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

Proc Natl Acad Sci U S A. 2020 Aug 25;117(34):20836-20847. doi: 10.1073/pnas.2006110117. Epub 2020 Aug 7.


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

Keywords: Staphylococcus aureus; bacterial competition; membrane-depolarizing toxin; type VII secretion system; zebrafish.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Bacterial / metabolism
  • Bacterial Proteins / metabolism
  • Cell Membrane / metabolism
  • Gene Expression Regulation, Bacterial / genetics
  • Membrane Proteins / metabolism
  • Multigene Family / genetics
  • Protein Transport / genetics
  • Proteomics
  • Staphylococcal Infections / microbiology
  • Staphylococcus aureus / metabolism*
  • Toxins, Biological / metabolism
  • Type VII Secretion Systems / metabolism*
  • Type VII Secretion Systems / physiology
  • Virulence / genetics
  • Zebrafish / microbiology


  • Antigens, Bacterial
  • Bacterial Proteins
  • Membrane Proteins
  • Toxins, Biological
  • Type VII Secretion Systems