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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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Microarray gene expression profiling of the tlo1Δ/tlo2Δ (tloΔΔ) mutant in YEPD and 10% serum.(A) A heat map generated in GeneSpring GX12 showing all 1.5 fold regulated genes in the tlo1Δ/tlo2Δ (tloΔΔ) mutant relative to wild type strain Wü284 during exponential growth in YEPD broth and following inoculation in 10% (v/v) serum (1 h and 3 h). The fold change (Log2) relative to wild-type is color coded as indicated in the lower panel. GO terms associated with up and down regulated clusters are indicated on the left. (B) A cartoon metabolic map of the tlo1Δ/tlo2Δ (tloΔΔ) mutant showing the changes in expression of genes involved in energy metabolism during growth in YEPD. Genes in green exhibit a 1.5-fold or greater reduction in expression relative to wild-type whereas those in red exhibited a 1.5-fold increase in expression. Genes in black were not significantly changed.
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pgen-1004658-g004: Microarray gene expression profiling of the tlo1Δ/tlo2Δ (tloΔΔ) mutant in YEPD and 10% serum.(A) A heat map generated in GeneSpring GX12 showing all 1.5 fold regulated genes in the tlo1Δ/tlo2Δ (tloΔΔ) mutant relative to wild type strain Wü284 during exponential growth in YEPD broth and following inoculation in 10% (v/v) serum (1 h and 3 h). The fold change (Log2) relative to wild-type is color coded as indicated in the lower panel. GO terms associated with up and down regulated clusters are indicated on the left. (B) A cartoon metabolic map of the tlo1Δ/tlo2Δ (tloΔΔ) mutant showing the changes in expression of genes involved in energy metabolism during growth in YEPD. Genes in green exhibit a 1.5-fold or greater reduction in expression relative to wild-type whereas those in red exhibited a 1.5-fold increase in expression. Genes in black were not significantly changed.

Mentions: Since Mediator is important for transcription regulation, we analysed RNA expression patterns in the tlo1Δ/tlo2Δ mutant relative to wild-type cells grown in nutrient-rich growth conditions (YEPD at 37°C) and grown in hyphal inducting conditions (water plus 10% serum, optimal for C. dubliniensis hypha formation). During exponential growth in YEPD, a total of 746 genes exhibited a 1.5-fold or greater increase in expression and 635 genes exhibited a 1.5-fold or greater reduction in expression (Q≤0.05; Figure 4A). This scale of differential gene expression observed in our tlo1Δ/tlo2Δ is similar to that seen in S. cerevisiae Mediator tail mutants [23], [24]. In the nutrient-rich YEPD broth, the tlo1Δ/tlo2Δ mutant exhibited a transcriptional profile that resembled a response to nutrient starvation (Figure 4B). The induced set of genes was enriched for processes associated with catabolism of alternative carbon and nitrogen sources such as N-acetyl-glucosamine (NAG1, NAG3, NAG4, NAG6), amino acids (e.g. GDH2, CAR1, PUT2, PUT1, LPD1, FDH1 and FDH3) and fatty acids. The tlo1Δ/tlo2Δ mutant cells also up-regulated key genes of gluconeogenesis (PCK1 and FBP1) and the glyoxylate cycle (ICL1 and MDH1) (Figure 4B). In concert with this, the tlo1Δ/tlo2Δ mutant also caused down-regulation of genes encoding glycolytic enzymes (PFK1, PFK2, FBA1, GPM1 and ENO1) and the glycolytic regulator TYE7. The tlo1Δ/tlo2Δ mutant also exhibited a greater than 2-fold decrease in expression of genes encoding proteins important for sulphur amino acid biosynthesis (e.g. SAM2, MET1, MET6, MET10, MET14, MET16) and ergosterol biosynthesis (ERG1, ERG9, ERG25). In addition, some hypha-specific genes were induced in the tlo1Δ/tlo2Δ mutant grown in YEPD. This included IHD1, RBT5 and SAP7 (induced in C. dubliniensis hyphae) as well as several regulators of biofilm and hyphal growth (BCR1, NRG1, SFL1, TEC1 and EED1).


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)

Microarray gene expression profiling of the tlo1Δ/tlo2Δ (tloΔΔ) mutant in YEPD and 10% serum.(A) A heat map generated in GeneSpring GX12 showing all 1.5 fold regulated genes in the tlo1Δ/tlo2Δ (tloΔΔ) mutant relative to wild type strain Wü284 during exponential growth in YEPD broth and following inoculation in 10% (v/v) serum (1 h and 3 h). The fold change (Log2) relative to wild-type is color coded as indicated in the lower panel. GO terms associated with up and down regulated clusters are indicated on the left. (B) A cartoon metabolic map of the tlo1Δ/tlo2Δ (tloΔΔ) mutant showing the changes in expression of genes involved in energy metabolism during growth in YEPD. Genes in green exhibit a 1.5-fold or greater reduction in expression relative to wild-type whereas those in red exhibited a 1.5-fold increase in expression. Genes in black were not significantly changed.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214616&req=5

pgen-1004658-g004: Microarray gene expression profiling of the tlo1Δ/tlo2Δ (tloΔΔ) mutant in YEPD and 10% serum.(A) A heat map generated in GeneSpring GX12 showing all 1.5 fold regulated genes in the tlo1Δ/tlo2Δ (tloΔΔ) mutant relative to wild type strain Wü284 during exponential growth in YEPD broth and following inoculation in 10% (v/v) serum (1 h and 3 h). The fold change (Log2) relative to wild-type is color coded as indicated in the lower panel. GO terms associated with up and down regulated clusters are indicated on the left. (B) A cartoon metabolic map of the tlo1Δ/tlo2Δ (tloΔΔ) mutant showing the changes in expression of genes involved in energy metabolism during growth in YEPD. Genes in green exhibit a 1.5-fold or greater reduction in expression relative to wild-type whereas those in red exhibited a 1.5-fold increase in expression. Genes in black were not significantly changed.
Mentions: Since Mediator is important for transcription regulation, we analysed RNA expression patterns in the tlo1Δ/tlo2Δ mutant relative to wild-type cells grown in nutrient-rich growth conditions (YEPD at 37°C) and grown in hyphal inducting conditions (water plus 10% serum, optimal for C. dubliniensis hypha formation). During exponential growth in YEPD, a total of 746 genes exhibited a 1.5-fold or greater increase in expression and 635 genes exhibited a 1.5-fold or greater reduction in expression (Q≤0.05; Figure 4A). This scale of differential gene expression observed in our tlo1Δ/tlo2Δ is similar to that seen in S. cerevisiae Mediator tail mutants [23], [24]. In the nutrient-rich YEPD broth, the tlo1Δ/tlo2Δ mutant exhibited a transcriptional profile that resembled a response to nutrient starvation (Figure 4B). The induced set of genes was enriched for processes associated with catabolism of alternative carbon and nitrogen sources such as N-acetyl-glucosamine (NAG1, NAG3, NAG4, NAG6), amino acids (e.g. GDH2, CAR1, PUT2, PUT1, LPD1, FDH1 and FDH3) and fatty acids. The tlo1Δ/tlo2Δ mutant cells also up-regulated key genes of gluconeogenesis (PCK1 and FBP1) and the glyoxylate cycle (ICL1 and MDH1) (Figure 4B). In concert with this, the tlo1Δ/tlo2Δ mutant also caused down-regulation of genes encoding glycolytic enzymes (PFK1, PFK2, FBA1, GPM1 and ENO1) and the glycolytic regulator TYE7. The tlo1Δ/tlo2Δ mutant also exhibited a greater than 2-fold decrease in expression of genes encoding proteins important for sulphur amino acid biosynthesis (e.g. SAM2, MET1, MET6, MET10, MET14, MET16) and ergosterol biosynthesis (ERG1, ERG9, ERG25). In addition, some hypha-specific genes were induced in the tlo1Δ/tlo2Δ mutant grown in YEPD. This included IHD1, RBT5 and SAP7 (induced in C. dubliniensis hyphae) as well as several regulators of biofilm and hyphal growth (BCR1, NRG1, SFL1, TEC1 and EED1).

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