Septins and K63 chains form separate bacterial microdomains during autophagy of entrapped Shigella

During host cell invasion, Shigella escapes to the cytosol and polymerizes actin for cell-to-cell spread. To restrict cell-to-cell spread, host cells employ cell-autonomous immune responses including antibacterial autophagy and septin cage entrapment. How septins interact with autophagy to target Shigella to destruction is poorly understood. Here, we employed a correlative light and cryo-soft X-ray tomography (cryo-SXT) pipeline to study Shigella septin cage entrapment in its near native state. Quantitative cryo-SXT showed that Shigella fragments mitochondria and enabled visualization of X-ray dense structures ([~]30 nm resolution) surrounding Shigella entrapped in septin cages. Using Airyscan confocal microscopy, we observed Lysine 63 (K63)-linked ubiquitin chains decorating septin caged entrapped Shigella. Remarkably, septins and K63 chains form separate bacterial microdomains, indicating they are recruited separately during antibacterial autophagy. Cryo-SXT and live cell imaging revealed an interaction between septins and LC3B-positive membranes during autophagy of Shigella. Together, these findings demonstrate how septin caged Shigella are targeted to autophagy and provide fundamental insights into autophagy-cytoskeleton interactions.