title: A novel C-terminal region within the multicargo type III secretion chaperone CesT contributes to effector secretion.
abstract: The enteropathogenic Escherichia coli (EPEC) multicargo chaperone CesT interacts with at least 10 effector proteins and is central to pathogenesis. CesT has been implicated in coordinating effector hierarchy, although the mechanisms behind this regulation are poorly understood. To address this question, we set out to functionally characterize CesT with respect to roles in (i) effector binding, (ii) effector recruitment to the type III secretion system (T3SS), and (iii) effector translocation into host cells. A CesT variant expression library was screened in EPEC using a newly developed semi-high-throughput secretion assay. Among many deficient CesT variants, a predominant number were localized to a novel CesT C-terminal region. These CesT C-terminal variants exhibited normal effector binding yet reduced effector secretion levels. Structural correlation and thermal spectroscopy analyses of purified CesT variants implicated multiple surface-exposed residues, a terminal helix region, and a flexible C-terminal triple-serine stretch in effector secretion. Site-directed mutagenesis of the flexible CesT C-terminal triple-serine sequence produced differential effector secretion, implicating this region in secretion events. Infection assays further indicated that the C-terminal region of CesT was important for NleA translocation into host cells but was dispensable for Tir translocation. The findings implicate the CesT C terminus in effector secretion and contribute to a model for multiple-cargo chaperone function and effector translocation into host cells during infection.
authors.affiliation: Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Thangadurai
authors.initials: T
authors.lastname: Ramu
authors.forename: Madhulika Esther
authors.initials: ME
authors.lastname: Prasad
authors.forename: Erica
authors.initials: E
authors.lastname: Connors
authors.forename: Amit
authors.initials: A
authors.lastname: Mishra
authors.forename: Jenny-Lee
authors.initials: JL
authors.lastname: Thomassin
authors.forename: Jason
authors.initials: J
authors.lastname: Leblanc
authors.forename: Jan K
authors.initials: JK
authors.lastname: Rainey
authors.forename: Nikhil A
authors.initials: NA
authors.lastname: Thomas
mesh_terms: D000483:Alleles; D019943:Amino Acid Substitution; D001430:Bacteriocins; D019295:Computational Biology; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D014644:Genetic Variation; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D010455:Peptides; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment
chemical_list: D001430:Bacteriocins; C122044:CesT protein, E coli; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D019151:Peptide Library; D010455:Peptides; C110490:mutacin II prepeptide, bacteria