Gene expression response to the antifungal compound sampangine in C. albicans

Sampangine, a plant-derived alkaloid found in the Annonaceae family, exhibits strong inhibitory activity against the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus. In the present study, transcriptional profiling experiments coupled with the analysis of mutants were performed in an effort to elucidate its mechanism of action. Using Saccharomyces cerevisiae as a model organism, we show that sampangine produces a transcriptional response indicative of hypoxia, altering the expression of genes known to respond to low oxygen conditions. Several additional lines of evidence obtained suggest that these responses could involve effects on heme. First, the hem1 deletion mutant lacking the first enzyme in the heme biosynthetic pathway showed increased sensitivity to sampangine, and exogenously supplied hemin partially rescued the inhibitory activity of sampangine in wild-type cells. In addition, heterozygous mutants with deletions in genes involved in five out of eight steps in the heme biosynthetic pathway showed increased susceptibility to sampangine. Furthermore, spectral analysis of pyridine extracts indicated significant accumulation of free porphyrins in sampangine-treated cells. Transcriptional profiling experiments were also performed in C. albicans to investigate the response of a pathogenic fungal species to sampangine. Taking into account the known differences in the physiological responses of C. albicans and S. cerevisiae to low oxygen, significant correlations were observed between the two transcription profiles suggestive of heme-related defects. Our results indicate that the antifungal activity of the plant alkaloid sampangine is due, at least in part, to perturbations in the biosynthesis or metabolism of heme. Keywords: antifungal compound, transcriptional profiling, C. albicans C. albicans SC5314 cells at OD 0.2 were treated with either sampangine (SMP) at IC50 concentration (6.13 ug/ml), or solvent (0.25% DMSO), allowed to grow to OD 0.5, then harvested and frozen. Three biological replicate samples were analyzed for each treatment.

番荔枝科（Annonaceae）植物来源的生物碱桑加明（sampangine），对机会致病性真菌白色念珠菌（Candida albicans）、新型隐球菌（Cryptococcus neoformans）及烟曲霉（Aspergillus fumigatus）具有强效抑制活性。
本研究通过转录谱分析（transcriptional profiling）结合突变体分析，旨在阐明其作用机制。
本研究以酿酒酵母（Saccharomyces cerevisiae）为模式生物，结果显示桑加明可引发表征为缺氧的转录应答，改变已知响应低氧条件的基因的表达水平。
本研究获得的多项额外实验证据表明，此类应答可能涉及对血红素（heme）的作用。
其一，缺失血红素生物合成通路首个酶编码基因的hem1缺失突变体，对桑加明的敏感性显著升高；外源添加的氯化血红素（hemin）可部分逆转野生型细胞中桑加明的抑制活性。
此外，血红素生物合成通路8个步骤中5个步骤相关基因缺失的杂合突变体，对桑加明的易感性显著增强。
进一步而言，吡啶提取物的光谱分析显示，经桑加明处理的细胞中游离卟啉发生显著积累。
本研究亦在白色念珠菌（Candida albicans）中开展转录谱分析实验，以探究该致病性真菌对桑加明的应答反应。
结合已知的白色念珠菌与酿酒酵母对低氧的生理应答差异，二者的转录谱之间存在显著相关性，提示存在血红素相关的功能缺陷。
本研究结果表明，植物生物碱桑加明的抗真菌活性至少部分源于对血红素生物合成或代谢过程的扰动。
关键词：抗真菌化合物、转录谱分析（transcriptional profiling）、白色念珠菌（Candida albicans）
将光密度（OD）为0.2的白色念珠菌SC5314（Candida albicans SC5314）细胞分别用IC₅₀浓度（6.13 μg/ml）的桑加明（SMP）或溶剂（0.25%二甲基亚砜（DMSO））处理，待其生长至光密度（OD）为0.5时收集细胞并冷冻保存。每组处理均设置3个生物学重复样本进行分析。