Investigating the role of TspA, a novel substrate of the Staphylococcus aureus Type VII protein secretion system

Abstract

The Gram-positive bacterium Staphylococcus aureus is a major pathogen of humans and one of the leading causes of community- and hospital-acquired infections. The Type VII Secretion System (T7SS) is widespread in Gram-positive bacteria belonging to both the Actinobacteria and Firmicutes phyla. In commonly-studied strains of S. aureus the T7SS gene cluster is encoded by 12 genes, and it has been shown to play an important role in virulence and interbacterial competition. To date, very few T7SS-dependent substrates of known biological function have been identified. In this study an unbiased mass-spectrometry approach was used to identify candidate T7 substrate proteins. A previously uncharacterised protein, SAOUHSC_00584, was found in the culture supernatant in a T7-dependent manner. Cell fractionation and protease accessibility experiments demonstrated that SAOUHSC_00584 is a peripheral membrane protein that localises at the extracellular side of the membrane. The stability of SAOUHSC_00584 was shown to be dependent on the EssC componentof the T7SS. Structural modelling suggested that SAOUHSC_00584 shares homology to the pore-forming protein colicin Ia. The C-terminal domain of SAOUHSC_00584 was shown to have toxic activity when produced in the periplasm of Escherichia coli, and that it depolarised the cytoplasmic membrane. It was subsequently re-named TspA. TspA is encoded at a genetic locus with multiple DUF443 domain-encoding proteins. The neighbouring gene, SAOUHSC_00585 (tsaI) was shown to encode a membrane protein that confers immunity to the toxic activity of TspA. A zebrafish larva hindbrain infection model was developed to investigate biological roles of the T7SS and TspA. Using this model it was seen that there was a role for the T7SS and for TspA in S. aureus virulence. The model was also adapted to assess interbacterial competition in vivo. TspA was shown to contribute to intra-species growth inhibition. Thus TspA is a T7-secreted toxin with the unusual property of targeting both eukaryotes and prokaryotes.

Date of Award	2020
Original language	English
Sponsors	Biotechnology and Biological Sciences Research Council & BBSRC EASTBIO DTP Studentship
Supervisor	Tracy Palmer (Supervisor)