A membrane-depolarizing toxin substrate of the <i>Staphylococcus aureus</i> type VII secretion system mediates intraspecies competition

Abstract

The type VII protein secretion system (T7SS) is conserved across <i>Staphylococcus aureus</i> strains and plays important roles in virulence and interbacterial competition. To date, only one T7SS substrate protein, encoded in a subset of <i>S. aureus</i> 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 <i>S. aureus</i> strains as well as in <i>Listeria</i> and Enterococci. Heterologous expression of TspA from <i>S. aureus</i> strain RN6390 indicates its C-terminal domain is toxic when targeted to the <i>Escherichia coli</i> 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 <i>tspA</i> on the <i>S. aureus</i> chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of <i>tspA</i> leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and <i>S. aureus</i> strains encode multiple <i>tsaI</i> homologs at the <i>tspA</i> 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.