New insights into the structure and mode of action of Mo-CBP3, an antifungal chitin-binding protein of Moringa oleifera seeds.

Mo-CBP3 is a chitin-binding protein purified from Moringa oleifera Lam. seeds that displays inhibitory activity against phytopathogenic fungi. This study investigated the structural properties and the antifungal mode of action of this protein. To this end, circular dichroism spectroscopy, antifungal assays, measurements of the production of reactive oxygen species and microscopic analyses were utilized. Mo-CBP3 is composed of 30.3% α-helices, 16.3% β-sheets, 22.3% turns and 30.4% unordered forms. The Mo-CBP3 structure is highly stable and retains its antifungal activity regardless of temperature and pH. Fusarium solani was used as a model organism for studying the mechanisms by which this protein acts as an antifungal agent. Mo-CBP3 significantly inhibited spore germination and mycelial growth at 0.05 mg.mL-1. Mo-CBP3 has both fungistatic and fungicidal effects, depending on the concentration used. Binding of Mo-CBP3 to the fungal cell surface is achieved, at least in part, via electrostatic interactions, as salt was able to reduce its inhibitory effect. Mo-CBP3 induced the production of ROS and caused disorganization of both the cytoplasm and the plasma membrane in F. solani cells. Based on its high stability and specific toxicity, with broad-spectrum efficacy against important phytopathogenic fungi at low inhibitory concentrations but not to human cells, Mo-CBP3 has great potential for the development of new antifungal drugs or transgenic crops with enhanced resistance to phytopathogens

Structural properties of <i>Mo</i>-CBP₃.

<p>(A) Circular dichroism spectra (Far-UV) of <i>Mo</i>-CBP₃ (2.22 mM) in 20 mM sodium phosphate buffer, pH 7.0, using a rectangular quartz cuvette with a 0.1 cm path length. (B) Denaturing polyacrilamide gel electrophoresis (SDS-PAGE - 15% acrylamide gel) of <i>Mo-</i>CBP₃. Molecular mass standards are shown (in kDa) on the left; Lanes 1 and 2, <i>Mo-</i>CBP₃ (20 µg) in reducing (4 kDa and 8 kDa subunits) and non-reducing conditions (18 kDa), respectively.</p

Effect of pH on the conformation and antifungal activity of <i>Mo</i>-CBP₃.

<p>(A) Far-UV CD spectra of <i>Mo</i>-CBP₃ (2.22 mM) at various pH values. (B) Light micrographs of <i>F. solani</i> spores either in 20 mM sodium acetate-borate-phosphate buffer at different pH values (control) or incubated with <i>Mo</i>-CBP₃ (0.1 mg.mL⁻¹) prepared in these buffers. Trials were conducted for 24 h at 22°C in the dark. Bars: 2.5 µm.</p

Induction of reactive oxygen species (ROS) in <i>F. solani</i> spores.

<p>Cells were treated with 3,3′-diaminobenzidine (DAB) for ROS detection. Cells were previously incubated with (A) H₂O, (B) BSA (0.1 mg.mL⁻¹) or (C) <i>Mo</i>-CBP₃ (0.1 mg.mL⁻¹). Uptake of DAB is confirmed by the dark staining (reddish-brown) reaction in conidia, as indicated by arrows. Bars: 2.5 µm (A–C).</p

Transmission electron microscopy of <i>F. solani</i> cells.

<p>The cells were cultured either in the absence (A) and presence (B and C) of <i>Mo</i>-CBP₃ (0.05 mg.mL⁻¹). Star indicates condensation of the cytosolic content. Vacuole condensation (V) is also shown. Arrows indicate shrinkage of the cell wall. Bars: 0.5 µm (A–C).</p

Evaluation of the cytotoxic effect of <i>Mo</i>-CBP₃.

<p><i>In vitro</i> haemolytic activity of <i>Mo</i>-CBP₃ on human erythrocytes using concentrations ranging from 280 to 0.137 µM. Positive control (C+): 1% Triton X-100. Negative control (C–): 100 mM sodium phosphate buffer, pH 7.4, 150 mM NaCl.</p

Effect of temperature on the conformation and antifungal activity of <i>Mo</i>-CBP₃.

<p>(A) Far-UV CD spectra of <i>Mo</i>-CBP₃ (2.22 mM) at various temperatures. (B) CD spectra of <i>Mo</i>-CBP₃ (2.22 mM) after heating at 100°C for 60 min. (C) Light micrographs of <i>F. solani</i> spores in either the culture medium (control) or incubated with <i>Mo</i>-CBP₃ (0.1 mg.mL⁻¹) and either unheated or previously heated at 100°C for 60 min in a water bath. Trials were conducted for 24 h at 22°C in the dark. Bars: 2.5 µm.</p