2018 Fiscal Year Research-status Report

Identification of immunomodulation molecules in Mycobacterial ESX secretory systems

Research Project

Project/Area Number	18K15161
Research Institution	公益財団法人結核予防会結核研究所
Principal Investigator	郭姿君 (GuoTzーChun) 公益財団法人結核予防会結核研究所, 生体防御部病理科, 研究員 (00813065)
Project Period (FY)	2018-04-01 – 2020-03-31
Keywords	Mycobacteria / Immunomodulation / Dual RNAseq
Outline of Annual Research Achievements	To elucidate the functional role of ESX-5 secreted PE/PPE proteins, two BCG deletion mutants, △ppe26 and △ppe27, were generated by mutagenesis. Macrophage cells were subjected to infection with wild-type and two mutated BCG variants. Dual RNAseq was performed to unveil the altered gene expression both in the bacteria and infected host cells. Despite small fraction of bacterial RNAs in the infected cells, the RNAseq result showed good coverage of the bacterial genome, suggesting the approach of analyzing gene expression in the mycobacterial infected cell line is successfully established. Heatmap analysis indicates gene expression patterns between three BCG variants were distinguishable. A highly upregulated gene involved in the process of glycolysis in both deletion mutants is identified.
Current Status of Research Progress	Current Status of Research Progress 2: Research has progressed on the whole more than it was originally planned. Reason The plan of measuring cytokine release by Bio-Plex system was a bit delayed due to high cost of analyzing small number of samples per plate. Biological replicates will be performed and more supernatant derived from infected samples will be collected for Bio-Plex analysis all together to reduce the cost per sample.
Strategy for Future Research Activity	In the second year, the effort will be made to the NGS data analysis to identify the key molecules that play in immunomodulation during host-bacteira interaction. The RNAseq data will be validated by both real-time PCR and cytokine release assay using Bio-Plex system. Results will be summarized and manuscript will be prepared for publication in the second half part of the year.
Causes of Carryover	Additional NGS library preparation kits/ sequencing reagents/ cytokine release assay kits will be purchased in the second fiscal year.