The bZIP Transcription Factor Rca1p Is a Central Regulator of a Novel CO2 Sensing Pathway in Yeast

Like many organisms the fungal pathogen Candida albicans senses changes in the environmental CO2 concentration. This response involves two major proteins: adenylyl cyclase and carbonic anhydrase (CA). Here, we demonstrate that CA expression is tightly controlled by the availability of CO2 and identify the bZIP transcription factor Rca1p as the first CO2 regulator of CA expression in yeast. We show that Rca1p upregulates CA expression during contact with mammalian phagocytes and demonstrate that serine 124 is critical for Rca1p signaling, which occurs independently of adenylyl cyclase. ChIP-chip analysis and the identification of Rca1p orthologs in the model yeast Saccharomyces cerevisiae (Cst6p) point to the broad significance of this novel pathway in fungi. By using advanced microscopy we visualize for the first time the impact of CO2 build-up on gene expression in entire fungal populations with an exceptional level of detail. Our results present the bZIP protein Rca1p as the first fungal regulator of carbonic anhydrase, and reveal the existence of an adenylyl cyclase independent CO2 sensing pathway in yeast. Rca1p appears to regulate cellular metabolism in response to CO2 availability in environments as diverse as the phagosome, yeast communities or liquid culture

Environmental sensing in the fungal pathogen of humans candida glabrata

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Carbonic anhydrase activators: Activation of the beta-carbonic anhydrase from the pathogenic yeast Candida glabrata with amines and amino acids

The protein encoded by the NCE103 gene of Candida glabrata, a beta-carbonic anhydrase (CA, EC 4.2.1.1) designated as CgCA, was investigated for its activation with amines and amino acids. CgCA was weakly activated by amino acids such as l-/d-His, l-Phe, l-DOPA, and l-Trp and by histamine or dopamine (K(A)s of 21.2-37microM) but more effectively activated by d-Phe, d-DOPA, d-Trp as well as serotonin, pyridyl-alkylamines, aminoethyl-piperazine/morpholine (K(A)s of 10.1-16.7microM). The best activators were l-/d-Tyr, with activation constants of 7.1-9.5microM. This study may bring a better understanding of the catalytic/activation mechanisms of beta-CAs from pathogenic fung

Carbonic anhydrase inhibitors: Inhibition of the ?-class enzyme from the pathogenic yeast Candida glabrata with sulfonamides, sulfamates and sulfamides.

The fungal pathogen Candida glabrata encodes for a ?-carbonic anhydrase (CA, EC 4.2.1.1), CgNce103, recently discovered. Only anions have been investigated as CgNce103 inhibitors up until now. Here we report the first sulfonamides inhibition study of this enzyme. Simple sulfonamides showed weak or moderate CgNce103 inhibitory properties, whereas acetazolamide, and a series of 4-substituted ureido-benzene-sulfonamides, sulfamates and sulfamides showed effective CgNce103 inhibitory properties, with KIs in the range of 4.1–115 nM, being also ineffective as human CA II inhibitors. As there is significant resistance of C. glabrata clinical isolates to many classical antifungal agents, inhibition of the ?-CA from this organism may allow an interesting means of controlling the pathogen growth, eventually leading to antifungals with a novel mechanism of action

Carbonic anhydrase regulation and CO2 sensing in the fungal pathogen Candida glabrata involves a novel Rca1p ortholog

Carbon dioxide (CO2) is a ubiquitous gas present at 0.0391% in atmospheric air and 5.5% in human blood. It forms part of numerous carboxylation and decarboxylation reactions carried out in every cell. Carbonic anhydrases (CA) enhance the hydration of CO2 to generate bicarbonate, which is subsequently used in cellular metabolism. In microorganisms, including the yeasts Candida albicans and Saccharomyces cerevisiae, inactivation of CA leads to a growth defect in air, which is complemented in an atmosphere enriched with CO2. In this study we characterize the CA from the fungal pathogen of humans Candida glabrata, CgNce103p, and report a comparable phenotype following its inactivation. Furthermore, we show that expression of the C. glabrata CA is strongly regulated by environmental CO2 at both the protein and transcript level. Similar to what we have previously reported for C. albicans and S. cerevisiae, C. glabrata CA regulation by CO2 is independent from the cAMP-PKA pathway and requires the novel bZIP transcription factor CgRca1p. We show that CgRca1p is an ortholog of the transcription factors Rca1p from C. albicans and Cst6p from S. cerevisiae and prove that CA induction in low CO2 involves the conserved DNA-binding motif TGACGTCA located on this C. glabrata promoter. However, in contrast to what is found in C. albicans CgRca1p expression itself is not affected by CO2. Although our results suggest a high level of similarity between the CO2 sensing pathways from C. glabrata, S. cerevisiae and C. albicans, they also point out significant intrinsic differences

<i>RCA1</i> is involved in growth, cell wall structure, filamentation and is regulated by CO₂.

<p><b>A</b>) Generation time in YPD of <i>C. albicans</i> (left panel) and <i>S</i>. <i>cerevisiae</i> (right panel) control strain (black columns) and <i>RCA1</i> ortholog mutants (white columns) grown in air or 5.5% CO₂ (grey columns). <b>B)</b> Germ tube formation in response to 5% serum of <i>C. albicans</i> control strain (black columns) and the <i>rca1</i>Δ (white columns) grown in air. <b>C</b>) Sensitivity assay of <i>C. albicans</i> control strain and <i>rca1</i>Δ. <b>D</b>) qRT-PCR using specific primers for Ca<i>RCA1</i> and <i>CST6</i> on RNA extracted from <i>C. albicans</i> (top) and <i>S. cerevisiae</i> (bottom) control strains, CAI4+pSM2 and BY4741, grown in air (black columns) or air enriched with 5.5% CO₂ (white columns). Data are represented as mean +/− SD. Asterisk indicates statistical significance determined by two-sample <i>t</i> test (<i>P</i>≤0.05).</p

Serine 124 is involved in Rca1p function as an inducer of <i>NCE103</i>.

<p>Western blot with <i>C. albicans rca1</i>Δ complemented with a wild-type allele, an <i>RCA1</i> allele mutated in serine 124, serine 126 or serine 222.</p

Rca1p is associated to <i>NCE103</i> and cell wall structure genes.

<p><b>A</b>) Venn diagram of the Rca1p associated genes. <b>B</b>) Rca1p binds to the <i>NCE103</i> promoter. Representation of the normalized log₂-transformed signal intensities of RCA1-HA₃-tagged in air (top panel) and high CO₂ (bottom panel) compared to the untagged strain versus the corresponding position of each signal on <i>C. albicans</i> genomic regions. Log₂-transformed signal intensity values are indicated at the left of the <i>y</i>-axis, the reference is the value 0 (i.e., a binding ratio of 1). <b>C)</b> ChIP-qPCR of RCA1-HA₃ tagged strain versus untagged control in air and a 5.5% CO₂ environment normalized to <i>ACT1</i> level with primers designed to amplify the above identified binding region of Rca1p on the <i>NCE103</i> promoter. <b>D</b>) qRT-PCR carried out with primers for <i>C. albicans CHT2</i> (top) and <i>OCH1</i> (bottom) on total RNA extracted from the <i>C. albicans</i> control strain (black columns) and <i>rca1</i>Δ (white columns) grown in air. Data are represented as mean +/− SD. Asterisk indicates statistical significance determined by two-sample <i>t</i> test (<i>P</i>≤0.05).</p

Rca1p orthologs regulate <i>NCE103</i> expression in <i>S. cerevisiae</i> via a TGACGTCA binding motif.

<p><b>A</b>) Western blot with extracts from <i>S. cerevisiae Sc</i>NCE103−GFP+pRS316 control strain, <i>cst6</i>Δ mutant and the complemented strain (ScNCE103−GFP+<i>cst6</i>Δ+pRS316−CST6). <b>B</b>) qRT-PCR with specific primers were used to calculate the ratio of <i>NCE103</i> transcript between low (air) and high CO₂ (5.5%) in <i>S. cerevisiae</i> control strain (black column), <i>cst6</i>Δ mutant (white column) and point mutation in the promoter of <i>NCE103</i> (Sc<i>nce103Δ</i>+<i>ScNCE103</i>−<i>GFP</i>−<i>MUT</i>) (grey column). Data are represented as mean +/− SD. Asterisk indicates statistical significance determined by two-sample <i>t</i> test (<i>P</i>≤0.05).</p

<i>NCE103</i> is essential for growth of <i>C. albicans</i> and <i>S. cerevisiae</i> and its expression is controlled by the concentration of environmental CO₂.

<p>Inactivation of the β-carbonic anhydrase encoded by <i>NCE103</i> in (<b>A)</b><i>C. albicans,</i> (<b>B</b>) <i>S. cerevisiae</i> inhibits growth in ambient air (right set of pictures) but not in air enriched with 5.5% CO₂ (left set of pictures). All strains were incubated on YPD medium for 24 hours. <b>C</b>) Western blots from <i>C. albicans</i> (left) and <i>S. cerevisiae</i> (right). Yeast carbonic anhydase is present in higher quantity in air than air enriched with 5.5% CO₂ samples. <b>D</b>) qRT-PCR using <i>NCE103</i> specific primers and RNA extracted from <i>C. albicans</i> (left) and <i>S. cerevisiae</i> (right) grown in air (black columns) or air enriched with 5.5% CO₂ (white columns). <b>E</b>) Western blot (top) and qRT-PCR (bottom) relative to <i>C. albicans cyr1</i>Δ. Data are represented as mean +/− SD. Asterisk indicates statistical significance determined by two-sample <i>t</i> test (<i>P</i>≤0.05).</p