title: SepL, a protein required for enteropathogenic Escherichia coli type III translocation, interacts with secretion component SepD.
abstract: Enteropathogenic Escherichia coli (EPEC), an important cause of infantile diarrhoea in the developing world, disrupts host cell microvilli, causes actin rearrangements and attaches intimately to the host cell surface. This characteristic phenotype, referred to as the attaching and effacing (A/E) effect, is encoded on a 36 kb pathogenicity island called the locus of enterocyte effacement (LEE). The LEE includes genes involved in type III secretion and translocation, the eae gene encoding an outer membrane adhesin known as intimin, the tir gene for the translocated intimin receptor, a regulator and various genes of unknown function. Among this last group is sepL. To determine the role of SepL in EPEC pathogenesis, we constructed and tested a non-polar sepL mutant. We found that this sepL mutant is deficient for A/E and that it secretes markedly reduced quantities of those proteins involved in translocation (EspA, EspB and EspD), but normal levels of those proteins presumed to be effectors (Tir, EspF and EspG). Despite normal levels of secretion, the mutant strain was unable to translocate EspF and Tir into host cells and formed no EspA filaments. Fractionation studies revealed that SepL is a soluble cytoplasmic protein. Yeast two-hybrid and affinity purification studies indicated that SepL interacts with the LEE-encoded protein SepD. In contrast to SepL, we found that SepD is required for type III secretion of both translocation and effector proteins. Together, these results demonstrate that SepL has a unique role in type III secretion as a functional component of the translocation system that interacts with an essential element of the secretion machinery.
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201-1192, USA.
authors.forename: Colin B
authors.initials: CB
authors.lastname: O'Connell
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Creasey
authors.forename: Stuart
authors.initials: S
authors.lastname: Knutton
authors.forename: Simon
authors.initials: S
authors.lastname: Elliott
authors.forename: Lynette J
authors.initials: LJ
authors.lastname: Crowther
authors.forename: Wensheng
authors.initials: W
authors.lastname: Luo
authors.forename: M John
authors.initials: MJ
authors.lastname: Albert
authors.forename: James B
authors.initials: JB
authors.lastname: Kaper
authors.forename: Gad
authors.initials: G
authors.lastname: Frankel
authors.forename: Michael S
authors.initials: MS
authors.lastname: Donnenberg
mesh_terms: D001422:Bacterial Adhesion / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial; D009154:Mutation; D011485:Protein Binding; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D001425:Bacterial Outer Membrane Proteins; C087863:EaeB protein, E coli; D029968:Escherichia coli Proteins; C102051:EspA protein, E coli; C474481:EspD protein, E coli; C429282:EspG protein, E coli; D011956:Receptors, Cell Surface; C495684:SepD protein, E coli; C416081:SepL protein, E coli; C109287:Tir protein, E coli