Virus-like particle capture reveals coordination of actin remodeling during Shigella flexneri entry by host proteins

Shigella spp. are intracellular bacterial pathogens that enter the host via plasma membrane insertion of a type 3 secretion system (T3SS) translocon, which triggers signaling cascades that include modulation of cytoskeletal dynamics, resulting in bacterial uptake. To better understand translocon insertion-induced host processes, we adapted a method to capture in virus-like particles (VLP) host proteins that are recruited to the cytosolic face of natively delivered S. flexneri translocons. Proteomic analyses reveal enrichment of 14-3-3ζ, a signaling protein, and CAP2, a regulator of actin turnover. 14-3-3ζ and CAP2 are necessary for host entry by T3SS pathogens. 14-3-3ζ dimers function as molecular scaffolds in the formation of bacterial-associated membrane ruffles. Concurrently, CAP2 localizes to membrane ruffles and cooperates with 14-3-3ζ to enable the formation of membrane ruffles that function efficiently in bacterial uptake. The findings define a coordinated role for 14-3-3ζ and CAP2 in cytoskeletal dynamics during T3SS pathogen infection.