Excavation of biomarker candidates for the diagnosis of Talaromyces marneffei infection via genome-wide prediction and functional annotation of secreted proteins. Excavation of biomarker candidates for the diagnosis of Talaromyces marneffei infection via genome-wide prediction and functional annotation of secreted proteins

Talaromyces marneffei is the third most common infectious pathogen in AIDS patients and leads to the highest death rate in Guangxi, China. The lack of reliable biomarkers is one of the major obstacles in current clinical diagnosis, which largely contributes to this high mortality. Here, we present a study that aimed at identifying diagnostic biomarker candidates through genome-wide prediction and functional annotation of Talaromyces marneffei secreted proteins. A total of 584 secreted proteins then emerged, including 382 classical and 202 non-classical ones. Among them, 87 newly obtained functional annotations in this study. The annotated proteins were further evaluated by combining RNA profiling and homology comparison. Three proteins were ultimately highlighted as biomarker candidates with robust expression and remarkable specificity. The predicted phosphoinositide phospholipase C and the galactomannoprotein were suggested playing an interactive immune game though metabolism of arachidonic acid. And therefore, they hold promises in developing new tools for clinical diagnosis of Talaromyces marneffei, possibly also serve as molecular targets of future therapy. Overall design: Standard strain of T. marneffei ATCC18224 was obtained from American Type Culture Collection (ATCC) and cultured under yeast condition. In brief, fungus was expanded in 37℃ for 14 days, with 200-220 rpm rotation in RPMI 1640 medium plus 10% FBS (Fetal Bovine Serum). Fungus was then precipitated with centrifuge of 14000 g for 10 minutes60. RNA samples were then extracted and sequenced in GeneChem (China). Three independent biological replicates were set in parallel from culture to profiling. Reads were calculated and normalized for each unique transcript.

马尔尼菲篮状菌（Talaromyces marneffei）是艾滋病患者中第三常见的感染性病原体，在中国广西的致死率位居首位。当前临床诊断缺乏可靠的生物标志物，这是导致该高死亡率的主要障碍之一。本研究旨在通过全基因组预测并对马尔尼菲篮状菌的分泌蛋白进行功能注释，以筛选潜在的诊断生物标志物。最终共鉴定出584种分泌蛋白，其中382种为经典分泌蛋白，202种为非经典分泌蛋白。本研究首次为其中87种蛋白提供了功能注释。随后，结合RNA表达谱分析与同源比对，对注释后的蛋白进行了进一步评估。最终筛选出3种表达稳定且特异性显著的候选生物标志物。其中，预测的磷脂酰肌醇磷脂酶C与半乳甘露聚糖蛋白可能通过花生四烯酸代谢参与免疫交互过程，因此有望开发为马尔尼菲篮状菌临床诊断的新型工具，同时也可作为未来治疗的分子靶点。
总体实验设计：从美国典型培养物保藏中心（American Type Culture Collection, ATCC）获取马尔尼菲篮状菌标准菌株ATCC18224，置于酵母样培养条件下培养。具体而言，将真菌接种于添加10%胎牛血清（Fetal Bovine Serum, FBS）的RPMI 1640培养基中，于37℃、200~220 rpm振荡培养14天。随后以14000 g离心10分钟收集菌体。提取RNA样本并交由中国吉凯基因（GeneChem）进行测序。从培养到测序的全过程均设置3次独立生物学重复。对每个独特转录本的测序读段进行计数与标准化处理。