Casearia sylvestris essential oil and its fractions inhibit Candida albicans ABC transporters related to multidrug resistance (MDR)

Levy Tenorio Sousa Domingos Flaviane Gomes Pereira Daniel Clemente de Moraes Ronaldo Marquete Marco Eduardo do Nascimento Rocha Davyson de Lima Moreira Elisabeth Mansur Antonio Ferreira-Pereira About the authors

Abstract

ABC transporters constitute a superfamily of transmembrane proteins that act mediating the translocation of several substrates across the membrane, using the energy of ATP hydrolysis. This mechanism of unrelated substrates efflux (multidrug resistance) has been associated with several diseases and it is a problem in chemotherapy efficacy. Nowadays, approximately 25% of the prescription drugs in the world are derived from plants. Casearia sylvestris is commonly found in the Americas and different parts of this plant are popularly used to treat several diseases. Previous studies have also confirmed the biological activities of C. sylvestris, such as anti-tumor, anti-leishmania, and antifungal properties. Then, the propose of this study was demonstrate that fraction 1-6 of C. sylvestris, essential oil, was able to reverse the fluconazole resistance phenotype in the Saccharomyces cerevisiae model mediated by the heterologous protein CaCdr2p from Candida albicans. The MIC value of fraction 1-6 combined with fluconazole in the checkerboard assay decreased approximately 4-fold, suggesting a synergistic effect. In addition, fraction 1-6 increased intracellular rhodamine 6G accumulation from 17% to 49% in the presence of glucose. Data indicate that C. sylvestris fraction 1-6 is a potential reverser of the fluconazole resistance phenotype.

Key words:
ABC transporters; Atlantic Forest; fluconazole; sesquiterpenes; yeast

Palavras-chave:
transportadores ABC; Mata Atlântica; fluconazol; sesquiterpenos; levedura

Resumo

Os transportadores ABC constituem uma superfamília de proteínas transmembranares que atuam mediando a translocação de vários substratos através da membrana, utilizando a energia da hidrólise de ATP. Esse mecanismo de efluxo de substratos não relacionados (resistência a múltiplas drogas) tem sido associado a várias doenças e é um problema na eficácia da quimioterapia. Atualmente, aproximadamente 25% dos medicamentos prescritos no mundo são derivados de plantas. Casearia sylvestris é comumente encontrada nas Américas e diferentes partes desta planta são popularmente usadas para tratar várias doenças. Estudos anteriores também confirmaram as atividades biológicas de C. sylvestris, como propriedades antitumorais, anti-leishmania e antifúngica. Desta forma, a proposta deste trabalho foi demonstrar a fração 1-6 de C. sylvestris, o óleo essencial, foi capaz de reverter o fenótipo de resistência ao fluconazol no modelo de Saccharomyces cerevisiae mediado pela proteína heteróloga CaCdr2p de Candida albicans. O valor da CIM da fração 1-6 combinada com fluconazol no ensaio tipo tabuleiro de xadrez diminuiu aproximadamente 4 vezes, sugerindo um efeito sinérgico. Além disso, a fração 1-6 aumentou o acúmulo intracelular de rodamina 6G, de 17% para 49% na presença de glicose. Os dados indicam que a fração 1-6 de C. sylvestris é um potencial reversor do fenótipo de resistência ao fluconazol.

Introduction

Nowadays, mycoses are a serious problem in the world public health scenario, causing a high ratio of morbidity and mortality. The increase of the prevalence of fungal infections is mainly due to the large number of immunocompromised individuals, which are part of the risk group for opportunistic infections, especially those caused by fungi, including invasive fungal infections (IFI), such as candidiasis, cryptococcosis and aspergillosis, which are responsible for the death of around 1.5 million people per year, a number close to those caused by tuberculosis and malaria (Brown et al. 2012Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG & White TC (2012) Hidden killers: human fungal infections. Science Translational Medicine 4: 165rv113.; Vallabhaneni et al. 2016).

The similarity between mammalian cells and fungal cells embarrass the development of new antifungal drugs thus limiting the therapeutic arsenal for the treatment of fungal infections (Paul & Moye-Rowley 2014Paul S & Moye-Rowley WS (2014) Multidrug resistance in fungi: regulation of transporter-encoding gene expression. Frontiers in Physiology 5: 143.). Azole-type compounds are the most common antifungal agents, especially fluconazole, which has an important spectrum of action and effective pharmacokinetic properties (Vandeputte et al. 2012Vandeputte P, Ferrari S & Coste AT (2012) Antifungal resistance and new strategies to control fungal infections. International Journal of Microbiology 2012: 713687.). Since the 90s, fluconazole has been the “gold standard” in the treatment of fungal infections, and many countries have fluconazole as the only therapeutic choice (Kneale et al. 2016Kneale M, Bartholomew JS, Davies E & Denning DW (2016) Global access to antifungal therapy and its variable cost. Journal of Antimicrobial Chemotherapy 71: 3599-3606.). However, the indiscriminate and prolonged use of this chemotherapeutic over the years has increased the incidence of resistance to azole drugs (Sanglard 2002Sanglard D (2002) Resistance of human fungal pathogens to antifungal drugs. Current Opinion in Microbiology 5: 379-385.).

The major mechanism of resistance to fluconazole in Candida spp. is the overexpression of transmembrane transporters, especially those belonging to the ATP-binding cassette (ABC) superfamily (Holmes et al. 2008Holmes AR, Lin YH, Niimi K, Lamping E, Keniya M, Niimi M, Tanabe K, Monk BC & Cannon RD (2008) ABC transporter Cdr1p contributes more than Cdr2p does to fluconazole efflux in fluconazole-resistant Candida albicans clinical isolates. Antimicrobial Agents and Chemotherapy 52: 3851-3862.). These pumps are able to translocate structurally unrelated drugs using the hydrolysis of ATP as energy source. The increasing reduction of intracellular levels of antifungal drugs by efflux pumps is a critical mechanism which causes therapeutic failure in the treatment of fungal infections (Prasad & Goffeau 2012Prasad R & Goffeau A (2012) Yeast ATP-binding cassette transporters conferring multidrug resistance. Annual Review of Microbiology 66: 39-63.).

Inhibition of ABC transporters is a well-studied strategy to reverse the resistance mediated by these proteins (Cannon et al. 2009Cannon RD, Lamping E, Holmes AR, Niimi K, Baret PV, Keniya MV, Tanabe K, Niimi M, Goffeau A & Monk BC (2009) Efflux-mediated antifungal drug resistance. Clinical Microbiology Reviews 22: 291-321, Table of Contents.). Co-administration of an efflux pump inhibitor together with an antifungal, such as fluconazole, has a chemosensitizer function, enabling the antifungal drug to reach appropriate intracellular concentrations to perform its pharmacological activity (Lacka et al. 2015Lacka I, Konieczny MT, Bulakowska A, Kodedova M, Gaskova D, Maurya IK, Prasad R & Milewski S (2015) Chemosensitization of multidrug resistant Candida albicans by the oxathiolone fused chalcone derivatives. Frontiers in Microbiology 6: 783.). ABC transporters inhibitors have already been described in the literature, and may be obtained both from synthetic sources (Reis de Sa et al. 2014Reis de Sa LF, Toledo FT, de Sousa BA, Gonçalves AC, Tessis AC, Wendler EP, Comasseto JV, Santos AA & Ferreira-Pereira A (2014) Synthetic organotelluride compounds induce the reversal of Pdr5p mediated fluconazole resistance in Saccharomyces cerevisiae. BMC Microbiology 14: 201.) and natural products (Belofsky et al. 2013Belofsky G, Kolaczkowski M, Adams E, Schreiber J, Eisenberg V, Coleman CM, Zou Y & Ferreira D (2013) Fungal ABC transporter-associated activity of isoflavonoids from the root extract of Dalea formosa. Journal of Natural Products 76: 915-925.).

Casearia sylvestris Sw. (Salicaceae) is popularly known as “erva-de-lagarto”, “guaçatonga” or “tiú”, and can be found throughout the Brazilian territory. According to ethnobotanical surveys, this species has medicinal properties, including antiviral, antitumor and antifungal activities (Pereira et al. 2016Pereira F, Costa F, Marquete R, May B, Falcão D, Mansur E, Moreira D & Romanos M (2016) Antiherpes activities of the pure and nanoemulsion of essential oil from leaves of Casearia sylvestris Sw. (Salicaceae). International Journal of Green and Herbal Chemistry 5: 112-121.,b). According to the chemical profile of the essential oils from Casearia genus, sesquiterpenes represent the main fraction of the mixture, but monoterpenes are also identified. However, despite many phytochemical studies with this species, there are few reports about the chemical profile of essential oils. In previous report we demonstrated that pure essential oil from fresh leaves of C. sylvestris, rich in α-humulene, as well as its fraction 1-6 rich in 14-hydroxy-9-epi-β-caryophyllene have antifungal activity (Pereira et al. 2017aPereira FG, Marquete R, Domingos LT, Rocha MEN, Ferreira-Pereira A, Mansur E & Moreira DL (2017a) Antifungal activities of the essential oil and its fractions rich in sesquiterpenes from leaves of Casearia sylvestris Sw. Anais da Academia Brasileira de Ciências 89: 2817-2824.).

The non-pathogenic yeast Saccharomyces cerevisiae was the first eukaryote to have its genome completely sequenced, thus facilitating genetic modifications for several scientific studies (Goffeau et al. 1996Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H & Oliver SG (1996) Life with 6000 genes. Science 274: 546, 563-547.), including the construction of mutant organisms that overexpress CaCdr1p and CaCdr2p proteins of C. albicans involved in the fluconazole resistance phenotype. Due to these characteristics, the experimental model of S. cerevisiae expressing heterologous proteins is outstanding for the evaluation of new fungal ABC transporters inhibitors (Lamping et al. 2007Lamping E, Monk BC, Niimi K, Holmes AR, Tsao S, Tanabe K, Niimi M, Uehara Y & Cannon RD (2007) Characterization of three classes of membrane proteins involved in fungal azole resistance by functional hyperexpression in Saccharomyces cerevisiae. Eukaryotic Cell 6: 1150-1165.).

Finding new compounds that increase the antifungal potential of drugs, such as fluconazole, is important to provide alternatives to treat fungal infections, and overcome drug resistance. Thus, in the present work the effect of C. sylvestris essential oil fractions was evaluated using S. cerevisiae strains that overexpress ABC transporters originally found in C. albicans.

Material and Methods

Strains and culture conditions

Three strains of S. cerevisiae mutants were used: a fluconazole-sensitive strain, which does not express ABC transporters related to MDR phenotype (AD 1-8u-), and two strains that overexpress CaCdr1p (CaCDR1) and CaCdr2p (CaCDR2), which are proteins of C. albicans that confer resistance to xenobiotics (Lamping et al. 2007Lamping E, Monk BC, Niimi K, Holmes AR, Tsao S, Tanabe K, Niimi M, Uehara Y & Cannon RD (2007) Characterization of three classes of membrane proteins involved in fungal azole resistance by functional hyperexpression in Saccharomyces cerevisiae. Eukaryotic Cell 6: 1150-1165.). All strains were kindly provided by Drs. Richard Cannon and Brian Monk (University of Otago - New Zealand). Strains were grown overnight at 30 °C in YPD medium (1% Yeast extract, 2% Peptone and 2% Glucose) at 100 rpm and were collected at the exponential growth phase.

Essential oil and fraction 1-6 chemical analysis

The plant C. sylvestris Sw. (Salicaceae) was collected in Tijuca National Park (22°57’05.04”S, 43°17’10.09”W), Rio de Janeiro, Brazil (SISBIO license n. 38765-1/CGEN license n. 010105/2014-0). Plant identification was conducted by Dr. Ronaldo Marquete, and the herbarium voucher was deposited in the Botanical Garden Herbarium of Rio de Janeiro with registration number RB 570651.

Fresh leaves of plant were used to obtain the essential oil by hydrodistillation as described in a previous work (Pereira et al. 2017aPereira FG, Marquete R, Domingos LT, Rocha MEN, Ferreira-Pereira A, Mansur E & Moreira DL (2017a) Antifungal activities of the essential oil and its fractions rich in sesquiterpenes from leaves of Casearia sylvestris Sw. Anais da Academia Brasileira de Ciências 89: 2817-2824.). Essential oil was fractionated over silica gel column, resulting in fractions 1-6; 7-10; 11-13; 14-28; 29-56; 57-62; 87-88; 91-93, which that were analyzed by GC-MS (Pereira et al. 2017aPereira FG, Marquete R, Domingos LT, Rocha MEN, Ferreira-Pereira A, Mansur E & Moreira DL (2017a) Antifungal activities of the essential oil and its fractions rich in sesquiterpenes from leaves of Casearia sylvestris Sw. Anais da Academia Brasileira de Ciências 89: 2817-2824.). Pure essential oil and its fractions were used for the tests.

Agar diffusion chemosensitization assay

CaCDR1 and CaCDR2 cells (2.5 × 106 cells/ml) were inoculated onto the surface of YPD medium, in the presence or absence of subinhibitory concentrations of fluconazole. Then, 6 mm-diameter sterile disks of Whatman 3MM paper were placed on the surface of the medium and 5 µl of C. sylvestris essential oil fractions (10 mg/ ml) were added, as well as a control with dimethyl sulfoxide (DMSO) (Sigma Aldrich®, St. Louis, USA). Plates were incubated at 30 °C for 48 h for observation of the growth inhibition zones around the disks (Ricardo et al. 2009Ricardo E, Costa-de-Oliveira S, Silva Dias A, Guerra J, Rodrigues ACGA & Pina-Vaz CL (2009) Ibuprofen reverts antifungal resistance on Candida albicans showing overexpression of CDR genes. FEMS Yeast Research 9: 618-625.).

Fraction 1-6 susceptibility testing

The minimal inhibitory concentration (MIC) was determined according to Niimi et al. (2004)Niimi K, Harding DRK, Parshot R, King A, Lun DJ, Decottignies A, Niimi M, Lin S, Cannon RD, Goffeau A & Monk BC (2004) Chemosensitization of fluconazole resistance in Saccharomyces cerevisiae and pathogenic fungi by a D-octapeptide derivative. Antimicrobial Agents Chemotherapy 48: 1256-1271. with slight modifications, using a microdilution assay on 96-well microplates. CaCDR2 cells (5 × 104 cells/ml) were inoculated in YPD medium in the presence of several concentrations of the fraction 1-6 (ranging from 100 to 0.78 μg/ml). The plates were incubated at 30 °C for 48 h in a rotary shaker (100 rpm). Cellular growth was evaluated at 600 nm using a FLUOstar Optima microplate reader (BMG Labtech, Germany).

Checkerboard liquid susceptibility assay

CaCDR2 cells (5 × 104 cells/ml) were incubated in a 96-well plate in the presence of different combinations of fluconazole (UFJF, Juiz de Fora-MG, Brazil) and fraction 1-6. The plates were incubated at 30 °C for 48 h in a rotary shaker (100 rpm). Cellular growth was measured at 600 nm (Fluostar Optima, BMG Labtech, Offenburg, Germany). The type of interaction between the azole drug and fraction 1-6 was measured by fractional inhibition concentration index (FICI). The FICI values are interpreted as follows: synergism, < 0.5; indifferent or additive, 0.5-4.0; antagonism, > 4.0 (Mukherjee et al. 2005Mukherjee PK, Sheehan DJ, Hitchcock CA & Ghannoum MA (2005) Combination treatment of invasive fungal infections. Clinical Microbiology Reviews 18: 163-194.).

Intracellular accumulation of rhodamine 6G (R6G)

CaCDR2 cells at the exponential growth phase were incubated on ice for 2 h to decrease cellular energy reserves. Then, 5 × 106 cells were resuspended in ice-cold PBS, and incubated with 15 μM of R6G (Sigma Aldrich®, St. Louis, USA) at 30 °C for 40 min with agitation (100 rpm). Further, cells were incubated in the presence or absence of fraction 1-6 (12.5 μg/ml) at 30 °C for 1 h with agitation (100 rpm). Then, the efflux of rhodamine was started by adding glucose (2%) with subsequent incubation at 30 ºC for 30 min. Cells were centrifuged at 9,000 rpm for 2 min and resuspended in PBS for flow cytometry analysis. A total of 20,000 events were read using the FACSCalibur FL-2 filter (Becton Dickinson, California, USA) at the Multi-User Cytometry Unit -CCS- UFRJ and data were analyzed by Flowjo software (Reis de Sa et al. 2017Reis de Sá LF, Toledo FT, Gonçalves AC, Sousa BA, Santos AA, Brasil PF, Duarte da Silva VA , Tessis AC, Ramos JA, Carvalho MA, Lamping E & Ferreira-Pereira A (2017) Synthetic organotellurium compounds sensitize drug-resistant Candida albicans clinical isolates to fluconazole. Antimicrobial Agents and Chemotherapy 61: e01231-16.)

Statistical analysis

All experiments were performed at least three times, and the results were expressed as mean ± standard deviation, when applicable. Data were analyzed by Student’s t-test, and P-values lower than 0.05 were considered significant.

Results

Agar diffusion chemosensitization assay

Eight C. sylvestris essential oil fractions and the pure essential oil were tested for their abilities to chemosensitize the CaCDR2 strain to the antifungal drug fluconazole. None of the fractions presented antifungal activity alone or combined to fluconazole against CaCDR1 strain. On the contrary, when CaCDR2 strain was tested, it appeared a growth inhibition zone around the disk containing the fraction 1-6, suggesting an antifungal activity (Fig. 1a). Moreover, there was a significant increase in the growth inhibition zone when fraction 1-6 was combined to a sub-inhibitory concentration of fluconazole (Fig. 1b).

Figure 1
Agar diffusion chemosensitization assay with CaCDR2 strain. The resistant strain grown in the absence (a) or presence (b) of fluconazole (10.5 μg/ ml) and disks (1-9) impregnated with 5 μl of Casearia sylvestris essential oil and fractions (10 mg/ml), in addition to DMSO control. In the absence of fluconazole, 1 showed growth inhibition around the disk, while in the presence of antifungal the inhibition zone was even greater. Disk 1: fraction 1-6; disk 2: fraction 7-10; disk 3: fraction 11-13; disk 4: fraction 14-28; disk 5: fraction 29-56; disk 6: fraction 57-62; disk 7: fraction 87-88; disk 8: fraction 91-93; disk 9: pure essential oil and disk 10: DMSO.

Antifungal susceptibility testing

CaCDR2 strain showed a dose-response inhibition curve in the presence of different concentrations of the fraction 1-6 of C. sylvestris essential oil (Fig. 2). A complete inhibition of cell growth was observed at the concentration of 50 μg/ ml (MIC), with an IC50 of 20 μg/ml.

Figure 2
Minimum inhibitory concentration (MIC) of fraction 1-6 for Saccharomyces cerevisiae CaCDR2 strain. Cells were incubated at 30 °C for 48 h in the presence of serial dilutions of fraction 1-6. The inhibition of cell growth by fraction 1-6 was dose-dependent showing a MIC of 50 μg/ml and IC50 of 20 μg/ml. The data represent means of three independent experiments.

Checkerboard: the synergistic effect between fraction 1-6 and fluconazole

The MIC value of fraction 1-6 decreased from 50 μg/ml to 12.5 μg/ml when combined with fluconazole, while the MIC of fluconazole decreased from 75 μg/ml to 17.5 μg/ml when combined with fraction 1-6. The FICI value was 0.48, suggesting a synergistic interaction between the azole drug and the fraction 1-6 (Tab. 1).

Table 1
Checkerboard assay of CaCDR2 strain. Chemosensitization test was done from the growth of the fluconazole resistant strain in the presence of different combined concentrations of antifungal and fraction 1-6.

Flow cytometry analysis

In the absence of glucose, 98% of CaCDR2 cells were stained with rhodamine 6G (Fig. 3a), whereas only 17% of cells were labeled after glucose addition. However, in the presence of fraction 1-6 there was a decrease in the efflux of rhodamine 6G, and the number of stained cells increased by 32% as compared to untreated cells (Fig. 3b).

Discussion

Different parts of C. sylvestris are popularly used to treat several diseases, and previous studies have confirmed some of its biological activities, such as anti-tumor, anti-leishmania and antifungal (Ferreira et al. 2011Ferreira PM, Costa-Lotufo LV, Moraes MO, Barros FW, Martins AM, Cavalheiro AJ, Bolzani VS, Santos AG & Pessoa C (2011) Folk uses and pharmacological properties of Casearia sylvestris: a medicinal review. Anais da Academia Brasileira de Ciências 83: 1373-1384.; Moreira et al. 2019Moreira RRD, Santos AGD, Carvalho FA, Perego CH, Crevelin EJ, Crotti AEM, Cogo J, Cardoso MLC & Nakamura CV (2019) Antileishmanial activity of Melampodium divaricatum and Casearia sylvestris essential oils on Leishmania amazonensis. Revista do Instituto de Medicina Tropical de São Paulo 61: e33.; Pereira et al. 2017aPereira FG, Marquete R, Domingos LT, Rocha MEN, Ferreira-Pereira A, Mansur E & Moreira DL (2017a) Antifungal activities of the essential oil and its fractions rich in sesquiterpenes from leaves of Casearia sylvestris Sw. Anais da Academia Brasileira de Ciências 89: 2817-2824.).

The present study was aimed at evaluating the potential of C. sylvestris essential oil, mainly composed of α-humulene, spathulenol and α-copaene (Pereira et al. 2017aPereira FG, Marquete R, Domingos LT, Rocha MEN, Ferreira-Pereira A, Mansur E & Moreira DL (2017a) Antifungal activities of the essential oil and its fractions rich in sesquiterpenes from leaves of Casearia sylvestris Sw. Anais da Academia Brasileira de Ciências 89: 2817-2824.), and its fractions to revert the multidrug resistance phenotype of Saccharomyces cerevisiae strains that overexpress efflux pumps from Candida albicans. An advantage of using these organisms as model, instead of C. albicans clinical isolates, is the possibility of evaluating a specific efflux pump since we can use modified cells that overexpress only one type of ABC transporter, or even just the MFS transporter that is also related to multidrug resistance phenotype in yeast.

Initially, an agar-based disk diffusion screening assay was performed in order to evaluate the ability of pure essential oil and its fractions from C. sylvestris to enhance the antifungal activity of fluconazole. The results obtained with CaCDR2 strain have demonstrated the antifungal activity of the essential oil when used alone or in combination with fluconazole. In addition, fraction 1-6 displayed a significant activity among the other fractions, and also increased the diameter of zone of inhibition when combined with fluconazole. The microbroth dilution analysis revealed a dose-dependent relationship between S. cerevisiae growth and fraction 1-6 concentration, with a MIC value of 50 µg/ml. In a previous work, the antifungal activity of fraction 1-6 against a fluconazole-sensitive Saccharomyces cerevisiae strain, showed a MIC value of 62.5 µg/ ml (Pereira et al. 2017aPereira FG, Marquete R, Domingos LT, Rocha MEN, Ferreira-Pereira A, Mansur E & Moreira DL (2017a) Antifungal activities of the essential oil and its fractions rich in sesquiterpenes from leaves of Casearia sylvestris Sw. Anais da Academia Brasileira de Ciências 89: 2817-2824.). Comparing both results, it may be suggested that the major compound 14-hydroxy-9-epi-β-caryophyllene or set of compounds responsible for the antifungal effect of fraction 1-6 are not a substrate of CaCdr2p. A higher MIC value against CaCDR2 strain would be expected if the active compound of fraction 1-6 (14-hydroxy-9-epi-β-caryophyllene) was pumped out by this transporter, as observed with the terpenoid eucalyptal D (Xu et al. 2019Xu J, Liu R, Sun F, An L, Shang Z, Kong L & Yang M (2019) Eucalyptal D enhances the antifungal effect of fluconazole on fluconazole-resistant Candida albicans by competitively inhibiting efflux pump. Frontiers in Cellular and Infection Microbiology 9: 211.), and drugs like azoles, rhodamines, and tacrolimus (Niimi et al. 2012Niimi K, Harding DR, Holmes AR, Lamping E, Niimi M, Tyndall JD, Cannon RD & Monk BC (2012) Specific interactions between the Candida albicans ABC transporter Cdr1p ectodomain and a D-octapeptide derivative inhibitor. Molecular Microbiology 85: 747-767.).

The results obtained from the checkerboard test to evaluate the type of interaction between the fraction 1-6 and fluconazole against the growth of CaCDR2 strain showed a synergist interaction between the two, as the concentration of fluconazole needed to completely inhibit the growth of yeast cells decreased by approximately four times. The potential of sesquiterpenes (the major components of C. sylvestris essential oil and fraction 1-6) to enhance antifungal activity of fluconazole has already been reported. Khoury et al. (2019)Khoury M, El Beyrouthy M, Ouaini N, Eparvier V & Stien D (2019) Hirtellina lobelii DC. essential oil, its constituents, its combination with antimicrobial drugs and its mode of action. Fitoterapia 133: 130-136. observed that essential oil from leaves of Hirtellina lobelii (DC.) Dittrich (Asteraceae) showed a synergistic interaction with fluconazole and griseofulvin against Trichophyton rubrum (Castell.) Sabour, T. mentagrophytes (C.P. Robin) Sabour., T. violaceum Sabour. ex E. Bodin, T. soudanense Joyeux, and T. tonsurans Malmsten (Khoury et al. 2019). Sesquiterpenes are the main components of H. lobelii essential oil, and between them, α-bisabolol is the major one, corresponding to 34.5% of the composition of the oil. This compound was also able to enhance antifungal activity of fluconazole against C. albicans and C. tropicalis strains (Rodrigues et al. 2018Rodrigues FFG, Colares AV, Nonato CFA, Galvao-Rodrigues FF, Mota ML, Moraes Braga MFB & Costa J (2018) In vitro antimicrobial activity of the essential oil from Vanillosmopsis arborea Barker (Asteraceae) and its major constituent, alpha-bisabolol. Microbial Pathogenesis 125: 144-149.). Futhermore, the antifungal activity of essential oil obtained from leaves of Piper claussenianum (miq.) C.DC. (Piperaceae), in combination with fluconazole, has also been described. It has shown synergism activity with the azole drug against C. albicans strains, and it was suggested that this result was obtained due to the high concentration of the sesquiterpene nerolidol in the essential oil (Curvelo et al. 2014Curvelo JA, Marques AM, Barreto AL, Romanos MT, Portela MB, Kaplan MA & Soares RM (2014) A novel nerolidol-rich essential oil from Piper claussenianum modulates Candida albicans biofilm. Journal of Medical Microbiology 63: 697-702.). The synergistic effect between natural products from C. sylvestris and antimicrobial drugs has not been reported so far. Synergism between two or more drugs may occur by mechanisms unrelated to efflux pumps. For instance, fluconazole enhances berberine antifungal activity by increasing cell membrane permeability (Yang et al. 2018Yang Z, Wang Q, Ma K, Shi P, Liu W & Huang Z (2018) Fluconazole inhibits cellular ergosterol synthesis to confer synergism with berberine against yeast cells. Journal of Global Antimicrobial Resistance 13: 125-130.). To confirm that fraction 1-6 impairs CaCdr2p efflux activity, a flow cytometry assay was performed. Rhodamine 6G, a fluorescent substrate of candida ABC transporters (Maesaki et al. 1999Maesaki S, Marichal P, Vanden Bossche H, Sanglard D & Kohno S (1999) Rhodamine 6G efflux for the detection of CDR1-overexpressing azole-resistant Candida albicans strains. Journal of Antimicrobial Chemotherapy 44: 27-31.), was used to evaluate the effect of fraction 1-6 on CaCdr2p functioning. When the efflux pump is active, R6G is extruded from the intracellular environment, decreasing cellular fluorescence. The results obtained in the present work have demonstrated that fraction 1-6 significantly decreased R6G efflux, confirming that synergism between this fraction and fluconazole occurs due to the inhibition of CaCdr2p efflux.

In summary, this study highlighted the potential of C. sylvestris essential oil, specifically fraction 1-6, to be used in the future as an adjuvant in candidiasis therapy. Due to its ability of inhibiting CaCdr2p, the fraction could be used to revert the resistance phenotype of cells that overexpress this efflux pump, then improving the outcome of the treatment. Further studies will be conducted to evaluate the effect of fraction 1-6 combined with fluconazole against the growth of C. albicans clinical isolates.

Figure 3
Intracellular rhodamine 6G accumulation in the presence of fraction 1-6. Intracellular rhodamine 6G accumulation was measured after incubation of exponentially growing phase CaCDR2 cells for 1 h at 30 °C, without (a) or with (b) glucose. Even the presence of glucose, the fraction 1-6 was able to block the extrusion of rhodamine 6G decreasing the number of cells stained with the fluorescent substrate. Autofluorescence (red line); Untreated CaCDR2 cells (orange line); CaCDR2 cells treated with fraction 1-6 (green line); Negative efflux control with the AD 1-8u- cells (blue line).

Acknowledgedments

We would like to thank Ms. Geralda Rodrigues Almeida, for technical support. This research was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Brazil), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) -Finance Code 001.

References

  • Belofsky G, Kolaczkowski M, Adams E, Schreiber J, Eisenberg V, Coleman CM, Zou Y & Ferreira D (2013) Fungal ABC transporter-associated activity of isoflavonoids from the root extract of Dalea formosa. Journal of Natural Products 76: 915-925.
  • Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG & White TC (2012) Hidden killers: human fungal infections. Science Translational Medicine 4: 165rv113.
  • Cannon RD, Lamping E, Holmes AR, Niimi K, Baret PV, Keniya MV, Tanabe K, Niimi M, Goffeau A & Monk BC (2009) Efflux-mediated antifungal drug resistance. Clinical Microbiology Reviews 22: 291-321, Table of Contents.
  • Curvelo JA, Marques AM, Barreto AL, Romanos MT, Portela MB, Kaplan MA & Soares RM (2014) A novel nerolidol-rich essential oil from Piper claussenianum modulates Candida albicans biofilm. Journal of Medical Microbiology 63: 697-702.
  • Ferreira PM, Costa-Lotufo LV, Moraes MO, Barros FW, Martins AM, Cavalheiro AJ, Bolzani VS, Santos AG & Pessoa C (2011) Folk uses and pharmacological properties of Casearia sylvestris: a medicinal review. Anais da Academia Brasileira de Ciências 83: 1373-1384.
  • Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H & Oliver SG (1996) Life with 6000 genes. Science 274: 546, 563-547.
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Publication Dates

  • Publication in this collection
    09 July 2020
  • Date of issue
    2021

History

  • Received
    12 Apr 2020
  • Accepted
    20 May 2020
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