Metabolic Profiling of Candida auris, a Newly-Emerging Multi-Drug Resistant Candida Species, by GC-MS

Candida auris, a newly-emerging Candida species, is a serious global health threat due to its multi-drug resistant pattern, difficulty to diagnose, and the high mortality associated with its invasive and bloodstream infections. Unlike C. albicans, and C. dubliniensis which can form true hyphae, C. auris grows as yeast or pseudohyphae and is capable of developing biofilms. The reasons for the inability of C. auris to form true hyphae are currently unknown. Metabolites secreted by microorganisms, including Candida, are known as important factors in controlling morphogenesis and pathogenesis. Metabolic profiling of C. auris and C. albicans cultures was performed using gas chromatography–mass spectrometry (GC–MS). Compared to C. albicans, C. auris secreted several hyphae-inhibiting metabolites, including phenylethyl, benzyl and isoamyl alcohols. Furthermore, a biofilm-forming metabolite—tyrosol—was identified. On the other hand, several other biomarkers identified from C. auris but not from C. albicans cultures may be produced by the organism to overcome the host immune system or control fungal adaptations, and hence ease its invasion and infections. The results from this study are considered as the first identification of C. auris metabolic activities as a step forward to understand its virulence mechanisms.

prominent increase in palmitelaidic acid (1.8 ± 0.34), and at lower extent, benzyl alcohol (0.2 ± 0.06%) as well as isoamyl alcohol (0.57 ± 0.19%) was detected ( Figure 4A and Supplementary Table 1).   The secreted metabolites in CAU09 cultures were further compared at 4 and 16 h incubation periods ( Figure 2C and D). Both phenylethyl alcohol, and tyrosol, were increased by 10 and 4-fold, respectively, within 16 h of incubation versus 4 h cultures ( Figure 2C and D), while little changes were detected in the levels of benzyl and isoamyl alcohols. It is reported that the aforementioned alcohols including phenylethyl, benzyl and isoamyl alcohols are hyphae-inhibiting metabolites [3,12,23], while tyrosol is a filamentation and biofilm-forming metabolite [16]. The production of phenylethyl, benzyl and isoamyl alcohols is consistent with the yeast form of C. auris.
The aforementioned changes in metabolites were compared among four other C. auris strains along with C. albicans ATCC10231 (Heatmap in Figure 3, Figure 5A and Supplementary Table 1). A similar pattern was obtained in all other C. auris strains. Specifically, C. auris strains secreted high percentages of aromatic alcohols such as phenylethyl alcohol (~7-11%), benzyl alcohol (~0.1-0.2%), and at a lower extent, isoamyl alcohol. Palmitelaidic acid (~1-2%) was also detected in C. auris strains. In contrast, none of these metabolites was identified in C. albicans ATCC10231 except decanoic acid which was detected in very low amount (0.09 ± 0.023) ( Figure 5A and Supplementary  Table 1). These results indicate that both phenylethyl alcohol and tyrosol and to some extent palmitelaidic acid are likely critical C. auris morphogenic metabolites at the tested condition. The secreted metabolites in CAU09 cultures were further compared at 4 and 16 h incubation periods ( Figure 2C,D). Both phenylethyl alcohol, and tyrosol, were increased by 10 and 4-fold, respectively, within 16 h of incubation versus 4 h cultures ( Figure 2C,D), while little changes were detected in the levels of benzyl and isoamyl alcohols. It is reported that the aforementioned alcohols including phenylethyl, benzyl and isoamyl alcohols are hyphae-inhibiting metabolites [3,12,23], while tyrosol is a filamentation and biofilm-forming metabolite [16]. The production of phenylethyl, benzyl and isoamyl alcohols is consistent with the yeast form of C. auris.
The aforementioned changes in metabolites were compared among four other C. auris strains along with C. albicans ATCC10231 (Heatmap in Figure 3, Figure 5A and Supplementary Table S1). A similar pattern was obtained in all other C. auris strains. Specifically, C. auris strains secreted high percentages of aromatic alcohols such as phenylethyl alcohol (~7-11%), benzyl alcohol (~0.1-0.2%), and at a lower extent, isoamyl alcohol. Palmitelaidic acid (~1-2%) was also detected in C. auris strains. In contrast, none of these metabolites was identified in C. albicans ATCC10231 except decanoic acid which was detected in very low amount (0.09 ± 0.023) ( Figure 5A and Supplementary Table S1). These results indicate that both phenylethyl alcohol and tyrosol and to some extent palmitelaidic acid are likely critical C. auris morphogenic metabolites at the tested condition.     The data display the mean of the relative percentage ± standard error of the mean. The data was graphed using Box-and-Whiskers Plots and analyzed by one-way analysis of variance (ANOVA) using Bonferroni's Multiple Comparison Test. P value < 0.05 was considered as significant. The standard error represents the mean of 4 replicas of two independent experiments. The level of significance was indicated by asterisks.
Farnesol was absent in both C. auris and C. albicans cultures. The absence of farnesol in C. albicans culture is expected, since it is a yeast-to-hyphal inhibitory metabolite [15], and the growth condition favored hyphal growth. It is prudent to mention that C. auris harbors a homologue for C. albicans farnesyl synthase, the rate-limiting enzyme in farnesol biosynthesis [26], with 79% amino acid identity. Equally important, non-albicans species of Candida are known to produce ~8-35 times lower levels of farnesol when compared to C. albicans [27]. Therefore, the lack of detection of any farnesol could be due to lower limits of production consistent with what is seen with non-albicans species. This result indicated that farnesol expression was not required in C. auris culture condition under study and hence confirmed that other factors may be involved in maintaining the growth of C. auris as yeast form. Importantly, farnesol is known to have reversal effect on tyrosol [16]; and production of tyrosol by C. auris was favored at this condition, a condition suitable for biofilm development. Collectively, these results indicate that fundamental biological processes are under complex positive and negative control by environmental conditions in which these aromatic alcohols are secreted. Future studies focusing on gene expression analysis and investigation of transcription factors that govern yeast-to-hyphae switch might shed light on the reasons why C. auris lacks the ability to form true hyphae.
Decanoic, 10-undecenoic and palmitelaidic were acid metabolites identified in C. auris cultures that have never been detected by other Candida Spp. (Figure 3, 5A and Supplementary Table 1). This result raises the possibility of using these acid metabolites as biomarkers to aid in the organism diagnosis. The detected metabolites were also reported as hyphae-inhibitory substances [28][29][30], a situation may favor the growth of the organism in the yeast form. The metabolites produced by the organism for the purpose of protection and colonization. The quantity of each metabolite was represented as the relative percentage by measuring the area under the peak of each metabolite in relation to the total areas of all other metabolites detected in the extract. The data display the mean of the relative percentage ± standard error of the mean. The data was graphed using Box-and-Whiskers Plots and analyzed by one-way analysis of variance (ANOVA) using Bonferroni's Multiple Comparison Test. P value < 0.05 was considered as significant. The standard error represents the mean of 4 replicas of two independent experiments. The level of significance was indicated by asterisks.
Farnesol was absent in both C. auris and C. albicans cultures. The absence of farnesol in C. albicans culture is expected, since it is a yeast-to-hyphal inhibitory metabolite [15], and the growth condition favored hyphal growth. It is prudent to mention that C. auris harbors a homologue for C. albicans farnesyl synthase, the rate-limiting enzyme in farnesol biosynthesis [26], with 79% amino acid identity. Equally important, non-albicans species of Candida are known to produce~8-35 times lower levels of farnesol when compared to C. albicans [27]. Therefore, the lack of detection of any farnesol could be due to lower limits of production consistent with what is seen with non-albicans species. This result indicated that farnesol expression was not required in C. auris culture condition under study and hence confirmed that other factors may be involved in maintaining the growth of C. auris as yeast form. Importantly, farnesol is known to have reversal effect on tyrosol [16]; and production of tyrosol by C. auris was favored at this condition, a condition suitable for biofilm development. Collectively, these results indicate that fundamental biological processes are under complex positive and negative control by environmental conditions in which these aromatic alcohols are secreted. Future studies focusing on gene expression analysis and investigation of transcription factors that govern yeast-to-hyphae switch might shed light on the reasons why C. auris lacks the ability to form true hyphae.
Decanoic, 10-undecenoic and palmitelaidic were acid metabolites identified in C. auris cultures that have never been detected by other Candida Spp. (Figure 3, Figure 5A and Supplementary Table S1). This result raises the possibility of using these acid metabolites as biomarkers to aid in the organism diagnosis. The detected metabolites were also reported as hyphae-inhibitory substances [28][29][30], a situation may favor the growth of the organism in the yeast form.  The quantity of each metabolite was represented as the relative percentage by measuring the area under the peak of each metabolite in relation to the total areas of all other metabolites detected in the extract. The data display the mean of the relative percentage ± standard error of the mean. The data was analyzed by two-way analysis of variance (ANOVA). P value < 0.05 was considered as significant. The standard error represents the mean of 4 experimental replicates of two independent biological experiments. The level of significance was indicated by asterisks.

C. auris Produced Metabolic Fermentation Products, Known for Colonization and Invasion
Within the host, the pathogen must efficiently compete for nutrients with host cells [45] and its metabolism is affected in a niche-specific fashion [46]. Microbial pathogens produce metabolic products to overcome the host resistance mechanisms and hence allow the pathogen to colonize and invade. Such metabolic fermentation products may modulate the host immune system and change the body temperature or pH [47,48]. Compared to C. albicans, C. auris produced fermentation metabolic products including hexanoic acid (caproic acid), 2,3-butanediol [49], 2-propenoic acid (acrylic acid) [50], 3-hydroxypropanoic acid (hydracrylic acid) [51] and the yeast-specific fermentation product methionol [52] (Figure 3 and Supplementary Table S1).

Organisms and Culture Conditions
Five different C. auris clinical isolates were obtained from Centers for Disease Control and Prevention (CDC, Atlanta, GA, USA). These were CAU-01 (East Asian clade, ear), CAU-03 (African clade, blood), CAU-05 (South American clade, blood), CAU-07 (South Asian clade, blood), and CAU-09 (South Asian clade, bronchoalveolar lavage [BAL]). C. albicans reference strain ATCC10231 was used as a control. Candida isolates were allowed to grow separately in yeast nitrogen base (YNP) supplemented with 2% glucose at 37 • C for 16 h. Cells were washed with phosphate buffered saline (PBS), and the inoculums adjusted to 5 × 10 6 yeast/mL with RPMI-1640 (Sigma-Aldrich, Torrance, CA, USA) using hemacytometer [53]. The cultures were further incubated for 24 h at 37 • C in 125 mL Corning culture flasks, after which cell-free supernatants were collected by filtration using 0.2 µM Whatman filter paper.

Preparation of Samples for GC-MS Analysis
The filtered supernatants collected separately from each Candida isolate was extracted with chloroform (Fisher Scientific, Santa Clara, CA, USA). The chloroform layer was dehydrated over anhydrous sodium sulphate (Fisher Scientific) followed by evaporation using rotatory evaporator (Buchi, Essen, Germany). The residue collected from each extracted culture was dissolved in 500 µL chloroform prior to GC-MS injection. Furthermore, 100 µL of the chloroform extract was derivatized by adding 50 µL of N-trimethylsilyl-N-methyl trifluoroacetamide and trimethylchlorosilane (MSTFA + 1% TMS) followed by incubation at 50 • C for 30 min prior to GC-MS analysis.

GC-MS Spectrometry
GC-MS analysis was performed using a QP2010 gas chromatography-mass spectrometer (GC-2010 coupled with a GC-MS QP-2010 Ultra) equipped with an auto-sampler (AOC-20i+s) from Shimadzu (Tokyo, Japan), using Rtx-5ms column (30 m length × 0.25 mm inner diameter × 0.25 µm film thickness; Restek, Bellefonte, PA, USA). Helium (99.9% purity) was used as the carrier gas with the column flow rate of 1 mL/min. The column temperature regime was initially adjusted at 35 • C for 2 min; followed by an increase in a rate of 10 • C/min to reach 250 • C. The temperature was then increased by 20 • C/min until reaching 320 • C and kept for 23 min. The injection volume and injection temperature were 1 µL and 250 • C using splitless injection mode, respectively. The mass spectrometer operated in electron compact mode with electron energy of 70 eV. Both the ion source temperature and the interface temperature were set at 240 • C and 250 • C, respectively. The MS mode was set on scan mode starting from 35 to 450 m/z with a scan speed of 1428. Data collection and analysis were performed using MSD Enhanced Chemstation software (Shimadzu). Product spectra were identified by comparison of the measured fragmentation patterns to those found in the NIST 08 Mass Spectral Library.

Bioinformatics detection of farnesyl synthase protein in C. auris
A local BLAST database of C. auris genome was created using Basic Local Alignment Search Tool plus (BLAST+) [54]. Farnesyl synthase protein sequence (Accession #KGQ89011) from C. albicans was downloaded from NCBI and used to search the generated C. auris genome using tblastn command. The search revealed a sequence with 79% identity within 577 score.

Statistical analysis
The data was collected and graphed using either Excel to generate the heatmap or Graph Pad (5.04, La Jolla, CA, USA) for Windows to generate the Box-and-Whiskers Plots and group comparison. The quantification of metabolites extracts were analyzed by one-way analysis of variance (ANOVA) using Bonferroni's Multiple Comparison Test or two-way analysis of variance as indicated per each graph. P value < 0.05 was considered significant. The standard error represents the mean of 4 replicas of two independent experiments (two experimental replicates per each independent biological experiment). Each extract was divided into two and extracted separately followed by derivatization prior to GC-MS analysis.

Conclusions
Metabolites produced by microorganisms may be used as communication signals that allow the microorganisms to share information and hence provide some regulatory responses during infection. Metabolic profiling can be of great help to identify critical determinant of pathogens and hence can control disease progression. Candida spp. is known for secreting metabolites that control its morphogenesis. C. auris is a newly-emergent Candida species that is maintained in the yeast phenotype during its growing stages. Analysis of C. auris cultures by GC-MS showed that the fungus produces diverse hyphae-inhibiting metabolites in addition to biofilm-forming tyrosol that are distinct from C. albicans. The results provided in this research is the first to identify C. auris metabolic profiling; and thus can shed light on the virulence of this multi-drug resistant yeast and may eventually lead to the development of new strategies for in deep investigation including gene expression analysis and study of morphogenesis-regulatory pathways.