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. 2020 Jun 24;5(3):e00371-20.
doi: 10.1128/mSphere.00371-20.

Candida auris Phenotypic Heterogeneity Determines Pathogenicity In Vitro

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Candida auris Phenotypic Heterogeneity Determines Pathogenicity In Vitro

Jason L Brown et al. mSphere. .

Abstract

Candida auris is an enigmatic yeast that provides substantial global risk in health care facilities and intensive care units. A unique phenotype exhibited by certain isolates of C. auris is their ability to form small clusters of cells known as aggregates, which have been to a limited extent described in the context of pathogenic traits. In this study, we screened several nonaggregative and aggregative C. auris isolates for biofilm formation, where we observed a level of heterogeneity among the different phenotypes. Next, we utilized an RNA sequencing approach to investigate the transcriptional responses during biofilm formation of a nonaggregative and aggregative isolate of the initial pool. Observations from these analyses indicate unique transcriptional profiles in the two isolates, with several genes identified relating to proteins involved in adhesion and invasion of the host in other fungal species. From these findings, we investigated for the first time the fungal recognition and inflammatory responses of a three-dimensional skin epithelial model to these isolates. In these models, a wound was induced to mimic a portal of entry for C. auris We show that both phenotypes elicited minimal response in the model minus induction of the wound, yet in the wounded tissue, both phenotypes induced a greater response, with the aggregative isolate more proinflammatory. This capacity of aggregative C. auris biofilms to generate such responses in the wounded skin highlights how this opportunistic yeast is a high risk within the intensive care environment where susceptible patients have multiple indwelling lines.IMPORTANCECandida auris has recently emerged as an important cause of concern within health care environments due to its ability to persist and tolerate commonly used antiseptics and disinfectants, particularly when attached to a surface (biofilms). This yeast is able to colonize and subsequently infect patients, particularly those that are critically ill or immunosuppressed, which may result in death. We have undertaken analysis on two different phenotypic types of this yeast, using molecular and immunological tools to determine whether either of these has a greater ability to cause serious infections. We describe that both isolates exhibit largely different transcriptional profiles during biofilm development. Finally, we show that the inability to form small aggregates (or clusters) of cells has an adverse effect on the organism's immunostimulatory properties, suggesting that the nonaggregative phenotype may exhibit a certain level of immune evasion.

Keywords: Candida auris; aggregate; heterogeneity; host-pathogen interactions; in vitro skin model.

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Figures

FIG 1
FIG 1
Nonaggregative and aggregative Candida auris biofilm heterogeneity. Biomass and impedance measurements were used as measures of biofilm formation of 26 isolates of C. auris (n = 14 for the nonaggregative phenotype and n = 12 for the aggregative phenotype). For biomass assessment, 1 × 106 fungal cells were seeded in 96-well plates, and biofilm developed for 4 h or 24 h prior to crystal violet staining. Panels A and B show the differences in absorbance at 570 nm of the nonaggregative and aggregative phenotypes, respectively, at 4 h and 24 h. (C and D) The heatmaps show the average absorbance at 570 nm for each individual isolate at both time points. The formation of C. auris biofilms in real time was monitored using electron impedance measurements on the xCELLigence real-time cell analyzer. (E and F) Electron impedance measurements are presented as cell index for all isolates and for biofilms developed for 4 h and 24 h, respectively. (G and H) The heatmaps depict the mean cell index values for all nonaggregative (G) and aggregative (H) isolates. Red data points indicate the two isolates selected for further analyses in this study (NCPF 8973 and NCPF 8978). Paired Student’s t tests were used for statistical analyses, and statistically significant differences for data were determined and are indicated by bars and asterisks as follows: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
FIG 2
FIG 2
Transcriptional profile of nonaggregative and aggregative Candida auris during biofilm formation. RNA from planktonic cells and biofilms formed for 24 h of two isolates (NCPF 8973 and NCPF 8978) was used for RNA sequencing and transcriptome analyses as described in the text. (A and B) Venn diagrams depict upregulated genes in aggregative (A) or nonaggregative (B) phenotype in either the planktonic form (blue circle) or the biofilm form (red circle) or in both forms (blue and red circle). (C and D) Gene distribution of significantly upregulated genes in biofilm forms were grouped for gene ontology analysis. Genes upregulated that belong to the functional pathway cellular components (CC) are shown, while the other two pathways (biological processes [BP] and metabolic functions [MF], respectively) are included in Fig. S2 in the supplemental material. A cutoff twofold upregulation was used for gene ontology analysis using an adjusted P value of <0.05.
FIG 3
FIG 3
Cytotoxic and inflammatory effects of aggregative and nonaggregative Candida auris on skin epithelial models in vitro. For these analyses, a two- and three-dimensional skin epithelial model was used as schematically shown in Fig. 4. (A and B) First, cytotoxicity in the models was determined by quantifying the amount of lactate dehydrogenase (LDH) released by the human adult epidermal keratinocytes (HEKa) (A) and skin epidermis (B) following coculture with the aggregative (NCPF 8978) and nonaggregative (NCPF 8973) isolates of C. auris. For this, data were presented as fold change relative to the value for the PBS control. (C) To study the host response to C. auris, an RT2 profiler array containing genes associated with inflammation and fungal recognition was utilized to assess the transcriptional profile of the skin epidermis following stimulation. Data in the heatmap are presented as log2 fold change relative to the value for the PBS control. (D and E) Finally, expression of two virulence genes, ALS5 and SAP5, was determined in the isolates. Values are presented as percent expression relative to the fungus-specific housekeeping gene, β-actin. All epithelial cells or tissues were infected in triplicate, and statistical significance was determined from raw data threshold cycle (CT) values using unpaired Student’s t tests for comparison of two variables or one-way ANOVA with Tukey’s multiple-comparison posttest for more than two variables (*, P < 0.05; ** and §§, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
FIG 4
FIG 4
Schematic diagram depicting the experimental set up for the three-dimensional coculture of skin epidermis and Candida auris. A 17-day mature reconstructed human epidermis (RHE) on 0.5-cm2 inserts was purchased from Episkin (Skin Ethic). (A) Inserts were carefully lowered into 24-well plates containing maintenance medium supplied by the company. (B) To assess the host response to aggregative and nonaggregative C. auris, control and wounded tissue was cocultured with both isolates (NCPF 8973 and NCPF 8978). A total of 2 × 106 fungal cells in 100 μl PBS was added to the tissue and incubated overnight at 37°C and 5% CO2. For some tissues, prior to the addition of fungal inoculum, three scratch wounds were induced using a sterile 19-gauge needle across the surface of the tissue. For visual representation of phenotype, scanning electron microscopy images of 24-h biofilms are included in panel B, clearly showing the differences in cellular phenotypes between the two C. auris isolates. These images were taken at ×1,000 magnification as viewed under a JEOL JSM-6400 scanning electron microscope (samples processed as previously described [70]). Image created using Biorender.

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