Fungal adenylyl cyclase integrates CO2 sensing with cAMP signaling and virulence

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.     

Carbonic anhydrase and CO2 sensing during Cryptococcus neoformans growth, differentiation, and virulence

The gas carbon dioxide (CO2) plays a critical role in microbial and mammalian respiration, photosynthesis in algae and plants, chemoreception in insects, and even global warming . However, how CO2 is transported, sensed, and metabolized by microorganisms is largely not understood. For instance, CO2 is known to induce production of polysaccharide capsule virulence determinants in pathogenic bacteria and fungi via unknown mechanisms . Therefore, we studied CO2 actions in growth, differentiation, and virulence of the basidiomycetous human fungal pathogen Cryptococcus neoformans. The CAN2 gene encoding beta-carbonic anhydrase in C. neoformans was found to be essential for growth in environmental ambient conditions but dispensable for in vivo proliferation and virulence at the high CO2 levels in the host. The can2Delta mutant in vitro growth defect is largely attributable to defective fatty acid synthesis. CO2 was found to inhibit cell-cell fusion but not filamentation during sexual reproduction. The can2 mutation restored early mating events in high CO2 but not later steps (fruiting body formation, sporulation), indicating a major role for carbonic anhydrase and CO2/HCO3- in this developmental cascade leading to the production of infectious spores. Our studies illustrate diverse roles of an ancient enzyme class in enabling environmental survival of a ubiquitous human pathogen.     

Carbonic anhydrase metabolism is a key factor in the toxicity of CO2 and COS but not CS2 toward the flour beetle Tribolium castaneum [Coleoptera: Tenebrionidae

The analogues carbon dioxide (CO(2)), carbonyl sulfide (COS) and carbon disulfide (CS(2)) have been useful as substrate probes for enzyme activities. Here we explored the affinity of the enzyme carbonic anhydrase for its natural substrate CO(2), as well as COS and CS(2) (1) by in vitro kinetic metabolism studies using pure enzyme and (2) through mortality bioassay of insects exposed to toxic levels of each of the gases during carbonic anhydrase inhibition. Hydrolysis of COS to form hydrogen sulfide was catalysed rapidly showing parameters K(m) 1.86 mM and K(cat) 41 s(-1) at 25 degrees C; however, the specificity constant (K(cat)/K(m)) was 4000-fold lower than the reported value for carbonic anhydrase-catalysed hydration of CO(2). Carbonic anhydrase-mediated CS(2) metabolism was a further 65,000-fold lower than COS. Both results demonstrate the deactivating effect toward the enzyme of sulfur substitution for oxygen in the molecule. We also investigated the role of carbonic anhydrases in CO(2), COS and CS(2) toxicity using a specific inhibitor, acetazolamide, administered to Tribolium castaneum (Herbst) larvae via the diet. CO(2) toxicity was greatly enhanced by up to seven-fold in acetazolamide-treated larvae indicating that carbonic anhydrases are a key protective enzyme in elevated CO(2) concentrations. Conversely, mortality was reduced by up to 12-fold in acetazolamide-treated larvae exposed to COS due to reduced formation of toxic hydrogen sulfide. CS(2) toxicity was unaffected by acetazolamide. These results show that carbonic anhydrase has a key role in toxicity of the substrates CO(2) and COS but not CS(2), despite minor differences in chemical formulae.     

Indispensability of the Escherichia coli carbonic anhydrases YadF and CynT in cell proliferation at a low CO2 partial pressure

We found that a carbonic anhydrase, YadF, is essential for cell growth in the absence of another carbonic anhydrase, CynT, in Escherichia coli. However, mutant strains lacking both of them grew at high CO2 concentrations (5%), where non-enzymatic mechanisms generate HCO3-. This suggests that these carbonic anhydrases are essential because they maintain HCO3- levels at ambient CO2 concentrations.     

碳酸酐酶的生理功能、多样性及其在CO2捕集中的应用

碳酸酐酶（carbonic anhydrase）作为一种活性中心含有锌离子的金属酶,能够可逆催化CO2生成碳酸氢盐的水合反应,该反应在生物体内承担着多样的生理学功能,具有高度的生物学意义。除广泛存在于真核生物以外,该酶在淡水、海水、嗜常温、嗜热、厌氧、好氧、致病、产酸、自养、异养等多种原核微生物中也有广泛的分布,并参与光合作用、呼吸作用和以CO2作为底物的反应,维持生理pH以及离子转运等生理过程。近年来,随着温室效应的日益加剧．生物固定CO2作为该酶的一种全新应用引起了研究者的广泛关注。回顾了碳酸酐酶作为催化剂参与CO2固定过程的历史、现状和最新发现,同时展望了未来应用的趋势。     

Expression of proteins encoded by the Escherichia coli cyn operon: carbon dioxide-enhanced degradation of carbonic anhydrase.

Cyanase catalyzes the reaction of cyanate with bicarbonate to give 2CO2. The cynS gene encoding cyanase, together with the cynT gene for carbonic anhydrase, is part of the cyn operon, the expression of which is induced in Escherichia coli by cyanate. The physiological role of carbonic anhydrase is to prevent depletion of cellular bicarbonate during cyanate decomposition due to loss of CO2 (M.B. Guilloton, A.F. Lamblin, E. I. Kozliak, M. Gerami-Nejad, C. Tu, D. Silverman, P.M. Anderson, and J.A. Fuchs, J. Bacteriol. 175:1443-1451, 1993). A delta cynT mutant strain was extremely sensitive to inhibition of growth by cyanate and did not catalyze decomposition of cyanate (even though an active cyanase was expressed) when grown at a low pCO2 (in air) but had a Cyn+ phenotype at a high pCO2. Here the expression of these two enzymes in this unusual system for cyanate degradation was characterized in more detail. Both enzymes were found to be located in the cytosol and to be present at approximately equal levels in the presence of cyanate. A delta cynT mutant strain could be complemented with high levels of expressed human carbonic anhydrase II; however, the mutant defect was not completely abolished, perhaps because the E. coli carbonic anhydrase is significantly less susceptible to inhibition by cyanate than mammalian carbonic anhydrases. The induced E. coli carbonic anhydrase appears to be particularly adapted to its function in cyanate degradation. Active cyanase remained in cells grown in the presence of either low or high pCO2 after the inducer cyanate was depleted; in contrast, carbonic anhydrase protein was degraded very rapidly (minutes) at a high pCO2 but much more slowly (hours) at a low pCO2. A physiological significance of these observations is suggested by the observation that expression of carbonic anhydrase at a high pCO2 decreased the growth rate.     

Proton transport by phosphate diffusion--a mechanism of facilitated CO2 transfer

We have measured CO2 fluxes across phosphate solutions at different carbonic anhydrase concentrations, bicarbonate concentration gradients, phosphate concentrations, and mobilities. Temperature was 22-25 degrees C, the pH of the phosphate solutions was 7.0-7.3. We found that under physiological conditions of pH and pCO2 a facilitated diffusion of CO2 occurs in addition to free diffusion when (a) sufficient carbonic anhydrase is present, and (b) a concentration gradient of HCO3- is established along with a pCO2 gradient, and (c) the phosphate buffer has a mobility comparable to that of bicarbonate. When the phosphate was immobilized by attaching 0.25-mm-long cellulose particles, no facilitation of CO2 diffusion was detectable. A mechanism of facilitated CO2 diffusion in phosphate solutions analogous to that in albumin solutions was proposed on the basis of these findings: bicarbonate diffusion together with a facilitated proton transport by phosphate diffusion. A mathematical model of this mechanism was formulated. The CO2 fluxed predicted by the model agree quantitatively with the experimentally determined fluxes. It is concluded that a highly effective proton transport mechanism acts in solutions of mobile phosphate buffers. By this mechanism; CO2 transfer may be increased up to fivefold and proton transfer may be increased to 10,000-fold.     

Structure and Inhibition of the CO2-Sensing Carbonic Anhydrase Can2 from the Pathogenic Fungus Cryptococcus neoformans

In the pathogenic fungus Cryptococcus neoformans, a CO₂-sensing system is essential for survival in the natural environment (∼ 0.03% CO₂) and mediates the switch to virulent growth in the human host (∼ 5% CO₂). This system is composed of the carbonic anhydrase (CA) Can2, which catalyzes formation of bicarbonate, and the fungal, bicarbonate-stimulated adenylyl cyclase Cac1. The critical role of these enzymes for fungal metabolism and pathogenesis identifies them as targets for antifungal drugs. Here, we prove functional similarity of Can2 to the CA Nce103 from Candida albicans and describe its biochemical and structural characterization. The crystal structure of Can2 reveals that the enzyme belongs to the “plant-type” β-CAs but carries a unique N-terminal extension that can interact with the active-site entrance of the dimer. We further tested a panel of compounds, identifying nanomolar Can2 inhibitors, and present the structure of a Can2 complex with the inhibitor and product analog acetate, revealing insights into interactions with physiological ligands and inhibitors.     

Carbonic anhydrase in Escherichia coli. A product of the cyn operon.

The product of the cynT gene of the cyn operon in Escherichia coli has been identified as a carbonic anhydrase. The cyn operon also includes the gene cynS, encoding the enzyme cyanase. Cyanase catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. The carbonic anhydrase was isolated from an Escherichia coli strain overexpressing the cynT gene and characterized. The purified enzyme was shown to contain 1 Zn2+/subunit (24 kDa) and was found to behave as an oligomer in solution; the presence of bicarbonate resulted in partial dissociation of the oligomeric enzyme. The kinetic properties of the enzyme are similar to those of carbonic anhydrases from other species, including inhibition by sulfonamides and cyanate. The amino acid sequence shows a high degree of identity with the sequences of two plant carbonic anhydrases. but not with animal and algal carbonic anhydrases. Since carbon dioxide formed in the bicarbonate-dependent decomposition of cyanate diffuses out of the cell faster than it would be hydrated to bicarbonate, the apparent function of the induced carbonic anhydrase is to catalyze hydration of carbon dioxide and thus prevent depletion of cellular bicarbonate.     

Role of bicarbonate/CO2 in the inhibition of Escherichia coli growth by cyanate.

Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate to give two CO2 molecules. The gene for cyanase is part of the cyn operon, which includes cynT and cynS, encoding carbonic anhydrase and cyanase, respectively. Carbonic anhydrase functions to prevent depletion of cellular bicarbonate during cyanate decomposition (the product CO2 can diffuse out of the cell faster than noncatalyzed hydration back to bicarbonate). Addition of cyanate to the culture medium of a delta cynT mutant strain of E. coli (having a nonfunctional carbonic anhydrase) results in depletion of cellular bicarbonate, which leads to inhibition of growth and an inability to catalyze cyanate degradation. These effects can be overcome by aeration with a higher partial CO2 pressure (M. B. Guilloton, A. F. Lamblin, E. I. Kozliak, M. Gerami-Nejad, C. Tu, D. Silverman, P. M. Anderson, and J. A. Fuchs, J. Bacteriol. 175:1443-1451, 1993). The question considered here is why depletion of bicarbonate/CO2 due to the action of cyanase on cyanate in a delta cynT strain has such an inhibitory effect. Growth of wild-type E. coli in minimal medium under conditions of limited CO2 was severely inhibited, and this inhibition could be overcome by adding certain Krebs cycle intermediates, indicating that one consequence of limiting CO2 is inhibition of carboxylation reactions. However, supplementation of the growth medium with metabolites whose syntheses are known to depend on a carboxylation reaction was not effective in overcoming inhibition related to the bicarbonate deficiency induced in the delta cynT strain by addition of cyanate. Similar results were obtained with a deltacyn strain (since cyanase is absent, this strain does not develop a bicarbonate deficiency when cyanate is added); however, as with the deltacynT strain, a higher partial CO(2) pressure in the aerating gas or expression of carbonic anhydrase activity (which contributes to a higher intercellular concentration of bicarbonate/CO(2)) significantly reduced inhibition of growth. There appears to be competition between cyanate and bicarbonate/CO(2) at some unknown but very important site such that cyanate binding inhibits growth. These results suggest that bicarbonate/CO(2) plays a significant role in the growth of E. coli other than simply as a substrate for carboxylation reactions and that strains with mutations in the cyn operon provide a unique model system for studying aspects of the metabolism of bicarbonate/CO(2) and its regulation in bacteria.     

Carbonic anhydrase inhibitors: inhibition of the beta-class enzymes from the fungal pathogens Candida albicans and Cryptococcus neoformans with simple anions

The catalytic activity and inhibition of the beta-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Candida albicans (Nce103) and Cryptococcus neoformans (Can2) with inorganic anions such as halogenides, pseudohalogenides, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, perchlorate, sulfate were investigated. The two enzymes showed appreciable CO(2) hydrase activity (k(cat) in the range of (3.9-8.0)x10(5)s(-1), and k(cat)/K(m) in the range of (4.3-9.7)x10(7)M(-1)s(-1)). Can2 was weakly inhibited by cyanide and sulfamic acid (K(I)s of 8.22-13.56 mM), while all other anions displayed more potent inhibition. Nce103 was strongly inhibited by cyanide and carbonate (K(I)s of 10-11 microM), and weakly inhibited by sulfate, phenylboronic, and phenyl arsonic acid (K(I)s of 14.15-30.85 mM). These data demonstrate that pathogenic, fungal beta-CAs may be targets for the development of antifungals that have a novel mechanism of action.     

Investigation of the system copper(II) carbonic anhydrase and HCO3-/CO2

Copper(II) substituted human and bovine carbonic anhydrases B in the presence of bicarbonate have been investigated in solution through water-solvent proton nuclear magnetic resonance (nmr) at variable magnetic fields. HCO3-, contrary to all the other monoanionic inhibitors, partially reduces the water proton relaxation rates. This has been accounted for on the basis of the availability within the active cavity of two coordination positions partially overlapping. 13C-nmr measurements on both CO2 and HCO3- confirm that HCO3- binds the metal, whereas CO2 interacts with the paramagnetic center at nonbonding distance. The upper limit for the CO2 in equilibrium HCO3- interconversion has been estimated to be 10 sec-1.     

A plant-type (beta-class) carbonic anhydrase in the thermophilic methanoarchaeon Methanobacterium thermoautotrophicum.

Carbonic anhydrase, a zinc enzyme catalyzing the interconversion of carbon dioxide and bicarbonate, is nearly ubiquitous in the tissues of highly evolved eukaryotes. Here we report on the first known plant-type (beta-class) carbonic anhydrase in the archaea. The Methanobacterium thermoautotrophicum DeltaH cab gene was hyperexpressed in Escherichia coli, and the heterologously produced protein was purified 13-fold to apparent homogeneity. The enzyme, designated Cab, is thermostable at temperatures up to 75 degrees C. No esterase activity was detected with p-phenylacetate as the substrate. The enzyme is an apparent tetramer containing approximately one zinc per subunit, as determined by plasma emission spectroscopy. Cab has a CO(2) hydration activity with a k(cat) of 1.7 x 10(4) s(-1) and K(m) for CO(2) of 2.9 mM at pH 8.5 and 25 degrees C. Western blot analysis indicates that Cab (beta class) is expressed in M. thermoautotrophicum; moreover, a protein cross-reacting to antiserum raised against the gamma carbonic anhydrase from Methanosarcina thermophila was detected. These results show that beta-class carbonic anhydrases extend not only into the Archaea domain but also into the thermophilic prokaryotes.     

CO2 concentrating mechanisms in cyanobacteria: molecular components,their diversity and evolution

Cyanobacteria have evolved an extremely effective single-cell CO(2) concentrating mechanism (CCM). Recent molecular, biochemical and physiological studies have significantly extended current knowledge about the genes and protein components of this system and how they operate to elevate CO(2) around Rubisco during photosynthesis. The CCM components include at least four modes of active inorganic carbon uptake, including two bicarbonate transporters and two CO(2) uptake systems associated with the operation of specialized NDH-1 complexes. All these uptake systems serve to accumulate HCO(3)(-) in the cytosol of the cell, which is subsequently used by the Rubisco-containing carboxysome protein micro-compartment within the cell to elevate CO(2) around Rubisco. A specialized carbonic anhydrase is also generally present in this compartment. The recent availability of at least nine cyanobacterial genomes has made it possible to begin to undertake comparative genomics of the CCM in cyanobacteria. Analyses have revealed a number of surprising findings. Firstly, cyanobacteria have evolved two types of carboxysomes, correlated with the form of Rubisco present (Form 1A and 1B). Secondly, the two HCO(3)(-) and CO(2) transport systems are distributed variably, with some cyanobacteria (Prochlorococcus marinus species) appearing to lack CO(2) uptake systems entirely. Finally, there are multiple carbonic anhydrases in many cyanobacteria, but, surprisingly, several cyanobacterial genomes appear to lack any identifiable CA genes. A pathway for the evolution of CCM components is suggested.     

Insertional mutants of Chlamydomonas reinhardtii that require elevated CO(2) for survival

Aquatic photosynthetic organisms live in quite variable conditions of CO(2) availability. To survive in limiting CO(2) conditions, Chlamydomonas reinhardtii and other microalgae show adaptive changes, such as induction of a CO(2)-concentrating mechanism, changes in cell organization, increased photorespiratory enzyme activity, induction of periplasmic carbonic anhydrase and specific polypeptides (mitochondrial carbonic anhydrases and putative chloroplast carrier proteins), and transient down-regulation in the synthesis of Rubisco. The signal for acclimation to limiting CO(2) in C. reinhardtii is unidentified, and it is not known how they sense a change of CO(2) level. The limiting CO(2) signals must be transduced into the changes in gene expression observed during acclimation, so mutational analyses should be helpful for investigating the signal transduction pathway for low CO(2) acclimation. Eight independently isolated mutants of C. reinhardtii that require high CO(2) for photoautotrophic growth were tested by complementation group analysis. These mutants are likely to be defective in some aspects of the acclimation to low CO(2) because they differ from wild type in their growth and in the expression patterns of five low CO(2)-inducible genes (Cah1, Mca1, Mca2, Ccp1, and Ccp2). Two of the new mutants formed a single complementation group along with the previously described mutant cia-5, which appears to be defective in the signal transduction pathway for low CO(2) acclimation. The other mutations represent six additional, independent complementation groups.     

Catalytic properties and inhibition of Cd2+-carbonic anhydrases

Cd2+ derivatives of human carbonic anhydrases I and II and bovine red cell carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) have been prepared. The metal ion in these derivatives is readily displaced by Zn2+. The Cd2+-carbonic anhydrases have appreciable 4-nitrophenyl acetate hydrolase activities. These activities increase with pH as if dependent on the basic form of a group with pKa near 10. The Cd2+-carbonic anhydrases also have significant CO2 hydration activities. The Cd2+ derivatives are strongly inhibited by monovalent anions. In particular, I- is a much more potent inhibitor of the Cd2+ enzymes than of the native enzymes. Acetazolamide (5-acetylamido-1,3,4-thiadiazole 2-sulfonamide) is also a strong inhibitor although its affinity for the Cd2+ enzyme is less than its affinity for the native enzyme.     

Comparative properties of mammalian and insect carbonic anhydrases: effects of potassium and chloride on the rate of carbon dioxide hydration.

1. Carbonic anhydrase (E.C.4.2.1.1) catalysed CO2 hydration was studied with enzymes from mammalian and insect sources at CO2 concentrations of 7.6-30.8 mM. 2. At 0.01-0.15 M, potassium chloride (KCl) or choline chloride (ChCl) markedly inhibited all 8 mammalian enzymes studied. 3. Inhibition by KCl is always greater than that associated with ChCl. 4. KCl non-competitively inhibits and choline chloride competitively inhibits bovine carbonic anhydrase. 5. Carbonic anhydrase obtained from fat body, integumentary epithelium and midgut tissues of larval tobacco hornworms, Manduca sexta, is greatly stimulated by KCl and slightly inhibited by ChCl. 6. We propose that the effect of K+ on mammalian and insect carbonic anhydrases if fundamentally different.