Calcineurin activity in Fonsecaea pedrosoi: tacrolimus and cyclosporine A inhibited conidia growth, filamentation and showed synergism with itraconazole

Abstract

Fonsecaea pedrosoi is a melanized fungus that causes chromoblastomycosis (CBM), a tropical neglected disease responsible for chronic and disability-related subcutaneous mycosis. Given the challenging nature of CBM treatment, the study of new targets and novel bioactive drugs capable of improving patient life quality is urgent. In the present work, we detected a calcineurin activity in F. pedrosoi conidial form, employing primarily colorimetric, immunoblotting and flow cytometry assays. Our findings reveal that the calcineurin activity of F. pedrosoi was stimulated by Ca²⁺/calmodulin, inhibited by EGTA and specific inhibitors, such as tacrolimus (FK506) and cyclosporine A (CsA), and proved to be insensitive to okadaic acid. In addition, FK506 and CsA were able to affect the cellular viability and the fungal proliferation. This effect was corroborated by transmission electron microscopy that showed both calcineurin inhibitors promoted profound changes in the ultrastructure of conidia, causing mainly cytoplasm condensation and intense vacuolization that are clear indication of cell death. Our data indicated that FK506 exhibited the highest effectiveness, with a minimum inhibitory concentration (MIC) of 3.12 mg/L, whereas CsA required 15.6 mg/L to inhibit 100% of conidial growth. Interestingly, when both were combined with itraconazole, they demonstrated anti-F. pedrosoi activity, exhibiting a synergistic effect. Moreover, the fungal filamentation was affected after treatment with both calcineurin inhibitors. These data corroborate with other calcineurin studies in fungal cells and open up further discussions aiming to establish the role of this enzyme as a potential target for antifungal therapy against CBM infections.

Keywords: Antifungal drugs; Calcineurin inhibitors; Chromoblastomycosis; Combined therapy; Intracellular phosphatase activity.

Fig. 1

Calcineurin activity in F. pedrosoi conidia. (A) The conidial cell extract was incubated 1 h in the presence of calcium/calmodulin (Ca²⁺/CAM), 0.75 mM/313 µM) or EGTA (10 mM) in reaction medium containing 50 mM Tris-buffer, pH 7.5 and 10 mM p-nitrophenylphosphate (p-NPP), as substrate. (B) The cell extract was subjected to 12% SDS-PAGE. (a) F. pedrosoi whole homogenate silver-stained (b) diluted homogenate silver-stained (c) the proteins from SDS-PAGE were transferred to nitrocellulose membrane followed by sequential incubations with anti-calcineurin antibody and peroxidase-labeled anti-rabbit antibody and then revealed by chemiluminescence. The number on the right indicate the relative molecular masses expressed in kilodaltons (kDa). (C) The cell extract was incubated 1 h in the absence (control) or presence of 10 µg/mL tacrolimus (FK506), 100 µg/mL cyclosporine A (CsA) or 0.625 µM okadaic acid (OA), in reaction medium containing 50 mM Tris-buffer, pH 7.5, Ca.²⁺/CAM (0.75 mM/313 µM) and 10 mM p-NPP. (D) Conidia were pretreated for 15 h with calcineurin inhibitors, incubated 1 h in the presence of anti-calcineurin antibody and FITC-labeled anti-rabbit IgG antibody and analyzed using flow cytometry. Inset: Representative quantification showing fluorescent cells percentage (%FC) that was converted of the control values discounting the autofluorescence (AF). *p < 0.05, ****p < 0.0001

Fig. 2

Effect of calcineurin inhibitors on the growth of F. pedrosoi conidia. (A) Flow cytometry showing the fluorescence intensity of conidia untreated (control), heat-killed and treated for 15 h with tacrolimus (FK506) 10 µg/mL and cyclosporine A (CsA) 100 µg/mL and incubated with propidium iodide 10 µg/mL for 5 min. (B) The colony forming units (CFU) were determined after incubation of conidia for 15 h in the absence (control) or in the presence of FK506 10 µg/mL and CsA 100 µg/mL followed by plating 100 µL of each cellular suspension on SDA medium and incubation for 7 days at 26 °C.****p < 0.0001

Fig. 3

Effect of calcineurin inhibitors on conidia F. pedrosoi ultrastructure. Conidia were (A) untreated (control) and treated 15 h with (B) tacrolimus (FK506) 10 µg/mL and (C) cyclosporine A (CsA) 100 µg/mL, then the systems were fixed and processed for routine TEM and ultrathin sections observed under JEOL 1210 microscope. Control system shows the cell wall preserved and electrodense cytoplasm, while cells treated with FK506 and CsA exhibited extraction of cytoplasm with condensed material (➤) and intense vacuolization (*), indicative of cell death. Especially, after FK506 treatment the cells were more pigmented than control system. This inhibitor was able to detach the cell wall showing intense pigmentation (➨)

Fig. 4

Anti-F. pedrosoi activity of calcineurin inhibitors and their combination with antifungal drugs. (A) Conidia were incubated in RPMI 1640 medium pH 7.0 containing different concentrations of tacrolimus (FK506), cyclosporine A (CsA), itraconazole (ITC) and amphotericin B (AMB), then the minimum inhibitory concentration (MIC) values were determined as preconized by protocol M38-A2 of CLSI (2008), with some modifications. The MIC was considered as the lowest concentration capable of inhibiting 100% of fungal growth. The concentration that inhibits 50% (IC₅₀) of fungal proliferation was also determined. The interaction type of drug combinations was defined using the checkerboard method by calculating the fractional inhibitory concentration index (FICI) as detailed in Material and Methods. (B) Representative image of minimum fungicidal concentrations (MFC) assay of CsA (62.5—0.03 mg/L) and FK506 (25.0—0.012 mg/L) on F. pedrosoi growth. (A,B) Fungistatic effect was observed, since fungal cells were able to grow in the culture medium without inhibitors. ND, Not determined

Fig. 5

Effect of calcineurin inhibitors on F. pedrosoi filamentation. Conidia were added in 24-well microplates containing RPMI medium and treated with non-toxic concentrations (MIC and 2 × MIC) of tacrolimus (FK506) and cyclosporine A (CsA) for 72 h at 26 °C. The microscopy images show control systems of conidia at zero-time (T = 0) and after 72 h (T = 72 h) when the fungal mycelia were fully developed. The inhibition of F. pedrosoi filamentation by FK506 and CsA was revealed by hyphal branch growth reduction (➨) and moniliform hyphae detection (➤). Bar: 50 µm