The Anti-Biofilm Efficacy of Caffeic Acid Phenethyl Ester (CAPE) In Vitro and a Murine Model of Oral Candidiasis

Abstract

Candida albicans is the main fungal species associated with the development of oral candidiasis. Currently, therapeutic options for these infections are limited by the adverse effects of antifungal drugs and by the emergence of drug resistant strains. Thus, the development of new antifungal agents is needed for the prevention and treatment of oral Candida infections. Caffeic acid phenethyl ester (CAPE) is a natural compound from propolis polyphenolic groups that exhibits many pharmacological properties. In this study, we investigated whether CAPE can have antifungal and immunomodulatory effects on oral candidiasis. Preliminary tests to assess the antifungal activity of CAPE were performed using the Minimum Inhibitory Concentration (MIC) assay that demonstrated inhibition in a range from 16 to 32 μg/mL, confirming its antifungal activity on several C. albicans strains isolated from the oral cavity. Subsequently, we analyzed Candida spp biofilms formed in vitro, in which CAPE treatment at 5 x MIC caused a reduction of 68.5% in the total biomass and ~2.60 Log in the viable cell count (CFU/mL) in relation to the untreated biofilm (p<0.0001). Next, RNA was extracted from untreated and CAPE-treated biofilms and analyzed by real-time qPCR. A series of genes analyzed (ALS1, ECE1, EPA1, HWP1, YWP1, BCR1, BGR1, CPH1, EFG1, NDT80, ROB1, TEC1, UME6, SAP2, SAP5, PBL2, and LIP9) were downregulated by CAPE compared to the untreated control group (p<0.0001). In in vivo studies using Galleria mellonella, the treatment with CAPE prolonged survival of larvae infected by C. albicans by 44.5% (p < 0.05) and accompanied by a 2.07-fold increase in the number of hemocytes. Flow cytometry revealed the most prominent increases were in types P2 and P3 hemocytes, granular cells, which phagocytize pathogens. In addition, CAPE treatment decreased the fungal load in the hemolymph and stimulated the expression of antifungal peptide genes such as galiomicin and gallerimycin. The antifungal and immunomodulatory activities observed in G. mellonella were extended to a murine model of oral candidiasis, in which CAPE decreased the levels of C. albicans colonization (~2 log CFU/mL) in relation to the untreated control group. In addition, CAPE treatment significantly reduced pseudomembranous lesions, invasion of hyphae on epithelium surfaces, tissue damage and inflammatory infiltrate (p < 0.05). CAPE was also able to increase the expression of β-defensin 3 compared to the infected and untreated group by 3.91-fold (p < 0.0001). Taken together, these results show that CAPE has both antifungal and immunomodulatory effects, making it a promising natural antifungal agent for the treatment and prevention of candidiasis and shows impact to oral candidiasis.

Antifungal Activity of the Phenolic Compounds Ellagic Acid (EA) and Caffeic Acid Phenethyl Ester (CAPE) against Drug-Resistant Candida auris

“…Furthermore, to the best of our knowledge, this is the first study that investigated the anti-C. albicans properties of EA using C. elegans. Our findings corroborate with previous studies using different invertebrate models, since the protective effect of EA on a Drosophila melanogaster model and the efficacy of CAPE on G. mellonella and C. elegans models have already been described against C. albicans [21,33,85]. EA had a protective effect on Drosophila melanogaster from infection with C. albicans SC5314.…”

“…CAPE at 2× MIC (16 µg/mL) completely suppressed yeast-to-hyphal conversion without interfering with fungal growth. Microscopic images confirmed that CAPE inhibited hyphae formation, in agreement with other studies [85].…”

“…In agreement with the biofilm results, CAPE impaired C. auris and C. albicans adhesion to cultured human epithelial cells at 4× MIC. Similar results were found by De Barros et al (2021), which reported a reduction of 68.5% in the amount of total biomass of C. albicans biofilm when treated with CAPE at 5× MIC (80 µg/mL) [85]. De Vita et al (2014) reported that caffeic acid derivatives could inhibit C. albicans ATCC 10231 biofilms.…”

“…Survival rates of 45%, 33%, and 34% were observed for the groups that received 3.2, 6.4, and 32 µg/mL of EA, respectively [33]. De Barros et al (2021) found that the survival rate of G. mellonella larvae was 44.5% when treated with CAPE at 5 mg/kg, accompanied by a 2.07-fold increase in the number of hemocytes and stimulation of the expression of antifungal peptide genes galiomicin and gallerimycin, suggesting both antifungal and immunomodulatory activities [85]. We found that CAPE was not effective to protect C. elegans against C. albicans.…”

Antifungal and Anti-Biofilm Effects of Caffeic Acid Phenethyl Ester on Different Candida Species

“…However, few studies that have investigated the antifungal activity of CAPE as a single molecule or in combination with some antibiotics were found in the literature. De Barros and coworkers recently reported the ability of CAPE to inhibit the growth of both fluconazole-sensitive and fluconazole-resistant strains of C. albicans [ 23 ]. Moreover, Sun and coworkers found the synergistic effects of CAPE with caspofungin and fluconazole against C. albicans and fluconazole-resistant C. albicans , respectively [ 24 , 25 ].…”