Antifungal activity of Cardiospermum halicacabum L. (Sapindaceae) against Trichophyton rubrum occurs through molecular interaction with fungal Hsp90

Abstract

Introduction: Dermatophytosis is a superficial fungal infection limited to the stratum corneum of the epidermis, or to the hair and nails, and constitutes an important public health problem because of its high prevalence and associated morbidity. Dermatophyte fungi, especially 2 species, Trichophyton rubrum and Trichophyton mentagrophytes, are the predominant pathogens. Topical antifungal drugs, mainly azoles or allyamines, are currently used for the treatment of dermatophytoses, although in some cases, such as in nail and hair involvement, systemic treatment is required. However, therapeutic efficacy of current antifungal agents can be limited by their side effects, costs, and the emergence of drug resistance among fungi. Plant extracts represent a potential source of active antimicrobial agents, due to the presence of a variety of chemical bioactive compounds. In the present work, we evaluated in silico and in vitro the antifungal activity of an extract of the medicinal plant Cardiospermum halicacabum against T. rubrum suggesting a potential interaction with Hsp90 as playing an important role in both pathogenicity and drug susceptibility of T. rubrum.

Methods: We investigated in vitro the effect of different concentrations of C. halicacabum (from 500 to 31.25 µg) against a clinical isolate of T. rubrum. Furthermore, using a computational assessment, the interaction between different C. halicacabum active compounds and the fungal Hsp90 was also investigated.

Results: Our results indicate a clear-cut antifungal activity of the total plant extract at the highest concentrations (500 and 250 µg). Among all tested C. halicacabum compounds, the luteolin and rutin molecules have been identified in silico as the most important potential inhibitors of Hsp90. Based on these data, luteolin and rutin were also individually assessed for their antifungal activity. Results demonstrate that both substances display an antifungal effect, even if lower than that of the total plant extract.

Conclusion: Our data indicate a strong fungistatic effect of C. halicacabum against T. rubrum, suggesting its potential therapeutic efficacy in the treatment of dermatophytoses. Additionally, C. halicacabum compounds, and particularly luteolin and rutin, are all possible Hsp90 interactors, explaining their fungistatic activity.

Keywords: Cardiospermum halicacabum; Hsp90; Trichophyton rubrum; antifungal activity; dermatophytoses; luteolin; molecular docking; molecular modeling; rutin.

Figure 1

Structural model of T. rubrum Hsp90 dimeric structure generated using as a template the crystal structure of Hsp90 from Saccharomyces cerevisiae (PDB ID: 2CG9). Notes: Blue and red colors indicate the 2 monomers. This image has been generated using the Chimera program. Abbreviation: PDB, Protein Data Bank.

Figure 2

Disc diffusion assay of antifungal activity of Cardiospermum halicacabum against Trichophyton rubrum. Notes: Fungal culture was incubated at 30°C with different concentrations of C. halicacabum total extract, as described in the “Materials and methods” section (A). Amphotericin B at 12, 6, 3, and 1.5 µg/disc was used as positive control (B). As a negative control, PBS with 1% of methanol was used (C). After 48 hours of incubation, a clear inhibition zone was observed in the areas treated with 500 or 250 µg of Cardiospermum extract solution. One of 3 representative experiments is shown.

Figure 3

Schematic view of the best molecular docking complex between Trichophyton rubrum Hsp90 and ATP (left panel), rutin (central panel), and luteolin (right panel). Notes: The residues interacting through hydrogen bonds (green dashed lines) are shown as ball-and-stick images, while the residues in contact with the ligands are indicated by arches with rays. This image has been produced using the LigPlot+ software (European Bioinformatics Institute, Cambridgeshire, UK). Abbreviation: ATP, adenosine triphosphate.

Figure 4

Best molecular docking complex between Trichophyton rubrum Hsp90 and ATP (left panel), rutin (central panel), and luteolin (right panel). Notes: The β-strands are represented by violet arrows, while the α-helices and the loops are shown as orange spirals and light gray wires, respectively. The ligand molecules, hosted in the Hsp90 ATP-binding site, are indicated by stick models colored by atom type. This picture has been generated using the Chimera program. Abbreviation: ATP, adenosine triphosphate.

Figure 5

Disc diffusion assay of antifungal activity of luteolin and rutin against Trichophyton rubrum. Notes: Fungal cultures were incubated at 30°C with luteolin (A) or rutin (B), at 500, 250, 125, and 62.5 µg/disc. After 48 hours of incubation, a significant inhibition zone was observed in the area around the disc impregnated with 500 µg of each compound. One of 3 representative experiments is shown.