The dimorphic fungus Candida albicans is a commensal and opportunistic fungal pathogen of humans. It secretes at least four small lipophilic molecules, farnesol and three aromatic fusel alcohols. Farnesol has been identified as both a quorum sensing molecule (QSM) and a virulence factor. Our gas chromatography (GC)-based assay for these molecules exhibits high throughput, prevention of analyte loss by avoiding filtration and rotary evaporation, simultaneous cell lysis and analyte extraction by ethyl acetate, and the ability to compare whole cultures with their cell pellets and supernatants. Farnesol synthesis and secretion were separable phenomena and pellet:supernatant ratios for farnesol were high, up to 12:1. The assay was validated in terms of precision, specificity, ruggedness, accuracy, solution stability, detection limits (DL), quantitation limits (QL), and dynamic range. The DL for farnesol was 0.02 ng/µl (0.09 µM). Measurement quality was assessed by the relative error of the whole culture versus the sum of pellet and supernatant fractions (WPS). C. albicans strain SC5314 grown at 30 °C in complex and defined media (YPD and mRPMI) was assayed in biological triplicate 17 times over 3 days. Farnesol and the three aromatic fusel alcohols can be measured in the same assay. The levels of all four are greatly altered by the growth medium chosen. Significantly, the three fusel alcohols are synthesized during stationary phase, not during growth. They are secreted quickly without being retained in the cell pellet and may accumulate up to mM concentrations. Key points: • Quantitative analysis of both intra- and extracellular farnesol, and aromatic fusel oils. • High throughput, whole culture assay with simultaneous lysis and extraction. • Farnesol secretion and synthesis are distinct and separate events.
Keywords: Farnesol; Aromatic fusel alcohols; Candida albicans; Quorum sensing; Fungal signaling
Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s00253-022-12165-w.
The dimorphic fungus Candida albicans is a major commensal and opportunistic fungal pathogen of humans (Rai et al. [
Effective approaches to answer these questions require reliable, accurate, and cost-effective assays for farnesol measurement. The need for this straightforward goal is reinforced by the large variability in farnesol measurements reported by different laboratories. For instance, our lab typically reported 2–4 µM farnesol while Weber et al. ([
The strain used throughout method development and validation was C. albicans SC5314 (Hickman et al. [
Growth curves were performed in Fernbach flasks containing 1 L of growth medium aerated by shaking at 225 rpm (rounds per minute) at 30 °C. Following inoculation with overnight cultures to an initial OD
Experiments were performed on Agilent Technologies 7890 B gas chromatography system (Santa Clara, CA, USA) equipped with autosampler and flame ionization detector (FID). Chromatographic separation was performed on an HP-Innowax (Agilent 19091 N-133I) column with a film thickness of 0.25 µm, diameter of 0.25 mm, and length of 30 m. An injection volume of 2 µL was used (splitless) with hydrogen as carrier gas at an initial temperature of 90 °C ramped at a rate of 30 °C/min to a final temperature of 245 °C and held there for 7 min. Data files were batched and analyzed using OpenLab Services (Agilent Technologies) with software calculated response factors based on standard calibrator solutions. Quantitation settings were calculated using the peak area from the 17.2 ng/µL 1-tetradecanol internal standard.
A 4 mM 1-tetradecanol internal standard stock solution was prepared by dissolving 430 mg of 1-tetradecanol in 500 mL ethyl acetate. Two 100 × analyte standard solutions, a low concentration standard (standard A) and a high concentration standard (standard B) in methanol were prepared fresh weekly throughout this study. Standards A and B contained 4000 and 10,000 ng/µL methionol (3-(methylthio)-1-propanol, CAS: 505–10-2, Millipore-Sigma), 200 and 4000 ng/µL phenethyl alcohol (2-phenylethanol, CAS: 60–12-8, Millipore-Sigma), 100 and 500 ng/µL farnesol (trans,trans-farnesol 96%, CAS: 106–28-5, Millipore-Sigma), 1500 and 4500 ng/µL tyrosol (2-(4-hydroxyphenyl)ethanol, CAS: 501–94-0, Millipore-Sigma), and 500 and 2000 ng/µL tryptophol (3-(2-hydroxyethyl)indole, CAS: 526–55-6, Millipore-Sigma), respectively. Both standards were prepared using fresh 5000 × methionol, 10,000 × phenethyl alcohol, 1000 × tyrosol and tryptophol working stocks, and fresh 10,000 × (45 mM) farnesol. Internal standard stock solution was stable for at least 6 months and this stability was confirmed for each new extraction solution batch via extraction of blank media sample. All stock solutions were stored at − 20 °C.
Two ten mL aliquots were harvested from culture into glass screw cap test tubes (15 × 123 mm). One aliquot was directly extracted and measured for analyte concentrations; this we refer to as the whole culture (W). The second aliquot was centrifuged for 5 min at 2000 rpm in a JS7.5 swinging bucket rotor in a Beckman model J2-21 centrifuge (Beckman Coulter Life Sciences, Indianapolis, IN, USA). After centrifugation, the cell-free fraction was transferred into a clean tube; this we refer to as the supernatant fraction (S). The pelleted cells were reconstituted in 10 mL fresh medium by vortexing for 2 min. This sample is the pellet fraction (P). It is important that the pellet not be too tightly packed (only 2000 rpm for 5 min) so that the pelleted cells can be fully resuspended. The pellet and supernatant fractions were subsequently extracted and measured for their analyte concentrations. The relative error for these three values reflected the quality of fractionation and analyte measurement for each biological data point, using the following Eq. 1:
Graph
where W = whole culture in ng/µL, P = pellet fraction in ng/µL, and S = supernatant fraction in ng/µL. We treat W as the expected value and P + S as the observed value. The values are absolute because W − (P + S) can be negative. Figure 1 shows a single time point (24 h) for YPD and mRPMI grown cells, comparing the farnesol content of a whole culture versus the sum of the supernatant and pellet fractions. They are close to identical, meaning a whole-pellet-supernatant (WPS) relative error value from Eq. 1 of close to zero.
Extraction solution was prepared by adding 2 mL of 4 mM 1-tetradecanol stock solution to 98 mL ethyl acetate for a 1-tetradecanol assay concentration of 17.2 ng/µL. The extraction solution (1.5 mL) is added to 10 mL of sample in glass screw cap tubes. Each tube is covered with Duraseal® (Diversified Biotech, Boston, MA, USA), capped, and vortexed on high for 2 min inverting intermittently. Then 1.0 mL of 5 M NaCl is added per tube followed by vortexing for an additional 10 s and the extraction tubes are centrifuged at 3000 rpm for 12 min in a Beckman J2-21 centrifuge with a JS 7.5 swinging bucket rotor. The organic phase is removed with individual glass Pasteur pipets into 2 mL amber autosampler vials (Agilent Technologies 5182–0716) with PTFE/red silicone septa screw caps (Agilent Technologies 5185–5820) and 50 µl inserts (JG Finneran Associates, Vineland, NJ, USA, 4005BS-625) and stored for up to one week at − 20 °C until assayed by gas chromatography. An extraction ratio of 1.5 mL per 10 mL sample was used for all experiments in this paper in order to achieve optimal sensitivity. With the use of standard calibrator solutions and internal standard, assay volumes could be adjusted for experimental optimization.
Fifty mL of fresh GPP media in 250 mL flasks was spiked with 2 × standard A and treated as an inoculated culture by shaking at 225 rpm at 30 °C. The flasks were either covered with a foam plug or sealed with Duraseal® and Parafilm® (Bemis Flexible Packaging, Neenah, WI, USA). An aliquot of media was taken prior to shaking to establish a baseline and then the cell-free cultures were assayed at 24-, 48-, and 72-h time points. Percent reduction values are presented as the mean of four independent experiments ± standard deviation.
The binding of farnesol to seven filter types (all 25 mm in diameter) was measured by filtering 30 mL (3 × 10 mL) of GPP media containing 2 × standard A. A prefilter aliquot was taken for baseline values. All filters were purchased from Thermo Fisher Inc. (Waltham, MA, USA). They were the following: glass fiber GF/A Whatman 1820–024; Nylon 09-719C; surfactant-free cellulose acetate 190–9920; cellulose acetate Corning 430,636: polycarbonate Nucleopore Whatman 111,106; cellulose nitrate Whatman 7195–004; and mixed cellulose esters Celltreat Scientific Products 229,751. Each filter type was tested on 3 or 4 occasions in triplicate. Percent reduction values are presented as the mean of at least three independent experiments ± standard deviation. Both syringe and vacuum filtration were employed. GC analyte measurements were done following extraction.
Statistical analyses were performed using Microsoft Excel (Version 16.61, Microsoft Office, Las Vegas, NV, USA) and GraphPad Prism Software (Version 9.3.1, San Diego, CA, USA). All biological data are represented as mean ± SEM of at least 3 biological replicates unless otherwise stated. Differences between two groups were accessed by unpaired two-tailed Student's t-test. Differences were considered significant at p < 0.05. In cross comparing analytes for instrument validation, percent relative standard deviation (RSD%) is often reported.
The primary goal of this work was to develop a high-throughput, quantitative assay for farnesol and aromatic fusel oils (phenethyl alcohol, tyrosol and tryptophol) measured in whole cultures, as well as pellet and supernatant fractions. This assay can, for example, be used to assess the levels of farnesol produced on a per cell basis throughout a complete growth curve. Given the need for technical and biological replicates at each time point, using this assay throughout a complete growth curve requires several hundred farnesol measurements for each culture studied. Thus, we needed a simple, scalable assay superior to those we had employed originally (Hornby et al. [
Graph: Fig. 1 Workflow for quantitative analysis of farnesol and the aromatic fusel alcohols. Key steps include: (
To optimize analyte sensitivity, we compared sample volumes of 5 or 10 mL with extraction by 0.5–2.0 mL ethyl acetate, followed by 0.5–1.0 mL of 5 M NaCl or KCl, and various concentrations of 1-tetradecanol internal standard to give peak intensities close to those of the sample analytes, especially farnesol. Eliminating premeasurement steps now known to remove farnesol allowed us to use small sample and extraction volumes of 10 mL and 1.5 mL, respectively. Salt is added to decrease ethyl acetate aqueous solubility and improve the aqueous/ethyl acetate phase separation. The combination of a 10 mL sample with 1.5 mL of ethyl acetate and 1-tetradecanol as an internal standard at 17.2 ng/µL achieved the requisite cell lysis and phase separation, with appropriate GC signal intensities for biologically relevant concentrations of the analytes (Fig. 2). Microscopic examination following staining with trypan blue showed that > 99.9% of the yeast cells were killed by this treatment. This dual role for ethyl acetate is advantageous in that there is no time lag between cell lysis and extraction. To be sure that we had achieved complete cell lysis, we compared samples with and without a glass bead vortex step. Since no differences in trypan blue staining or analyte extraction were detected with and without vortexing, the glass bead step was omitted. The fusel alcohols derived from the branched-chain amino acids (isobutanol, isoamyl alcohol, and amyl alcohol) are more volatile than those derived from the aromatic amino acids and their GC peaks unfortunately are under the solvent peak for ethyl acetate.
Graph: Fig. 2 Representative GC chromatogram of standard A mix. The mix contains methionol (40 ng/μL), phenethyl alcohol (2 ng/μL), 1-tetradecanol (17.2 ng/μL), E,E-farnesol (1 ng/μL), tyrosol (15 ng/μL), and tryptophol (5 ng/μL)
We know that farnesol is volatile in that it is a common perfume ingredient (Nickerson et al. [
Table 1 Percent reduction in farnesol following evaporation. Data are the means of % farnesol loss ± SD of two independent experiments
Time (hours) Mean % loss farnesol ± SD Unsealed Sealed 24 26.41 ± 1.40 14.68 ± 0.66 48 43.53 ± 0.02 18.71 ± 0.45 72 56.40 ± 4.47 23.92 ± 2.74
Similarly, previous methods regularly included filtration to remove cells remaining after centrifugation without giving sufficient regard to the chemical composition of the filters and their ability to bind farnesol (Table 2). All seven of the filter types tested caused substantial reductions in the levels of farnesol detected in standard growth medium. The glass fiber filters gave the least reduction while the cellulose acetate filters gave the largest reduction (Table 2). Equivalent reductions were observed using vacuum filtration or syringe filtration (Table 2). This high level of removal by filtration was specific for farnesol in that none of the fusel alcohols tested showed greater than 5% reduction. Previous reports from this lab used cellulose nitrate filters (Hornby and Nickerson [
Table 2 Percent reduction in farnesol following filtration
Filter type Mean % reduction ± SEM Nb No filter 0 ± 1.82 3S GF/A (glass fiber) 27.61 ± 0.59 3V Nylon (0.22 µm) 55.86 ± 0.94 4S Cellulose acetate (0.22 µm)c 80.47 ± 0.76 3S Cellulose acetate (0.22 µm) 98.4 ± 0.20 3V Polycarbonate nucleopore (0.22 µm) 48.13 ± 0.83 3V Cellulose nitrate (0.22 µm) 71.06 ± 0.89 3V Mixed cellulose ester (0.22 µm) 52.96 ± 1.74 3S
Five general experiments for validation were employed: Instrument precision and analyte specificity were conducted with the analytes (standard A) in water; linearity and range followed by assay robustness and reproducibility were conducted with the analytes (standard A and standard B) in water and two growth media (YPD and RPMI); and extraction efficiency and accuracy were conducted on whole cell cultures and fractionated parts spiked with known amounts of the analytes (standard A and standard B). Details are described in the next five sections.
A typical GC chromatogram showed well separated peaks for all analytes and the 1-tetradecanol internal standard (Fig. 2). Ten replicate injections of analyte and 1-tetradecanol were conducted with a sample of standard A. All RSD% values for mean area and ratio of analyte to 1-tetradecanol internal standard (IS) were under 3% with an average area RSD% for all analytes of 0.60 ± 0.84 SD, and average analyte to IS ratio of 1.55 ± 0.58 SD (Supplemental Table S1). The GC instrument is precise for analyte measurement.
Chromatographic analyte separation was of little concern given the widely separate analyte retention times (Fig. 2). However, given the wide range of possible analyte concentrations, a series of 12 standard mixes containing biologically relevant concentrations were generated to detect any analyte signal interference. For each analyte the detection efficiency (analyte to IS ratio) was compared at both low and high concentrations, as defined by standard A and standard B, respectively (Supplemental Table S2-1). For instance, in the low comparison for phenethyl alcohol, the levels of phenethyl alcohol are determined when all 5 analytes are low (standard A) versus when only phenethyl alcohol is low, with the other four analytes at the standard B levels. Similarly, for the high comparison, the level of phenethyl alcohol when all 5 analytes are high (standard B) versus when only phenethyl alcohol is high, with the other four analytes at standard A levels. Each comparison, performed in triplicate, had %RSD for the analyte to 1-tetradecanol internal standard below 5% (Supplemental Table S2-2), suggesting that there is minimal to no signal interference within the biological range of these analytes, and that the assay is specific for analytes assessed.
A useful assay should work best for biologically relevant analyte concentrations. To determine the appropriate range for the fusel alcohols, C. albicans was grown in minimal media containing the amino acid precursor for that alcohol with and without ammonium tartrate. To determine the range for farnesol, the ratio of the farnesol area to IS area for cultures grown in YPD and mRPMI was assessed after 12 and 24 h, identifying values around 1 ng/μL (4.49 μM) farnesol (Fig. 3).
Graph: Fig. 3 Production of farnesol in YPD and mRPMI 1640 over 72 h. Farnesol production was accessed in A whole culture, B pellet fraction, C supernatant fraction, D OD600 normalized whole culture, E OD600 normalized pellet fraction, F OD600 normalized supernatant fraction, G pellet to supernatant ratio. Data are the means ± SEM of three biological replicates. Note the change in the time scale before and after 24 h for A, B, and C. Student's t-test was performed for comparison between YPD and mRPMI (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001). YPD is displayed in blue and mRPMI is displayed in red, respectively
To evaluate the linearity of each assay, standards A and B were diluted to make 8 concentrations of each analyte, exceeding the minimum suggestion of five by the International Council for Harmonization of Technical Requirements for Pharmaceutical for Human Use (ICH) (Bhardwaj et al. [
Table 3 Analyte observed and calculated linearity and dynamic range
Analyte Retention time Detection limit (ng/µL) Quantitation limit (ng/µL) Upper limit (ng/µL) Observed Calculated Observed Calculated Observed Water YPD RPMI Water YPD RPMI Water YPD RPMI Water YPD RPMI Methionol 3.30 6.25 0.80 1.00 4.05 2.91 2.41 12.50 3.87 0.40 12.27 8.82 7.31 150 Phenethyl alcohol 3.95 0.04 0.20 0.20 0.77 1.04 0.50 0.16 0.40 0.40 2.35 3.16 1.52 160 E,E-Farnesol 5.33 0 0.02 0.02 0.15 0.05 0.09 0.06 0.05 0.05 0.45 0.14 0.27 12 Tyrosol 7.31 0.04 0.90 0.90 2.30 1.48 1.51 0.16 4.48 0.45 6.98 4.48 4.58 45 Tryptophol 10.37 00.03 0.31 0.20 0.61 0.31 0.20 0.23 0.93 0.20 1.84 0.93 0.62 30
These QL values are for the calculated quantification limits. However, there were also many acceptable (< 0.20) WPS relative errors for earlier time points in the growth curves prior to QL being attained (Supplemental Table S4). These observed QL values suggest a broader dynamic range than those calculated. The observed QL values were defined by extracting 1:10, 1:50, and 1:100 dilutions of standards A and B in triplicate from each growth medium (Table 3). Concentrations with a relative error at or below 0.10 linearly compared to their undiluted standard were considered to be in the dynamic range with an observed QL. These values were 0.4 ng/μL for phenethyl alcohol in both YPD and mRPMI, and 0.05 ng/μL for farnesol in both YPD and mRPMI (Table 3). The tryptophol and tyrosol calculated QL and observed QL were the same for YPD grown cultures, but quite different for the mRPMI grown cultures (Table 3). These differences show the importance of examining and comparing both complex and chemically defined growth media. Finally, the observed detection limits are based on signal intensities that are greater than twofold above the noise levels for that analyte (Table 3).
To assure assay robustness and reproducibility standards A and B were prepared fresh weekly. To confirm this reproducibility, the analyte to 1-tetradecanol ratios of six independent preparations from different working stocks were done by three different researchers. Standards extracted from both complex and defined media showed average analyte RSD% values as follows: farnesol 6.21, phenethyl alcohol 5.27, tyrosol 3.51, tryptophol 3.74, and methionol 3.75 (Table 4). Thus, the standard preparations are reproducibly quantitative for multiple users and are suitable for routine use.
Table 4 Assay robustness and standard reproducibility
Standard Media Prep Methionol:IS ratio Phenethyl alcohol:IS ratio E,E-Farnesol:IS ratio Tyrosol:IS ratio Tryptophol:IS ratio A YPD 1 1.5134 0.8282 0.5085 1.1774 1.1662 2 1.5521 0.8245 0.5300 1.2173 1.2925 3 1.6070 0.8411 0.6115 1.2582 1.2170 4 1.6216 0.9644 0.5970 1.2770 1.2208 5 1.5991 0.8674 0.5886 1.2516 1.2715 6 1.5395 0.7934 0.4913 1.2416 1.2340 Average 1.5721 0.8532 0.5545 1.2372 1.2337 SD 0.0431 0.0596 0.0508 0.0353 0.0445 RSD% 2.74 6.98 9.17 2.85 3.61 RPMI 1 1.6404 0.8626 0.4868 1.3081 1.2215 2 1.7389 0.8710 0.5405 1.3115 1.2300 3 1.6789 0.8637 0.6178 1.2103 1.1078 4 1.7566 0.9971 0.5872 1.2865 1.0955 5 1.6542 0.8686 0.5565 1.1833 1.0873 6 1.6928 0.8454 0.5146 1.2334 1.1472 Average 1.6936 0.8847 0.5506 1.2555 1.1482 SD 0.0461 0.0558 0.0477 0.0541 0.0635 RSD% 2.72 6.31 8.66 4.31 5.53 B YPD 1 3.8134 15.1622 2.5819 3.5382 4.8995 2 3.8553 15.2778 2.6492 3.6579 5.1300 3 3.2805 14.5664 2.7555 3.5630 4.9772 4 3.6047 15.2476 2.9447 3.8655 5.1300 5 3.5648 16.2557 2.7498 3.6754 5.0096 6 3.7362 14.8853 2.6034 3.7141 5.2483 Average 3.6425 15.2325 2.7141 3.6690 5.0658 SD 0.2106 0.5689 0.1342 0.1176 0.1267 RSD% 5.78 3.73 4.95 3.21 2.50 RPMI 1 4.1139 15.3830 2.7038 3.8495 5.0325 2 4.0844 15.8329 2.6938 3.7712 5.0283 3 3.7374 15.0023 2.6991 3.8130 4.7374 4 4.0034 15.7146 2.8383 4.1199 4.7584 5 3.8584 16.8205 2.7188 3.7290 4.6892 6 4.0748 15.2868 2.6946 3.7732 4.9940 Average 3.9787 15.6733 2.7247 3.8427 4.8733 SD 0.1497 0.6367 0.0564 0.1419 0.1609 RSD% 3.76 4.06 2.07 3.69 3.30 Overall RSD% 3.75 5.27 6.21 3.51 3.74
An assay's accuracy and linearity can be determined by a spike recovery experiment wherein two cultures are compared, one of which has been spiked with a known standard while the other has not. How closely does the assay come to detecting the precise amount by which one exceeds the other? Also, given the vital importance of WPS relative error assessment in these assays, we wondered whether the % spike recoveries were equivalent for whole cultures, pellets, and supernatants. Thus, overnight YPD grown whole cultures and their pellet, and supernatant fractions were spiked in duplicate with both standard A and standard B and their individual recoveries were compared (Table 5). The overall percent analyte recoveries for both standards were: whole culture 97.08 ± 6.97, pellet 92.52 ± 8.02, and supernatant 102.81 ± 7.26 (Table 5). There was equivalent extraction and recovery for all three fractions and thus the whole cultures and fractions can be cross compared in analysis of their analyte measurement quality, i.e. their WPS relative error.
Table 5 % Spike recovery shows extraction efficiencies for whole cultures, pellets, and supernatants
Sample Standard Replicate Methionol Phenethyl alcohol E,E-Farnesol Tyrosol Tryptophol Whole culture A 1 100.19 93.79 99.88 82.87 82.34 2 101.95 97.43 109.87 88.35 89.34 B 1 96.88 102.38 99.06 98.96 102.94 2 98.65 93.88 97.89 100.01 104.92 Average 99.42 96.87 101.68 92.55 94.89 SD 2.16 4.05 5.52 8.33 10.86 Pellet fraction A 1 86.17 91.01 103.07 76.96 85.14 2 87.02 92.38 105.06 77.67 86.64 B 1 89.61 91.49 99.41 95.08 102.62 2 91.88 91.77 98.58 95.45 103.36 Average 88.67 91.66 101.53 86.29 94.44 SD 2.59 0.57 3.05 10.37 9.89 Supernatant fraction A 1 109.01 100.75 99.14 95.93 89.73 2 108.47 102.04 108.77 96.13 91.22 B 1 112.84 112.43 100.00 115.19 109.71 2 110.78 95.23 99.85 105.24 103.75 Average 107.77 102.61 101.94 103.12 98.60 SD 5.05 7.18 4.57 9.14 9.71
The data in Table 5 were collected at a single time point. It is also important to check whether these accuracy and % spike recovery considerations apply to cells at all stages of growth. Thus, four samples were taken roughly 12 h apart throughout the growth of the YPD- and mRPMI-grown cultures (Figs. 3 and 4) and examined for their % spike recovery. Under all conditions farnesol extraction efficiency and percent recovery was calculated at 74% or greater (Supplemental Table S5). At high cell densities, such as in stationary phase, phenethyl alcohol was produced at very high amounts (≻ 150 ng/µL, Fig. 4A) and the assay was unable to differentiate the 2 ng/µL of standard A spike phenethyl alcohol from unspiked samples (Supplemental Table S5); however, the larger analyte concentration of standard B was recovered at 72% or greater. These experiments indicate that, as expected, assay sensitivity decreases as analyte concentrations reach significantly high levels. Overall, we demonstrate efficient and overlapping recovery in the complex matrix of cell growth across the dynamic range of the assay for all analytes.
Graph: Fig. 4 Production of aromatic fusel alcohol in YPD and mRPMI 1640 over 72 h. Phenethyl alcohol (A), tyrosol (B), and tryptophol (C) were accessed in YPD (circles) and mRPMI (inverted triangle) between the whole culture (red), pellet fraction (blue), and supernatant fraction (green). Data are the means ± SEM of three biological replicates. Student's t-test was performed for comparison of whole culture fusel alcohol amounts between YPD and mRPMI (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001)
When considering the physiological significance of secreted molecules which are probably signaling molecules, it is important to quantify their concentrations at all stages of cell growth. Thus, we followed the production of farnesol and the aromatic fusel alcohols in triplicate for both YPD and mRPMI at 30 °C for 3 days (Figs. 3 and 4). Concentrations of the 4 analytes were measured in the whole culture, cell pellet, and supernatant at 90 min intervals over the growth phase (6 to 24 h) and at 12-h intervals thereafter. YPD and RPMI are two very different media commonly used for the growth of C. albicans. RPMI is a chemically defined medium containing all the amino acids, vitamins, and inorganic components typically needed by yeasts while YPD is a complex medium containing glucose, peptone, and yeast extract. The mRPMI replicates were supplemented with a mineral mix and buffered with 60 mM MOPS, pH 7.0. YPD was chosen to be part of this comparison because the chemical nature of its components, especially the peptone, would be focused at an ethyl acetate/aqueous interface, possibly interfering with analyte extraction and phase separation. However, the percent spike recoveries for the two growth media were comparable, with averages ± SD within 5% of each other (Supplemental Table S5). The secretion patterns varied greatly depending on the growth media (Figs. 3 and 4), as did the pellet:supernatant ratios (Fig. 3G) and the maximum analyte concentrations achieved (Table 6). These analyses, which are a direct consequence of the capabilities of our assay system, are the first to compare pellet and supernatant concentrations directly. They show that production and secretion are distinct and separable phenomena.
Table 6 Mean maximum analyte concentrations achieved in YPD and RPMI media over 72 h
Analyte YPD mRPMI 27.5 ± 2.7μM 46.6 ± 5.4 μM Phenethyl alcohol 1263.9 ± 26 μM 220.7 ± 15.8 μM Tyrosol 197.2 ± 30.2 μM 133.0 ± 18.5 μM Tryptophol 165.4 ± 7.1 μM 22.52 ± 0.8 μM Methionol Below QL Below QL
The farnesol production data are shown in Fig. 3. The whole culture, pellet, and supernatant values are in Fig. 3A–C, respectively, and these values are normalized to cell growth (OD
Figure 4 shows the aromatic fusel alcohol production data for whole culture, pellet, and supernatant fractions of both YPD and mRPMI grown cells. Data for phenethyl alcohol, tyrosol, and tryptophol are shown in Fig. 4A–C, respectively. Fusel alcohols are well known in yeasts as the byproducts of using amino acids as a less preferred nitrogen source, following removal of the nitrogen by a transaminase followed by the action of a decarboxylase, a dehydrogenase, and secretion (Hazelwood et al. [
The reliability of comparing whole culture values with the sum of the pellet and supernatant fractions (Eq. 1) is shown in Table 7. There are a total of 306 assay samples with values above their respective QL (Table 3) as follows: phenethyl alcohol 191 (62%), farnesol 233 (76%), tyrosol 121 (40%), and tryptophol 185 (60%) (Supplemental Table S4, green highlight). Methionol is left out of analysis because only 5 samples were above QL. Out of the 102 biological data points, all three assay samples were above their QL thresholds a total of: phenethyl alcohol 28 (27%), farnesol 72 (71%), tyrosol 0, and tryptophol 30 (29%). The average WPS relative error of each biological data point when all three assay samples were above the analyte QL are as follows: phenethyl alcohol 0.08 ± 0.05 SD, farnesol 0.11 ± 0.09 SD, tryptophol 0.08 ± 0.04 SD (Table 7). The standard deviation of the average relative error for farnesol is slightly skewed towards higher values given that 63 (88%) of the WPS data points within the dynamic range were less than 0.20, and 2 were above 0.30. We recommend that future applications of this method use a maximum WPS relative error value of 0.20. This cutoff point is especially important for single biological data points, such as a single extraction of a 24-h culture, while a higher cutoff point may be granted to time points that are supported by other data, such as those in a growth curve. All data points in the triplicate biological experiments for both YPD and mRPMI within the dynamic range are included here to display ruggedness (Table 7).
Table 7 Assay statistics; total assay samples 306, WPS biological data points 102. DR; dynamic range
Analyte Assay samples in DR ( WPS all in DR ( WPS RE Methionol 5 (2%) 0 NA Phenethyl alcohol 191 (62%) 28 (27%) 0.08 ± 0.05 E,E-Farnesol 233 (76%) 72 (71%) 0.11 ± 0.09 Tyrosol 121 (40%) 0 NA Tryptophol 185 (60%) 30 (29%) 0.08 ± 0.04
The overall measurement quality of this assay is displayed in the WPS relative error values for each analyte (Eq. 1) at each biological data point. The method was fully validated in terms of precision, specificity, ruggedness, accuracy, solution stability, detection limits, quantitation limits, and overall system suitability. Key features include the following: (
Detailed time courses following farnesol and the fusel alcohols during batch culture have not been presented before. The experiments presented as proof of principle for the usefulness of this assay compared analyte production (total, pellet and supernatant) in two different growth media over a 72-h time course (Figs. 3 and 4). The assays are quantitatively reliable in that they identify and avoid traps which previously caused major underestimates of the farnesol present. For instance, previous reports (Nickerson et al. [
During log growth, the per cell farnesol levels in the pellets increased ca. sixfold for mRPMI grown cells and threefold for YPD grown cells (Fig. 3E) whereas the OD normalized supernatant levels showed only a slight increase for the mRPMI cells and no increase for the YPD cells (Fig. 3F). When considering the physiological significance of a secreted molecule, it is important that a QSM should be secreted equivalently at all stages of cell growth so that the external concentration accurately reflects total cell number or cell mass (Hornby et al. [
A distinctive feature of the two time courses shown in Fig. 3A is the marked drop in farnesol during stationary phase. This decrease was expected because a similar drop in QSM activity had been observed in our original publication (Hornby et al. [
We anticipate that the availability of this reliable and accurate assay will permit a multitude of farnesol-directed questions to be addressed. A possible list includes the following: (
We thank Rebecca Roston and Zach Shomo for use and assistance with gas chromatography system. We thank Katheryn Hanford for statistical guidance.
CHTB: conceptualization, methodology, validation, investigation, writing — original draft, writing — review and editing, visualization, formal analysis; DJG: validation, investigation, visualization, formal analysis, writing — review and editing; JJK: validation, investigation, writing — review and editing; ALA: resources, supervision, writing — review and editing; KWN: conceptualization, methodology, resources, supervision, writing — original draft, writing — review and editing, project administration, funding acquisition.
This work was supported by Ann L. Kelsall and the Farnesol and Candida albicans Research Fund, University of Nebraska Foundation.
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Growth curve raw data supplied in supplementary materials.
This article does not contain any studies with human participants or animals performed by any of the authors.
The authors declare no competing interests.
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