Overexpression of Ca<i>HSP12</i> in <i>C. albicans</i>.

<p>(A) qRT-PCR analysis of the <i>CaHSP12</i> transcripts in HSP12OE. The level of transcripts was normalized to <i>ACT1</i>. The error bars represent the S.D. of triplicate independent reactions (B) Overexpression of Ca<i>HSP12</i> induced cell clumping. The control CAI4+pFM2 and HSP12OE were grown at pH 7. (C) Overexpression of <i>CaHSP12</i> promoted cell aggregation which was independent from filamentation. CAI4+pFM2 and HSP12OE were grown at pH 4 for 4 h. Aggregation was then measured. The graphs were plotted by the percentage of cells sedimented against time. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042894#s3" target="_blank">Results</a> represent the means of three biological replicates with S.D. *<i>P</i> value<0.05, versus control strain, two-sided unpaired student t-test. (D) Overexpression of Ca<i>HSP12</i> enhanced cell adhesion at pH 4 or pH 7. HSP12OE and CAI4+pFM2 were grown on the flat-bottomed 96-well polystyrene plates and incubated at 37°C for 24 h. The adherent cells were quantified using the XTT reduction assay. The error bars were calculated from the S.D. of the triplicates. *<i>P</i> value<0.01, versus control strain, two-sided unpaired student t-test. (E) Overexpression of Ca<i>HSP12</i> promoted filamentation at pH 7. The percentage of the germ tube formation was counted every 30 min. The results presented are the means of three biological replicates with the S.D. **<i>P</i> value<0.01, * <i>P</i> value>0.05 versus control strains, two-sided unpaired student t-test. (F) Overexpression of Ca<i>HSP12</i> impacts on farnesol susceptibility. Cells were spotted onto 5% serum YEPD plates supplemented with or without 100 µm farnesol. Scale bar, 200 µm. (G) CAI4+pFM2 and HSP12OE were incubated in YNB supplemented with 5% serum with or without 100 µM farnesol. Germ tube formation was quantified every 30 min. The error bars were calculated from the S.D. of the triplicates. **<i>P</i> value<0.01, *<i>P</i> value>0.05 versus control strains, two-sided unpaired student t-test. (G) Overexpression of Ca<i>HSP12</i> increases susceptibility to azole antifungal drugs. 10-fold dilutions were spotted onto YNB plates containing 4 µg ml⁻¹ itraconazole, ketoconazole and fluconzaole. YNB plates supplemented with 1% chloroform, methanol and DMSO act as control.</p

Expression of <i>Ca</i>Hsp12p in <i>C. albicans</i> mutant strains.

<p>(A) <i>Ca</i>Hsp12p expression is regulated by the Hog1p stress response and cAMP-PKA signalling pathway. <i>Ca</i>Hsp12p was isolated from mutant strains after growing until mid-log phase and its level analyzed by Western blot. Equal protein loading was assessed by probing the blot with anti-actin antibody. H: anti-<i>Ca</i>Hsp12p; A: anti-actin; RDU: relative densitometry units. (B) <i>EFG1</i> is required for the induction of <i>Ca</i>Hsp12p in response to heat shock and oxidative stress. Western blot show the level of <i>Ca</i>Hsp12p in the <i>efg1</i> mutant in unstressed condition (US) or following exposure to heat shock from 37°C to 45°C (HS), osmotic stress, 0.3 M NaCl, (OS) or oxidative stress, 1 mM H₂O₂, (XS). Equal protein loading was assessed by probing the blot with anti-actin antibody. H: anti-<i>Ca</i>Hsp12p; A: anti-actin; RDU: relative densitometry units.</p

<i>HSP12</i> differs among yeast species.

<p>(A) Two Ca<i>HSP12</i> genes have been identified in <i>C. albicans</i> SC5314 and five clinical isolates by Southern blot. (B) Both alleles of Ca<i>HSP12</i> are transcriptionally expressed. The transcription level of Ca<i>HSP12</i> is assessed by qRT-PCR of total RNA obtained from the strain with absence of one Ca<i>HSP12</i> gene (HSP12KO2) and its parental strain (BWP17). The error bars represent the S.D. of triplicate independent reactions. <i>P</i> value<0.01, two-sided unpaired student t-test. (<b>C</b>) 5′ RACE analysis of Ca<i>HSP12</i>. Two DNA bands (band 1 and band 2) with the expected size above 250 bp were sequenced. The sequencing shows that the 5′ untranslated region (in lowercase) contains 29 bp nucleotides and only the second start codon (underlined) of <i>CaHSP12</i> can be identified. M: 1 kb DNA ladder; -ve: negative control of PCR without template; <i>HSP12</i>: 5′RLM-RACE PCR product of <i>CaHSP12</i>.</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

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 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

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

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