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Tight junctional inhibition of entry of Toxoplasma gondii into MDCK cells
H W Nam,J H Youn,D J Kim and W Y Choi
Department of Parasitology, Catholic University Medical College, Seoul 137-701, Korea.
Abstract
Various conditions of cultures were performed to investigate the role of tight junctions formed between adjacent MDCK cells on the entry of Toxoplasma. When MDCK cells were cocultured with excess number of Toxoplasma at the seeding density of 1 × 105, 3 × 105, and 5 × 105 cells/ml for 4 days, the number of intracellular parasites decreased rapidly as the host cells reached saturation density, i.e., the formation of tight junctions. When the concentration of calcium in the media (1.8 mM in general) was shifted to 5 µM that resulted in the elimination of tight junction, the penetration of Toxoplasma increased about 2-fold (p < 0.05) in the saturated culture, while that of non-saturated culture decreased by half. Trypsin-EDTA which was treated to conquer the tight junctions of saturated culture favored the entry of Toxoplasma about 2.5-fold (p < 0.05) compared to the non-treated, while that of non-saturated culture decreased to about one fifth. It was suggested that the tight junctions of epithelial cells play a role as a barrier for the entry of Toxoplasma and Toxoplasma penetrate into host cells through membrane structure-specific, i.e., certain kind of receptors present on the basolateral rather than apical surface of MDCK cells.
Figures
Fig. 1 Host cell entry of Toxoplasma into MDCK cells. MDCK cells and parasites were cocultured for 60 min and intracellular parasites were counted on 100 cells after staining with Giemsa solution.
Fig. 2 Cell number-dependent entry of Toxoplasma. MDCK cells were seeded at densities of 1×105(-◦-), 3×105(-•-), and 5×105(-□-) cells/ml. After incubation for 60 min with excess number of parasites intracellular parasites were counted.
Fig. 3 Photographs of MDCK cells penetrated by Toxoplasma. The number of Toxoplasma (black dots in the cytoplasms but not in nuclei of MDCK cells) decreased rapidly as the host cell reached saturation density. 1T1, at day 1; 1T2, at day 2; 1T3, at day 3; and 1T4, at day 4 after seeding with density of 1×105 cells/ml. 3T1, at day 1; 3T2, at day 2; 3T3, at day 3; and 3T4, at day 4 after seeding with density of 3×105 cells/ml. And 5T1, at day 1; 5T2, at day 2; 5T3, at day 3; and 5T4, at day 4 after seeding with density of 5×105 cells/ml.
Fig. 4 Effect of calcium depletion on the entry of Toxoplasma. MDCK cells were seeded with densities of 1×105(□) and 5×105(▩) cells/ml, grown in normal media or LC media, and then Toxoplasma was added.
Fig. 5 Effect of trypsin-EDTA on the entry of Toxoplasma. MDCK cells were seeded with densities of 1×105(□) and 5×105(▩) cells/ml, treated with trypsin-EDTA or not, and then Toxoplasma was added.
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