Biological Properties of a Novel Multifunctional Host Defense Peptide from the Skin Secretion of the Chaco Tree Frog, Boana raniceps

Abstract

In recent years, the number of new antimicrobial drugs launched on the market has decreased considerably even though there has been an increase in the number of resistant microbial strains. Thus, antimicrobial resistance has become a serious public health problem. Amphibian skin secretions are a rich source of host defense peptides, which generally are cationic and hydrophobic molecules, with a broad-spectrum of activity. In this study, one novel multifunctional defense peptide was isolated from the skin secretion of the Chaco tree frog, Boana raniceps. Figainin 2 (¹FLGAILKIGHALAKTVLPMVTNAFKPKQ²⁸) is cationic and hydrophobic, adopts an α-helical structure in 50% (v/v) trifluoroethanol (TFE), and is thermally stable. This peptide exhibited activity against Gram-negative and Gram-positive pathogenic bacteria arboviruses, T. cruzi epimastigotes; however, it did not show activity against yeasts. Figainin 2 also showed antiproliferative activity on cancer cells, is moderately active on human erythrocytes, and activates the oxidative burst in human neutrophils.

Keywords: Boana raniceps; amphibian; antimicrobial peptide; multifunctional defense peptide; neutrophils; skin secretion; structural analysis.

Figure 1

(A) Chromatographic profile of B. raniceps crude skin secretion fractionated by RP-HPLC performed on a Shimadzu LC system using a Vydac C₈ column. (B) Rechromatography of the marked fraction (with antibacterial activity) by RP-HPLC using a Shim-pack VP-ODS C₁₈ column.

Figure 2

MALDI-TOF mass spectrum of Figainin 2. The insert showed the presence of sodium and potassium adducts +22 Da and +38 Da, respectively.

Figure 3

Sequence alignment of Br22 to other putative host defense peptides (HDPs) from B. raniceps: The highlighted letters and asterisk indicate identical amino acids.

Figure 4

(A) Schiffer–Edmunson prediction of Figainin 2 showing the hydrophilic face and the nonpolar and hydrophobic face (in yellow). (B) Secondary structure model using I-Tasser server: Hydrophobic amino acids are marked in yellow.

Figure 5

(A) Far-UV Circular Dichroism (CD) spectra of Figainin 2 at 25 °C. Solid intense black line shows the CD spectrum in water. Slim solid, dashed, and dotted lines represent the spectra in 10%, 30%, and 50% (v/v) trifluoroethanol (TFE), respectively. The red shift of molar ellipticity from 200 nm (solid line) to 208 and 222 nm (dotted and dashed lines) are observed as a function of TFE addition (10–50%). (B) Effect of temperature from 25 °C to 95 °C on secondary structure of Figainin 2 in TFE 50% (v/v): The decreased dichroic bands at 222 and 208 nm are shown as a function of temperature, indicating the changes of the α-helix structure contents (Table 3). The isodichroic point of about 203 nm is indicated by the arrow. Insert: Dichroic spectra of the renaturation process of Figainin 2 in TFE 50% (v/v).

Figure 6

Hemolytic effects of Figainin 2 in human erythrocytes: Triton X-100 1% (v/v) was used as positive control (100% hemolysis), and Tris-saline was used as negative control (0% hemolysis). Data points show mean ± SD.

Figure 7

Cytotoxic activity of Figainin 2 against cancer cell lines (A) B16F10 (IC₅₀ = 12.8 µM) and (B) MCF-7 (IC₅₀ = 15.3 µM): Data points show mean ± SD.

Figure 8

Dose-response curves of Figainin 2: The X-axis indicates the log of Figainin 2 concentration (molar); the right Y-axis shows the normalized antiviral activity in percentage (in black), which represents the inhibition of infection in relation to positive control; and the right Y-axis shows the cell ratio or cell survival (in red). (A) chikungunya virus (CHIKV) 181/25, EC₅₀ = 17.9 μM, CC₅₀ = 49.0 μM, and SI = 2.88; (B) Dengue serotype 4 virus, EC₅₀ = 20.8 μM, CC₅₀ = 50.1 μM, and SI = 2.41; and (C) Yellow Fever Virus, EC₅₀ = 21.8 μM, CC₅₀ = 26.4 μM, and SI = 1.21.

Figure 9

Reactive oxygen species (ROS) generation in human neutrophils in response to control (HBSS), fMLP (0.1 µM), and Figainin 2 (8 µM): Differences between the samples were determined by one-way ANOVA with Tukey’s posttest. * p < 0.05 compared to control.