P085

1:Laboratoire d'Optique et Biosciences (LOB), Ecole Polytechnique, Inserm U1182, CNRS UMR7645, Institut Polytechnique de Paris, 91120 Palaiseau, France.; 2:Université Paris-Saclay, Gif-sur-Yvette, France.; 3:CNRS, AgroParisTech, Ecologie Société Evolution, Université Paris-Saclay, 91190, Gif sur Yvette, France

The rise of drug-resistant Mycobacterium tuberculosis (Mtb), including multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains, poses a serious global health threat. Bedaquiline, the only new tuberculosis (TB) drug approved in the past 50 years, is already facing resistance, with up to 47% of post-treatment strains showing acquired mutations. Unlike many pathogens, Mtb develops antimicrobial resistance (AMR) solely through spontaneous mutations, not horizontal gene transfer. Understanding the mechanisms underlying this mutagenesis is crucial to designing new intervention strategies. This study focuses on DNA repair, a key process balancing genome stability and mutation-driven adaptation. Our long-term goal is to assess the endonuclease NucS, part of the Mismatch Repair (MMR) system, as a potential target for “anti-evolution” strategies to slow resistance emergence. As a first step, we identified and characterized NucS interaction partners and their functional role in Mtb’s DNA repair pathways. Using a yeast two-hybrid system screening, we identified two NucS partners, Mku and UvrA. We then constructed single and double gene deletion mutants in Mycobacterium smegmatis, a model for Mtb, and assessed mutation rates and competitive fitness to validate their interplay. Our results suggest NucS functions in two distinct contexts: in coordination with Mku, it likely couples MMR with double-strand break repair; with UvrA, it appears to support nucleotide excision repair of UV-induced lesions. These findings highlight the central role of NucS and its partners in maintaining genomic stability. They also support the feasibility of targeting DNA repair to limit AMR evolution, offering a novel avenue for durable TB control strategies.