Silver(I) 1,10-Phenanthroline Complexes Are Active against Fonsecaea pedrosoi Viability and Negatively Modulate Its Potential Virulence Attributes

Abstract

The genus Fonsecaea is one of the etiological agents of chromoblastomycosis (CBM), a chronic subcutaneous disease that is difficult to treat. This work aimed to evaluate the effects of copper(II), manganese(II) and silver(I) complexes coordinated with 1,10-phenanthroline (phen)/1,10-phenanthroline-5,6-dione (phendione) on Fonsecaea spp. Our results revealed that most of these complexes were able to inhibit F. pedrosoi, F. monophora and F. nubica conidial viability with minimum inhibitory concentration (MIC) values ranging from 0.6 to 100 µM. The most effective complexes against F. pedrosoi planktonic conidial cells, the main etiologic agent of CBM, were [Ag(phen)₂]ClO₄ and [Ag₂(3,6,9-tdda)(phen)₄].EtOH, (tdda: 3,6,9-trioxaundecanedioate), displaying MIC values equal to 1.2 and 0.6 µM, respectively. These complexes were effective in reducing the viability of F. pedrosoi biofilm formation and maturation. Silver(I)-tdda-phen, combined with itraconazole, reduced the viability and extracellular matrix during F. pedrosoi biofilm development. Moreover, both silver(I) complexes inhibited either metallo- or aspartic-type peptidase activities of F. pedrosoi as well as its conidia into mycelia transformation and melanin production. In addition, the complexes induced the production of intracellular reactive oxygen species in F. pedrosoi. Taken together, our data corroborate the antifungal action of metal-phen complexes, showing they represent a therapeutic option for fungal infections, including CBM.

Keywords: antifungal activity; chromoblastomycosis; metal-based drugs; virulence factors.

Figure 1

Effect of phen and its silver complexes on the viability of F. pedrosoi biofilm formation and maturation. (A) In a 96-well polystyrene microplate, conidia were treated immediately with different concentrations of phen and its silver complexes, and incubated in RPMI medium for 72 h at 37 °C; (B) Conidia were added in another microplate to form biofilm and, just after 72 h, the test compounds were supplemented and the systems incubated for an extra 48 h; and (C) Conidia were incubated for 72 h with a combination of non-inhibitory concentrations of itraconazole and the complexes 12 (2×MIC) and 14 (4×MIC). The cell viability and the minimum inhibitory concentration values of fungal biofilms (bMIC) were assessed using XTT reduction assay after reading at 490 nm [52]. Non-treated conidia were also included as control systems. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Effect of phen and its silver complexes on F. pedrosoi biomass and extracellular matrix during biofilm formation. (A,B) Conidia were added to 96-well microplates containing RPMI medium and non-cytotoxic concentrations of each compound. (C,D) Conidia were treated with a combination of non-inhibitory concentrations of itraconazole and the complexes 12 (½ MIC) and 14 (2×MIC). The systems were incubated for 72 h at 37 °C and then biofilm biomass (A,C) and extracellular matrix (B,D) were quantified by incorporation of crystal violet and safranin, respectively. Systems containing only non-treated conidia were also prepared (control). * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 3

Effect of silver complexes on F. pedrosoi differentiation. (A) Control systems, conidia (⮞) in RPMI medium before (Time zero, T = 0) and after 48 h of incubation (T = 48 h), when filamentous form (►) was produced; (B) Conidia treated with ¼ and ½ MIC of complex 12 had the ability to branch (*) and form hyphae (►) inhibited after 48 h. In the MIC concentration, germ tubes (→) and conidia (⮞) were especially observed; and (C) Complex 14 (¼ and ½ MIC) inhibited hyphae formation, but moniliform cells (🡆) were found. While in the MIC and 2×MIC values, germ tubes (→) and conidia (⮞) were the most often detected. Bar: 10 µM. (Inset): The graph shows the viability of conidia after treatment with complexes using XTT assay [52]. **** p < 0.0001.

Figure 4

Effect of silver-phen complexes on F. pedrosoi melanin production. Fluorescence microscopy of fungal cells grown in the absence (control) and in the presence of silver complexes at different concentrations for 5 days at 26 °C, and then labeled with 6D2 mAb followed by incubation with anti-mouse IgM-Alexa 488 and calcofluor white (CW), as detailed in Material and Methods. Images were obtained using differential interference contrast (DIC) and fluorescence microscopy. Scale Bar: 50 µM. Note that all the cells treated with non-inhibitory concentration of ¼×MIC of both silver complexes were stained with calcofluor; however, most of them were not labeled with 6D2 mAb, showing that these cells had their melanin production inhibited.

Figure 5

Conidia were incubated for 24 h at 26 °C in RPMI medium supplemented with distinct non-inhibitory concentrations of complexes 12 and 14. The resorufin fluorescence was determined using Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit and detected after reading in the fluorimeter. Antimycin A (AA) was used as positive control. ** p < 0.01, *** p < 0.001.