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

Proteoglycans (PG) which glycoconjugates predominantly expressed on the cell surface and consists of glycosaminoglycans (GAGs) are linked to core proteins. The initial step of the assembly GAG regulated by the enzyme β-D-xylosyltransferase encoded in mammals with XylT1 / XylT2 genes. PG is essential for cell interaction with other cells and with the extracellular matrix. 

Numerous studies have highlighted the role of PG in bacterial adhesion, invasion, and immune response. In this work, we investigated the role of PG in Salmonella enterica serovar Typhimurium (S. Typhimurium) infection of epithelial cells. gentamicin protection test and chloroquine resistance are applied to judge the invasion and replication in wild-type S. Typhimurium and xylosyltransferase-deficiency (ΔXylT2) Chinese hamster ovary (CHO) cells lacking PG. 

We found that S. Typhimurium embrace and attack the cells of WT and CHO CHO ΔXylT2 at comparable rates. However, 24 hours after infection, cells proteoglycan-deficient CHO tube  ΔXylT2 significantly more colonized with S. Typhimurium compared with WT CHO cells. proteoglycans dependent phenotype can be rescued by the addition of PG in cell culture media, as well as by complementation of genes XylT2.exosome Chloroquine resistance assay and immunostaining revealed that in the absence of PG, significantly less bacteria associated with Salmonella-containing vacuoles (SCVs) for re-distribution of gentamicin endocytosis.

 Inhibition of endo-lysosome fusion by specific inhibitor of phosphatidylinositol phosphate kinase PIKfyve significantly increased burden of S. Typhimurium in CHO cells ΔXylT2 shows the important role of PG to PIKfyve dependent vesicle fusion which is modulated by Salmonella to establish an infection. Overall, our results suggest that PG affect intracellular survival of Salmonella in the epithelial cells via modulation PIKfyve dependent endo-lysosomal fusion.

Nanofabrication and intracellular Raman photocatalytic Live Cell Imaging with functionalized AFM probe.

The atomic force microscope (AFM) is an effective platform for in vitro manipulation and analysis of living cells in medicine and biology. To introduce additional new features and functionality into the conventional AFM system, we investigated the photocatalytic nanofabrication and intracellular Raman imaging of living cells by using functionalized AFM probe. 

Here, we examined the effects of speed grooves on the perforation of the cell membrane of living cells based on a very local HeLa by photochemical Feline oxidation catalytic titanium dioxide (TiO2) -functionalized of an AFM probe. On the basis of force-distance curves obtained during the indentation process, the possibility of perforation of the cell membrane, Agarose  penetrating power, and cell viability was determined quantitatively. 

In addition, we explore the possibility of intracellular tip-enhanced Raman spectroscopy (TERS) imaging of molecular dynamics in living cells through functionalized AFM probe with silver nanoparticles in homemade Raman system integrated with inverted microscope. We successfully demonstrated that intracellular TERS imaging has the potential to visualize the different distinctive features in the Raman spectrum between the nucleus and cytoplasm of single living cells and to analyze the dynamic behavior of biomolecules in living cells.