonal click-chemistry activity-based protein profiling (CC-ABPP) of newly synthesized oxadiazolone (OX), cyclophostin, and cyclipostins (CyC) probes, we report the direct capture of target proteins in Mycobacterium abscessus growing under carbon excess and nitrogen-deprived in vitro conditions that promote triacylglycerol production and ILI formation. This approach led to the identification of a set of 65 enzymes potentially involved in global processes related to ILI anabolism. Among these, the long-chain-fatty-acid--CoA ligase MAB_1978c/FadD15 has been validated as a pivotal enzyme that colocalizes on ILI and as a major contributor in ILI formation in M. abscessus.