PepSAVI-MS Reveals a Proline-rich Antimicrobial Peptide in Amaranthus tricolor

Abstract

Traditional medicinal plants are a rich source of antimicrobials; however, the bioactive peptide constituents of most ethnobotanical species remain largely unexplored. Herein, PepSAVI-MS, a mass spectrometry-based peptidomics pipeline, was implemented for antimicrobial peptide (AMP) discovery in the medicinal plant Amaranthus tricolor. This investigation revealed a novel 1.7 kDa AMP with strong activity against Escherichia coli ATCC 25922, deemed Atr-AMP1. Initial efforts to determine the sequence of Atr-AMP1 utilized chemical derivatization and enzymatic digestion to provide information about specific residues and post-translational modifications. EThcD (electron-transfer/higher-energy collision dissociation) produced extensive backbone fragmentation and facilitated de novo sequencing, the results of which were consistent with orthogonal characterization experiments. Additionally, multistage HCD (higher-energy collisional dissociation) facilitated discrimination between isobaric leucine and isoleucine. These results revealed a positively charged proline-rich peptide present in a heterogeneous population of multiple peptidoforms, possessing several post-translational modifications including a disulfide bond, methionine oxidation, and proline hydroxylation. Additional bioactivity screening of a simplified fraction containing Atr-AMP1 revealed activity against Staphylococcus aureus LAC, demonstrating activity against both a Gram-negative and a Gram-positive bacterial species unlike many known short chain proline-rich antimicrobial peptides.

Antimicrobial peptides: mechanism of action, activity and clinical potential

“…This is because that: (1) as a component of the innate immune system, production of AMPs by the host cells requires less time and energy than antibody synthesis by the acquired immunity; (2) these small molecule peptides can reach the target faster than immunoglobulins; and (3) some eukaryotes lack of lymphocyte-based immune system, such as insects mainly rely on the synthesis of a series of antibacterial compounds to remove invading microorganisms [ 16 ]. Since the first AMP was discovered in the American silkworm chrysalis, a large number of AMPs have been widely found in various organisms, including microorganisms [ 17 ], plants [ 18 ], invertebrates [ 19 ], fish [ 20 ], amphibians [ 21 ], reptiles [ 22 ], birds [ 23 ] and mammals [ 24 ].…”

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

“…V. inconspicua library fraction activity was assessed with a simple 8 straight-channel microfluidic chip ( Figure 1 ) to explore the merits of a microfluidic bioassay format. Whereas traditional “bulk” assays (e.g., 96-well plate) often require a minimum of 50 μL of sample mixture in each well (Sarker et al, 2007 ; Teh et al, 2017 ; Fowler et al, 2018 ) (10 μL extract, 40 μL of media and cells for each replicate) (Kirkpatrick et al, 2017 ; Parsley et al, 2018 , 2019 ; Moyer et al, 2019 ), microfluidic devices utilize sample sizes in the nanoliter/picoliter range. However, connectors and interfaces between microfluidic channels/chambers and injection sites can generate significant dead volume, requiring excess sample to ensure complete filling of microfluidic compartments.…”

“…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).…”

Proteomic response of Escherichia coli to a membrane lytic and iron chelating truncated Amaranthus tricolor defensin

“…Defensins previously predicted from the 1000 Plant Project transcriptome (1000 Plant Project sample ID: XSSD) [ 49 , 50 ] of Amaranthus tricolor using Cysmotif Searcher ( https://github.com/fallandar/cysmotifsearcher ) [ 21 , 51 ] were aligned with ClustalOmega (1.2.4) [ 52 ]. The synthesis of Atr-DEF2(G39-C54) was accomplished using a semi automated flow chemistry instrument built in-house [ 53 , 54 ]. In brief, synthesis was performed using 200 mg of Fmoc-Cys (Trt)-2-chlorotrityl resin (0.77 mmol/g, 200–400 mesh).…”

“…E. coli 25922 and K. pneumoniae VK148 assays were conducted in triplicate as previously described [ 54 ]. Atr-DEF2(G39-C54) is prone in precipitation when added to bacterial culture.…”