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Telomeric ORFs (TLOs) in Candida spp. Encode mediator subunits that regulate distinct virulence traits.

Haran J, Boyle H, Hokamp K, Yeomans T, Liu Z, Church M, Fleming AB, Anderson MZ, Berman J, Myers LC, Sullivan DJ, Moran GP - PLoS Genet. (2014)

Bottom Line: Complementation with TLO1 also had a greater effect on doubling times in galactose broth.The different abilities of TLO1 and TLO2 to restore wild-type functions was supported by transcript profiling studies that showed that only TLO1 restored expression of hypha-specific genes (UME6, SOD5) and galactose utilisation genes (GAL1 and GAL10), whereas TLO2 restored repression of starvation-induced gene transcription.Thus, Tlo/Med2 paralogs encoding Mediator subunits regulate different virulence properties in Candida spp. and their expansion may account for the increased adaptability of C. albicans relative to other Candida species.

View Article: PubMed Central - PubMed

Affiliation: Division of Oral Biosciences, Dublin Dental University Hospital, University of Dublin, Trinity College Dublin, Dublin, Ireland.

ABSTRACT
The TLO genes are a family of telomere-associated ORFs in the fungal pathogens Candida albicans and C. dubliniensis that encode a subunit of the Mediator complex with homology to Med2. The more virulent pathogen C. albicans has 15 copies of the gene whereas the less pathogenic species C. dubliniensis has only two (CdTLO1 and CdTLO2). In this study we used C. dubliniensis as a model to investigate the role of TLO genes in regulating virulence and also to determine whether TLO paralogs have evolved to regulate distinct functions. A C. dubliniensis tlo1Δ/tlo2Δ mutant is unable to form true hyphae, has longer doubling times in galactose broth, is more susceptible to oxidative stress and forms increased levels of biofilm. Transcript profiling of the tlo1Δ/tlo2Δ mutant revealed increased expression of starvation responses in rich medium and retarded expression of hypha-induced transcripts in serum. ChIP studies indicated that Tlo1 binds to many ORFs including genes that exhibit high and low expression levels under the conditions analyzed. The altered expression of these genes in the tlo1Δ/tlo2Δ mutant indicates roles for Tlo proteins in transcriptional activation and repression. Complementation of the tlo1Δ/tlo2Δ mutant with TLO1, but not TLO2, restored wild-type filamentous growth, whereas only TLO2 fully suppressed biofilm growth. Complementation with TLO1 also had a greater effect on doubling times in galactose broth. The different abilities of TLO1 and TLO2 to restore wild-type functions was supported by transcript profiling studies that showed that only TLO1 restored expression of hypha-specific genes (UME6, SOD5) and galactose utilisation genes (GAL1 and GAL10), whereas TLO2 restored repression of starvation-induced gene transcription. Thus, Tlo/Med2 paralogs encoding Mediator subunits regulate different virulence properties in Candida spp. and their expansion may account for the increased adaptability of C. albicans relative to other Candida species.

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Deletion of TLOs has different effects on biofilm formation compared to a med31Δ mutant.(A) Gene Set Enrichment Analysis (GSEA) plot showing differential expression of genes down-regulated in a C. albicans med31Δ mutant in the tlo1Δ/tlo2Δ mutant (i.e. they are upregulated in the tlo1Δ/tlo2Δ mutant) (B) Biofilm formation was assessed in the indicated strains on plastic surfaces at 37°C in the presence or absence of 5% CO2 as indicated. Adherent biomass determined by an XTT reduction assay. The experiment was carried out in triplicate on three separate occasions (n = 9) and error bars represent the standard deviation from the mean. P values above figure are the result of one-way ANOVA with Tukey's post hoc test.
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pgen-1004658-g007: Deletion of TLOs has different effects on biofilm formation compared to a med31Δ mutant.(A) Gene Set Enrichment Analysis (GSEA) plot showing differential expression of genes down-regulated in a C. albicans med31Δ mutant in the tlo1Δ/tlo2Δ mutant (i.e. they are upregulated in the tlo1Δ/tlo2Δ mutant) (B) Biofilm formation was assessed in the indicated strains on plastic surfaces at 37°C in the presence or absence of 5% CO2 as indicated. Adherent biomass determined by an XTT reduction assay. The experiment was carried out in triplicate on three separate occasions (n = 9) and error bars represent the standard deviation from the mean. P values above figure are the result of one-way ANOVA with Tukey's post hoc test.

Mentions: Uwamahoro et al.[15] showed that in C. albicans deletion of MED31, encoding a middle subunit of Mediator affected cytokinesis, filamentous growth and biofilm formation. Similarly, the C. dubliniensis tlo1Δ/tlo2Δ mutant described here also grew as chains of cells, typical of mutants with defects in cytokinesis (Figure S3A). However, the C. albicans med31Δ mutant was capable of filamentous growth in response to serum, whereas the C. dubliniensis tlo1Δ/tlo2Δ mutant is incapable of forming true hyphae in serum. GSEA analysis of our C. dubliniensis tlo1Δ/tlo2Δ mutant transcript data identified a significant enrichment for genes that are also affected in a C. albicans med31Δ mutant [15]. Interestingly, while the tlo1Δ/tlo2Δ and the med31Δ deletions affected similar genes, the C. dubliniensis tlo1Δ/tlo2Δ mutant showed increased expression of genes that were both Med31-activated and -repressed in C. albicans (Figure 7A). Inspection of these differentially expressed genes identified several genes required for biofilm formation that were downregulated in the C. albicans med31Δ mutant and induced in the C. dubliniensis tlo1Δ/tlo2Δ mutant, including ALS1, TEC1 and SUC1. Uwamahoro et al.[15] showed that reduced ALS1 expression in the C. albicans med31Δ mutant was largely responsible for the defect in biofilm formation. As the C. dubliniensis tlo1Δ/tlo2Δ mutant exhibited increased expression of ALS1, we examined whether this mutant was affected in biofilm formation. In contrast to the med31Δ mutant phenotype, deletion of TLO1 and TLO2 in C. dubliniensis enhanced biofilm growth on plastic surfaces (Figure 7B). In this case, complementation with TLO2 reduced the amount of biofilm formation more than complementation with TLO1 (ANOVA P<0.05) (Figure 7B). Thus the mediator middle domain has a different effect on biofilm formation than does the mediator tail domain.


Telomeric ORFs (TLOs) in Candida spp. Encode mediator subunits that regulate distinct virulence traits.

Haran J, Boyle H, Hokamp K, Yeomans T, Liu Z, Church M, Fleming AB, Anderson MZ, Berman J, Myers LC, Sullivan DJ, Moran GP - PLoS Genet. (2014)

Deletion of TLOs has different effects on biofilm formation compared to a med31Δ mutant.(A) Gene Set Enrichment Analysis (GSEA) plot showing differential expression of genes down-regulated in a C. albicans med31Δ mutant in the tlo1Δ/tlo2Δ mutant (i.e. they are upregulated in the tlo1Δ/tlo2Δ mutant) (B) Biofilm formation was assessed in the indicated strains on plastic surfaces at 37°C in the presence or absence of 5% CO2 as indicated. Adherent biomass determined by an XTT reduction assay. The experiment was carried out in triplicate on three separate occasions (n = 9) and error bars represent the standard deviation from the mean. P values above figure are the result of one-way ANOVA with Tukey's post hoc test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214616&req=5

pgen-1004658-g007: Deletion of TLOs has different effects on biofilm formation compared to a med31Δ mutant.(A) Gene Set Enrichment Analysis (GSEA) plot showing differential expression of genes down-regulated in a C. albicans med31Δ mutant in the tlo1Δ/tlo2Δ mutant (i.e. they are upregulated in the tlo1Δ/tlo2Δ mutant) (B) Biofilm formation was assessed in the indicated strains on plastic surfaces at 37°C in the presence or absence of 5% CO2 as indicated. Adherent biomass determined by an XTT reduction assay. The experiment was carried out in triplicate on three separate occasions (n = 9) and error bars represent the standard deviation from the mean. P values above figure are the result of one-way ANOVA with Tukey's post hoc test.
Mentions: Uwamahoro et al.[15] showed that in C. albicans deletion of MED31, encoding a middle subunit of Mediator affected cytokinesis, filamentous growth and biofilm formation. Similarly, the C. dubliniensis tlo1Δ/tlo2Δ mutant described here also grew as chains of cells, typical of mutants with defects in cytokinesis (Figure S3A). However, the C. albicans med31Δ mutant was capable of filamentous growth in response to serum, whereas the C. dubliniensis tlo1Δ/tlo2Δ mutant is incapable of forming true hyphae in serum. GSEA analysis of our C. dubliniensis tlo1Δ/tlo2Δ mutant transcript data identified a significant enrichment for genes that are also affected in a C. albicans med31Δ mutant [15]. Interestingly, while the tlo1Δ/tlo2Δ and the med31Δ deletions affected similar genes, the C. dubliniensis tlo1Δ/tlo2Δ mutant showed increased expression of genes that were both Med31-activated and -repressed in C. albicans (Figure 7A). Inspection of these differentially expressed genes identified several genes required for biofilm formation that were downregulated in the C. albicans med31Δ mutant and induced in the C. dubliniensis tlo1Δ/tlo2Δ mutant, including ALS1, TEC1 and SUC1. Uwamahoro et al.[15] showed that reduced ALS1 expression in the C. albicans med31Δ mutant was largely responsible for the defect in biofilm formation. As the C. dubliniensis tlo1Δ/tlo2Δ mutant exhibited increased expression of ALS1, we examined whether this mutant was affected in biofilm formation. In contrast to the med31Δ mutant phenotype, deletion of TLO1 and TLO2 in C. dubliniensis enhanced biofilm growth on plastic surfaces (Figure 7B). In this case, complementation with TLO2 reduced the amount of biofilm formation more than complementation with TLO1 (ANOVA P<0.05) (Figure 7B). Thus the mediator middle domain has a different effect on biofilm formation than does the mediator tail domain.

Bottom Line: Complementation with TLO1 also had a greater effect on doubling times in galactose broth.The different abilities of TLO1 and TLO2 to restore wild-type functions was supported by transcript profiling studies that showed that only TLO1 restored expression of hypha-specific genes (UME6, SOD5) and galactose utilisation genes (GAL1 and GAL10), whereas TLO2 restored repression of starvation-induced gene transcription.Thus, Tlo/Med2 paralogs encoding Mediator subunits regulate different virulence properties in Candida spp. and their expansion may account for the increased adaptability of C. albicans relative to other Candida species.

View Article: PubMed Central - PubMed

Affiliation: Division of Oral Biosciences, Dublin Dental University Hospital, University of Dublin, Trinity College Dublin, Dublin, Ireland.

ABSTRACT
The TLO genes are a family of telomere-associated ORFs in the fungal pathogens Candida albicans and C. dubliniensis that encode a subunit of the Mediator complex with homology to Med2. The more virulent pathogen C. albicans has 15 copies of the gene whereas the less pathogenic species C. dubliniensis has only two (CdTLO1 and CdTLO2). In this study we used C. dubliniensis as a model to investigate the role of TLO genes in regulating virulence and also to determine whether TLO paralogs have evolved to regulate distinct functions. A C. dubliniensis tlo1Δ/tlo2Δ mutant is unable to form true hyphae, has longer doubling times in galactose broth, is more susceptible to oxidative stress and forms increased levels of biofilm. Transcript profiling of the tlo1Δ/tlo2Δ mutant revealed increased expression of starvation responses in rich medium and retarded expression of hypha-induced transcripts in serum. ChIP studies indicated that Tlo1 binds to many ORFs including genes that exhibit high and low expression levels under the conditions analyzed. The altered expression of these genes in the tlo1Δ/tlo2Δ mutant indicates roles for Tlo proteins in transcriptional activation and repression. Complementation of the tlo1Δ/tlo2Δ mutant with TLO1, but not TLO2, restored wild-type filamentous growth, whereas only TLO2 fully suppressed biofilm growth. Complementation with TLO1 also had a greater effect on doubling times in galactose broth. The different abilities of TLO1 and TLO2 to restore wild-type functions was supported by transcript profiling studies that showed that only TLO1 restored expression of hypha-specific genes (UME6, SOD5) and galactose utilisation genes (GAL1 and GAL10), whereas TLO2 restored repression of starvation-induced gene transcription. Thus, Tlo/Med2 paralogs encoding Mediator subunits regulate different virulence properties in Candida spp. and their expansion may account for the increased adaptability of C. albicans relative to other Candida species.

Show MeSH
Related in: MedlinePlus