Cyclosporine Affects the Main Virulence Factors of Cryptococcus neoformans In Vitro

Abstract

This study aimed to investigate the effects of cyclosporine on the morphology, cell wall structure, and secretion characteristics of Cryptococcus neoformans. The minimum inhibitory concentration (MIC) of cyclosporine was found to be 2 µM (2.4 µg/mL) for the H99 strain. Yeast cells treated with cyclosporine at half the MIC showed altered morphology, including irregular shapes and elongated projections, without an effect on cell metabolism. Cyclosporine treatment resulted in an 18-fold increase in chitin and an 8-fold increase in lipid bodies, demonstrating changes in the fungal cell wall structure. Cyclosporine also reduced cell body and polysaccharide capsule diameters, with a significant reduction in urease secretion in C. neoformans cultures. Additionally, the study showed that cyclosporine increased the viscosity of secreted polysaccharides and reduced the electronegativity and conductance of cells. The findings suggest that cyclosporine has significant effects on C. neoformans morphology, cell wall structure, and secretion, which could have implications for the development of new antifungal agents.

Keywords: Cryptococcus neoformans; calcineurin biology; capsule; cyclosporine; virulence.

Figure 1

Effects of cyclosporine on cryptococcal cells. (A) Morphological alterations of C. neoformans induced by cyclosporine. Yeasts were stained with Indian ink to show their polysaccharide capsules. Scale bar: 5 µm. (B) Effect of cyclosporine on the metabolic activity of C. neoformans, measured using the XTT test. Statistical significance was determined via a t-test, which did not find statistical differences (p-value = 0.1778).

Figure 2

Effects of cyclosporine on chitin production by Cryptococcus neoformans. (A) Fluorescence electron microscopy of untreated and treated cells with 1 µM cyclosporine. BF: bright field; Uvitex2B: dye used for marking chitin. Scale bar: 10 µm. (B) Analysis of the mean fluorescence intensity (MFI) of untreated C. neoformans cells and those treated with 1 µM cyclosporine and labeled with Uvitex2B, a chitin cell wall marker. Statistical significance was determined via a t-test (* p-value = 0.04).

Figure 3

Analysis of the mean fluorescence intensity (MFI) of untreated C. neoformans cells and those treated with 1 µM of cyclosporine and stained with Nile red, a lipid body marker. Statistical significance was determined via a t-test (** p-value = 0.007).

Figure 4

Measurements of cellular bodies (A), capsules (B), and secreted carbohydrates (C) of C. neoformans cells treated and untreated with 1 µM of cyclosporine. **** p-value < 0.001, * p-value = 0.02, ** p-value = 0.0075. Student’s t test was used for statistical analysis.

Figure 5

Calculation of the effective diameter (A) and size distribution (B) of PS secreted by C. neoformans in cells treated and not treated with 1 µM of cyclosporine. * p-value = 0.0499. Statistical significance was determined using Student’s t test. (C) Passive microrheology of secreted polysaccharides (PS) from C. neoformans in the presence and absence of cyclosporine. Elastic modulus G′(Pa), left; viscous modulus G”(Pa), middle; and viscous complex Ƞ*(mPa•s), right, were measured.

Figure 6

Effect of cyclosporine on zeta potential (A) and conductance (B) of C. neoformans cells. * p-value = 0.03 and **** p-value < 0.001; statistical significance determined by t test. Effect of cyclosporine on the zeta potential (C) and conductance (D) of PSs from C. neoformans. **** p-value < 0.001; statistical significance determined using Student’s t test.

Figure 7

Urease production, measured according to optical density (OD) at 559 nm, of C. neoformans previously treated or not with 1 µM cyclosporine in Christensen’s urea broth. C. albicans was used as negative control. ** p-value = 0.0038 and **** p-value < 0.00001; statistical significance was determined using Kruskal–Wallis test.

Figure 8

Phospholipase secretion by C. neoformans treated and not treated with 1 µM of cyclosporine. Statistical significance determined by t test without significant differences.