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Suppl. Movies for Scherer et al "Redundant Trojan horse and endothelial-circulatory mechanisms for host-mediated spread of Candida albicans yeast "

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posted on 31.01.2020 by Robert Wheeler, Allison Scherer, Bailey Blair, Jieun Park, Brittany Seman, Joshua Kelley

Suppl. Movies for Scherer et al "Redundant Trojan horse and endothelial-circulatory mechanisms for host-mediated spread of Candida albicans yeast "

Movie S1. As described in Fig. 2A. Tg(mpeg1:GAL4/UAS:nfsb-mCherry)/Tg(mpx:EGFP) larvae were infected with yeast-locked C. albicans and scored for recruitment of phagocytes and dissemination of yeast at 24 hpi. A time-lapse of a larva with recruitment was taken over the course of 7 hours starting at ~32 hpi. Neutrophils and macrophages were observed moving within and away from the infection site carrying yeast.

Movie S2. As described in Fig. 2A, but from an independent experiment. Tg(mpeg1:GAL4/UAS:nfsb-mCherry)/Tg(mpx:EGFP) larvae were infected with yeast-locked C. albicans and scored for recruitment of phagocytes and dissemination of yeast at 24 hpi. A time-lapse of fish with recruitment was taken over the course of 7 hours starting at ~32 hpi. Neutrophils and macrophages were observed moving within and away from the infection site carrying yeast.

Movie S3. As described in Fig. 2C. Tg(mpeg1:GAL4)x(UAS:Kaede) larvae were infected with yeast-locked C. albicans and scored for recruitment of macrophages and dissemination of yeast at 24 hpi. Macrophages near the infection site of larvae with immune recruitment were photo-switched at 24 hpi. A time-lapse movie was taken over the course of 7 hours starting at ~46 hpi. A photo-converted macrophage moves out of the blood stream into tail tissue.

Movie S4. As described in Fig. 2D. Tg(mpeg1:GAL4)x(UAS:Kaede) larvae were infected with yeast-locked C. albicans and scored for recruitment of macrophages and dissemination of yeast at 24 hpi. Macrophages near the infection site of larvae with immune recruitment were photo-switched at 24 hpi. A time-lapse movie taken over the course of 7 hours starting at ~32 hpi. Intracellular yeast growth and apparent NLE events are highlighted.

Movie S5. Tg(mpx:EGFP) larvae were infected with hypofilamentous C. albicans strain cph1∆/∆efg1∆/∆-dTomato in the yolk as described and imaged at 44 hpi by time-lapse microscopy. Neutrophils (green) interacting with yeast (red) as they move down the tail tissue inside a blood vessel.

Movie S6. As described in Fig. 4D. Tg(tnfα:EGFP) larvae with green fluorescence with tnfα expression were given control liposomes and infected with a yeast-locked C. albicans as described. Z series demonstrating typical tnfα expression at the infection site in a wild type larva.

Movie S7. As described in Fig. 4D. Tg(tnfα:EGFP) larvae with green fluorescence with tnfα expression were given clodronate liposomes and infected with a yeast-locked C. albicans as described. Z series demonstrating background tnfα expression in a wild type larva.

Movie S8. As described in Fig. 4E. Rac2-D57N/Tg(tnfα:EGFP) larvae with red fluorescent neutrophils and green fluorescence with tnfα expression were given control liposomes and infected with a yeast-locked C. albicans as described. Z series demonstrating typical tnfα expression at the infection site in a Rac2/D57N larva.

Movie S9. As described in Fig. 4E. Rac2-D57N/Tg(tnfα:EGFP) larvae with red fluorescent neutrophils and green fluorescence with tnfα expression were given clodronate liposomes and infected with a yeast-locked C. albicans as described. Z series demonstrating background tnfα expression in a Rac2/D57N larva.

Movie S10. As described in Fig. 4. Rac2-D57N/Tg(tnfα:EGFP) larvae with red fluorescent neutrophils and green fluorescence with tnfα expression were given control liposomes and infected with a yeast-locked C. albicans as described. Time-lapse movie taken over the course of 7 hours starting at ~32 hpi. Macrophage-like cells turn on tnfa expression as they move closer to the site of infection.

Movie S11. As described in Fig. 4. Rac2-D57N/Tg(tnfα:EGFP) larvae with red fluorescent neutrophils and green fluorescence with tnfα expression were given clodronate liposomes and infected with a yeast-locked C. albicans as described. Time-lapse movie taken over the course of 7 hours starting at ~32 hpi. There is an absence of phagocytes turning on tnfa expression.

Movie S12. As described in Fig. S10. Tg(mpeg:GAL4/UAS:nfsb-mCherry)/Tg(mpx:EGFP) larvae were bathed with 2 μM terfenadine/DMSO vehicle following infection with NRG1OEX-iRFP. Representative movie of infected larvae at ~32 hpi treated with 2 μM terfenadine, which blocks blood flow.

Movie S13. As described in Fig. S10. Tg(mpeg:GAL4/UAS:nfsb-mCherry)/Tg(mpx:EGFP) larvae were bathed in 0.1 mg/ml valproic acid/E3 water vehicle following infection with NRG1OEX-iRFP. Representative movie of infected larvae at ~32 hpi treated with 0.1 mg/ml valproic acid, which blocks blood flow.

Movie S14. As described in Fig. 5G. Tg(fli1:EGFP) larvae, with GFP-expressing endothelial cells, were infected with NRG1OEX-iRFP as previously described. A vehicle-treated Tg(fli1:EGFP) fish with intact blood flow was imaged at ~32 hpi by time-lapse on the confocal.

Movie S15. As described in Fig. 5G. Tg(fli1:EGFP) larvae, with GFP-expressing endothelial cells, were infected with NRG1OEX-iRFP as previously described. A vehicle-treated Tg(fli1:EGFP) fish with intact blood flow was imaged at ~32 hpi by time-lapse on the confocal.

Movie S16. As described in Fig. 7. RAC2-D57N/Tg(tnfα:GFP) larvae were injected with control liposomes and infected with the wild type CAF2-iRFP C. albicans as described for the yeast-locked infections. Fish were imaged by confocal microscopy between 32 and 40 hpi.

Movie S17. As described in Fig. 7. RAC2-D57N/Tg(tnfα:GFP) larvae were injected with clodronate liposomes and infected with the wild type CAF2-iRFP C. albicans as described for the yeast-locked infections. Fish were imaged by confocal microscopy between 32 and 40 hpi.

Movie S18. As described in Fig. 7. Tg(mpeg:GAL4/UAS:nfsb-mCherry)/Tg(mpx:EGFP) larvae were infected with the wild type Caf2-iRFP C. albicans as described for the yeast-locked infections. Fish were imaged by confocal microscopy between 32 and 40 hpi.

Funding

Intravital study of innate immunity to mucosal candidiasis

National Institute of Allergy and Infectious Diseases

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Genetics and visualization of innate host response to C. albicans infection in vi

National Institute of Allergy and Infectious Diseases

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Hatch Project # ME0-21821, USDA

Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Disease

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