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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

Uncoupling of mitochondrial oxidative phosphorylation stimulates biofilm matrix production.The wild type stain DAY185 was used for these experiments. (A) qPCR analysis of the expression of the indicated mitochondrial biogenesis genes in 48 h grown biofilm samples upon treatment with CCCP. Error bars are ± standard errors of the average of three biological replicates. P values are as follows: ** <0.01, * < 0.05. (B) SEM of biofilms in the presence or absence of 20 μM CCCP. The ECM is indicated with black arrows. Scale bar = 10 μm. (C) The susceptibility to zymolyase of CCCP-treated biofilms grown in RPMI-MOPS was determined by crystal violet staining. Results were calculated from three biological repeats in technical duplicates. Shown are the averages and the standard errors. P value is * < 0.05.
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pgen.1005590.g008: Uncoupling of mitochondrial oxidative phosphorylation stimulates biofilm matrix production.The wild type stain DAY185 was used for these experiments. (A) qPCR analysis of the expression of the indicated mitochondrial biogenesis genes in 48 h grown biofilm samples upon treatment with CCCP. Error bars are ± standard errors of the average of three biological replicates. P values are as follows: ** <0.01, * < 0.05. (B) SEM of biofilms in the presence or absence of 20 μM CCCP. The ECM is indicated with black arrows. Scale bar = 10 μm. (C) The susceptibility to zymolyase of CCCP-treated biofilms grown in RPMI-MOPS was determined by crystal violet staining. Results were calculated from three biological repeats in technical duplicates. Shown are the averages and the standard errors. P value is * < 0.05.

Mentions: One of the main stresses experienced by biofilm cells is hypoxia [50,51], and Ccr4 has been recently implicated in the response of C. albicans to hypoxia [52]. We therefore hypothesized that, additional to regulation of factors with roles in matrix carbohydrate accumulation, changes to mitochondrial activity in ccr4Δ/Δ biofilms, and potentially hypoxic adaptation might be responsible for the observed biofilm phenotypes of the mutant. Treatment with CCCP, which uncouples mitochondrial oxidative phosphorylation, mimics the early effects of hypoxia on the level of the genome-wide transcriptional response [52]. Therefore, we analyzed the structural features of biofilms in the presence of CCCP. For these experiments we predominantly used RPMI medium, because it supported biofilm growth significantly better than Spider medium upon mitochondrial inhibition. The dose of 20 μM CCCP is in line with previous studies in S. cerevisiae [53], and was effective in causing mitochondrial stress: similarly to ccr4Δ/Δ biofilms, treatment with CCCP led to up-regulation of mRNAs with mitochondrial functions (Fig 8A), which is a known consequence of mitochondrial perturbation [29,54]. CCCP had an effect on biofilm growth (S10 Fig), but nevertheless a complex biofilm that structurally resembled the wild type formed (Fig 8B). Similar to deletion of CCR4, treatment of biofilms with CCCP led to the accumulation of extracellular matrix (Fig 8B), and somewhat greater stability upon treatment with zymolyase (Fig 8C). These results support the proposition that mitochondrial dysfunction triggers ECM accumulation in biofilms.


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)

Uncoupling of mitochondrial oxidative phosphorylation stimulates biofilm matrix production.The wild type stain DAY185 was used for these experiments. (A) qPCR analysis of the expression of the indicated mitochondrial biogenesis genes in 48 h grown biofilm samples upon treatment with CCCP. Error bars are ± standard errors of the average of three biological replicates. P values are as follows: ** <0.01, * < 0.05. (B) SEM of biofilms in the presence or absence of 20 μM CCCP. The ECM is indicated with black arrows. Scale bar = 10 μm. (C) The susceptibility to zymolyase of CCCP-treated biofilms grown in RPMI-MOPS was determined by crystal violet staining. Results were calculated from three biological repeats in technical duplicates. Shown are the averages and the standard errors. P value is * < 0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608769&req=5

pgen.1005590.g008: Uncoupling of mitochondrial oxidative phosphorylation stimulates biofilm matrix production.The wild type stain DAY185 was used for these experiments. (A) qPCR analysis of the expression of the indicated mitochondrial biogenesis genes in 48 h grown biofilm samples upon treatment with CCCP. Error bars are ± standard errors of the average of three biological replicates. P values are as follows: ** <0.01, * < 0.05. (B) SEM of biofilms in the presence or absence of 20 μM CCCP. The ECM is indicated with black arrows. Scale bar = 10 μm. (C) The susceptibility to zymolyase of CCCP-treated biofilms grown in RPMI-MOPS was determined by crystal violet staining. Results were calculated from three biological repeats in technical duplicates. Shown are the averages and the standard errors. P value is * < 0.05.
Mentions: One of the main stresses experienced by biofilm cells is hypoxia [50,51], and Ccr4 has been recently implicated in the response of C. albicans to hypoxia [52]. We therefore hypothesized that, additional to regulation of factors with roles in matrix carbohydrate accumulation, changes to mitochondrial activity in ccr4Δ/Δ biofilms, and potentially hypoxic adaptation might be responsible for the observed biofilm phenotypes of the mutant. Treatment with CCCP, which uncouples mitochondrial oxidative phosphorylation, mimics the early effects of hypoxia on the level of the genome-wide transcriptional response [52]. Therefore, we analyzed the structural features of biofilms in the presence of CCCP. For these experiments we predominantly used RPMI medium, because it supported biofilm growth significantly better than Spider medium upon mitochondrial inhibition. The dose of 20 μM CCCP is in line with previous studies in S. cerevisiae [53], and was effective in causing mitochondrial stress: similarly to ccr4Δ/Δ biofilms, treatment with CCCP led to up-regulation of mRNAs with mitochondrial functions (Fig 8A), which is a known consequence of mitochondrial perturbation [29,54]. CCCP had an effect on biofilm growth (S10 Fig), but nevertheless a complex biofilm that structurally resembled the wild type formed (Fig 8B). Similar to deletion of CCR4, treatment of biofilms with CCCP led to the accumulation of extracellular matrix (Fig 8B), and somewhat greater stability upon treatment with zymolyase (Fig 8C). These results support the proposition that mitochondrial dysfunction triggers ECM accumulation in biofilms.

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