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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

The 3′ UTR landscape of the C. albicans transcriptome.(A) Comparison of 3′ UTRs as determined by our study and Bruno et al [42]. Of the 3′ UTR that are called by both technologies, 84.5% are within 100 bases (r = 0.4684 (p much < 0.001, n = 2697. Of note, the correlation is highly significant because of the high numbers and would be classed as of moderate strength). Where there is a difference, it is due to filtering differences: we have used the most abundant 3′ UTR, whereas Bruno et al used the longest 3′ UTR for which there was evidence, including minor alternative 3′ UTR isoforms. (B) Graph showing 3′ UTRs are overall shorter in C. albicans than S. cerevisiae. The global positions of polyadenylation in the forward direction and, where it exists, the position of any anti-parallel overlapping adenylated RNA running in the reverse direction. Note any effect of filtering is avoided by this approach as all adenylation sites are utilized in this comparison (370997 and 201547 sites in C. albicans and S. cerevisiae respectively). (C) Comparison between the 3′ UTR lengths of the 3552 orthologous genes between C. albicans and S. cerevisiae. Genes annotated to GO “Mitochondrion” are labeled in red. (D) Comparison of the distance of the Puf3 binding site in putative mRNA targets conserved between S. cerevisiae and C. albicans relative to the closest coding sequence (CDS). (E) Comparison of the distance of the Puf3 binding site in putative mRNA targets conserved between S. cerevisiae and C. albicans relative to the polyadenylation site (PA).
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pgen.1005590.g002: The 3′ UTR landscape of the C. albicans transcriptome.(A) Comparison of 3′ UTRs as determined by our study and Bruno et al [42]. Of the 3′ UTR that are called by both technologies, 84.5% are within 100 bases (r = 0.4684 (p much < 0.001, n = 2697. Of note, the correlation is highly significant because of the high numbers and would be classed as of moderate strength). Where there is a difference, it is due to filtering differences: we have used the most abundant 3′ UTR, whereas Bruno et al used the longest 3′ UTR for which there was evidence, including minor alternative 3′ UTR isoforms. (B) Graph showing 3′ UTRs are overall shorter in C. albicans than S. cerevisiae. The global positions of polyadenylation in the forward direction and, where it exists, the position of any anti-parallel overlapping adenylated RNA running in the reverse direction. Note any effect of filtering is avoided by this approach as all adenylation sites are utilized in this comparison (370997 and 201547 sites in C. albicans and S. cerevisiae respectively). (C) Comparison between the 3′ UTR lengths of the 3552 orthologous genes between C. albicans and S. cerevisiae. Genes annotated to GO “Mitochondrion” are labeled in red. (D) Comparison of the distance of the Puf3 binding site in putative mRNA targets conserved between S. cerevisiae and C. albicans relative to the closest coding sequence (CDS). (E) Comparison of the distance of the Puf3 binding site in putative mRNA targets conserved between S. cerevisiae and C. albicans relative to the polyadenylation site (PA).

Mentions: To better define the Puf3-regulon in C. albicans, we performed a bioinformatics search for the (C/U/A)(A/G/C/U)UGUA(A/C/U)AUA recognition element in 3′ UTRs genome-wide. Firstly, we precisely defined the landscape of 3′ UTRs across the C. albicans transcriptome with a new 3′ sequencing technology that we developed called PAT-seq [41]. The 3' UTRs were called based on the most highly expressed peak within 400 bases of the end of the coding sequence, and not lying within a following gene on the same strand. Previous RNA-seq data has been informative in determining 3′ UTRs of C. albicans transcripts [42], and our mapping correlates well with the study of Bruno et al (Fig 2A). The apparent extension in length of 3′ UTRs in the Bruno et al data [42] is due to alternative adenylation that is present at lower abundance than the major peak called by our approach. The distinction between alternative 3′ UTR length isoforms is not easily extracted from regular RNA-seq, but is sensitively detected by PAT-seq. Moreover, with our technology we mapped 4862 3′ UTRs (or 78% of the transcriptome), and could map an additional 2006 3′ UTRs, beyond the annotations published by Bruno et al (S2 Dataset). Files to display 3′ UTR positions (including alternate isoforms where they exist) in CGD gbrowse are available (see Materials and Methods). We performed an equivalent analysis in S. cerevisiae, where we could map 5402 3′ UTRs. Comparison of positions of adenylation with S. cerevisiae showed that in C. albicans 3′ UTRs are overall shorter. The offset between convergent and overlapping 3′ UTRs is also slightly shorter, with both of these features reflecting a higher level of compaction of the C. albicans genome (Fig 2B). Despite this global similarity between 3′ UTR length distributions, the absolute 3′ UTR length in orthologous genes is not conserved between the two species (Fig 2C). Analysis of gene ontology enrichment (GO Function) in the C. albicans dataset, in windows of 50 bases of 3′ UTR length and at p < 0.0001, identified enrichment of very broad GO terms, such as “binding”, “anion binding”, “ATP binding”, “transferase activity”, “pyrophosphatase activity”, with one exception being “structural constituent of the ribosome” in the 50–99 bases 3′ UTR length group (S3 Dataset). An equivalent analysis of the S. cerevisiae dataset similarly showed broad terms, with the exception being “electron carrier activity” that mapped to 3′ UTRs longer than 200 bases (S3 Dataset). To further address if there is conservation of 3′ UTR lengths between the two yeasts related to gene function, we mapped genes with mitochondrial functions in Fig 2C (shown as red dots). However, no correlation was seen in regards to 3′ UTR lengths for mitochondria-related transcript between the two yeast species.


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)

The 3′ UTR landscape of the C. albicans transcriptome.(A) Comparison of 3′ UTRs as determined by our study and Bruno et al [42]. Of the 3′ UTR that are called by both technologies, 84.5% are within 100 bases (r = 0.4684 (p much < 0.001, n = 2697. Of note, the correlation is highly significant because of the high numbers and would be classed as of moderate strength). Where there is a difference, it is due to filtering differences: we have used the most abundant 3′ UTR, whereas Bruno et al used the longest 3′ UTR for which there was evidence, including minor alternative 3′ UTR isoforms. (B) Graph showing 3′ UTRs are overall shorter in C. albicans than S. cerevisiae. The global positions of polyadenylation in the forward direction and, where it exists, the position of any anti-parallel overlapping adenylated RNA running in the reverse direction. Note any effect of filtering is avoided by this approach as all adenylation sites are utilized in this comparison (370997 and 201547 sites in C. albicans and S. cerevisiae respectively). (C) Comparison between the 3′ UTR lengths of the 3552 orthologous genes between C. albicans and S. cerevisiae. Genes annotated to GO “Mitochondrion” are labeled in red. (D) Comparison of the distance of the Puf3 binding site in putative mRNA targets conserved between S. cerevisiae and C. albicans relative to the closest coding sequence (CDS). (E) Comparison of the distance of the Puf3 binding site in putative mRNA targets conserved between S. cerevisiae and C. albicans relative to the polyadenylation site (PA).
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Related In: Results  -  Collection

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pgen.1005590.g002: The 3′ UTR landscape of the C. albicans transcriptome.(A) Comparison of 3′ UTRs as determined by our study and Bruno et al [42]. Of the 3′ UTR that are called by both technologies, 84.5% are within 100 bases (r = 0.4684 (p much < 0.001, n = 2697. Of note, the correlation is highly significant because of the high numbers and would be classed as of moderate strength). Where there is a difference, it is due to filtering differences: we have used the most abundant 3′ UTR, whereas Bruno et al used the longest 3′ UTR for which there was evidence, including minor alternative 3′ UTR isoforms. (B) Graph showing 3′ UTRs are overall shorter in C. albicans than S. cerevisiae. The global positions of polyadenylation in the forward direction and, where it exists, the position of any anti-parallel overlapping adenylated RNA running in the reverse direction. Note any effect of filtering is avoided by this approach as all adenylation sites are utilized in this comparison (370997 and 201547 sites in C. albicans and S. cerevisiae respectively). (C) Comparison between the 3′ UTR lengths of the 3552 orthologous genes between C. albicans and S. cerevisiae. Genes annotated to GO “Mitochondrion” are labeled in red. (D) Comparison of the distance of the Puf3 binding site in putative mRNA targets conserved between S. cerevisiae and C. albicans relative to the closest coding sequence (CDS). (E) Comparison of the distance of the Puf3 binding site in putative mRNA targets conserved between S. cerevisiae and C. albicans relative to the polyadenylation site (PA).
Mentions: To better define the Puf3-regulon in C. albicans, we performed a bioinformatics search for the (C/U/A)(A/G/C/U)UGUA(A/C/U)AUA recognition element in 3′ UTRs genome-wide. Firstly, we precisely defined the landscape of 3′ UTRs across the C. albicans transcriptome with a new 3′ sequencing technology that we developed called PAT-seq [41]. The 3' UTRs were called based on the most highly expressed peak within 400 bases of the end of the coding sequence, and not lying within a following gene on the same strand. Previous RNA-seq data has been informative in determining 3′ UTRs of C. albicans transcripts [42], and our mapping correlates well with the study of Bruno et al (Fig 2A). The apparent extension in length of 3′ UTRs in the Bruno et al data [42] is due to alternative adenylation that is present at lower abundance than the major peak called by our approach. The distinction between alternative 3′ UTR length isoforms is not easily extracted from regular RNA-seq, but is sensitively detected by PAT-seq. Moreover, with our technology we mapped 4862 3′ UTRs (or 78% of the transcriptome), and could map an additional 2006 3′ UTRs, beyond the annotations published by Bruno et al (S2 Dataset). Files to display 3′ UTR positions (including alternate isoforms where they exist) in CGD gbrowse are available (see Materials and Methods). We performed an equivalent analysis in S. cerevisiae, where we could map 5402 3′ UTRs. Comparison of positions of adenylation with S. cerevisiae showed that in C. albicans 3′ UTRs are overall shorter. The offset between convergent and overlapping 3′ UTRs is also slightly shorter, with both of these features reflecting a higher level of compaction of the C. albicans genome (Fig 2B). Despite this global similarity between 3′ UTR length distributions, the absolute 3′ UTR length in orthologous genes is not conserved between the two species (Fig 2C). Analysis of gene ontology enrichment (GO Function) in the C. albicans dataset, in windows of 50 bases of 3′ UTR length and at p < 0.0001, identified enrichment of very broad GO terms, such as “binding”, “anion binding”, “ATP binding”, “transferase activity”, “pyrophosphatase activity”, with one exception being “structural constituent of the ribosome” in the 50–99 bases 3′ UTR length group (S3 Dataset). An equivalent analysis of the S. cerevisiae dataset similarly showed broad terms, with the exception being “electron carrier activity” that mapped to 3′ UTRs longer than 200 bases (S3 Dataset). To further address if there is conservation of 3′ UTR lengths between the two yeasts related to gene function, we mapped genes with mitochondrial functions in Fig 2C (shown as red dots). However, no correlation was seen in regards to 3′ UTR lengths for mitochondria-related transcript between the two yeast species.

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