Biology subjects

Anes, E.

Publications and source records attributed to Anes, E..

3 recordsLinked to original sources

bioRxiv2025

Genetic interference of distinctive Mycobacterium tuberculosis peptidoglycan modifications enhances β-lactam susceptibility and reveals expression-sensitive host immune dynamics

The high mortality associated with tuberculosis (TB), alongside the lack of efficient therapeutics against emerging multidrug-resistant Mycobacterium tuberculosis (Mtb) strains, emphasizes the need to identify novel antitubercular targets. Mycobacterial peptidoglycan, displaying characteristic modifications comprising the amidation of D-iso-glutamate (D-iGlu) and the N-glycolylation of muramic acid, is a promising therapeutic target. The genes encoding the enzymes mediating these PG modifications (murT/gatD and namH) were silenced in Mtb using CRISPR interference (CRISPRi) to investigate their impact on {beta}-lactam susceptibility and host immune responses. First, qRT-PCR confirmed successful target mRNA knockdown, with variable repression efficiency based on the selected sgRNA, PAM strength, and target site. Phenotypic characterization through spotting dilution and growth curve assays corroborated the essentiality of D-iGlu amidation for mycobacterial survival, in contrast to the N-glycolylation of muramic acid. Moreover, susceptibility assays showed that both PG modifications contribute to {beta}-lactam resistance, with sgRNA2-mediated murT knockdown substantially increasing {beta}-lactam and isoniazid susceptibility. Furthermore, checkerboard assays showed reductions in the minimum fractional inhibitory concentration index (FICImin) value for AMX/MEM+CLA and EMB combinations following the depletion of both PG modifications, with significant differences observed upon namH knockdown. Additionally, D-iGlu amidation was uncovered as a determinant of Mtb survival within THP-1-derived macrophages at 6 days post-infection. Infection of THP-1-derived macrophages with MurT/GatD-depleted Mtb upregulated IL-1{beta} and downregulated IL-10, whereas NamH depletion caused upregulation of both IL-1{beta} and IL-10. Altogether, our findings unveiled the potential of targeting these PG modifications for the development of innovative therapeutic regimens against TB.

microbiology↗

bioRxiv2025

Effects of CwlM, a peptidoglycan synthesis regulator, on beta-lactam resistance and host-pathogen interactions

BackgroundThe emergence and spread of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (Mtb) urge the development of novel drugs and efficient therapeutic programs. A recent study aiming to uncover differential beta-lactam susceptibility phenotypes in clinical strains of Mtb found that the M237V substitution in cwlM (Rv3915) was associated with increased susceptibility to amoxicillin. Considering that Mycobacterium smegmatis (Msm) is a widely used surrogate model for Mtb, we constructed a cwlM knockdown mutant in Msm using CRISPR interference (CRISPRi) to elucidate the role of CwlM in beta-lactam susceptibility and intracellular survival. ResultsQuantitative RT-PCR assays confirmed the successful repression of cwlM, while the phenotyping assays confirmed the essentiality of CwlM-related processes for mycobacterial viability. Collectively, the antibiotic susceptibility assays suggested that CwlMSMEG may promote beta-lactam resistance, particularly to meropenem and cefotaxime. Moreover, CwlMSMEG was found to support M. smegmatis intracellular survival within THP-1-derived macrophages. To address conflicting reports regarding its predicted peptidoglycan (PG) hydrolase activity, we purified recombinant CwlMTB. The Micrococcus luteus-derived PG-based zymogram indicated that CwlMTB lacks PG-hydrolytic activity, suggesting it might act as a regulator of PG biosynthesis instead. ConclusionsOur findings indicate that CwlM contributes to beta-lactam resistance and intracellular survival, regardless of lacking detectable PG-hydrolytic activity. Overall, CwlM was found to be essential and highly vulnerable, highlighting its potential as a therapeutic target that warrants further investigation.

microbiology↗

bioRxiv2023

Ethambutol and Meropenem/Clavulanate Synergy Promotes Enhanced Extracellular and Intracellular Killing of Mycobacterium tuberculosis

Increasing evidence supports the repositioning of beta-lactams for tuberculosis (TB) therapy. However, additional research on the interaction of these drugs with conventional anti-TB agents is still warranted. Since the complex cell envelope of Mycobacterium tuberculosis (Mtb) may pose an additional obstacle to the diffusion of beta-lactams, an improved activity upon combination with drugs that inhibit the synthesis of outer cell wall elements is particularly relevant. In this context, we aimed to determine potential synergies between beta-lactams and the antimycobacterial drugs ethambutol and isoniazid. This was followed by experiments that aimed to confirm if the increased antimicrobial effects remained within the intracellular milieu and if they promoted heightened immune responses. Results of checkerboard assays with H37Rv and eight clinical isolates, including four drug-resistant Mtb strains, exposed that only the treatments containing ethambutol and beta-lactams achieved synergistic effects, while the standard ethambutol and isoniazid association failed to produce synergy in any of the tested isolates. In Mtb-infected THP-1 macrophages, combinations of ethambutol with increasing meropenem concentrations consistently displayed superior killing activities over the individual antibiotics. Flow cytometry with BODIPY FL vancomycin, which binds directly to the peptidoglycan, confirmed an increased exposure of this layer after co-treatment. This was reinforced by the high IL-1{beta} secretion levels found in infected macrophages after incubation with concentrations of meropenem above 5 mg/L, which indicated an exposure of the host innate response sensors to pathogen-associated molecular patterns in the PG. Our findings show that the proposed impaired access of beta-lactams to periplasmic transpeptidases is counteracted by concomitant administration with ethambutol. The efficiency of this combination may be attributed to the synchronized inhibition of arabinogalactan and peptidoglycan synthesis, two key cell wall components. Given that beta-lactams exhibit a time-dependent bactericidal activity, a more effective pathogen recognition and killing prompted by this association may be highly beneficial to optimize TB regimens containing carbapenems.

microbiology↗