SHIGELLA FLEXNERI REQUIRES THE HOST SCAFFOLD PROTEIN RACK1 TO MODULATE ACTIN POLYMERIZATION, PROMOTING INFECTION

Abstract

Shigella flexneri, the causative agent of the acute diarrheal disease shigellosis, manipulates the host actin cytoskeleton to infect intestinal epithelial cells (ECs). Shigella secretes bacterial effectors into the EC cytoplasm, inducing actin polymerization at the entry site. After cell invasion, Shigella escapes the endocytic vacuole accessing the cytoplasm, where the pathogen polymerizes an actin tail acquiring actin-mediated motility. Propelled by the actin tail, Shigella generates a bacterium-containing membrane protrusion that infects neighbouring cells. The receptor for activated C kinase 1 (RACK1) is a scaffolding protein that provides a platform for protein-protein interactions playing pivotal roles in host cell homeostasis. However, the role of RACK1 in bacterial pathogenesis is unclear. RACK1 binds to the focal adhesion kinase and actin-binding proteins, allowing proper focal adhesion assembly and cell migration; thus, I hypothesized that RACK1 promotes Shigellamediated induction of actin polymerization. Live-cell microscopy and automated image data analysis were used to characterize the role of RACK1 in Shigella infection. I found that RACK1 silencing in HeLa cells reduced the yield of Shigella recovered from within ECs, due to cell invasion impairment and cell-to-cell spreading inhibition. RACK1 was recruited to the entry focus promoting Shigella-mediated induction of actin polymerization and internalization. RACK1 also promoted actin tail polymerization and actin-mediated motility, resulting in effective cell-to-cell spreading. In contrast, RACK1 silencing did not affect Shigella’s escape from the vacuole or intracellular replication. Moreover, RACK1 depletion in Drosophila melanogaster enterocytes reduced mortality of Shigella-fed flies. Furthermore, RACK1 silencing inhibited jasplakinolide-induced actin polymerization and reduced actin turnover in membrane ruffles but not in stress fibres. In conclusion, I report a novel function of RACK1 promoting Shigella invasion and spreading in ECs and colonization of Drosophila’s intestine. My findings also suggest RACK1 function in actin polymerization is not restricted to Shigella infection.