Viola “inconspicua” No More: An Analysis of Antibacterial Cyclotides

Abstract

The emergence of rapidly evolving multidrug-resistant pathogens and a deficit of new compounds entering the clinical pipeline necessitate the exploration of alternative sources of antimicrobial therapeutics. Cyclotides revealed in Viola spp. are a class of highly stable, cyclic, and disulfidebound peptides with diverse intrinsic bioactivities. Herein we have identified a novel complement of 42 putative cyclotide masses in the plant species Viola inconspicua. Cyclotide-containing fractions of a V. inconspicua peptide library revealed potent bioactivities against the Gramnegative bacteria Escherichia coli ATCC 25922 and the highly virulent and multidrug-resistant Klebsiella pneumoniae VK148. As such, six previously uncharacterized cyclotides, cycloviolacins I1-6 (cyI1-cyI6), were prioritized for molecular characterization. Cyclotides cyI3-cyI6 contain a novel "TLNGNPGA" motif in the highly variable loop six region, expanding the already substantial sequence diversity of this peptide class. Library fractions comprised of cyclotides cyI3-cyI6 exhibited MIC values of 18 and 35 μM against E. coli and K. pneumoniae, respectively, whereas isolated cyI3 killed ~50% of E. coli at 60 μM and isolated cyI4 demonstrated no killing at concentrations >60 μM against both pathogens. This work expands the repertoire of bioactive cyclotides found in Viola spp. and highlights the potential of these antibacterial cyclic peptides.

Implementation of Microfluidics for Antimicrobial Susceptibility Assays: Issues and Optimization Requirements

“…Cyclotide-containing peptide libraries consistently demonstrate potent antibacterial activity in 96-well plate assay format (Parsley et al, 2019 ), and were thus utilized for comparison analyses of plate-based and microfluidics activity assays. V. inconspicua library fractions 34–39 were analyzed for peptidyl constituents via LC-MS and chosen for the high intensity UV traces and late retention time characteristic of cyclotide species.…”

“…V. inconspicua library fractions 34-39 were analyzed for peptidyl constituents via LC-MS and chosen for the high intensity UV traces and late retention time characteristic of cyclotide species. Mass spectral (MS1) analysis revealed that all library fractions contain masses corresponding to cyclotides or putative cyclotide species previously identified in V. inconspicua (Figure 2) (Parsley et al, 2019). Highly abundant masses present in the V. inconspicua library fractions are consistent with the masses of the fully characterized cyclotides cyO8 (fractions 38-39), cyO9 (fractions 35-38), viba 11 (fractions 35-36), cyI1 (fractions 35-36), cyI2 (fractions 38-39), and cyO8/cyO9 oxidized variants (fractions 34/38).…”

“…Plant material was extracted and peptide library prepared as previously described (Kirkpatrick et al, 2017 ; Parsley et al, 2019 ) ( Figure 1 ). Briefly, V. inconspicua leaf tissue was harvested, ground under liquid nitrogen, and extracted into 10% acetic acid (1:3 w/v, 4 h, 4 °C, with stirring).…”

“…To address this gap, PepSAVI-MS was developed to identify low abundance bioactive peptides in complex natural product extracts (Kirkpatrick et al, 2017 , 2018a , b ; Moyer et al, 2019 ; Parsley et al, 2019 ). PepSAVI-MS is a top-down peptidomics approach that leverages modern mass spectrometry and relies on traditional bioassays for bioactivity characterization (e.g., 96-well plate or disk-diffusion).…”

“…Herein, a microfluidics-based bioactivity assay is compared with that of a traditional bulk 96-well plate in order to assess its potential to elevate PepSAVI-MS AMP identification and biological characterization. A natural product peptide library with previously reported antibacterial bioactivity (Parsley et al, 2019 ) was generated from the cyclotide-expressing botanical species Viola inconspicua and screened against Escherichia coli ATCC 25922. Bioactivity is determined through a polymethylmethacrylate (PMMA) microfluidic platform coupled with confocal brightfield microscopy and compared to that of polypropylene (PP) and polystyrene (PS) plate-based assays using both optical density and resazurin-based fluorescence measurements.…”