The early transcriptional and post-transcriptional responses to fluconazole in sensitive and resistant Candida albicans (5P-Seq)

Candida albicans is a leading cause of fungal infections in immunocompromised patients. Management of candidemia relies on a few antifungal agents, with fluconazole being first line therapy. The emergence of fluconazole-resistant strains highlights the pressing need to improve our molecular understanding of the drug response mechanisms. By sequencing the 5'P mRNA degradation intermediates, we show that co-translational mRNA decay is common in C. albicans and characterize how in vivo 5´-3´ exonuclease degradation trails the last translating ribosome. Thus, the study of the 5'P mRNA degradome (5PSeq) offers a simple and affordable way to measure ribosome dynamics and identify codon specific ribosome stalls in response to drugs and amino acid deprivation. Building upon this, we combine RNA-Seq and 5PSeq to study the early response of sensitive and resistant C. albicans isolates to fluconazole. Our results highlight that transcriptional responses, rather than changes in ribosome dynamics, are the main driver of Candida resistance to fluconazole. Overall design: 5' phosphate sequencing (5Pseq) of Candida albicans SC5314, PLC124, MAY7 and MAY478 treated with fluconazole (1 µg/ml, 1xMIC) for 30 min versus control (2% DMSO). Candida SC5314 treated with cycloheximide (0.067 mg/ml) for 15 min, and proline-arginine-ornithine depletion experiments with corresponding controls.

白色念珠菌（Candida albicans）是免疫受损患者真菌感染的主要致病菌之一。念珠菌血症（candidemia）的临床治疗依赖少数抗真菌药物，其中氟康唑（fluconazole）为一线疗法。氟康唑耐药菌株的出现，凸显了深入解析药物应答分子机制的迫切需求。通过对5'P mRNA降解中间产物进行测序，本研究证实共翻译mRNA降解在白色念珠菌中普遍存在，并阐明了体内5'-3'核酸外切酶（5´-3´ exonuclease）的降解过程尾随最后一个正在翻译的核糖体的具体机制。因此，针对5'P mRNA降解组（5'P mRNA degradome，即5PSeq）的研究，可为检测核糖体动态变化、鉴定药物及氨基酸剥夺应答下的密码子特异性核糖体停滞位点提供一种简便且经济的手段。基于此，本研究结合RNA测序（RNA-Seq）与5PSeq，探究敏感与耐药白色念珠菌菌株对氟康唑的早期应答反应。研究结果显示，转录应答而非核糖体动态变化，是白色念珠菌对氟康唑产生耐药性的核心驱动因素。总体实验设计：对经氟康唑（1 μg/ml，1倍最低抑菌浓度（1xMIC））处理30分钟的白色念珠菌SC5314、PLC124、MAY7及MAY478菌株，以2%二甲基亚砜（DMSO）作为空白对照，开展5'磷酸测序（5Pseq）；同时设置经环己酰亚胺（cycloheximide，0.067 mg/ml）处理15分钟的白色念珠菌SC5314组，以及脯氨酸-精氨酸-鸟氨酸缺失实验及其对应的对照组。