The transmembrane domain of the Staphylococcus aureus ESAT-6 component EssB mediates interaction with the integral membr
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ORIGINAL PAPER
The transmembrane domain of the Staphylococcus aureus ESAT-6 component EssB mediates interaction with the integral membrane protein EsaA, facilitating partially regulated secretion in a heterologous host Manar M. Ahmed1 · Khaled M. Aboshanab2 · Yasser M. Ragab3 · Dominique M. Missiakas4 · Khaled A. Aly1 Received: 3 December 2017 / Revised: 24 April 2018 / Accepted: 24 April 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract The ESAT-6-like secretion system (ESS) of Staphylococcus aureus plays a significant role in persistent infections. EssB is a highly conserved bitopic ESS protein comprising a cytosolic N-terminus, single transmembrane helix and a C-terminus located on the trans-side of the membrane. Six systematic truncations covering various domains of EssB were constructed, followed by bacterial two-hybrid screening of their interaction with EsaA, another conserved integral membrane component of the ESS pathway. Results show that the transmembrane domain of EssB is critical for heterodimerization with EsaA. In vivo crosslinking followed by Western blot analysis revealed high molecular weight species when wild-type EssB and EsaA were crosslinked, but this band was not detected in the absence of the transmembrane domain of EssB. Heterologous overproduction of EssB, EsaA and five other components of the ESS pathway in Escherichia coli BL21(DE3), followed by fractionation experiments led to a remarkable increase in the periplasmic protein content, suggesting the assembly of partially regulated secretion mechanism. These data identify the transmembrane domain of EssB as indispensable for interaction with EsaA, thereby facilitating protein secretion across bacterial membranes in a fashion that requires other components of the ESS pathway. Keywords EssB–EsaA interaction · Bacterial two-hybrid · T7SS · ESAT-6
Introduction Staphylococcus aureus poses a serious threat to the global healthcare system due to its significant pathogenic potential (Thammavongsa et al. 2015; Ventola 2015). Naturally, Communicated by Djamel DRIDER. * Khaled A. Aly [email protected] 1
Department of Microbiology and Immunology, Faculty of Pharmacy and Pharmaceutical Industries, Sinai University, El‑Masaeed, El‑Arish, North Sinai, Egypt
2
Department of Microbiology and Immunology, Faculty of Pharmacy, Ain Shams University, Cairo 11566, Egypt
3
Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
4
Department of Microbiology, University of Chicago, 920 East 58 Street, Chicago, IL 60637, USA
S. aureus colonizes one-third of the global population as a commensal, mostly inhabiting their skins and nares (van Belkum et al. 2009). Quite often however, this commensal lifestyle transitions into a pathogenic one, resulting in skin and soft tissue infections, but also in more invasive attacks that can be life threatening, such as bacteremia, sepsis and pneumonia (David and Daum 2010; Thammavongsa et al. 2015). Staphylococcal invasiveness is accompanie
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