Enteropathogenic E. coli relies on collaboration between the formin mDia1 and the Arp2/3 complex for actin pedestal biogenesis and maintenance

Enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC) are closely related extracellular pathogens that reorganize host cell actin into “pedestals” beneath the tightly adherent bacteria. This pedestal-forming activity is both a critical step in pathogenesis, and it makes EPEC and EHEC useful models for studying the actin rearrangements that underlie membrane protrusions. To generate pedestals, EPEC relies on the tyrosine phosphorylated bacterial effector protein Tir to bind host adaptor proteins that recruit N-WASP, a nucleation-promoting factor that activates the Arp2/3 complex to drive actin polymerization. In contrast, EHEC depends on the effector EspFU to multimerize N-WASP and promote Arp2/3 activation. Although these core pathways of pedestal assembly are well-characterized, the contributions of additional actin nucleation factors are unknown. We investigated potential cooperation between the Arp2/3 complex and other classes of nucleators using chemical inhibitors, siRNAs, and knockout cell lines. We found that inhibition of formins impairs actin pedestal assembly, motility, and cellular colonization for bacteria using the EPEC, but not the EHEC, pathway of actin polymerization. We also identified mDia1 as the formin contributing to EPEC pedestal assembly, as its expression level positively correlates with the efficiency of pedestal formation, and it localizes to the base of pedestals both during their initiation and once they have reached steady state. Collectively, our data suggest that mDia1 enhances EPEC pedestal biogenesis and maintenance by generating seed filaments to be used by the N-WASP-Arp2/3-dependent actin nucleation machinery and by sustaining Src-mediated phosphorylation of Tir.肠致病性大肠埃希菌（EPEC）与肠出血性大肠埃希菌（EHEC）是两类紧密相关的胞外致病菌，可将宿主细胞肌动蛋白重排为紧密黏附于细菌下方的"肌动蛋白基座（pedestals）"。该基座形成能力既是病原菌致病过程中的关键步骤，也使得EPEC和EHEC成为研究膜突起相关肌动蛋白重排机制的理想模型。为形成肌动蛋白基座，EPEC依赖经酪氨酸磷酸化的细菌效应蛋白Tir结合宿主衔接蛋白，进而招募成核促进因子N-WASP——后者可激活Arp2/3复合物（Arp2/3 complex）以驱动肌动蛋白聚合。与之相反，EHEC则通过效应蛋白EspFU使N-WASP多聚化，从而促进Arp2/3复合物的激活。尽管目前对基座组装的核心通路已有充分解析，但其他肌动蛋白成核因子在此过程中的具体贡献仍未明确。本研究借助化学抑制剂、小干扰RNA（siRNA）及基因敲除细胞系，探究了Arp2/3复合物与其他类型成核因子之间的潜在协同作用。研究发现，抑制Formin蛋白家族（Formins）可破坏采用EPEC肌动蛋白聚合通路的细菌的肌动蛋白基座组装、运动能力及细胞定殖能力，但对采用EHEC通路的细菌无此影响。本研究还鉴定出mDia1是参与EPEC基座组装的Formin家族成员：其表达水平与基座形成效率呈正相关，且在基座形成的起始阶段与稳态阶段均定位于基座底部。综上，本研究数据表明，mDia1可通过两种途径增强EPEC基座的生物发生与维持：一是生成肌动蛋白种子丝，供N-WASP-Arp2/3依赖的肌动蛋白成核机制使用；二是维持Src介导的Tir磷酸化过程。