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Infection and Immunity, July 2000, p. 4344-4348, Vol. 68, No. 7
Department of Biochemistry and Molecular
Biology and Biotechnology Laboratory, University of British Columbia,
Vancouver, British Columbia, V6T 1Z3, Canada
Received 27 January 2000/Returned for modification 20 March
2000/Accepted 5 April 2000
Enteropathogenic Escherichia 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.
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Mechanical Fractionation Reveals Structural
Requirements for Enteropathogenic Escherichia coli Tir
Insertion into Host Membranes
*
Corresponding author. Mailing address: Biotechnology
Laboratory, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. Phone: (604) 822-2210. Fax: (604) 822-9830. E-mail: bfinlay{at}unixg.ubc.ca.
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