Calcineurin is essential for virulence in Candida albicans

Abstract

Calcineurin is a conserved Ca(2+)-calmodulin-activated, serine/threonine-specific protein phosphatase that regulates a variety of physiological processes, e.g., cell cycle progression, polarized growth, and adaptation to salt and alkaline pH stresses. In the pathogenic yeast Cryptococcus neoformans, calcineurin is also essential for growth at 37 degrees C and virulence. To investigate whether calcineurin plays a role in the virulence of Candida albicans, the major fungal pathogen of humans, we constructed C. albicans mutants in which both alleles of the CMP1 gene, encoding the calcineurin catalytic subunit, were deleted. The C. albicans Delta cmp1 mutants displayed hypersensitivity to elevated Na(+), Li(+), and Mn(2+) concentrations and to alkaline pH, phenotypes that have been described after calcineurin inactivation in the related yeast Saccharomyces cerevisiae. Unlike S. cerevisiae calcineurin mutants, which exhibit reduced susceptibility to high Ca(2+) concentrations, growth of C. albicans was inhibited in the presence of 300 mM CaCl(2) after the deletion of CMP1, demonstrating that there are also differences in calcineurin-mediated cellular responses between these two yeast species. In contrast to C. neoformans, inactivation of calcineurin did not cause temperature sensitivity in C. albicans. In addition, hyphal growth, an important virulence attribute of C. albicans, was not impaired in the Delta cmp1 mutants under a variety of inducing conditions. Nevertheless, the virulence of the mutants was strongly attenuated in a mouse model of systemic candidiasis, demonstrating that calcineurin signaling is essential for virulence in C. albicans.

FIG. 1.

Alignment of the calcineurin catalytic subunits Cna1p and Cna2p from S. cerevisiae (Sc) and Cmp1p from C. albicans (Ca). The amino acid sequences of the proteins are displayed in standard single-letter code using the BOXSHADE program (http://bioweb.pasteur.fr/seqanal/interfaces/boxshade.html). Identical residues are on solid backgrounds, and similar residues are on shaded backgrounds. Amino acid positions are shown on the right. The highly conserved calcineurin B binding, calmodulin binding, and autoinhibitory domains are indicated by labeled bars, and their delineation is according to the work of Odom et al. (43).

FIG. 2.

Construction of C. albicans cmp1 deletion mutants and complemented strains. (A) Structure of the deletion cassette from plasmid pCMP1M2 (top) and genomic structure of the CMP1 locus in the parent strain, CAI4 (bottom). The CMP1 coding region is represented by the open arrow, and the upstream and downstream sequences are represented by solid lines. Details of the URA3 flipper (shaded rectangle bordered by FRT sites [solid arrows]) have been presented elsewhere (39). The 34-bp FRT sites are not drawn to scale. The DNA fragment used for Southern hybridization analysis of the mutants is represented by the thick bar (probe 1). (B) Structures of the DNA fragments from pCMP1K1 (top) and pCMP1M4 (bottom), which were used for reintegration of an intact CMP1 copy (open arrow) or only the URA3 marker (shaded arrows), respectively, into one of the inactivated cmp1 alleles (middle). The ACT1 transcription termination sequence (ACT1T) is indicated by the solid diamond. Only relevant restriction sites are given. B, BamHI; Bg, BglII; K, KpnI; P, PstI; ScI, SacI; ScII, SacII; Sl, SalI; X, XbaI; Xh, XhoI. The restriction sites shown in parentheses were destroyed by the cloning procedure. The CMP1 internal fragment used for Southern hybridization analysis of the strains is indicated by the thick bar (probe 2). (C) Southern hybridization of BglII-digested genomic DNAs of the parent strain, CAI4, and mutant derivatives with the CMP1 probe 1. The sizes of the hybridizing fragments (in kilobases) are given on the left side of the blot, and their identities are indicated on the right. (D) Rehybridization of the same blot with a CMP1 internal fragment (probe 2).

FIG. 3.

Deletion of CMP1 causes hypersensitivity to cationic and alkaline stresses. Serial 10-fold dilutions of the indicated strains were spotted on a YPD plate or YPD plates containing NaCl (1.8 M for URA3⁺ strains and 1.0 M for ura3 mutant strains), 0.3 M LiCl, 10 mM MnSO₄, or 0.3 M CaCl₂ and incubated at 30°C for 2 days. Alkaline pH sensitivity was tested on YPD plates buffered at pH 8.0.

FIG. 4.

Deletion of CMP1 does not cause temperature sensitivity in C. albicans. The indicated strains were streaked on YPD plates and grown for 2 days at 30 or 37°C or for 7 days at 43°C. The upper row shows the growth of URA3 strains, and the lower row shows the growth of ura3 strains, on uridine-supplemented plates.

FIG. 5.

Filamentous growth of C. albicans Δcmp1 mutants and control strains on hypha-inducing solid media. The colonies were photographed after 2 or 5 days as indicated.

FIG. 6.

Hyphal growth of C. albicans Δcmp1 mutants and control strains in liquid media at 37°C. (A) Microscopic appearance of the indicated strains after 7 h in Lee's medium (pH 7.0), 7 h in Spider medium, 8 h in SLAD medium, or 4 h in RPMI medium with 10% FCS (serum). (B) Aggregation phenotypes of Δcmp1 mutants after overnight growth in Lee's medium (pH 7.0). The cultures were briefly shaken and allowed to settle for 1 min before the photograph was taken.

FIG. 7.

C. albicans Δcmp1 mutants are avirulent. (A) Survival curves of mice intravenously infected with 5 × 10⁵ cells of the Δcmp1 mutant strains CMP1M5A and -B or the respective complemented strains, CMP1MK1A and -B. (B) Macrographs of kidneys from animals infected with strains CMP1MK1B (Δcmp1 + CMP1) (left) or CMP1M5B (Δcmp1) (right). The duration of infection is indicated for each frame (d, day p.i.). (C) Fungal organ burden for kidneys. Shown are average numbers of CFU (+ standard deviations) after infection with 5 × 10⁵ cells of either the Δcmp1 mutant strain CMP1M5B (open bars) or the complemented strain CMP1MK1B (solid bars) calculated for two or three pairs of kidneys. n.d., not detectable.