In Vitro Antifungal Activity of Chimeric Peptides Derived from Bovine Lactoferricin and Buforin II against Cryptococcus neoformans var. grubii

Abstract

Cryptococcosis is associated with high rates of morbidity and mortality. The limited number of antifungal agents, their toxicity, and the difficulty of these molecules in crossing the blood-brain barrier have made the exploration of new therapeutic candidates against Cryptococcus neoformans a priority task. To optimize the antimicrobial functionality and improve the physicochemical properties of AMPs, chemical strategies include combinations of peptide fragments into one. This study aimed to evaluate the binding of the minimum activity motif of bovine lactoferricin (LfcinB) and buforin II (BFII) against C. neoformans var. grubii. The antifungal activity against these chimeras was evaluated against (i) the reference strain H99, (ii) three Colombian clinical strains, and (iii) eleven mutant strains, with the aim of evaluating the possible antifungal target. We found high activity against these strains, with a MIC between 6.25 and 12.5 µg/mL. Studies were carried out to evaluate the effect of the combination of fluconazole treatments, finding a synergistic effect. Finally, when fibroblast cells were treated with 12.5 µg/mL of the chimeras, a viability of more than 65% was found. The results obtained in this study identify these chimeras as potential antifungal molecules for future therapeutic applications against cryptococcosis.

Keywords: Cryptococcus neoformans; antifungals; antimicrobial peptides; bovine lactoferricin; buforin II; chimeric peptides; cryptococcosis.

Figure 1

Chimeric peptides time–kill curve against C. neoformans var. grubii. (a,b) H99 strain; (c,d) clinical isolate 2807; (e,f) clinical isolate 3279; and (g,h) clinical isolate 2643. The asterisks represent the significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001) among the strains exposed to the different concentrations and the strains without treatment (growth control or positive control); not significant (ns). RPMI medium without yeast and peptide (sterility control or negative).

Figure 1

Chimeric peptides time–kill curve against C. neoformans var. grubii. (a,b) H99 strain; (c,d) clinical isolate 2807; (e,f) clinical isolate 3279; and (g,h) clinical isolate 2643. The asterisks represent the significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001) among the strains exposed to the different concentrations and the strains without treatment (growth control or positive control); not significant (ns). RPMI medium without yeast and peptide (sterility control or negative).

Figure 2

Cell viability assay of C. neoformans var. grubii after treatment (72 h) with C6 at 3.125 μg/mL, 6.25 μg/mL 12.5 μg/mL, and 25 μg/mL.

Figure 3

Cytotoxic effect of peptides on the mouse fibroblast cell line L929. Chimera C5: RRWQWR-Ahx-KLLKKLLK (black and white triangle shape) and chimera C6: KKWQWK-Ahx-RLLRRLLR (black box shape).