Open AccessMechanical Fractionation Reveals Structural Requirements for Enteropathogenic Escherichia coli Tir Insertion into Host Membranes
Author(s) -
Annick Gauthier,
Myriam de Grado,
B. Brett Finlay
Publication year - 2000
Publication title -
infection and immunity
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.508
H-Index - 220
eISSN - 1070-6313
pISSN - 0019-9567
DOI - 10.1128/iai.68.7.4344-4348.2000
Subject(s) - biology , enteropathogenic escherichia coli , intimin , escherichia coli , lysis , secretion , hela , transmembrane protein , microbiology and biotechnology , cell membrane , membrane , cell fractionation , bacterial outer membrane , cell , enterobacteriaceae , biochemistry , receptor , gene
EnteropathogenicEscherichia coli (EPEC) inserts its receptor for intimate adherence (Tir) into host cell membranes by using a type III secretion system. Detergents are frequently used to fractionate infected host cells to investigate bacterial protein delivery into mammalian cells. In this study, we found that the Triton X-100-soluble membrane fraction from EPEC-infected HeLa cells was contaminated with bacterial proteins. We therefore applied a mechanical method of cell lysis and ultracentrifugation to fractionate infected HeLa cells to investigate the biology and biochemistry of Tir delivery and translocation. This method demonstrates that the translocation of Tir into the host cell membrane requires its transmembrane domains, but not tyrosine phosphorylation or binding to Tir's ligand, intimin.