Coiled-coil domain of enteropathogenic Escherichia coli type III secreted protein EspD is involved in EspA filament-mediated cell attachment and hemolysis

Many animal and plant pathogens use type III secretion systems to secrete k ey virulence factors, some directly into the host cell cytosol. However, th e basis for such protein translocation has yet to be fully elucidated for a ny type III secretion system. We have previously shown that in enteropathog enic and enterohemorrhagic Escherichia coli the type III secreted protein E spA is assembled into a filamentous organelle that attaches the bacterium t o the plasma membrane of the host cell. Formation of EspA filaments is depe ndent on expression of another type Ln: secreted protein, EspD. The carboxy terminus of EspD, a protein involved in formation of the translocation por e in the host cell membrane, is predicted to adopt a coiled-coil conformati on with 99% probability. Here, we demonstrate EspD-EspD protein interaction using the yeast two-hybrid system and column overlays. Nonconservative tri ple amino acid substitutions of specific EspD carboxy-terminal residues gen erated an enteropathogenic E. coli mutant that was attenuated in its abilit y to induce attaching and effacing lesions on HEp-2 cells. Although the mut ation had no effect on EspA filament biosynthesis, it also resulted in redu ced binding to and reduced hemolysis of red blood cells. These results segr egate, for the first time, functional domains of EspD that control EspA fil ament length from EspD-mediated cell attachment and pore formation.