AUTHOR=Galeev Alibek , Suwandi Abdulhadi , Bakker Hans , Oktiviyari Ade , Routier Françoise H. , Krone Lena , Hensel Michael , Grassl Guntram A. 

TITLE=Proteoglycan-Dependent Endo-Lysosomal Fusion Affects Intracellular Survival of Salmonella Typhimurium in Epithelial Cells

JOURNAL=Frontiers in Immunology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2020.00731

DOI=10.3389/fimmu.2020.00731

ISSN=1664-3224

ABSTRACT=<p>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 β-D-xylosyltransferase enzymes encoded in mammals by the <italic>XylT1/XylT2</italic> genes. 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 in <italic>Salmonella enterica</italic> serovar Typhimurium (<italic>S</italic>. Typhimurium) infection of epithelial cells. Gentamicin protection and chloroquine resistance assays were applied to assess invasion and replication of <italic>S</italic>. Typhimurium in wild-type and xylosyltransferase-deficient (Δ<italic>XylT2</italic>) Chinese hamster ovary (CHO) cells lacking PGs. We found that <italic>S</italic>. Typhimurium adheres to and invades CHO WT and CHO Δ<italic>XylT2</italic> cells at comparable levels. However, 24 h after infection, proteoglycan-deficient CHO Δ<italic>XylT2</italic> cells are significantly less colonized by <italic>S</italic>. 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 the <italic>XylT2</italic> gene. Chloroquine resistance assay and immunostaining revealed that in the absence of PGs, significantly less bacteria are associated with <italic>Salmonella</italic>-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 increased <italic>S</italic>. Typhimurium burden in CHO Δ<italic>XylT2</italic> cells demonstrating an important role of PGs for PIKfyve dependent vesicle fusion which is modulated by <italic>Salmonella</italic> to establish infection. Overall, our results demonstrate that PGs influence survival of intracellular <italic>Salmonella</italic> in epithelial cells via modulation of PIKfyve-dependent endo-lysosomal fusion.</p>