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

2021 Fiscal Year Research-status Report

• Project/Area Number: 20K08832
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: アブケセーサ イマド 国立研究開発法人理化学研究所, 生命医科学研究センター, 上級研究員 (10749906)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Keywords: Mycology / Galleria mellonella / Mycetoma / Eumycetoma / Antifungal / Madurella mycetomatis / RNA-Seq

Outline of Annual Research Achievements

1-Madurella mycetomatis genome strain was used to infect Galleria mellonella larvae. The larvae were infected with a final concentration of 4mg wet weight fungus per larvae.2-Larvae were treated at 4, 24 and 48 hours after infection, with itraconazole 400 mg/kg and ravuconazole 300 mg/kg. These concentrations were chosen as they resemble the concentrations used in human for itraconazole.3-We determined the toxicity of these drugs for the larvae. For that we treated uninfected larvae and monitor their survival for 5 days. At this stage no toxicity was noted in thedrug concentrations tested.
4-Larvae were infected with M. mycetomatis via the last left proleg and treated with [400 mg/kg] itraconazole or [300 mg/kg] ravuconazole for three days.5-Survival of the larvae was monitored for a duration of 10 days after infection.6-Histology slides have been prepared, stained with HE& Grocott, for grain counts analysis.7-RNA isolation was performed according the previously described methodology. In short, Larval content was harvested and frozen using liquid nitrogen, followed by mechanical crushing using a pestle and mortar. The crushed powder was collected and used for RNA extracted, using the TRIZOL Chloramphenicol method.8-RNA QC was performed using the Nanodrop and measured all RNA standard QC values. Samples were stored at -80oC.9-In total we have prepared 72 samples for RNA-Seq library preparation.For each sample type we have four timepoints and in each timepoint we have triplicate therefore. All samples passed RNA QC check.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

We recovered from the delay for some tasks in FY2020 (due to the lock-dwon and pandemic)

Strategy for Future Research Activity

1- Histology slides have been prepared, stained with HE& Grocott, for grain counts analysis. Grain counts are ongoing according previous established methodology [PMID: 29698504].
2- Determination of the pharmacokinetics(PK) will be performed according previously methodology at time points 5 min, 30 min, 1h, 2h, 4h, 6h and 24h [PMID: 28992315]. Currently Hemolymph of larvae, treated with the respective concentrations, is stored for further processing. This will be continued in the near future.
3- RNA-Seq library preparation
4- Sequencing of RNA library

Causes of Carryover

Due to the impact of the COVID-19 lockdown, we asked to Incurring FY2021 budget to FY2022. The Incurring will be used for RNA-Seq library preparation, Library QC and RNA sequencing as planned.