A plant peptide with dual activity against multidrug-resistant bacterial and fungal pathogens

Abstract

Multidrug-resistant (MDR) bacteria pose a major threat to public health, and additional sources of antibacterial candidates are urgently needed. Noncanonical peptides (NCPs), derived from noncanonical small open reading frames, represent small biological molecules with important roles in biology. However, the antibacterial activity of NCPs remains largely unknown. Here, we discovered a plant-derived noncanonical antibacterial peptide (NCBP1) against both Gram-positive and Gram-negative bacteria. NCBP1 is composed of 11 amino acid residues with cationic surface potential and favorable safety and stability. Mechanistic studies revealed that NCBP1 displayed antibacterial activity by targeting phosphatidylglycerol and cardiolipin in bacterial membrane, resulting in membrane damage and dysfunction. Notably, NCBP1 showed promising efficacy in mice. Furthermore, NCBP1 effectively inhibited the growth of plant fungal pathogens and enhanced disease resistance in maize. Our results demonstrate the unexplored antimicrobial potential of plant-derived NCPs and provide an accessible source for the discovery of antimicrobial substances against MDR bacterial and fungal pathogens.

Fig. 1.. Noncanonical antibacterial peptides in maize.

(A) Classification of ABPs. (B) Broad-spectrum antibacterial activity of NCBP1. Agar diffusion assay of NCBP1 (0.4 μmol per disk) against MDR bacterial strains. The inhibition zones of NCBP1 are shown in red circles. Scale bars, 5 mm. (C) The corresponding inhibition zone diameters of NCBP1. Data are presented as mean ± SDs (n = 3 biological replicates). (D) The location of NCBP1 derived from maize genome. The distances between NCBP1 (red) and the adjacent genes (blue, BRN1 and DnaJ) are shown between two black dotted lines. The start site of NCBP1 and the direction of transcription of the adjacent genes are indicated by arrows. (E) Chemical structure of NCBP1. Cationic amino acid residues are marked in blue. (F) The three-dimensional structure of NCBP1 predicted by AlphaFold2. Positive charges, negative charges, and hydrophobic groups are shown in blue, red, and white, respectively. (G) Phylogenetic analysis of NCBP1 orthologous peptides. The phylogenetic tree is constructed using the Construct/Test Neighbor-Joining method. NCBP1 and GRPSp are indicated in red and blue font, respectively. The tree scale is 0.1.

Fig. 2.. Rapid bactericidal activity of NCBP1.

(A) Growth curves of S. cohnii L5 in the presence of NCBP1 at 1× and 2× MIC. PBS and vancomycin (VAN, 2× MIC) were used as negative and positive control, respectively. (B) Time-killing kinetics of NCBP1 against S. cohnii L5 [1 × 10⁷ colony-forming units (CFUs)/ml] at 4× and 10× MIC. PBS and VAN (10× MIC) were used as controls. (C) Normalized CD spectra of NCBP1 (0.1 mM) in PBS (black), 40 mM SDS (purple), and 50% TFE (yellow). (D) Amino acid sequences of NCBP1 derivatives. Cationic amino acid residues are in blue, and alanine is shown in red. (E) Antibacterial activities of NCBP1 and the derivatives. Scale bar, 1 cm. (F) Bacterial density of S. cohnii treated with NCBP1 and derivatives. PBS was used as the control. Data are presented as mean ± SDs (n = 3 biological replicates). P values were calculated using one-way analysis of variance (ANOVA). ***P < 0.001.

Fig. 3.. NCBP1 targeting bacterial membrane.

(A) Agar diffusion assay of NCBP1 (0.1 μmol) against S. cohnii L5 in the presence of PE, PG, CL, and PC (0.25 μmol). The chemical structures of the four phospholipids are shown (right). Scale bars, 10 mm. (B) The corresponding inhibition zone diameters (mm) of NCBP1 under the application of phospholipids against S. cohnii L5. (C) Fold-increased MICs of NCBP1 against S. cohnii L5 in the presence of phospholipids (0 to 64 μg/ml) using checkerboard microdilution assays. (D) MST analysis of the interaction between NCBP1 and PG (left) and PE (right). The equilibrium dissociation constant (K_d) value is shown. (E) Leakage of calcein dye from liposome that mimics bacterial membrane after treatment of NCBP1. Triton X-100 was used as a positive control. (F) Dynamic curves of the membrane permeability probed with PI for S. cohnii L5 under NCBP1 treatments. Experiments were performed as three biologically independent replicates. Nisin served as the positive control, and PBS was used as the negative control. (G) Fluorescence images of S. cohnii L5 under NCBP1 treatments. The live cells are stained green by Calcein-AM, whereas the dead cells are stained red by PI. Scale bars, 10 μm. (H) Morphological changes in S. cohnii L5 (1 × 10¹⁰ CFUs/ml) using SEM and TEM under the treatments of NCBP1 (160 μM) for 30, 60, 240, and 480 min. PBS was used as the control. The arrows indicate membrane disruption (red), the leakage of intracellular contents (blue), and the wrinkling of cell surface (yellow), respectively. Scale bars, 0.5 μm. Data are presented as mean ± SDs (n = 3 biological replicates). P values were calculated using one-way ANOVA. NS, no significance; ***P < 0.001.

Fig. 4.. Mechanism of action of NCBP1 on bacterial membrane disruption.

(A) Membrane potential of S. cohnii L5 treated with NCBP1 (16, 32, and 64 μM). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and PBS were used as a positive and a negative control, respectively. (B) Extracellular (left) and intracellular ATP level (right) in S. cohnii L5 treated with NCBP1, with Nisin as the positive control and PBS as the negative control. (C) Total cellular ROS content in S. cohnii L5 cells treated with NCBP1. Rosup was used as a positive control, and PBS was used as a negative control. (D) PCA of S. cohnii L5 under the treatment of NCBP1 at 0.5× MIC (green), 4× MIC (purple), and 10× MIC (yellow) based on RNA-seq analysis. (E) GSEA plot of the genes related to the membrane under 0.5× (left), 4× (middle), and 10× MIC (right) of NCBP1 treatments. GO, gene ontology; NES, normalized enrichment score; NOM P value, normalized P value. (F) Volcano plot for the genes in S. cohnii L5 treated with 4× MIC of NCBP1. Expressed genes with fold changes of more than 16 under both treatments (4× and 10× MIC) of NCBP1, are marked in yellow (membrane repair), purple (stress response), and blue (energy metabolism). (G) Scheme of the mode of action of NCBP1. NCBP1 rapidly kills bacteria by directly causing membrane dysfunction. Data are presented as mean values ± SDs (n = 3 biological replicates). P values were calculated using one-way ANOVA. **P < 0.01, ***P < 0.001.

Fig. 5.. Safety and antibacterial properties of NCBP1 in vivo.

(A) Hemolytic activity of NCBP1. Triton X-100 was used as a positive control. Data are presented as mean ± SDs (n = 3 biological replicates). (B) Cell viability of HEp-2, A549, Vero, and H9c2 cells treated with NCBP1 based on the water-soluble tetrazolium salt-1 (WST-1) assay. (C) Survival rates of the mice after 5 days (n = 5 mice per group) following intravenous injections of PBS or NCBP1 at doses of 50, 100, and 150 mg/kg. (D) Residual antibacterial activity of NCBP1 at temperatures ranging from 25° to 100°C for 1 and 2 hours. S. cohnii L5 was used as the tested strain. (E) Residual antibacterial activity of NCBP1 at different pH levels ranging from 2 to 12 at 37°C for 1 and 2 hours. (F) Scheme of the experimental protocol of the mouse skin wound infection model and mouse peritonitis-sepsis model. (G) Representative images of the wounds. Scale bars, 5 mm. (H) Changes of wound areas under the treatment of NCBP1 on days 2, 4, 6, 8, and 10. (I) Survival rates of mice in the peritonitis-sepsis model (n = 6). P values were calculated using the two-sided log-rank (Mantel-Cox) test. (J) Effect of NCBP1 (55 and 110 mg/kg) or vancomycin (VAN, 110 mg/kg) on bacterial survival in different organs of mice (n = 4). P values were calculated using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.. NCBP1 enhances broad-spectrum disease resistance in maize.

(A) Images of B. maydis treated with 0 (control), 50, 75, 100, 150, and 200 μM NCBP1 at 12, 36, and 60 hours postinoculation (hpi). Scale bars, 5 mm. (B) The inhibition rates (%) of NCBP1 on B. maydis after 36 hours of treatment at concentrations of 0 (control), 50, 75, 100, 150, and 200 μM. (C) Images of F. verticillioides treated with 0 (control), 50, 75, 100, 150, and 200 μM NCBP1 at 12, 36, and 60 hpi. Scale bars, 5 mm. (D) The inhibition rates (%) of NCBP1 on F. verticillioides after 36 hours of treatment at concentrations of 0 (control), 50, 75, 100, 150, and 200 μM. (E) Representative images of southern corn leaf blight caused by B. maydis in NCBP1-treated and control plants. Photos were taken 4 days after inoculation (DAI). Scale bar, 1 cm. (F) Statistical analysis of the percentage of necrotic leaf area in NCBP1-treated and control plants infected with B. maydis. (G) Quantification of B. maydis DNA in NCBP1-treated and control plants. Maize Ef1a was used as the internal control. (H) Representative images of seed rot caused by F. verticillioides in NCBP1-treated and control plants. Photos were taken at 7 DAI. Scale bar, 0.5 cm. (I) Statistical analysis of the percentage of diseased seed area in NCBP1-treated and control plants infected with F. verticillioides. (J) Quantification of F. verticillioides levels in the seeds by quantitative PCR. Maize Ef1a was used as the internal control. P values were calculated using Student’s t test. *P < 0.05, ***P < 0.001.