Structural and functional studies of the enteropathogenic Escherichia coli type III needle complex protein EscJ.
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 beta-sheet flanked by two alpha-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.
Crepin
Valérie F
VF
Centres for Molecular Microbiology and Infection, Imperial College London, London SW7 2AZ, UK.
Prasannan
Sunil
S
Shaw
Rob K
RK
Wilson
Rebecca K
RK
Creasey
Elizabeth
E
Abe
Cecilia M
CM
Knutton
Stuart
S
Frankel
Gad
G
Matthews
Steve
S
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure; D017124:Conserved Sequence; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D017353:Gene Deletion; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics / Q000502:physiology; D017386:Sequence Homology, Amino Acid
D029968:Escherichia coli Proteins; D046911:Macromolecular Substances