Identification and characterisation of novel families of ‘reverse’ substrates of the Staphylococcus aureus Type VII Secretion System

Abstract

The type VII secretion system (T7SS) is found in many Gram positive bacteria. In Staphylococcus aureus, the T7SSb secretes large protein toxins, facilitated by small helix-turn-helix partner proteins. To date, all T7SSb toxins share a common architecture - the N-terminus forms a helix-turn-helix domain, required for secretion, with a C-terminal functional domain. Previously, a candidate T7SSb substrate, SAOUHSC_00406, was identified by proteomic analysis of the S. aureus RN6390 secretome. From the work in this thesis, SAOUHSC_00406 has been renamed TslA. TslA has the reverse structural organisation to known T7SSb substrates with the helical domain at the C-terminus and a predicted lipase domain at the N-terminus. It is encoded in a conserved gene cluster, with at least one DUF576 lipoprotein encoded upstream, and two small helix-turn-helix proteins encoded downstream. The helix-turnhelix proteins are predicted to facilitate secretion of TslA while the DUF576 lipoprotein is predicted to provide immunity from lipase activity, and so the latter has been named TilA. Here I show that that TslA is indeed secreted in a T7SSb-dependent manner, facilitated by the two small helix-turn-helix proteins. I demonstrate that the TslA N-terminus is a phospholipase domain, with activity dependent on a catalytic triad composed of conserved Ser, Asp and His residues. Substitution of any of these residues inhibits lipase activity, both in vitro and in vivo. TilA binds with high affinity to the lipase domain of TslA, blocking its activity. A strain lacking all immunity protein copies is sensitive to killing by TslA, which causes disruption of the target cell membrane, confirming that it has antibacterial activity. Using bioinformatic techniques two further novel families of predicted lipases are identified which share a similar ‘reversed’ domain architecture. The work described here reveals a new paradigm for substrate targeting to the T7SSb.