Fungal Adenylyl Cyclase Integrates CO₂ Sensing with cAMP Signaling and Virulence

Summary

The ascomycete Candida albicans is the most common fungal pathogen in immunocompromised patients [1]. Its ability to change morphology, from yeast to filamentous forms, in response to host environmental cues is important for virulence [2–5]. Filamentation is mediated by second messengers such as cyclic adenosine 3′,5′-monophosphate (cAMP) synthesized by adenylyl cyclase [4]. The distantly related basidiomycete Cryptococcus neoformans is an encapsulated yeast that predominantly infects the central nervous system in immunocompromised patients [6–8]. Similar to the morphological change in C. albicans, capsule biosynthesis in C. neoformans, a major virulence attribute, is also dependent upon adenylyl cyclase activity [7]. 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.

Results and Discussion

CO₂ Is a Powerful Inducer of Filamentation that Requires Adenylyl Cyclase, Bypasses Ras, and Is Independent of C. albicans Aquaporin

Much of the success of C. albicans as a pathogen is attributed to its ability to adapt to the diverse microbial habitats in the mammalian host. Indeed, various mammalian environmental cues (i.e., pH of 7.4, an ambient temperature of 37°C, serum) induce a reversible transition from yeast-like to filamentous growth form that is critical for virulence [2–5]. Several regulators of this highly controlled developmental program have been identified. For example, filamentation in response to serum involves Ras activation of the adenylyl cyclase/cAMP-signaling pathway [4, 9, 10]. In fact, in C. albicans, adenylyl cyclase (AC) is essential for morphogenesis in response to all known physiological signals [4].

In contrast, involvement of a critical physiological signal, CO₂ concentration, has not been investigated in the pathogenesis of C. albicans [11]. In mammals, the CO₂ concentration is more than 150-fold higher (5%) than it is in atmospheric air (0.033%) [12, 13]. Consequently, fungal pathogens, including C. albicans, are exposed to dramatically different CO₂ concentrations during superficial infections compared with invasion of the blood. We found that 5% CO₂ induces filamentation, predominantly in the form of pseudohyphal development, or invasion of the underlying agar in 208 independent isolates of C. albicans (Figures 1A and 1B) but not other yeast species (51 Candida dubliniensis, 45 Candida glabrata, 22 Candida parapsilosis, 11 Candida krusei, and 4 Saccharomyces cerevisiae) [14]. C. albicans filamentation in response to CO₂ was observed on seven different media (YNB, YEPD, chocolate agar, Columbia blood agar, medium M199, and DMEM), including a matrix made up of distilled water and 2% agar, and it was independent of pH; C. albicans responded to 5% CO₂ at both pH 7 and pH 4 although filamentation rates were higher at pH 7 (80% at pH 7 versus 30% at pH 4). These observations are consistent with the findings of Mock et al. [11] who demonstrated that 25 mM bicarbonate induces filamentation in C. albicans.

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.

To determine if CO₂-mediated agar invasion and filamentation were dependent on signaling proteins known to govern filamentation in response to other stimuli, i.e., Ras or AC, we exposed mutant strains of C. albicans that lacked either RAS1 or the AC-encoding gene CDC35 to 5% CO₂ [4, 9]. Exposure to 5% CO₂ induced the ras1Δ/ras1Δ strain to invade the underlying agar and form a filamentous colony fringe (Figures 1B and 1C). In contrast, the cdc35Δ/cdc35Δ strain lacking AC was refractory to 5% CO₂ and failed to invade the agar or filament (Figures 1B and 1C). Thus, CO₂ signaling in C. albicans bypasses Ras but requires AC.

In mammalian cells, cAMP is synthesized by transmembrane adenylyl cyclases (tmACs) and soluble adenylyl cyclase (sAC) [15]. In contrast to tmAC, sAC is activated by physiological concentrations of bicarbonate [15, 16]. Bicarbonate-responsive sAC-like ACs are also found in bacteria [15], revealing the link between bicarbonate sensing and the second messenger cAMP to be among the most evolutionarily ancient signaling pathways. Bicarbonate stimulation of mammalian and bacterial sAC-like cyclases is direct [15] and works by inducing a conformational change, which facilitates catalysis but has little effect on affinity for substrate ATP [17].

Transport of CO₂ across biological membranes by aquaporin water channels has been reported for both plant and mammalian cells [18]. C. albicans contains a single aquaporin gene (AQY1) that encodes a functional water channel [19]. To determine if Aqy1 was required for CO₂-mediated polymorphism, we exposed the C. albicans aqy1Δ/aqy1Δ mutant to 5% CO₂. We found that this strain was not restricted in its ability to filament compared to wild-type (Figure 1D).

Nce103 Is a Carbonic Anhydrase that Is Essential for Pathological Growth in Niches where Sufficient CO₂ Is Not Supplied by the Host

Cellular effects of CO₂ can be mediated via its hydrated form, bicarbonate. Bicarbonate is spontaneously formed from CO₂ in solution, but this reaction is greatly accelerated by a ubiquitous family of carbonic anhydrases. We cloned the C. albicans NCE103 gene showing similarities to NCE103 from S. cerevisiae encoding a β-class carbonic anhydrase [20]. Stopped-flow experiments with purified C. albicans Nce103 demonstrated carbonic anhydrase activity of the protein and confirmed inhibition by the carbonic anhydrase inhibitor ethoxyzolamide (Figure 2A).

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).

To investigate the role of Nce103 in growth and virulence, we deleted NCE103 in two separate strain backgrounds (CAI4 and BWP17). Mutant strains in either background showed similar phenotypes. C. albicans TK1 and TK1a (nce103Δ/nce103Δ in a CAI4 or BWP17 background) grew at 5% CO₂ but failed to grow in air (Figure 2B and Supplemental Data available with this article online). Reintroduction of a single copy of C. albicans NCE103, yielding TK2 and TK2a (nce103Δ/nce103Δ + NCE103), restored the ability to grow and filament in air. We also found that C. albicans senses CO₂ over a range of concentrations and that 0.5% already partially complemented the growth defect of TK1 (Supplemental Data).

The inability of TK1 to grow in air may be due to limiting amounts of bicarbonate known to serve as a substrate for several cellular carboxylases important to metabolism. However, addition of various cellular metabolites and carbon sources, including citrate, succinate, oxalacetate, malate, a-ketoglutarate, arginine, adenine, and oleate, failed to complement the growth defect of TK1 (Supplemental Data and data not shown). Interestingly, in an accompanying paper in this issue of Current Biology, Bahn et al. succeeded in complementing the growth defect of a C. neoformans carbonic anhydrase mutant with the fatty acid palmitate [27].

CO₂ concentrations in skin will be lowered due to equilibration with the atmosphere, yet fungi including Candida must exploit these lower levels during epithelial infections. Consistently, elevating CO₂ concentrations at the skin surface of patients have been reported to aggravate Candida infections [21]. When tested in an experimental model of atmospheric pathogenicity using human oral epithelium, the nce103Δ/nce103Δ mutant failed to inflict damage to the epithelium, while the control strain, with a single copy of C. albicans NCE103 (nce103Δ/nce103Δ + NCE103), invaded the epithelium (Figure 2C). When the experiment was repeated in an atmosphere containing 5% CO₂, both the nce103Δ/nce103Δ null mutant and the reconstituted strain exhibited a comparable degree of tissue damage (Figure 2C). We also tested the carbonic anhydrase mutants in an intravenous model of systemic candidiasis, in which Candida is exposed to the higher partial pressures of CO₂ present in blood. Injection of either control (TK2) or mutant (TK1) strain resulted in death of 50% of mice by day 4 and 100% by day 9 or day 12 postinfection (Figure 2D); thus, there was no discernible difference due to the absence of NCE103. Further comparison of TK1 and TK2 in two additional CO₂-rich host niches (a pulmonary model of invasive candidiasis in immunosuppressed mice and a vaginal model of candidiasis) did not reveal any virulence defects of the nce103Δ/nce103Δ mutant TK1 (Supplemental Data). Therefore, not only can CO₂ function as a differentiation factor for C. albicans, but its conversion to bicarbonate via carbonic anhydrase is essential for causing damage in niches where sufficient CO₂ is not supplied by the host.

The Catalytic Domain of C. albicans AC Is Sufficient to Induce CO₂-Mediated Filamentation

To explore whether the link between CO₂ and the AC might be direct, we expressed either the entire CDC35 coding region or a fragment encoding amino acids 1166–1571, containing the presumptive catalytic domain, under the control of the TEF2 promoter in the cdc35Δ/cdc35Δ strain CR276. Expression of either full-length or truncated Cdc35 restored CO₂-mediated filamentation in CR276 (Figure 3A). Thus, the Cdc35 catalytic domain is sufficient to support CO₂ sensing.

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.

C. neoformans AC Restores the Ability to Sense CO₂ in the C. albicans cdc35Δ/cdc35Δ Mutant

A signal capable of inducing capsule biosynthesis in C. neoformans is elevated carbon dioxide (CO₂), but the link between this ubiquitous by-product of cellular metabolism and C. neoformans AC has remained unknown [8]. To determine whether CO₂ sensing is conserved in fungal pathogens, we expressed the catalytic domain of the C. neoformans AC [7] in the C. albicans cdc35Δ/cdc35Δ strain. A CAC1 fragment encoding amino acids 1721–2271 restored the ability of the C. albicans cdc35Δ/cdc35Δ strain to filament upon exposure to 5% CO₂ (Figure 3B). Thus, ACs from other, distantly related, fungi can restore the ability to sense CO₂, suggesting that the link between cAMP and CO₂ sensing may be a general feature of pathogenic fungi.

ACs from C. albicans and C. neoformans Are CO₂ Chemosensors

We previously demonstrated that mammalian and bacterial sAC-like cyclases are directly stimulated by physiological levels of bicarbonate [15], but fungal ACs, including the C. albicans and C. neoformans cyclases, are only modestly related to sAC-like ACs [16]. To test whether fungal cyclases might also directly sense CO₂/HCO₃^–, we purified recombinant C. albicans Cdc35 and C. neoformans Cac1 proteins. Purified C. albicans cyclase was stimulated more than 20-fold with a median effective concentration (EC₅₀) of 3.5 ± 0.3 mM bicarbonate (Figure 4A), and C. neoformans AC was stimulated nearly 2-fold with an EC₅₀ of 4.1 ± 0.1 mM bicarbonate (Figure 4B). These dose-response relationships reveal why 5% CO₂, which corresponds to ~25 mM bicarbonate (at pH 7.4) and would maximally stimulate both fungal ACs, was sufficient to induce the filamentous transition in C. albicans. In contrast, in skin where diffusion into the atmosphere results in significantly lower endogenous CO₂ concentrations, the C. albicans cyclase would require carbonic anhydrase activity to achieve sufficiently high bicarbonate concentrations to support its activity. When measuring intracellular cAMP changes in the presence and absence of CO₂ with a number of different protocols, we found that the resting intracellular cAMP levels were extremely low, and the CO₂-induced changes we observed were too small to be statistically significant (data not shown).

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).

Bicarbonate regulation of cAMP synthesis provides a mechanism for the CO₂-dependent filamentation in C. albicans and CO₂-dependent capsule biosynthesis in C. neoformans. Morphogenesis in the fungal pathogen Coccidioides immitis is positively regulated by physiological CO₂ concentrations [22], suggesting that this fungal pathogen may also be dependent upon a bicarbonate-regulated AC for virulence. Conservation among distantly related fungi, along with the presence of bicarbonate-sensitive ACs in animals and prokaryotes [15, 16, 23], establish CO₂/HCO₃^– chemosensing via the second messenger cAMP as one of the most ancient and widely conserved signaling pathways in biology. Furthermore, our finding that a carbonic anhydrase is essential for growth and virulence in specific niches where the CO₂ concentration is limiting, in conjunction with reports that assign a role in virulence to bacterial carbonic anhydrase (Salmonella enteritidis) [24], identifies the CO₂-chemosensing pathway as a prime target for the development of new antimicrobial agents.