Role played by the WD domain of ATG16L1 in defense against Salmonella infection in vivo

Abstract

Non-canonical autophagy, or LC3-associated phagocytosis (LAP), conjugates autophagy protein LC3 to endo-lysosome compartments to facilitate delivery of extracellular materials to lysosomes. The WD domain of autophagy protein ATG16L1 is required for conjugation of LC3 to endo-lysosomes during LAP, but is not required for LC3 conjugation during autophagy. Mice lacking the WD domain of ATG16L1 (WD mice) are therefore defective in LAP, but can activate autophagy to maintain tissue homeostasis. This study has used WD mice to determine the role played by the WD domain of ATG16L1 during S. Typhimurium infection "in vivo". S. Typhimurium showed increased replication and virulence in δWD mice characterized by high mortality rate, severe weight loss, and enhanced dissemination of bacteria and lymphocytes to liver and spleen. Crosses with LysMcre and villincre mice showed that expression of the WD domain of ATG16L1 in intestinal epithelial cells, rather than myeloid cells, protected against S. Typhimurium. SopF is an S. Typhimurium SPI-1 T3SS effector thought to increase virulence by blocking the binding of the WD domain of ATG16L1 to the endo-lysosome v-ATPase to prevent recruitment of LC3 to Salmonella containing vacuoles. A SopF knockout S. Typhimurium strain (JH3009δSOPF), generated by lambda recombination, showed reduced virulence in control mice, but both wild type S. Typhimurium and JH3009δSOPF showed increased virulence in mice lacking the WD domain. This suggested that the WD domain restricted S. Typhimurium replication by additional pathways that are independent of the WD-v-ATPase axis blocked by Sop-F. Fluorescent probes and biosensors showed that cells and tissues of WD mice had raised intracellular cholesterol that accumulated at sites of replication. Replication was reduced by cholesterol-lowering drugs. Taken together the results suggest that WD domain of ATG16L1 restricts S. Typhimurium replication in intestinal epithelial cells partly by reducing intracellular cellular cholesterol levels