A membrane-depolarizing toxin substrate of theStaphylococcus aureustype VII secretion system mediates intraspecies competition

Abstract

The type VII protein secretion system (T7SS) is conserved acrossStaphylococcus aureusstrains and plays important roles in virulence and interbacterial competition. To date, only one T7SS substrate protein, encoded in a subset ofS. aureusgenomes, 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 allS. aureusstrains as well as inListeriaand Enterococci. Heterologous expression of TspA fromS. aureusstrain RN6390 indicates its C-terminal domain is toxic when targeted to theEscherichia coliperiplasm 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 totspAon theS. aureuschromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion oftspAleads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic andS. aureusstrains encode multipletsaIhomologs at thetspAlocus, 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.