LC3-associated phagocytosis (LAP) is a non-canonical autophagy pathway regulated by Rubicon, with an emerging role in immune homeostasis and antifungal host defence. Aspergillus cell wall melanin protects conidia (spores) from killing by phagocytes and promotes pathogenicity through blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent activation of LAP. However, the signalling regulating LAP upstream of Rubicon and the mechanism of melanin-induced inhibition of this pathway remain incompletely understood. Herein, we identify a Ca2+ signalling pathway that depends on intracellular Ca2+ sources from endoplasmic reticulum, endoplasmic reticulum–phagosome communication, Ca2+ release from phagosome lumen and calmodulin (CaM) recruitment, as a master regulator of Rubicon, the phagocyte NADPH oxidase NOX2 and other molecular components of LAP. Furthermore, we provide genetic evidence for the physiological importance of Ca2+–CaM signalling in aspergillosis. Finally, we demonstrate that Ca2+ sequestration by Aspergillus melanin inside the phagosome abrogates activation of Ca2+–CaM signalling to inhibit LAP. These findings reveal the important role of Ca2+–CaM signalling in antifungal immunity and identify an immunological function of Ca2+ binding by melanin pigments with broad physiological implications beyond fungal disease pathogenesis.
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The authors would like to thank N. Tavernarakis for helpful suggestions and G. Garinis and A. Eliopoulos for providing antibodies and reagents. The authors are grateful to G. Chalepakis, E. Papadogiorgaki and other members of the electron microscopy facility at UOC.
I.K.’s work is supported by the Onassis Foundation under the ‘Special Grant and Support Program for Scholars’ Association Members’ (Grant no. R ZM 003-1/2016-2017); G.C. was supported by grants from the Greek State Scholarship Foundation (I.K.Y.), the Hellenic General Secretariat for Research and Technology-Excellence program (ARISTEIA) and a Research Grant from Institut Mérieux; J.P.L. was supported by European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement 260338 ALLFUN and ANR-10-BLAN-1309 HYDROPHOBIN, and the Association Vaincre La Mucoviscidose (RF20140501052/1/1/141); H.F. and N.M.N. were supported by the project FROnTHERA (NORTE-01-0145-FEDER-000023), supported by Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e Tecnologia (FCT) project SPARTAN (PTDC/CTM-BIO/4388/2014), funded through the PIDDAC Program. A.C. and C.C. were supported by NORTE 2020, under the Portugal 2020 Partnership Agreement, through the ERDF (NORTE-01-0145-FEDER-000013), and by FCT (IF/00735/2014 and SFRH/BPD/96176/2013). G.S.D. and J.L.F. were supported by NIH grant AI-106269. K.J.K-C is supported by the Division of Intramural Research (DIR), NIAID, NIH.
The authors declare no competing interests.
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Supplementary Figures 1–19, Supplementary Tables 1 and 2
Primary human monocytes loaded with Fluo4 and then infected with conidia of the melanin-competent parental strain (Ku80) of A. fumigatus at a MOI of 10:1. Internalization of conidia was followed at 5 s intervals. A representative frame of this video is shown in Fig. 4b (top panel). A video of 1 of 12 experiments performed independently with similar results is shown
Primary human monocytes loaded with Fluo4 and then infected with conidia of melanin-competent ΔrodA mutant of A. fumigatus at a MOI of 10:1. Internalization of conidia was followed at 5 s intervals. A representative frame of this video is shown in Fig. 4b (middle panel). Video of 1 of 5 experiments performed independently with similar results is shown
Primary human monocytes loaded with Fluo4 and then infected with conidia of melanin-deficient (albino) ΔrodA/pksP mutant of A. fumigatus at a MOI of 10:1. Internalization of conidia was followed at 5 s intervals. A representative frame of this video is shown in Fig. 4b (lower panel). Video of 1 of 12 experiments performed independently with similar results is shown
Primary human monocytes loaded with Fluo4 and then infected with conidia of melanin-deficient (albino) 24 ΔrodA/pksP mutant of A. fumigatus. Internalization of conidia was followed at 5 s intervals. A representative frame from a cell with peri-phagosomal Ca2+ ring in the middle of the optical field of this video is shown in Fig. 4c. Video of 1 of 12 experiments performed independently with similar results is shown
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Kyrmizi, I., Ferreira, H., Carvalho, A. et al. Calcium sequestration by fungal melanin inhibits calcium–calmodulin signalling to prevent LC3-associated phagocytosis. Nat Microbiol 3, 791–803 (2018). https://doi.org/10.1038/s41564-018-0167-x
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