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

Date Published: December 14, 2018

Publisher: Public Library of Science

Author(s): Katrina B. Velle, Kenneth G. Campellone, Mathieu Coureuil.


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.

Partial Text

Bacteria and viruses have historically been useful tools for studying the regulation of cytoskeletal dynamics [1], as several intracellular pathogens rearrange host actin into comet tails, which propel them through the cytosol [2] and/or promote their transmission from cell-to-cell [3]. Pathogen motility is frequently driven by activation of the Arp2/3 complex, a ubiquitous actin nucleator, through either bacterial [4, 5] or host [6] actin nucleation-promoting factors (NPFs), although how different classes of nucleators cooperate in cells is not well understood. Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are also capable of reorganizing host actin via the Arp2/3 complex, but these pathogens remain extracellular to form actin-rich protrusions of the plasma membrane called pedestals [7, 8]. Actin pedestals promote “surfing” motility [9, 10], which is important for cell-to-cell spread [11]. Because EPEC and EHEC activate the host actin nucleation machinery from an extracellular location, they represent ideal models for studying the transmembrane signaling mechanisms, cytoskeletal dynamics, and nucleator cooperation that underlie cellular protrusions [12].

Pathogens such as Listeria and Shigella are often employed as tools to better understand actin dynamics and uncover new pathways and regulators of actin assembly, yet their utility for modeling actin polymerization at the plasma membrane is limited by the fact that they are cytosolic. By remaining extracellular throughout infection, EPEC and EHEC represent ideal models to study actin rearrangements triggered by transmembrane signaling cascades [12]. While the core pathways of EHEC and EPEC pedestal assembly have been characterized to some degree [81], the potential contributions of actin nucleation factors outside of the Arp2/3 complex and WASP-family have never been directly assessed. Given that the coordinated actions of multiple nucleators orchestrate a variety of cellular functions, including lamellipodia formation and cell motility [39–44], and that both the Arp2/3 complex and formins participate in pathogen-induced protrusions [51–53, 82], we examined whether some form of nucleator cooperation exists in EPEC and EHEC pedestals. Our results indicate that the formin mDia1 contributes to Arp2/3 complex-mediated actin assembly in the pedestals of EPEC but not EHEC. Our findings also support a model in which mDia1 participates in the biogenesis and maintenance of EPEC pedestals by both providing filaments that can be used by the Arp2/3 complex for branched nucleation (Fig 9A) and by promoting tyrosine kinase activation and Tir phosphorylation (Fig 9B).




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