Unambiguous identification of dimorphic fungal pathogens: a case study on an Emmonsia investigated by high resolution electrospray OrbitrapTM mass spectrometry J. Freeke1,2, A. Jamalian Nasrabadi1, K. Dukik1, L. Moreno1, B. Gerrits van den Ende1, C. Kenyon3, J.B. Stielow1,2, G.S. de Hoog1 1CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands, 2Thermo Fisher Scientific, Landsmeer, The Netherlands, 3Institute of Sexually Transmitted Infections, Institute of Tropical Medicine, Antwerp, Belgium EV0972 Mycology

Overview Results

30000 all results passing filter Purpose: Determine which protein markers can unambiguously identify common to all a newly discovered species (Emmonsia viz. africana) causing I II cutaneous infections in HIV patients. ) D a 20000 s ( Methods: Two outbreak causing strains in yeast and filamentous states a m

were investigated by phylogeny estimation, next generation d

sequencing (NGS) and top down proteomics. c t e t e 10000 D e Results: Protein markers can provide robust identification of Emmonsia viz. africana, with superior species discrimination, from phylogenetically close taxa nested in . 0 CBS1 136260 2 CBS3 1367304 Introduction filamentous | yeastConditfilamentousion | yeast Dimorphic fungi nested in the family of Ajellomycetaceae (e.g. FIGURE 4. Summary of protein masses detected. capsulatum) are amongst the most hazardous fungal Intact Intact pathogens known to medicine. Within a patient the adopts Mass Mass Unique To Emmonsia Species Superfamily or Identified Protein (Blast) the yeast form predominantly, while under standard culture conditions Theoretical Observed (% similarity to known protein) (room temperature) it displays a filamentous morphology. Additionally, (Da) (Da) Common to Emmonsia spp. and their dimorphism can cause safety issues in the clinical lab where 7766.23 Ribosomal protein S28e PFAM01200 1 7766.23 dermatitidis 40S Ribosomal protein S28, Ajellomyces Unique to Ajellomyces capsulatus (99%) upon initial culture before identification they may resemble yeast 7752.21 capsulatus PFAM01200 (mass discrepancy) species resulting in potential exposure. In this study we characterise Protein of unknown function (DUF2611); the proteome of a dimorphic Emmonsia species responsible for a 2 7822.10 7822.10 This family is conserved in the Dikarya of unique (93%) recent outbreak in South Africa which caused disseminated Fungi PFAM11022 FIGURE 2. Emmonsia disease presentation in HIV+ patients (I), macro- Ubiquitin-60S ribosomal protein L40, Common to many fungal species 3 8220.42 8464.55 cutaneous disease as yeast phase in tissue in HIV+ patients, and micromorphological characters of filamentous phase for strain Neurospora tetrasperma PFAM00240 (mass discrepancy) Ubiquitin, Naumovozyma dairenensis Common to many fungal species including an isolated case of infection in an immunocompetent CBS 136260 (II). Microscopy images courtesy of Peiying Feng 8650.64 4 8551.57 PFAM00240 (mass discrepancy) individual, which now can be recognised as another, yet emerging (CBS-KNAW). Polyubiquitin (Fragment), Fusarium Common to many fungal species 8923.76 97 novel systemic pathogen [1,2]. oxysporum PFAM00240 (mass discrepancy) Mitochondrial F1F0 ATP synthase subunit Common to Ajellomyces dermatitidis and Emmonsia sp. nov. (HIV-) 5 11188.80 10254.42 [1] N Engl J Med 2013; 369:1416-1424 var.crescens (wrongly classified) F, Ajellomyces dermatitidis PFAM10791 Emmonsia spp. (mass discrepancy)

Emmonsia parva var. parva (wrongly classified) [2] N Engl J Med 2014; 370:283-284 80 6 10586.40 ATP synthase E chain PFAM05680 unique (93%) Emmonsia parva var. parva (wrongly classified) 10586.38 ATP synthase E chain, Paracoccidioides Common to Paracoccidiodes brasiliensis Histoplasma spp. 7 10595.40 99 Emmonsia similis brasiliensis PFAM05680 and Emmonsia spp. (mass discrepancy) Methods 100 Ubiquinol-cytochrome C reductase Fungal population Protein extract Thermo Scientific Emmonsia coralliformis 8 10838.50 10838.50 unique (93%) 100 Buffer: formic acid, Emmonsia parva var. crescens complex PFAM02939 n g acetonitrile, N ano LC Q Exactive Plus 96 water Emmonsia parva var. parva Separation Protein mode 97 9 11739.90 11739.90 Acyl CoA binding protein PFAM00887 unique (88%) Data dependent TopN 100 Emmonsia pasteuriana MSMS method HCD activation (22) Emmonsia viz. africana (HIV +) CBS 136260 LC: Thermo Scientific 96 10 14183.80 14183.93 Histone 2A PFAM00125 unique (99%) 100

s e q u n c i EASY nLCII Emmonsia viz. africana (HIV +) CBS 136730 Column: Thermo 95 A SDA Ubiquinol-cytochrome C reductase unique (95%) Scientific EASYSpray Emmonsia sp. nov. N o o 11 14353.50 14333.58 CBS 136260at 24 C & 37 C Accucore C4 100

D complex 14kD subunit PFAM02271 (mass discrepancy) o o Gradient: 5-60% Emmonsia beijingensis CBS 136730at 24 C & 37 C acetonitrile over 60 mins MMSS 92 300nL/min 90 FIGURE 5. Summary of proteins identified in all conditions and Paracoccidioides brasiliensis Step 1: Protein Molecular Weight 81 their discriminatory power for identifying this Emmonsia species Coccidioides immitis 100

Coccidioides psadasii Step 2: Protein Sequencing and Identification Extremely 0.09 informative fragment FIGURE 3. Single locus phylogeny estimation (identity confirmation only) Organism ID, MS//MSS spectrum Mixtures, using the universal fungal DNA barcode ITS via RaxMl (v.8.1.11) Virulence, Software: Resistance Thermo Scientific employing a GTR-MIX model. Novel disease causing species are ProSight PC v3.0 highlighted in bold, branches with more than five terminal leaves were FIGURE 1. Schematic of Top Down proteomics workflow for collapsed. [email protected] Emmonsia strain analysis