Extended Data Fig. 1. A heat stable and hyphae-associated Mucorales extract damages mammalian host cells in vitro.

(a) R. delemar caused time dependent alveolar epithelial cell damage (n=9 wells/time point, pooled from three independent experiments). Data are median ± interquartile range. (b) Heat-killed R. delemar hyphae showed ~50% damage to mammalian cells compared to ~100% damage caused by living hyphae (n=6 wells/group, pooled from three independent experiments). Data are median ± interquartile range. Statistical analysis was performed by using Mann-Whitney non-parametric (two-tailed) test comparing live vs killed hyphae. (c) Extracts from comparable wet weight of R. delemar hyphae/spores, or hyphae, but not spores, damaged alveolar epithelial cells (n=6 wells/group, pooled from three independent experiments). Data are median ± interquartile range. Statistical analysis was performed by using Mann-Whitney non-parametric (two-tailed) test comparing spores vs spore/hyphae or hyphae. (d) Disrupted pellet from Mucorales germlings containing the cell-associated fraction was compared to live or heat-killed cells in causing injury to HUVECs (n= 3 wells/group, pooled from three independent experiments). Data are median ± interquartile range. (e) Fungal hyphae from representative clinical Mucorales isolates ground in liquid nitrogen and extracted with mammalian cell culture caused significant A549 alveolar epithelial cell damage (n= 3 wells/Mucorales, pooled from three independent experiments). Data are median ± interquartile range. (f) IgG anti-R. delemar toxin but not normal rabbit IgG (50 μg/ml) blocked host cell damage caused by heat-killed hyphae from different Mucorales (n=8 or 9 replicates/treatment/Mucorales, pooled from three independent experiments). Data presented as median ± interquartile range. Statistical analysis was performed by Mann-Whitney non-parametric (two-tailed) test comparing IgG anti-toxin vs. without IgG or normal rabbit IgG.