Inhibitory Effect of Morin Against Candida albicans Pathogenicity and Virulence Factor Production: An in vitro and in vivo Approaches

Abstract

Candida albicans is considered an exclusive etiologic agent of candidiasis, a very common fungal infection in human. The expression of virulence factors contributes highly to the pathogenicity of C. albicans. These factors include biofilm formation, yeast-to-hyphal transition, adhesins, aspartyl proteases, and phospholipases secretion. Moreover, resistance development is a critical issue for the therapeutic failure of antifungal agents against systemic candidiasis. To circumvent resistance development, the present study investigated the virulence targeted therapeutic activity of the phyto-bioactive compound morin against C. albicans. Morin is a natural compound commonly found in medicinal plants and widely used in the pharmaceutical and cosmetic products/industries. The present study explicated the significant inhibitory potential of morin against biofilm formation and other virulence factors' production, such as yeast-hyphal formation, phospholipase, and exopolymeric substances, in C. albicans. Further, qPCR analysis confirmed the downregulation of biofilm and virlence-related genes in C. albicans upon morin treatment, which is in correspondence with the in vitro bioassays. Further, the docking analysis revealed that morin shows strong affinity with Hwp-1 protein, which regulates the expression of biofilm and hyphal formation in C. albicans and, thereby, abolishes fungal pathogenicity. Moreover, the anti-infective potential of morin against C. albicans-associated systemic candidiasis is confirmed through an in vivo approach using biomedical model organism zebrafish (Danio rerio). The outcomes of the in vivo study demonstrate that the morin treatment effectively rescues animals from C. albicans infections and extends their survival rate by inhibiting the internal colonization of C. albicans. Histopathology analysis revealed extensive candidiasis-related pathognomonic changes in the gills, intestine, and kidney of animals infected with C. albicans, while no extensive abnormalities were observed in morin-treated animals. The results evidenced that morin has the ability to protect against the pathognomonic effect and histopathological lesions caused by C. albicans infection in zebrafish. Thus, the present study suggests that the utilization of morin could act as a potent therapeutic medication for C. albicans instigated candidiasis.

Keywords: Candida albicans; anti-biofilm; anti-infective; anti-virulence; morin; systemic candidiasis; zebrafish.

FIGURE 1

(A) Effect of morin on growth and biofilm of C. albicans. Data are presented as means ± SD. *Indicates statistical significance (p < 0.05). (B) Inhibitory effect of morin on C. albicans exopolysaccharide production. Data are presented as means ± SD. *Indicates the statistical significance (p < 0.05). (C) Effect of morin on cell surface hydrophobicity of C. albicans. Data are presented as means ± SD. *Indicates statistical significance (p < 0.05).

FIGURE 2

Microscopic analysis of C. albicans biofilms in the absence and presence of morin at MBIC. (A) Light microscope. (B) Confocal laser scanning microscope. (C) Scaning electron microscope analysis.

FIGURE 3

(A) Growth curve analysis. The graph represents the effect of morin (at MBIC) on the growth dynamics of C. albicans. (B) FTIR spectra of EPS samples extracted from morin-treated (at MBIC) and untreated C. albicans. FT-IR spectra showed variations in regions such as (a) mixed regions of polysaccharides and nucleic acids (1300-900 cm^–1). (b) proteins (1700–1500 cm^–1),

FIGURE 4

Effect of morin on filamentous morphology. Hyphal morphology was significantly reduced at MBIC concentration (at MBIC).

FIGURE 5

(A) Activity of morin against C. albicans in an agar invasion assay. (B) Effect of morin on C. albicans protease production at the increasing concentration (37.5, 75, and 150μg/ml). Data are presented as means ± SD. *Indicates the statistical significance (p < 0.05). (C) Effect of morin (at MBIC) on C. albicans phospholipase production.

FIGURE 6

(A) Effect of morin (at MBIC) on C. albicans catalase production. (B) Quantitative analysis of the catalase inhibitory activity of morin (at MBIC).

FIGURE 7

Gene expression analysis of C. albicans in the presence and absence of morin (at MBIC). Data are presented as means ± SD. *Indicates statistical significance (p < 0.05).

FIGURE 8

Docking analysis shows the binding pattern and amino acid interactions of morin (−6.1 kcal/mol) with Hwp1 receptor in C. albicans for possible inhibition of adhesion as well as biofilm formation.

FIGURE 9

(A) Surival percentage of zebrafish in the presence of morin at different concentrations. (B) Survival percentage of C. albicans-infected zebrafish upon morin treatment (75 μg/ml) up to 96 h. Data are presented as means ± SD. *Indicates statistical significance (p < 0.05).

FIGURE 10

Histopathology analysis of the Gills (A), Intestine (B), and Kidney (C) sections of uninfected control, C. albicans infected control and morin treated zebrafishes.