The Greater Celandine: Identification and Characterization of an Antimicrobial Peptide from Chelidonium majus

Abstract

Chelidonium majus, known as Greater Celandine, is a latex-bearing plant that has been leveraged for its anticancer and antimicrobial properties. Herein, C. majus aerial tissue is mined for the presence of antimicrobial peptides. A highly abundant cysteine-rich peptide with a length of 25 amino acids, deemed CM-AMP1, is characterized through multiple mass spectrometric approaches. Electron-activated dissociation is leveraged to differentiate between isoleucine and leucine residues and complement conventional collision-induced dissociation to gain full sequence coverage of the full-length peptide. CM-AMP1 shares little sequence similarity with any proteins in publicly available databases, highlighting the novelty of its cysteine landscape and core motif. The presence of three disulfide bonds in the native peptide confers proteolytic stability, and antimicrobial activity is greatly decreased upon the alkylation of the cysteine residues. Synthetic variants of CM-AMP1 are used to confirm the activity of the full-length sequence and the core motif. To assess the biological impact, E. coli was grown in a sublethal concentration of CM-AMP1 and quantitative proteomics was used to identify proteins produced by the bacteria under stress, ultimately suggesting a membrane lytic antimicrobial mechanism of action. This study integrates multiple analytical methods for molecular and biological characterization of a unique antimicrobial peptide identified from C. majus.

Figure 1.

Bioactivity profile of C. majus peptide library. A) Antibiotic activity of C. majus reversed-phase fractions (n = 3) assayed against E. coli ATCC 25922 and S. aureus LAC. Activity was calculated relative to ampicillin and erythromycin controls for E. coli and S. aureus, respectively. Error bars represent +/− 1 standard deviation away from the mean values. B) Total ion chromatogram (TIC) of fraction 24. Inset represents the MS1 level of the most abundant peak at t = 12.72 min (red dashed line).

Figure 2.

Sequence elucidation of CM-AMP1 enabled by complementary fragmentation strategies. A) Comparison of fragmentation spectra achieved by collision-induced dissociation (CID, top) and electron-activated dissociation (EAD, bottom) of the Cys-alkylated CM-AMP1 precursor (m/z = 655.31, M = 3271.51). B) Sequence coverage of the intact CM-AMP1 peptide (top). Cumulative observed fragments across precursor charge states +3 – +6 were counted at each position for both fragmentation strategies (bottom).

Figure 3.

Leucine / Isoleucine determination in CM-AMP1. A) Overview of neutral losses occurring from electron-activated dissociation (EAD) of Leu and Ile amino acids. B) EAD fragmentation spectrum of LysC digested CM-AMP1, and resulting sequence assignment.

Figure 4.

Proteolytic stability of CM-AMP1 against Proteinase K. Extracted ion chromatograms (XICs) of the +5 charge state of CM-AMP1 before and after 10 min of exposure to Proteinase K. Disulfide-bound CM-AMP1 (top) is compared to CM-AMP1 with reduced and alkylated Cys residues.

Figure 5.

Bioactivity of synthetic CM-AMP1 and CM-AMP1 core. A) Sequence of sCM-AMP1 and sCM-AMP1 core analogs. Cysteine residues are highlighted in red. B) Antibiotic activity of sCM-AMP1 and the sCM-AMP1 variant at different concentrations (n = 3) assayed against E. coli ATCC 25922. Activity was calculated relative to ampicillin controls (100 μg / mL). Error bars represent +/− 1 standard deviation away from the mean values.

Figure 6.

Biological characterization of CM-AMP1 against E. coli. A) Volcano plot visualizing global proteomic changes in the E. coli proteome following treatment with 5 μg / mL linear reduced CM-AMP1. Vertical lines represent the cutoff used to determine significant proteomic changes (|log₂(FC)| > 1). Horizontal lines represent the threshold used to determine significant false discovery rates determined by an FDR-corrected two-sided t-test (-log₁₀(q-value) > 1.3). B) Outer membrane permeabilization assay with NPN after treatment with synthetic peptide. % NPN uptake is calculated relative to the positive (Polymyxin B, PmB) and negative (H₂O + 0.1% DMSO) controls.