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

The Gram-positive bacterium <i>Staphylococcus aureus</i> 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 <i>S. aureus</i> 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 <i>Escherichia coli</i>, 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, <i>SAOUHSC_00585</i> (<i>tsaI</i>) 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 <i>S. aureus</i> virulence. The model was also adapted to assess interbacterial competition <i>in vivo</i>. 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.<br/>

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

University of Dundee

Year dc:date.issued

2020

Author dc:creator

• Ulhuq, Fatima

Advisor dc:contributor.advisor

• Palmer, Tracy

Language dc:language

eng

Identifier

oai:discovery.dundee.ac.uk:studenttheses/4ac77951-a5bd-4913-9205-8faed5d7ff0e

OAI identifier oai:identifier

oai:discovery.dundee.ac.uk:studenttheses/4ac77951-a5bd-4913-9205-8faed5d7ff0e