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Calcium sequestration by fungal melanin inhibits calcium–calmodulin signalling to prevent LC3-associated phagocytosis

Nature Microbiology volume 3, pages 791–803 (2018)Cite this article

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Abstract

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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Fig. 1: Ca2+–CaM signalling regulates A. fumigatus LAP.
Fig. 2: CaM phagosome recruitment is dependent on endoplasmic reticulum Ca2+ sources.
Fig. 3: Ca2+ sequestration inside the phagosome abrogates CaM recruitment and inhibits LAP.
Fig. 4: A. fumigatus cell wall melanin inhibits peri-phagosomal Ca2+ accumulation to the phagosome.
Fig. 5: Ca2+ sequestration by melanin inside the phagosome blocks CaM dependent activation of LAP.
Fig. 6: CALM1 gene polymorphism CC is associated with increased risk for invasive aspergillosis in a high-risk group of haematopoietic stem-cell recipients.

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Acknowledgements

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.

Author information

Author notes

  1. These authors contributed equally: Helena Ferreira, Agostinho Carvalho.

Authors and Affiliations

  1. Department of Medicine, University of Crete, Heraklion, Crete, Greece

    Irene Kyrmizi, Tonia Akoumianaki, Kostas Stylianou, George Samonis & Georgios Chamilos

  2. Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion, Crete, Greece

    Irene Kyrmizi & Georgios Chamilos

  3. 3B’s Research Group - Biomaterials, Biodegradables and Biomimetics, Guimarães, Portugal

    Helena Ferreira & Nuno M. Neves

  4. ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal

    Helena Ferreira, Agostinho Carvalho, Cristina Cunha & Nuno M. Neves

  5. Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal

    Agostinho Carvalho & Cristina Cunha

  6. Department of Chemistry, University of Cincinnati/Agilent Technologies Metallomics Center of the Americas, University of Cincinnati, Cincinnati, OH, USA

    Julio Alberto Landero Figueroa

  7. Department of Chemistry, University of Crete, Heraklion, Crete, Greece

    Pavlos Zarmpas & Nikolaos Mihalopoulos

  8. Division of Infectious Diseases, College of Medicine, University of Cincinnati, Cincinnati, OH, USA

    George S. Deepe Jr

  9. Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Lisbon, Portugal

    João F. Lacerda

  10. Serviço de Hematologia e Transplantação de Medula, Hospital de Santa Maria, Lisbon, Portugal

    João F. Lacerda

  11. Serviço de Transplantação de Medula Óssea (STMO), Instituto Português de Oncologia do Porto, Porto, Portugal

    António Campos Jr

  12. Department of Infectious Diseases, The University of Texas, MD Anderson Cancer Center, Austin, TX, USA

    Dimitrios P. Kontoyiannis

  13. Molecular Microbiology Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, USA

    Kyung J. Kwon-Chung

  14. INSERM-U1149, CNRS-ERL8252, Centre de Recherche sur l’Inflammation, Paris, France

    Jamel El-Benna

  15. Université Paris Diderot, Sorbonne Paris Cité, Laboratoire d’Excellence Inflamex, DHU FIRE, Faculté de Médecine, Site Xavier Bichat, Paris, France

    Jamel El-Benna

  16. Unité des Aspergillus, Institut Pasteur, Paris, France

    Isabel Valsecchi, Anne Beauvais & Jean-Paul Latge

  17. Department of Molecular and Applied Microbiology, Leibniz-Institute for Natural Product Research and Infection Biology (HKI) and Friedrich Schiller University, Jena, Germany

    Axel A. Brakhage

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Contributions

I.K. designed, performed and analysed most of the experiments in this study, established protocols for Ca2+ imaging and phagosome processing and participated in the writing of the manuscript. A.C. and C.C. performed experiments on analysis of CaLM1 SNPs in clinical samples. J.F.L. and A.C.Jr. provided patient samples and clinical information. L.F.J.A. performed ICP-MS measurements of Ca2+ content of phagosomes; P.Z. and N.P. performed Ion Chromatography Analysis of Ca2+ binding affinity of A. fumigatus conidia; T.A., G.S., D.P.K., G.S.D. Jr and A.B. analysed data and provided suggestions throughout the study. K.S. performed electron microscopy studies. J.E. and K.J.K. provided reagents and analysed data. H.F. and N.M.N. developed, produced and characterized the DTPA-PEI-coated conidia; A.B. generated A. fumigatus purified melanin; I.V and J.P.L. generated A. fumigatus mutants and provided reagents, analysed data and provided discussions and suggestions throughout the study. G.C. conceived and supervised the study, performed experiments, was involved in the design and evaluation of all experiments, and wrote the manuscript along with comments from co-authors.

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Correspondence to Georgios Chamilos.

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Supplementary information

Supplementary Information

Supplementary Figures 1–19, Supplementary Tables 1 and 2

Reporting Summary

Supplementary Video 1

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

Supplementary Video 2

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

Supplementary Video 3

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

Supplementary Video 4

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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