Transcription profile of <i>C. albicans</i> and <i>S. cerevisiae</i> in xylose

<p>A) From microarray experiments with <i>C. albicans</i> grown in xylose, 175 genes have at least a 2-fold transcription increase in xylose when compared to cells in dextrose. The 44 genes with more than a 5-fold induction in xylose (SX) are shown with their corresponding value. Data for <i>S. cerevisiae</i> is shown for comparison, and in gray-shade if no reciprocal best-hit ortholog is found. B) Transcription modulation of the <i>S. cerevisiae</i> sugar transporters in xylose. The data from the microarray experiments in xylose (SX) and in no-sugar condition (S) is shown for 18 members of the sugar transporter family, relative to cells in dextrose. <i>HXT5</i> and <i>HXT2</i> are likely induced due to the absence of dextrose (glucose). <i>HXT6</i>, <i>HXT7</i> and <i>HXT4</i> are induced specifically by the presence of xylose. <i>HXT4</i>, <i>HXT1</i> and <i>HXT3</i> show a transcriptional reduction only for the no-sugar condition (S), likely due to the absence of sugar. A summary for the transporters description from SGD is reported in the ‘SGD description’ column.</p

The xylose reductase and xylitol dehydrogenase genes from <i>S. cerevisiae</i> complement <i>C. albicans</i> deletion mutants of the equivalent genes.

<p>(A) <i>S. cerevisiae </i><i>GRE3</i> (CA255), and (B) <i>S. cerevisiae </i><i>SOR1</i> (CDH139) complement the <i>C. albicans </i><i>gre3</i> and <i>xyl2</i> deletion mutants respectively. Strains were grown aerobically at 30°C in 2% xylose (SX) or 2% xylitol (ST). The optical density was measured over a period of 14 days, n=3.</p

The xylose isomerase gene <i>XYLA</i> from <i>Piromyces</i><i>sp.</i> complements a double <i>gre3/xyl2</i> deletion or single <i>gre3</i> deletion in <i>C. albicans</i>.

<p>Growth analysis in synthetic medium (SC) with 2% xylose as the sole carbon source for <i>C. albicans</i> strains: <i>gre3-9</i> (<i>gre3</i> deletion), <i>gre3/xyl2-3</i> (<i>gre3 </i><i>xyl2</i> double deletion), CDH128 (<i>gre3/xyl2-3</i> with the integrated <i>Pir</i>.<i>XYLA</i>) and CDH140 (<i>gre3-9</i> with the integrated <i>Pir</i>. <i>XYLA</i>). The optical density was measured over a period of 6 days, n=3. </p

<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

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

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

The bZIP transcription factor Rca1p is a regulator of CO₂ signaling in <i>C. albicans</i>.

<p><b>A</b>) Western blots with protein extract from the <i>C. albicans</i> control strain, <i>rca1</i>Δ and <i>rca1</i> complemented strains. <b>B</b>) qRT-PCR using <i>NCE103</i> specific primers and RNA extracted from the above strains grown in air (black columns) or air enriched with 5.5% CO₂ (white columns). <b>C</b>) qRT-PCR with <i>NCE103</i> specific primers were used to calculate the ratio of transcript between cells phagocyted and cells grown <i>in vitro</i> in the control (black column) or <i>rca1</i>Δ strains (white 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

Carbonic anhydrase is differentially expressed in <i>S. cerevisiae</i> populations.

<p><b>A</b>) Pictures of <i>S. cerevisiae Sc</i>NCE103-GFP cells grown in YPD for 4h in air (right panel) or air supplemented with 5% CO₂ (left panel). Picture magnification x60, bar corresponds to 20 µm. <b>B</b>) Cross section of a ScNCE103-GFP colony grown in ambient air (first panel) or in air enriched with 5.5% CO₂ (second panel), ScNCE103-GFP+<i>cst6Δ</i> (third panel) BY4741+pTEF-GFP (fourth panel) were grown in air. Bar corresponds to 100 µm.</p