Activity of Scorpion Venom-Derived Antifungal Peptides against Planktonic Cells of Candida spp. and Cryptococcus neoformans and Candida albicans Biofilms

Abstract

The incidence of fungal infections has been increasing in the last decades, while the number of available antifungal classes remains the same. The natural and acquired resistance of some fungal species to available therapies, associated with the high toxicity of these drugs on the present scenario and makes an imperative of the search for new, more efficient and less toxic therapeutic choices. Antimicrobial peptides (AMPs) are a potential class of antimicrobial drugs consisting of evolutionarily conserved multifunctional molecules with both microbicidal and immunomodulatory properties being part of the innate immune response of diverse organisms. In this study, we evaluated 11 scorpion-venom derived non-disulfide-bridged peptides against Cryptococcus neoformans and Candida spp., which are important human pathogens. Seven of them, including two novel molecules, showed activity against both genera with minimum inhibitory concentration values ranging from 3.12 to 200 μM and an analogous activity against Candida albicans biofilms. Most of the peptides presented low hemolytic and cytotoxic activity against mammalian cells. Modifications in the primary peptide sequence, as revealed by in silico and circular dichroism analyses of the most promising peptides, underscored the importance of cationicity for their antimicrobial activity as well as the amphipathicity of these molecules and their tendency to form alpha helices. This is the first report of scorpion-derived AMPs against C. neoformans and our results underline the potential of scorpion venom as a source of antimicrobials. Further characterization of their mechanism of action, followed by molecular optimization to decrease their cytotoxicity and increase antimicrobial activity, is needed to fully clarify their real potential as antifungals.

Keywords: Candida albicans; Candida spp.; Cryptococcus neoformans; antifungal drugs; antimicrobial peptides; scorpions venom.

FIGURE 1

Comparative analysis of ToAP2, ToAP1, ToAP3, ToAP4 against previously described AMPs. Conserved amino acid residues are marked bold. Asterisks indicate peptides with amidated C-termini. (A,B) Multiple sequence alignment of ToAP2 mature peptide with known AMPs from scorpion and ant venoms such as Ponericin-W-like 32.1 [UniProt: P0CI91], Ponericin-W-like 32.2 [UniProt: P0CI92], Css54 [UniProt: P0DL41], Pandinin 2 [UniProt: P83240], Heterin-2 [UniProt: A0A0C4G5K0], Ponericin-W1 [UniProt: P82423], Ponericin-W2 [UniProt: P82424], and Ponericin-W5 [UniProt: P82427] and their consensus sequence logo. (C,D) Multiple sequence alignment of ToAP1 mature peptide with known antimicrobial peptides from scorpion venom such as NDBP-4.23 [UniProt: Q5G8B5/S6D3A7], Mucroporin [UniProt: B9UIY3], Imcroporin [UniProt: C7B247], Stigmurin [GenBank: JK483709], AP6_TITCO [UniProt: Q5G8B3], TsAP-1 [UniProt: S6CWV8], AamAP1 [UniProt: G8YYA5], AamAP2 [UniProt: G8YYA6], BmKb1 [UniProt: Q718F4], and AcrAP1 [UniProt: A0A0A1I6E7] and their consensus sequence logo.

FIGURE 2

Helical wheel projections of the α-helical structured peptides. The projections were generated at the web server for this tool (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi). Diamonds, hydrophobic residues; circles, hydrophilic residues, non-charged; pentagons, positively charged residues; triangles, negatively charged residues.

FIGURE 3

Hemolytic activity of peptides against human erythrocytes. (A) ToAP2 and ToAP2S1, (B) ToAP1, (C) NDBP-4.23, and (D) NDBP-5.7.

FIGURE 4

Cytotoxicity of peptides against peritoneal macrophages of BALB/c mice. (A) ToAP2, (B) ToAP1, (C) NDBP-4.23, and (D) NDBP-5.7.

FIGURE 5

Effects of the peptides ToAP2, NDBP-4.23, ToAP1, NDBP-5.7, and amphotericin B on C. albicans biofilm formation. Effects of the peptides (A) and Amp B (B) on the initial adherence and effects of the peptides (C) and Amp B (D) on the mature stage. Results are expressed in terms of biofilm viability as XTT-reduction assay readings percentage, normalized by the control groups. The experiments were performed at least twice on different days. Statistical analyses: ANOVA and Turkey post-test (^∗∗∗∗P ≤ 0.0001; ^∗∗∗P ≤ 0.001; ^∗∗P ≤ 0.01; ^∗P ≤ 0.05). Mean ± SEM.

FIGURE 6

FAR-UV CD spectra of peptides in water and 10, 30, 50% TFE. (A) Spectra of ToAP2 presenting a red shift from 200 nm to 208 and 222 nm in the presence of TFE; (B) Spectra of ToAP2S1 showing an increase of the dichroic signals at 208 and 222 nm from water to TFE.