Haran John, Boyle Hannah, Hokamp Karsten, Yeomans Tim, Liu Zhongle, Church Michael, Fleming Alastair B, Anderson Matthew Z, Berman Judith, Myers Lawrence C, Sullivan Derek J, Moran Gary P
Division of Oral Biosciences, Dublin Dental University Hospital, University of Dublin, Trinity College Dublin, Dublin, Ireland.
School of Genetics and Microbiology, University of Dublin, Trinity College Dublin, Dublin, Ireland.
PLoS Genet. 2014 Oct 30;10(10):e1004658. doi: 10.1371/journal.pgen.1004658. eCollection 2014 Oct.
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 null 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.
TLO基因是白色念珠菌和都柏林念珠菌这两种真菌病原体中与端粒相关的开放阅读框家族，它们编码一种与Med2具有同源性的中介体复合物亚基。致病性更强的病原体白色念珠菌有该基因的15个拷贝，而致病性较弱的都柏林念珠菌只有两个（CdTLO1和CdTLO2）。在本研究中，我们以都柏林念珠菌为模型，研究TLO基因在调节毒力中的作用，并确定TLO旁系同源物是否已进化以调节不同的功能。都柏林念珠菌tlo1Δ/tlo2Δ突变体无法形成真正的菌丝，在半乳糖肉汤中的倍增时间更长，对氧化应激更敏感，且形成的生物膜水平增加。tlo1Δ/tlo2Δ突变体的转录谱分析显示，在丰富培养基中饥饿反应的表达增加，而在血清中菌丝诱导转录本的表达延迟。染色质免疫沉淀研究表明，Tlo1与许多开放阅读框结合，包括在分析条件下表现出高表达和低表达水平的基因。这些基因在tlo1Δ/tlo2Δ缺失突变体中的表达改变表明Tlo蛋白在转录激活和抑制中发挥作用。用TLO1而不是TLO2对tlo1Δ/tlo2Δ突变体进行互补，恢复了野生型丝状生长，而只有TLO2完全抑制了生物膜生长。用TLO1互补对半乳糖肉汤中的倍增时间也有更大影响。转录谱分析研究支持了TLO1和TLO2恢复野生型功能的不同能力，该研究表明只有TLO1恢复了菌丝特异性基因（UME6、SOD5）和半乳糖利用基因（GAL1和GAL10）的表达，而TLO2恢复了饥饿诱导基因转录的抑制。因此，编码中介体亚基的Tlo/Med2旁系同源物调节念珠菌属中的不同毒力特性，它们的扩增可能解释了白色念珠菌相对于其他念珠菌属物种适应性增强的原因。
