Effect of nystatin on invasion of <i>Serratia grimesii</i> and <i>Serratia proteamaculans</i> bacteria into epithelial cells

Abstract

BACKGROUND: Bacteria use various endocytic pathways during entering non-phagocytic cells. The involvement of caveolae/lipid rafts in bacterial invasion has been demonstrated for many bacterial pathogens. However, for bacteria of the genus Serratia, the involvement of membrane microdomains in the process of bacterial internalization has been poorly studied. AIM: To evaluate the involvement of caveolae/lipid rafts in the invasion of S. grimesii and S. proteamaculans bacteria into non-phagocytic epithelial Caco-2 and M-HeLa cells using nystatin. MATERIALS AND METHODS: M-HeLa and Caco-2 epithelial cells were incubated with 50 μM nystatin for 1 hour at 37 ºC, after which they were infected with the bacteria S. grimesii strain 30063 and S. proteamaculans strain 94, the multiplicity of infection was 100 bacteria per cell. The number of intracellular bacteria was assessed using gentamicin protection assay. The level of caveolin-1 in cells was visualized using confocal microscopy and Western blotting. The expression of Toll-like receptors genes were measured by real-time RT-PCR. RESULTS: Treatment of epithelial cells with nystatin reduces the internalization of S. grimesii and S. proteamaculans into M-HeLa cells by 30% and does not affect penetration into Caco-2 cells. At the same time, nystatin does not affect the redistribution / the integrity impairment of lipid rafts and does not lead to the cytoskeleton reorganization of eukaryotic cells. The addition of nystatin increases the level of caveolin-1 in M-HeLa cells (caveolin-1 is not expressed in Caco-2), which leads to a change plasma membrane fluidity. Nystatin promotes the secretion of proinflammatory cytokines interleukin-6 and interleukin-8 in both cell lines. Infection of M-HeLa cells pretreated with nystatin with the studied bacteria leads to an increase in the expression of tlr2 and tlr4 genes, but does not exceed the level of their expression in control samples. Therefore, it is impossible to speak unambiguously about the participation of Toll-like receptors in the invasion of Serratia bacteria. CONCLUSIONS: The results obtained suggest that the interaction of bacteria with eukaryotic cells induces the expression of caveolin-1, which leads to a change plasma membrane components mobility. This may be due to the fact that β1-integrin is involved in the invasion of the studied bacteria, which should be stabilized at the plasma membrane upon binding of the ligand due to the formation of a cholesterol- and sphingolipid-rich membrane microenvironment.