Abstract
Enteropathogenic Escherichia coli (EPEC) is an extracellular pathogen that tightly adheres to host cells by forming "actin pedestals" beneath the bacteria, a critical step in pathogenesis. EPEC injects effector proteins that manipulate host cell signaling cascades to trigger pedestal assembly. We have recently shown that one such effector, EspG, hijacks p21-activated kinase (PAK) and sustains its activated state to drive the cytoskeletal changes necessary for attachment of the pathogen to target cells. This EspG subversion of PAK required active Rho family small GTPases in the host cell. Here we show that EPEC itself promotes the activation of Rho GTPases by recruiting Frabin, a host guanine nucleotide exchange factor (GEF) for the Rho GTPase Cdc42. Cells devoid of Frabin showed significantly lower EPEC-induced PAK activation, pedestal formation, and bacterial attachment. Frabin recruitment to sites of EPEC attachment was driven by EspG and required localized enrichment of phosphatidylinositol 4,5-bisphosphate (PIP2) and host Arf6. Our findings identify Frabin as a key target for EPEC to ensure the activation status of cellular GTPases required for actin pedestal formation.IMPORTANCE Enteropathogenic Escherichia coli (EPEC) is a leading cause of diarrhea in children, especially in the developing world. EPEC initiates infection by attaching to cells in the host intestine, triggering the formation of actin-rich "pedestal" structures directly beneath the adherent pathogen. These bacteria inject their own receptor into host cells, which upon binding to a protein on the pathogen surface triggers pedestal formation. Multiple other proteins are also delivered into the cells of the host intestine, which work together to hijack host signaling pathways to drive pedestal production. Here we show how EPEC hijacks a host protein, Frabin, which creates the conditions in the cell necessary for the pathogen to manipulate a specific pathway that promotes pedestal formation. This provides new insights into this essential early stage in disease caused by EPEC.
Highlights
The results presented here allow us to propose a model by which Enteropathogenic Escherichia coli (EPEC) ensures that target cells contain the high levels of localized active Rho GTPases necessary for subversion of p21-activated kinase (PAK) signaling (Figure 5D)
The initial attachment of EPEC leads to the accumulation of PIP2 directly beneath the adherent bacteria
The coincident PIP2 and EspG-Arf6 lead to enrichment of Frabin, triggering local activation of Cdc42
Summary
Lipid bilayers designed to further mimic the site of action of EspG by including PIP2 (phosphatidylinositol 4,5 bisphosphate), known to be enriched at sites of EPEC attachment to host cells[23], recruited active, phosphorylated PAK (PAK-P) and a small amount of Cdc42 This was significantly enhanced by the presence of EspG, which lead to efficient recruitment of both Cdc and phosphorylated PAK (Figure 2C). As seen for PAK phosphorylation above, pedestal length and bacterial attachment showed a small further decrease upon inhibition of Rac in Frabin cells, and only a partial reduction in WT Hap cells (Figure 3D and 3E) This data shows that EPEC generates the level of active Cdc in infected cells required for subsequent hijacking of PAK via the host GEF Frabin. This suggests that EspG can enhance recruitment of Frabin without interacting with Arf, and does not act to protect active Arf from inactivation
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