Fig. 1: Experimental workflow, burden of infection on G. mellonella larvae, and quality of transcriptomics data.

A Schematic illustration of the time course experiment design and transcriptomic assays used for high-throughput RNA profiling. Total RNA was extracted at five time points (three biological replicates each at each time point). At point 0 h, RNA was extracted from healthy G. mellonella larvae (host) and cultured M. mycetomatis strain (mm55) (pathogen) to serve as non-infected controls. After the inoculation of G. mellonella larvae with M. mycetomatis, RNA was extracted at 4, 24, 72, and 168 h post-infection (Left panel). Measure of the growth of M. mycetomatis cultured pathogen on YANAAI agar and YNAAI agar containing 500 M of the iron chelator 2′2-bipiridyl to study the impact of the FeCl₂, FeCl₃, apo- and holo-transferrin, and apo- and holoferritin on the growth (Middle panel). Impact of iron on the infection in the model. Grain development was monitored for 10 days, and different measures were performed. Total RNA was extracted at time 0, 4, 24, and 72 h, and RNA-Seq performed (Right panel). B To evaluate the burden of infection in the host, the melanisation of the hemolymph, the number of grains, and the grains size were measured and plotted in violin plots for each time point. In each of the violins, the box indicates the interquartile range (first quartile(Q1), third quartile(Q3), and the median value). The highest values are noted at t = 72 h (the maximum burden of infection). Non-infected larvae were included as controls. The data shown is an example based on observations from three evaluated biological replicates. The difference in the total number of grains or the total grain size observed on the respective time points was determined using the Mann–Whitney U test in GraphPad Prism 8. A p value > 0.05 was deemed significant (Methods). C Grain formation in the host was visualized 40 times magnified using H&E staining and light microscopy. Yellow arrows point towards the grain inside the capsule. Based on the characteristics of the grain development observed at each time point, the grains were defined as early, pre-mature, mature, and late grains. In the early grains, the cement material is not formed, and hemocytes are present between hyphae. In the pre-mature grains, hemolymph is forming, but still, individual hemocytes can be noted within the forming cement material. In the mature grain, the cement material is completely formed, and a capsule surrounds the grain. In the late grain, you see the capsule disappearing and an influx of hemocytes towards the grain. D t-SNE clustering: Host and pathogen RNA-Seq libraries mapped to G. mellonella genome assembly ASM364042v2. The replicates at each time point are shown as colored dots. The t-SNE plot shows the clustering of the RNA-Seq libraries mapped to the host genome. E Pathogen transcripts detected in host RNA-Seq after infection (three biological replicates were used at each time point, total n = 15). The line plot shows the number of pathogen reads detected in the host RNA-Seq data after infection, the largest number of the pathogen transcripts detected at time 72 h (mature grain) (lines are colored by time point). Data in the line plot are represented as mapped sequenced reads ± SEM. F RNA-Seq vs. LQ-ssCAGE mapped sequence reads correlation. The correlation matrix shows the Spearman correlation of the RNA-Seq and LQ-ssCAGE reads between replicates. Source data are provided as a Source Data file (B–E). Source data are available in the Gene Expression Omnibus (GEO) under accession numbers GSE213329 and GSE213332(F).