Candida Auris Phenotypic Heterogeneity Determines Pathogenicity in Vitro

bioRxiv preprint doi: https://doi.org/10.1101/2020.04.20.052399; this version posted April 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Candida auris phenotypic heterogeneity determines pathogenicity in vitro

3 Jason L Brown1, Chris Delaney1, Bryn Short1, Mark C Butcher1, Emily

4 McKloud1, Craig Williams1,3, Ryan Kean1,2, Gordon Ramage1*

5 1 Glasgow Biofilm Research Network, Glasgow Dental School, School of Medicine,

6 College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow,

7 UK, G12 8TA

8 2Department of Life Sciences, School of Health and Life Sciences, Glasgow

9 Caledonian University, Glasgow, UK, G4 0BA

10 3Microbiology Department, Lancaster Royal Infirmary & University of Lancaster

12 *Author for correspondence

13 [email protected]

15 Key words;

16 Candida auris, aggregate, host-pathogen interactions, in vitro skin model

1 Abstract

2 Candida auris is an enigmatic yeast that provides substantial global risk in

3 healthcare facilities and intensive care units. A unique phenotype exhibited by

4 certain isolates of C. auris is their ability to form small clusters of cells known as

5 aggregates, which have been to a limited extent described in the context of

6 pathogenic traits. In this study, we screened several non-aggregative and

7 aggregative C. auris isolates for biofilm formation, where we observed a level of

8 heterogeneity amongst the different phenotypes. Next, we utilised an RNA-

9 sequencing approach to investigate the transcriptional responses during biofilm

10 formation of a non-aggregative and aggregative isolate of the initial pool.

11 Observations from these analyses indicate unique transcriptional profiles in the two

12 isolates, with several genes identified relating to proteins involved in adhesion and

13 invasion of the host in other fungal species. From these findings we investigated for

14 the first time the fungal recognition and inflammatory responses of a three-

15 dimensional skin epithelial model to these isolates. In these models, a wound was

16 induced to mimic a portal of entry for C. auris. We show both phenotypes elicited

17 minimal response in the model minus induction of the wound, yet in the wounded

18 tissue both phenotypes induced a greater response, with the aggregative isolate

19 more pro-inflammatory. This capacity of aggregative C. auris biofilms to generate

20 such responses in the wounded skin highlights how this opportunistic yeast is a high

21 risk within the intensive care environment where susceptible patients have multiple

22 indwelling lines.

24 Importance

1 Candida auris has recently emerged as an important cause of concern within

2 healthcare environments due to its ability to persist and tolerate commonly used

3 antiseptics and disinfectants, particularly when surface attached (biofilms). This

4 yeast is able to colonise and subsequently infect patients, particularly those that are

5 critically ill or immunosuppressed, which may result in death. We have undertaken

6 analysis on two different types of this yeast, using molecular and immunological tools

7 to determine whether either of these has a greater ability to cause serious infections.

8 We describe that both isolates exhibit largely different transcriptional profiles during

9 biofilm development. Finally, we show that the inability to form small aggregates (or

10 clusters) of cells has an adverse effect on the organisms immuno-stimulatory

11 properties, suggestive the non-aggregative phenotype may exhibit a certain level of

12 immune evasion.

1 Introduction

2 C. auris is a nosocomial pathogen first identified in 2009 [1]. To date, this multidrug

3 resistant organism has been identified in over 40 countries on 6 different continents,

4 providing a substantial global risk in healthcare facilities and intensive care units [2-

5 4]. It is postulated that the emergence of C. auris may have coincided with climate

6 change based on its particular attributes, resulting in an thermotolerant organism

7 with the ability to persist in the environment before transmission to humans [5].

8 A unique pathogenic trait exhibited by some isolates of C. auris is their ability to form

9 aggregates (Agg) [6-8]. Despite the well-documented prevalence of C. auris

10 worldwide, relatively little is known about the Agg phenotype of the organism. The

11 existence of four geographically and phylogenetically distinct clades of the organism

12 [2], and a fifth recently proposed [9], has restricted a definitive profiling of C. auris

13 pathogenic mechanism of these aggregates in regard to biofilm forming capabilities,

14 drug resistance pathways and interactions with the host. Of the publications that

15 exist, these have documented characteristic pathogenic traits for both phenotypes in

16 vitro and in vivo [6, 10, 11]. Others have shown that the Agg phenotype is inducible

17 under certain conditions [7, 8], whilst histological analyses of murine models have

18 shown that aggregates can accumulate in organs following C. auris infection [7, 12,

19 13]. Therefore, further studies are required to investigate this characteristic Agg

20 phenomenon in C. auris isolates to fully comprehend the pathogenic pathways of the

21 organism, and to understand how such mechanisms may differ from their non-Agg

22 counterparts.

23 Limited evidence also exists for studies investigating the interactions of C. auris with

24 components of the host, although several in vivo models have been employed to

1 document the virulence of C. auris. Of these, Galleria mellonella larvae infection

2 models and murine models of invasive candidiasis have shown varying survival rates

3 post-infection with C. auris [6, 8, 10, 12-15], reaffirming that genetic variability

4 amongst clades impacts the organisms virulence. However, such studies have been

5 limited in investigating the host immune response to the organism. Recently,

6 Johnson et al (2019) utilised a Zebrafish (Danio rerio) model to monitor C. auris-host

7 cell interactions in vivo. This work highlighted that C. auris (strain B11203 Indian

8 isolate phylogenetically part of the South Asian or India/Pakistan clade [2], which

9 appeared to exhibit a non-Agg phenotype) was resistant to neutrophil-mediated

10 killing, suggesting that the organism has the ability to persist incognito in the host

11 [16]. In vitro studies have shown interactions between C. auris and epithelial tissue,

12 emphasizing that the organism can persist on skin. In this study, Horton and

13 colleagues (2020) demonstrated that C. auris (B11203 strain, as above) formed

14 high-burden biofilms on porcine skin biopsies in the presence of an artificial sweat

15 medium [17]. To date, no studies have investigated the host inflammatory response

16 to the non-Agg and/or Agg phenotype.

17 In this study, we sought to investigate the level of heterogeneity amongst different

18 non-Agg and Agg isolates. We deemed this pertinent given that such traits of

19 heterogeneity amongst isolates have previously been described for other Candida

20 species, significantly impacting clinical outcomes and mortality rates [18]. To further

21 investigate this Agg versus non-Agg phenotype, transcriptome analyses were

22 performed on planktonic cells and biofilms of two selected isolates from the initial

23 pool. Upon completion of these analyses, we discovered that several genes

24 associated with cell membrane and/or cell wall proteins (e.g. cellular components)

25 were upregulated in the Agg biofilm. Such unique transcriptional profiles in respect to

1 the cellular components led us to investigate the host response following stimulation

2 with both C. auris phenotypes in vitro. For this, a two- and three-dimensional skin

3 wound model was employed to investigate the epithelial response to the Agg and

4 non-Agg isolates of C. auris. Both skin wound models exhibited different profiles to

5 both isolates indicating unique fungal recognition and/or host response to the Agg

6 and non-Agg phenotype. Interestingly, there was minimal response by the host to C.

7 auris without induction of the wound, suggestive the organism relies on loss of tissue

8 integrity to become invasive.

10 Methods

11 Microbial growth and standardisation

12 For in vitro biofilm biomass assessment, a pool of aggregating (Agg; n=12) and

13 single-celled, non-aggregative (non-Agg; n=14) C. auris clinical isolates (gifted by Dr

14 Andrew Borman and Dr Elizabeth Johnson, Public Health England, UK). All C. auris

15 isolates were stored in MicrobankTM beads (Pro-lab Diagnostics, UK) prior to use.

16 Each isolate was grown on Sabouraud dextrose (SAB) agar (Oxoid, UK) at 30˚C for

17 24-48 h then stored at 4˚C prior to propagation in yeast peptone dextrose (YPD;

18 Sigma-Aldrich, UK) medium overnight (16 h) at 30˚C, gently shaking at 200 rpm.

19 Cells were pelleted by centrifugation (3,000 x g) then washed two times in phosphate

20 buffered saline (PBS). Cells were then standardised to desired concentration

21 following counting using a haemocytometer, then resuspended in selected media for

22 each assay, as described within.

23 C. auris isolate phenotypes were determined visually by suspending one colony in 1

24 mL (PBS, Sigma-Aldrich, UK). Isolates were termed as ‘aggregators’ if the added

1 colony did not disperse upon mixing in PBS. For RNA sequencing and transcriptional

2 analysis of C. auris biofilms and co-culture systems, one Agg (NCPF 8978) and non-

3 Agg (NCPF 8973) isolate was used.

5 Biofilm growth and biomass assessment

6 Fungal cells were adjusted to 1 x 106 cells/mL in Roswell Parks Memorial Institute

7 (RPMI) media (Sigma-Aldrich, UK) and biofilms formed for 4 or 24 hours at 37˚C in

8 flat-bottom wells of 96-well plates (Corning, UK). Appropriate media controls were

9 included on each plate to test for contamination. Following incubation, biofilms were

10 washed gently once in PBS to remove any non-adhered cells. The biomass of each

11 biofilm was determined via 0.05% crystal violet (CV) staining as described previously

12 [19]. Absorbance of the CV stain was measured spectrophotometrically at 570nm in

13 a microtiter plate reader (FLUOStar Omega, BMG Labtech, UK).

15 Monitoring the growth of Candida auris biofilms in real time

16 The xCELLigence real time cell analyser (RTCA, ACEA Bioscience Inc, San Diego,

17 CA) was used to monitor the formation of C. auris biofilms in real time using electron

18 impedance measurements (presented as cell index, CI) which is directly related to

19 cell attachment and proliferation. In brief, the E-plate containing 100 µL of pre-heated

20 RPMI medium was loaded into the RTCA which had been placed in the incubator 2 h

21 prior to the experiment to test media impedance and electrode connectivity. Cultures

22 of each C. auris isolate used in this study were standardised to 2 x 106 CFU/mL and

23 added to the E-plate in 100 µL aliquots in triplicate. Appropriate media controls

24 minus inoculum were also included in triplicate. Biofilm formation was measured over

1 24 hours with CI readings taken every 5 minutes. Normalised CI values were

2 exported from the RTCA software and analysed in GraphPad Prism (version 8;

3 GraphPad Software Inc., La Jolla, CA). More detailed descriptions for this technology

4 can be found elsewhere [20].

6 Two-dimensional monolayer co-culture model

7 Adult human epidermal keratinocytes (HEKa) cells (Invitrogen, GIBCO®, UK) were

8 used for two-dimensional co-culture experiments. Frozen stocks of HEKa cells (1 x

9 106 cells/ml; passage number lower than 10) were revived and seeded in T-75 tissue

10 culture flasks (Corning, UK) in media 154 (Thermo-Fisher, UK) supplemented with

11 100 U/ml of pen/strep and human keratinocyte growth supplement (HKGS) (Thermo-

12 Fisher, UK). The flasks were incubated at 37°C (5 % CO2) and media was changed

13 every 48 h until the cells reached 80-90% confluence [21]. Confluent cells were

14 passaged using 0.05% trypsin EDTA (Sigma-Aldrich, UK) and the enzymatic

15 reaction inhibited using trypsin neutralizer solution (Sigma-Aldrich, UK). Passaged

16 cells were then seeded into 24-well plates (Corning, UK) at final concentration 2 x

17 105 cells/ml. After 24 to 48 h the cells reached the adequate confluence for C. auris

18 co-culture experiments as described below.

20 Three-dimensional human epidermis co-culture model

21 Reconstituted human epidermis (RHE) used for 3D co-culture experiments was

22 purchased from Episkin (Skin Ethic™, Lyon, France http://www.episkin.com). RHE

23 was formed from normal human keratinocytes cultured on an inert polycarbonate

24 filter at the air-liquid interface, in a chemically defined medium grown to 17-day

1 maturity. This model is histologically similar to in vivo human epidermis. Upon arrival

2 and prior to experimental set-up, RHE was incubated with maintenance media in 24-

o 3 well plates (Corning, UK) for 24 h, 5% CO2 at 37 C. Maintenance media was

4 replaced then the co-culture three-dimensional system was set up as described

5 below.

7 Wound model in two-dimensional and three-dimensional co-culture systems

8 HEKa cell monolayers were scratched using a similar method to as previously

9 described to mimic a wound model [22, 23]. Briefly, monolayers were grown to

10 confluence as described above then three parallel scratches were introduced across

11 the surface using a 100 μL pipette tip prior to inoculation with C. auris. For RHE, a

12 19-gauge needle was used to scratch the tissue. For all co-culture experiments, Agg

13 C. auris NCPF 8978 and non-Agg C. auris NCPF 8973 were grown as described

14 above, then standardised to 2 x 106/mL (multiplicity of infection of 10 to HEKa cells;

15 MOI 10, and as previously described for Candida-tissue co-culture [24]). For the two-

16 dimensional system, 2 x 106/mL C. auris was prepared in 500µL supplemented

17 media 154 and added directly to the confluent HEKa cells. For the three-dimensional

18 co-culture model, 2 x 106/mL C. auris was prepared in 100µL of sterile PBS and this

19 suspension was added directly to the RHE tissue. All experiments were conducted

20 for 24 hr at 5% CO2. Infected non-scratched HEKa cells and RHE tissue were used

21 as controls e.g., no wound, and uninoculated co-cultured cells or tissues were also

22 included for all experiments. All control and wounded models were infected in

23 triplicate with both isolates of C. auris.

1 Histological staining of skin epidermis

2 Following co-culture with C. auris, epithelial tissue was carefully cut from the 0.5 cm2

3 insert using a 19-gauge needle and washed three times in sterile PBS to remove

4 non-adherent cells in a similar manner as previously described [25], as summarized

5 in the schematic in Figure 1. Tissue was then fixed in 10% neutral-buffered formalin

6 prior to embedding in paraffin. A Finnesse ME+ microtome (Thermo Scientific, UK)

7 was used to cut 2 μm sections and tissue sections stained with haematoxylin and

8 eosin, or with the fungal specific Periodic acid-Schiff (PAS) reagents and counter-

9 stained with haematoxylin.

11 Epithelial cell viability

12 To assess any cytotoxic effects of C. auris on HEKa cells and RHE tissue, a Pierce

13 lactate dehydrogenase (LDH) Cytotoxicity Assay Kit (Thermo Scientific; UK) was

14 used according to the manufacturers’ instructions. Following co-culture, cell or tissue

15 spent media was assayed using the above kit to quantify the level of LDH release as

16 a measure of host cellular disruption.

18 Differential gene expression analysis

19 HEKa cells and RHE tissue following co-culture were lysed in RLT lysis buffer

20 (Qiagen Ltd, UK) containing 0.01% (v/v) β-2-mercaptoethanol (β2ME) before bead

21 beating. All RNA was extracted using the RNeasy Mini Kit according to

22 manufacturers’ instructions (Qiagen Ltd, UK) and quantified using a NanoDrop 1000

23 spectrophotometer [Thermo Scientific, UK]. RNA was converted to complementary

1 DNA (cDNA) using the High Capacity RNA to cDNA kit (Life Technologies, UK) as

2 per the manufacturer’s instructions. Gene expression was assessed using SYBR

3 GreenER based-quantitative polymerase chain reaction (qPCR) or RT2 profiler arrays

4 (Qiagen Ltd, UK). For SYBR GreenER based-qPCR analyses, the following PCR

5 thermal profiles was used; holding stage at 50˚C for 2 minutes, followed by

6 denaturation stage at 95˚C for 10 minutes and then 40 cycles of 95˚C for 3 seconds

7 and 60˚C for 15 seconds. qPCR plates were run on the StepOnePlus™ Real-Time

8 PCR System. The following primer sequences were used for SYBR GreenER based-

9 qPCR analyses of host cells; GAPDH, forward primer, 5’ to 3’,

10 CAAGGCTGAGAACGGGAAG and reverse primer, 5’ to 3’,

11 GGTGGTGAAGACGCCAGT [26]; IL8, forward primer, 5’ to 3’,

12 CAGAGACAGCAGAGCACACAA and reverse primer, 5’ to 3’,

13 TTAGCACTCCTTGGCAAAAC [27]. For gene expression analyses of C. auris,

14 primers for adhesin gene ALS5 and proteinase gene SAP5 were used as follows;

15 ALS5; forward primer, 5’ to 3’, ATACCAGGGTCGGTAGCAGT and reverse primer,

16 5’ to 3’, CTATCTTCGCCGCTTGGGAT and SAP5; forward primer, 5’ to 3’,

17 GGATGCAGCTCTTCCTGGTT and reverse primer, 5’ to 3’,

18 CTTCCAGTTTGCGGTTGTGG. For other gene expression analyses of RHE tissue,

19 a custom designed RT2 Profiler PCR array was compiled containing primers for

20 genes associated with inflammation and fungal recognition or stimulation of host

21 tissue. For these arrays, the following thermal cycle was used on the MxProP

22 Quantitative PCR machine; 10 minutes at 95oC followed by 40 cycles of 15 seconds

23 at 95oC and 60 seconds at 60oC, and data assembled using MxProP 3000 software

24 (Stratagene, Netherlands). Expression levels for all genes of interest were

25 normalized to the housekeeping gene, β-actin for C. auris gene expression and

1 GAPDH for mammalian cells, according to the 2-ΔCt method, and then quantified

2 using the 2-ΔΔCt method [28].

4 RNA sequencing

5 For RNA sequencing of C. auris biofilms, RNA was extracted from 24-h C. auris

6 biofilms as described previously [29]. In brief, biofilms were grown as above on

7 Thermanox coverslips (Thermo-Fisher, UK) in 24 well plates (Corning, UK). Biofilms

8 were removed from coverslips by sonication at 35 kHz for 10 minutes in a sonic bath

9 in 1 ml of PBS and the sonicate transferred to a 2.0 ml RNase-free bead beating

10 tube (Sigma-Aldrich, UK). Cells were homogenized in TRIzol ™ (Invitrogen, UK) with

11 0.5 mm glass beads using a BeadBug microtube homogeniser for a total of 90

12 seconds (Benchmark-Scientific, USA). RNA was then extracted as described above

13 using the RNeasy Mini Kit according to manufacturers’ instructions (Qiagen Ltd, UK).

14 Following extraction, RNA quality and quantity were determined using a Bioanalyzer

15 (Agilent, USA), where a minimum RNA integrity number and quantity of 7 and 2.5 µg,

16 respectively were obtained for each sample. Annotation of data following sample

17 submission to Edinburgh Genomics (http://genomics.ed.ac.uk/) was completed as

18 previously described [30]. Briefly, raw fastq reads were trimmed and aligned to the

19 Candida auris representative genome B8441 using Hisat2 [31]. Reads were then

20 processed and assembled de novo using the Trinity assembly pipeline [32]. Trinotate

21 and Blast2Go were utilised to assign gene IDs to homologous sequences using

22 BLAST and Interpro [33, 34]. Differential expression analysis was performed

23 according to DESeq2 pipeline and functional over representation was determined

24 using GOseq [35, 36] within R. Additionally visualisation of overrepresented

1 pathways were drawn within R. Raw data files for these analyses are deposited

2 under accession no. PRJNA477447

3 (https://www.ncbi.nlm.nih.gov/bioproject/477447).

5 Statistical analysis

6 Statistical analyses were performed using GraphPad Prism. Two-tailed paired or

7 unpaired Student’s t-tests were used to compare the means of two samples as

8 stated within, or one-way analysis of variance (ANOVA) to compare the means of

9 more than two samples. Tukey’s post-test was applied to the p value to account for

10 multiple comparisons of the data. P-values of <0.05 were considered statistically

11 significant.

13 Results

14 The Agg phenotype is a unique trait of C. auris, one that can influences the

15 organisms pathogenic traits in vitro and in vivo [6, 10, 11]. To corroborate these

16 previous observations, differences in early and late biofilm formation were assessed

17 between non-Agg and Agg isolates of C. auris. A total of 26 non-Agg and Agg C.

18 auris clinical isolates were screened during early (4 h) and mature (24 h) biofilm

19 growth stages (Figure 2A and B). Both sets of non-Agg and Agg C. auris isolates

20 formed biofilms in a time-dependant manner as assessed by crystal violet staining.

21 With the exception of NCPF 8993 (non-Agg) and NCPF 8996 (Agg), all isolates

22 formed biofilms with greater biomass after 24 hours culture compared to 4 hours

23 (Figure 2C and D). Although, the Agg phenotype tended to form biofilms with greater

24 biomass at both 4 h and 24 h, we observed no statistical differences when

1 collectively comparing all non-aggregating and aggregating isolates of C. auris at

2 either timepoint (Supplementary Figure 1A and B). From this data, it was clear that

3 some isolates formed biofilms with greater biomass than others, suggestive of a

4 certain level of heterogeneity between isolates of Agg and non-Agg phenotype, in

5 line with previous observations [6, 10, 37]. Similar trends of biofilm heterogeneity

6 were observed when monitoring biofilm formation via impedance measurements in

7 real time at both timepoints in non-Agg and Agg phenotype (Figure 2E-H and

8 Supplementary 1C and D). These data support corroboration of these unique

9 phenotypes.

10 To further study the pathogenic and biofilm-forming characteristics of Agg and non-

11 Agg C. auris, transcriptional profiling of 24-hour planktonic vs biofilm phenotypes

12 was performed. For these studies, two clinical isolates from the initial pool tested

13 were selected for analysis (non-Agg NCPF 8973 and Agg NCPF 8978, indicated by

14 the red points in Figure 2). Firstly, we found that a total of 701 genes were

15 upregulated in planktonic and/or biofilm form of Agg compared to the non-Agg

16 phenotype, of which 450 genes were upregulated in the biofilm state (Figure 3A).

17 Conversely, less genes (430) were upregulated in non-Agg C. auris in the planktonic,

18 biofilm or both states compared to Agg C. auris counterparts, with 190 genes up-

19 regulated in the biofilm form (Figure 3B). In order to understand the functional

20 processes related to differentially expressed genes, a cut-off of 2-fold up-regulation

21 was used for gene ontology (GO) analysis (adjusted P value of < 0.05). Up-regulated

22 genes in non-Agg vs. Agg C. auris biofilms normalized to planktonic cell expression

23 involved three functional classes; biological processes (BP), cellular components

24 (CC) and metabolic functions (MF) (Supplementary Figure 2A and B). Interestingly,

25 most genes upregulated in the Agg biofilm belonged to the CC functional class; over

1 40 genes related to membrane and cell wall constituents were upregulated in the

2 Agg form compared to the non-Agg phenotype (Figure 3C). Several genes

3 associated with fungal cell wall proteins were upregulated in the Agg biofilms

4 including TSA1, ECM33, MP65 and PHR1 (Supplementary Figure 2C). Moreover,

5 included in these 40 genes were members of the ALS family of adhesins such as

6 ALS1. On the contrary, in the non-Agg biofilms, the greatest changes in expression

7 were observed for genes belonging to functional classes of BP and MF

8 (Supplementary Figure 2C). Only a small number of genes belonging to cellular

9 components were upregulated in the non-Agg compared to Agg biofilm (Figure 3D).

10 The observed differences in the transcriptional profiles of genes belonging to the CC

11 of C. auris could impact the ability of the host to recognize the two phenotypes. Key

12 cellular components such as ALS proteins of other Candida species, such as

13 Candida albicans play important roles in aiding host colonization and orchestrating

14 the innate immune response [38, 39]. However, it is currently unknown whether

15 similar mechanisms exist for C. auris. Therefore, the following section investigates

16 whether a non-Agg or Agg phenotype dictates the response by the host to C. auris.

17 For this, a two- and three-dimensional skin epithelial model was employed to study

18 the host response to the two C. auris used above. For both co-culture skin systems,

19 a wound was induced to mimic the possible entry site of patients for C. auris in

20 healthcare environments. In the two-dimensional model containing adult human

21 epidermal keratinocytes (HEKa) cells, both isolates were significantly more cytotoxic

22 to host cells following induction of the wound (Figure 4A; **** p < 0.001). Intriguingly,

23 Agg C. auris was significantly more cytotoxic than non-Agg form in the wound model

24 (Figure 4A; §§ p < 0.01). It is noteworthy that wounded monolayers minus inoculum

25 were comparable to untreated monolayers (data not shown), suggestive that

1 induction of the wound did not induce cytotoxic effects on the cells. A similar trend in

2 cytotoxicity was observed between the two isolates in the three-dimensional model

3 (Episkin, SkinEthicTM reconstructed human epidermis; RHE) although this did not

4 reach statistical significance (Figure 4B). Interestingly, cytotoxicity of Agg and non-

5 Agg C. auris were comparable in both co-culture models minus wounds.

6 To further study the host response in the three-dimensional system, a transcriptional

7 response was investigated using a RT2 profiler array containing primers specific for

8 genes associated with inflammatory responses and/or fungal recognition (Figure

9 4C). Upon investigation, it was evident that the greatest changes in gene expression

10 were observed in the wound models for both C. auris isolates. Importantly, induction

11 of the wound minus C. auris did not significantly alter the expression of any of the

12 genes arrayed. In the wound model, pro-inflammatory cytokines TNFα and IL1β (*

13 p<0.05) were significantly upregulated in RHE tissue cultured with non-Agg C. auris.

14 Conversely, the Agg phenotype of C. auris induced the greatest changes in RHE; the

15 expression of 8 of the 11 genes profiled (IL1β, *p<0.05; TNF, IL6, CAMP, ** p<0.01;

16 CFS2, CFS3, CLEC7A, *** p<0.001; TLR4, **** p<0.0001) were all significantly

17 upregulated following co-culture compared to the untreated tissue.

18 In the three-dimensional co-culture system, it was evident from histological and

19 fungal specific Periodic acid-Schiff (PAS) staining that the both isolates of C. auris

20 had adhered to the peripheral keratinised layer of the RHE tissue (Supplementary

21 Figure 3). Uninfected tissue displayed a well-organized multi-layered structure

22 characteristic of skin epidermis in vivo (Supplementary Figure 3A). However, in

23 infected tissue, there was no sign of C. auris infiltration into the tissue

24 (Supplementary Figure 3A and B) as seen with previous publications of skin tissue

25 infection models with invasive C. albicans [24, 40]. Unfortunately, loss of tissue

1 integrity in the wounded model rendered them unsuitable for histological staining

2 therefore at this juncture we were unable to visualize whether C. auris invaded the

3 tissue at the wound site. Nonetheless, given that the expression of several important

4 cell membrane and wall proteins are differentially regulated in non-Agg and Agg

5 phenotypes of C. auris biofilms, we finally wanted to assess the expression of two

6 key virulence factor genes belonging to the ALS and SAP families, respectively.

7 ALS5 and SAP5 were both up-regulated in the Agg phenotype compared to the non-

8 Agg C. auris isolate in the three-dimensional tissue model (Figure 4D and 4E). Such

9 a response in the Agg isolate may begin to elucidate the mechanism by which this

10 phenotype generated a greater inflammatory response within the tissue.

12 Discussion

13 Results from this study further indicates that the Agg phenotype of C. auris

14 determines its pathogenicity in vitro. This phenotype, first reported by Borman et al

15 (2016), characterised in certain isolates by the formation of individual yeast cells

16 mixed with large aggregations in planktonic form [6]. This aggregating behaviour was

17 later shown to affect biofilm formation, antifungal susceptibility and virulence of the

18 organism [6, 10, 11, 37]. Moreover, aggregation has recently been found to be an

19 inducible trait triggered by sub-inhibitory concentrations of triazole and echinocandin

20 antifungals, suggestive that treatment regimens must be carefully considered to

21 combat C. auris dependant on its Agg phenotype [8]. In this study, we report a

22 previously described RNA-sequencing approach [30] in order to compare the

23 transcriptional responses of one non-Agg (NCPF 8973) and one Agg (NPCF 8978)

24 isolate of C. auris during formation of biofilms from planktonic cells. These analyses

1 indicated that several key cell membrane and cell wall components were upregulated

2 in Agg biofilms, many of which involved in cell adhesion to abiotic surfaces and host

3 cells. These observations led us to pose the question: how would the host respond

4 to the two phenotypes? As such, we document for the first time an investigation into

5 the host skin epithelial response to Agg and non-Agg C. auris.

6 Given the clear differences in virulence traits of the non-Agg and Agg phenotype, we

7 deemed it pertinent to study in greater detail the transcriptional profiles of one non-

8 Agg and one Agg C. auris. These two isolates displayed the heterogeneity exhibited

9 by other Agg and non-Agg isolates, with the NCPF 8973 isolate forming a denser

10 biofilm after 24 h (as shown here in Figure 2 by the red data points, and elsewhere

11 [10]). However, irrespective of the biofilm-forming capabilities, transcriptome

12 analyses showed that an increased number of genes were upregulated in the Agg

13 compared to the non-Agg biofilms (450 vs 194 genes, respectively; Figure 3),

14 suggestive that the clustering of aggregated cells greatly impacts the transcriptome

15 of the organism during the formation of a biofilm. Of these differential responses in

16 the two biofilm phenotypes, a vast number of genes upregulated in the Agg biofilm

17 belonged to CC as assessed using GO analyses Specifically, several cell wall genes

18 were upregulated in the Agg phenotype e.g., TSA1, ECM33, MP65, ALS1 and

19 PHR1. In C. albicans, ECM33 and MP65 have been implemented as important

20 proteins in maintenance of fungal cell wall integrity, biofilm formation and stress

21 responses [41-43]. Furthermore, the ALS family of adhesin proteins and PHR family

22 of extracellular transglycosylases also function as important regulators of biofilm

23 formation in C. albicans [44, 45]. For example, loss of function of PHR and ALS

24 proteins results in impaired adhesion and biofilm development [46, 47]. It must be

25 noted here that such comparisons between findings on C. auris and C. albicans

1 biofilms must be taken with a certain degree of caution given the lack of true hyphal

2 formation of C. auris isolates grown in biofilms [6], although such a trait can be

3 induced under certain conditions [7, 14]. Nevertheless, such in-depth analyses, as

4 those documented herein, may begin to explain the mechanisms behind aggregate

5 formation in biofilms of some C. auris isolates.

6 Most of the aforementioned genes (TSA1, ECM33, MP65, ALS1 and PHR1) have

7 multiple functions in C. albicans pathogenicity, particularly in biofilm formation as

8 discussed above. In addition, most also play key roles in attachment and/or survival

9 within the host in other fungal species. For example, TSA1, which encodes for a

10 protein called thiol-specific antioxidant 1, has been identified in C. albicans and

11 Cryptococcus neoformans, and functions as an important stress response regulator

12 in unfavourable oxidative environments [48, 49]; potentially those generated by the

13 host[50]. In C. albicans, ECM33, MP65 and PHR1 have been shown to be important

14 genes necessary for production of proteins involved in adherence and invasion of

15 host cells [41-43, 46]. For example, in similar three-dimensional reconstituted skin

16 and oral epithelial co-culture models, heterozygous mutants of ECM33 and PHR1

17 displayed clear deficiencies in penetration of epithelial cell layers leading to reduced

18 tissue invasion and subsequent cellular damage [42, 46]. Finally, another gene

19 upregulated in Agg C. auris biofilms was ALS1, a member of the ALS family of

20 adhesin proteins. This cohort of adhesin proteins which contains at least 8 members

21 in C. albicans are well documented virulence factors, particularly in host-pathogen

22 interactions in vitro and in vivo [44, 51, 52]. Concerning the results shown in this

23 study, the role of ALS1, which encodes for the protein Als1p, in Candida-host

24 interactions remains unclear. As such, contradictory reports state a role for this

25 adhesion in attachment to epithelial cells. Kamai and colleagues (2002) showed that

1 heterozygous knockouts of ALS1 were unable to colonize oral tissues of mice in vivo

2 and ex vivo as efficiently as wildtype strains or knockouts coupled with the Als1p

3 reinstated [53]. On the contrary, it was shown that attachment of the C. albicans

4 ALS1 null mutant to oral epithelial cells was no different than wildtype controls,

5 suggestive its role in adhesion was not as important as other ALS family members

6 [54]. Nevertheless, as discussed briefly above, such comparisons between C. auris

7 and C. albicans interactions with host cells must be tentatively compared given the

8 differences in the morphological forms of the two species. In particular, the transition

9 from yeast to hyphae in C. albicans is essential for tissue invasion of mucosal

10 surfaces. For example, fungal invasion mechanisms have been identified that show

11 morphological changes from yeast, to pseudohyphae, to hyphae during the process

12 of adhesion and invasion in C. albicans in epithelial tissue [55, 56]. This process is

13 even essential for the discrimination of commensal and pathogenic forms of C.

14 albicans by the host [57]. Given its inability to form hyphae under normal

15 physiological conditions, how C. auris can invade epithelial tissue is unknown. It is

16 possible that the organism has adapted slightly different invasive mechanisms.

17 However, at this juncture, further studies are necessary to elucidate such

18 mechanisms.

19 It has been postulated that C. auris exhibits a level of immune evasion to bypass our

20 immunological defences [4]. Recent work by Johnson et al (2019) showed that C.

21 auris were resistant to neutrophil-mediated killing by failing to stimulate neutrophil

22 elastase trap (NET) formation both in vitro and in vivo in a zebrafish infection model

23 [16]. Another study found that viable C. auris did not induce an inflammatory

24 response in human peripheral blood mononuclear cells (PBMCs), although fungal

25 cells were recognized and engulfed by human monocyte-derived macrophages.

1 Conversely, such host responses were significantly greater against other Candida

2 species such as Candida tropicalis, Candida guilliermondii and Candida krusei [58].

3 It is apparent from these studies that the host recognizes yet fails to generate an

4 effective immune response against C. auris. From the results described here, it is

5 evident that C. auris is not cytotoxic nor pro-inflammatory to intact skin epithelial cells

6 or epidermis tissue. Only following induction of a wound in these models did C. auris

7 elicit any significant response by the host. From this, it could be suggested that

8 under normal immunological conditions, the organism is non-invasive, and any

9 immune response from the host is minimal. These postulations have been confirmed

10 elsewhere, whereby immunocompetent mice were more resistant to C. auris

11 infection than immunocompromised mice [15]. Such findings have been seen in

12 humans; invasive C. auris infections generally occur in critically ill patients with

13 serious underlying medical conditions resulting in haematological deficiencies and/or

14 immunosuppression [59-61]. In the context of this study, the observed non-invasive

15 phenotype of C. auris may simply be due to lack of hyphal formation, whereby yeast

16 cells colonize the periphery of the skin yet do not invade the underlying layers unless

17 in the presence of a wound. Indeed, similar observations have been made

18 elsewhere. Horton et al (2020) recently showed that C. auris forms layers of cells on

19 the periphery of porcine skin ex vivo, however, these structures of C. auris are

20 devoid of pseudohyphae or hyphae [17].

21 A limitation from previous studies is the failure investigate the host response to the

22 Agg phenotype expressed by certain isolates. We and others have previously shown

23 that the non-Agg isolate of C. auris are more virulent in G. mellonella larvae models

24 than the Agg counterparts, possibly resulting from enhanced dissemination rates of

25 the single cells [6, 10]. Here, the Agg NCPF 8978 isolate was considerably more

1 cytotoxic and pro-inflammatory than non-Agg NCPF 8973 in the two- and three-

2 dimensional skin models. At this juncture it is unknown why such a response occurs

3 in the host. Murine model of candidiasis have shown that C. auris can accumulate in

4 the kidney of the mice in the form of aggregates [7, 12, 13], suggestive that

5 aggregation may occur in vivo to enable persistence and survival. However, to date,

6 no studies have investigated the host response to such C. auris aggregates in vivo. It

7 could simply be that the Agg phenotype generates a cluster of cells with increased

8 pathogenic traits to induce a greater host response than single cells. This was

9 confirmed by the upregulation of two key adhesin and proteinase genes, ALS5 and

10 SAP5, in the Agg NCPF 8978 isolate compared to non-Agg NCPF 8973 in the skin

11 epidermis model. Interestingly, similar observations have been described in biofilm-

12 dispersed single cells vs. aggregates in model bacteria such as Pseudomonas

13 aeruginosa. As such, dispersed aggregates of P. aeruginosa possess enhanced

14 antibiotic resistance traits, likely due to encapsulation by extracellular matrix, and

15 greater immune evasion techniques over dispersed single cells[62-64]. Conversely, it

16 could be argued that the single cell phenotype of C. auris exhibits a level of immune

17 evasion (as postulated elsewhere [16, 65]), which may explain the lack of response

18 by the host to this phenotype. Future studies must continue to investigate the unique

19 Agg phenotype of C. auris to fully clarify the organisms’ pathogenic mechanisms.

20 These investigations must consider interactions between C. auris and other

21 organisms that comprise the skin and/or wound microbiomes, which may function as

22 important beacons for host invasion of C. auris.

23 The identification of five geographically and phylogenetically distinct clades of C.

24 auris [2, 9], containing isolates capable of forming aggregates with enhanced drug

25 resistance, has meant that unravelling the pathogenic mechanisms employed by C.

1 auris in vitro and in vivo remains extremely difficult. This study has highlighted

2 different pathogenic signatures of Agg and non-Agg forms of C. auris in biofilms and

3 during host invasion in vitro. Of course, given the level of heterogeneity amongst

4 isolates, in-depth analyses from this work are limited to one Agg and one-Agg

5 isolate. As such, future studies must continue to investigate these unique phenotypic

6 traits of different Agg and non-Agg isolates of C. auris to fully understand the

7 persistence of this nosocomial pathogen in the healthcare environment, and whether

8 such traits are comparable amongst the diverse isolates.

10 Acknowledgements

11 The authors would like to thank the Glasgow Imaging Facility (University of Glasgow)

12 and Margaret Mullin for assistance in scanning electron microscopic techniques. The

13 authors would like to acknowledge funding support of the BBSRC Industrial

14 GlaxoSmithKline CASE PhD studentship for Christopher Delaney (BB/P504567/1).

16 Declaration of interest

17 The authors declare no conflicts of interest.

19 References

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14 Figure legends

15 Figure 1 – Schematic diagram depicting the experimental set up for the three-

16 dimensional co culture of skin epidermis and Candida auris. A 17- day mature

17 reconstructed human epidermis (RHE) on 0.5 cm2 inserts was purchased from

18 Episkin (Skin Ethic™). Inserts were carefully lowered into 24- well plates containing

19 maintenance media supplied by the company (panel A). To assess the host

20 response to aggregative and non-aggregative C. auris, control and wounded tissue

21 was co-cultured with both isolates (NCPF 8973 and NCPF 8978) (panel B). A total of

22 2 x 106 fungal cells in 100 µL PBS was added to the tissue and incubated overnight

o 23 at 37 C, 5% CO2. For some tissues, prior to addition of fungal inoculum, three

24 scratch wounds were induced using a sterile 19 G needle across the surface of the

25 tissue. For visual representation of phenotype, scanning electron microscopy images

26 included in panel B from 24 h biofilms, clearly showing the differences in cellular

27 phenotypes between the two C. auris isolates. These images were taken at x 1000

28 magnification as viewed under a JEOL JSM-6400 scanning electron microscope

29 (samples processed as previously described [66]).

1 Figure 2 – Non-aggregative and aggregative Candida auris biofilm

2 heterogeneity. Biomass and impedance measurements were used as measures of

3 biofilm formation of 26 isolates of C. auris (n=14 for non-aggregative and n=12 for

4 aggregative phenotype). For biomass assessment, 1 x 106 fungal cells were seeded

5 in 96-well plates and biofilm developed for 4 h or 24 h prior to crystal violet staining.

6 Panels A and B show the differences in absorbance at 570nm of the non-

7 aggregative and aggregative, at 4 h and 24 h, respectively. The heatmap shows the

8 average absorbance at 570nm for each individual isolate at both timepoints (C and

9 D). The formation of C. auris biofilms in real time was monitored using electron

10 impedance measurements on the xCELLigence real time cell analyser. Electron

11 impedance measurements are presented as cell index for all isolates, for biofilms

12 developed for 4 h and 24 h, respectively (E and F). The heatmap depicts the mean

13 cell index values for all non-aggregative (G) and aggregative (H) isolates. Red data

14 points indicate the two isolates selected for further analyses in this study (NCPF

15 8973 and NCPF 8978). Paired Student’s t-tests were used for statistical analyses

16 and significance between data determined at * p < 0.05 (** p < 0.01, *** p < 0.001,

17 **** p < 0.0001).

18 Figure 3 – Transcriptional profile of non-aggregative and aggregative Candida

19 auris during biofilm formation. RNA from planktonic cells and biofilms formed for

20 24 h of two isolates (NCPF 8973 and NCPF 8978) was used for RNA sequencing

21 and transcriptome analyses as described in the text. Venn diagrams in panels A and

22 B depict up-regulated genes in non-aggregative (A) or aggregative (B) phenotype in

23 either planktonic form (blue circle), biofilm form (red circle) or in both forms (blue and

24 red circle). In panels C and D, gene distribution of significantly upregulated genes in

25 biofilm forms were grouped for gene ontology analysis. Genes upregulated that

1 belong to the functional pathway cellular components are shown, whilst all other

2 pathways are included in supplementary Figure 2. A cut-off of 2-fold up-regulation

3 was used for gene ontology analysis using an adjusted P value of < 0.05.

4 Figure 4 – Cytotoxic and inflammatory effects of aggregative and non-

5 aggregative Candida auris on skin epithelial models in vitro. For these analyses,

6 a two- and three-dimensional skin epithelial model was used as schematically

7 described in Figure 1. Firstly, cytotoxicity in the models were determined by

8 quantifying the amount of lactate dehydrogenase (LDH) released by the human adult

9 epidermal keratinocytes (HEKa) (A) and skin epidermis (B) following co-culture with

10 the aggregative (NCPF 8978) and non-aggregative (NCPF 8973) isolates of C. auris.

11 For this, data was presented as fold change relative to PBS control. To study the

12 host response to C. auris, a RT2 profiler array containing genes associated with

13 inflammation and fungal recognition was utilised to assess the transcriptional profile

14 of the skin epidermis following stimulation (C). Data in the heatmap presented as

15 Log2 fold change relative to PBS control. Finally, expression of two virulence genes,

16 ALS5 and SAP5 was determined in the isolates infected on the tissue, values

17 presented as % expression relative to the fungal specific house-keeping gene, β-

18 actin (D and E). All epithelial cells or tissues were infected in triplicate and statistical

19 significance determined from raw data CT values using unpaired Student’s t-tests for

20 comparison of two variables or one-way ANOVA with Tukey’s multiple comparison

21 post-test for more than two variables (* p < 0.05, ** and §§ p < 0.01, *** p < 0.001,

22 **** p < 0.0001).

29 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.20.052399; this version posted April 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.20.052399; this version posted April 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Up in Agg Up in Non-Agg

A bioRxiv preprint doi: https://doi.org/10.1101/2020.04.20.052399B; this version posted April 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Biofilm specific terms (Agg) cellular components C plasma membrane

membrane

fungal−type cell wall

P.value

m r

C 0.0025 e C 0.0020 T cell surface 0.0015 O 0.0010 G 0.0005

integral component of plasma membrane

anchored component of membrane

hyphal cell wall

0 5 10 15 20 25 Genes Biofilm specific terms (non-Agg) cellular components

D peroxisome

glycine cleavage complex

P.value

m r

C 0.014 e C 0.012 T 0.010 O 0.008 G 0.006

myelin sheath

glyoxysome

0 2 4 6 8 Genes bioRxiv preprint doi: https://doi.org/10.1101/2020.04.20.052399; this version posted April 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.