Identification of Dimerization Interaction of Certain Type VII Secretion System Membrane Proteins in Staphylococcus aureus: A new Approach for Virulence Control

Abstract

The genome of S. aureus encodes type VII secretion system (T7SS), which contributes to full virulence of the organism. T7SS is a specialized secretion system consisting of at least 12 genes that encode twelve protein products categorized under cytosolic to membrane-associated proteins. Some cytosolic components of the T7SS are secreted effectors that play an important role in staphylococcal establishment in host tissues. Little is known about how the translocation machinery is organized or the translocation mechanism of these ESS effectors across the staphylococcal envelope. In this study, genomic and proteomic approaches were taken in order to identify key membrane protein-protein interactions in the T7SS that can potentially perturb the functional mechanism of the ESS pathway. Towards addressing such objective, this study concerns with identifying the nature of interaction between two integral membrane proteins: EssB and EsaA (EssF) in type VII secretion system of S. aureus ESS pathway. EssB and EsaA are two integral membrane proteins that were shown highly conserved in the T7SS/ESS conserved module of various S. aureus strains and other Gram-positive firmicutes. Bacterial two-hybrid screening under three different growth conditions, LB/X-gal, MacConkey/maltose, M63/maltose gar media and in vivo crosslinking experiments using 1% formaldehyde crosslinker have directly validated an interaction pattern between these two membrane components of the ESS pathway. EssB is a bitopic membrane protein consisting of a transmembrane domain that resides at the center of the protein with two soluble domains namely, N and C domains. In addition, systematic truncations were constructed in