Discovering a new pathway involved in methionine repair in the bacterial envelope 

The reactive species of oxygen (ROS) and chlorine (RCS) damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine (Met) is converted to methionine sulfoxide (Met-O), which can cause a loss of biological activity. To rescue proteins with Met-O residues, cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts. However, no Msr had been identified in the periplasm of important human pathogens, including Escherichia coli, Salmonella Typhimurium and Pseudomonas aeruginosa. This was surprising as the periplasm is in immediate contact with the environment, and therefore particularly exposed to the toxic ROS and RCS generated by the host cells. During my thesis, I discovered and characterized, in collaboration with Benjamin Ezraty, a senior scientist in the lab of Frédéric Barras (CNRS), a new reducing enzymatic system rescuing periplasmic proteins from methionine oxidation. This system, which we named MsrPQ, is widely conserved throughout Gram-negative bacteria and protects proteins present in the bacterial cell envelope from bleach stress. The characterization of the MsrPQ system led to a number of major findings. First, we showed that the MsrPQ system reduces Met-O residues by uniquely involving a molybdopterin-based chemical mechanism. Second, we found that MsrPQ is able to reduce both the S and R isoforms of Met-O, unlike the other enzymes able to repair Met-O residues. Thus, MsrPQ is the first non-stereospecific Msr system identified. Third, by combining proteomics and bacterial genetics, we showed that MsrPQ is able to repair a wide range of periplasmic proteins, including the primary periplasmic chaperone SurA and the lipoprotein Pal. These results are consistent with the fact that MsrPQ is required to maintain the integrity of the envelope under bleach stress. Finally, we provided evidence that the reducing equivalents used by MsrPQ to reduce Met-O residues originate from the respiratory chain, revealing a novel connection between cellular metabolism and protein quality control.