Mapping the Madurella mycetomatis pathogen and host responses during and after antifungal treatment to identify prognostic therapeutic markers for mycetoma

绘制抗真菌治疗期间和之后的足菌肿病原体和宿主反应，以确定足菌肿的预后治疗标志物

• 批准号: 20K08832
• 负责人: アブケセーサ イマド
• 金额: $ 2.75万
• 依托单位: Institute of Physical and Chemical Research
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2020
• 资助国家: 日本
• 起止时间: 2020-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-20K08832/
• 关键词: Mycology; Eumycetoma; Galleria mellonella; Antifungal; Madurella mycetomatis; Transcriptomics; Mycetoma; RNA-Seq; Tropical disease

During FY2022 we completed two major tasks.First, the library preparations and sequencing for transcriptomics analysis was successfully completed.Second, pharmacokinetics of antifungal agents in Galleria mellonella larvae. For the trsanscriptomics analysis, total RNA was extracted from six type of samples at 4 timepoints (6hr,30hr,72hr, and 168hr) in triplicates (4x3 =12) the type of samples are (Healthy G. mellonella larvae,infected G. mellonella larvae with M. mycetomatis, Healthy G. mellonella larvae treated with itraconazole,infected G. mellonella larvae with M. mycetomatis treated with itraconazole (n=12),Healthy G. mellonella larvae treated with ravuconazole and infected Galleria mellonella larvae with M. mycetomatis treated with ravuconazole.Total of 72 RNA-seq libraries were prepared and sequenced. Since we have extra RNA aliquot reaming, we prepared and sequenced LQ-ssCAGE (low quantity single strand CAGE) libraries from the same samples (n=72).Raw sequenced reads mapped against the G. mellonella larvae (host) genome and then against the pathogen M. mycetomatis genome. We obtained a high mapping ratio according to RNA-seq standards.As for the pharmacokinetics(PK) of antifungal agents in G. mellonella larvae,The PK of itraconazole was determined after the administration of a single dose to uninfected G. mellonella larvae. Concentrations of antifungal agents were present during 24hr after injection and above the MIC. The AUC24h for itraconazole (61.9% of human AUC) was lower in G. mellonella larvae than in humans.

在2022财年期间，我们完成了两项主要工作。第一，成功地完成了转录分析的文库准备和测序。第二，抗真菌药物在梅隆格氏杆菌幼虫体内的药代动力学研究。从6小时、30小时、72小时和168小时四个时间点(4x3=12)的6种样本中提取总RNA，样本类型为(健康梅氏杆菌幼虫、感染梅氏支原体的梅氏杆菌幼虫、伊曲康唑处理的健康梅隆幼虫、伊曲康唑处理的梅氏杆菌感染的梅氏杆菌幼虫(n=12)、雷武康唑处理的梅氏微囊虫健康幼虫和雷公藤处理的梅氏杆菌感染的梅氏杆菌幼虫)。由于我们有额外的RNA等量扩增，我们从相同的样本(n=72)中制备并测序了LQ-ssCAGE(Low Quantity Single Strand Cage)文库。对于抗真菌药物在梅氏革兰氏菌幼虫体内的药代动力学(PK)，伊曲康唑在未感染的梅氏革兰氏菌幼虫中单次给药后的药代动力学(PK)测定。抗真菌药物的浓度在注射后24小时内存在，并高于MIC。伊曲康唑在梅氏革兰氏菌幼虫体内的24小时AUC(占人AUC的61.9%)低于人。

项目成果

Erasmus University Medical Center(オランダ)

伊拉斯姆斯大学医学中心（荷兰）