TY - JOUR T1 - Cytosolic Extract Induces Tir Translocation and Pedestals in EPEC-Infected Red Blood Cells A1 - Swimm, Alyson I A1 - Kalman, Daniel Y1 - 2008/01/18 N2 - Author SummaryEnteropathogenic Escherichia coli (EPEC) is a diarrheagenic enteric pathogen that attaches to host cells and forms actin-filled membranous protrusions called pedestals. Pedestal formation is initiated when the EPEC virulence factor, Tir, is translocated into the host cell via bacterial Type III secretion (T3S) and inserted into the plasma membrane, initiating a signaling cascade that results in actin polymerization beneath attached bacteria. We have developed a cytoplasmic extract-based system in permeabilized cells to study these early events in EPEC pathogenesis, many of which are not easily studied in intact cells. We have taken advantage of the observation that EPEC fail to form pedestals on red blood cells (RBC). We report that low calcium triggers T3S of Tir into RBC, indicating how the T3S apparatus may sense entry into the host cytoplasm. Additionally, insertion of Tir into the host membrane depends upon host cytoplasmic components, a requirement not previously recognized or accessible to experimental manipulation in intact cells. Finally, cytoplasmic extract reconstitutes actin polymerization beneath attached bacteria using signaling molecules required in intact cells. We are currently purifying the components that mediate these processes. Together, these experiments show how functional biochemistry approaches can reveal novel roles for cytoplasmic factors in host–pathogen interactions. JF - PLOS Pathogens JA - PLOS Pathogens VL - 4 IS - 1 UR - https://doi.org/10.1371/journal.ppat.0040004 SP - e4 EP - PB - Public Library of Science M3 - doi:10.1371/journal.ppat.0040004 ER -