Novel Peptide-Based Inhibitors for Microtubule Polymerization in Phytophthora capsici

Abstract

The plant disease Phytophthora blight, caused by the oomycete pathogen Phytophthora capsici, is responsible for major economic losses in pepper production. Microtubules have been an attractive target for many antifungal agents as they are involved in key cellular events such as cell proliferation, signaling, and migration in eukaryotic cells. In order to design a novel biocompatible inhibitor, we screened and identified inhibitory peptides against alpha- and beta-tubulin of P. capsici using a phage display method. The identified peptides displayed a higher binding affinity (nanomolar range) and improved specificity toward P. capsici alpha- and beta-tubulin in comparison to Homo sapiens tubulin as evaluated by fluorometric analysis. One peptide demonstrated the high inhibitory effect on microtubule formation with a nanomolar range of IC₅₀ values, which were much lower than a well-known chemical inhibitor-benomyl (IC₅₀ = 500 µM). Based on these results, this peptide can be employed to further develop promising candidates for novel antifungal agents against Phytophthora blight.

Keywords: microtubules; peptide; phage display; phytophthora blight; phytophthora capsici.

Figure 1

The SDS-PAGE analysis of the purification of P. capsici and H. sapiens α, β-tubulin. Electrophoresis was performed using a 12% polyacrylamide gel, and the staining of proteins was carried out using Coomassie blue R-250.

Figure 2

Polymerization of P. capsici α, β-tubulin. (A) The rate of the polymerization reaction was monitored by light scattering at 350 nm; as the polymerization progresses, the turbidity of reactant increases. (B) Time-dependent analysis of polymerization was confirmed by SDS-PAGE analysis. Due to the combining of α, β-tubulin for polymerization, both tubulins are consumed and the band intensity at 55 and 57 kDa decreases over time.

Figure 3

The relative activity of microtubule polymerization of P. capsici as a function of the fixed concentration of inhibitory phages. A fixed concentration of each phage (10 nM) was treated initially with the polymerization assay mixture for 40 min of reaction time. The percent inhibition of the peptides was as follows; α_P1, 28%; α_P2, 54%; α_P3, 32%; α_P4, 19%; α_P5, 5%; α_P8, 4%; β_P1, 21%; β_P2, 2%.

Figure 4

Determination of peptide specificities (A) α_P1, (B) α_P2, (C) α_P3, and (D) β_P1. Binding reactions were performed with a constant concentration of 2% BSA, 5 μg each of α, β-tubulin of P. capsici and H. sapiens in a 96-well white plate as a target and various concentrations of each peptide (0–400 nM). The specificities of the bound peptides were measured using a fluorometric method and were plotted against the concentration of the peptides used in the binding reaction. The data are representative of one of three independent experiments, and the specificities were obtained from nonlinear fitting of the saturation–binding curves.

Figure 5

The relative activity of microtubule polymerization of (A) P. capsici and (B) H. sapiens as a function of increased concentration of inhibitory peptides. The activity for P. capsici yielded the following values of IC₅₀; α_P1 (■), 2.69 μM; α_P2 (●), 802 nM; α_P3 (▲), 1.24 μM; β_P1 (▼), 6.91 μM.

Figure 6

Western blot analysis to confirm the inhibition of microtubule polymerization using anti-His-antibody (rabbit monoclonal antibody, 1:1000 dilution in bovine serum albumin in Tris-buffered saline (TBST)). Reaction products were resolved on 12% SDS-PAGE, followed by Western blot analysis.

Figure 7

TEM images of polymerized P. capsici microtubules. α, β-tubulin were polymerized for 40 min. (A) Microtubules were observed without peptide. (B) Amorphous morphology of microtubules occurred predominantly with 1 μM of α_P2. (C) Ten micromolar α_P2 completely inhibited the formation of microtubules. As the concentration of peptide increased, the extent of polymerization was inhibited. The scale bar corresponds to 300 nm.