Next Article in Journal
Investigation of Staphylococcus aureus Biofilm-Associated Toxin as a Potential Squamous Cell Carcinoma Therapeutic
Previous Article in Journal
Inhibiting F-Actin Polymerization Impairs the Internalization of Moraxella catarrhalis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microorganisms
Volume 12
Issue 2
10.3390/microorganisms12020292
microorganisms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Candida albicans Infection Disrupts the Metabolism of Vaginal Epithelial Cells and Inhibits Cellular Glycolysis

by
Yanni Zhao
1,2,†,
Pengjiao Wang
1,2,†,
Xiaodong Sun
1,2,
Mei Zhao
1,2,
Yixuan Chen
1,2 and
Xiuli Gao
1,2,*
1
State Key Laboratory of Functions and Applications of Medicinal Plants, School of Pharmacy, Guizhou Medical University, Guiyang 550031, China
2
Microbiology and Biochemical Pharmaceutical Engineering Research Center, Guizhou Provincial Department of Education, Guizhou Medical University, Guiyang 550031, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Microorganisms 2024, 12(2), 292; https://doi.org/10.3390/microorganisms12020292
Submission received: 6 January 2024 / Revised: 17 January 2024 / Accepted: 22 January 2024 / Published: 30 January 2024
(This article belongs to the Section Molecular Microbiology and Immunology)
Browse Figures
Versions Notes

Abstract

:
Vulvovaginal candidiasis (VVC) is a common gynecologic disorder caused by fungal infections of the vaginal mucosa, with the most common pathogen being Candida albicans (C. albicans). Exploring metabolite changes in the disease process facilitates further discovery of targets for disease treatment. However, studies on the metabolic changes caused by C. albicans are still lacking. In this study, we used C. albicans-infected vaginal epithelial cells to construct an in vitro model of VVC, analyzed the metabolites by UHPLC-Q-Exactive MS, and screened the potential metabolites based on metabolomics. The results showed that C. albicans infection resulted in significant up-regulation of D-arabitol, palmitic acid, adenosine, etc.; significant down-regulation of lactic acid, nicotinamide (NAM), nicotinate (NA), etc.; and disruption of amino acid metabolism, and that these significantly altered metabolites might be potential therapeutic targets of VVC. Further experiments showed that C. albicans infection led to a decrease in glycolytic enzymes in damaged cells, inhibiting glycolysis and leading to significant alterations in glycolytic metabolites. The present study explored the potential metabolites of VVC induced by C. albicans infection based on metabolomics and verified the inhibitory effect of C. albicans on vaginal epithelial cell glycolysis, which is valuable for the diagnosis and treatment of VVC.

1. Introduction

VVC is the second most common inflammatory disease of the vagina after bacterial vaginitis. It often presents clinically with symptoms such as vulvar itching, vaginal pain, and increased discharge. According to foreign epidemiological studies, about 75% of women of reproductive age have the disease once in their lifetime, and about 50% have it twice or more [1], which seriously affects women’s health and quality of life. The main pathogens of VVC are Candida spp., including Candida albicans, Candida smoothus, and Candida tropicalis [2]. C. albicans is the most common pathogen in women with VVC and can be isolated in 75% to 90% of cases [3].
Because of its high prevalence and recurrence rate, timely diagnosis of VVC is particularly important for the control of disease progression. We can determine whether a patient is infected with C. albicans by observing the clinical symptoms or performing fungal cultures [4]. However, these methods often do not allow us to make a quick and accurate judgment. In recent years, metabolomics has been widely used to study key metabolites of diseases, and it is an effective tool for diagnosis and treatment of diseases [5]. However, studies on disease metabolites for VVC induced by C. albicans infection are still lacking. In this study, we infected the human vaginal epithelial cells, which are commonly used for in vitro vaginitis studies, with C. albicans, the most common pathogen causing VVC, to construct an in vitro model of VVC, analyzed the metabolites using UHPLC-Q-Exactive MS, and explored the differential metabolites based on metabolomics.
C. albicans is a commensal and opportunistic pathogen that can cause various diseases when the body’s immunity is out of balance, most commonly superficial infections of the vaginal mucosa, esophagus, and skin [6]. The pathogenic mechanisms reported for C. albicans include mainly the changes in C. albicans itself during infection, as well as the effects on epithelial cells. C. albicans adhering to the vaginal mucosa changes its morphology from a free yeast state to an invasive hyphae state [7], facilitating its further colonization and adhesion to cells and penetrating the barrier into the deeper tissues. The invasion of epithelial cells by C. albicans can affect the cellular immune response by activating signaling pathways such as TLR and MAPK [8]. In addition, C. albicans secretes virulence factors that enhance invasion and infection of cells, including cell wall adhesins (ALS, EPA, HWP1), secreted aspartyl proteinases (Saps), candidalysin, and phospholipases [9]. Saps and candidalysin are considered the most important virulence factors. Saps promote the adhesion of C. albicans and facilitate hyphae penetration into tissues by hydrolyzing host histones. The interaction of high concentrations of candidalysin with cell membranes can cause pore-like structures in the cell membrane, leading to cell membrane damage [10,11].
In addition, previous studies have shown that during C. albicans infection of cells, the cytoarchitecture of vaginal epithelial cells is damaged by C. albicans hyphae and virulence factors [12], and it can be inferred that this would lead to a massive release of the cellular damage marker lactate dehydrogenase (LDH) [13]. A large loss of LDH, an important enzyme in glycolysis, may result in the inhibition of glycolysis in vaginal epithelial cells. Glycolysis is an essential metabolic pathway for the production of ATP and the activation of immune cells [14], and its reaction intermediates are involved in various biosynthetic pathways, which are essential for maintaining normal cellular life activities [15,16]. However, there is insufficient evidence that C. albicans affects the glycolysis of vaginal epithelial cells, and there is a lack of studies exploring the mechanism of action of C. albicans on vaginal epithelial cells from the metabolomic perspective. Therefore, we examined the metabolic effects of C. albicans on vaginal epithelial cells by detecting changes in cellular metabolites, key metabolites, and enzymes involved in glycolysis and observed whether cellular glycolysis levels were suppressed in infection, which could be valuable in further exploring the pathogenesis of C. albicans.

2. Materials and Methods

2.1. Cell Culture

The human vaginal epithelial cells (VK2/E6E7, CRL-2616, ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle medium (DMEM, PM150223, Procell Life Sciences & Technology, Wuhan, China) added with 1% penicillin–streptomycin–gentamicin mixed solution (Solarbio, Beijing, China) and 10% FBS (VivaCell Biosciences, Shanghai, China) and maintained at 37 °C in a humidified atmosphere with 5% CO2. C. albicans (BNCC186382, BeNa Culture Collection, Beijing, China) was cultured 24 h aerobically in liquid YM medium (0.3% w/v yeast extract, 0.3% w/v malt extract, 1% w/v dextrose, and 0.5% w/v peptone) at 37 °C, collected and resuspended in PBS, and stored at 4 °C for use.

2.2. Groups and Treatment

Three groups were set up for this experiment: control group, model group, and amphotericin B (AmB) group. C. albicans cells were cocultured with vaginal epithelial cells for 24 h of infection to simulate an in vitro model of VVC [6]. The cells were seeded in cell culture flasks and incubated at 37 °C. After 24 h, the medium was discarded, and DMEM was added to the control group; DMEM containing C. albicans was added to the model and AmB groups. After 24 h, the medium was discarded, and DMEM was added to the control and model groups; DMEM containing 0.5 μg/mL AmB was added to the AmB group. After 24 h, the supernatant was collected and stored at −80 °C for glucose, pyruvic acid, lactic acid, LDH, and inflammatory factors assay, and the cultures were washed with PBS and stored at −80 °C for metabolism analysis.

2.3. Metabolomics Analysis

The cultures were added with 1mL 80% methanol aqueous solution, ground in the freeze grinder, and centrifuged at 4 °C, 14,000 rpm for 15 min. The supernatant was evaporated with nitrogen and re-dissolved by adding 50% methanol aqueous solution to precipitate the protein fully, and then centrifuged at 4 °C, 14,000 rpm for 15 min, and collected the supernatant for UHPLC-Q-Exactive Plus Orbitrap MS (Thermo Fisher Scientific, Waltham, MA, USA) analysis.
UHPLC-Q-Exactive Plus Orbitrap MS equipped with Thermo Hypersil GOLD column (150 × 2.1 mm2, 1.9 μm, 40 °C) was used to detect the samples (injection volume: 4 μL). The flow rate was 0.300 mL·min−1. The 19 min gradient (equate A: H2O, 0.1% formic acid; equate B: acetonitrile, 0.1% formic acid) was set as follows: 2% B at 0–2.5 min; 2–40% B at 2.5–5 min; 40–100% B at 5–12 min; 100% B at 12–16 min; 100–2% B at 16–16.1 min; 2% B at 16.1–19 min. The mass spectrometry conditions were set as follows: ion source, HESI; ion spray voltage, 3500/2800V (+/−); capillary temperature, 320 °C; probe heater temperature, 350 °C; S-Lens RF Level, 50.

2.4. Scanning Electron Microscopy

To further judge the damage to vaginal epithelial cells, we used scanning electron microscopy to observe the morphology of the cells. The cell crawls attached in the 12-well plates were washed with PBS, then added with electron microscope fixative (2.5% glutaraldehyde), fixed at room temperature for 2 h, and stored at 4 °C. The samples were dehydrated with different gradients of ethanol (30, 50, 70, 80, 90, 95, and 100%), dried, and sputtered with gold plating. The scanning electron microscope (JSM-IT700HR, JEOL, Tokyo, Japan) was used to observe and acquire images.

2.5. Cellular Supernatant Index Assay

In order to detect the content of glycolysis-related metabolites and LDH in the culture supernatant, we processed the supernatant after centrifugation with the glucose assay kit (glucose oxidase method), pyruvic acid, lactic acid, and LDH assay kits (NJJCBIO, Nanjing, China). The inflammatory factors were detected by human interleukin-6 (IL-6) and human interleukin-8 (IL-8) ELISA kit (4 A Biotech, Beijing, China) according to the instructions. Finally, the absorbance was detected with a microplate reader.

2.6. Immunofluorescence Staining

Immunofluorescence assays were used to detect the expression of enzymes involved in glycolysis in vaginal epithelial cells. The samples were washed with PBS and fixed in 4% paraformaldehyde solution for 20 min. The cells were placed in 0.3% Triton X-100 solution for 30 min after washing with PBS and blocked with 5% goat serum at room temperature for 1 h. The cells were incubated in rabbit anti-human LDHA antibody or mouse anti-human PKM2 antibody (1:200; Proteintech Group, Inc., Wuhan, China) overnight at 4 °C, washed with PBS, and incubated in FITC AffiniPure goat anti-rabbit IgG (H + L) secondary antibody (1:50; PUMOKE, Wuhan, China) or Alexa Fluor 594 AffiniPure goat anti-mouse IgG (H + L) secondary antibody (1:50; Yeasen Biotech Co., Ltd., Shanghai, China) for 1 h while protected from light. After washing with PBS, 30 μL of antifade mounting medium with DAPI (Beyotime Biotechnology Co., Ltd., Shanghai, China) was added. The confocal laser scanning microscope (LSM 710, ZEISS, Oberkochen, Germany) was used to observe and acquire images, and fluorescence intensity was measured using Image J 1.5.3.

2.7. Statistical Analysis

Thermo Compound Discover 2.0 and Xcalibur were used for initial identification and relative quantification of metabolites; SIMCA 14.1 software was used to perform principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA), and permutation tests; and bioinformatics (https://www.bioinformatics.com.cn/) was used to perform heatmap analysis of metabolites. We used one-way ANOVA and Student’s t-test to analyze the experimental data and plotted them with GraphPad Prism 9.5.1. We considered a p-value of less than 0.05 a significant difference.

3. Results

3.1. C. albicans Infection Disrupts the Metabolism of Vaginal Epithelial Cells

In order to study the changes in metabolites after C. albicans infection of vaginal epithelial cells, we performed metabolomics studies on the cultures. Three groups were set up to explore the disease metabolites of VVC by comparing the model group with the control group, while the target metabolites for the treatment of VVC were studied by comparing the AmB group with the model group. As shown in the PCA plot (Figure 1A) and OPLS-DA (Figure 1B), the metabolic profile data of the control, model, and AmB groups exhibited the characteristics of separation between groups and proximity within groups, which indicated that the difference between groups was obvious, and the experimental methods were stable. The OPLS-DA models were tested for 200 permutations, respectively. The results (Figure 1C) showed that the values of R2 and Q2 on the left side were consistently lower than those on the right side and that Q2 regression lines intersected the negative semiaxis of y, indicating that the models were stable and reliable and did not suffer from overfitting.
Differential metabolites were screened by setting threshold conditions (fold change value greater than 2 or less than 0.5, p-value less than 0.05, variable importance in the projection value greater than 1). As shown in Figure 2, 50 endogenous metabolites were significantly changed in the model group compared to the control group, of which 19 metabolites were up-regulated (D-arabitol, lysoPC(18:2(9z,12z)), betaine, palmitic acid, adenosine, etc.), and 31 metabolites were down-regulated (lactic acid, NAM, NA, phenylalanine, etc.). These metabolites may be potential disease biomarkers for VVC caused by C. albicans infection.

3.2. Changes in Metabolites after Administration of Antifungal Therapy

Amphotericin B is a polyene antifungal drug commonly used in clinical practice to treat vaginitis, endocarditis, and other fungal infections caused by C. albicans infection [17,18]. In this study, amphotericin B was used as an anticandidal-infection strategy to observe whether there is a tendency for the changes in metabolites to return to the control group after controlling the course of the fungal infection and whether the glycolysis of vaginal epithelial cells can return to normal. After administration of AmB for antifungal treatment, 28 metabolites (Table 1) tended to revert to the control group, and we considered these 28 reverse-regulated metabolites as potential biomarkers representing effective control of the VVC pathologic process. Of these 28 metabolites, 15 metabolites (lysoPC(18:2(9z,12z)), adenylsuccinic acid, indole, palmitic acid, 2-furoic acid, guanosine monophosphate, betaine, cysteinylglycine, pipecolic acid, guanine, pyruvic acid, ophthalmic acid, D-arabitol, diacetyl, cis-aconitic acid) were positively correlated with C. albicans infection, and 13 metabolites (thiamine, NAM, NA, hypoxanthine, N-acetyl-L-aspartic acid, inosine, uracil, allantoic acid, Beta-citryl-L-glutamic acid, lactic acid, 2-phosphoglyceric acid, phosphoenolpyruvic acid, octadecanamide) were negatively correlated with infection status.
Previous studies have shown that decreased lactic acid was a common feature of reproductive tract infections [19]. In our experiment, the lactic acid content of the model group was significantly reduced, along with significant changes in the levels of pyruvic acid, phosphoenolpyruvic acid, 2-phosphoglyceric, NAM, NA, etc. (Figure 3). The significant changes in the levels of these substances, which were essential metabolites involved in glycolysis, suggested that C. albicans infection may cause a disturbance in the glycolysis response of vaginal epithelial cells.

3.3. C. albicans Infection Disrupted Glycolysis-Related Metabolites and Enzymes

To verify the inhibitory effect of C. albicans on cellular glycolysis, we observed the damage to cells’ membranes by scanning electron microscopy (Figure 4) and detected the contents of glucose, pyruvic acid, lactic acid, and LDH in the supernatant with a microplate reader (Figure 5). From the scanning electron microscopy results, it can be seen that the cells in the control group had a full and naturally spreading morphology, while the cells in the model group were penetrated and invaded by hyphae, and their surfaces were severely damaged, with ruptured cell membranes, wrinkled morphology, and disappeared pseudopods. In addition, the experimental results showed that the glucose content in the supernatant was significantly reduced compared with the control group. After administration of antifungal therapy, the growth of C. albicans was inhibited, and the glucose content was increased considerably. In addition, the pyruvic acid content was elevated, and the lactic acid content was decreased in the model group, which was consistent with the results of metabolomics. LDH, an important metabolic enzyme for glycolysis, as a marker of cell damage, was significantly elevated in the supernatant of the model group, indicating that the cell damage was severe. A large amount of leakage of LDH in the cytoplasm occurred, and the glycolysis of the cells was inhibited.
To further demonstrate the inhibition of glycolysis in vaginal epithelial cells, we examined the key glycolytic metabolizing enzymes, LDHA and PKM2, using immunofluorescence. As shown in Figure 6, the green fluorescence intensity represented the expression degree of LDHA in cells, the red fluorescence intensity represented the expression degree of PKM2 in cells, and the blue fluorescence represented the nucleus. Compared with the control group, the levels of LDHA and PKM2 were significantly lower in the model group, indicating that the glycolysis of infected cells was inhibited; after the administration of antifungal treatment, the levels of these two enzymes were close to those of the control group, indicating that the antifungal treatment restored the cells to the normal level of glycolysis.

4. Discussion

Exploring metabolites of diseases is vital for disease prevention, diagnosis, and treatment. Metabolomics, as an important method for detecting endogenous small molecule metabolites, can be used to detect metabolites in body fluids, tissues, and cells for efficient and accurate diagnosis of diseases, and it is often used in the study of various diseases, including cancer, cardiovascular disease, genital inflammation, etc. Gynecological diseases are often closely linked to metabolic disorders, such as cervical cancer, cervicitis, and vaginitis [20,21].
Camilla Ceccarani et al. performed a metabolomic study of vaginal secretions from VVC patients using 1H-NMR. They found more than 10 differential metabolites, including lactic acid, phenylalanine, taurine, etc., in samples from VVC patients compared with the healthy group [19]. In our study, we performed metabolomic analysis of an in vitro model of VVC using UHPLC-Q-exact MS, and we found that a total of 50 metabolites were significantly altered in the model group, and these metabolites may be disease biomarkers of VVC. Specifically, we found that lactic acid and phenylalanine levels were also significantly down-regulated in the model group. This further suggested that lactic acid and phenylalanine could be used as disease markers for VVC. In addition, previous studies have shown that D-arabitol is the biomarker for C. albicans infection, and GC-MS was used to detect the D-arabitol and L-arabitol ratio in urine to determine the presence of C. albicans infection [22]. In our experiments, the D-arabitol level was significantly elevated in the model group and significantly decreased after drug administration, suggesting that D-arabitol can be used as one of the biomarkers for diagnosing VVC.
In addition, these significantly altered potential metabolites we have identified are closely linked to disease. Lysophosphatidylcholine is considered a deleterious pro-inflammatory mediator, with levels elevated considerably when inflammation occurs in the body [23], and has been implicated as a biomarker for cervical cancer [24]. Palmitic acid, a saturated fatty acid, activates toll-like receptors to promote inflammation [25]. Under hypoxic and inflammatory conditions, adenosine levels are significantly elevated to inhibit inflammation [26], but persistently high adenosine levels favor tumor cell growth, promoting cancer [27]. Thiamine is a kind of B vitamin that has an important effect on cellular energy metabolism, and a deficiency can cause a decrease in ATP production and lead to cell death [28]. Betaine (trimethylglycine) is an osmoprotective agent that is anti-inflammatory and regulates energy metabolism [29], but a positive correlation has been reported between betaine concentrations and reproductive inflammation [21]. Maintaining normal levels of amino acids is important for health, and disturbances in amino acid metabolism often accompany genital inflammation. In the present study, significant changes in amino acids were observed in the model group, including phenylalanine, L-homoserine, cysteinylglycine, L-glutamine, L-arginine, N-acetyl-L-aspartic acid, Beta-citryl-L-glutamic acid, and L-aspartic acid. This may be due to the secretion of the virulence factor SAPs by C. albicans, which allows the hydrolysis of various proteins in the vaginal epithelium, leading to disturbed amino acid metabolism in the VVC [19].
In particular, we found that metabolites involved in the glycolysis, nicotinate, and nicotinamide metabolism pathways were significantly altered in the VVC model, including 2-phosphoglyceric acid, phosphoenolpyruvic acid, pyruvic acid, lactic acid, NAM, and NA.
Lactic acid is essential for maintaining the stability of the intravaginal environment. Lactic acid produced by vaginal epithelial cells and lactic acid produced by Lactobacillus maintains the pH of the vaginal environment against the growth of harmful microorganisms [30]. Some studies have shown that lactic acid inhibits the growth of C. albicans [31]. The innate immune mechanism of vaginal epithelial cells plays a crucial role in antibacterial infections [32]. Notably, lactic acid can further modulate local immunity within the vagina by stimulating the production of anti-inflammatory factors by human vaginal epithelial cells and inhibiting the production of inflammatory factors (IL-6 and IL-8) triggered by toll-like receptors [33]. This was corroborated by the ELISA results (Figure 7), which showed that the levels of the inflammatory factors IL-6 and IL-8 in the cell supernatants were significantly lower in the control and AmB groups, which had normal lactic acid levels, than in the model group, which had lower lactic acid levels.
As shown in Figure 8, in the hypoxic vaginal cavity, vaginal epithelial cells can produce ATP by anaerobic glycolysis and produce lactic acid to diffuse into the vaginal cavity. Briefly, glucose is metabolized to a series of glycolytic intermediates such as glucose-6-phosphate, glyceraldehyde 3-phosphate, 2-phosphoglyceric acid, etc. 2-phosphoglyceric acid is converted to phosphoenolpyruvic acid, which PKM2 catalyzes to produce pyruvic acid, and eventually pyruvic acid is catalyzed by LDH to produce lactic acid [15,33]. Compared with the control group, the glucose content in the supernatant of the model group was significantly lower, which implied that C. albicans infection depleted a large amount of glucose in the culture system, which may lead to a decrease in the amount of glucose that enters vaginal epithelial cells to participate in glycolysis. In addition, after the cells were attacked by C. albicans, the expression of LDHA and PKM2 was inhibited, which prevented normal glycolysis in the cells. Thus, the production of metabolites of glycolysis was reduced. The metabolomics results showed a significant decrease in 2-phosphoglyceric acid, phosphoenolpyruvic acid, and lactic acid and an increase in pyruvic acid in the model group, which was consistent with the results of pyruvic acid and lactic acid detection in the culture medium. The increased pyruvic acid content in the model group may be due to the accumulation of pyruvic acid as a result of the significant reduction in LDH preventing its normal conversion to lactic acid.
NADH is oxidized to nicotinamide adenine dinucleotide (NAD+) during the LDHA-catalyzed conversion of pyruvic acid to lactic acid [34]. NAD+ is an essential coenzyme in cellular metabolism and is indispensable for glycolysis. NAM and NA are both precursors of NAD+, producing NAD+ to maintain cellular homeostasis. NAM and NA are both B vitamins necessary for living cells, and NA deficiency can lead to mucosal inflammation [35]. NAM and glycolysis are closely linked. As an inhibitor of PARP-1, it avoids the blocking effect of glycolysis caused by PARP-1. In addition, glycolysis produces NAD+, which produces NAM in the presence of enzymes [36]. NAM has been shown to reduce oxidative stress and has anti-inflammatory effects [37]. NAM is reported to have significant anti-C. albicans activity and can synergize with amphotericin B to play a common antifungal role [38,39]. In our study, the levels of NAM and NA were significantly lower in the model group, which may be attributed to the reduced expression of LDHA in the cells and the inhibition of anaerobic glycolysis, reducing NADH conversion to NAD+.
It can be seen that metabolites such as lactic acid, NAM, and NA, which are closely related to glycolysis, are essential for maintaining the health of the human intravaginal environment and that C. albicans infection causes disruptions in cellular glycolysis, which disrupts the balance of these natural protective factors, allowing the body’s biological protective effects to be adversely affected.

5. Conclusions

In this study, we revealed the metabolic disorders and inhibition of glycolysis in vaginal epithelial cells induced by C. albicans infection through metabolomics and biological validation, which provided a reference for the clinical diagnosis and treatment of VVC caused by C. albicans infection. In addition, our findings provide new insight for further exploration of C. albicans pathogenesis.

Author Contributions

Conceptualization, Y.Z. and P.W.; Software, Y.Z., P.W. and X.S.; Validation, Y.Z. and X.S.; Formal analysis, Y.Z., M.Z. and Y.C.; Investigation, Y.Z.; Resources, X.S.; Data curation, X.S. and M.Z.; Writing—original draft, Y.Z. and P.W.; Writing—review & editing, Y.Z., P.W., X.S., M.Z., Y.C. and X.G.; Visualization, X.G.; Supervision, X.G.; Project administration, P.W., Y.C. and X.G. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Science and Technology Department of Guizhou Province ([2021]414).

Data Availability Statement

The data presented in this study are available upon request from the corresponding author.

Acknowledgments

The authors are sincerely grateful to Guizhou Changsheng Pharmaceutical Co., Ltd. for supporting our research and to Bo Hu for her assistance with the research.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Dovnik, A.; Golle, A.; Novak, D.; Arko, D.; Takač, I. Treatment of vulvovaginal candidiasis: A review of the literature. Acta Dermatovenerol. Alp. Pannonica Adriat. 2015, 24, 5–7. [Google Scholar] [CrossRef]
  2. Gonçalves, B.; Ferreira, C.; Alves, C.T.; Henriques, M.; Azeredo, J.; Silva, S. Vulvovaginal candidiasis: Epidemiology, microbiology and risk factors. Crit. Rev. Microbiol. 2016, 42, 905–927. [Google Scholar] [CrossRef]
  3. Rosati, D.; Bruno, M.; Jaeger, M.; Ten Oever, J.; Netea, M.G. Recurrent Vulvovaginal Candidiasis: An Immunological Perspective. Microorganisms 2020, 8, 144. [Google Scholar] [CrossRef]
  4. Baron, E.J.; Miller, J.M.; Weinstein, M.P.; Richter, S.S.; Gilligan, P.H.; Thomson, R.B., Jr.; Bourbeau, P.; Carroll, K.C.; Kehl, S.C.; Dunne, W.M.; et al. A guide to utilization of the microbiology laboratory for diagnosis of infectious diseases: 2013 recommendations by the Infectious Diseases Society of America (IDSA) and the American Society for Microbiology (ASM)(a). Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am. 2013, 57, e22–e121. [Google Scholar] [CrossRef]
  5. Rinschen, M.M.; Ivanisevic, J.; Giera, M.; Siuzdak, G. Identification of bioactive metabolites using activity metabolomics. Nat. Rev. Mol. Cell Biol. 2019, 20, 353–367. [Google Scholar] [CrossRef]
  6. Shroff, A.; Sequeira, R.; Reddy, K.V.R. Human vaginal epithelial cells augment autophagy marker genes in response to Candida albicans infection. Am. J. Reprod. Immunol. 2017, 77, e12639. [Google Scholar] [CrossRef]
  7. Roselletti, E.; Perito, S.; Sabbatini, S.; Monari, C.; Vecchiarelli, A. Vaginal Epithelial Cells Discriminate Between Yeast and Hyphae of Candida albicans in Women Who Are Colonized or Have Vaginal Candidiasis. J. Infect. Dis. 2019, 220, 1645–1654. [Google Scholar] [CrossRef] [PubMed]
  8. Lopes, J.P.; Lionakis, M.S. Pathogenesis and virulence of Candida albicans. Virulence 2022, 13, 89–121. [Google Scholar] [CrossRef] [PubMed]
  9. Modrzewska, B.; Kurnatowski, P. Adherence of Candida sp. to host tissues and cells as one of its pathogenicity features. Ann. Parasitol. 2015, 61, 3–9. [Google Scholar]
  10. Dabiri, S.; Shams-Ghahfarokhi, M.; Razzaghi-Abyaneh, M. Comparative analysis of proteinase, phospholipase, hydrophobicity and biofilm forming ability in Candida species isolated from clinical specimens. J. Mycol. Med. 2018, 28, 437–442. [Google Scholar] [CrossRef] [PubMed]
  11. Kadry, A.A.; El-Ganiny, A.M.; El-Baz, A.M. Relationship between Sap prevalence and biofilm formation among resistant clinical isolates of Candida albicans. Afr. Health Sci. 2018, 18, 1166–1174. [Google Scholar] [CrossRef] [PubMed]
  12. Niu, X.X.; Li, T.; Zhang, X.; Wang, S.X.; Liu, Z.H. Lactobacillus crispatus Modulates Vaginal Epithelial Cell Innate Response to Candida albicans. Chin. Med. J. 2017, 130, 273–279. [Google Scholar] [CrossRef] [PubMed]
  13. Osis, G.; Traylor, A.M.; Black, L.M.; Spangler, D.; George, J.F.; Zarjou, A.; Verlander, J.W.; Agarwal, A. Expression of lactate dehydrogenase A and B isoforms in the mouse kidney. Am. J. Physiol. Renal Physiol. 2021, 320, F706–F718. [Google Scholar] [CrossRef] [PubMed]
  14. Liu, S.; Liao, S.; Liang, L.; Deng, J.; Zhou, Y. The relationship between CD4(+) T cell glycolysis and their functions. Trends Endocrinol. Metab. 2023, 34, 345–360. [Google Scholar] [CrossRef] [PubMed]
  15. Chandel, N.S. Glycolysis. Cold Spring Harb. Perspect. Biol. 2021, 13, a040535. [Google Scholar] [CrossRef] [PubMed]
  16. Yin, Y.; Choi, S.C.; Xu, Z.; Perry, D.J.; Seay, H.; Croker, B.P.; Sobel, E.S.; Brusko, T.M.; Morel, L. Normalization of CD4+ T cell metabolism reverses lupus. Sci. Transl. Med. 2015, 7, 274ra218. [Google Scholar] [CrossRef]
  17. Desai, J.V.; Urban, A.; Swaim, D.Z.; Colton, B.; Kibathi, L.W.; Ferrè, E.M.N.; Stratton, P.; Merideth, M.A.; Hunsberger, S.; Matkovits, T.; et al. Efficacy of Cochleated Amphotericin B in Mouse and Human Mucocutaneous Candidiasis. Antimicrob. Agents Chemother. 2022, 66, e0030822. [Google Scholar] [CrossRef]
  18. Bezerra, L.S.; Silva, J.A.D.; Santos-Veloso, M.A.O.; Lima, S.G.; Chaves-Markman, Â.V.; Jucá, M.B. Antifungal Efficacy of Amphotericin B in Candida Albicans Endocarditis Therapy: Systematic Review. Braz. J. Cardiovasc. Surg. 2020, 35, 789–796. [Google Scholar] [CrossRef]
  19. Ceccarani, C.; Foschi, C.; Parolin, C.; D’Antuono, A.; Gaspari, V.; Consolandi, C.; Laghi, L.; Camboni, T.; Vitali, B.; Severgnini, M.; et al. Diversity of vaginal microbiome and metabolome during genital infections. Sci. Rep. 2019, 9, 14095. [Google Scholar] [CrossRef]
  20. Wishart, D.S. Metabolomics for Investigating Physiological and Pathophysiological Processes. Physiol. Rev. 2019, 99, 1819–1875. [Google Scholar] [CrossRef]
  21. Ilhan, Z.E.; Łaniewski, P.; Thomas, N.; Roe, D.J.; Chase, D.M.; Herbst-Kralovetz, M.M. Deciphering the complex interplay between microbiota, HPV, inflammation and cancer through cervicovaginal metabolic profiling. EBioMedicine 2019, 44, 675–690. [Google Scholar] [CrossRef]
  22. Sigmundsdóttir, G.; Christensson, B.; Björklund, L.J.; Håkansson, K.; Pehrson, C.; Larsson, L. Urine D-arabinitol/L-arabinitol ratio in diagnosis of invasive candidiasis in newborn infants. J. Clin. Microbiol. 2000, 38, 3039–3042. [Google Scholar] [CrossRef]
  23. Knuplez, E.; Marsche, G. An Updated Review of Pro- and Anti-Inflammatory Properties of Plasma Lysophosphatidylcholines in the Vascular System. Int. J. Mol. Sci. 2020, 21, 4501. [Google Scholar] [CrossRef]
  24. Yang, K.; Xia, B.; Wang, W.; Cheng, J.; Yin, M.; Xie, H.; Li, J.; Ma, L.; Yang, C.; Li, A.; et al. A Comprehensive Analysis of Metabolomics and Transcriptomics in Cervical Cancer. Sci. Rep. 2017, 7, 43353. [Google Scholar] [CrossRef]
  25. Korbecki, J.; Bajdak-Rusinek, K. The effect of palmitic acid on inflammatory response in macrophages: An overview of molecular mechanisms. Inflamm. Res. 2019, 68, 915–932. [Google Scholar] [CrossRef]
  26. Li, X.; Berg, N.K.; Mills, T.; Zhang, K.; Eltzschig, H.K.; Yuan, X. Adenosine at the Interphase of Hypoxia and Inflammation in Lung Injury. Front. Immunol. 2020, 11, 604944. [Google Scholar] [CrossRef] [PubMed]
  27. Antonioli, L.; Blandizzi, C.; Pacher, P.; Haskó, G. Immunity, inflammation and cancer: A leading role for adenosine. Nat. Rev. Cancer 2013, 13, 842–857. [Google Scholar] [CrossRef]
  28. Smith, T.J.; Johnson, C.R.; Koshy, R.; Hess, S.Y.; Qureshi, U.A.; Mynak, M.L.; Fischer, P.R. Thiamine deficiency disorders: A clinical perspective. Ann. N. Y. Acad. Sci. 2021, 1498, 9–28. [Google Scholar] [CrossRef]
  29. Zhao, G.; He, F.; Wu, C.; Li, P.; Li, N.; Deng, J.; Zhu, G.; Ren, W.; Peng, Y. Betaine in Inflammation: Mechanistic Aspects and Applications. Front. Immunol. 2018, 9, 1070. [Google Scholar] [CrossRef] [PubMed]
  30. Tachedjian, G.; Aldunate, M.; Bradshaw, C.S.; Cone, R.A. The role of lactic acid production by probiotic Lactobacillus species in vaginal health. Res. Microbiol. 2017, 168, 782–792. [Google Scholar] [CrossRef] [PubMed]
  31. Köhler, G.A.; Assefa, S.; Reid, G. Probiotic interference of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 with the opportunistic fungal pathogen Candida albicans. Infect. Dis. Obstet. Gynecol. 2012, 2012, 636474. [Google Scholar] [CrossRef]
  32. Cassone, A. Vulvovaginal Candida albicans infections: Pathogenesis, immunity and vaccine prospects. BJOG 2015, 122, 785–794. [Google Scholar] [CrossRef]
  33. Baldewijns, S.; Sillen, M.; Palmans, I.; Vandecruys, P.; Van Dijck, P.; Demuyser, L. The Role of Fatty Acid Metabolites in Vaginal Health and Disease: Application to Candidiasis. Front. Microbiol. 2021, 12, 705779. [Google Scholar] [CrossRef] [PubMed]
  34. Lagarde, D.; Jeanson, Y.; Portais, J.C.; Galinier, A.; Ader, I.; Casteilla, L.; Carrière, A. Lactate Fluxes and Plasticity of Adipose Tissues: A Redox Perspective. Front. Physiol. 2021, 12, 689747. [Google Scholar] [CrossRef]
  35. Chu, X.; Raju, R.P. Regulation of NAD(+) metabolism in aging and disease. Metabolism 2022, 126, 154923. [Google Scholar] [CrossRef]
  36. Fania, L.; Mazzanti, C.; Campione, E.; Candi, E.; Abeni, D.; Dellambra, E. Role of Nicotinamide in Genomic Stability and Skin Cancer Chemoprevention. Int. J. Mol. Sci. 2019, 20, 5946. [Google Scholar] [CrossRef]
  37. Mitchell, S.J.; Bernier, M.; Aon, M.A.; Cortassa, S.; Kim, E.Y.; Fang, E.F.; Palacios, H.H.; Ali, A.; Navas-Enamorado, I.; Di Francesco, A.; et al. Nicotinamide Improves Aspects of Healthspan, but Not Lifespan, in Mice. Cell Metab. 2018, 27, 667–676.e664. [Google Scholar] [CrossRef]
  38. Xing, X.; Liao, Z.; Tan, F.; Zhu, Z.; Jiang, Y.; Cao, Y. Effect of Nicotinamide Against Candida albicans. Front. Microbiol. 2019, 10, 595. [Google Scholar] [CrossRef] [PubMed]
  39. Yan, Y.; Liao, Z.; Shen, J.; Zhu, Z.; Cao, Y. Nicotinamide potentiates amphotericin B activity against Candida albicans. Virulence 2022, 13, 1533–1542. [Google Scholar] [CrossRef]
Microorganisms 12 00292 g001
Figure 1. Metabolomics analysis. (A) PCA score plot, n = 6. (B,C) OPLS−DA score plot and permutation diagrams (control vs. model; model vs. AmB), n = 6.
Figure 1. Metabolomics analysis. (A) PCA score plot, n = 6. (B,C) OPLS−DA score plot and permutation diagrams (control vs. model; model vs. AmB), n = 6.
Microorganisms 12 00292 g001
Microorganisms 12 00292 g002
Figure 2. Heatmap of 50 differential metabolites in the model group compared to the control group.
Figure 2. Heatmap of 50 differential metabolites in the model group compared to the control group.
Microorganisms 12 00292 g002
Microorganisms 12 00292 g003
Figure 3. There were significant changes in glycolysis-related metabolites in the model group compared to the control group, and the levels returned to normal after administration. *** p < 0.001.
Figure 3. There were significant changes in glycolysis-related metabolites in the model group compared to the control group, and the levels returned to normal after administration. *** p < 0.001.
Microorganisms 12 00292 g003
Microorganisms 12 00292 g004
Figure 4. C. albicans infection leads to cell membrane damage.
Figure 4. C. albicans infection leads to cell membrane damage.
Microorganisms 12 00292 g004
Microorganisms 12 00292 g005
Figure 5. The glycolysis-related metabolites and enzyme (glucose, pyruvic acid, lactic acid, and LDH) levels in the supernatant. *** p < 0.001.
Figure 5. The glycolysis-related metabolites and enzyme (glucose, pyruvic acid, lactic acid, and LDH) levels in the supernatant. *** p < 0.001.
Microorganisms 12 00292 g005
Microorganisms 12 00292 g006
Figure 6. LDHA and PKM2 expression levels in vaginal epithelial cells. ** p < 0.01; *** p < 0.001.
Figure 6. LDHA and PKM2 expression levels in vaginal epithelial cells. ** p < 0.01; *** p < 0.001.
Microorganisms 12 00292 g006
Microorganisms 12 00292 g007
Figure 7. The inflammatory factors’ (IL-6, IL-8) levels in the supernatant. **** p < 0.0001.
Figure 7. The inflammatory factors’ (IL-6, IL-8) levels in the supernatant. **** p < 0.0001.
Microorganisms 12 00292 g007
Microorganisms 12 00292 g008
Figure 8. Schematic representation of the metabolic effects of C. albicans on vaginal epithelial cells. The green borders represent down-regulation of metabolite levels and red borders represent up-regulation of metabolite levels; red arrows represent the glycolysis and blue arrows represent the nicotinate and nicotinamide metabolism.
Figure 8. Schematic representation of the metabolic effects of C. albicans on vaginal epithelial cells. The green borders represent down-regulation of metabolite levels and red borders represent up-regulation of metabolite levels; red arrows represent the glycolysis and blue arrows represent the nicotinate and nicotinamide metabolism.
Microorganisms 12 00292 g008
Table 1. Significant reversal of regulation of 28 differential metabolites after administration of antifungal therapy.( The “↑” symbol represents an increase in metabolite levels and the “↓” symbol represents a decrease in metabolite levels.)
Table 1. Significant reversal of regulation of 28 differential metabolites after administration of antifungal therapy.( The “↑” symbol represents an increase in metabolite levels and the “↓” symbol represents a decrease in metabolite levels.)
No.NameHMDBFormulam/zRT (Min)Model vs. ControlAmB vs. Model
1LysoPC (18:2(9Z,12Z))HMDB0010386C26H50NO7P519.3310.71↑↓
2Adenylsuccinic acidHMDB0000536C14H18N5O11P463.072.74↑↓
3IndoleHMDB0000738C8H7N117.065.67↑↓
4Palmitic acidHMDB0000220C16H32O2273.278.91↑↓
52-Furoic acidHMDB0000617C5H4O3112.011.47↑↓
6Guanosine monophosphateHMDB0001397C10H14N5O8P381.071.69↑↓
7BetaineHMDB0000043C5H11NO2117.081.06↑↓
8CysteinylglycineHMDB0000078C5H10N2O3S161.011.45↑↓
9Pipecolic acidHMDB0000070C6H11NO2147.091.45↑↓
10GuanineHMDB0000132C5H5N5O134.021.69↑↓
11Pyruvic acidHMDB0000243C3H4O3106.031.53↑↓
12Ophthalmic acidHMDB0005765C11H19N3O6289.131.51↑↓
13D-ArabitolHMDB0000568C5H12O5152.071.02↑↓
14DiacetylHMDB0003407C4H6O2104.051.49↑↓
15cis-Aconitic acidHMDB0000072C6H6O6174.021.08↑↓
16ThiamineHMDB0000235C12H16N4OS264.101.22↓↑
17NicotinamideHMDB0001406C6H6N2O122.051.43↓↑
18HypoxanthineHMDB0000157C5H4N4O136.041.43↓↑
19N-Acetyl-L-aspartic acidHMDB0000812C6H9NO5175.051.12↓↑
20InosineHMDB0000195C10H12N4O5268.081.70↓↑
21UracilHMDB0000300C4H4N2O295.001.50↓↑
22Nicotinic acidHMDB0001488C6H5NO2123.031.18↓↑
23Allantoic acidHMDB0001209C4H8N4O4176.050.92↓↑
24Beta-Citryl-L-glutamic acidHMDB0013220C11H15NO10321.071.60↓↑
25L-Lactic acidHMDB0000190C3H6O390.031.43↓↑
262-Phosphoglyceric acidHMDB0000362C3H7O7P185.991.26↓↑
27Phosphoenolpyruvic acidHMDB0000263C3H5O6P167.981.39↓↑
28OctadecanamideHMDB0034146C18H37NO283.2914.69↓↑
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Zhao, Y.; Wang, P.; Sun, X.; Zhao, M.; Chen, Y.; Gao, X. Candida albicans Infection Disrupts the Metabolism of Vaginal Epithelial Cells and Inhibits Cellular Glycolysis. Microorganisms 2024, 12, 292. https://doi.org/10.3390/microorganisms12020292

AMA Style

Zhao Y, Wang P, Sun X, Zhao M, Chen Y, Gao X. Candida albicans Infection Disrupts the Metabolism of Vaginal Epithelial Cells and Inhibits Cellular Glycolysis. Microorganisms. 2024; 12(2):292. https://doi.org/10.3390/microorganisms12020292

Chicago/Turabian Style

Zhao, Yanni, Pengjiao Wang, Xiaodong Sun, Mei Zhao, Yixuan Chen, and Xiuli Gao. 2024. "Candida albicans Infection Disrupts the Metabolism of Vaginal Epithelial Cells and Inhibits Cellular Glycolysis" Microorganisms 12, no. 2: 292. https://doi.org/10.3390/microorganisms12020292

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop