The calcineruin inhibitor cyclosporine a synergistically enhances the susceptibility of Candida albicans biofilms to fluconazole by multiple mechanisms

Jia et al. BMC Microbiology (2016) 16:113 DOI 10.1186/s12866-016-0728-1 RESEARCH ARTICLE Open Access The calcineruin inhibitor cyclosporine a synergistically enhances the susceptibility of Candida albicans biofilms to fluconazole by multiple mechanisms Wei Jia1,2, Haiyun Zhang3, Caiyun Li4, Gang Li1,2, Xiaoming Liu1* and Jun Wei1,2* Abstract Background: 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. Results: 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). Conclusion: 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. Keywords: Candida albicans, Biofilm, Calcineurin inhibitor, Cyclosporine A, Fluconazole Background 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 * Correspondence: ; 1 Ningxia Key laboratory of Clinical and Pathogenic Microbiology, the General Hospital of Ningxia Medical University, Yinchuan, Ningxia 750004, China Full list of author information is available at the end of the article 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 © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jia et al. BMC Microbiology (2016) 16:113 (ERG11), an essential enzyme in the ergosterol biosynthetic pathway of C. albican [5]. 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 [6]. 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 [24]. 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. Results Biofilm-produc (...truncated)