IRSp53 links the enterohemorrhagic E. coli effectors Tir and EspFU for actin pedestal formation

Area of Science:

• Microbiology
• Cell Biology
• Pathogenesis

Background:

• Pathogenic E. coli strains like EHEC utilize bacterial effectors to manipulate host cell actin polymerization, forming actin pedestals essential for infection.
• Enteropathogenic E. coli (EPEC) typically uses tyrosine kinase signaling, while EHEC O157:H7 employs the N-WASP activator EspF(U) (TccP).
• The precise host factors mediating EHEC-induced actin assembly via Tir and EspF(U) remained largely unidentified.

Purpose of the Study:

• To identify the host cell factors that link bacterial intimin receptor Tir and the effector EspF(U) during EHEC-induced actin pedestal formation.
• To elucidate the role of IRSp53 family proteins in the EHEC actin pedestal assembly pathway.

Main Methods:

• Co-immunoprecipitation assays to detect protein-protein interactions between bacterial effectors and host factors.
• Immunofluorescence microscopy to visualize the colocalization of bacterial effectors, host factors, and actin in pedestals.
• Genetic manipulation (loss-of-function studies) to assess the necessity of IRSp53 in actin assembly.

Main Results:

• IRSp53 family members were identified as direct interaction partners of both Tir and EspF(U).
• IRSp53 colocalizes with EspF(U) and N-WASP within actin pedestals formed by EHEC.
• IRSp53 targeting to pedestals depends on Tir residues 454-463, but not on EspF(U) or N-WASP.
• Genetic ablation of IRSp53 significantly impaired EHEC-mediated actin assembly.

Conclusions:

• IRSp53 family proteins are essential host factors that bridge bacterial Tir and EspF(U) signaling in EHEC pedestal formation.
• IRSp53 acts as a critical link in the EHEC pathogenesis pathway, mediating host actin polymerization.
• This study reveals a novel mechanism of bacterial manipulation of host cell cytoskeleton by EHEC O157:H7.