Time Course Analysis of Candida albicans Metabolites during Biofilm

Article pubs.acs.org/jpr

Time Course Analysis of Candida albicans Metabolites during Biofilm Development ZhenYu Zhu,† Hui Wang,† QingHua Shang, YuanYing Jiang, YingYing Cao,* and YiFeng Chai* School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, P. R. China S Supporting Information *

ABSTRACT: Biofilm-associated infections are difficult to treat because of their decreased susceptibility to antimicrobial therapy. Candida albicans is the most common fungal pathogen associated with colonization and biofilm formation on the surfaces of indwelling medical devices which show intrinsic resistance to many commonly used antifungal agents. In this study, a metabonomic method using gas chromatography−mass spectrometry (GC/MS) was developed to characterize metabolic profiles during the whole biofilm developmental phases compared to the planktonic mode in C. albicans. Thirty-one differentially produced metabolites between the biofilm and planktonic specimens at each time point were identified, and they were mainly involved in the tricarboxylic acid (TCA) cycle, lipid synthesis, amino acid metabolism, glycolysis, and oxidative stress. Further experiments showed that lack of trehalose, one of the metabolites differentially produced between biofilm and planktonic cells, resulted in abnormal biofilm formation and increased sensitivity to amphotericin B and miconazole. This study provides a systemic view of the metabolic pattern during the development of C. albicans biofilms, indicating that multicomponent, phasespecific mechanisms are operative in the process of biofilm formation. KEYWORDS: Candida albicans, biofilm, metabolite, trehalose, drug resistance

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INTRODUCTION Candida albicans is the most common fungal pathogen involved in life-threatening systemic infections. Predisposing factors for C. albicans infections include immunosuppressive therapy, antibiotic therapy, human immunodeficiency virus infection, diabetes, and old age.1 In addition, structured microbial communities attached to surfaces, commonly referred to as biofilms, have increasingly been found to be the source of C. albicans infections. Biomaterials such as stents, shunts, prostheses, implants, pacemakers, and various types of catheters have all been shown to facilitate C. albicans colonization and biofilm formation.2 Biofilms are spatially organized heterogeneous communities of cells embedded within an extracellular matrix (ECM). Biofilm formation includes three developmental phases: the early phase (0−11 h), involving adhesion of fungal cells to the substrate; the intermediate phase (12−30 h), during which the blastospores coaggregate and proliferate, forming communities while producing a carbohydrate-rich ECM; and the maturation phase (31−72 h), in which the fungal cells are completely encased in a thick ECM. It is apparent that cells in a biofilm represent an epigenetic modification of the cellular state compared to their planktonic counterparts, with changes in cellular morphology, cell-to-cell communication, and gene expression, as well as with the production of an ECM.3 In comparison with planktonic cells, biofilm cells display unique phenotypic traits, the most outstanding of which is that they are © XXXX American Chemical Society

notoriously resistant to both antimicrobial agents and host immune factors. It is reported that C. albicans biofilms are resistant to a variety of clinical antifungal agents, such as amphotericin B (AmB) and azoles.4 Extensive research has focused on the mechanisms of drug resistance in C. albicans biofilms. It has been reported that increased cell density, increased expression of drug efflux pumps, decreased ergosterol content and metabolic activity, elevated β-1,3 glucan levels in the cell wall and biofilm matrix, signaling mediated by protein kinase C (PKC) and the protein phosphatase calcineurin, as well as “persister cells” may all contribute to biofilm-associated drug resistance. Although microarrays and proteomics, along with many other approaches, have been successfully used to clarify mechanisms underlying biofilm formation and drug resistance, a clear answer to this clinically important question remains elusive.5 Metabolomics has been widely used in the fields of microbiology, botany, and disease diagnosis, and it is commonly defined as “the quantitative measurement of the dynamic multiparametric response of a living system to pathophysiological stimuli or genetic manipulations”. It is a powerful tool both for complementing other “omic” studies and Special Issue: Systems Biology of the Gut Microbiome Received: May 17, 2012

A

dx.doi.org/10.1021/pr300447k | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

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for investigating the immense metabolic diversity.6 Metabolomics has been used to study the effects of antibiotics in Mycobacterium tuberculosis and compare ethanol and ironlimitation stress in Staphylococcus aureus.7 Additionally, we previously characterized the metabolic profile of DOX-induced cardiomyopathy in mice using gas chromatography−mass spectrometry (GC/MS).8 However, the application of metabolomics to a fungal infection, especially Candidiasis, is a largely unexplored area. Here we present a GC/MS-based metabolomics study of the biofilm and planktonic modes of C. albicans growth. The dynamic multiparametric response and metabolite profiles were analyzed in both the biofilm and planktonic forms of C. albicans at each growth phase. These analyses led to a description of metabolic fingerprinting and the changes in metabolites that suggest the mechanisms responsible for the phenotypic changes that occur during C. albicans biofilm development and drug resistance.

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introduced into the wells of 96-well tissue culture plates (Corning Incorporated, Corning, NY) and incubated for 1 h at 37 °C. Following the initial 1 h adhesion, the medium was aspirated, and nonadherent cells were removed and fresh RPMI-1640 was then added to the adherent cells to allow biofilm formation at 37 °C. Isolation of Metabolites from Biofilm and Planktonic Cultures

Metabolites from C. albicans SC5314 planktonic and biofilm cells were obtained as previously described by other groups with slight modifications.9 Briefly, at the end of the incubation periods, either planktonic or resuspended biofilm cells were washed rapidly with ultrapure sterilized water which was precooled. The washing step was finished less than 1 min before metabolite extraction. Then each sample was resuspended in 1 mL of boiling water for 15 min and frozen to −80 °C. The resulting solution underwent three repeated freeze− thaw cycles for 10 min in a −80 °C refrigerator and on hot water (60 °C) alternatively. After centrifuging at 10,000g for 5 min at 4 °C, the supernatant was transferred to a filter cap and centrifuged again at 10,000g for 5 min at 4 °C. The filtrate was collected, prefrozen in a −20 °C refrigerator, and freeze-dried with a freeze-dryer.

MATERIALS AND METHODS

Strains and Culture Conditions

Stock cultures of C. albicans strain SC5314 (Table 1) were routinely maintained on Sabouraud dextrose agar, and Table 1. C. albicans Strains Used in This Study strain

parent

SC5314 CAI4-EXP

CAI4

tps1Δ-EXP tps1Δ-TPS1

tps1Δ tps1Δ

genotype wild type ura3△::immm434/ ura3△::immm434::URA3 tps1△::hisG/tps1△::hisG::URA3 tps1△::hisG/tps1△::hisG::TPS1-URA3

Sample Derivatization for GC-MS Analysis

The cell extracts were dried in a freeze-dryer overnight. For derivatization, 30 μL of O-ethylhydroxylamine hydrochloride (sigma) in pyridine (20 mg/mL) was added as the first derivatizing agent. The mixture was incubated at 40 °C for 90 min. Similarly, 70 μL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA, Sigma) as the secondary derivatizing agent was added and incubated at 40 °C for 50 min. Then, the supernatant was collected and added with 100 μL of heptane.

ref 27 28 28 28

propagated in yeast peptone dextrose (YPD) medium (1% w/v yeast extract, 2% w/v peptone, and 2% w/v dextrose). A batch of medium was inoculated from YPD agar plates containing freshly grown C. albicans and incubated overnight in an orbital shaker at 30 °C. C. albicans grew in the buddingyeast phase under these conditions. For the experiments on tps1Δ mutant (tps1Δ-EXP), TPS1 reintroduced (tps1Δ-TPS1), and wild-type CAI4-EXP) strains, the cells were cultured in YPGal medium (1% w/v yeast extract, 2% w/v peptone, and 2% w/v galactose) or on YPGal agar plates. Prior to use in biofilm experiments, blastospores were harvested and washed twice in sterile phosphate-buffered saline (PBS; 10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride [pH 7.4]). The cells were then suspended in RPMI1640 medium (Gibco, USA) supplemented with L-glutamine and buffered with morpholinepropanesulfonic acid (MOPS), counted in a hemocytometer, and adjusted to the desired cell density (1 × 106 cells/mL). For the formation of biofilms used for metabolites isolation, 20 mL of standardized C. albicans SC5314 cells (1 × 106 cells/ mL) were introduced into the 250-mL tissue culture flasks (Corning Incorporated, Corning, NY) and incubated for 1 h at 37 °C. Following the initial 1 h adhesion, the medium was aspirated, nonadherent cells were removed, and fresh RPMI1640 was added to the adherent cells to allow biofilm formation with shaking at 50 rpm. The cells were harvested at 6, 12, 18, 24, 36, 48, and 72 h. The age-matched planktonic cultures were obtained in parallel through inoculation in 250-mL flasks and orbital shakers at the same temperature, and they reached the stationary growth phase at 24 h. For tps1Δ-EXP, tps1Δ-TPS1, and CAI4-EXP strains, 100 μL standardized cells were

GC/MS Analysis

The derivatized extract was analyzed on a Thermo Trace Ultra/ DSQIIGC/MS. One microliter of derivatized sample was injected in a splitless mode onto a TR-5MS capillary column (30 m × 0.25 mm × 0.25 m). The injection temperature and the interface temperature were set to 250 °C. The temperature of the quadrupole and the ion source temperature were adjusted to 150 and 230 °C, respectively. The initial GC oven temperature was 50 °C for 5 min and increased by 15 °C min−1 to 270 °C, and held for 10 min. Helium was used as the carrier at a flow rate of 1 mL/min. The solvent delay was set to 4 min. Measurement was performed under electron impact (EI) ionization (70 eV) in the full scan mode (m/z 15−800). Identification of the compounds was searched against the mainlab library and reference standards. Data Processing

Data in instrument-specific format were converted to CDF format files. The program XCMS was implemented in R language for nonlinear alignment of the data in the time domain and automatic integration and extraction of the peak intensities. XCMS parameters were default settings except for the following: fwhm = 4, bw = 5, and snthersh = 5. The output data were imported into Microsoft Excel 2010, where data were calibrated by the biomass dry weight of each sample, and the ratio of the intensity of mass ions to that of the internal standard fragment ion (m/z 130.08 as the most abundance fragment ion for the silylation derivative of α-aminobutyrate) was calculated. B



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Figure 1. Typical GC/MS chromatograms of C. albicans SC5314 biofilm (A) and planktonic cells (B).

Antifungal Agent Treatment and XTT Reduction Assay

Owing to variability and complexity, such as multiple fragment ions from single compounds in GC-MS data inherently, directly concatenating the matrices of processedMS data is suboptimal, as this may result in a matrix with an unfavorable ratio of samples to variables. It is necessary to use the properly reduced matrix to conduct multivariate statistical analysis. A simple strategy was untargeted filtration of ion peaks using our in-house scripts in MATLAB 7.0 (The MathWorks, Inc., USA), where the most abundant fragment ion with the same retention time 1 (the time bin is 0.01 min) was retained and the other ions were excluded.

Biofilms formed in 96-well microtiter plates were rinsed with PBS and treated with AmB or miconazole for 24 h at 37 °C. The metabolic activity of sessile cells was measured by assaying 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction, a reaction catalyzed by mitochondrial dehydrogenases. Briefly, adherent cells were washed twice with PBS and then incubated with 0.5 mg/mL XTT and 1 mM menadione in PBS at 37 °C for 90 min. A490 was determined using a microtiter plate reader. The effects of the antifungal agents were expressed as a percentage relative to the metabolic activity of the untreated biofilms.

Statistical Analysis

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Multivariate statistical analyses, such as principal components analysis (PCA) and partial least-squares discriminant analysis (PLS-DA), were performed with SIMCA-P software version 11.0 (Umetrics AB, Umea, Sweden). It should be noted that data were log transformed for multivariate analysis to eliminate the unit error. The significance was expressed by using one-way ANOVA of SPSS software. P values less than 0.05 were considered significant. Significant peak changes between samples were confirmed by manual quantification by calculating the area under the peak from raw chromatograms.

RESULTS AND DISCUSSION

Grouping and Comparison of Biofilms and Planktonic Cells

GC/MS analysis of endogenous metabolites in the cellular samples was conducted with the MSTFA derivatization. Typical total ion chromatograms of cell samples of biofilm- and planktonic-grown cells are shown in Figure 1. After aligning mass ions using XCMS software, 1094 ion peaks were obtained, where a few of these fragment ion peaks were found from the same silylation derivatives of endogenous metabolites. By untargeted filtration of ion peaks (which was described in the Materials and Methods section), the data were simplified, and 203 ion peaks were obtained. Principal component analysis (PCA), an unsupervised method, was used to explore correlations between biofilm- and planktonicgrown cells during each growth process. The biofilm samples at each time point were clearly separated, whereas the planktonic samples did not separate at 18 and 36 h. The partial leastsquares (PLS) method, a traditional supervised method, was applied to separate the planktonic samples, showing that they were separated clearly in the 3D scatter plot (Figure 2). The calculated R2 and Q2 values, which indicate goodness of fit and cross validation predictive ability for PCA and PLS-DA, respectively, are shown in Table 2. To distinguish biofilm- and planktonic-grown cells at each time point separately, both the

Biomass Dry Weight Measurement

At the end of the incubation periods, 5 mL of either planktonic or resuspended biofilm cells was filtered through filters (0.45 μm) and washed three times with ultrapure sterilized water. Cells were dried at room temperature to constant weight and weighed at least four times. Scanning Electron Microscopy (SEM)

Biofilms were examined by SEM after sample processing by a freeze-drying technique to improve preservation of the biofilm matrix, fixed with glutaraldehyde (2.5%, v/v, in 0.1 M cacodylate buffer, pH 7.0), washed gently three times in distilled water, and then plunged into a liquid propane/ isopentane mixture before freeze-drying under vacuum. The samples were finally coated with gold with a Polaron coater and viewed under a Philips 500 scanning electron microscope. C



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Figure 2. PLS score plot derived from planktonic (P) and biofilm models (B). (A) Score plot of planktonic model. (B) 3D score plot of planktonic model. (C) Score plot of biofilm model. (D) Score plot of planktonic and biofilm models. The time of growth was from 6 h to 72 h in sequence. The direction of the arrows in each figure represents the growth trend of the corresponding model.

value, several variables were first selected as candidates of potential biomarkers. Some of these variables were found to be from the same metabolites. After merging the variables from the identical metabolites, fewer variables were collected. And to accurately select potential biomarkers worthy of preferential study in the next step, these differential metabolites were validated using one-way analysis of variance (ANOVA) with Tukey post hoc test performed by SPSS software. The critical p-value was set to 0.05 for significantly differential variables in

PCA and PLS-DA models were used, and the values indicate that there were statistically significant differences between the samples. The statistics for these models are also given in Table 2. The variable influence in the projection (VIP) which could be generated after PLS-DA processing indicated each metabolite's significance to the model. A metabolite with a VIP greater than 1 was considered to have a statistically significant contribution to the model. According to the VIP D



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Table 2. Calculated R2 and Q2 Values of PCA and PLS-DA by the Biofilm and Planktonic Models model

A

type

R2Xa

6h 6h 12 h 12 h 18 h 18 h 24 h 24 h 36 h 36 h 48 h 48 h 72 h 72 h biofilm(6−72 h) biofilm(6−72 h) planktonic(6−72 h) planktonic(6−72 h) total(6−72 h)

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3

PCA-X PLS-DA PCA-X PLS-DA PCA-X PLS-DA PCA-X PLS-DA PCA-X PLS-DA PCA-X PLS-DA PCA-X PLS-DA PCA-X PLS-DA PCA-X PLS-DA PCA-X

0.708 0.777 0.769 0.743 0.737 0.728 0.778 0.761 0.667 0.636 0.776 0.837 0.875 0.927 0.707 0.69 0.726 0.735 0.770

R2Ya 0.975 0.995 0.970 0.967 0.978 0.995 0.999 0.381 0.515

listed metabolites were mainly involved in the TCA cycle, lipid synthesis, amino acids metabolism, glycolysis, and oxidative stress. Compared with the other phases, more metabolites were upregulated in biofilms in the intermediate phase (12−24 h), especially at the 12 h time point when 21 metabolites were upregulated. This result is consistent with the report from Yeater et al., whose microarray data showed that more genes were upregulated in biofilms at the 12 h time point than at the other time points assayed.5i Indeed, the 12 h time point marks the start of the synthesis of biomass (ECM) that occurs during biofilm development, during which more materials are needed to support the large increase in biofilm biomass, such as sugars and amino acids.

Q2(cum) 0.476 0.942 0.595 0.982 0.471 0.897 0.631 0.921 0.410 0.934 0.607 0.981 0.778 0.992 0.658 0.358 0.596 0.465 0.721

TCA Cycle

The TCA cycle is a key component of the metabolic pathway in all aerobic organisms to generate energy. In eukaryotic cells, the TCA cycle occurs in the mitochondrial matrix. Here the levels of four pivotal intermediates of TCA cycle metabolites, succinate, fumarate, citrate, and malate, were decreased in mature biofilms (36, 48, and 72 h) compared to planktonic cells, with citrate also decreased in early and intermediate biofilms. A similar pattern of downregulation was observed for lactate, which can participate in the TCA cycle indirectly by being converted to pyruvate, the precursor of oxaloacetate. These results demonstrate that the TCA cycle was inhibited in mature biofilms. This can be explained by the fact that mature biofilms contain low oxygen, resulting in reduced respiration. Recently Bonhomme et al. demonstrated that mature biofilms represented a hypoxic environment which would limit respiration and hence the TCA cycle.10 Reduced metabolic activities may reflect energy and nutrient conservation to avoid stress inducers. In a previous study on proteomic analysis of C. albicans biofilm, Seneviratne et al. reported that biofilm had much less ROS activity than planktonic cells, which could be explained by the up-regulated proteins conferring antioxidant capacity.11 Since the primary source of endogenous ROS (normal byproducts of energy production in respiring cells) is the leakage of electron from the mitochondrial respiratory chain, down-regulation of TCA-related metabolites found in this study indicates that mitochondrial respiration was inhibited, which may explain the reduced ROS level in mature biofilms.

a

Ideal values are close to 1, but this is rare and unlikely in biological systems. For the PCA and PLS-DA models, these values indicate statistically significant differences between the samples.

this study. In addition, the criteria were further restricted to features with an average intensity difference of at least 1.5-fold (high or low) between different group samples. According to this algorithm, we found (1) significantly differential variables which were considered as differential metabolites in biofilm or planktonic-grown cells during their total growth process and also (2) potential biomarkers in biofilm or planktonic cells at each time point at last. Summary of Metabolomic Analysis

The main goal of this study was to identify the small molecule metabolites that were differentially produced in C. albicans during biofilm develpoment compared to planktonic cells. In order to distinguish the differences of metabolites in biofilm and planktonic cells better, seven different time points representing important developmental periods during biofilm formation were examined. Biofilms were studied at early phase (6 h) when antifungal drug resistance was first observed, intermediate phase (12−24 h) when ECM formation was underway, and mature phase (36−72 h) when the biofilm structure became mature. The planktonic specimens were collected at the same time points as the biofilm specimens. Metabolite profiles were analyzed (a) between the biofilm and planktonic forms of C. albicans at each time point (Table 3) and (b) as a function of time in the biofilm and planktonic cultures separately (Figure 3). These comparisons would provide a longitudinal view of processes that occur in the specimens, and the results of analysis as shown in Table 3 and Figure 3 can be combined to produce a visual summary of metabolites production in the biofilm and planktonic specimens, giving a description of temporal changes in metabolite levels that suggest the cellular mechanisms responsible for the phenotypic changes that occur during biofilm development. Differentially produced (P < 0.05) metabolites were considered significant. Table 3 lists 31 differentially produced metabolites between the biofilm and planktonic specimens at each time point. The

Lipid Synthesis

Lipids, in addition to being structural and metabolic components of yeast cells, also appear to be responsible for drug resistance in C. albicans. Alteration in the sterol composition is responsible for increased azole resistance of C. albicans biofilms.5c Phosphatidylinositol (PI), a key yeast phospholipid, has been shown to contribute to antifungal resistance12 and pathogenesis.13 In this study, we observed changes in the levels of several compounds essential for phospholipids synthesis, including myo-inositol, serine, glycerol, phosphate, and glycerophosphate. Inositol, a precursor for building PI, was upregulated in the whole growth phase of biofilms compared to planktonic cells. This is consistent with our previous microarray analysis showing that INO1, encoding a key enzyme in inositol biosynthesis, was down-regulated in farnesol-inhibited biofilms.14 Serine, a nutrient specific for phosphatidylserine (PS) synthesis, was upregulated in intermediate and mature biofilms. A recent study revealed that the lipid profiles of biofilms differed significantly from planktonic cells. Compared to planktonic cells, biofilms contained high E



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Table 3. Metabolites with Statistical Differences between Biofilm and Planktonic Cells at Each Time Pointa metabolite

6h

fold

12 h

fold

18 h

fold

24 h

TCA Cycle − − − − − ↓ − − − ↓ Lipid Synthesis 2.19 ↑ 1.72 ↑ 4.50 ↑ 2.31 ↑

fold

36 h

fold

48 h

fold

72 h

fold

− − 0.87 − 0.85

↓ ↓ ↓ ↓ ↓

0.44 0.44 0.40 0.33 0.35

↓ ↓ ↓ ↓ ↓

0.75 0.75 0.40 0.36 0.19

↓ ↓ ↓ ↓ ↓

0.75 0.57 0.26 0.38 0.39

7.35 4.90 99.94 17.67

↑ ↓ ↓ ↓

1.47 0.95 0.81 0.84

↑ ↓ ↑ ↑

1.61 0.78 8.26 1.77

↑ ↓ ↑ −

10.60 0.62 4.75 −

− − ↓ − ↓

− − 0.53 − 0.38

− − ↓ − ↓

− − 0.73 − 0.71

− − − − −

myo-inositol ↑ glycerophosphate ↑ glycerol ↑ phosphate ↑ Amino Acid Metabolism alanine − aspartate − asparagine − N-acetyl- glutamate − γ- aminobutyrate ↑ glutamate ↑ pyroglutamate ↑ isoleucine ↑ valine − glycine − sarcosine ↑ serine − threonine − phenylalanine − Glycolysis glucose − galactose ↑ phosphomannose − mannose − Oxidative Stress trehalose ↑ Others arabinitol − mannitol − putrescine −

2.58 3.80 5.02 5.81

↑ ↑ − ↑

1.36 2.96 − 1.45

↑ ↑ ↑ ↑

− − − − 7.15 1393.41 5.62 12.01 − − 9.81 − − −

↑ ↑ ↑ − ↑ ↑ ↑ ↑ ↑ ↑ − ↑ ↑ ↑

3.28 12.35 2.54 − 1.46 1.14 2.54 4.55 8.26 2.97 − 2.95 3.59 12.52

↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ − ↑ ↑ ↑

3.34 1.62 1.63 2.56 1.84 2.59 1.87 2.83 4.12 4.19 − 3.94 4.01 4.14

↑ − ↑ − ↑ ↑ − ↑ − ↑ ↑ ↑ ↑ −

12.51 − 9.15 − 21.88 2.19 − 227.65 − 28.71 107.85 4.97 3.77 −

− − − − ↓ ↓ ↓ − ↑ ↑ − ↑ ↑ ↑

− − − − 0.72 0.73 0.60 − 1.60 1.58 − 1.84 1.72 1.40

↑ ↑ ↑ ↑ − ↓ ↑ ↑ ↑ ↑ − ↑ ↑ −

1.03 2.53 5.95 3.88 − 0.73 2.20 39.25 4.97 3.88 − 3.09 2.75 −

↑ ↑ ↑ ↑ ↑ ↓ ↑ ↑ ↑ ↑ − ↑ − −

2.41 2.73 5.31 2.78 1.26 0.87 4.73 28.16 9.70 9.46 − 2.30 − −

− 1.80 − −

↑ ↑ ↑ ↑

5.22 1.20 9.96 3.53

− ↑ − −

− 1.47 − −

↑ ↑ − ↑

103.85 1.86 − 164.81

↑ ↑ − ↑

17.66 1.42 − 6.24

↑ ↑ − −

6.16 1.50 − −

− ↑ − −

− 1.16 − −

17.99

−

−

−

−

↑

3.15

↓

0.45

↓

0.59

↓

0.12

− − −

− ↑ ↑

− 2.93 7.11

↓ − −

0.75 − −

↓ − ↑

0.87 − 662.33

− − −

− − −

↑ ↑ ↑

1.62 2.98 16.51

↑ − ↑

1.99 − 9.62

succinate fumarate citrate malate lactate

a Note: ↑, upregulated in biofilm; ↓, downregulated in biofilm; −, no difference between biofilm and planktonic cells. The additional data are listed in the Supporting Information.

levels of phospholipids, including PI and PS.15 Here the high levels of inositol and serine can be explained by the increased need for PI and PS biosynthesis in biofilms. Glycerol, phosphate, and glycerophosphate are essential components for general phospholipid synthesis. These three compounds were all upregulated in early and intermediate biofilm, whereas glycerophosphate was downregulated and glycerol and phosphate levels fluctuated in mature biofilms. Besides the role in lipid synthesis, glycerol also acts as a compatible solute and plays a central role in osmoadaptation. In yeast, the spontaneous intracellular accumulation of glycerol is one characteristic feature of defense response to osmotic stress. Time-course analysis showed that the highest glycerol level was in intermediate biofilms (24 h) compared to the other time points during biofilm development. In addition, the concentration of glycerol in biofilm at this time point was over 99-fold higher that that in planktonic cells, indicating a high capacity for osmotic stress defense in biofilm.

Amino Acids

It was found in this study that various amino acids, including alanine, aspartate, asparagine, threonine, valine, and glycine, were upregulated almost during the whole process of biofilm development compared to planktonic cells, and most of these upregulations were initiated at 12 h and maintained throughout the process (Table 3). This result is consistent with previous studies which revealed that genes involved in amino acid biosynthesis were up-regulated in the transcriptional analysis of C. albicans biofilms compared to planktonic cells. In addition, the importance of amino acid biosynthesis in biofilm formation has been described previously. Gcn4p, a general regulator of amino acid biosynthesis, was shown to be required for normal biofilm formation.5i,16 Additionally, many nutrients, including amino acids, can be metabolized into TCA intermediates and enter this cycle. Reduced TCA cycle activity would affect the consumption of corresponding nutrients. The increased amino acids levels in biofilms imply a reduced consumption of amino acids due to the inhibited TCA cycle activity. F



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al.16 reported that increased expression of MET3, MET10, CYS3, and CYS4 was detected in the intermediate (12 h) biofilm. In our experiments, the difference of sulfur amino acids between biofilm and planktonic cells was not observed. However, amino acids involved in the biosynthesis of glutathione, which is not only an important intracellular antioxidant conferring protection against reactive oxygen species but also serves as a storage and transport form of cysteine, were differentially regulated between biofilm and planktonic cells. These metabolites included 5-oxoproline, glycine, glutamate, and 4-aminobutyrate. They showed similar upregulated patterns in early and intermediate biofilms (6−24 h) compared to planktonic cells, whereas these compounds were differentially regulated in mature biofilm, with glycine remaining upregulated; 5-oxoproline and 4-aminobutyrate fluctuating; and glutamate downregulated. Glutathione can be synthesized from the precursor amino acid cysteine, glycine, and glutamate. Transpeptidation of glutathione produced γglutamylamine, which can be converted to 5-oxoproline by the enzyme γ-glutamylcyclotransferase. The 5-oxoproline formed in this reaction can be converted to glutamate. Glutathione plays a key role in cellular resistance against oxidative damage through the detoxification of the radicals and naturally occurring deleterious compounds. As inhibition of mitochondrial respiration resulting from decreased TCA cycle may explain the reduced ROS level in mature biofilms, increased synthesis of intracellular glutathione as represented by the upregulation of these compounds may contribute to low ROS activity in early and intermediate biofilms compared to planktonic cells. Recently, Zhu et al. reported that farnesol, a quorum sensing molecule in C. albicans with the activity of biofilm prevention, can conjugate with glutathione. Glutathione conjugates act as substrates for ATP-dependent ABC transporters and are extruded from cells, resulting in total glutathione depletion, oxidative stress, and ultimately cell death.19

Figure 3. Colors in metabolite arrays represent x-fold change in the metabolite concentration during the time course of biofilm (A) development or planktonic culture (B). Red indicates increased concentration levels of metabolites; green indicates decreased concentration levels of metabolites.

Biofilm development is accompanied with the large increase in biomass that is, in part, due to the accumulation of large amounts of carbohydrate-rich ECM, a specific aspect of biofilms predominantly consisting of carbohydrate and protein. It has been proposed that drug resistance of C. albicans biofilms may be significantly enhanced by increasing the production of ECM.5e The synthesis of ECM begins in the intermediate biofilm, which is the same phase as the initiation of amino acid upregulation. Thus, here the abundance of amino acids might be propitious to the formation of ECM. In regard to this, previous studies also showed that monosaccharides were essential components of ECM, and the major component in C. albicans is glucose (32%). Galactose contributes 3% of the dry weight of the extracellular polymeric material of C. albicans biofilm.17 In our experiment, three monosaccharides (glucose, galactose, and mannose) were upregulated in biofilms. Among them, galactose was upregulated during the whole course of biofilm development. Glucose and mannose were upregulated in intermediate and mature biofilms. In addition, phosphomannose was upregulated in 12 h biofilm compared to planktonic cells. This result is in agreement with the previous transcriptional analysis using microarray which showed that genes encoding glucose transporters (HGT1, HGT2, HGT14) and genes regulated by galactose (GAL1, GAL1) were overexpressed in biofilms compared to planktonic cells.16 An emphasis on the sulfur amino acid (mainly referred to as methionine and cysteine) biosynthetic pathway was also observed in previous studies that transcriptionally profiled C. albicans biofilm.5g,18 The sulfur assimilation pathway has been found to be up-regulated in early C. albicans biofilm. Nett et

Putrescine

Putrescine is one of the polyamines which are basic molecules required for cellular growth and differentiation in many organisms. In mammals and fungi, putrescine is produced from ornithine initiated by ornithine decarboxylase (ODC). It was reported that ODC activity in quiescent cells is extremely low but strongly induced by a wide variety of stimuli, such as during fungal spore germination.20 ODC inhibitors inhibit the yeast-hypha transition in C. albicans.21 Herrero et al. reported that polyamine levels control the switch from the yeast form to a filamentous pattern. In their studies, Candida mutants behaved as polyamine auxotrophs and grew exclusively in the yeast form at low polyamine levels (0.01 mM putrescine) under all conditions tested and an increase in the polyamine concentration (10 mM putrescine) restored the capacity to switch from the yeast to the filamentous form.22 Mature Candida biofilms are marked by a basal layer of yeast cells with subsequent layering of filamentous morphotypes and extensive matrix production. Previous studies have reported differential regulation of hyphal-associated genes required for hyphal morphology transition.3b,16 In our study, the putrescine concentration did not undergo obvious change in planktonic cells. However, its level was higher in 12, 24, and 48 h biofilms compared to other time points. By comparing biofilm and planktonic cells during the total growth process, the putrescine concentration was higher in 12, 24, 48, and 72 h biofilms. So higher putrescine in C. albicans biofilm may contribute to the G



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conversion of yeast to the filamentous form, which is important for biofilm formation. This result is consistent with a previous report on microarray analysis of C. albicans biofilm, in which ODC, a key regulator of putrescine bioynthesis, and PTK2, encoding a protein kinase required for efficient polyamine uptake, were overexpressed in biofilms compared to the planktonic mode.5g Trehalose

Recently, many studies proved that trehalose, besides its primary role in the reservation of carbohydrates, played an important part in yeast protection against a variety of stresses. This sugar is found in bacteria, fungi, plants, and invertebrates but not in mammals. In yeast, trehalose acts both as a main reserve of carbohydrates and as a cellular protector against a variety of nutritional and/or environmental stress challenges (oxidative, heat shock, osmotic and/or saline stress, and xenobiotics), increasing cell resistance to such insults.23 It was found in our study that trehalose was apparently upregulated in biofilm at 6 and 24 h while it was downregulated at 36, 48, and 72 h compared to planktonic cells. Time-course analysis showed that the highest trehalose level was observed in an early phase (6 h) and remained at this high level at 12 h compared to the other time points during biofilm development, which is different from the planktonic mode where the highest trehalose level was observed at the 12 h time point, then declined, and increased again at 72 h. Previous work on C. albicans pointed out a specific role of trehalose in cellular protection against oxidative stress, and the trehalose biosynthesis pathway was considered as an antifungal target. C. albicans tps1Δ mutant cells, which were deficient in trehalose synthesis, were extremely sensitive to H2 O 2 exposure accompanied with enhanced intracellular ROS.24 Here the fact that trehalose was accumulated in early and intermediate biofilms but not in mature biofilms could indicate that its use as an antioxidant is more prominent in early and intermediate biofilms. The decrease of trehalose in mature biofilm might be due to its degradation into glucose to provide energy, when extracellular sources are scarce.

Figure 4. Effects of trehalose accumulation on C. albicans biofilm formation and drug resistance. (A) The wild-type (CAI4-EXP), tps1Δ null mutant (tps1Δ-EXP), and TPS1 reintroduced (tps1Δ-TPS1) cells were cultured in biofilm mode for 48 h and observed by scanning electron microscopy. The C. albicans strains cultured in the biofilm mode for different time were treated with 1 μg/mL amphotericin B (B) or 4 μg/mL miconazole (C) for 24 h at 37 °C. The metabolic activity of biofilms was assessed using an XTT assay. The data were shown as percentages of biofilm growth relative to growth of untreated wild-type biofilm and were mean values ± SD from three independent experiments. * indicates P < 0.01 compared with values from the control CAI4-EXP cells.

composition are involved in the resistance phenotype in the intermediate and mature phases of biofilm development, and elevated β-1,3 glucan mainly refers to mature biofilm.5c,d In recent years, production of intracellular ROS is considered as a mechanism for antifungal agents such as AmB and azoles. A report from González-Párraga et al. showed that AmB could induce trehalose accumulation in the planktonic mode of C. albicans cells, and a tps1Δ mutant was severely sensitive to AmB.26 The fact that trehalose plays an important role in oxidative stress defense and sensitivity to AmB could indicate that increased intracellular trehalose contributes to drug resistance in early and intermediate phase biofilms. Subsequently, we further examined the growth behavior of biofilm in the presence of antifungal agents by detecting metabolic activities with an XTT reduction assay. Consistent with its planktonic mode, the tps1Δ mutant biofilm was more sensitive to AmB than the wild-type biofilm (Figure 4B), while these two strains showed similar metabolic activities in the absence of AmB (data not shown). It should be noted that the differences in AmB sensitivity between tps1Δ mutant and wild-type cells were extremely obvious in early and intermediate biofilms (6 to 24 h), which is in agreement with the increased accumulation of trehalose in these two phases of biofilms. Similar results were observed upon miconazole treatment (Figure 4C), indicating a general role of trehalose in the drug resistance of C. albicans biofilm.

Lack of Trehalose Affects Biofilm Formation and Drug Resistance

We further investigated the role of trehalose in biofilm development using a tps1Δ mutant. As expected, in contrast to the high trehalose content in wild-type cells, biofilm formed by the tps1Δ mutant failed to accumulate trehalose (data not shown). Scanning electron microscopy (SEM) showed that the biofilms formed by tps1Δ mutant were predominantly composed of pseudohyphae and yeast cells, while the biofilm formed by wild-type strain exhibited a typical three-dimensional structure and was mainly composed of long tubular hyphae (Figure 4A). Since the ability to switch between yeast to hyphal forms is an important feature for C. albicans to form normal biofilms,3b here the defective biofilm formed by tps1Δ mutant was consistent with a previous report that tps1Δ mutant was defective in hyphae formation.25 The acquisition of high-level drug resistance is a unique feature of the biofilm. C. albicans biofilm is resistant to commonly used antifungal agents, notably the azoles and polyenes that target membrane sterols. Previous studies showed that multicomponent, phase-specific mechanisms are operative in antifungal resistance of fungal biofilms. For example, efflux pumps contribute to azole resistance in early phase C. albicans biofilms but not in later phases, while changes in sterol H



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Figure 5. Schematic overview of differences in metabolite change between biofilm and planktonic cells. The differently produced metabolites are mainly involved in tricarboxylic acid (TCA) cycle, lipid synthesis, amino acids metabolism, glycolysis, or gluconeogenesis. Metabolites in red represent those metabolites that always remained at a high level in biofilms; metabolites in blue represent those metabolites that always remained at a high level in planktonic cells; metabolites in green represent those metabolites that fluctuated during the whole process of cell growth.

Author Contributions

In conclusion, our metabolomic study presented here revealed the changes in metabolites production during biofilm development compared to planktonic cells. Specific subsets of metabolites including TCA cycle, lipid synthesis, amino acids metabolism, glycolysis, and oxidative stress were observed at different phases of biofilm formation (Figure 5), which suggests that multicomponent and phase-specific mechanisms are operative in the development of C. albicans biofilms. Further experiments revealed that the biosynthesis of trehalose, one of the differentially produced metabolites, played an important role in biofilm formation and drug resistance.

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†

These authors contributed equally to this work

Funding

This work was supported by the National Natural Science Foundation of China (No. 30870105). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor William A. Fonzi for kindly providing the C. albicans strains SC5314. We thank Professor Carlos Gancedo for kindly providing the C. albicans tps1Δ strain.

ASSOCIATED CONTENT

S Supporting Information *

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This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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*Y.Y.C.: phone, +86-21-81871331; fax, 8621-81871331; e-mail, [email protected]. Y.F.C.: phone, +86-2181871201; fax, 8621-81871201; e-mail, [email protected]. I



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Time Course Analysis of Candida albicans Metabolites during Biofilm

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