The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes

Mohamed I Abdelwahab Hassan; Monique Keller; Michael Hillger; Ulrike Binder; Stefanie Reuter; Kristina Herold; Anusha Telagathoti; Hans-Martin Dahse; Saiedeh Wicht; Nora Trinks; Sandor Nietzsche; Tanja Deckert-Gaudig; Volker Deckert; Ralf Mrowka; Ulrich Terpitz; Hans Peter Saluz; Kerstin Voigt

doi:10.1016/j.csbj.2021.01.023

The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes

Comput Struct Biotechnol J. 2021 Jan 20:19:880-896. doi: 10.1016/j.csbj.2021.01.023. eCollection 2021.

Authors

Mohamed I Abdelwahab Hassan^{1

2

3}, Monique Keller², Michael Hillger², Ulrike Binder⁴, Stefanie Reuter^{5

6}, Kristina Herold⁵, Anusha Telagathoti¹, Hans-Martin Dahse¹, Saiedeh Wicht⁷, Nora Trinks⁷, Sandor Nietzsche⁸, Tanja Deckert-Gaudig⁹, Volker Deckert^{9

10

11}, Ralf Mrowka^{5

6}, Ulrich Terpitz⁷, Hans Peter Saluz¹, Kerstin Voigt^{1

2}

Affiliations

¹ Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute (HKI), Jena, Germany.
² Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany.
³ Pests & Plant Protection Department, National Research Centre, 33rd El Buhouth St. (Postal code: 12622) Dokki, Giza, Egypt.
⁴ Department of Hygiene, Microbiology and Public Health, Institute of Hygiene and Medical Microbiology, Medical University Innsbruck, Schöpfstrasse 41/2, 6020 Innsbruck, Tirol, Austria.
⁵ Experimental Nephrology Group, KIM III, Universitätsklinikum Jena, Jena, Germany.
⁶ ThIMEDOP-Thüringer Innovationszentrum für Medizintechnik-Lösungen, Universitätsklinikum Jena, Jena, Germany.
⁷ Department of Biotechnology and Biophysics, Julius Maximilian University of Würzburg, Biocenter - Am Hubland, Würzburg, Germany.
⁸ Elektronenmikroskopisches Zentrum, Universitätsklinikum Jena, Jena, Germany.
⁹ Leibniz Institute of Photonic Technology (IPHT), Albert-Einstein-Str. 9, 07745 Jena, Germany.
¹⁰ Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller University, Helmholtzweg 4, 07743 Jena, Germany.
¹¹ Institute of Quantum Science and Engineering, Texas A&M University, College Station, TX 77843-4242, USA.

Abstract

Fungal infections caused by the ancient lineage Mucorales are emerging and increasingly reported in humans. Comprehensive surveys on promising attributes from a multitude of possible virulence factors are limited and so far, focused on Mucor and Rhizopus. This study addresses a systematic approach to monitor phagocytosis after physical and enzymatic modification of the outer spore wall of Lichtheimia corymbifera, one of the major causative agents of mucormycosis. Episporic modifications were performed and their consequences on phagocytosis, intracellular survival and virulence by murine alveolar macrophages and in an invertebrate infection model were elucidated. While depletion of lipids did not affect the phagocytosis of both strains, delipidation led to attenuation of LCA strain but appears to be dispensable for infection with LCV strain in the settings used in this study. Combined glucano-proteolytic treatment was necessary to achieve a significant decrease of virulence of the LCV strain in Galleria mellonella during maintenance of the full potential for spore germination as shown by a novel automated germination assay. Proteolytic and glucanolytic treatments largely increased phagocytosis compared to alive resting and swollen spores. Whilst resting spores barely (1-2%) fuse to lysosomes after invagination in to phagosomes, spore trypsinization led to a 10-fold increase of phagolysosomal fusion as measured by intracellular acidification. This is the first report of a polyphasic measurement of the consequences of episporic modification of a mucormycotic pathogen in spore germination, spore surface ultrastructure, phagocytosis, stimulation of Toll-like receptors (TLRs), phagolysosomal fusion and intracellular acidification, apoptosis, generation of reactive oxygen species (ROS) and virulence.

Keywords: AFM, Atomic Force Microscopy; Atomic Force Microscopy (AFM); CD14, Cluster of differentiation 14; CFW, Calcofluor white; Galleria mellonella; HEK, human embryonic kidney; HSI, Hyperspectral imaging; Hyperspectral imaging (HIS); IPS, Insect physiological saline; Intracellular survival; LCA, Lichtheimia corymbifera attenuated; LCV, Lichtheimia corymbifera virulent; MD-2, Myeloid Differentiation factor 2; MH-S, Murine alveolar macrophages; MM6, Acute monocytic leukemia derived human monocyte Mono-Mac-6; Monocytes; NF-κB, Nuclear factor 'kappa-light-chain-enhancer' of activated B-cells; PBS, Phosphate buffer saline solution; PI, Phagocytosis index; ROS, Reactive oxygen species; TEM, Transmission Electron Microscopy; TLRs, Toll like receptors; Transmission Electron Microscopy (TEM).