Mechanical fractionation reveals structural requirements for enteropathogenic Escherichia coli Tir insertion into host membranes

Enteropathogenic Escherichia coli (EPEC) inserts its receptor for intimate adherence (Tir) into host cell membranes by using a type III secretion syst em, Detergents are frequently used to fractionate infected host cells to in vestigate bacterial protein delivery into mammalian cells. In this study, w e 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 inf ected HeLa cells to investigate the biology and biochemistry of Tir deliver y and translocation. This method demonstrates that the translocation of Tir into the host cell membrane requires its transmembrane domains, but not ty rosine phosphorylation or binding to Tir's ligand, intimin.