The enteropathogenic E. coli receptor Tir remains non‐ubiquitylated to enable the bacteria to remain extracellular

Cells use clathrin‐mediated endocytosis as one strategy to internalize particles from the extracellular compartment. This mechanism of internalization requires the use of a ligand and receptor for the internalization event to occur. Recent work suggests that in order for large particles to enter non‐phagocytic cells their receptors must become ubiquitylated. Ubiquitylation involves the posttranslational modification of a protein by ubiquitin. Enteropathogenic E. coli (EPEC) is an extracellular microbe that injects its own receptor, called the translocated intimin receptor (Tir), into epithelial cells. Surprisingly clathrin‐associated components are recruited to the site of EPEC/host cell contact, but somehow clathrin's internalization function is blocked. We hypothesized that the EPEC receptor Tir remains non‐ubiquitylated to ensure that EPEC remains extracellular. To test this hypothesis we used immunoprecipitations and western blotting of EPEC infected epithelial cells to examine the ubiquitylation status of EPEC Tir and found that it does not become ubiquitylated. To functionally test our hypothesis we forced EPEC Tir to be ubiquitylated by directly fusing ubiquitin to EPEC Tir. This fusion was transfected into epithelial cells and the cells infected with EPEC containing a non‐functional Tir, resulting in the bacteria being internalized. Our work demonstrates that during normal EPEC infections, the blockage of Tir receptor ubiquitylation contributes to EPEC remaining extracellular.