Convergent Evolution of Calcineurin Pathway Roles in Thermotolerance and Virulence in Candida glabrata

Abstract

Candida glabrata is an emerging human fungal pathogen that is frequently drug tolerant, resulting in difficulties in treatment and a higher mortality in immunocompromised patients. The calcium-activated protein phosphatase calcineurin plays critical roles in controlling drug tolerance, hyphal growth, and virulence in diverse fungal pathogens via distinct mechanisms involving survival in serum or growth at host temperature (37° and higher). Here, we comprehensively studied the calcineurin signaling cascade in C. glabrata and found novel and uncharacterized functions of calcineurin and its downstream target Crz1 in governing thermotolerance, intracellular architecture, and pathogenesis in murine ocular, urinary tract, and systemic infections. This represents a second independent origin of a role for calcineurin in thermotolerant growth of a major human fungal pathogen, distinct from that which arose independently in Cryptococcus neoformans. Calcineurin also promotes survival of C. glabrata in serum via mechanisms distinct from C. albicans and thereby enables establishment of tissue colonization in a murine systemic infection model. To understand calcineurin signaling in detail, we performed global transcript profiling analysis and identified calcineurin- and Crz1-dependent genes in C. glabrata involved in cell wall biosynthesis, heat shock responses, and calcineurin function. Regulators of calcineurin (RCN) are a novel family of calcineurin modifiers, and two members of this family were identified in C. glabrata: Rcn1 and Rcn2. Our studies demonstrate that Rcn2 expression is controlled by calcineurin and Crz1 to function as a feedback inhibitor of calcineurin in a circuit required for calcium tolerance in C. glabrata. In contrast, the calcineurin regulator Rcn1 activates calcineurin signaling. Interestingly, neither Rcn1 nor Rcn2 is required for virulence in a murine systemic infection model. Taken together, our findings show that calcineurin signaling plays critical roles in thermotolerance and virulence, and that Rcn1 and Rcn2 have opposing functions in controlling calcineurin signaling in C. glabrata.

Keywords: Crz1; ER stress; Rcn1; Rcn2; calcium; calmodulin; cell wall integrity; drug tolerance; ocular infection; pH homeostasis; phosphatase; thermotolerance; urinary tract infection; virulence.

Figure 1

Calcineurin pathway is required for thermotolerance in C. glabrata. (A) Pharmacological inhibition of calcineurin phenocopies genetic deletion of calcineurin. Cells were grown overnight in YPD at 24°, 5-fold serially diluted, and spotted onto YPD medium containing FK506 or cyclosporin A (CsA), and incubated at the indicated temperatures for 48 hr. Strains tested were wild-type (CBS138), cna1 mutants (YC67 and YC98), cnb1 mutants (YC191 and YC193), and crz1 mutants (YC267 and YC182). (B) The growth kinetics of C. glabrata wild-type and mutant strains at 24°. Cells were grown overnight at 24°, washed twice with dH₂O, diluted to 0.2 OD/ml in fresh liquid YPD medium, and incubated at 24° with shaking at 250 rpm. The OD₆₀₀ of cultures was measured at 0, 3, 6, 9, 24, and 30 hr. The experiments were performed in triplicate, and data were plotted using Prism 5.03. Strains tested were wild-type (CBS138), cna1 mutant (YC98), cnb1 mutant (YC193), and crz1 mutant (YC182). (C) Calcineurin is required for survival at 40°. Cells were grown overnight in YPD at 24°, 5-fold serially diluted, spotted onto YPD. The plate was incubated at 40° for 48 hr, then transferred to 24° for 48 hr incubation. (D) Calcineurin mutant cells exhibit a shrunken cell morphology at 40°. Cells were grown overnight in YPD medium at 24°, washed twice with dH₂O, diluted to 0.5 OD₆₀₀/ml in fresh liquid YPD medium, and incubated at 24° or 40° with shaking at 250 rpm for 4 hr. Strains tested were wild-type (CBS138), cna1 mutant (YC98), cnb1 mutant (YC193), and crz1 mutant (YC182). The images were taken at 100×. Scale bar = 5 µm. (E) An osmotic stabilizer rescued temperature-sensitive phenotypes of calcineurin mutants from C. glabrata, but not from C. neoformans. Cells were grown overnight in YPD at 24°, 5-fold serially diluted, spotted onto YPD medium containing 1 M sorbitol, and incubated at the temperatures indicated for 48 hr. C. neoformans strains tested were wild-type (H99) and cna1 mutant (KK1).

Figure 2

TEM images of C. glabrata grown at 37°. Calcineurin mutants (cna1 and cnb1) and crz1 mutants display aberrant cell membrane structure (red arrowheads) compared with wild-type. (A–C) wild-type (CBS138). (D–F) cna1 mutant (YC98). (G–I) cnb1 mutant (YC193). (J–L) crz1 mutant (YC182). The cna1 and cnb1 mutants display the most aberrant phenotypes at 37° and could not be recovered from 40° for TEM analysis. Scale bar = 500 nm (A, D, G, J) and 100 nm (B, C, E, F, H, I, K, L). Column one comprises low-magnification images displaying the overall cell structure. Column two displays a second image, at higher magnification, representative of the cell membrane structure of the cell in column one. Column three displays a high-magnification view of the cell membrane of one additional cell.

Figure 3

C. glabrata calcineurin plays critical roles in tissue colonization of murine urinary tract and systemic infection models. (A) The fungal burden in the kidneys was determined at day 7 after challenge with 3 × 10⁷ cells (in 30 μl) via urinary tract infection. Five female C3H/HeJ mice per strain (except four mice with one crz1 mutant due to a death following anesthesia with pentobarbital) were used. The P value between wild-type (WT) and mutants is shown. Strains tested were wild-type (CBS138); cna1 mutants (YC67 and YC98); cnb1 mutants (YC191 and YC193); and crz1 mutants (YC267 and YC182). (B) Mouse TLR4 is not required for protection from C. glabrata urinary tract infection. The fungal burden in the kidneys and bladders was determined at day 7 after challenging C3H/HeJ (group of five animals) or C3H/HeOuJ (group of eight animals) mice with 3 × 10⁷ C. glabrata wild-type (CBS138) cells via urinary tract infections. The P value between C3H/HeJ and C3H/HeOuJ mice is shown. (C) The fungal burden in the spleen and kidneys was determined at day 7 after challenge with 4 × 10⁷ cells (in 200 µl) via lateral tail vein injection. Ten male CD1 mice per strain were used. The P value between WT and mutants is shown.

Figure 4

Calcineurin pathway mutants are unable to establish murine ocular infections. Representative clinical photographs of corneas of immunocompetent ICR mice 8 days after challenge with 10⁶ cells of C. glabrata wild-type (Cg WT; CBS138), cna1 (YC98), cnb1 (YC193), crz1 (YC182), C. albicans wild-type (Ca WT; SC5314), or mock inoculation with PBS. All strains were grown overnight at 25° and diluted with PBS prior to topical application. Fungal keratitis, indicated by blue arrowheads, was seen only in animals infected with C. glabrata CBS138 (43%) or C. albicans SC5314 (100%). No infection with the calcineurin pathway mutants resulted in visible keratitis in mice.

Figure 5

Calcineurin controls ER stress tolerance, cell wall integrity, and pH homeostasis in C. glabrata. (A) Calcineurin mutants are hypersensitive to ER stress chemicals and cell wall integrity-damaging agents. Cells were grown overnight in YPD at 24°, 5-fold serially diluted, and spotted onto YPD medium containing tunicamycin (TM), dithiothreitol (DTT), calcofluor white (CFW), or Congo red (CR), and incubated at 24° for 48 hr. (B) Calcineurin and crz1 mutants are hypersensitive to the cell membrane integrity-damaging agent sodium dodecyl sulfate (SDS). Cells were grown overnight in YPD at 24°, 5-fold serially diluted, and spotted onto YPD medium containing SDS and incubated at indicated temperatures for 48 hr. (C) Calcineurin is required for growth at acidic and alkaline environments. Cells were grown overnight in YPD at 24°, 5-fold serially diluted, and spotted onto YPD medium containing 150 mM HEPES buffered at pH 2 or 5. For pH 8 medium, YPD was buffered with 150 mM pH 9 HEPES (85 ml of YPD plus 15 ml of 1M HEPES at pH 9) due to slightly acidic YPD medium. The pHs of solid media were confirmed with pH indicator strips.

Figure 6

Calcineurin, but not Crz1, is required for optimal growth in serum. (A) Cells were grown overnight in YPD at 24°, 5-fold serially diluted, spotted onto synthetic complete (SC) medium ± 40% fetal bovine serum, and incubated at 24° for 4 days. (B) Time-killing curve of wild-type (CBS138), cna1 (YC98), cnb1 (YC193), and crz1 (YC182) strains grown in 100% serum. Cells surviving at 24° with shaking at 250 rpm after 0, 3, 6, 9, and 24 hr were serially diluted onto YPD medium, and CFUs were counted after 48 hr incubation at 24°. The data are represented as means ± SD from triplicate experiments. (C) Calcineurin mutants cannot proliferate as well as the wild-type strain or crz1 mutant in serum. Two microliters of 1 OD₆₀₀/ml cells was added to 100 μl of 100% serum or YPD medium in 96-well polystyrene plates, and the OD₆₀₀ was measured after stationary incubation at 24° for 24 hr. The data are represented as means ± SD from triplicate experiments. Asterisk represents P < 0.001 compared with the wild-type.

Figure 7

Calcineurin is required for azole and echinocandin tolerance in C. glabrata. (A, B) Calcineurin mutants are hypersensitive to azoles and echinocandins. Cells were grown overnight in YPD at 24°, 5-fold serially diluted, spotted onto YPD medium containing fluconazole (FLC), posaconazole (PSC), ketoconazole (KTC), micafungin (MCF), caspofungin (CSF), or anidulafungin (ANF), and incubated at 24° for 48 hr. (C, D) Disk diffusion assays were used to determine fluconazole and micafungin susceptibility of wild-type and mutant strains. Cells were grown overnight at 24°, and 0.1 OD₆₀₀ (in 100 μl) was spread on the surface of YPD medium. A disk containing 20 μg of fluconazole (C) or 50 ng of micafungin (D) was placed on the surface of the medium. The plates were incubated at 24° for 24 hr. (E, F) Time-killing curve of wild-type and cna1 (YC98), cnb1 (YC193), and crz1 (YC182) mutants in YPD medium ± fluconazole (E) or micafungin (F). The experiments were performed at 24°. The data are represented as means ± SD from triplicate experiments.

Figure 8

Genome-wide analysis of calcineurin- and Crz1-dependent targets in C. glabrata. Strains were grown overnight at 24° and washed twice with dH₂O. Cells were diluted to 0.2 OD/ml in YPD and incubated at 24° for 3 hr. For the wild-type strain, cells in log-phase were diluted to 0.2 OD/ml (10 ml) in YPD in the presence or absence of FK506 (1 μg/ml). cna1 (YC98) and crz1 (YC182) mutants were diluted to 0.2 OD/ml (10 ml) in YPD. Following 3 hr incubation at 37°/250 rpm, cells were collected for total RNA extraction, microarray experiments, and subsequent analysis. (A) Calcineurin pathway up- and downregulated genes in a C. glabrata wild-type strain treated with FK506, cna1, and crz1 mutants. (B) The overlaps between regulated genes for an FK506-treated wild-type strain, cna1, and crz1 mutants are depicted as Venn diagrams.

Figure 9

Genes regulated by the calcineurin pathway in C. glabrata. (A) List of 34 genes regulated by calcineurin and Crz1. There are 30 genes that were upregulated by calcineurin and Crz1, 3 genes downregulated by calcineurin and Crz1, and 1 gene (LIP5) upregulated by calcineurin but downregulated by Crz1. The criteria used to classify genes as upregulated or downregulated in the microarray experiments is a cutoff Q value < 0.001. (B) The transcription of CNA1 and CRZ1 was abolished in the cna1 (YC98) and crz1 (YC182) mutants, respectively. Real-time RT-PCR was used to measure the expression level of each gene. (C) Real-time RT-PCR was used to independently assess the expression levels detected in microarray analyses for YPS5 and RCN2 in wild-type, FK506-treated wild-type, cna1 mutant (YC98), and crz1 mutant (YC182). Statistically significant differences (P < 0.05 based on ANOVA, Bonferroni’s multiple comparison tests) compared with the wild-type are indicated by the asterisks.

Figure 10

Divergent roles of the calcineurin signaling cascade in stress responses. (A) Rcn2 and Rcn1 have divergent roles in stress responses. Strain preparations were carried out as described above. The plates were incubated at 30° for 48 hr. Strains tested were wild-type (CBS138), rcn2 mutants (YC525 and YC531), rcn1 mutants (YC553 and YC556), and the rcn2 rcn1 double mutant (YC594). (B) Rcn1 contributes to thermotolerance. The plates were incubated at temperatures indicated for 24 hr. (C) Rcn2 acts as a negative regulator of calcineurin signaling in response to calcium stress. Strains spotted onto YPD medium ± CaCl₂ (0.4 M) and/or FK506 (1 μg/ml) were incubated at 30° for 48 hr. (D) Calcineurin mutants exhibit modest resistance to calcium stress and are rescued by calcium at elevated temperatures. Cells were grown overnight in YPD at 24°, 5-fold serially diluted, spotted onto YPD medium in the absence or presence of 0.4 M CaCl₂, and incubated at the indicated temperatures for 48 hr. (E) The fungal burden in the spleens and kidneys was determined on day 7 after challenge with 4 × 10⁷ C. glabrata wild-type or mutant cells (in 200 µl) via lateral tail vein injection. Ten male CD1 mice per strain were used.

Figure 11

Proposed roles of the calcineurin signaling cascade in stress responses, drug tolerance, and virulence in C. glabrata. Calcineurin and Crz1 are required for various stress responses, including thermotolerance, ER stress, echinocandin tolerance, cell membrane integrity, and virulence. Calcineurin also exhibits Crz1-independent functions, including pH homeostasis and optimal growth in serum. Calcineurin (Cna1 or Cnb1) suppresses calcium tolerance, whereas Crz1 inhibits azole tolerance. Regulator of calcineurin 1 (Rcn1) is an endogenous positive regulator of calcineurin that participates in the control of multiple cellular processes, including thermotolerance, ER stress, and echinocandin tolerance. Rcn2 acts as a feedback inhibitor of calcineurin function in calcium tolerance. Solid arrows represent promoting functions, and bar-headed arrows represent inhibitory functions within the calcineurin signaling cascade. The dotted arrow represents function as seen in S. cerevisiae, but undetermined in C. glabrata.