Abstract
Autophagy defends cells against proliferation of bacteria such as Salmonella in the cytosol. After escape from a damaged Salmonella‐containing vacuole (SCV) exposing luminal glycans that bind to Galectin‐8, the host cell ubiquitination machinery deposits a dense layer of ubiquitin around the cytosolic bacteria. The nature and spatial distribution of this ubiquitin coat in relation to other autophagy‐related membranes are unknown. Using transmission electron microscopy, we determined the exact localisation of ubiquitin, the ruptured SCV membrane and phagophores around cytosolic Salmonella. Ubiquitin was not predominantly present on the Salmonella surface, but enriched on the fragmented SCV. Cytosolic bacteria without SCVs were less efficiently targeted by phagophores. Single bacteria were contained in single phagophores but multiple bacteria could be within large autophagic vacuoles reaching 30 μm in circumference. These large phagophores followed the contour of the engulfed bacteria, they were frequently in close association with endoplasmic reticulum membranes and, within them, remnants of the SCV were seen associated with each engulfed particle. Our data suggest that the Salmonella SCV has a major role in the formation of autophagic phagophores and highlight evolutionary conserved parallel mechanisms between xenophagy and mitophagy with the fragmented SCV and the damaged outer mitochondrial membrane serving similar functions.
Highlights
Autophagy is a catabolic pathway to maintain cellular homeostasis by generating nutrients under starvation conditions or by eliminating damaged organelles and dysfunctional cellular components as a quality control mechanism.[1,2] This pathway is important for the innate immunity response to infection by targeting intracellular bacteria residing in phagosomes, damaged vacuoles and the cytosol.[3,4] TheNicholas T
Our results demonstrate the exact topology of host and Salmonella membranes, ubiquitinated target proteins and components of the autophagy machinery and provide evidence that Salmonella-containing vacuole (SCV) membrane proteins are ubiquitination targets that are recognised by selective autophagy receptors, such as TAX1BP1 leading to the assembly of phagophores
To further analyse whether the formation of a phagophore around cytosolic bacteria overlaps with the presence of Galectin8-positive SCV membrane fragments, mouse embryonic fibroblasts (MEFs) which transiently express GFP-Galectin-8 were infected with Salmonella-Cherry for 1 hour and processed for immunoelectron micros to label the localization of Galectin-8 with anti-GFP
Summary
Autophagy is a catabolic pathway to maintain cellular homeostasis by generating nutrients under starvation conditions or by eliminating damaged organelles and dysfunctional cellular components as a quality control mechanism.[1,2] This pathway is important for the innate immunity response to infection by targeting intracellular bacteria residing in phagosomes, damaged vacuoles and the cytosol.[3,4] The. Defence process to restrict and eliminate cytosolic bacteria is activated through several parallel events.[5,6,7] A recently discovered pathway of direct targeting of intracellular pathogens inside the vacuole is through recruitment of ATG16L1 by the vacuolar-ATPase onto the bacteria-surrounding membrane, which is blocked by the bacterial effector SopF.[8] In addition, an early “eat-me” signal in the form of lectins can be found on the inside of the ruptured SCV and detected by Galectin-8 as early as 1 hour after infection.[9] This Galectin-8 recognised signal coincides with formation of a ubiquitin coat surrounding Salmonella at early time points. These findings highlight the similarity between mitophagy and xenophagy; in the former, proteins associated with the outer mitochondrial membrane, which is reminiscent of the SCV, are ubiquitinated to promote clearance of mitochondria via autophagy
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