MM MANSOUR<sup>1

1</sup>laboratory of microbiology and molecular genetics

The aim of this project is to understand the molecular mechanism and the physiological role of toxinantitoxin systems (TAS) in mycobacteria. We propose to characterize the three HigBA systems of M.tuberculosis, focusing mainly on the toxins, which belong to the large RelE superfamily of ribosome-dependent RNases known to have very diverse mechanisms and substrate specificities. We will investigate possible crosstalk and cooperation among these systems, as well as their functional interplay with the SecB-like chaperone of the atypical tripartite HigB1-HigA1-Chaperone system (TAC) of Mtb, known to specifically control the HigB1 toxin inhibition cascade. A comprehensive analysis of the three Mtb-HigB cellular targets and the precise cleavage sites within these targets will be determined using the non-phosphorylated EMOTE methodology, which will allow the exact mapping of 5’-OH toxin cleaved sites in mRNA on a genome-wide scale (with PV and LF partners). Next, we will examine whether Mtb-HigB cleavage requires translating ribosome and possibly explore the nature of secondary structures of mRNA around cleavage sites (with LF). Finally, antibiotic profiling in the presence of each Mtb-HigB toxins will be performed and correlated with the identification of cellular toxin targets by nEMOTE (with PV). Such entirely novel approach will hopefully reveal interesting pathways for antibiotic tolerance/sensibility involving toxins, as well as compounds that block or stimulate toxins activity in vivo. If successful, the most interesting targets will be selected for further genetic studies.