Unmethylated Cyc1 downregulates hyphal specific genes and upregulates hyphal suppressors in Candida albicans. Unmethylated Cyc1 downregulates hyphal specific genes and upregulates hyphal suppressors in Candida albicans

Our genetic screen reveals that deletion of CTM1, which abolishes the lysine trimethylation of cytochrome c (Cyc1), results in inhibition of hyphal morphogenesis in Candida albicans. Similar results are observed in the unmethylatable Cyc1 mutant (cyc1K79A). To elucidate how unmethylated Cyc1 inhibits hyphal growth, we performed RNA-Seq analysis by comparing WT (BWP17), ctm1∆/∆, and cyc1K79A cells grown in yeast and hyphal condition. Consistent with previous published data, many hyphal specific genes (HSGs), such as ALS3, ECE1, HWP1, and UME6, are upregulated while three major hyphal suppressor genes, TUP1, NRG1, and RFG1, are downregulated when WT cells switch from yeast to hyphal growth. Similar changes are observed in ctm1Δ/Δ and cyc1K79A cells upon hyphal induction, even though most mutant cells maintain yeast morphology throughout the induction. Further comparisons reveal that the basal transcriptional levels of HSGs are much lower in ctm1Δ/Δ and cyc1K79A cells than those in WT cells. Upon hyphal induction, the levels of HSGs in ctm1Δ/Δ and cyc1K79A cells increase but still remain lower than their basal levels in WT cells. In contrast, the hyphal suppressor genes (especially NRG1) exhibit much higher basal transcriptional levels in ctm1Δ/Δ and cyc1K79A cells than in WT cells. Their transcriptional levels reduce upon hyphal induction but still remain higher than the basal levels in WT cells. Together, these data suggest that unmethylated Cyc1 inhibits hyphal morphogenesis via transcriptional regulation of HSGs and hyphal suppressor genes. Overall design: Three C. albicans strains, including WT (BWP17), ctm1∆/∆, and cyc1K79A, were grown in YPD at 30°C overnight and subjected to hyphal induction with 10% fetal bovine serine (FBS) and incubation at 37°C for 1-2 h. Three types of samples (overnight yeast cultures, 1 h and 2 h hyphal induction cultures) from each strains were taken for RNA-Seq. We then performed gene expression profiling analysis using data obtained from RNA-Seq of there different strains at three timeppoints.

本研究通过遗传筛选发现，敲除CTM1可消除细胞色素c（cytochrome c, Cyc1）的赖氨酸三甲基化修饰，进而抑制白色念珠菌（Candida albicans）的菌丝形态发生（hyphal morphogenesis）。在无法发生甲基化的Cyc1突变体（cyc1K79A）中，也得到了一致的实验结果。
为阐明未甲基化的Cyc1如何抑制菌丝生长，本研究以酵母态及菌丝态条件下培养的野生型（WT, BWP17）、ctm1Δ/Δ突变株及cyc1K79A突变株为材料开展RNA测序（RNA-Seq）分析。与已发表的前期研究结果一致，当野生型菌株从酵母态转换为菌丝态时，大量菌丝特异性基因（HSGs，如ALS3、ECE1、HWP1及UME6）呈上调表达，而三个核心菌丝抑制基因（TUP1、NRG1及RFG1）则呈下调表达。在菌丝诱导条件下，ctm1Δ/Δ与cyc1K79A突变株也呈现出类似的基因表达变化，尽管多数突变株在诱导全程中始终维持酵母态形态。
进一步的对比分析显示，ctm1Δ/Δ与cyc1K79A突变株中，HSGs的基础转录水平显著低于野生型菌株。经菌丝诱导后，突变株中HSGs的转录水平虽有所上升，但仍低于野生型菌株中的基础转录水平。与之相反，ctm1Δ/Δ与cyc1K79A突变株中，菌丝抑制基因（尤其是NRG1）的基础转录水平显著高于野生型菌株。经菌丝诱导后，此类基因的转录水平虽有所下调，但仍高于野生型菌株中的基础转录水平。
综上，上述实验结果表明，未甲基化的Cyc1可通过调控HSGs与菌丝抑制基因的转录水平，进而抑制白色念珠菌的菌丝形态发生。
实验设计：本研究选取3株白色念珠菌，分别为野生型（WT, BWP17）、ctm1Δ/Δ突变株及cyc1K79A突变株。将上述菌株于30℃的YPD培养基中过夜培养，随后添加10%胎牛血清（FBS）进行菌丝诱导，并置于37℃条件下培养1~2小时。分别收集每株菌株的三类样本：过夜酵母培养物、诱导1小时及2小时的菌丝培养物，用于RNA-Seq测序。本研究基于3株菌株在3个时间点的RNA-Seq测序数据开展基因表达谱分析。