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Structural and functional studies of the enteropathogenic Escherichia coli type III needle complex protein EscJ
Author(s) -
Crepin Valérie F.,
Prasannan Sunil,
Shaw Rob K.,
Wilson Rebecca K.,
Creasey Elizabeth,
Abe Cecilia M.,
Knutton Stuart,
Frankel Gad,
Matthews Steve
Publication year - 2005
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2005.04508.x
Subject(s) - periplasmic space , biology , enteropathogenic escherichia coli , secretion , effector , inner membrane , escherichia coli , virulence , type three secretion system , bacterial outer membrane , mutant , cytosol , microbiology and biotechnology , biochemistry , membrane , gene , enzyme
Summary The type III secretion system (TTSS) is a macromolecular structure that spans the cell wall of Gram‐negative bacterial pathogens, enabling delivery of virulence effector proteins directly to the membranes and cytosol of host eukaryotic cells. TTSS consists of a conserved needle complex (NC) that is composed of sets of inner and outer membranes rings connected by a periplasmic rod. Enteropathogenic Escherichia coli (EPEC) is an extracellular diarrhoeagenic pathogen that uses TTSS to induce actin polymerization and colonizes the intestinal epithelium. In EPEC, EscJ is predicted to be targeted to the periplasm, in a sec ‐dependent manner, and to bridge the TTSS membrane‐associated rings. In this study we determined the global fold of EscJ using Nuclear Magnetic Resonance spectroscopy. We show that EscJ comprises two subdomains (D1 – amino acid residues 1–55 in the mature protein, and D2 – amino acid residues 90–170), each comprising a three‐stranded β‐sheet flanked by two α‐helices. A flexible region (residues 60–85) couples the structured regions D1 and D2. Periplasmic overexpression of EscJ D1 and EscJ D2 in a single escJ mutant bacterium failed to restore protein secretion activity, suggesting that the flexible linker is essential for the rod function. In contrast, periplasmic overexpression of EscJ D1 and EscJ D2 in the same wild‐type bacterium had a dominant‐negative phenotype suggesting defective assembly of the TTSS and protein translocation.