Repetitive N-WASP–Binding Elements of the Enterohemorrhagic Escherichia coli Effector EspFU Synergistically Activate Actin Assembly

PLoS Pathogens

Michael K. Rosen¹  Hui-Chun Cheng¹  John M. Leong²  Douglas Robbins²  Kenneth G. Campellone³  Anosha D. Siripala³  Matthew D. Welch³  Emma J. McGhie⁴  Richard D. Hayward⁴  Vassilis Koronakis⁴ 

[1] Department of Biochemistry and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America;Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America;Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California United States of America;Department of Pathology, University of Cambridge, Cambridge, United Kingdom

关键词: Actins;    Actin polymerization;    Polymerization;    Cell membranes;    Membrane proteins;    Membrane fusion;    Enterohaemorrhagic Escherichia coli;    Immunoblotting;   

DOI  :  10.1371/journal.ppat.1000191

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

Enterohemorrhagic Escherichia coli (EHEC) generate F-actin–rich adhesion pedestals by delivering effector proteins into mammalian cells. These effectors include the translocated receptor Tir, along with EspFU, a protein that associates indirectly with Tir and contains multiple peptide repeats that stimulate actin polymerization. In vitro, the EspFU repeat region is capable of binding and activating recombinant derivatives of N-WASP, a host actin nucleation-promoting factor. In spite of the identification of these important bacterial and host factors, the underlying mechanisms of how EHEC so potently exploits the native actin assembly machinery have not been clearly defined. Here we show that Tir and EspFU are sufficient for actin pedestal formation in cultured cells. Experimental clustering of Tir-EspFU fusion proteins indicates that the central role of the cytoplasmic portion of Tir is to promote clustering of the repeat region of EspFU. Whereas clustering of a single EspFU repeat is sufficient to bind N-WASP and generate pedestals on cultured cells, multi-repeat EspFU derivatives promote actin assembly more efficiently. Moreover, the EspFU repeats activate a protein complex containing N-WASP and the actin-binding protein WIP in a synergistic fashion in vitro, further suggesting that the repeats cooperate to stimulate actin polymerization in vivo. One explanation for repeat synergy is that simultaneous engagement of multiple N-WASP molecules can enhance its ability to interact with the actin nucleating Arp2/3 complex. These findings define the minimal set of bacterial effectors required for pedestal formation and the elements within those effectors that contribute to actin assembly via N-WASP-Arp2/3–mediated signaling pathways.

【 授权许可】

CC BY