A membrane-depolarizing toxin substrate of the type VII secretion system mediates intraspecies competition.

The type VII protein secretion system (T7SS) is conserved across strains and plays important roles in virulence and interbacterial competition. To date, only one T7SS substrate protein, encoded in a subset of genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all strains as well as in and Enterococci. Heterologous expression of TspA from strain RN6390 indicates its C-terminal domain is toxic when targeted to the periplasm and that it depolarizes the cytoplasmic membrane. The membrane-depolarizing activity is alleviated by coproduction of the membrane-bound TsaI immunity protein, which is encoded adjacent to on the chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and strains encode multiple homologs at the locus, suggestive of additional roles in intraspecies competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologs.