A large soluble domain of the Staphylococcus aureus ESAT-6 Virulence Factor EsaA is stable in the absence of its cognate transmembrane domains

Document Type : Original Article

Authors

1 Department of Microbiology and Immunology, Faculty of Pharmacy and Pharmaceutical Industries, Sinai University, El-Arish, North Sinai, Egypt.

2 Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt.

3 Department of Microbiology and Immunology, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt.

Abstract

Staphylococcus (s.) aureus is a commensal microorganism with a significant threat to human health. S. aureus harbors secretion systems that are utilized to exploit host cells through the secretion of a subset of virulence proteins that cause serious illness. The ESAT6-like or Type VII Secretion System (T7SS) contributes to S. aureus virulence. The T7SS secretion system encodes at least twelve proteins categorized as cytosolic, membrane-associated or secreted. Little is known about the exact components of the translocation machinery or translocation mechanism of T7SS substrates across the staphylococcal envelope, but translocation of T7SS substrates across bacterial membranes requires four membrane proteins: EsaA and EssA, B, and C. Topology predictions of EsaA suggest six transmembrane domains with large soluble stretch, likely exposed into trans side of the membrane. Whether the large soluble stretch of EsaA is stable without the need for other transmembrane domains, a recombinant 6x-His-tagged soluble domain of EsaA was overproduced in Escherichia (E.) coli BL21(DE3) cells. This was followed by affinity purification of the tagged EsaA soluble over a Fast Protein Liquid Chromatography-operated nickel column to apparent homogeneity. SDS-PAGE of the affinity-purified soluble stretch without its cognate transmembrane domains revealed a strong signal, suggesting an independent role for that domain in mediating protein-protein interactions within the ESAT-6 secretion system.

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