Isolation of a membrane protein complex for type VII secretion in Staphylococcus aureus

Abstract

The ESAT6-like secretion system (ESS) of Staphylococcus aureus promotes effector protein transport across the bacterial envelope. Genes in the ESS cluster are required for S. aureus establishment of persistent abscess lesions and the modulation of immune responses during bloodstream infections. However, the biochemical functions of most of the ESS gene products, specifically the identity of secretion machine components, are unknown. Earlier work demonstrated that deletion of essB, which encodes a membrane protein, abolishes S. aureus ESS secretion. Lossof- function mutations truncating the essB gene product cause dominant-negative phenotypes on ESS secretion, suggesting that EssB is a central component of the secretion machinery. To test this prediction, we purified native and affinity-tagged EssB from staphylococcal membranes via dodecyl-maltoside extraction, identifying a complex assembled from five proteins, EsaA, EssA, EssB, EssD, and EsxA. All five proteins are essential for secretion, as knockout mutations in the corresponding genes abolish ESS transport. Biochemical and bacterial two-hybrid analyses revealed a direct interaction between EssB and EsaA that, by engaging a mobile machine component, EsxA, may also recruit EssA and EssD.