Calcineurin is essential for Candida albicans survival in serum and virulence

Abstract

Calcineurin is a calcium-activated protein phosphatase that is the target of the immunosuppressants cyclosporin A and FK506. In T cells, calcineurin controls nuclear import of the NF-AT transcription factor and gene activation. In plants and fungi, calcineurin functions in stress responses (e.g., temperature, cations, and pH) and is necessary for the virulence of the fungal pathogen Cryptococcus neoformans. Here we show that calcineurin is also required for the virulence of another major fungus that is pathogenic to humans, Candida albicans. C. albicans calcineurin mutants had significantly reduced virulence in a murine model of systemic infection. In contrast to its role in C. neoformans, calcineurin was not required for C. albicans survival at 37 degrees C. Moreover, C. albicans calcineurin mutant strains exhibited no defects in known Candida virulence traits associated with host invasion, including filamentous growth, germ tube formation, and adherence to and injury of mammalian cells. C. albicans calcineurin mutant strains failed to colonize and grow in the kidneys of infected animals and were unable to survive when exposed to serum in vitro. Our studies illustrate that calcineurin has evolved to control aspects of the virulence of two divergent fungal pathogens via distinct mechanisms that can be targeted to achieve broad-spectrum antifungal action.

FIG. 1.

Calcineurin is essential for the virulence of C. albicans. The wild-type strain CAF2 (diamonds), the cnb1/cnb1 calcineurin B mutants JRB64 (squares) and JRB71 (triangles), and the cnb1/cnb1+ CNB1 calcineurin B reconstituted strain MCC85 (multiplication signs) were used to infect groups of 5 (A) or 10 (B) mice each by lateral tail vein injection, and survival was monitored over time. The survival of mice infected with calcineurin mutant strains was significantly different from that of mice infected with the wild-type strain or the reconstituted mutant strain (P < 0.05), except between JRB64 and MCC85 in panel A. No significant difference was noted between the two calcineurin mutant strains or between the wild-type and reconstituted strains in either experiment (P > 0.05).

FIG. 2.

Calcineurin is not required for growth of C. albicans at 37°C. Wild-type and calcineurin B mutant strains of C. albicans and C. neoformans were grown at 30 and 37°C on rich medium for 72 h and photographed.

FIG. 3.

Calcineurin is not required for germ tube formation or adherence to epithelial cells. (A) Wild-type (WT) (SC5314), calcineurin mutant (cnb1/cnb1) (JRB64), and calcineurin reconstituted (cnb1/cnb1+ CNB1) (MCC85) strains were grown in rich liquid medium with 10% FBS at 37°C for 2.5 h, and germ tubes produced by all three strains were visualized by Nomarski optics and photographed at a magnification of ×200. At this stage, 89.6% of wild-type, 82.5% of calcineurin mutant, and 87.0% of reconstituted cells had procuced germ tubes (only the difference between the data for wild-type and calcineurin mutant cells was statistically significant [P = 0.03]). (B) A total of 5 × 10⁶ wild-type (SC5314) or calcineurin mutant (JRB64) cells were incubated with 5 × 10⁴ human BECs. Cells were Gram stained, visualized with a Zeiss Axioscop 2 plus microscope, and photographed at a magnification of ×400. Arrows indicate representative C. albicans cells attached to epithelial cells. Bars, 25 μm.

FIG. 4.

Calcineurin mutants exhibit a modest defect in endothelial cell wounding. A marker-matched wild-type strain, DAY185, was compared with the calcineurin B cnb1/cnb1 mutant strain JRB64 and the cnb1/cnb1 + CNB1 reconstituted strain MCC85 for the ability to wound endothelial cells, as measured by ⁵¹Cr release. The rim101/rim101 mutant served as a control based on its established defect in endothelial cell wounding. The level of wounding by the wild-type strain was taken as 100%. Error bars, standard errors of the means for six replicate tests.

FIG. 5.

Calcineurin is important for renal infiltration during C. albicans infection. Histological analysis of renal tissue 1 day postinfection demonstrated a marked decrease in C. albicans levels in the renal parenchyma of animals infected with the cnb1/cnb1 calcineurin mutant strain. Kidneys of animals infected with the wild-type strain (WT) (left) had multiple nodules of infection approximately 75 to 250 μm in diameter, whereas kidneys of animals infected with the cnb1/cnb1 calcineurin mutant (JRB64) (right) contained fewer fungi, which were typically present as single cells or in small nodules of infection (arrow).

FIG. 6.

Calcineurin is required for C. albicans survival in serum. A wild-type strain (WT) (diamonds), calcineurin mutant (cnb1/cnb1) strains JRB64 (squares) and JRB71 (triangles), and the calcineurin reconstituted (cnb1/cnb1 + CNB1) strain MCC85 (multiplication signs) were tested for survival in 100% FBS. The fold change in population size was determined by comparing the CFU at each time point with the initial CFU. Values below the dashed line represent a fold change below 1, indicating cell death. (A) WT, calcineurin mutant (cnb1/cnb1), and reconstituted (cnb1/cnb1 + CNB1) strains at ∼2,500 cells/ml were incubated in 100% FBS at 30°C. Aliquots were collected at 3, 6, and 9 h postincubation and grown on YPD medium for 18 h at 37°C. The CFU at each time point was compared to the CFU at time zero and was recorded as fold population change. Error bars, standard errors of the means for three to six repetitions. (B) Aliquots of cultures grown for 24 h in serum were grown on YPD medium for 12 h. CFU was measured and compared to CFU at time zero. The difference between the calcineurin mutant and both the WT and reconstituted strains was statistically significant (P < 0.05), whereas there was no statistically significant difference between the WT and reconstituted strains (P > 0.05). Error bars, standard errors of the means for six repetitions.

FIG. 7.

Medium supplementation restores growth of calcineurin mutants in serum. (A) Wild-type (SC5314) (shaded bars), calcineurin mutant (JRB64) (hatched bars), and reconstituted (MCC85) (open bars) strains were grown for 24 h at 30°C in serum diluted 1:1 with PBS or YPD, or in YPD diluted 1:1 with PBS. Statistically significant differences were noted between the wild-type strain and both the calcineurin mutant strain (P < 0.005) and the reconstituted strain (P = 0.006) but not between the calcineurin mutant and reconstituted strains (P > 0.05) in the serum-PBS experiment. Also, no significantly significant difference in growth was found between the samples in the serum-YPD and YPD-PBS experiments (P > 0.05). (B) Wild-type (WT) (SC5314) and calcineurin mutant (cnb1/cnb1) (JRB64) strains were grown for 24 h at 30°C in serum either alone or supplemented with either 5.95 mg of niacin/ml or 5.3 mg of thiamine/ml. Fold changes in population size were measured by comparing the CFU at the conclusion of the experiments with the initial CFU. Differences in growth between the wild-type and calcineurin mutant strains in both the serum-alone (P < 0.001) and thiamine addition (P = 0.004) experiments were statistically significant. Values below the dashed line represent a fold change below 1, indicating cell death.