P343 Novel hydrophobic binding surface proteins are instrumental for phagocytosis of Lichtheimia corymbifera by macrophages

POSTER SESSION 3, SEPTEMBER 23, 2022, 12:30 PM - 1:30 PM: OBJECTIVES: Perform proteomic analysis of the spore surface alongside secretome analysis of two strains of Lichtheimia corymbifera. Evaluate interaction, recognition, and phagocytosis of Lichtheimia spores by murine alveolar macrophages. METHODS: Two strains of L. corymbifera (JMRC: FSU: 09682 and JMRC: FSU: 10164) were used in this study. For phagocytosis assay, the spores were labeled with 0.1 mg/ml Fluorescein isothiocyanate (FITC) (Sigma Aldrich Chemie) in 0.1 M Na2CO3 for 30 min 146 at 30°C. Murine alveolar macrophages MH-S (ATCC:CRL-2019) were cultivated in RPMI-1640 (Sigma, 30-2001) supplemented. Identification of the surface proteins of L. corymbifera was carried out as described before with minor modification.(1) The supernatants were stored at −80°C for liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Secreted proteins of L. corymbifera were determined as described in a previous study with minor modification(2). The raw files generated by the LC-MS/MS were further processed by the software Proteome Discoverer v1.4.0.288 (Thermo). Tandem mass spectra were searched against the NCBI L. corymbifera protein database.(3) Approximately 10 000 MH-S cells were cultivated onto 96-well microplates (NUNC 163320) in 100 μL RPMI-1640 medium (Sigma, 30-2001). Images were acquired by the Zeiss Axio Observer 7 Spinning Disk Confocal Microscope (ZEISS, Jena, Germany) and processed with ZEN 2.1 Software (ZEISS) by 63x objective lens. RESULTS: Abundant surface proteins were found which serve as Candidates for secretome analysis. A total of 113 proteins were identified. Thirty proteins were confirmed to be on the spore surface based on the presence of signal peptides(3). The following proteins were predominantly identified: Spore coat protein (CotH), hydrophobic surface binding protein A (HsbA), aconitase, ricin-like lectin, two transition elongation factors, multi-copper oxidase, heat shock protein 70 family (Hsp70), malate synthase, putative allergen Candidates, etc. These proteins were heterologsly overexpressed in yeast. Successful overexpression was confirmed by LC-MS/MS. The yeast mutants were subjected to phagocytosis assays (Fig. 1). The role of surface proteins in macrophages is discussed. CONCLUSION: The surface proteins are instrumental for recognition of L. corymbifera by macrophages and for intracellular survival in macrophages. The role of surface proteins is discussed in the light of evolution from environmental to human pathogenic fungus. SOURCES: 1. Voltersen V, Blango MG, Herrmann S, et al. Proteome Analysis Reveals the Conidial Surface Protein CcpA Essential for Virulence of the Pathogenic Fungus Aspergillus fumigatus. mBio. 2018;9(5). doi:10.1128/MBIO.01557-18. 2. Thürich J, Meichsner D, Furch ACU, et al. Arabidopsis thaliana responds to colonisation of Piriformospora indica by secretion of symbiosis-specific proteins. PLOS ONE. 2018;13(12):e0209658. doi:10.1371/JOURNAL. PONE.0209658. 3. Schwartze VU, Winter S, Shelest E, et al. Gene Expansion Shapes Genome Architecture in the Human Pathogen Lichtheimia corymbifera: An Evolutionary Genomics Analysis in the Ancient Terrestrial Mucorales (Mucoromycotina). PLOS Genetics. 2014;10(8):e1004496. doi:10.1371/JOURNAL.PGEN.1004496.