Journal: Plant Biotechnology Journal

Article Title: High‐yield, plant‐based production of an antimicrobial peptide with potent activity in a mouse model

doi: 10.1111/pbi.14460

Figure Lengend Snippet: Expression of AMP1 for apoplast targeting in N. benthamiana . (a) Schematic representation of the AMP1 expression cassette designed for apoplast sequestration using the backbone of the pMDC43 vector. e35S, enhancer region of 35S promoter; 35S, cauliflower mosaic virus 35S promoter; a synthetic 5′UTR namely the 5S0 [a synthetic untranslated region (UTR)‐appended at the 5′ designated with the number ‘0’] and 3′CPMV ( Cowpea mosaic virus UTR); PS, signal sequence sourced from phaseolin from Phaseolus vulgaris for apoplast secretion; bdSUMO Eu1 , mutated SUMO domain; HA, hemagglutinin epitope; nos , nopaline synthase terminator. (b) Analysis of apoplast wash fluid with SDS–PAGE. The apoplast wash fluid was purified using Streptactin beads, separated on SDS–PAGE, and gels were stained with Coomassie Brilliant blue. (c) Immunoblot analysis of PS‐SUMO‐AMP1 in apoplast wash fluid. Separated proteins were transferred to a polyvinylidene difluoride membrane (PVDF) and probed with a monoclonal rat anti‐HA antibody (1:1000) and goat anti‐rat IgG (1:4000) to detect PS‐bdSUMO Eu1 ‐AMP1 (∼64.9 kDa). (d) Reversed‐phase liquid chromatography separation of peptides. The lyophilized extract, after SUMO protease cleavage, was separated on a C8 column for a 60‐min run reaction. (e) Tricine–SDS–PAGE analysis of peptide. Peptide separated using RP‐HPLC (dual absorbance at 215/280 nm) was analysed on 12% tricine‐SDS–PAGE and stained with Coomassie Brilliant blue solution containing 40% methanol and 4% formaldehyde. (f) Mass spectrometry sequencing of peptide. The MS/MS spectrum of plant‐purified AMP1 and the amino acid sequence of the peptide is shown with observed b ions (represent product when the charge is retained on the N‐terminus) and y ions (represent product when the charge is retained on the C‐terminus) mapped.

Article Snippet: As controls, the vehicle 5% dextrose (Sigma Aldrich, Cat no. DX0145), synthetic AMP1 (purchased from a commercial source GenScript) and Vancomycin hydrochloride (Sigma Aldrich, Cat no. PHR1732) were used.

Techniques: Expressing, Plasmid Preparation, Virus, Sequencing, SDS Page, Purification, Staining, Western Blot, Membrane, Reversed-phase Chromatography, Mass Spectrometry, Tandem Mass Spectroscopy

Journal: Plant Biotechnology Journal

Article Title: High‐yield, plant‐based production of an antimicrobial peptide with potent activity in a mouse model

doi: 10.1111/pbi.14460

Figure Lengend Snippet: Antibacterial activity of plant‐purified AMP1. (a and b) Bacterial growth inhibition assay in the presence of plant‐purified and synthetic AMP1. Nearly 10 6 cells colony‐forming units (CFU)/mL of E. coli PI7 (grown in LB containing 8 μg mL −1 meropenem) and MRSA USA300 (grown in tryptic soy broth containing 10 μg mL −1 chloramphenicol) were treated with 100–1.56 μg mL −1 of both plant‐purified and synthetic AMP1 in cation‐adjusted Mueller‐Hinton broth for 24 h. Bacterial growth inhibition was measured at OD 600 using a TECAN Infinite 200 PRO series. Two independent experiments were performed in triplicate, and the data are represented as mean ± SD. (c and d) NPN assay for outer‐membrane permeabilization of E. coli PI7 and MRSA USA300 induced by synthetic and plant‐purified AMPs. Profiles show a rapid increase in fluorescence emission intensity, followed by a slow decay. Measurements were conducted on a white, 96‐well plate using a TECAN Infinite 200 PRO series with excitation and emission wavelengths set to 340 and 405 nm, respectively, following the experimental procedure outlined in the Materials and Methods. Two independent experiments were performed in triplicate, and the data are represented as mean ± SD.

Article Snippet: As controls, the vehicle 5% dextrose (Sigma Aldrich, Cat no. DX0145), synthetic AMP1 (purchased from a commercial source GenScript) and Vancomycin hydrochloride (Sigma Aldrich, Cat no. PHR1732) were used.

Techniques: Activity Assay, Purification, Growth Inhibition Assay, Inhibition, NPN Assay, Membrane, Fluorescence

Journal: Plant Biotechnology Journal

Article Title: High‐yield, plant‐based production of an antimicrobial peptide with potent activity in a mouse model

doi: 10.1111/pbi.14460

Figure Lengend Snippet: Biocompatibility assessment of purified AMP1 on human dermal fibroblasts in terms of HDF cell viability, proliferation and metabolic activity. (a and b) Live/dead fluorescence staining of human dermal fibroblasts (HDFs) at 12 (a) and 24 h (b) post‐treatment with peptide concentrations of 30 and 50 μg mL −1 . Untreated HDFs were used as a control. Live cells show green fluorescence, while dead cells have red fluorescence. Scale bars, 100 μm. (c) The metabolic index of treated and untreated HDFs measured by directly estimating the ATP levels using a luciferase‐based CellTiter‐Glo reagent. Statistical significance was calculated using a standard two‐tailed paired t‐test ( n = 3 biological replicates). (d) Confocal microscopy images of HDFs after cytoskeleton staining of actin fibres (red) and nuclei (blue) at 12 h. Scale bars, 100 μm. (e) The organization of F‐Actin stress fibres within the HDF cells at 24 h. Scale bars, 50 μm. (f) Close‐ups of F‐Actin stress fibres within untreated and treated HDF cells at 24 h of treatment. Scale bars, 20 μm. Three independent microscopy experiments were performed with similar results. (g) Analysis of the purified peptide sample using polymerase chain reaction (PCR) to identify the presence of any bacterial‐specific 16S rRNA sequence. Positive controls consisted of whole genomic DNA from E. coli MG1655 and A. tumefaciens GV3101. The labelling is as follows: M for 1 kb Plus ladder, S for peptide sample and NTC for negative control.

Article Snippet: As controls, the vehicle 5% dextrose (Sigma Aldrich, Cat no. DX0145), synthetic AMP1 (purchased from a commercial source GenScript) and Vancomycin hydrochloride (Sigma Aldrich, Cat no. PHR1732) were used.

Techniques: Purification, Activity Assay, Fluorescence, Staining, Control, Luciferase, Two Tailed Test, Confocal Microscopy, Microscopy, Polymerase Chain Reaction, Sequencing, Negative Control

Journal: Plant Biotechnology Journal

Article Title: High‐yield, plant‐based production of an antimicrobial peptide with potent activity in a mouse model

doi: 10.1111/pbi.14460

Figure Lengend Snippet: Anti‐bacterial activity of purified AMP1 against a bacterial pathogen in a mouse infection model. (a) Schematic representation of the skin abscess model used to assess the anti‐bacterial activity of purified AMP1 ( n = 6 vehicle‐treated group; n = 6 purified AMP1‐treated group; n = 6 synthetic AMP1‐treated group and n = 6 vancomycin hydrochloride‐treated group). (b) Weight of the mice was consistently monitored over the course of the 1‐, 5‐ and 8‐day experiments to eliminate the possibility of any potential toxic effects from the purified peptide. Statistical analysis was performed using a standard two‐tailed paired t ‐test. (c) Representative IVIS images of the whole body of mice acquired 72 h after infection with luminescent MRSA tagged with the lux operon, using an IVIS Spectrum imaging system. For the treated group, the purified peptide was injected 60 min after bacterial inoculation. Synthetic AMP1 at a dose of 7 mg Kg −1 and vancomycin hydrochloride at a dose of 150 mg Kg −1 were used as controls. (d) Quantification of luminescence signal expressed as total flux (photons/s) in the region of interest (ROI) of bacterial load of the mice. The final ROI was calculated by subtracting the average background ROI from the measurement ROI. (e) Treatment with the peptide leads to decreased MRSA USA300 bacterial counts compared with the control group (not treated with the peptide). Statistical significance in d and e was calculated using a one‐way ANOVA (Tukey's multiple comparison tests), * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: As controls, the vehicle 5% dextrose (Sigma Aldrich, Cat no. DX0145), synthetic AMP1 (purchased from a commercial source GenScript) and Vancomycin hydrochloride (Sigma Aldrich, Cat no. PHR1732) were used.

Techniques: Activity Assay, Purification, Infection, Two Tailed Test, Imaging, Injection, Control, Comparison

Journal: Plant Biotechnology Journal

Article Title: High‐yield, plant‐based production of an antimicrobial peptide with potent activity in a mouse model

doi: 10.1111/pbi.14460

Figure Lengend Snippet: Summary of upstream and downstream production costs

Article Snippet: As controls, the vehicle 5% dextrose (Sigma Aldrich, Cat no. DX0145), synthetic AMP1 (purchased from a commercial source GenScript) and Vancomycin hydrochloride (Sigma Aldrich, Cat no. PHR1732) were used.

Techniques: