Cryptococcus neoformans senses CO2 through the carbonic anhydrase Can2 and the adenylyl cyclase Cac1

Abstract

Cryptococcus neoformans, a fungal pathogen of humans, causes fatal meningitis in immunocompromised patients. Its virulence is mainly determined by the elaboration of a polysaccharide capsule surrounding its cell wall. During its life, C. neoformans is confronted with and responds to dramatic variations in CO2 concentrations; one important morphological change triggered by the shift from its natural habitat (0.033% CO2) to infected hosts (5% CO2) is the induction of capsule biosynthesis. In cells, CO2 is hydrated to bicarbonate in a spontaneous reaction that is accelerated by carbonic anhydrases. Here we show that C. neoformans contains two beta-class carbonic anhydrases, Can1 and Can2. We further demonstrate that CAN2, but not CAN1, is abundantly expressed and essential for the growth of C. neoformans in its natural environment, where CO2 concentrations are limiting. Structural studies reveal that Can2 forms a homodimer in solution. Our data reveal Can2 to be the main carbonic anhydrase and suggest a physiological role for bicarbonate during C. neoformans growth. Bicarbonate directly activates the C. neoformans Cac1 adenylyl cyclase required for capsule synthesis. We show that this specific activation is optimal at physiological pH.

FIG. 1.

C. neoformans CAN1 and CAN2 encode β-class carbonic anhydrases. The figure shows an alignment of sequences of β-class carbonic anhydrases from Saccharomyces cerevisiae, Candida glabrata, Candida albicans, Schizosaccharomyces pombe, Aspergillus nidulans, C. neoformans, and Ustilago maydis. Identical residues are highlighted in dark gray, and conserved amino acids are shown in light gray. The four conserved residues that are important for zinc binding are labeled with asterisks.

FIG. 2.

Can2 is expressed and functional in E. coli. (A) The coding region of CAN2 was cloned and expressed in a carbonic anhydrase mutant of E. coli (strain EDCM636). The transformants were grown at 37°C for 24 h in either air or 5% CO₂. (B) Can2 was expressed as a GST fusion protein in E. coli BL21, purified using glutathione-Sepharose columns, allowed to migrate through a sodium dodecyl sulfate-polyacrylamide gel, and stained with Coomassie blue. Fragment molecular masses are indicated in kDa.

FIG. 3.

Can2 has carbonic anhydrase activity. (A) The biochemical properties of Can2 were analyzed by an electrometric procedure. Can2 was purified as a GST fusion protein and assayed for enzymatic activity (46 μg/ml). Bovine carbonic anhydrase (2.3 μg/ml) and expressed GST (46 μg/ml) were used as controls. One Wilbur-Anderson (W-A) unit causes the pH of the buffer to drop from 8.3 to 6.3 in 1 minute at 0°C. The slopes of the fitted lines give specific activities of 5,600 and 28 U/mg for bovine CA and GST-Can2, respectively. Each data point represents the mean value of activity, expressed in W-A units, for each volume of enzyme tested in triplicate. Error bars indicate standard errors of the means. (B) Measurements of enzymatic activity were repeated as described above, using 2 mg/ml (60 U) purified Can2 in the presence or absence of the carbonic anhydrase inhibitor ethoxyzolamide (1 to 1,000 nM ethoxyzolamide). Bovine carbonic anhydrase (40 U/ml) was used as a positive control. Data points represent the mean percentage of inhibition for each concentration of ethoxyzolamide tested in triplicate, with error bars indicating standard errors. The hyperbolic curves fit to the data indicate 50% inhibitory concentrations of ∼50 nM and ∼100 μM for bovine CA and GST-Can2, respectively.

FIG. 4.

Size-exclusion chromatography profile of Can2 on a Superdex 200 column. The arrows indicate elution positions for marker proteins of the indicated molecular masses. The Can2 elution peak is found at the position expected for a 52-kDa homodimer of Can2 monomers (26 kDa).

FIG. 5.

Homology model for the structure of Can2. (A) Ribbon representation of the modeled homodimeric structure of Can2. The two monomers are colored blue (monomer A) and red (monomer B), respectively. The N and C termini as well as the active-site zinc ions are labeled. (B) Electrostatic surface of the modeled Can2 structure. Blue indicates positive charges, red indicates negative charges, and gray indicates hydrophobic areas. The tilted view reveals a mainly hydrophobic groove on top of the enzyme. (C) Surface of the Can2 dimer colored according to sequence conservation. Blue indicates high amino acid conservation, green indicates medium conservation, and red indicates high variation.

FIG. 6.

Growth inhibition of C. neoformans by ethoxyzolamide. Tenfold serial dilutions of C. neoformans cells were spotted (10⁵ to 10 cells) on YPD agar containing 4% dimethyl sulfoxide (control) or 3 mM ethoxyzolamide dissolved in 4% dimethyl sulfoxide and grown in 0.033% or 5% CO₂.

FIG. 7.

Disruption of CAN2 by biolistics, using the nourseothricin resistance gene as a marker. C. neoformans strain KN99 (MATa) was transformed by biolistics. The wild-type strain (NE241 [MATα]), two can2 deletion mutants (NE417 and NE418 [MATα can2Δ::NAT1]), and one revertant (NE425 [MATα can2Δ::NAT1 HYG1::CAN2]) were screened for the ability to grow on YPD medium (pH 6.7 and 7.4) at 37°C in air or a physiological concentration of CO₂ (0.033% and 5%, respectively).

FIG. 8.

Restoration of CO₂-dependent filamentation of C. albicans by a truncated form of C. neoformans adenylyl cyclase Cac1. The fragment encoding amino acids 1825 to 2271 of the C. neoformans adenylyl cyclase gene CAC1 was expressed in C. albicans (strain CR276) grown in Dulbecco's modified Eagle's medium, pH 7, in 0.033% or 5% CO₂ at 37°C for 24 h. Cells were photographed at a magnification of ×70. Strain CR276 of C. albicans harboring pFM-2 was used a control.

FIG. 9.

Cyclase activity of purified C. neoformans adenylyl cyclase Cac1. Cac1 was produced as a GST fusion protein and assayed for enzymatic activity at the indicated concentrations of NaHCO₃, pH 7.5, in 10 mM ATP and 10 mM MgCl₂ (A) or in the presence (dashed line) or absence (solid line) of 8 mM NaHCO₃ at the indicated pHs (B). Data are expressed as nanomoles of cAMP formed per minute per milligram of protein, and values are averages of duplicate determinations. The results shown are representative of experiments that were repeated at least three times.

FIG. 10.

Proposed model for CO₂ sensing in C. neoformans. The double line represents the C. neoformans cell wall. Can2, carbonic anhydrase; Cac1, adenylyl cyclase; HCO3⁻, bicarbonate; cAMP, cyclic AMP.