Deciphering the mechanistic details of Mycobacterium tuberculosis nucleotide excision repair and related proteins as key players of bacterial persistence and adaptation: structure-based insights for anti-evolution drug design

Tuberculosis (TB) disease is a global health threat caused by Mycobacterium tuberculosis (MTB), counting 1.6 million deaths in 2021. This health emergency is even more alarming due to the occurrence of Multi-Drug-Resistant (MDR) MTB strains. During its life cycle, MTB is exposed to DNA damaging stresses that could compromise the establishment, the containment and the reactivation of the infection. Considering the high cytotoxic and promutagenic potential of DNA damage, DNA repair systems are key elements for mycobacterial survival, and they are involved on the development of antibiotic resistance Although some progress has been made towards the functional characterization of mycobacterial DNA repair enzymes it is still based on homology studies, so that the biochemical and structural haracterization of DNA repair systems is largely incomplete. Among different DNA repair pathways, Nucleotide Excision Repair (NER) represents one of the major molecular machineries that controls chromosome stability in all living species. In Eubacteria, the first steps of this pathway involves three components, namely the UvrA, UvrB and UvrC proteins that act in a multi-step mode functional to the lesion sensing and the damage removal. UvrA interacts with UvrB and scans the DNA searching for the damage, but the stoichiometry of UvrAUvrB complex and the damage recognition process is still unclear. Our preliminary unpublished data sheds light and guides the future research on this aspects. Furthermore, little is known about the dynamic of UvrC recruitment; however, recent publications suggest the formation of a repairsome between UvrA, UvrB and UvrC. MTB genome also encodes for a putative DNA-alkyltransferase-like protein (MtATL) that recruits the NER proteins to the damage in other organisms. Our preliminary results together with the literature data suggest an alternative role of ATL in MTB acting as a secreted tool involved in the host DNA repair. Thus, it represents a novel pathogenic mechanism promoting the host cell survival and the preservation of the MTB niche until a high bacterial burden. Considering these premises, the milestones of this project are: i) the description of the molecular architecture of Uvr protein complexes by means of an integrated biochemical and structural approach; ii) the identification of the molecular determinants for the damage recognition performed by UvrA; and iii) the characterization of MtATL as a protecting factors for the host’s DNA integrity through an interactome analysis aimed at revealing human DNA repair proteins, that are recruited to the damage through an ATL-dependent mechanism. The results of this proposal will pave the way for further drug design in order to identify active molecules that disrupt key protein-protein and protein-DNA interactions in MTB NER for the development of “anti-evolution” drugs interfering with genome integrity and with the acquisition of antibiotic resistance through chromosomal mutations.