The Set3/Hos2 Histone Deacetylase Complex Attenuates cAMP/PKA Signaling to Regulate Morphogenesis and Virulence of Candida albicans

Candida albicans, like other pleiomorphic fungal pathogens, is able to undergo a reversible transition between single yeast-like cells and multicellular filaments. This morphogenetic process has long been considered as a key fungal virulence factor. Here, we identify the evolutionarily conserved Set3/Hos2 histone deacetylase complex (Set3C) as a crucial repressor of the yeast-to-filament transition. Cells lacking core components of the Set3C are able to maintain all developmental phases, but are hypersusceptible to filamentation-inducing signals, because of a hyperactive cAMP/Protein Kinase A signaling pathway. Strikingly, Set3C-mediated control of filamentation is required for virulence in vivo, since set3Δ/Δ cells display strongly attenuated virulence in a mouse model of systemic infection. Importantly, the inhibition of histone deacetylase activity by trichostatin A exclusively phenocopies the absence of a functional Set3C, but not of any other histone deacetylase gene. Hence, our work supports a paradigm for manipulating morphogenesis in C. albicans through alternative antifungal therapeutic strategies

Cells lacking Hat1 show reduced virulence but persist in mouse kidneys.

<p>(A) Reduced growth rate of the <i>hat1</i>Δ/Δ strain was determined by measuring the OD₆₀₀ of cells growing in YPD at 30°C. (B) Cells lacking Hat1 are not cleared efficiently from kidneys. At the indicated time points, fungal burdens in kidneys of mice infected with <i>C</i>. <i>albicans</i> strains were determined and expressed as CFUs per gram kidney. Groups of 5–10 mice were analyzed at each time point and statistical significance was determined using the non-parametric Mann-Whitney-test. n.s.: not significant, *P<0.05 and **P<0.01 relative to the corresponding wild-type. (C) <i>hat1</i>Δ/Δ cells are defective in killing the host. Survival of mice infected with the indicated strains was monitored over 32 days post infection (p.i.). The data are presented as Kaplan-Meier survival curves. Groups of 6 mice were infected per <i>C</i>. <i>albicans</i> strain. Statistical significance was determined using the Log-rank test. ns: not significant; (D) Fungal burdens in kidneys of surviving mice from panel C were determined and expressed as CFUs per gram organ. One mouse infected with the <i>hat1</i>Δ/Δ strain was able to clear <i>Candida</i>. (E) The <i>cac2</i>Δ/Δ strain is not cleared efficiently from kidneys. Experiment was performed as described in (B). Groups of 4–5 mice were analyzed at each time point. (F) Infection with <i>hat1</i>Δ/Δ cells causes reduced kidney damage. Urea levels were determined in sera of infected mice at day 3 and 7 post infection. n.s.: not significant, *P<0.05, **P<0.01 relative to the wild-type (Student's t-test).</p

Specific functional gene groups are upregulated in cells lacking Hat1.

<p>(A) GO terms enriched among 2-fold significantly upregulated genes in logarithmically growing <i>hat1</i>Δ/Δ cells are shown. (B) The plot shows GO terms found within genes significantly upregulated in the <i>hat1</i>Δ/Δ and <i>rtt109</i>Δ/Δ strains only. (C) GO terms enriched within genes significantly upregulated in the <i>hat1</i>Δ/Δ and <i>cac2</i>Δ/Δ strains only. (D) The panel shows GO terms found among genes significantly upregulated in the <i>hat1</i>Δ/Δ mutant only and not in the <i>rtt109</i>Δ/Δ and the <i>cac2</i>Δ/Δ strains. (E) GO terms enriched among significantly upregulated genes in <i>hat1</i>Δ/Δ cells after treatment with H₂O₂ are shown. (F) The plot shows GO terms found within genes significantly upregulated in the <i>hat1</i>Δ/Δ strain only and not in the <i>rtt109</i>Δ/Δ and the <i>cac2</i>Δ/Δ strains upon H₂O₂ treatment. (A-F) The corresponding p-values for the enrichment (empty bars) and the percentage of genes changed within the GO group (filled bars) are presented. The absolute number of regulated genes within a GO group is presented in brackets. Groups containing identical genes are depicted in the same color. Significantly regulated genes were defined by a p-value <0.05.</p

Deletion of <i>HAT1</i> and <i>HAT2</i> increases oxidative stress resistance and azole tolerance.

<p>(A) Cells lacking Hat1 or Hat2 show increased resistance to H₂O₂. Lack of both genes mimics the corresponding single deletion strains. (B) Deletion of <i>HAT1</i> increases resistance to <i>tert</i>-butyl hydroperoxide (tBOOH). Lack of Rtt109 does not affect tBOOH sensitivity. (C) Loss of Hat1 causes reduced susceptibility to voriconazole (Voric.) and itraconazole (Itrac.). Deletion of <i>HAT2</i> or <i>HAT1</i> and <i>HAT2</i> mimics loss of Hat1. (D) Deletion of <i>RTT109</i> or <i>RAD52</i> does not increase voriconazole tolerance. (A-D) Fivefold serial dilutions of the indicated strains were spotted on agar plates containing the indicated substances and pictures were taken after incubation at 30°C for 3 days.</p

Lack of histone chaperones mimics deletion of <i>HAT1</i>.

<p>(A) Loss of Cac2 increases H₂O₂ resistance. Deletion of <i>RTT106</i> or <i>HIR1</i> does not affect susceptibility to hydrogen peroxide. Fivefold serial dilutions of the indicated strains were spotted on agar plates containing the indicated substances and pictures were taken after incubation at 30°C for 3 days. (B) Deletion of <i>HAT1</i> or <i>CAC2</i> increases survival to transient hydrogen peroxide treatment. Exponentially growing cells were treated with the indicated concentrations of H₂O₂ for 2 hours. Cells were plated and colonies counted after 3 days of incubation on YPD plates at 30°C to determine viability. Data are shown as mean + SD from three independent experiments. (C) Deletion of <i>HIR1</i> reduces voriconazole (Voric.) susceptibility. The <i>hat1hir1</i>Δ/Δ double deletion strain mimics lack of Hat1. Loss of Cac2 has only a minor effect and deletion of <i>RTT106</i> does not alter azole susceptibility. Experiment was performed as described in (A). (D) Increased azole tolerance of <i>hat1</i>Δ/Δ, <i>hir1</i>Δ/Δ and <i>hat1hir1</i>Δ/Δ was confirmed using a liquid growth inhibition assay. Logarithmically growing cells were diluted into medium containing the indicated concentrations of voriconazole (Voric.) and incubated at 30°C for 18 hours. OD₆₀₀ was determined and growth inhibition relative to untreated samples was calculated. Data are shown as mean + SD from three independent experiments. (E) Lack of Spt6 reduces H₂O₂ susceptibility. Experiment was performed as described in (B). Cells were treated with 10 mM H₂O₂. Data are shown as mean + SD from two independent experiments. (F) Deletion of <i>SPT6</i> increases H₂O₂ resistance and azole tolerance. Fivefold serial dilutions of the indicated strains were spotted on agar plates containing the indicated substances and pictures were taken after incubation at 30°C for 5 days. (G) Reduction of histone gene dosage decreases H₂O₂ and azole susceptibility. Experiment was performed as described in (A). (B, D, E) *P<0.05, **P<0.01 and ***P<0.001 relative to the corresponding wild-type (Student's t-test).</p

Deletion of <i>HAT1</i> primarily leads to upregulation of genes.

<p>(A) Lack of Hat1 causes mainly induction of genes in logarithmically growing cells. Each dot corresponds to one protein-coding gene. The fold change in RNA expression between untreated wild-type and <i>hat1</i>Δ/Δ cells (y-axis) is plotted against the expression level of each gene in this dataset (x-axis). Differentially expressed genes in the <i>hat1</i>Δ/Δ mutant are depicted in red. logCPM: log2 counts per million reads; logFC: log2 fold change; (B+C) Loss of Cac2 or Rtt109 causes almost exclusively upregulation of genes in logarithmically growing cells. Plots were created as described in (A). (D) Venn diagram showing the overlaps of upregulated genes in the <i>hat1</i>Δ/Δ, <i>cac2</i>Δ/Δ and <i>rtt109</i>Δ/Δ mutants in the absence of H₂O₂. (E) Venn diagram showing the overlaps of upregulated genes in the <i>hat1</i>Δ/Δ, <i>cac2</i>Δ/Δ and <i>rtt109</i>Δ/Δ mutants upon treatment with H₂O₂. (F) H₂O₂ repressed genes are upregulated in the <i>hat1</i>Δ/Δ mutant upon peroxide treatment. Each dot corresponds to one protein-coding gene. The -fold change in RNA expression between H₂O₂ treated wild-type and <i>hat1</i>Δ/Δ strains (y-axis) is plotted against the fold change between the wild-type without and with treatment (x-axis). Differentially expressed genes in the <i>hat1</i>Δ/Δ mutant are depicted in red. logFC: log2 fold change; (A-F) Differentially regulated genes were defined by a fold change > = 2 and p-value <0.05.</p

Higher ROS detoxification capacity of <i>hat1</i>Δ/Δ cells causes resistance to neutrophil killing.

<p>(A) Superoxide dismutases Sod4 and Sod5 are induced in <i>hat1</i>Δ/Δ cells. Expression levels of <i>SOD4</i> and <i>SOD5</i> in logarithmically growing cells were detected by RT-qPCR. Transcript levels were normalized to the expression level of the reference gene (RG) <i>RIP1</i>. Data are shown as mean + SD from 3 independent experiments. (B) Infection of macrophages with <i>hat1</i>Δ/Δ cells causes reduced ROS accumulation. ROS levels were determined by measuring luminol-dependent chemiluminescence [relative luciferase units (RLU) min^-1 per 1000 immune cells] in 2.5 min intervals during interaction of the indicated <i>C</i>. <i>albicans</i> strains with bone marrow-derived murine macrophages (BMDMs). One representative experiment is shown. Data were reproduced in three independent experiments. (C) Quantification of total ROS release upon interaction with BMDMs. Experiment was performed as described in (B). The area under the curve within 90 min of interaction was calculated. Data are shown as mean + SD from three independent experiments. (D) Cells lacking Hat1 show increased survival to neutrophil killing. Survival of <i>C</i>. <i>albicans</i> cells upon one hour interaction with murine bone marrow neutrophils was determined by plating and CFU counting. Data are shown as mean + SD from three independent experiments. (A-D) *P<0.05, **P<0.01 relative to the wild-type (Student's t-test).</p

Resistance phenotypes caused by loss of Hat1 are specific for <i>C</i>. <i>albicans</i>.

<p>(A) Deletion of <i>HAT1</i> in <i>S</i>. <i>cerevisiae</i> (a), <i>C</i>. <i>glabrata</i> (b) and <i>S</i>. <i>pombe</i> (c) has no effect on H₂O₂ resistance. Exponentially growing cells were treated with 5 mM (a), 50 mM (b) or 20 mM (c) H₂O₂ for 2 hours. Cells were plated and colonies counted after 3 days of incubation on YPD plates at 30°C to determine viability. Data are shown as mean + SD from three independent experiments. (B) Lack of Hat1 in <i>S</i>. <i>cerevisiae</i> (a) and <i>C</i>. <i>glabrata</i> (b) does not increase azole tolerance. Deletion of Hat1 in <i>S</i>. <i>pombe</i> reduces susceptibility to voriconazole (c). Logarithmically growing cells were diluted into medium containing 150 ng/ml (a), 1000 ng/ml (b) or 800 ng/ml (c) voriconazole and incubated at 30°C for 24 hours. OD₆₀₀ was determined and growth inhibition relative to untreated samples was calculated. Data are shown as mean + SD from three independent experiments. (C) <i>C</i>. <i>parapsilosis</i> (a) and <i>C</i>. <i>tropicalis</i> (b) <i>hat1</i>Δ/Δ cells show increased resistance to H₂O₂. Experiment was performed as described in (A). H₂O₂ concentrations were 50 mM (a) and 20 mM (b). (D) Loss of Hat1 in <i>C</i>. <i>parapsilosis</i> (a) and <i>C</i>. <i>tropicalis</i> (b) reduces susceptibility to voriconazole. Experiment was performed as described in (B). For <i>C</i>. <i>parapsilosis</i> cells were incubated for 41 hours prior to OD₆₀₀ measurement. Voriconazole concentrations were 50 ng/ml (a) and 200 ng/ml (b). (A-D) *P<0.05, **P<0.01 and ***P<0.001 relative to the corresponding wild-type (Student's t-test).</p

Lack of Hat1 accelerates induction of oxidative stress genes.

<p>(A) Catalase induction rate is strongly increased in <i>hat1</i>Δ/Δ cells. <i>CAT1</i> expression levels were measured by RT-qPCR after induction with 1.6 mM H₂O₂ at the indicated time points. Transcript levels were normalized to the expression level of the reference gene (RG) <i>PAT1</i>. Data are shown as mean + SD from 3 independent experiments. (B) Histone density at the <i>CAT1</i> locus is reduced in cells lacking Hat1. Histone H3 occupancy was determined by ChIP at the <i>CAT1</i> promoter region (a) and the CDS (b). (C) Loss of Hat1 leads to increased RNAPII recruitment at the <i>CAT1</i> locus. RNAPII levels were determined by ChIP at the <i>CAT1</i> CDS. (D) Induction rate of glutathione-utilizing enzymes is increased in hat1Δ/Δ cells. <i>GPX1</i> (a) and <i>GST1</i> (b) expression levels were determined by RT-qPCR at the indicated time points. Experiment was performed as described in (A). (E) Lack of Hat1 leads to increased RNAPII recruitment at the <i>GPX1</i> and <i>GST1</i> loci. RNAPII levels were determined by ChIP at the <i>GPX1</i> (a) and <i>GST1</i> (b) genes. (F+G) Loss of Cac2 increases the induction rate of both <i>GPX1</i> and <i>GST1</i> following H₂O₂ treatment. Experimental conditions were used as described in (A).</p