Host PIK3C3 promotes Shigella flexneri spread from cell to cell through vacuole formation

Abstract

Shigella flexneri is a human intracellular pathogen responsible for bacillary dysentery (bloody diarrhea). S. flexneri invades colonic epithelial cells and spreads from cell to cell, leading to massive epithelial cell fenestration, a critical determinant of pathogenesis. Cell- to-cell spread relies on actin-based motility, which leads to formation of membrane protrusions, as bacteria project into adjacent cells. Membrane protrusions resolve into intermediate structures termed vacuole-like protrusions (VLPs), which remain attached to the primary infected cell by a membranous tether. The resolution of the membranous tether leads to formation of double-membrane vacuoles (DMVs), from which S. flexneri escapes to gain access to the cytosol of adjacent cells. Here, we identify the class III PI3K family member PIK3C3 as a critical determinant of S. flexneri cell-to-cell spread. Inhibition of PIK3C3 decreased the size of infection foci formed by S. flexneri in HT-29 cells. Tracking experiments using live-fluorescence confocal microscopy showed that PIK3C3 is required for efficient resolution of VLPs into DMVs. PIK3C3-dependent accumulation of PtdIns(3)P at the VLP membrane in adjacent cells correlated with the transient recruitment of the membrane scission machinery component Dynamin 2 at the neck of VLPs at the time of DMV formation. By contrast, Listeria monocytogenes did not form VLPs and protrusions resolved directly into DMVs. However, PIK3C3 was also required for L. monocytogenes dissemination, but at the stage of vacuole escape. Finally, we showed that PIK3C3 inhibition decreased S. flexneri dissemination in the infant rabbit model of shigellosis. We propose a model of Shigella dissemination in which vacuole formation relies on the PIK3C3-dependent accumulation of PtdIns(3)P at the VLP stage of cell-to-cell spread, thereby supporting the resolution of VLPs into DMVs through recruitment of the membrane scission machinery component, DNM2. Author summaryShigella flexneri is an intracellular pathogen causing bacillary dysentery, a disease responsible for more than 200,000 deaths each year in the world. With the lack of efficient vaccines and the dramatic increase in multi-drug-resistant clinical isolates, it is critical to better understand Shigella pathogenesis to suggest new therapeutic treatments. Previous studies demonstrated that invasion of epithelial cells in the human colon and subsequent spread from cell to cell are critical determinants of pathogenesis. Cell-to-cell spread relies on manipulation of the host cell actin cytoskeleton supporting bacterial movement in the cytosol of infected cells. At cell-cell contacts, bacteria project into adjacent cells through formation of membrane protrusions that resolve into vacuoles from which the bacteria escape to gain access to the cytosol of adjacent cells. Here, we show that the host cell kinase PIK3C3 is critical for efficient cell-to-cell spread through resolution of protrusions into vacuoles. Our work suggests that inhibitors of PIK3C3 may represent novel avenues of therapeutic intervention in shigellosis.