Encrypted Antimicrobial Peptides from Plant Proteins

Abstract

Examples of bioactive peptides derived from internal sequences of proteins are known for decades. The great majority of these findings appear to be fortuitous rather than the result of a deliberate and methodological-based enterprise. In the present work, we describe the identification and the biological activities of novel antimicrobial peptides unveiled as internal fragments of various plant proteins founded on our hypothesis-driven search strategy. All putative encrypted antimicrobial peptides were selected based upon their physicochemical properties that were iteratively selected by an in-house computer program named Kamal. The selected peptides were chemically synthesized and evaluated for their interaction with model membranes. Sixteen of these peptides showed antimicrobial activity against human and/or plant pathogens, some with a wide spectrum of activity presenting similar or superior inhibition efficacy when compared to classical antimicrobial peptides (AMPs). These original and previously unforeseen molecules constitute a broader and undisputable set of evidences produced by our group that illustrate how the intragenic concept is a workable reality and should be carefully explored not only for microbicidal agents but also for many other biological functions.

Figure 1

Physicochemical parameters and helical wheel plot of the IAPs Tc02, Tc06 and Tc08. Quantile box plots of the net charge, molecular mass (Da), isoelectric point, average hydrophobicity (TM scale), Hydrophobic moment (TM scale), Aggregation (Nav4SS parameter from Aggrescan), GOR IV alpha helix, extended configuration and coil structures was generated for 10.000 randomly selected peptides ranging from 16–22 amino acids from T. cacao genome. Color bars in each quantile plot represent the physicochemical parameters for the IAPs Tc02 (red), Tc06 (blue) and Tc08 (green). Helical wheel plots for the same IAPs are also represented, demonstrating that once structured as α-helical segments, these peptides show an amphiphilic character similar to classical AMPs. Hydrophobic residues are represented in green, positively charged residues are represented in red, negatively charged residues are represented in blue and polar uncharged residues in light blue. Tc02, Tc06 and Tc08 were selected based on their tested antimicrobial activities (Tables 4 and 5) to illustrate physicochemical parameters of the filtered IAPs when compared to randomly selected peptides from T. cacao. Physicochemical properties and helical wheel plots of the remaining IAPs are shown on Supplementary Material 01.

Figure 2

Circular Dichroism (CD) spectra of IAPs. Far-UV CD scans were performed to evaluate the secondary structure of IAPs in buffer alone (40 μM solution peptide in phosphate buffer), represented as a black line, and after the addition of 2 mM DMPC, represented as a red line, or in the presence of 2 mM 2:1 DMPC:DMPG, blue line. Tc02 and Gr02 dichroic bands indicate that both peptides are already partially structured as α-helical segments in buffer, and the addition of LUVs induces further structuration. Tc08, Gr01 and DS01 are randomly structured in buffer, only showing α-helix dichroic bands in the presence of LUVs.

Figure 3

Heating thermal scans of DMPC and 2:1 DMPC:DMPG LUVs enriched with 4 mol% IAPs. Non two-state model fitting of P′_β → L_α phase transition of a solution of 0.5 mM of (1) DMPC or (2) 2:1 DMPC:DMPG LUVs enriched with 4 mol% of Tc02 (a), Tc06 (b), Tc08 (c), Gr01 (d), Gr02 (e) or DS01 (f). Black line corresponds to experimental data. Red line corresponds to model fitting.

Figure 4

Moniliophthora perniciosa basidiospores germination test. After 4 hours of growth, the basidiospores were visualized. (a) Without peptides; (b) Tc02, 16 μM; (c) Tc05, 256 μM; (d) Tc06, 64 μM; (e). Tc08, 128 μM; (f) Tc10, 64 μM; (g). Asc-8, 16 μM; (h). DS01, 256 μM. The concentrations described for each peptide correspond to the minimum concentrations required to inhibit at least 95% basidiospore germination.

Figure 5

Ramularia areola infection test. Disease severity index ranging from 1 (without symptoms) and 5 (over 50% of leaf area showing symptoms) in both abaxial (a1) and adaxial surface (a2). Areolate mildew severity in cotton leaves after treatment with a commercial fungicide (PrioriXtra) and different concentrations of IAPs Gr01 (b1) and Tc06 (b2). Gr01 and Tc06 showed comparable results to fungicide at concentrations starting at 16 µM and 32 µM, respectively.