Investigating the secretion of a nuclease toxin and its inhibition by a cognate immunity protein

Abstract

The type VIIb protein secretion system (T7SSb) of Staphylococcus aureus is involved in virulence and interbacterial competition. Secretion of a nuclease toxin (EsaD) by the T7SSb mediates interbacterial competition. The EsaD nuclease toxin is neutralised by a cognate immunity protein, EsaG. I present a model of the EsaD nuclease domain in complex with its cognate immunity protein determined by X-ray crystallography. Natural variation that is found in this nuclease-immunity pairing across strains is presented in relation to the interaction interfaces between EsaD and EsaG. A study on the activity of the EsaD nuclease domain is also presented. EsaD interacts with a specific chaperone and small WXG100-like proteins that are typical of T7SS. I present biochemistry and AlphaFold2 predictions of complexes of EsaD with these secretion partners and reflect on the implications these have on the T7SSb mechanism and compare with the more well studied mycobacterial T7SSa. Also presented in this thesis is a phosphoproteomics study on whole cells and membrane proteins of S. aureus to investigate the possibility that the T7SSb could be controlled post-translationally by phosphorylation. Substrates of the sole serine-threonine protein kinase of S. aureus (PknB) were investigated and I find a range of targets important in S. aureus biology.