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Integration of Posttranscriptional Gene Networks into Metabolic Adaptation and Biofilm Maturation in Candida albicans.

Verma-Gaur J, Qu Y, Harrison PF, Lo TL, Quenault T, Dagley MJ, Bellousoff M, Powell DR, Beilharz TH, Traven A - PLoS Genet. (2015)

Bottom Line: The extracellular matrix is critical for antifungal resistance and immune evasion, and yet of all biofilm maturation pathways extracellular matrix biogenesis is the least understood.We propose a model in which the hypoxic biofilm environment is sensed by regulators such as Ccr4 to orchestrate metabolic adaptation, as well as the regulation of extracellular matrix production by impacting on the expression of matrix-related cell wall genes.Therefore metabolic changes in biofilms might be intimately linked to a key biofilm maturation mechanism that ultimately results in untreatable fungal disease.

View Article: PubMed Central - PubMed

Affiliation: Infection and Immunity Program, Biomedicine Discovery Institute and the Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

ABSTRACT
The yeast Candida albicans is a human commensal and opportunistic pathogen. Although both commensalism and pathogenesis depend on metabolic adaptation, the regulatory pathways that mediate metabolic processes in C. albicans are incompletely defined. For example, metabolic change is a major feature that distinguishes community growth of C. albicans in biofilms compared to suspension cultures, but how metabolic adaptation is functionally interfaced with the structural and gene regulatory changes that drive biofilm maturation remains to be fully understood. We show here that the RNA binding protein Puf3 regulates a posttranscriptional mRNA network in C. albicans that impacts on mitochondrial biogenesis, and provide the first functional data suggesting evolutionary rewiring of posttranscriptional gene regulation between the model yeast Saccharomyces cerevisiae and C. albicans. A proportion of the Puf3 mRNA network is differentially expressed in biofilms, and by using a mutant in the mRNA deadenylase CCR4 (the enzyme recruited to mRNAs by Puf3 to control transcript stability) we show that posttranscriptional regulation is important for mitochondrial regulation in biofilms. Inactivation of CCR4 or dis-regulation of mitochondrial activity led to altered biofilm structure and over-production of extracellular matrix material. The extracellular matrix is critical for antifungal resistance and immune evasion, and yet of all biofilm maturation pathways extracellular matrix biogenesis is the least understood. We propose a model in which the hypoxic biofilm environment is sensed by regulators such as Ccr4 to orchestrate metabolic adaptation, as well as the regulation of extracellular matrix production by impacting on the expression of matrix-related cell wall genes. Therefore metabolic changes in biofilms might be intimately linked to a key biofilm maturation mechanism that ultimately results in untreatable fungal disease.

No MeSH data available.


Related in: MedlinePlus

Effects of CCR4 on the extracellular matrix and biofilm stability.(A) Quantification of total extracellular matrix (ECM) in C. albicans biofilms. Biofilms were grown in Spider medium and collected after 48 h. The proportion of ECM was calculated relative to the total biofilm biomass (cells + ECM), as determined by dry weight measurement (see Materials and Methods). Results were calculated from three biological repeats in technical triplicates. Shown are the averages and the standard errors. P value of the difference between WT and ccr4Δ/Δ is shown as * <0.05. (B) Biofilms were grown in 96-well microtiter plates in RPMI-MOPS for 24 h and then exposed to zymolyase (20T) prepared in RPMI-MOPS + 0.9% NaCl (1:1 ratio) for 24 h. The remaining biomass was quantified by crystal violet staining. Results were calculated from three biological repeats in technical duplicates. Shown are the averages and the standard errors. P value of the difference between WT and ccr4Δ/Δ is shown as * < 0.05. (C) Matrix 1,3 β-glucan was quantified from biofilms grown in Spider medium as described in Materials and Methods. The yield of 1,3 β-glucan was calculated as the weight of 1,3 β-glucan (pg) per 1 μg of biofilm cells. Results were calculated from 6 biological repeats. Shown are the averages and the standard errors. Differences: WT versus ccr4Δ/Δ, p = 0.067; ccr4Δ/Δ + CCR4 versus ccr4Δ/Δ, p = 0.062. (D) qPCR analysis for the indicated transcripts was performed on biofilms grown for 48 h in Spider medium and data normalized to SCR1 RNA. Error bars are ± standard errors of the average of 3 biological replicates. P values are as follows: ** <0.01 or * <0.05.
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pgen.1005590.g007: Effects of CCR4 on the extracellular matrix and biofilm stability.(A) Quantification of total extracellular matrix (ECM) in C. albicans biofilms. Biofilms were grown in Spider medium and collected after 48 h. The proportion of ECM was calculated relative to the total biofilm biomass (cells + ECM), as determined by dry weight measurement (see Materials and Methods). Results were calculated from three biological repeats in technical triplicates. Shown are the averages and the standard errors. P value of the difference between WT and ccr4Δ/Δ is shown as * <0.05. (B) Biofilms were grown in 96-well microtiter plates in RPMI-MOPS for 24 h and then exposed to zymolyase (20T) prepared in RPMI-MOPS + 0.9% NaCl (1:1 ratio) for 24 h. The remaining biomass was quantified by crystal violet staining. Results were calculated from three biological repeats in technical duplicates. Shown are the averages and the standard errors. P value of the difference between WT and ccr4Δ/Δ is shown as * < 0.05. (C) Matrix 1,3 β-glucan was quantified from biofilms grown in Spider medium as described in Materials and Methods. The yield of 1,3 β-glucan was calculated as the weight of 1,3 β-glucan (pg) per 1 μg of biofilm cells. Results were calculated from 6 biological repeats. Shown are the averages and the standard errors. Differences: WT versus ccr4Δ/Δ, p = 0.067; ccr4Δ/Δ + CCR4 versus ccr4Δ/Δ, p = 0.062. (D) qPCR analysis for the indicated transcripts was performed on biofilms grown for 48 h in Spider medium and data normalized to SCR1 RNA. Error bars are ± standard errors of the average of 3 biological replicates. P values are as follows: ** <0.01 or * <0.05.

Mentions: (A) qPCR analysis of the expression of mitochondrial biogenesis genes in biofilms grown for 48 h in Spider medium. SCR1 RNA was used for normalization. Error bars are ± standard errors of the average of 3 biological replicates. P values are as follows: *** <0.001, ** <0.01, * <0.05. Additional genes are shown in S7 Fig. (B) Scanning electron microscopy of biofilms formed by C. albicans wild type (DAY185), ccr4Δ/Δ and pop2Δ/Δ mutants and their respective complemented strains. Mature biofilms (48 h) grown in Spider medium were assessed. Experiments were repeated at least twice and inset boxes are regional amplifications. Scale bar = 10 μm. Similar results were obtained when biofilms were grown in RPMI-MOPS or YNB (S9 Fig). (C) The data for RPMI-MOPS are from the control samples in the experiments performed to assess biofilm susceptibility to zymolyase (Fig 7B). Biofilms were grown for 48 h in the indicated media. Total biofilm biomass was determined by staining with crystal violet. Bars represent averages ± standard errors from three biological replicates. P value of the difference between WT versus ccr4Δ/Δ is shown as ** <0.01 in RPMI-MOPS and *<0.05 in Spider media. (D) Metabolic activity of C. albicans biofilms was determined using the XTT reduction assay. Results were calculated from three independently grown biofilms for each of the strains assayed in technical duplicates. The error bar represents standard errors. P value of the difference between WT and ccr4Δ/Δ is shown as * <0.05.


Integration of Posttranscriptional Gene Networks into Metabolic Adaptation and Biofilm Maturation in Candida albicans.

Verma-Gaur J, Qu Y, Harrison PF, Lo TL, Quenault T, Dagley MJ, Bellousoff M, Powell DR, Beilharz TH, Traven A - PLoS Genet. (2015)

Effects of CCR4 on the extracellular matrix and biofilm stability.(A) Quantification of total extracellular matrix (ECM) in C. albicans biofilms. Biofilms were grown in Spider medium and collected after 48 h. The proportion of ECM was calculated relative to the total biofilm biomass (cells + ECM), as determined by dry weight measurement (see Materials and Methods). Results were calculated from three biological repeats in technical triplicates. Shown are the averages and the standard errors. P value of the difference between WT and ccr4Δ/Δ is shown as * <0.05. (B) Biofilms were grown in 96-well microtiter plates in RPMI-MOPS for 24 h and then exposed to zymolyase (20T) prepared in RPMI-MOPS + 0.9% NaCl (1:1 ratio) for 24 h. The remaining biomass was quantified by crystal violet staining. Results were calculated from three biological repeats in technical duplicates. Shown are the averages and the standard errors. P value of the difference between WT and ccr4Δ/Δ is shown as * < 0.05. (C) Matrix 1,3 β-glucan was quantified from biofilms grown in Spider medium as described in Materials and Methods. The yield of 1,3 β-glucan was calculated as the weight of 1,3 β-glucan (pg) per 1 μg of biofilm cells. Results were calculated from 6 biological repeats. Shown are the averages and the standard errors. Differences: WT versus ccr4Δ/Δ, p = 0.067; ccr4Δ/Δ + CCR4 versus ccr4Δ/Δ, p = 0.062. (D) qPCR analysis for the indicated transcripts was performed on biofilms grown for 48 h in Spider medium and data normalized to SCR1 RNA. Error bars are ± standard errors of the average of 3 biological replicates. P values are as follows: ** <0.01 or * <0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608769&req=5

pgen.1005590.g007: Effects of CCR4 on the extracellular matrix and biofilm stability.(A) Quantification of total extracellular matrix (ECM) in C. albicans biofilms. Biofilms were grown in Spider medium and collected after 48 h. The proportion of ECM was calculated relative to the total biofilm biomass (cells + ECM), as determined by dry weight measurement (see Materials and Methods). Results were calculated from three biological repeats in technical triplicates. Shown are the averages and the standard errors. P value of the difference between WT and ccr4Δ/Δ is shown as * <0.05. (B) Biofilms were grown in 96-well microtiter plates in RPMI-MOPS for 24 h and then exposed to zymolyase (20T) prepared in RPMI-MOPS + 0.9% NaCl (1:1 ratio) for 24 h. The remaining biomass was quantified by crystal violet staining. Results were calculated from three biological repeats in technical duplicates. Shown are the averages and the standard errors. P value of the difference between WT and ccr4Δ/Δ is shown as * < 0.05. (C) Matrix 1,3 β-glucan was quantified from biofilms grown in Spider medium as described in Materials and Methods. The yield of 1,3 β-glucan was calculated as the weight of 1,3 β-glucan (pg) per 1 μg of biofilm cells. Results were calculated from 6 biological repeats. Shown are the averages and the standard errors. Differences: WT versus ccr4Δ/Δ, p = 0.067; ccr4Δ/Δ + CCR4 versus ccr4Δ/Δ, p = 0.062. (D) qPCR analysis for the indicated transcripts was performed on biofilms grown for 48 h in Spider medium and data normalized to SCR1 RNA. Error bars are ± standard errors of the average of 3 biological replicates. P values are as follows: ** <0.01 or * <0.05.
Mentions: (A) qPCR analysis of the expression of mitochondrial biogenesis genes in biofilms grown for 48 h in Spider medium. SCR1 RNA was used for normalization. Error bars are ± standard errors of the average of 3 biological replicates. P values are as follows: *** <0.001, ** <0.01, * <0.05. Additional genes are shown in S7 Fig. (B) Scanning electron microscopy of biofilms formed by C. albicans wild type (DAY185), ccr4Δ/Δ and pop2Δ/Δ mutants and their respective complemented strains. Mature biofilms (48 h) grown in Spider medium were assessed. Experiments were repeated at least twice and inset boxes are regional amplifications. Scale bar = 10 μm. Similar results were obtained when biofilms were grown in RPMI-MOPS or YNB (S9 Fig). (C) The data for RPMI-MOPS are from the control samples in the experiments performed to assess biofilm susceptibility to zymolyase (Fig 7B). Biofilms were grown for 48 h in the indicated media. Total biofilm biomass was determined by staining with crystal violet. Bars represent averages ± standard errors from three biological replicates. P value of the difference between WT versus ccr4Δ/Δ is shown as ** <0.01 in RPMI-MOPS and *<0.05 in Spider media. (D) Metabolic activity of C. albicans biofilms was determined using the XTT reduction assay. Results were calculated from three independently grown biofilms for each of the strains assayed in technical duplicates. The error bar represents standard errors. P value of the difference between WT and ccr4Δ/Δ is shown as * <0.05.

Bottom Line: The extracellular matrix is critical for antifungal resistance and immune evasion, and yet of all biofilm maturation pathways extracellular matrix biogenesis is the least understood.We propose a model in which the hypoxic biofilm environment is sensed by regulators such as Ccr4 to orchestrate metabolic adaptation, as well as the regulation of extracellular matrix production by impacting on the expression of matrix-related cell wall genes.Therefore metabolic changes in biofilms might be intimately linked to a key biofilm maturation mechanism that ultimately results in untreatable fungal disease.

View Article: PubMed Central - PubMed

Affiliation: Infection and Immunity Program, Biomedicine Discovery Institute and the Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

ABSTRACT
The yeast Candida albicans is a human commensal and opportunistic pathogen. Although both commensalism and pathogenesis depend on metabolic adaptation, the regulatory pathways that mediate metabolic processes in C. albicans are incompletely defined. For example, metabolic change is a major feature that distinguishes community growth of C. albicans in biofilms compared to suspension cultures, but how metabolic adaptation is functionally interfaced with the structural and gene regulatory changes that drive biofilm maturation remains to be fully understood. We show here that the RNA binding protein Puf3 regulates a posttranscriptional mRNA network in C. albicans that impacts on mitochondrial biogenesis, and provide the first functional data suggesting evolutionary rewiring of posttranscriptional gene regulation between the model yeast Saccharomyces cerevisiae and C. albicans. A proportion of the Puf3 mRNA network is differentially expressed in biofilms, and by using a mutant in the mRNA deadenylase CCR4 (the enzyme recruited to mRNAs by Puf3 to control transcript stability) we show that posttranscriptional regulation is important for mitochondrial regulation in biofilms. Inactivation of CCR4 or dis-regulation of mitochondrial activity led to altered biofilm structure and over-production of extracellular matrix material. The extracellular matrix is critical for antifungal resistance and immune evasion, and yet of all biofilm maturation pathways extracellular matrix biogenesis is the least understood. We propose a model in which the hypoxic biofilm environment is sensed by regulators such as Ccr4 to orchestrate metabolic adaptation, as well as the regulation of extracellular matrix production by impacting on the expression of matrix-related cell wall genes. Therefore metabolic changes in biofilms might be intimately linked to a key biofilm maturation mechanism that ultimately results in untreatable fungal disease.

No MeSH data available.


Related in: MedlinePlus