The vacuolar Ca²(+) exchanger Vcx1 is involved in calcineurin-dependent Ca²(+) tolerance and virulence in Cryptococcus neoformans

Abstract

Cryptococcus neoformans is an encapsulated yeast that causes a life-threatening meningoencephalitis in immunocompromised individuals. The ability to survive and proliferate at the human body temperature is an essential virulence attribute of this pathogen. This trait is controlled in part by the Ca²(+)-calcineurin pathway, which senses and utilizes cytosolic calcium for signaling. In the present study, the identification of the C. neoformans gene VCX1, which encodes a vacuolar calcium exchanger, is reported. The VCX1 knockout results in hypersensitivity to the calcineurin inhibitor cyclosporine A at 35°C, but not at 30°C. Furthermore, high concentrations of CaCl₂ lead to growth inhibition of the vcx1 mutant strain only in the presence of cyclosporine A, indicating that Vcx1 acts in parallel with calcineurin. The loss of VCX1 does not influence cell wall integrity or capsule size but decreases secretion of the major capsular polysaccharide glucuronoxylomannan (GXM) in culture supernatants.Vcx1 also influences C. neoformans phagocytosis by murine macrophages and is required for full virulence in mice. Analysis of cellular distribution by confocal microscopy confirmed the vacuolar localization of Vcx1 in C. neoformans cells.

Fig. 1.

Results of the in silico analysis of the C. neoformans Vcx1 ortholog. (A) Phylogenetic analysis results, applying the neighbor-joining method and including Vcx1 sequences from distinct eukaryotic organisms, as follows: S. cerevisiae (NCBI accession number NP_010155.1), C. albicans (NCBI accession number XP_711893.1), A. fumigatus (NCBI accession number XP_750174.2), S. pombe (NCBI accession number O59768), C. cinereus (Broad Institute accession number CC1G_02539), U. maydis (Broad Institute accession number UM_05132), A. thaliana (NCBI accession number Q945S5), O. sativa (NCBI accession number NP_00105030.1), and C. neoformans (Broad Institute accession number CNAG_00025.1). The bar marker indicates the genetic distance, which is proportional to the number of amino acid substitutions. Bootstrap values obtained with 1,000 resamplings are shown at the nodes. (B) Domain architecture in the Vcx1 orthologs of different eukaryotic organisms (with two-letter species abbreviations for each Vcx1; species are the same as those listed in panel A). The sodium/calcium exchanger superfamily (Na_Ca_ex; pfam01699) domain, a characteristic domain in Ca²⁺ exchangers, is represented by gray boxes. The transmembrane domains are represented by arrowheads. The lengths (in amino acids) of each of the sequence proteins are indicated on the right.

Fig. 2.

The vcx1 mutant displays calcineurin-dependent Ca²⁺ sensitivity. Ten-fold serial dilutions of WT H99, vcx1 mutant (vcx1Δ), and vcx1::VCX1-complemented (vcx1Δ::VCX1) cells were plated in YPD agar containing 100 μg/ml of CsA (A), 100 or 200 mM CaCl₂ (B), 50 mM CaCl₂ amended with 100 μg/ml of CsA (C), 4 mM MnCl₂ or 50 μM CdCl₂ (D), or 4 mM MnCl₂ or 50 μM CdCl₂ amended with 100 μg/ml of CsA (E). The plates were incubated for 2 days at 30°C, 35°C, or 37°C, as indicated. As control, cells were grown in YPD agar only.

Fig. 3.

Disruption of VCX1 does not influence capsule size but decreases extracellular GXM secretion. (A) Fluorescence microscopy of WT, vcx1 mutant, and vcx1::VCX1-complemented mutant cells stained with the lectin WGA (red spots), calcofluor white (blue staining), and the monoclonal antibody 18B7 (green), to visualize GlcNAc oligomers, cell wall, and GXM, respectively. Bars, 10 μm. (B) Relative capsule sizes of WT and vcx1 mutant cells. (C) Content of extracellular GXM in culture supernatants of WT, vcx1 mutant, and vcx1::VCX1-complemented mutant cells, as determined by ELISA. *, P < 0.05. (D) Content of extracellular GXM in culture supernatants of WT, vcx1 mutant, and vcx1::VCX1-complemented mutant cells incubated with different concentrations of CsA, as determined by ELISA.

Fig. 4.

VCX1 influences C. neoformans phagocytosis by macrophages and is required for full virulence in mice. (A) CFU counts after macrophage infection with WT, vcx1 mutant, or vcx1::VCX1-complemented strains. *, P < 0.05. (B) Rates of phagocytosis of WT, vcx1 mutant, and complemented cells as measured by flow cytometry. (C) Virulence assay of WT, vcx1 mutant, and vcx1::VCX1-complemented strains in an intranasal inhalation infection model with BALB/c mice.

Fig. 5.

Vcx1 localizes to the vacuoles of C. neoformans cells, as shown via confocal microscopy of Vcx1-mCherry fusion strain cells. The mCherry-tagged Vcx1 (red) colocalizes with vacuole organelles, which appear as a depression in the DIC image. Bars, 5 μm.

Fig. 6.

Disruption of VCX1 influences expression of other calcium transporters in C. neoformans. The relative expression levels of different C. neoformans calcium transporters (CCH1, ECA1, PMR1, and PMC1) in WT and vcx1 mutant cells were quantified by RT-PCR. The measured quantity of the mRNA in each of the samples was normalized using the threshold cycle (C_T) values obtained for the actin gene. The accession numbers for the C. neoformans orthologs of PMR1 and PMC1 from the Broad Institute database are CNAG_05135.2 and CNAG_01232.2, respectively. Data shown are means ± standard deviations. *, P < 0.05.

Fig. 7.

Disruption of VCX1 influences the relative levels of intracellular calcium concentration in C. neoformans. The relative levels of intracellular calcium in WT and vcx1 mutant cells were determined using the calcium-sensitive dye Fura-2-AM. The relative Ca²⁺ concentration was determined based on the fluorescence ratio after dual-wavelength excitation (fluorescence intensity at 340 nm [FI₃₄₀]/FI₃₈₀). The control was fresh YPD medium. Data shown are means ± standard deviations. **, P < 0.05; ***, P < 0.01.