IRSp53 Links the Enterohemorrhagic E. coli Effectors Tir and EspFU for Actin Pedestal Formation

Abstract

Actin pedestal formation by pathogenic E. coli requires signaling by the bacterial intimin receptor Tir, which induces host cell actin polymerization mediated by N-WASP and the Arp2/3 complex. Whereas canonical enteropathogenic E. coli (EPEC) recruit these actin regulators through tyrosine kinase signaling cascades, enterohemorrhagic E. coli (EHEC) O157:H7 employ the bacterial effector EspF(U) (TccP), a potent N-WASP activator. Here, we show that IRSp53 family members, key regulators of membrane and actin dynamics, directly interact with both Tir and EspF(U). IRSp53 colocalizes with EspF(U) and N-WASP in actin pedestals. In addition, targeting of IRSp53 is independent of EspF(U) and N-WASP but requires Tir residues 454-463, previously shown to be essential for EspF(U)-dependent actin assembly. Genetic and functional loss of IRSp53 abrogates actin assembly mediated by EHEC. Collectively, these data indentify IRSp53 family proteins as the missing host cell factors linking bacterial Tir and EspF(U) in EHEC pedestal formation.

Membrane-deforming Proteins Play Distinct Roles in Actin Pedestal Biogenesis by Enterohemorrhagic Escherichia coli

“…The best characterized of these interactions are with the I-BAR proteins IRSp53 (14) and IRTKS (13). In fact, a single 47-residue EspF U repeat binds to the SH3 domain of IRTKS with a much higher affinity (ϳ500 nM) than typical proline-rich sequences (43).…”

“…Given that TOCA1 and FBP17 from the F-BAR family (Fig. 4), along with IRSp53 and IRTKS from the I-BAR family (13,14) are recruited to EspF U -driven actin pedestals, we next sought to compare the abilities of the SH3 domains from these proteins to activate the N-WASP/WIP complex. Therefore, we measured the effects of the GST-SH3 domains on actin polymerization in vitro.…”

“…In fact, these proline-rich motifs are involved in binding to the SH3 domain-containing I-BAR proteins IRSp53 and IRTKS (13,14). However, the ability of these and other BAR protein relatives to regulate N-WASP during pedestal biogenesis has not been explored.…”

“…Tir acts as a plasma membrane receptor for intimin, an adhesin expressed on the bacterial surface, and intimin-Tir interactions result in clustering of the C-terminal cytoplasmic domain of Tir. A 12-residue peptide within this portion of Tir harbors its essential signaling function (10 -12), and it binds to the I-BAR (inverse-BAR) domains of the membrane-deforming host cell proteins IRSp53 and IRTKS (13,14). SH3 domains from IRSp53 and IRTKS, in turn, recruit EspF U (13,14), which possesses multiple 47-residue proline-rich peptide repeats (EspF U (R1-6)) (15,16).…”

“…A 12-residue peptide within this portion of Tir harbors its essential signaling function (10 -12), and it binds to the I-BAR (inverse-BAR) domains of the membrane-deforming host cell proteins IRSp53 and IRTKS (13,14). SH3 domains from IRSp53 and IRTKS, in turn, recruit EspF U (13,14), which possesses multiple 47-residue proline-rich peptide repeats (EspF U (R1-6)) (15,16). This repeat region is the most crucial bacterial component of the signaling machinery that triggers actin pedestal biogenesis, because a Tir-EspF U hybrid protein in which the C terminus of Tir is replaced with the EspF U repeats is fully capable of driving pedestal assembly (9).…”