WIPF2 promotesShigella flexneriactin‐based motility and cell‐to‐cell spread

Abstract

Shigella flexneri is an intracellular pathogen that disseminates in colonic epithelial cells through actin‐based motility and formation of membrane protrusions at cell–cell contacts, that project into adjacent cells and resolve into vacuoles, from which the pathogen escapes, thereby achieving cell‐to‐cell spread. Actin nucleation at the bacterial pole relies on the recruitment of the nucleation‐promoting factor N‐WASP, which activates the actin nucleator ARP2/3. In cells, the vast majority of N‐WASP exists as a complex with WIP. The involvement of WIP in N‐WASP‐dependent actin‐based motility of various pathogens, including vaccinia virus and S. flexneri, has been highly controversial. Here, we show that WIPF2 was the only WIP family member expressed in the human colonic epithelial cell line HT‐29, and its depletion impaired S. flexneri dissemination. WIPF2 depletion increased the number of cytosolic bacteria lacking actin tails (non‐motile) and decreased the velocity of motile bacteria. This correlated with a decrease in the recruitment of N‐WASP to the bacterial pole, and among N‐WASP‐positive bacteria, a decrease in actin tail‐positive bacteria, suggesting that WIPF2 is required for N‐WASP recruitment and activation at the bacterial pole. In addition, when motile bacteria formed protrusions, WIPF2 depletion decreased the number of membrane protrusions that successfully resolved into vacuoles.

Shigella flexneri subverts host polarized exocytosis to enhance cell‐to‐cell spread

“…As previously reported (Schuch et al, 1999), the mxiM-/pBAD-mxiM+ strain entered into HeLa cells similarly to WT Shigella when cultured in broth containing arabinose, but was defective in entry when grown in medium lacking arabinose (Figure 2a). For spreading studies, we used an established microscopy-based assay that measures the number of infected host cells in infection foci resulting from bacterial dissemination (Dowd, Mortuza, Bhalla et al, 2020;Gianfelice et al, 2015;Kuehl et al, 2014;Michard et al, 2019;Polle et al, 2014;Rigano et al, 2014). Because cell-to-cell spread of Shigella was reported to be inefficient in the HeLa subclone HeLa 229 (CCL-2.1 from the American Type Culture Collection) Itami et al, 1998), we verified that spread occurs efficiently in our HeLa cell line, which was HeLa CCL-2 (Experimental Procedures).…”

“…These studies were a variation of an established microscopy-based assay that measures the sizes of infection foci resulting from bacterial dissemination (Dowd, Mortuza, Bhalla et al, 2020;Gianfelice et al, 2015;Kuehl et al, 2014;Michard et al, 2019;Polle et al, 2014;Rigano et al, 2014). For experiments involving HeLa cells that were not transfected with siRNAs (Figures S1 and 2b), cells were grown to confluency for approximately 72 hr on sterile coverslips in 6-well plates prior to infection with Shigella strains at a multiplicity of infection (MOI) of approximately 2.5 bacteria per host cell (2.5:1).…”

Localization of host endocytic and actin‐associated proteins during Shigella flexneri intracellular motility and intercellular spreading

“…To accomplish this, cytoplasmic bacteria utilize their polarized surface effector IcsA to bind to and activate N‐WASp, a host actin nucleation‐promoting factor (Egile et al, 1999; Krokowski et al, 2019; Robbins et al, 2001; Rossi et al, 2017; Sandlin et al, 1995; Suzuki et al, 1998). Through N‐WASp and an assortment of host actin‐associated proteins, S. flexneri co‐opts the Arp2/3 complex to assemble, at one bacterial pole, a slender branched actin‐rich comet/rocket tail which propels the bacteria throughout the host cytoplasm (Dhanda, Yang, & Guttman, 2021; Egile et al, 1999; Michard et al, 2019; Suzuki et al, 1998). When a motile microbe interacts with the host cell plasma membrane, the propulsive forces generated by the actin tail in conjunction with other host processes exploited by the bacteria, such as exocytosis and membrane tensioning, result in the formation of an actin‐rich bacterial‐led membrane protrusion (Duncan‐Lowey et al, 2020; Gouin et al, 1999; Herath et al, 2021; Kadurugamuwa et al, 1991; Sansonetti et al, 1991; see also Agaisee, 2016 for a review).…”