Septins and K63 chains form separate bacterial microdomains during autophagy of entrappedShigella

Abstract

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.

High-content high-resolution microscopy and deep learning assisted analysis reveals host and bacterial heterogeneity duringShigellainfection

“…S. flexneri have been shown to interact with septins during infection. These interactions include septin ring-like structures surrounding phagocytic cups, autophagosomes, actin tails and protrusions, all being morphologically diverse structures that are normally scored by hand (Krokowski et al, 2018; Lobato-Márquez et al, 2023; Mostowy et al, 2010). To study septin-associated bacteria using high-content microscopy, we designed a tailored analysis pipeline for the automatic and unbiased identification of SEPT7-bacteria interactions ( Figure 3 ).…”

Soft X-ray Microscopy in Cell Biology: Current Status, Contributions and Prospects

“…Изучение изменений на уровне отдельных органелл, происходящих в клетках при инфекции Shigella flexneri, с использованием комбинации флуоресцентной микроскопии и ктМРД показало, что при инфекции в клетках происходит фрагментация митохондрий [53]. Корреляция данных световой микроскопии и ктМРД позволила визуализировать «ловушку» из септинов (белков, участвующих в ремоделировании мембран, цитоскелета и в инкапсуляции внутриклеточных патогенов [54]) вокруг клеток шигеллы, а также ее тесную связь с аутофагосомой.…”