Limits...
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.

Show MeSH

Related in: MedlinePlus

Reintroduction of TLO1 or TLO2 in the tlo1Δ/tlo2Δ (tloΔΔ) mutant restores wild-type levels of expression of overlapping and distinct sets of genes.(A) A graph plotting the change in expression of genes up regulated (red lines) or down regulated (blue lines) in the tlo1Δ/tlo2Δ (tloΔΔ) mutant following complementation with either TLO1 (+TLO1) or TLO2 (+TLO2) in YEPD or (B) 10% (v/v) serum. (C) Heat map of selected genes that were differentially expressed in the tlo1Δ/tlo2Δ (tloΔΔ) mutant during growth in YEPD broth (C) or 10% (v/v) serum (D) that exhibited a TLO1 or TLO2 specific restoration to wild-type in expression. Colors in the first column (tlo1Δ/tlo2Δ) represent the fold change in expression of the indicated genes in tloΔΔ vs wild-type; colors in columns 2 and 3 (+TLO1 and +TLO2) represent the fold change in expression of the indicated genes following reintegration of TLO1 or TLO2 versus tloΔΔ. (E to H) QRT-PCR of the indicated genes was carried out following 30, 60 and 180 min incubation in 10% (v/v) serum at 37°C. Gene expression was determined using Sybr green technology and gene-specific levels are expressed relative to ACT1. Primer sequences are listed in Table S5.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4214616&req=5

pgen-1004658-g005: Reintroduction of TLO1 or TLO2 in the tlo1Δ/tlo2Δ (tloΔΔ) mutant restores wild-type levels of expression of overlapping and distinct sets of genes.(A) A graph plotting the change in expression of genes up regulated (red lines) or down regulated (blue lines) in the tlo1Δ/tlo2Δ (tloΔΔ) mutant following complementation with either TLO1 (+TLO1) or TLO2 (+TLO2) in YEPD or (B) 10% (v/v) serum. (C) Heat map of selected genes that were differentially expressed in the tlo1Δ/tlo2Δ (tloΔΔ) mutant during growth in YEPD broth (C) or 10% (v/v) serum (D) that exhibited a TLO1 or TLO2 specific restoration to wild-type in expression. Colors in the first column (tlo1Δ/tlo2Δ) represent the fold change in expression of the indicated genes in tloΔΔ vs wild-type; colors in columns 2 and 3 (+TLO1 and +TLO2) represent the fold change in expression of the indicated genes following reintegration of TLO1 or TLO2 versus tloΔΔ. (E to H) QRT-PCR of the indicated genes was carried out following 30, 60 and 180 min incubation in 10% (v/v) serum at 37°C. Gene expression was determined using Sybr green technology and gene-specific levels are expressed relative to ACT1. Primer sequences are listed in Table S5.

Mentions: In order to investigate whether TLO1 and TLO2 regulate similar or different sets of genes, we analysed the transcript profiles of the TLO1 and TLO2 complemented mutants rather than the tlo1Δ and tlo2Δ single mutants, as the complemented strains exhibited equivalent expression of TLO1 or TLO2 (Figure 1B, 1C). Both TLO1 and TLO2 largely restored the transcript profile of the tlo1Δ/tlo2Δ mutant back to wild-type levels in both YEPD and 10% serum (Figure 5A, 5B). However, some TLO-specific transcript patterns were also detectable. Sixty-one genes were regulated in a TLO-specific manner in YEPD (Table S1). In comparison to the TLO1-complemented strain, the TLO2-complemented strain showed a greater reduction in expression of many tlo1Δ/tlo2Δ induced genes, including many hyphal genes (IHD1, SAP7 and EED1), negative regulators of hyphal growth (NRG1 and SFL1) and some starvation-induced genes such as CAR1, GDH3, NAG3 and NAG4 (Figure 5C). The TLO2 complemented strain was also better at restoring wild-type levels of SOD6 expression relative to the TLO1 complemented strain (Figure 5C).


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)

Reintroduction of TLO1 or TLO2 in the tlo1Δ/tlo2Δ (tloΔΔ) mutant restores wild-type levels of expression of overlapping and distinct sets of genes.(A) A graph plotting the change in expression of genes up regulated (red lines) or down regulated (blue lines) in the tlo1Δ/tlo2Δ (tloΔΔ) mutant following complementation with either TLO1 (+TLO1) or TLO2 (+TLO2) in YEPD or (B) 10% (v/v) serum. (C) Heat map of selected genes that were differentially expressed in the tlo1Δ/tlo2Δ (tloΔΔ) mutant during growth in YEPD broth (C) or 10% (v/v) serum (D) that exhibited a TLO1 or TLO2 specific restoration to wild-type in expression. Colors in the first column (tlo1Δ/tlo2Δ) represent the fold change in expression of the indicated genes in tloΔΔ vs wild-type; colors in columns 2 and 3 (+TLO1 and +TLO2) represent the fold change in expression of the indicated genes following reintegration of TLO1 or TLO2 versus tloΔΔ. (E to H) QRT-PCR of the indicated genes was carried out following 30, 60 and 180 min incubation in 10% (v/v) serum at 37°C. Gene expression was determined using Sybr green technology and gene-specific levels are expressed relative to ACT1. Primer sequences are listed in Table S5.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004658-g005: Reintroduction of TLO1 or TLO2 in the tlo1Δ/tlo2Δ (tloΔΔ) mutant restores wild-type levels of expression of overlapping and distinct sets of genes.(A) A graph plotting the change in expression of genes up regulated (red lines) or down regulated (blue lines) in the tlo1Δ/tlo2Δ (tloΔΔ) mutant following complementation with either TLO1 (+TLO1) or TLO2 (+TLO2) in YEPD or (B) 10% (v/v) serum. (C) Heat map of selected genes that were differentially expressed in the tlo1Δ/tlo2Δ (tloΔΔ) mutant during growth in YEPD broth (C) or 10% (v/v) serum (D) that exhibited a TLO1 or TLO2 specific restoration to wild-type in expression. Colors in the first column (tlo1Δ/tlo2Δ) represent the fold change in expression of the indicated genes in tloΔΔ vs wild-type; colors in columns 2 and 3 (+TLO1 and +TLO2) represent the fold change in expression of the indicated genes following reintegration of TLO1 or TLO2 versus tloΔΔ. (E to H) QRT-PCR of the indicated genes was carried out following 30, 60 and 180 min incubation in 10% (v/v) serum at 37°C. Gene expression was determined using Sybr green technology and gene-specific levels are expressed relative to ACT1. Primer sequences are listed in Table S5.
Mentions: In order to investigate whether TLO1 and TLO2 regulate similar or different sets of genes, we analysed the transcript profiles of the TLO1 and TLO2 complemented mutants rather than the tlo1Δ and tlo2Δ single mutants, as the complemented strains exhibited equivalent expression of TLO1 or TLO2 (Figure 1B, 1C). Both TLO1 and TLO2 largely restored the transcript profile of the tlo1Δ/tlo2Δ mutant back to wild-type levels in both YEPD and 10% serum (Figure 5A, 5B). However, some TLO-specific transcript patterns were also detectable. Sixty-one genes were regulated in a TLO-specific manner in YEPD (Table S1). In comparison to the TLO1-complemented strain, the TLO2-complemented strain showed a greater reduction in expression of many tlo1Δ/tlo2Δ induced genes, including many hyphal genes (IHD1, SAP7 and EED1), negative regulators of hyphal growth (NRG1 and SFL1) and some starvation-induced genes such as CAR1, GDH3, NAG3 and NAG4 (Figure 5C). The TLO2 complemented strain was also better at restoring wild-type levels of SOD6 expression relative to the TLO1 complemented strain (Figure 5C).

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