2022 Fiscal Year Research-status Report
Mapping the Madurella mycetomatis pathogen and host responses during and after antifungal treatment to identify prognostic therapeutic markers for mycetoma
| Project/Area Number |
20K08832
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| Research Institution | Institute of Physical and Chemical Research |
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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)
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| Project Period (FY) |
2020-04-01 – 2024-03-31
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| Keywords | Mycology / Eumycetoma / Galleria mellonella / Antifungal / Madurella mycetomatis / Transcriptomics |
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| Outline of Annual Research Achievements |
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.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
All planned tasks in FY2022 successfully completed. Tasks which was impacted by lock-down during FY2020-21 were recovered.Final computation and statistical analysis already started in FY2022.
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| Strategy for Future Research Activity |
The progress we made during FY2023 will enable us to be processed with differential gene expression analysis of host and pathogen genes followed by pathways enrichment analysis to find host and pathogen enriched pathways during treatment with antifungal agents.
For LQ-ssCAGE dataset, we will confirm the reproducibility of the RNA-seq results.
As addition outcome of this project, we will use LQ-ssCAGE to call promoters and enhancers of Galleria mellonella (host) and Madurella mycetomatis (pathogen). This will be an important contribution to the annotation of host and pathogen genomes.
After completing the above analysis, we will start to draft at least two manuscripts. One for the host response to the antifungal treatment and the second manuscript will be an atlas for promoters and enhancers of invertebrate Galleria mellonella.
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