The calcineruin inhibitor cyclosporine a synergistically enhances the susceptibility of Candida albicans biofilms to fluconazole by multiple mechanisms
© The Author(s). 2016
Received: 8 December 2015
Accepted: 6 June 2016
Published: 18 June 2016
Biofilms produced by Candida albicans (C. albicans) are intrinsically resistant to fungicidal agents, which are a main cause of the pathogenesis of catheter infections. Several lines of evidence have demonstrated that calcineurin inhibitor FK506 or cyclosporine A (CsA) can remarkably enhance the antifungal activity of fluconazole (FLC) against biofilm-producing C. albicans strain infections. The aim of present study is thus to interrogate the mechanism underpinning the synergistic effect of FLC and calcineurin inhibitors.
Twenty four clinical C. albicans strains isolated from bloodstream showed a distinct capacity of biofilm formation. A combination of calcineurin inhibitor CsA and FLC exhibited a dose-dependent synergistic antifungal effect on the growth and biofilm formation of C. albicans isolates as determined by a XTT assay and fluorescent microscopy assay. The synergistic effect was accompanied with a significantly down-regulated expression of adhesion-related genes ALS3, hypha-related genes HWP1, ABC transporter drug-resistant genes CDR1 and MDR1, and FLC targeting gene, encoding sterol 14alpha-demethylase (ERG11) in clinical C. albicans isolates. Furthermore, an addition of CsA significantly reduced the cellular surface hydrophobicity but increased intracellular calcium concentration as determined by a flow cytometry assay (p < 0.05).
The results presented in this report demonstrated that the synergistic effect of CsA and FLC on inhibited C. albicans biofilm formation and enhanced susceptibility to FLC was in part through a mechanism involved in suppressing the expression of biofilm related and drug-resistant genes, and reducing cellular surface hydrophobicity, as well as evoking intracellular calcium concentration.
The infection of Candida albicans (C. albicans) continues to be a major cause of high mortality among immunocompromised and hospitalized patients, and the bloodstream Candida infection has been listed as the third most common causes of nosocomial bacteremia and the most common etiologic agent of fungal-related biofilm infection [1, 2]. With an ability to form biofilm seen in the most microorganisms, a formation of C. albican biofilm not only provides a protection from environmental stress, but it also allows a horizontal transfer of genes that potentially encode antibiotic resistance, sequentially enhances the resistance of microorganisms to an antimicrobial agent by up to 1000-fold greater than that needed for a treatment of their planktonic counterparts [3, 4].
Fluconazole (FLC) is a member of the azole class, organic compounds posses a five-membered heterocyclic ring with two double bonds, which is the most commonly used first-line agent in the prevention and treatment for patients with candidemia or suspected invasive candidiasis, through a mechanism by which the FLC is able to functionally target encoding sterol 14alpha-demethylase (ERG11), an essential enzyme in the ergosterol biosynthetic pathway of C. albican . However, FLC was found to be ineffective in treatment of C. albicans biofilm, and the formation of biofilm has been demonstrated to contribute to the failure of anti-fungal treatment, including FLC and other agents, which has been attributed to a compromise in C. albicans cell membrane integrity caused by reduced sterols . Intriguingly, mounting evidence has revealed that the antifungal activity of FLC in C. albicans biofilm killing could be synergistically enhanced when it was employed in a combination with some antibiotics or immunosuppressants [7–14]. Among them, the calcineurin inhibitors, such as cyclosporine A (CsA) and FK506 have spurred an increased interest [14–19].
Calcineurin is a Ca2+-calmodulin-activated phosphatase, which is a multifunctional regulator with functions in governing fungal stress responses, physiological and cell cycle progression, biofilm formation, antifungal resistance, virulence and pathogenesis, and is essential for C. albicans survival during membrane stress [20–23]. Several lines of evidence have uncovered that C. albicans was resistant to calcineurin inhibitors of CsA and FK506, despite some fungal species were susceptible to these agents. Notably, a combination of either CsA or FK506 with the fluconazole exhibited a synergistic anti-fungal activity to both of planktonic and biofilm C. albicans [14–17, 20, 24]. Particularly, the calcineurin inhibitor CsA was recently found to be able to enhance the susceptibility of biofilm-producing C. albicans to fluconazole . These results implied that targeting calcineurin signaling using a combination of calcineurin inhibitor FK506 or CsA and FLC might be a promising antifungal strategy for prevention and treatment of biofilm C. albicans infection. However, the underlying mechanism by which a calcineurin inhibitor enhances the susceptibility of C. albicans to the most common antifungal agent, FLC has yet been fully understood.
In the present report, we aimed to interrogate the molecular mechanism of calcineurin inhibitor CsA in enhancing the susceptibility of biofilm-producing C. albicans to FLC by accessing its impacts on the alterations of the expression of drug-transporters and adhesion associated genes, cellular surface hydrophobicity (CSH) and intracellular calcium ([Ca(2+)]) levels. Our results demonstrated that an addition of CsA led an enhanced susceptibility of C. albicans to FLC in part through a mechanism by down-regulating the expression of genes associated to ABC transporter and adhesion, a decrease of CSH and an increased intracellular calcium ([Ca(2+)]) level.
Biofilm-producing capacity of clinical Candida albicans isolates
CsA enhances the susceptibility of clinical biofilm-producing Candida albicans to fluconazole
A combination of FLC and CsA alters C. albicans the expression of drug-resistant genes
CsA and FLC synergistically reduce cellular surface hydrophobicity of the C. albicans strain
CsA and FLC synergistically increases intracellular calcium in C. albicans
Candida albicans is an important nosocomial infectious agent, and an infection of biofilm-producing Candida albicans among immunocompromised patients remains a clinical challenge. In this regard, the use of medical devices such as central venous catheters (CVC’s) and prostheses is a well-known risk factor to induce biofilm formation [31, 32]. Despite a significant advance in our knowledge such as the molecular mechanism of C. albicans biofilm formation has been made over the past decade, there is no ideal therapeutic method for bloodstream infections caused by biofilm-producing Candida albicans in clinical practice .
Accumulating evidences have revealed that a formation of biofilm of C. albicans could enhance the resistance of this fungi species to most of the commonly used antifungal agents [1, 34, 35]. Therefore, it is urgent to discover novel antifungal agents or regimens based on new drug targets for the treatment of bloodstream infections, particularly an infection of MDR-biofilm-producing C. albicans. With this respect, several lines of evidence have uncovered that a combination of calcineurin inhibitor, such as FK506 and CsA, or antibiotics could synergistically enhance the susceptibility of biofilm-producing C. albicans to the first-line antifungal agent FLC [8, 10–14, 17, 18]. In the present report, we also demonstrated that the calcineurin inhibitor CsA had a potential to increase the susceptibility of clinical biofilm-producing C. albicans to FLC by suppressing their abilities to form biofilm, and inhibiting the expression of genes related to cell adhesion, hyphal formation and drug-transportators, as well as decreasing cellular surface hydrophobicity and increasing intracellular calcium concentration.
Previous studies have reported that biofilms formed by C. albicans strains that isolated from bloodstreams displayed phenotypes associated with drug-resistance and pathogenicity . Therefore, we aimed to morphologically assess biomasses of C. albicans clinical isolates using the crystal violet staining, and found distinct capacities of biofilm formations of clinical Candida albicans bloodstream isolates, suggesting that the biofilm producing capacity may have an implication of clinical significance. Interestingly, an addition of CsA was verified to be able synergistically increase the susceptibility of these isolates to FLC, along with a down-regulation of the expression of ALS3, HWP1, CDR1, MDR, ERG11 genes. Among them, ALS3 are members of the agglutinin-like sequence (ALS) gene family that encodes cell-wall glycoproteins , and both ALS and HWP1 genes are highly expressed in hyphae and play essential roles in the yeast-to-hypha morphological transition of C. albicans, in which the ALS3 contributes cell adhesions, and HWP1 mediates cell substrate and cell-cell interactions in biofilms [37–39]. Therefore, a combination of CsA and FLC-induced down-regulation of these genes might contribute to the anti-biofilm effect by targeting the three known stages for biofilm formation: adhesion to biomaterial surfaces, growth to form an anchoring layer, and morphological transition to form a complex three-dimensional structure [40, 41]. Of note, no alteration or marginal changes of the expression of these genes was found in cells treated with CsA and FLC alone in this study, indicating that the CsA or FLC had limited effect on biofilm growth of C. albicans. Equally noteworthy, FLC alone exhibited a limited effect on ERG11 gene expression, which may be in part due to that HBF isolates were more resistant to FLC than LBF strains, and more abundant ERG11 transcripts were to reported to be detected in FLC-resistant CA strains .
The azoles are generally fungistatic agents for treatment and prevention of C. albicans infections . However, azole resistant biofilm-producing C. albicans infections were frequently observed in clinic settings, which have been attributed to interactions of multiple mechanisms including the alteration of ERG11 gene expression . ERG11 gene encodes the 14α-demethylase enzyme which has an effect on ergosterol biosynthesis, and an up-regulated expression of this gene in biofilm C. albicans isolates may explain their resistance to azole . In agreement with this notion, exposing biofilm-producing C. albicans isolates to a combination of CsA and FLC caused adown-regulation of ergosterol biosynthesis-related gene ERG11, which implied an underlying mechanism by which calcineurin inhibitors have potentials to enhance the susceptibility of biofilm-producing C. albicans to FLC . It has been previously demonstrated that the highly frequent azole resistance in C.albicans strains was in part attributed to an increased efflux of drug mediated mostly by the ATP-binding cassette (ABC) and the major facilitator superfamily (MFS) transporters [46, 47]. In this context, the expression of genes encoding both types of efflux pumps was up-regulated during the course of biofilm formation and development in C. albicans . Controversially, a later study by Marchetti et al. suggested that a synergistic antifungal effect of cyclosporine and FLC in C. albicans was multidrug efflux transporter genes CDR1, CDR2, MDR1 and FLU1 independent . Inconsistent with this finding, we found that there was a significant down-regulation of efflux transporter genes CDR1 and MDR1 in clinical biofilm-producing C. albicans isolates treated with a combination of CsA and FLC, suggesting that the CsA-mediated increase of susceptibility of biofilm-producing C. albicans to FLC is at least in part through a mechanism by suppressing the expression of these drug transporter genes.
The cell surface hydrophobicity (CSH) of Candida species has an implication in the adhesion and biofilm formation of the organisms on epithelial cells or medical device [26, 49], which is also associated with the fungicidal resistance [50–52]. For instance, a FLC resistant C. tropicalis strain exhibited a significantly more hydrophobic, greater adherence and higher capacity of biofilm formation on polystyrene surface relative to its parent strain that susceptible to FLC, along with an increased expression of MDR1 and ERG11 genes and enhanced virulence in mice . The discrepancy of CSH and biofilm formation capacity between FLC-susceptible and resistant strains was also recently reported in C. albicans cells cultured with different media in presence or absence of FLC . In this regard, C. albicans cells dispersed from mature biofilms were more hydrophobic than those dispersed from earlier development stages of biofilms, and C. albicans isolates with high capacity of biofilm formation displayed an increased CSH relative to those with lower biofilm formation potential . In agreement with these findings, our result also indicated that calcineurin inhibitor CsA could enhance susceptibility of biofilm-producing C. albicans isolates to FLC and prevent cell adhesion on polystyrene surface and biofilm formation (with CSH as the indicator) in part by decreasing CSH.
Ca2+ burst is a common cellular response of C. albicans cells in response to an environmental stress . It is often along with an activation of calcineurin signaling pathways, in which the calcineurin is required for survival in serum, virulence, and resistance to azole antifungals, in part via its downstream target, Crz1 transcription factor [53–56]. In the present study, a significant Ca2+ burst was observed in cells exposed to the combination of CsA and FLC. Of note, either FLC or CsA alone showed an ability to decrease intracellular calcium concentration, however a combination of these two agents had a synergistic effect on increase but not decrease of intracellular calcium ([Ca(2+)]) levels. Such CsA-evoked intracellular calcium concentration might disturbed the calcium homeostasis and influenced the cell survival, which may partially explain the potential of CsA to enhance the effectiveness of FLC against the clinical biofilm-producing C. albicans. In addition, intracellular calcium was related to biofilm formation. For example, in a study on plant-pathogenic bacterium, Xylella fastidiosa, Cruz et al. demonstrated that intracellular calcium played a role in biofilm formation, which was related to the initial surface and cell-to-cell attachment and colonization stages of biofilm establishment, and was depended on functions of fimbrial structures .
In the present study, we provided additional evidences that calcineurin inhibitors (such as CsA) were able to enhance the susceptibility of C. albicans clinical biofilm-producing isolates to the most commonly used fungicidal agent, fluconazole (FLC). Mechanistically, CsA could synergistically suppress the expression of adhesion-related genes ALS3, hypha-related genes HWP1, ABC transporter drug-resistant genes CDR1 and MDR1, and FLC targeting gene ERG11 in biofilm producing C. albicans. In addition, a combination of CsA and FLC also could synergistically reduce cellular surface hydrophobicity (CSH) and increase intracellular calcium concentration in biofilm-producing C albicans isolates. Together with other studies, these results clearly suggest a combination of calcineurin inhibitor and fluconazole may prove to be a novel and effective therapeutic option, which warrants for further investigation.
Candida albicans strains and culture and identification
Candida albicans 1strain SC5314 was purchased from American Type Culture Collection (Mannasas, VA, USA). 24 FLC sensitive C. albicans clinical strains were isolated from bloodstream samples and collected from the department of laboratory medicine of the General Hospital of Ningxia Medical University between September 2014 and January 2015, which were identified by harnessing the VITEK-2 COMPACT fully automated microbiological system. The C. albicans strains were routinely grown in YPD liquid medium (20 g of glucose per liter, 10 g of yeast extract, 20 g of peptone) at 30 °C with 5%CO2 atmosphere . All strains had normal and comparable growth rates. Human blood samples were collected with a protocol approved by the Ethic Committee for the Conduct of Human Research at Ningxia Medical University (NXMU-2016-092). Written consent was obtained from every individual according to the Ethic Committee for the Conduct of Human Research protocol.
Characterization of Candida albicans biofilm formation
Candida albicans cells were grown in YPD overnight at 37 °C and resuspended in RPMI buffered with HEPES at a concentration of 1.0 × 106 cells/mL prior to be applied for biofilm formation culturing. The biofilm model was established using a method described in a previous study . Briefly, an 100 μL of above cell suspension was seeded in a flat-bottomed 96 well plates with and incubated at 37 °C at with 5%CO2 atmosphere for 24 h or until formation of mature biofilms, and biomass of each isolate was assessed in terms of the crystal violet (cv) assay by determining the distribution of biomass using the value of OD570nm as previously reported . A C. albicans isolate with a less than the 1st quartile (Q1) was grouped as having low biofilm formation (LBF) capacity, and a isolate with a biomass greater than the 3rd quartile (Q3) was considered isolates with high biofilm formation (HBF) ability, and an isolate that lay in between Q1 and Q2 was a deemed strain with intermediate biofilm formation (IBF Q2) potency (Fig. 1a) . After the culturing or treatment, harvested the cells by washing and scratching off from the culture wells, the cell suspension was then centrifuged for harvesting cell pellet.
Test of antifungal susceptibility of biofilm-producing C. albicans isolates
The antifungal susceptibility of biofilm-producing C. albicans was ascertained by determine minimum inhibitory concentration (MIC) of fungal cells on 24 h preformed biofilms, as previously described in flat-bottomed, 96 well microtitre plates . The MIC was determined at 80 % inhibition of fungal cells using an XTT (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-caboxanilide) metabolic reduction assay [61, 62]. The tested range of concentrations of agents was 2 μg/mL to 1024 μg/mL for FLC, and 9.3 μg/mL to 300 μg/mL for CsA. Combinations of these two agents were prepared in a chequerboard format as previously reported . All C. albicans strains were tested in duplicate for three independent experiments.
Fluorescence microscope assay
In order to morphologically observe the formation and integrity of C. albicans biofilm, biofilms cultured under different conditions were stained with 50 μg/mL FITC-conA, and imaged using a fluorescent microscopy.
Quantitative reverse transcriptional PCR (qRT-PCR)
Primer sequences used in this study
Primer sequences (5’-3’)
Cellular surface hydrophobicity assay
Since a hyphal form of C. albicans showed higher affinity for hydrocarbon than the yeast form, and the adherence of these fungus to hydrophobic surfaces increased when its morphology was changed from the yeast form to the hyphal form , C. albicans cellular surface hydrophobicity (CSH) was assessed using a water-hydrocarbon two-phase assay as described previously . Briefly, C. albicans isolates were standardized to 1 × 106 cells/mL in RPMI-1640 and 24 h at 37 °C and washed twice with PBS. C. albicans biofilms were scraped off to obtain a cell suspension (OD600nm, 1.0 mL in YPD medium). Then, 1.2 mL of cell suspension was transferred into a clean glass tube for each group and overlayed with 0.3 mL of octane. The cell suspension was incubated at 30 °C for 10 min prior the aqueous phase to be measured OD600nm . CSH was calculated using a formula as ([OD600nm of control - OD600nm of test]/OD600nm of control) × 100 % as previously described .
Detection of intracellular calcium ([Ca(2+)]) level
Candida albicans biofilms with different treatments were stained with 5 μmol/L of calcium-sensitive indicator Fluo-3/AM (Invitrogen, USA) in light proof at 37 °C for 30 min. The cells were then washed three times with D-Hanks buffer (Invitrogen, USA). The calcium levels were determined by flow cytometry in a FACScan flow cytometer (Becton Dickinson, USA) using a parameter of the excitation/emission wave lengths (485 nm/530 nm) with reading sensitivity level at 8 .
All data were recorded and analyzed by using the WHONET software (version 5.6). The statistical analysis was processed with the Statistical Package for the Social Sciences (SPSS) software (SPSS, version 18.0, Chicago, IL, USA). The changes of MICs for MDR C. albicans isolates between FLC or CsA alone and a combination of them were compared with a t-test analysis. Data were represented as the mean ± SD. A p < 0.05 was defined as a statistical significance.
ABC, ATP-binding cassette; ALS3, agglutinin-like sequence 3; C. albicans: Candida albicans; CDR1, candlda drug resistance 1; CsA, cyclosporine A; CSH, cellular surface hydrophobicity; CVC, central venous catheters; ERG11,encoding sterol 14alpha-demethylase; FLC, fluconazole; HBF, high biofilm formation; HWP1, hyphal wall protein 1; IBF, intermediate biofilm formation; LBF, low biofilm formation; MDR1, multidrug resistance 1; XTT, 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-carboxanilide
The authors thank Ms. Yuying Zhang, Mr. Fei Han, Mr. Anquan Shang and Mr. Jiali Yang for their valuable discussion and assistance.
This work was supported by competing grants from the Ningxia Key Laboratory of Clinical and Pathogenic Microbiology for WJ and JW (LCPM201501-I and LCPM201502-I). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Availability of data and materials
All the data supporting our findings is contained within the manuscript.
WJ, XL and JW conceived and designed the experiments; JW, HZ, CY and GL analyzed the data and drafted the manuscript; HZ, CY and GL performed experiments and acquired data; WJ, HZ and GL collected samples; JW and XL interpreted data and critically revised the manuscript. All authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Human blood samples were collected with a protocol approved by the Ethic Committee for the Conduct of Human Research at Ningxia Medical University (NXMU-2016-092). Written consent was obtained from every individual according to the Ethic Committee for the Conduct of Human Research protocol.
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