The mycoparasitic yeast Saccharomycopsis schoenii predates and kills multi-drug resistant Candida auris

Candida auris has recently emerged as a multi-drug resistant fungal pathogen that poses a serious global health threat, especially for patients in hospital intensive care units (ICUs). C. auris can colonize human skin and can spread by physical contact or contaminated surfaces and equipment. Here, we show that the mycoparasitic yeast Saccharomycopsis schoenii efficiently kills both sensitive and multi-drug resistant isolates of C. auris belonging to the same clade, as well as clinical isolates of other pathogenic species of the Candida genus suggesting novel approaches for biocontrol.

SCIenTIfIC REPORTS | (2018) 8:14959 | DOI: 10.1038/s41598-018-33199-z microorganisms, rare feature of being unable to assimilate sulfate as their sole source of sulfur. Recently, we reported the lack of eight genes in the sulfate assimilation pathway in draft genomes of Saccharomycopsis fodiens and Saccharomycopsis fermentans 15,16 .
Here we show that S. schoenii efficiently attacks and kills a range of pathogenic Candida species, including the newly emerged human pathogenic fungus C. auris. We follow the predation process using time lapse microscopy in combination with fluorescent dyes. Efficient predation as shown here could be useful for biocontrol purposes in either clinical settings for skin clearance or in agricultural settings for combatting plant pathogens.

Results and Discussion
In this study, we prospected the use of a predatory yeast, Saccharomycopsis schoenii, as a potential biocontrol agent against human fungal pathogens of the Candida clade with a focus on C. auris. To this end we confronted multiple drug resistant strains including C. auris NCPF8985#20, a multi-drug resistant isolate from the South Asian clade (India), with S. schoenii (Supplementary Table 1). Equal numbers of dimorphic S. schoenii and ovoid C. auris NCPF8985#20 cells were seeded on minimal media agar on microscopy slides to offer solid support for a potential S. schoenii interaction. This minimal media lacked methionine and thus did not support proliferation of S. schoenii in pure culture. We found that S. schoenii attacked C. auris cells upon contact and killed prey cells using specialized penetration pegs ( Fig. 1; Supplementary Movies 1-3). The chitin-staining fluorescent dye Calcofluor White, revealed septa at the bases of penetration pegs indicating the sites of entry ( Fig. 1a; Supplementary Movies 1 and 2). Within minutes after S. schoenii cells generated penetration pegs, C. auris cells started to vacuolarize, take up dyes such as propidium iodide that are not permeable to living cells and then collapse, presumably because of feeding and material transfer to the predating S. schoenii cell ( Fig. 1a; Supplementary Movies 1-3). We prepared Transmission Electron Microscopy (TEM) images of interactions between S. schoenii and C. auris after 1 h of co-culture on minimal media (Fig. 1b-e) and found that C. auris cells attacked by S. schoenii cells were necrotic (Fig. 1b). Penetration pegs were directed at prey cells (Fig. 1c) and cell wall interactions led to the formation of penetration peg start sites (# Fig. 1d). Ultimately this led to degradation of the prey cell wall (Fig. 1e). After killing of prey cells, penetration pegs did not grow further or develop into buds or daughter cells We determined rates of killing over a 6 h time-course using morphology and/or propidium iodide staining (PI) stain ( Supplementary Figs 1 and 2). Several prey cells were found to accumulate PI staining upon predation, however, many prey cells were apparently killed without being stained by PI. In these cases, killed prey cells were "flattened" and or shrunken in size. This resulted in the death of around 34% of C. auris cells within a period of 6 h of co-culture with S. schoenii (Fig. 2, middle panel; Supplementary Table 2). As a control, almost none of the C. auris cells (0.6%) had died after 6 h when cultured alone under identical experimental conditions. To examine if predator-prey interactions differ with different C. auris isolates that exhibit variable drug resistance phenotypes, we analysed predator-prey interactions in three additional C. auris isolates (Supplementary Table 1). Furthermore, to elucidate host range of predator-prey interactions within Candida species we included clinical isolates of Candida albicans, Candida glabrata, Candida lusitaniae, Candida parapsilosis and Candida tropicalis in this analysis. For reference, we used Saccharomyces cerevisiae and Schizosaccharomyces pombe, two previously known prey species of S. schoenii 14 . All isolates of Candida species tested, including several drug resistant C. auris strains, were susceptible to predation by S. schoenii (Fig. 2 and Supplementary Table 2).
Collectively, these results demonstrate that the predator yeast S. schoenii provides a novel opportunity to develop biocontrol methods for skin disinfection. Saccharomycopsis predator are unique within the Saccharomycetes in displaying predatory behaviour. Thus these yeasts may harbor potential as biocontrol agents of other fungi including human and plant pathogens. Based on genome survey sequencing, Saccharomycopsis yeasts, like the distantly related filamentous ascomycete Trichoderma, harbor multi-gene families of proteases and chitinases 15,16 (our unpublished data). These multi-gene families probably represent a resource for the identification of lytic enzymes that have the potential to generate novel antifungal compounds.

Microscopy. Imaging was performed using the PerkinElmer UltraVIEW VoX Spinning Disk Confocal
Microscope controlled by Volocity software. Images for movies were captured 2-4 times/min for up to 2 h, using the Nikon Perfect Focus System to autofocus. For kill curve analyses, three frames were captured every hour per species and time point. FIJI/ImageJ 17 was used for image processing and analysis. Drift in movies frames was corrected using the macro NMS fixTranslation v1 and the plugin Image Stabiliser. For kill curve analyses, individual cells were counted using the Cell counter plugin.
For TEM images, S. schoenii and C. auris cells were separately pre-cultured to log phase in YPD media, then washed and mixed together at equal ratios. A total 1*10 8 cells were seeded on SD media solidified with 2% agarose. After 1 h of co-culture, cells were scraped off, washed and pelleted. High Pressure Freezing was carried out using a Leica EM PACT 2 (Leica Microsystems, Milton Keynes, UK) and samples were freeze substituted in a Leica AFS 2. Freeze substitution was carried out using the following program: −95 °C to −90 °C for 30 h with 2% OsO 4 in acetone, −90 °C for 10 h with 2% OsO 4 in acetone, −90 °C to −30 °C for 8 h with 2% OsO 4 in acetone, −30 °C to −10 °C for 1 h with acetone, −10 °C to 4 °C for 1 h in acetone, 4 °C to 20 °C for 1 h in acetone. Samples were then removed and placed in 10% Spurr's (TAAB, UK): acetone for 72 h, followed by 30% Spurr's overnight, 50% Spurr's for 8 h, 70% Spurr's overnight, 90% Spurr's for 8 h and embedded in Spurr's resin at 60 °C for at least 24 h. Ultrathin sections were cut to 90 µm using a diamond knife (Diatome Ltd, Switzerland) onto copper grids (TAAB, UK) using a Leica UC6 and were contrast stained with uranyl acetate and lead citrate in a Leica AC20. Samples were imaged on a JEM 1400 plus (JEOL UK) Transmission Electron Microscope and captured using an AMT UltraVUE camera (AMT, USA). All relevant data are available from the authors.