Magnesium Ion Acts as a Signal for Capsule Induction in Cryptococcus neoformans

Abstract

Cryptococcal meningitis caused by Cryptococcus neoformans, is a common opportunistic neural infection in immunocompromised individuals. Cryptococcus meningitis is associated with fungal burden with larger capsule size in cerebrospinal fluid (CSF). To understand the role of CSF constituents in capsule enlargement, we have evaluated the effect of artificial CSF on capsule induction in comparison with various other capsule inducing media. Two different strains of C. neoformans, an environmental and a clinical isolates were used in the present study. While comparing the various capsule inducing media for the two different strains of C. neoformans, it was observed that the capsule growth was significantly increased when grown in artificial CSF at pH 5.5, temperature 34°C for ATCC C. neoformans and 37°C for Clinical C. neoformans and with an incubation period of 72 h. In addition, artificial CSF supports biofilm formation in C. neoformans. While investigating the individual components of artificial CSF, we found that Mg(2+) ions influence the capsule growth in both environmental and clinical strains of C. neoformans. To confirm our results we studied the expression of four major CAP genes namely, CAP10, CAP59, CAP60, and CAP64 in various capsule inducing media and in different concentrations of Mg(2+) and Ca(2+). Our results on gene expression suggest that, Mg(2+) does have an effect on CAP gene expression, which are important for capsule biosynthesis and virulence. Our findings on the role of Mg(2+) ion as a signal for capsule induction will promote a way to elucidate the control mechanisms for capsule biosynthesis in C. neoformans.

Keywords: CAP gene; Cryptococcus neoformans; biofilm; capsule; magnesium ions.

FIGURE 1

Comparison of capsule induction in ATCC C. neoformans in different media (ACSF, ACSF+FCS, DMEM, MOPS, and PDB) at various pH, temperature, and incubation period. The percentage of the capsule thickness [(total size^∗ – cell size)/(total size^∗) × 100] for ≥8 cells per group is shown. Bars represent standard errors (p < 0.0001). The greater capsule growth was observed in ACSF media (70%) at pH 5.5, 37°C, 72 h, followed by DMEM 60%, ACSF+FCS 55%, MOPS 53%, and PDB 47%. ^∗Total size = capsule size + cell size.

FIGURE 2

Comparison of capsule growth in clinical C. neoformans in different media (ACSF, ACSF+FCS, DMEM, MOPS, and PDB) at various pH, temperature, and incubation period. The higher capsule induction was observed in ACSF media at pH 5.5, 37°C, 72 h, which is followed by ACSF 70%, ACSF+FCS 56%, DMEM 55%, MOPS 53%, and PDB 49%. The cell size, capsule size, and percentage of the capsule thickness for ≥8 cells per group are shown. Bars represent standard errors (p < 0.0001).

FIGURE 3

Effect of pH and temperature on capsule induction in ATCC C. neoformans in different media at 72 h. Left Y-axis represents size (in μm) for cell and capsule, compared with capsule thickness (in %) through right Y-axis. The cell size, capsule size and percentage of the capsule thickness for ≥8 cells per group are shown. Bars represent standard errors (p < 0.0001).

FIGURE 4

Effect of pH and temperature on capsule induction in clinical C. neoformans in different media at 72 h. Left Y-axis represents size (in μm) for cell and capsule, compared with capsule thickness (in %) through right Y-axis. The cell size, capsule size and percentage of the capsule thickness for ≥8 cells per group are shown. Bars represent standard errors (p < 0.0001).

FIGURE 5

Effect of ACSF salts [Magnesium (Mg²⁺), Calcium (Ca²⁺), Potassium (K⁺), Phosphate (PO₄^3-) salts were supplemented with 0.1% glucose] on capsule induction in (A) ATCC C. neoformans at 34°C, 72 h, pH 5.5; (B) clinical C. neoformans at 37°C, 72 h, pH 5.5. 0.1% glucose used as a control. Left Y-axis represents size (in μm) for cell and capsule, compared with capsule thickness (%) through right Y-axis. The cell size, capsule size, and percentage of the capsule thickness for ≥8 cells per group are shown. Bars represent standard errors (p < 0.0001); G, Glucose.

FIGURE 6

Effect of Mg²⁺ with phosphate buffer, MOPS and PDB on capsule induction with increasing Mg²⁺ concentration (0.0 – 4.0 mM) in ATCC C. neoformans at 34°C, 72 h, pH 5.5 (A); clinical C. neoformans at 37°C, 72 h, pH 5.5 (B); 0.1% glucose used as a control. Y-axis represents capsule thickness (in %). The higher capsule size was observed with phosphate buffer + 2.0 mM Mg²⁺ in ATCC C. neoformans (67%) whereas in clinical C. neoformans (58%) sample, PDB + 0.5 mM Mg²⁺. The cell size, capsule size and percentage of the capsule thickness for ≥8 cells per group is shown. Bars represent standard errors (p < 0.0001).

FIGURE 7

ATCC C. neoformans capsule induction in different media. (A) Capsule induction in different media; (B) Capsule induction in individual ACSF salts; (C) Capsule induction in different media supplemented with Mg²⁺ ion.

FIGURE 8

Clinical C. neoformans capsule induction in different media. (A) Capsule induction in different media; (B) Capsule induction in individual ACSF salts; (C) Capsule induction in different media supplemented with Mg²⁺ ion.

FIGURE 9

Biofilm formation on coverslip with different media. Biofilm observed at 40× light microscope by India ink stain. Images of a mature biofilm show yeast cells and capsular polysaccharide.

FIGURE 10

Capsule associated gene expression. CAP gene expression of ATCC C. neoformans: (A) CAP10 gene; (B) CAP59 gene; (C) CAP60 gene; (D) CAP64 gene; (E) β-actin gene. CAP gene expression clinical C. neoformans: (A1) CAP10 gene; (B1) CAP59 gene; (C1) CAP60 gene; (D1) CAP64 gene; (E1) β-actin gene.

FIGURE 11

Proposed role of Mg²⁺ in capsule regulation in C. neoformans.