Figure 7

Comparative virulence study of BWP17 and CaGPI15Hz with epithelial cells. Phagocytosis of yeast (A,B) and Hyphal (C,D) form of BWP17 and CaGPI15Hz, respectively, by LA-4 cells. LA-4 cells were cultured on coverslips overnight for adherence and then cells were co-cultured with CFSE labelled BWP17 and CaGPI15Hz for 3 h (A,B) & 18 h (C,D) respectively followed by fixation with PFA and were mounted and examined using a confocal laser scanning microscope (Magnification 60X). Scale bar represents 20 µm. (E) Hyphae formation by C. albicans. The hyphae length was quantified using Nikon NIS element software. (F) CFU assay after incubation with LA-4 cells. LA-4 cells (0.1 million) were seeded in 48 well cell culture plate and kept for adherence followed by addition of the indicated C. albicans strains at MOI 1:5 for 18 h. The LA-4 cells were lysed and plated on YEPD plates kept at 30 °C for 24 h. The colony forming units obtained were quantified and plotted. (G) Uptake of BWP17 and CaGPI15Hz by LA-4 cells. LA-4 cells (0.3 million) were seeded in 24 well cell culture plate and allowed to adhere overnight in CO2 incubator at 37 °C. Cells were co-cultured with CFSE labelled BWP17 and CaGPI15Hz at MOI 1:1 (as control) & 1:5 for 3 h & 18 h and processed as discussed in Methods. The uptake of the C. albicans strains was calculated and plotted on the basis of percentage of cells taking up yeast form of BWP17 and CaGPI15Hz. (H) Live cell recovery of LA-4 cells. LA-4 cells (0.3 million) were seeded in 24 well cell culture plate. After adherence, C. albicans at MOI 1:1 & 1:5 for 3 h & 18 h were added to these cultures. Cells were trypsinized, washed and live cell recovery was calculated by trypan blue exclusion method. (I) C. albicans induced LA-4 cells pyroptosis. LA-4 cells (50,000) were seeded in 96 well cell culture plate. BWP17 and CaGPI15Hz at MOI 1:5 were added for 3 h and 18 h. 50 µl of supernatant were transferred to another 96 well cell culture plate followed by addition of substrate reagent for 30 min and then the reaction was stopped. (J) C. albicans induced LA-4 cells apoptosis. LA-4 cells (0.3 million) were seeded in 24 well cell culture plate and allowed to adhere overnight in CO₂ incubator at 37 °C. BWP17 and CaGPI15Hz at MOI 1:5 were added for 3 h and 18 h and processed as discussed in Methods. Cells were harvested and 1 µl Annexin V APC was added. Each point represents mean ± SEM of values obtained from three independent assays. *p ≤ 0.05, **p ≤ 0.005 and ***p ≤ 0.0005 represent statistically significant difference between control and treated cells, ns is no statistically significant difference. The significance of any difference was calculated by using one-tailed distribution in a two-sample equal variance student’s t test.