Hedera rhombea inhibits the biofilm formation of Candida, thereby increases the susceptibility to antifungal agent, and reduces infection

Daseul Kim; Ki-young Kim

doi:10.1371/journal.pone.0258108

Abstract

Candida is an opportunistic pathogen and a common cause of fungal infections worldwide. Anti-fungal use against Candida infections has resulted in the appearance of resistant strains. The limited choice of anti-fungal therapy means alternative strategies are needed to control fungal infectious diseases. The aim of this study was to evaluate the inhibition of Candida biofilm formation by Hedera rhombea (Korean name: songak) extract. Biofilm formation was assessed using the crystal violet assay which showed a dose dependent reduction in the presence of extract with the biofilm formation inhibitory concentration of C. albicans (IC₅₀ = 12.5μg/ml), C. tropicalis var. tropicalis (IC₅₀ = 25μg/ml), C. parapsilosis var. parapsilosis (IC₅₀ = 6.25μg/ml), C. glabrata (IC₅₀ = 6.25μg/ml), C. tropicalis (IC₅₀ = 12.5μg/ml), and C. parapsilosis (IC₅₀ = 12.5μg/ml) without directly reducing Candida growth. Treatment with 6.25μg/mL of extract increased the antifungal susceptibility to miconazole from 32% decreasing of fungal growth to 98.8% of that based on the fungal growth assay. Treatment of extract dose-dependently reduced the dimorphic transition of Candida based on the dimorphic transition assay and treatment of 3.125μg/mL of extract completely blocked the adherence of Candida to the HaCaT cells. To know the molecular mechanisms of biofilm formation inhibition by extract, qRT-PCR analysis was done, and the extract was found to dose dependently reduce the expression of hyphal-associated genes (ALS3, ECE1, HWP1, PGA50, and PBR1), extracellular matrix genes (GSC1, ZAP1, ADH5, and CSH1), Ras1-cAMP-PKA pathway genes (CYR1, EFG1, and RAS1), Cph2-Tec1 pathway gene (TEC1) and MAP kinases pathway gene (HST7). In this study, Hedera rhombea extract showed inhibition of fungal biofilm formation, activation of antifungal susceptibility, and reduction of infection. These results suggest that fungal biofilm formation is good screen for developing the antifungal adjuvant and Hedera rhombea extract should be a good candidate against biofilm-related fungal infection.

Citation: Kim D, Kim K-y (2021) Hedera rhombea inhibits the biofilm formation of Candida, thereby increases the susceptibility to antifungal agent, and reduces infection. PLoS ONE 16(10): e0258108. https://doi.org/10.1371/journal.pone.0258108

Editor: Roy Aziz Khalaf, Lebanese American University, LEBANON

Received: March 2, 2021; Accepted: September 17, 2021; Published: October 6, 2021

Copyright: © 2021 Kim, Kim. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This research was funded by the GRRC Program of Gyeonggi province [GRRC-KyungHee2020(B04)], Republic of Korea.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Candida albicans is an opportunistic pathogen which is responsible for systemic infections in immunocompromised patients. C. albicans can persist inside the host and can be aided by drug resistance traits which often lead to failure of therapeutic strategies [1]. One of the features of Candida species pathogenesis is their ability to form biofilms, and nosocomial infections are often related to the ability to produce biofilm on mucosal surfaces and implanted medical devices [2–5].

The formation of biofilms involves multiple interconnected signaling pathways [6–14], and is a finely controlled process that involves attachment to surface and embedment in the exopolymer extracellular matrix [15–18]. The biofilm matrix acts to structure microbial communities and includes sessile cells that are frequently much more resistant to antifungal agents. In fact, adherent C. albicans cells without specific drug resistant gene expression are up to 1,000 times more resistant to common antifungal agents than planktonic cells [19]. Therefore, the biofilm of C. albicans is a reservoir of viable fungal cells that can potentially cause systemic infections, with a mortality rate of around 40–60% [20, 21]. Efforts are being made to develop alternative strategies to eradicate biofilm-related infections [22, 23]. Medicinal plants are used for diverse traditional methods to treat cancer, infection, fever, asthma, and many other diseases. Herbal medicines usually have fewer side effects compared to over-the-counter medicines. Accordingly, medicinal plants should be a new provenance of replacement remedies to treat Candida infectious diseases [24–26].

Hedera rhombea is a species of ivy (genus Hedera) that is native to the coast and some islands of East Asia [27, 28]. In oriental medicine, H. rhombea is mainly used for arthritis, low back pain, hepatitis, high blood pressure, hemostasis, anti-rheumatism, facial paralysis, jaundice anti-inflammatory action, hypertension, and antitumor [27–29].

In this study, H. rhombea extract showed anti-biofilm formation activity against several fungi including C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis. Interestingly, the activity of the extract also increased susceptibility to antibiotics. These results suggest that H. rhombea extract can be used as a potential anti-fungal adjuvant to control the biofilm-related infection.

Materials and methods

Strains

The Candida strains used in this study are listed in Table 1. All strains were stored in 20% glycerol at −70°C and cultured in YPD plates [peptone 20 g/L (BD Difco, Belgium), yeast extract 10 g/L (BD Difco, Belgium) and 2% glucose (w/v) (Daejung, Korea)].

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Table 1. Strains used in this study.

https://doi.org/10.1371/journal.pone.0258108.t001

H. rhombea extraction

The leaf of H. rhombea was obtained from Jeju island. The extracts were produced using distilled water in 3L containing 300g of the sample at 80°C for 8 h, concentrate at 40°C using a rotary evaporator, and freeze-dried. 10mg of plant extract powder was dissolved in 1mL dimethyl sulfoxide for the experiments [15, 24–28, 30].

Inhibition of biofilm formation

H. rhombea extract ranging from 6.25 to 100μg/mL were prepared in 96-well flat-bottomed plates (SPL, Korea). Wells without test compounds served as controls (DMSO concentration of 0.1%). 1 × 10⁶ CFU/mL of C. albicans suspension were prepared in RPMI 1640 medium [26, 31–34, 36–40]. Then 100μl of the solution was inoculated into 96-well flat-bottomed plates. After incubation at 37°C for 24h, non-adherent cells were removed by washing with PBS and then 100ul of 1% aqueous crystal violet was applied for 30 minutes. Each well was washed three times with PBS and instantly de-stained with 150μl of 30% acetic acid for 15min. The absorbance was measured at 595nm with a microplate reader (Bio Tek Instruments, Korea). The experiments were performed in triplicate (Fig 1).

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Fig 1. Inhibition of Canddia spp. biofilm formation by H. rhombea extract.

The biofilm formation of (A) C. albicans, (B) C. tropicalis var. tropicalis, (C) C. parapsilosis var. parapsilosis, (D) C. glabrata, (E) C. tropicalis, and (F) C. parapsilosis was induced in YPD media supplemented with 10% fetal bovine serum with the indicated concentrations of H. rhombea extract at 37°C for 24h.

https://doi.org/10.1371/journal.pone.0258108.g001

Combinatorial antifungal effects of H. rhombea extract with antifungal agents

C. albicans were grown overnight in YPD diluted to 1 × 10⁶ cells/mL. The induction of biofilm formation was performed as described above. Miconazole (3.125μg/mL), magnoflorine (3.125μg/mL) and dioscin (3.125μg/mL) alone or with the indicated concentration of H. rhombea extract were added and incubated at 37°C for 24 h [26, 30–34]. The experiments were performed in triplicate.

Dimorphic transition of C. albicans

C. albicans were grown overnight in YPD medium. 1 × 10⁶ cells/mL of Candida with or without extracts were incubated in RPMI 1640 medium and YPD media supplemented with 10% fetal bovine serum medium to induce dimorphic transition at 37°C for 4 h. RPMI without sodium barcarbonate and with glutamine buffered with MOPS [3-(N-morpholino) propanesulfonic acid] to pH 7. Inhibition quantification of the yeast-to-hyphal-form transition was accomplished by counting the number of hyphae cells in the population as previously described [26, 32–40]. More than 1,000 cells were counted for each well in duplicate, and all assays were repeated five times. Representative results of images were obtained using a fluorescence microscope (EVOS® FL, ThermoFisher Scientific, Waltham, MA, USA). The experiments were performed in triplicate.

Candida adherence test

A human epithelial keratinocyte HaCaT cells were maintained in DMEM supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO₂.

HaCaT cells (0.5×10⁶ cells per wall) were grown to confluence on 24 well plates for 24 h. DMEM were drained and then plates were cautiously washed three times with PBS to remove nonadherent cell. 1×10⁶ cell/mL C. albicans mixed with H. rhombea extract ranged 1.56–25μg/mL concentration was treated into each well. The 24 well plates were incubated at 37°C for 24 h. Representative results of images were obtained using a microscope [32, 34]. The experiments were performed in triplicate.

qRT-PCR analysis

C. albicans was grown overnight in YPD and diluted to 1 × 10⁶ cells/mL. The diluted suspension with 6.25–100μg/mL of H. rhombea extract was incubated RPMI 1640 at 37°C for 24 h with shaking. Total RNA was isolated using TRIzol reagent (Life Technology, Thermo Fisher Scientific, USA) according to the manufacturer’s instruction and the reverse transcriptase (NanoHelix, Korea) reaction was prepared using 1 μg of RNA to obtain cDNA. qRT-PCR was carried out with the 2X SybrGreen qPCR Mater Mix (CellSafe, Korea). The transcript level of detected genes was calculated using the formula 2-^ΔΔCT. Primer sequences used are listed in Table 2. ACT1 was used as internal control [26, 30–34, 38–40]. The experiments were performed in triplicate.

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Table 2. Primers for C. albicans genes used in this study.

https://doi.org/10.1371/journal.pone.0258108.t002

MTT assay

The cytotoxicity of H. rhombea extract against HaCaT and THP-1 were tested by a slightly modified MTT assay [26, 32–34]. Briefly, 1×10⁴ HaCaT and THP-1 in DMEM and RPMI1640 medium, respectively were added to each well containing the indicated concentration of extract and incubated for 24 h. Cell viability was calculated by optical density (OD₅₄₀) values measured using a microplate reader (BioTek Instruments, Korea) and is reported as the percentage of the vehicle control [32]. The experiments were performed in triplicate.

Growth inhibition assay for C. albicans

Fungal culture was prepared with the fresh YPD to 1 × 10⁶ cells/mL of C. albicans [30–33, 40]. 100μg/mL of H. rhombea extract was added and then incubated at 37°C. The growth was evaluated by measuring OD₆₀₀ using microplate reader after 0, 1, 2, 4, 8, 12, and 24 h [30–32]. The experiments were performed in triplicate.

Statistical analysis

All experiments were performed at least three times and data were presented as the ± mean S.D.

Result

Inhibition of Candida biofilm formation by the treatment of H. rhombea extract

Biofilm is especially important for the fungi to survive and infect. H. rhombea was used to test whether it blocked fungal biofilm formation or not. H. rhombea extract dose-dependently inhibited the Candida biofilm formation in all the tested strains (Table 1) with the IC₅₀ value of approximately 6.25μg/mL for C. albicans (Fig 1A).

IC₅₀ values of other strains were 6.25μg/mL (C. parapsilosis var. parapsilosis and C. glabrata), 12.5μg/mL (C. tropicalis and C. parapsilosis), and 25μg/mL (C. tropicalis var. tropicalis) (Fig 1B, 1C, 1D, 1E and 1F).

H. rhombea extract increased the susceptibility to an antifungal agent against C. albicans

H. rhombea extract increased the susceptibility of antifungal agents against C. albicans. Treatment of 3.125μg/mL of extract increased the susceptibility to miconazole with 99% of fungal growth inhibition from 38% inhibition by miconazole treatment. The extract also increased the susceptibility to plant-derived antifungal candidates including magnoflorine (99% growth inhibition by 6.25μg/mL of extract compared with 29% growth inhibition by 3.125ug/ml of only magnoflorine treatment) and dioscin (99% growth inhibition by 6.25μg/mL of extract compared with 39% growth inhibition by 3.125ug/ml of only dioscin treatment) (Fig 2A, 2B and 2C).

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Fig 2. H. rhombea extracts increased the susceptibility of C. albicans to miconazole etc.

H. rhombea extract (6.25μg/mL) increased the susceptibility to miconazole (3.125μg/mL) (A), magnoflorine (3.125μg/mL) (B) and dioscin (3.125μg/mL) (c) against C. albicans. The biofilm formation was induced for 24 h by growing of C. albicans in YPD media supplemented with 10% fetal bovine serum. MCZ: Miconazole, MF: Magnoflorine, Do: Dioscin, HR: H. rhombea extract.

https://doi.org/10.1371/journal.pone.0258108.g002

H. rhombea extract blocked dimorphic transition from yeast to hyphae form

The yeast-to-hyphae conversion is an important virulence property of C. albicans. The formation of hyphae aids the subsequent invasive growth of C. albicans to penetrate host tissues and lead to the establishment of systemic infection [7]. Extract tested whether the dimorphic transition was influenced and found 1.56μg/mL of the extract significantly inhibited hyphae formation in RPMI 1640 or a 10% FBS YPD medium, and extract with higher than 6.25μg/mL completely blocked the hyphae formation (Fig 3A, 3B, 3C and 3D).

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Fig 3. Inhibition of C. albicans dimorphic transition by H. rhombea extract in different hyphal-inducing media.

(A) C. albicans dimorphic transition was induced using RPMI 1640 and images of C. albicans cells (B) were obtained using a microscope. A: C. albicans without extract, B: C. albicans with 1.56μg/mL of extract treated, C: C. albicans with 6.25μg/mL of extract treated, D: C. albicans with 25μg/mL of extract treated. (C) C. albicans dimorphic transition was induced using 10% FBS YPD medium and images of C. albicans cells (D) were obtained using a microscope. A: C. albicans without extract, B: C. albicans with 1.56μg/mL of extract treated, C: C. albicans with 6.25μg/mL of extract treated, D: C. albicans with 25μg/mL of extract treated.

https://doi.org/10.1371/journal.pone.0258108.g003

H. rhombea extract reduced fungal adherence to the HaCaT cells

Fungal biofilm formation on device-associated infection is an important medical problem. H. rhombea extract showed dose-dependently reduced adhesion of the fungi to the human HaCaT cells with 90% reduction by treatment of 1.56μg/mL of extract (Fig 4).

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Fig 4. H. rhombea extract inhibited adhesion of C. albicans to HaCaT cells.

C. albicans (1 × 10⁶ cells/mL) with indicated concentration of H. rhombea extract were incubated at 37°C for 24 h and Candida cells that were remaining on the HaCaT cells were counted.

https://doi.org/10.1371/journal.pone.0258108.g004

H. rhombea extract inhibited the expression of biofilm formation and infection related genes

To understand the molecular basis of H. rhombea extract of inhibition of biofilm formation and thereby reducing the infection of Candida, the expression of genes related to biofilm formation, hyphae growth, and cell adhesion was tested by qRT-PCR. The expression of biofilm formation related genes [CAN2 (IC₅₀ = 1.56μg/mL), EHT1 (IC₅₀ = 1.56μg/mL), TPO4 (IC₅₀ = 1.56μg/mL), and OPT7 (IC₅₀ = 1.56μg/mL)], Ras1-cAMP-PKA pathway related genes [RAS1 (IC₅₀ = 1.56μg/mL), EFG1 (IC₅₀ = 1.56μg/mL), TEC1 (IC₅₀ = 3.125μg/mL), HST7 (IC₅₀ = 3.125μg/mL), and CYR1 (IC₅₀ = 1.56μg/mL)], hyphal-specific genes [ALS3 (IC₅₀ = 1.56μg/mL), ECE1 (IC₅₀ = 3.125μg/mL), and HWP1 (IC₅₀ = 3.125μg/mL)] and extracellular matrix-related genes [GSC1 (IC₅₀ = 1.56μg/mL), ADH5 (IC₅₀ = 3.125μg/mL), and CSH1 (IC₅₀ = 1.56μg/mL)] were significantly decreased by treatment of extract (Fig 5A, 5B, 5C and 5D).

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Fig 5. H. rhombea extract inhibited the expression of genes related to biofilm formation and virulence of C. albicans.

H. rhombea extract reduced the expression of genes related with biofilm formation (A), Ras1-cAMP-Efg1 pathways (B), hyphal-specific (C) and extracellular matrix (D).

https://doi.org/10.1371/journal.pone.0258108.g005

Total RNA was extracted from C. albicans treated with the indicated concentration of H. rhombea extract using RNA extraction kit, converted to cDNA, and analyzed by qPCR with the respective primers.

H. rhombea extract did not affect the growth of human originated cell

The cytotoxic effects of H. rhombea extract on HaCaT cells and macrophage THP-1 were checked using an MTT assay. H. rhombea extract has no significant cytotoxic effect on both macrophage THP-1 and HaCaT cells (Fig 6A and 6B).

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Fig 6. H. rhombea extract did not show any toxicity against the human skin cells.

Cytotoxicity of H. rhombea extract against HaCaT cells (A) and THP-1 cells (B). Each cell (10⁴ per well) was incubated with the indicated concentration of H. rhombea extract in 96-well for 24 h and the cell viability was evaluated by MTT assay.

https://doi.org/10.1371/journal.pone.0258108.g006

H. rhombea extract did not inhibit the Candida growth

Several antifungal agents inhibit fungal biofilm formation because they can kill the fungi and indirectly decrease biofilm formation. Extract of H. rhombea have no effect on the growth of C. albicans after 24h of incubation and that suggested the biofilm formation inhibition of extract is not because of the reduced growth of Candida (Fig 7).

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Fig 7. H. rhombea extract did not inhibit the growth of C. albicans.

C. albicans (1 × 10⁶ cells/mL) with 100 μg/mL of H. rhombea extract were incubated at 30°C for 24 h.

https://doi.org/10.1371/journal.pone.0258108.g007

Discussion

Many fungi including Candida live in and on the human body, but when Candida begins to grow uncontrollably, it can cause an infection known as candidiasis. In fact, Candida is the most common cause of fungal infections in humans [41]. Antifungal drugs can cause side effects and resistance, and there have been multiple recent reports of resistance including in the emerging problematic organism Candida auris [42–44]. Outbreak response is complicated by the limited treatment options and inadequate disinfection strategies. So new approaches with a new target for the anti-fungal agents are required.

In the present study, H. rhombea extract was tested for inhibition of Candida biofilm formation and showed strong anti-biofilm formation activity against all tested Candida species including C. albicans, C. glabrata, C. tropicalis, and C. parapsilosis (Fig 1).

Even though the mechanism of Candida biofilm formation is diverse depending on the species, H. rhombea extract inhibited Candida biofilm formation that suggests there should be a common mechanism to induce the biofilm among the Candida species, but further studies must be conducted. Moreover, H. rhombea increased the antifungal activity of miconazole, magnoflorine, and dioscin (Fig 2), and reduced Candida infection (Figs 3 and 4), these results confirmed that biofilm formation is related to the susceptibility of antifungal agents and fungal infection, but further studies should be undertaken. In the case of the H. rhombea we tested, no inhibition of growth was observed. However, in the experiments in the reference literature [29], H. rhombea extract showed inhibition of growth. The reason might be the differences in the method of extracting plants.

Based on the gene expression analysis, the expression of genes related to biofilm formation, hyphae growth, and cell adhesion was significantly reduced by treatment with H. rhombea extract. The biofilm formation is tightly related to adherence to Candida and the early step is the attachment mechanism. C. albicans biofilm formation is determined by various transcription factors including BCR1, EFG1, TEC1, and NDT80 that function as components in several pathways and influence adherence of Candida, suggesting that even though biofilm formation was initially tested, adherence or infection factors could be also regulated by treatment of H. rhombea extract. Further studies, including the precise target determination of H. rhombea extract, effector components, and in vivo testing must be carried out.

In conclusion, H. rhombea extract inhibited C. albicans biofilm formation, increased the antifungal activity of antibiotics and putative antifungal agents, and decreased fungal adherence to the host cell. Therefore, H. rhombea extract could be a good treatment option for biofilm-forming fungal infections.

Supporting information

S1 Data.

https://doi.org/10.1371/journal.pone.0258108.s001

(XLSX)

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Figures

Abstract

Introduction

Materials and methods

Strains

H. rhombea extraction

Inhibition of biofilm formation

Combinatorial antifungal effects of H. rhombea extract with antifungal agents

Dimorphic transition of C. albicans

Candida adherence test

qRT-PCR analysis

MTT assay

Growth inhibition assay for C. albicans

Statistical analysis

Result

Inhibition of Candida biofilm formation by the treatment of H. rhombea extract

H. rhombea extract increased the susceptibility to an antifungal agent against C. albicans

H. rhombea extract blocked dimorphic transition from yeast to hyphae form

H. rhombea extract reduced fungal adherence to the HaCaT cells

H. rhombea extract inhibited the expression of biofilm formation and infection related genes

H. rhombea extract did not affect the growth of human originated cell

H. rhombea extract did not inhibit the Candida growth

Discussion

Supporting information

S1 Data.

References