Fungal adenylyl cyclase integrates CO2 sensing with cAMP signaling and virulence

Abstract

The ascomycete Candida albicans is the most common fungal pathogen in immunocompromised patients . Its ability to change morphology, from yeast to filamentous forms, in response to host environmental cues is important for virulence . Filamentation is mediated by second messengers such as cyclic adenosine 3',5'-monophosphate (cAMP) synthesized by adenylyl cyclase . The distantly related basidiomycete Cryptococcus neoformans is an encapsulated yeast that predominantly infects the central nervous system in immunocompromised patients . Similar to the morphological change in C. albicans, capsule biosynthesis in C. neoformans, a major virulence attribute, is also dependent upon adenylyl cyclase activity . Here we demonstrate that physiological concentrations of CO2/HCO3- induce filamentation in C. albicans by direct stimulation of cyclase activity. Furthermore, we show that CO2/HCO3- equilibration by carbonic anhydrase is essential for pathogenesis of C. albicans in niches where the available CO2 is limited. We also demonstrate that adenylyl cyclase from C. neoformans is sensitive to physiological concentrations of CO2/HCO3-. These data demonstrate that the link between cAMP signaling and CO2/HCO3- sensing is conserved in fungi and reveal CO2 sensing to be an important mediator of fungal pathogenesis. Novel therapeutic agents could target this pathway at several levels to control fungal infections.

Figure 1. CO₂ Sensing Requires Adenylyl Cyclase, Bypasses Ras, and Is Independent of C. albicans Aquaporin

(A) Wild-type strain SC5314 grown on DMEM medium (pH 7) in air (left) or 5% CO₂ (right). Cells were incubated for 24 hr at 37°C and photographed at ×70 magnification. The pH of the medium was adjusted to pH 7.0 using 150 mM HEPES as previously described [25, 26]. (B) 1 × 10⁵ C. albicans cells from strains SC5314, CDH107 [6] (ras1Δ/ras1Δ), and CR216 [4] (cdc35Δ/cdc35Δ) were spotted onto DMEM medium. Cells were incubated for 48 hr at 37°C in either air (top 2 rows) or 5% CO₂ (bottom 2 rows). Cells were photographed before and after washing. Scale bars equal 1 mm. (C) C. albicans cells from strains CDH107 (ras1Δ/ras1Δ) (top) and CR216 (cdc35Δ/cdc35Δ) (bottom) grown on DMEM medium. Cells were incubated for 12 hr at 37°C in either air or 5% CO₂ and photographed at ×70 magnification. (D) Wild-type strain SC5314 (left) and JCO188 (aqy1Δ/aqy1Δ, right) [20] were grown on DMEM medium in 5% CO₂. Cells were incubated for 24 hr at 37°C and photographed at ×70 magnification.

Figure 2. Nce103 Is a Carbonic Anhydrase that Is Required for Atmospheric Pathogenicity

(A) Biochemical analysis of Nce103 function by stopped flow. Left: spontaneous chemical hydration of CO₂ to bicarbonate (black line); reaction catalyzed by Nce103 (red line); reaction catalyzed by bovine carbonic anhydrase (green line). Right: spontaneous chemical hydration of CO₂ to bicarbonate (black line); reaction catalyzed by Nce103 (red line); inhibition of C. albicans Nce103 activity by 0.5 mM ethoxyzolamide (blue line). The reaction from CO₂ to HCO₃^– imposes a decrease of the pH. The pH-sensitive indicator m-cresol purple changes color from purple to yellow and therefore the absorption at 578 nm decreases. The absorbance in the catalyzed reaction decreases faster than in the noncatalyzed reaction. (B) C. albicans TK1 (nce103Δ/nce103Δ) and reconstituted TK2 (nce103Δ/nce103Δ + NCE103) were either spotted (1 × 10⁵ cells) (top) or streaked (bottom) onto DMEM medium and incubated in air (left four panels) or 5% CO₂ (right four panels) at 37°C for 48 hr. Streaked cells were photographed at ×70 magnification. Scale bars equal 1 mm. (C) Human reconstituted epithelium was infected with C. albicans TK1 (nce103Δ/nce103Δ) and reconstituted TK2 (nce103Δ/nce103Δ + NCE103) and incubated in air (left) or 5% CO₂ (right) at 37°C for 24 hr and photographed at ×100 magnification. C. albicans TK1 can invade the epithelium only in the presence of 5% CO₂, while TK2 causes tissue damage in air as well as 5% CO₂. (D) Balb/c mice (n = 5) were intraveneously infected with 2 × 10⁵ viable blastoconidia of TK1 (white squares) and TK2 (black squares).

Figure 3. Truncated Adenylyl Cyclase Restores CO₂ Sensing

C. albicans CR276-V (cdc35Δ/cdc35Δ + TEF2_pr); JR-3 (cdc35Δ/cdc35Δ + TEF2_pr::CDC35_1166-1571) expressing truncated Cdc35; CDC35-H (cdc35Δ/cdc35Δ + TEF2_pr::CDC35) expressing full-length Cdc35 (A), and TK4 (cdc35Δ/cdc35Δ + TEF2_pr::CAC1_1721-2271) expressing truncated C. neoformans Cac1 (B) were grown on DMEM (bottom two images) medium in air (left column) or 5% CO₂ (right column). Cells were incubated for 24 hr at 37°C and photographed at ×70 magnification.

Figure 4. Bicarbonate Activates C. albicans and C. neoformans Adenylyl Cyclases

Purified C.albicans Cdc35_1166-1571 (A) and C. neoformans Cac1_1721-2271 (B) were assayed in the presence of the indicated concentrations of NaHCO₃ with 10 mM ATP and 40 mM MgCl₂. Data are expressed as picomoles of cAMP formed per minute per milligram of protein, and values are averages of triplicate determinations. Shown are representative experiments repeated at least three times. Error bars indicate standard error from the mean (SEM).