Proteoglycan-Dependent Endo-Lysosomal Fusion Affects Intracellular Survival ofSalmonellaTyphimurium in Epithelial Cells

Abstract

Proteoglycans (PGs) are glycoconjugates which are predominately expressed on cell surfaces and consist of glycosaminoglycans (GAGs) linked to a core protein. An initial step of GAGs assembly is governed by the beta-D-xylosyltransferase enzymes encoded in mammals by theXylT1/XylT2genes. PGs are essential for the interaction of a cell with other cells as well as with the extracellular matrix. A number of studies highlighted a role of PGs in bacterial adhesion, invasion, and immune response. In this work, we investigated a role of PGs inSalmonella entericaserovar Typhimurium (S. Typhimurium) infection of epithelial cells. Gentamicin protection and chloroquine resistance assays were applied to assess invasion and replication ofS. Typhimurium in wild-type and xylosyltransferase-deficient (Delta XylT2) Chinese hamster ovary (CHO) cells lacking PGs. We found thatS. Typhimurium adheres to and invades CHO WT and CHO Delta XylT2cells at comparable levels. However, 24 h after infection, proteoglycan-deficient CHO Delta XylT2cells are significantly less colonized byS. Typhimurium compared to CHO WT cells. This proteoglycan-dependent phenotype could be rescued by addition of PGs to the cell culture medium, as well as by complementation of theXylT2gene. Chloroquine resistance assay and immunostaining revealed that in the absence of PGs, significantly less bacteria are associated withSalmonella-containing vacuoles (SCVs) due to a re-distribution of endocytosed gentamicin. Inhibition of endo-lysosomal fusion by a specific inhibitor of phosphatidylinositol phosphate kinase PIKfyve significantly increasedS. Typhimurium burden in CHO Delta XylT2cells demonstrating an important role of PGs for PIKfyve dependent vesicle fusion which is modulated bySalmonellato establish infection. Overall, our results demonstrate that PGs influence survival of intracellularSalmonellain epithelial cells via modulation of PIKfyve-dependent endo-lysosomal fusion.