Abstract

   The biofilm formation of Candida albicans, a major human fungal
   pathogen, represents a crucial virulence factor during candidiasis.
   Eicosapentaenoic acid (EPA), a polyunsaturated fatty acid, has emerged
   as a potential antibiofilm agent against C. albicans. Herein, we aim to
   investigate the antifungal effect of EPA (1 mM) on the mature biofilm
   of C. albicans and explore the underlying mechanism. Crystal violet and
   XTT assays showed that EPA exerted a strong inhibitory efficacy on
   preformed biofilms in C. albicans. Biofilm architecture and cell
   viability were observed using scanning electron microscopy and confocal
   laser scanning microscopy, indicating that EPA could block the
   yeast-to-hypha transition and damage the structure, thereby exhibiting
   antibiofilm activity. RNA sequencing analysis revealed that EPA
   treatment led to the downregulation of genes associated with hyphal
   formation and biofilm development. From the signaling pathway
   perspective, EPA regulated the C. albicans biofilms involving two
   signaling pathways, namely, Ras1-cAMP-PKA and Cek-MAPK pathways.
   Additionally, the EPA could effectively reduce the production of key
   messenger cAMP in the Ras1-cAMP-PKA pathway. Interestingly, in response
   to EPA, ergosterol biosynthesis-related genes were down-regulated,
   indicating EPA as antifungal agent might reduce the risk of developing
   drug resistance. The findings of this study highlight the potential of
   EPA as an alternative or adjunctive antibiofilm agent against C.
   albicans-related infections.

   Keywords: Candida albicans, Candidemia, Biofilm, EPA, Transcriptome

Highlights

     * •
       EPA exhibits potent antibiofilm activity against mature biofilms of
       Candida albicans by inhibiting yeast-to-hypha transition.
     * •
       Transcriptomic analysis reveals that EPA modulates key signaling
       pathways to affect C. albicans biofilm.
     * •
       These findings demonstrate that EPA might be an alternative or
       adjunct therapeutic agent against C. albicans-related infections.

1. Introduction

   Candidemia, an important nosocomial bloodstream infection associated
   with Candida spp., is most commonly caused by C. albicans [[39]1]. It
   poses a significant threat worldwide, in terms of substantial mortality
   and high hospital costs [[40][2], [41][3], [42][4]]. The ability of C.
   albicans to persist as a biofilm on medical devices is a crucial
   virulence factor, which has been linked with poor clinical outcomes
   [[43][5], [44][6], [45][7]]. Biofilms formed by C. albicans are complex
   and structured microbial communities, consisting of yeast, hyphae, and
   pseudohyphae surrounded by a protective extracellular matrix [[46]8].
   These biofilms provide a robust defense mechanism, significantly
   enhancing tolerance against conventional antifungal agents and the host
   immune system [[47]9]. Therefore, there is an urgent need for
   innovative antibiofilm treatment strategies to address this critical
   challenge.

   Eicosapentaenoic acid (EPA), a member of the omega-3 polyunsaturated
   fatty acid family and one of the essential fatty acids for human
   health, has emerged as a promising antimicrobial agent [[48]10]. Recent
   studies have reported that EPA can inhibit biofilm development by
   several microorganisms, including Enterococcus faecium [[49]11],
   Staphylococcus aureus [[50]12,[51]13], Staphylococcus epidermidis
   [[52]13], Streptococcus mutans [[53]14], and some oral pathogenic
   bacteria [[54]15,[55]16].

   Our previous research determined a significant antagonistic effect of
   EPA against C. albicans biofilms [[56]17]. Notably, even mature
   biofilms, typically recalcitrant to antimicrobial agents, were
   effectively eradicated by 1 mM EPA. Furthermore, other studies have
   reported the antifungal properties of EPA on C. albicans. For example,
   Thibane et al. demonstrated that EPA could cause apoptosis in C.
   albicans biofilms, as indicated by a decrease in mitochondrial membrane
   potential and the occurrence of nuclear condensation and fragmentation
   [[57]18]. Mokoena et al. discovered the EPA impacted C. albicans in
   Caenorhabditis elegans due to the inhibition of hyphal formation and
   stimulation of the host immune response [[58]19]. However, the
   transcriptome-based biofilm-related changes remain to be elucidated.

   A previous study suggested that medium-chain fatty acids mimicked the
   quorum-sensing molecule farnesol, and thus, caused physiological
   changes of fungal dimorphism, biofilm formation, and even cell death of
   C. albicans [[59]20]. Farnesol, a by-product in the ergosterol
   biosynthetic process of C. albicans, has been shown to inhibit
   filamentation and biofilm formation primarily by regulating complex
   signaling pathways, one of the more well-studied interactions is the
   inhibitory role of farnesol on the cyclic AMP (cAMP) signaling pathway
   [[60]21,[61]22]. In addition, the mitogen-activated protein kinase
   (MAPK) signaling pathway is also a major regulatory pathway in
   controlling biofilm formation in C. albicans (mediated by the Cek1/2,
   Mkc1, or Hog 1 MAPK) [[62]23,[63]24]. It remains unknown whether EPA
   possesses antibiofilm activity against C. albicans potentially through
   analogous mechanisms as farnesol.

   Therefore, this study aimed to elucidate the mechanism of EPA-mediated
   disruption of C. albicans biofilms using phenotypic and transcriptomic
   analysis. Delineating the mechanism of EPA-mediated disruption of C.
   albicans biofilms has the potential to inform the development of
   targeted therapeutic strategies for Candida-related biofilm infections.

2. Material and methods

2.1. Strains and growth conditions

   This study used 31 clinical isolates of pre-characterized C. albicans
   with strong biofilm-forming ability from candidemia [[64]17] and a
   reference strain C. albicans ATCC 90028. All C. albicans strains were
   routinely refreshed from the frozen stocks at −20 °C and inoculated at
   least twice onto Sabouraud Dextrose Agar (SDA) at 35 °C for 24 h before
   all experiments.

2.2. Reagents

   EPA (20:5; ω-3) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
   EPA was prepared as 2 M stock solutions in dimethyl sulfoxide (DMSO)
   and stored at −20 °C. Working solutions were prepared by diluting the
   stock with RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham,
   MA, USA). Our preliminary results show that DMSO at <0.1 % did not
   affect the growth or biofilm formation of C. albicans.

2.3. The minimum inhibitory concentration assay of EPA

   The minimum inhibitory concentration (MIC) of EPA on planktonic cells
   of C. albicans was determined using the microdilution method. According
   to Clinical and Laboratory Standards Institute (CLSI) protocols
   [[65]25], the initial concentration of the C. albicans suspension was
   adjusted to 1 × 10^3 CFU/mL in the RPMI 1640 medium. EPA was diluted in
   RPMI medium and tested at concentrations ranging from 0.125 mM to 1 mM.
   Following a 24 h incubation period at 35 °C, the MIC was determined
   visually as the lowest concentration at which there was a 50 % decrease
   in the growth of planktonic C. albicans. The assays were carried out in
   triplicate via three independent experiments.

2.4. Biofilm formation of C. albicans

   The biofilms of C. albicans were prepared as described previously
   [[66]17]. In brief, C. albicans cells were collected from overnight
   cultures and diluted to 1 × 10^5 CFU/mL with RPMI 1640 medium (Gibco,
   Thermo Fisher Scientific, Waltham, MA, USA). The C. albicans suspension
   was added into sterile 96-well round-bottomed polystyrene plates
   (Corning, NY, USA) of 200 μL each well, and the plates were incubated
   at 35 °C in an atmosphere of 5 % CO[2] for 24 h.

   After incubation, the non-adherent cells were removed by washing gently
   with phosphate-buffered saline (PBS; pH 7.2) twice. Then, 200 μL of
   fresh RPMI 1640 medium with or without 1 mM EPA (Sigma-Aldrich; St.
   Louis, MO, USA) was added to the above microtiter plates to detect
   whether EPA impacts an established biofilm. RPMI 1640 medium
   supplemented with 0.1 % DMSO was used as a solvent control. After
   another incubation at 35 °C for 4 h or 24 h, the medium was aspirated,
   and non-adherent cells were removed by washing the biofilms with 200 μL
   of PBS twice.

2.5. Determination of the biomass of C. albicans biofilms

   The C. albicans total biofilm mass was determined with the crystal
   violet (CV) assay previously described by Gulati et al. [[67]26].
   Briefly, the biofilms were fixed with 200 μL methanol for 15 min,
   stained with 200 μL crystal violet (0.2 %) for 20 min, and washed with
   distilled water. Then, the bound crystal violet was extracted with
   200 μL of 33 % acetic acid. 150 μL of the obtained solutions were
   transferred to a new flat bottom 96-well plate, and the OD[630 nm]
   values were measured using a Multiskan EX microplate photometer (Thermo
   Fisher Scientific, Waltham, MA, United States). The CV assays were
   performed in three independent biological replicates, each treatment in
   triplicates.

2.6. Determination of the metabolic activity of C. albicans biofilms

   The metabolic activity was assessed with the XTT
   [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanili
   de] reduction assay as previously described by Manoharan et al.
   [[68]27]. An XTT Cell Proliferation Kit II (Roche, Mannheim, Germany)
   was used according to the manufacturer's instructions. 200 μL of XTT
   solution was added to each well and incubated in the dark for 4 h at
   35 °C. After incubation, 150 μL of the solutions were transferred to a
   new 96-well plate to measure the OD[450 nm] values. The XTT assays were
   performed in three independent biological replicates, each treatment in
   triplicates.

2.7. Observations of the biofilm structure of C. albicans

   The effect of EPA on the biofilm structure of C. albicans was examined
   by scanning electron microscopy (SEM) and confocal laser scanning
   microscopy (CLSM). Biofilms were formed on cover glass (Solarbio,
   Beijing, China) deposited in 6-well plates (Corning, NY, USA) (3 mL of
   cell suspension per well). After 24 h of incubation, the cover glass
   was washed twice with PBS and put in an empty microplate well. 3 mL of
   fresh RPMI 1640 medium with or without 1 mM EPA was added and incubated
   for 24 h at the same culture conditions.

   For SEM, the biofilms were fixed in 2.5 % glutaraldehyde for 5 h at
   4 °C, then were dehydrated in a sequential-graded ethanol (30 %, 50 %,
   70 %, 80 %, 90 %, and 100 %), and then two times with 100 % ethanol for
   15 min. Finally, the samples were sputter-coated with gold and observed
   with an SU8020 scanning electron microscope (Hitachi, Japan) [[69]11].

   For CLSM, the biofilms were stained with a live/dead viability kit
   (Invitrogen, CA, United States). The stain was prepared by diluting
   3 μL of SYTO 9 and 3 μL of propidium iodide (PI) in 1.0 mL of
   filter-sterilized water. About 500 μL of staining solution was added to
   each biofilm sample. Samples were incubated for 30 min at room
   temperature in the dark. The biofilm architecture was then analyzed by
   a confocal laser scanning microscope (Leica TCS SP5, Germany). The
   excitation/emission was 488/498 nm for SYTO 9 and 535/617 nm for PI.
   Five random fields of view per experimental condition were imaged
   [[70]28].

2.8. Transcriptomic analysis

2.8.1. RNA extraction

   The total RNA was extracted from the EPA-treated and untreated biofilms
   of C. albicans isolate (ATCC 90028 and X27) using an RNAprep Pure Plant
   Kit (TIANGEN, Beijing, China) according to the manufacturer's
   instructions. The concentration and integrity of extracted RNA were
   assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific,
   USA) and Bioanalyzer 2100 system (Agilent Technologies, CA, USA).

2.8.2. RNA sequencing and analysis

   RNA sequencing (RNA-Seq) was performed by Novogene Co., Ltd (Beijing,
   China) with the Illumina NovaSeq 6000 platform (Illumina, USA) and
   paired-end reads with an average length of 150 bp were obtained. The
   raw sequence data have been deposited into the SRA database under the
   accession number [71]PRJNA1137062
   ([72]https://www.ncbi.nlm.nih.gov/sra/PRJNA1137062).

   For RNA-seq analysis, raw paired-end reads were filtered using the
   Fastp program v0.19.7 under default parameters. Clean reads were mapped
   to the genome of C. albicans SC5314, retrieved from the Candida Genome
   Database (CGD) ([73]www.candidagenome.org) using Hisat2 v2.0.5.
   Fragments per kilobase of transcript per million fragments mapped
   (FPKM) of each gene were calculated by FeatureCounts v1.5.0-p3. Gene
   expression analysis was performed using the DESeq2 R package (v1.20.0).
   The threshold of significantly differential expression genes (DEGs)
   between samples was the value of |Log[2] (fold change)| ≥ 1 and false
   discovery rate (FDR) < 0.05.

   Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes
   and Genomes (KEGG) pathway enrichment analysis of DEGs were performed
   with the ClusterProfiler (v3.8.1). Data represent three independent
   biological replicates for each condition.

2.9. Determination of intracellular cAMP levels

   The intracellular cAMP concentration was measured with a Cyclic AMP
   Select ELISA kit (Cayman Chemical, United States) following the
   manufacturer's instructions. Briefly, the biofilms of C. albicans were
   washed twice with pre-cooled PBS. Then, biofilm cells were scraped from
   the plates and resuspended in 3 mL of 0.1 M HCl. Following sonication,
   the supernatants were collected by centrifugation at 1500g for 10 min
   and transferred to a fresh tube. Then, half of each sample was used to
   determine the total protein concentration with a Bradford 1 × dye
   reagent (Solarbio, Beijing, China), and the other half was used to
   measure the cAMP level with the ELISA kit. The intracellular cAMP
   concentrations were converted to picomoles per milligram of protein
   [[74]29].

2.10. Statistical analysis

   Statistical analysis was performed using the IBM SPSS Statistics 20.0
   software program (IBM, Armonk, NY, USA). Data were expressed as
   means ± standard deviation (SD) of at least three independent
   experiments. A statistical comparison between the two groups was
   performed using the Student's t-test. A P-value <0.05 was considered
   statistically significant.

3. Results

3.1. Determination of MIC of EPA on planktonic cells of C. albicans

   In this study, results showed the MICs of EPA against C. albicans were
   ≥1 mM, determined by the microdilution method to evaluate the
   antifungal activity of the EPA.

3.2. EPA affected preformed biofilm of C. albicans

   The effect of EPA on the 24 h pre-formed biofilms biomass of 32 strains
   of C. albicans (a reference strain ATCC 90028 and 31 clinical isolates
   from candidemia) was evaluated. These strains have exhibited strong
   biofilm-forming abilities in our previous studies. The results showed
   EPA (1 mM) had a destructive effect on the 24 h pre-formed biofilms of
   90.63 % (29/32) strains. The OD[630 nm] values of C. albicans biofilms
   after EPA or RPMI 1640 medium treated 24 h were detected using the CV
   assay, as shown in [75]Fig. 1A. The OD[630 nm] for the EPA group
   (0.923 ± 0.273) was lower than that for the control group
   (1.096 ± 0.272). The difference was statistically significant
   (P < 0.05). Among the clinical isolates, EPA had the strongest impact
   on C. albicans X27, and the biofilm eradication rate was 46.02 %.

Fig. 1.

   [76]Fig. 1
   [77]Open in a new tab

   The disruptive effect of EPA on 24 h pre-formed biofilms of C.
   albicans. (A) CV assay to assess the antibiofilm activity of EPA
   against 32 isolates of C. albicans. (B) Biofilms biomass of C. albicans
   ATCC 90028 and X27 treated by EPA after 4 h and 24 h. (C) Biofilms
   metabolic activity of C. albicans ATCC 90028 and X27 treated by EPA
   after 4 h and 24 h. Data are presented as means ± SD. ∗P < 0.05,
   ∗∗P < 0.01, compared to the control group.

   After it was established that EPA had a destructive effect on the
   biofilm of most C. albicans strains, C. albicans ATCC 90028 and X27
   were selected for subsequent studies to clarify the mechanism. The
   biomass and metabolic activity of the biofilm were assessed at 4 h and
   24 h post-treatment with either EPA or the control, using the CV and
   XTT assays, respectively ([78]Fig. 1B and C). At 4 h, the biomass
   (0.794 ± 0.059 vs. 1.042 ± 0.025) and metabolic activity (2.253 ± 0.080
   vs. 2.656 ± 0.094) of the biofilm decreased in the EPA group of C.
   albicans X27 compared with the control group, while the ATCC 90028 was
   not affected. By 24 h, the biomass and metabolic activity of both C.
   albicans ATCC 90028 (1.011 ± 0.028 vs. 1.300 ± 0.086; 2.105 ± 0.045 vs.
   2.551 ± 0.132) and X27 (0.717 ± 0.101 vs. 1.439 ± 0.165; 1.455 ± 0.113
   vs. 2.450 ± 0.124) biofilms had significantly decreased. The
   differences were statistically significant (P < 0.01).

3.3. EPA affected C. albicans morphology in biofilms

   SEM provides a detailed visualization of the structure of C. albicans
   biofilms ([79]Fig. 2). C. albicans biofilms treated with RPMI 1640
   medium (control) formed dense and organized structures arranged in
   cellular multilayers, the biofilm consisted of mixtures of
   pseudohyphae/hyphae and a few yeast cells ([80]Fig. 2A–C). In contrast,
   EPA-treated biofilms exhibited uneven distribution, mainly yeast-form
   cells ([81]Fig. 2B–D).

Fig. 2.

   [82]Fig. 2
   [83]Open in a new tab

   Scanning electron microscopy (SEM) images of C. albicans biofilms
   treated with RPMI 1640 medium (control) or EPA. (A) Control group, C.
   albicans ATCC 90028. (B) EPA group, C. albicans ATCC 90028. (C) Control
   group, C. albicans X27. (D) EPA group, C. albicans X27. Scale
   bar = 20 μm.

   CLSM was used to observe the cell viability of the biofilm treated with
   or without EPA. C. albicans cells with intact cell membranes emitted
   green fluorescence upon staining with SYTO 9, whereas cells with
   damaged membranes emitted red fluorescence upon staining with PI. As
   shown in [84]Fig. 3, CLSM images displayed obvious changes in cell
   membrane integrity of C. albicans exposed to EPA compared with those of
   the control group, and the structural destruction in most of the
   biofilm was evident following EPA treatment.

Fig. 3.

   [85]Fig. 3
   [86]Open in a new tab

   Confocal laser scanning microscopy (CLSM) images of C. albicans
   biofilms treated with RPMI 1640 medium (control) or EPA. (A) Control
   group, C. albicans ATCC 90028. (B) EPA group, C. albicans ATCC 90028.
   (C) Control group, C. albicans X27. (D) EPA group, C. albicans X27.
   Green fluorescence denotes labeling with SYTO 9 for live cells, and red
   fluorescence denotes labeling with PI for dead cells. Scale
   bar = 25 μm. (For interpretation of the references to colour in this