Bifunctional compounds do not enhance phagocytosis of conidia by

wild-type zebrafish neutrophils.

A) Diagram of a 72 hpf zebrafish larva indicating the site of inoculation and the site of

analysis (i). (ii) Collapsed z-stack (maximum intensity) representative image showing

conidia (blue, Hoechst) and neutrophils (green, GFP) in the caudal venous plexus of

Tg(mpx:GFP/mpeg1:mCherry) embryos injected with spi1-MO at the one-cell stage

and infected with A. fumigatus conidia at 72 hpi. Scale: 100 μm. iii) highermagnification

of neutrophils and conidia indicating extracellular conidia (open yellow

arrowhead) and examples of phagocytosis by neutrophils (filled yellow arrowheads).

Scale: 20 μm.

B) Graph shows neutrophil (N) and macrophage (M) counts in each caudal venous plexus

field of view (FOV) for spi1-MO morphant larvae injected with treated and control

conidia.

C) Graph shows conidia counts per FOV for each treatment group.

D) Graph shows the percent of neutrophils containing conidia at 2 hpi for each treatment

group.

E) Graph shows the number of phagocytosed conidia per FOV at 2 hpi for each treatment

group. Each point represents an infected larva.

Treatment with bifunctional compounds does not enhance the

neutrophil-dependent suppression of fungal hyphae in wild-type zebrafish.

A) Representative images of spi1 (i) and spi1/csf3r (ii) morpholino-injected zebrafish

larvae infected with A. fumigatus at 1 dpi. Different A. fumigatus growth forms are

indicated by open yellow arrowheads.

B) Graph showing the proportion of surviving infected embryos with germinating conidia

or fully-developed hyphae at 1 dpi. Error bars: Mean + SEM. N = 10 larva per group

per experiment, data collated from ≥ 3 experiments

Bifunctional compounds enhance phagocytosis of A. fumigatus conidia by zebrafish neutrophils expressing human FPR1. (A) (i) Diagram of experimental approach: Calcofluor-stained A. fumigatus conidia (blue) are co-delivered with treatments into the circulation via the Duct of Cuvier at 3 days post-fertilization. Imaging is performed at 2 h post-infection at a distal site, the caudal venous plexus, which is rich in leukocytes. (ii) Example image of 3 dpf irf8-MO treated Tg(mpx:EGFP) larva (GFP-labeled neutrophils) with mosaic expression of human FPR1 (traced by mCherry, red fluorescence), 2 h following delivery of calcofluor-labeled (blue fluorescent) A. fumigatus conidia. A GFP/mCherry co-labeled leukocyte containing phagocytosed conidia is indicated in magnified panel (open white arrowhead). Off-target expression of the transgene was also observed in tissues including the somites (full white arrowhead). (B) Treatment with C-001 and C-016 resulted in significantly increased phagocytosis of conidia by GFP/mCherry+ (human FPR1-expressing) neutrophils compared to GFP(only) wild-type cells. Each point represents an infected larva. N ≥ 40 larva scored per condition. Data collated from multiple experiments. Statistics: two-tailed T-test. *p ≤ 0.05, ***p ≤ 0.001.