Bacterial toxin-antitoxin systems (TAs) are ubiquitous genetic elements found on chromosomes and mobile genetic elements. TAs consist of an operon encoding a toxin that inhibits cell growth and an antitoxin that counteracts the toxin. Typically, the toxins disrupt essential functions of the host bacterium, including translation, replication or peptidoglycan synthesis. Several biological roles associated with TAs have been demonstrated, including stabilising mobile genetic elements that confer resistance to antibiotics, providing bacterial immunity against phages and contributing to the virulence and persistence of pathogenic bacteria. Toxins use a variety of strategies and mechanisms to control bacterial growth, offering interesting alternatives to traditional antibiotics. However, the development of inovative antibacterial strategies requires a thorough understanding of the mechanisms of inhibition, expression and regulatory control of TAs. The Patho-TOX project focuses on the conserved Rosmer TA family (RmrTA), which has recently been identified as a potential defence mechanism against phages. RmrA antitoxins are paired with different RmrT toxins that generally show no detectable similarity to other protein families. RmrA proteins combine a DNA-binding HTH domain with a metallopeptidase-like ImmA domain, the function of which has not yet been elucidated. The fine-tuning of RmrT activity is critical for bacterial survival. Remarkably, some RmrT toxins have been shown to be responsible for the essentiality of ClpXP in bacteria such as Streptococcus pneumoniae and Escherichia coli, thus suggesting that RmrT toxins are kept inactive by the ClpXP AAA+ protease. The aim of this project is to reveal the cellular functions and activation mechanisms of chromosomal RmrTA systems in three major human pathogens, namely E. coli, S. pneumoniae, and Staphylococcus aureus. The project combines genetic, biochemical, and structural approaches to understand (i) the molecular mechanisms that cause bacterial growth inhibition by RmrT toxins, (ii) the roles of the RmrTA components in the transcriptional regulation of the rmrTA operon and in bacterial physiology, and (iii) how the AAA+ ClpXP protease control the activation of RmrT toxins, both in vitro and in vivo in response to relevant stress. This project will provide an in-depth analysis of novel toxin-mediated growth inhibition mechanisms and TA activation networks, which could be used to develop new strategies to combat bacterial pathogens and antibiotic resistance.