Limits...
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

C. albicans Puf3 is a repressor of mRNA stability in glucose and lactate growth conditions.(A) A cartoon depicting the location of primers used for specific amplification of the MET3p-driven COX23 and MRPL25 genes. Detection of this allele was specific, as demonstrated in S3 Fig. (B) qPCR showing time dependent decay of COX23 and MRPL25 genes following transcriptional shutdown of MET3p in glucose. Fold expression was represented as ratio of expression levels for each time point after dividing with the expression levels at time 0. Decay curves were obtained with the nonlinear regression (curve fit) method using the exponential, one phase decay equation in the GraphPad “Prism 6” software. The half-life (T1/2) was also calculated using this equation by plotting the decay curve of 3–4 biological replicates separately, and is shown as the average ± standard error. (C) The experiments were performed as described in (B), but with lactate as the carbon source. Data represent the average and standard error from 4 biological replicates. (D) The cartoon depicts mutations in the core and -2C positions of the Puf3 binding motif in the COX23 3′ UTR. Decay curves are from wild type C. albicans strains expressing either a COX23 construct with either the wild type or the mutant Puf3 recognition element. The experiments were performed as in (B). Strains were grown in lactate media and shown are results of 2 biological replicates. (E) The experiment was performed as in (C), but concomitant with addition of methionine and cysteine to inhibit transcription, the temperature was raised to 37°C. Shown are results of 3 biological repeats.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4608769&req=5

pgen.1005590.g004: C. albicans Puf3 is a repressor of mRNA stability in glucose and lactate growth conditions.(A) A cartoon depicting the location of primers used for specific amplification of the MET3p-driven COX23 and MRPL25 genes. Detection of this allele was specific, as demonstrated in S3 Fig. (B) qPCR showing time dependent decay of COX23 and MRPL25 genes following transcriptional shutdown of MET3p in glucose. Fold expression was represented as ratio of expression levels for each time point after dividing with the expression levels at time 0. Decay curves were obtained with the nonlinear regression (curve fit) method using the exponential, one phase decay equation in the GraphPad “Prism 6” software. The half-life (T1/2) was also calculated using this equation by plotting the decay curve of 3–4 biological replicates separately, and is shown as the average ± standard error. (C) The experiments were performed as described in (B), but with lactate as the carbon source. Data represent the average and standard error from 4 biological replicates. (D) The cartoon depicts mutations in the core and -2C positions of the Puf3 binding motif in the COX23 3′ UTR. Decay curves are from wild type C. albicans strains expressing either a COX23 construct with either the wild type or the mutant Puf3 recognition element. The experiments were performed as in (B). Strains were grown in lactate media and shown are results of 2 biological replicates. (E) The experiment was performed as in (C), but concomitant with addition of methionine and cysteine to inhibit transcription, the temperature was raised to 37°C. Shown are results of 3 biological repeats.

Mentions: PUF proteins negatively impact on mRNA stability by recruiting deadenylases such as Ccr4, which digest the poly(A) tail and initiate decay, reviewed in [38]. Therefore, we next tested mRNA half-lives in the absence of Puf3 in C. albicans. Attempts to use the transcriptional inhibitor 1,10 phenantroline in C. albicans failed (no repression of gene transcription was observed for a prolonged time, S2A Fig). Another transcriptional inhibitor, thiolutin, was effective in inhibiting transcription only at very high doses, and had a stabilizing effect on mRNA (S2B and S2C Fig). To circumvent these technical problems with transcriptional inhibitors, we placed two Puf3 targets, MRPL25 encoding a mitochondrial ribosomal subunit, and COX23 encoding a cytochrome c oxidase assembly factor, under the repressible MET3 promoter. In this set up, transcription is “on” in the absence of methionine and cysteine in the medium, whereas addition of methionine and cysteine results in rapid repression. In these strains, only one copy of the Puf3-dependent gene is placed under the MET3 promoter, and therefore primers were used to specifically detect this copy and not the endogenous gene (Fig 4A). This strategy was chosen over deleting the second copy because of the high impact of mitochondrial mutations on C. albicans fitness due to its petite negative nature [44]. Control experiments showed that the detection of the MET3p-controlled transcripts of MRPL25 and COX23 by quantitative PCR experiments was highly specific (S3 Fig), and these strains had no observable growth defects in any of the carbon sources tested (S4 Fig).


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)

C. albicans Puf3 is a repressor of mRNA stability in glucose and lactate growth conditions.(A) A cartoon depicting the location of primers used for specific amplification of the MET3p-driven COX23 and MRPL25 genes. Detection of this allele was specific, as demonstrated in S3 Fig. (B) qPCR showing time dependent decay of COX23 and MRPL25 genes following transcriptional shutdown of MET3p in glucose. Fold expression was represented as ratio of expression levels for each time point after dividing with the expression levels at time 0. Decay curves were obtained with the nonlinear regression (curve fit) method using the exponential, one phase decay equation in the GraphPad “Prism 6” software. The half-life (T1/2) was also calculated using this equation by plotting the decay curve of 3–4 biological replicates separately, and is shown as the average ± standard error. (C) The experiments were performed as described in (B), but with lactate as the carbon source. Data represent the average and standard error from 4 biological replicates. (D) The cartoon depicts mutations in the core and -2C positions of the Puf3 binding motif in the COX23 3′ UTR. Decay curves are from wild type C. albicans strains expressing either a COX23 construct with either the wild type or the mutant Puf3 recognition element. The experiments were performed as in (B). Strains were grown in lactate media and shown are results of 2 biological replicates. (E) The experiment was performed as in (C), but concomitant with addition of methionine and cysteine to inhibit transcription, the temperature was raised to 37°C. Shown are results of 3 biological repeats.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005590.g004: C. albicans Puf3 is a repressor of mRNA stability in glucose and lactate growth conditions.(A) A cartoon depicting the location of primers used for specific amplification of the MET3p-driven COX23 and MRPL25 genes. Detection of this allele was specific, as demonstrated in S3 Fig. (B) qPCR showing time dependent decay of COX23 and MRPL25 genes following transcriptional shutdown of MET3p in glucose. Fold expression was represented as ratio of expression levels for each time point after dividing with the expression levels at time 0. Decay curves were obtained with the nonlinear regression (curve fit) method using the exponential, one phase decay equation in the GraphPad “Prism 6” software. The half-life (T1/2) was also calculated using this equation by plotting the decay curve of 3–4 biological replicates separately, and is shown as the average ± standard error. (C) The experiments were performed as described in (B), but with lactate as the carbon source. Data represent the average and standard error from 4 biological replicates. (D) The cartoon depicts mutations in the core and -2C positions of the Puf3 binding motif in the COX23 3′ UTR. Decay curves are from wild type C. albicans strains expressing either a COX23 construct with either the wild type or the mutant Puf3 recognition element. The experiments were performed as in (B). Strains were grown in lactate media and shown are results of 2 biological replicates. (E) The experiment was performed as in (C), but concomitant with addition of methionine and cysteine to inhibit transcription, the temperature was raised to 37°C. Shown are results of 3 biological repeats.
Mentions: PUF proteins negatively impact on mRNA stability by recruiting deadenylases such as Ccr4, which digest the poly(A) tail and initiate decay, reviewed in [38]. Therefore, we next tested mRNA half-lives in the absence of Puf3 in C. albicans. Attempts to use the transcriptional inhibitor 1,10 phenantroline in C. albicans failed (no repression of gene transcription was observed for a prolonged time, S2A Fig). Another transcriptional inhibitor, thiolutin, was effective in inhibiting transcription only at very high doses, and had a stabilizing effect on mRNA (S2B and S2C Fig). To circumvent these technical problems with transcriptional inhibitors, we placed two Puf3 targets, MRPL25 encoding a mitochondrial ribosomal subunit, and COX23 encoding a cytochrome c oxidase assembly factor, under the repressible MET3 promoter. In this set up, transcription is “on” in the absence of methionine and cysteine in the medium, whereas addition of methionine and cysteine results in rapid repression. In these strains, only one copy of the Puf3-dependent gene is placed under the MET3 promoter, and therefore primers were used to specifically detect this copy and not the endogenous gene (Fig 4A). This strategy was chosen over deleting the second copy because of the high impact of mitochondrial mutations on C. albicans fitness due to its petite negative nature [44]. Control experiments showed that the detection of the MET3p-controlled transcripts of MRPL25 and COX23 by quantitative PCR experiments was highly specific (S3 Fig), and these strains had no observable growth defects in any of the carbon sources tested (S4 Fig).

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