A family of T6SS antibacterial effectors related to L,D-transpeptidases targets the Peptidoglycan

Type VI secretion systems (T6SSs) are contractile nanomachines widely used by bacteria to intoxicate competitors. Salmonella Typhimurium encodes a T6SS within the Salmonella pathogenicity island 6 (SPI-6) that is used during competition against species of the gut microbiota. We characterized a new SPI-6 T6SS antibacterial effector named Tlde1 (type VI L,D-transpeptidase effector 1). Tlde1 is toxic in target-cell periplasm and its toxicity is neutralized by co-expression with immunity protein Tldi1 (type VI L,D-transpeptidase immunity 1). Time-lapse microscopy revealed that intoxicated cells display altered cell division and lose cell envelope integrity. Bioinformatics analysis showed that Tlde1 is evolutionarily related to L,D-transpeptidases. Point mutations on conserved histidine121 and cysteine131 residues eliminated toxicity. Co-incubation of purified recombinant Tlde1 and peptidoglycan tetrapeptides showed that Tlde1 displays both L,D-carboxypeptidase activity by cleaving GM-tetrapeptides between meso-diaminopimelic acid3 and D-alanine4, and L,D-transpeptidase exchange activity by replacing D-alanine4 for a non-canonical D-amino acid. Tlde1 constitutes a new family of T6SS effectors widespread in Proteobacteria. This work increases our knowledge about the bacterial effectors used in interbacterial competitions and provides molecular insight into a new mechanism of bacterial antagonism.