Cell Membrane-Interrupting Antimicrobial Peptides from Isatis indigotica Fortune Isolated by a Bacillus subtilis Expression System

Abstract

The situation of drug resistance has become more complicated due to the scarcity of plant resistance genes, and overcoming this challenge is imperative. Isatis indigotica has been used for the treatment of wounds, viral infections, and inflammation for centuries. Antimicrobial peptides (AMPs) are found in all classes of life ranging from prokaryotes to eukaryotes. To identify AMPs, I. indigotica was explored using a novel, sensitive, and high-throughput Bacillus subtilis screening system. We found that IiR515 and IiR915 exhibited significant antimicrobial activities against a variety of bacterial (Xanthomonas oryzae, Ralstonia solanacearum, Clavibacter michiganensis, and C. fangii) and fungal (Phytophthora capsici and Botrytis cinerea) pathogens. Scanning electron microscope and cytometric analysis revealed the possible mechanism of these peptides, which was to target and disrupt the bacterial cell membrane. This model was also supported by membrane fluidity and electrical potential analyses. Hemolytic activity assays revealed that these peptides may act as a potential source for clinical medicine development. In conclusion, the plant-derived novel AMPs IiR515 and IiR915 are effective biocontrol agents and can be used as raw materials in the drug discovery field.

Prediction and Characterization of Cationic Arginine-Rich Plant Antimicrobial Peptide SM-985 From Teosinte (Zea mays ssp. mexicana)

“…However, in recent years, many studies have used the cDNA library method to clone AMPs (Ronèeviae et al, 2018;Wu et al, 2019;Tavares et al, 2020) due to its advantages such as enhanced fragments of efficiently transcribed genes and cloned AMPs from cDNA library, which can be actively manufactured via bacterial expression systems (Sharma et al, 2014) due to the lack of introns that might pose a problem when the target is manufactured as a eukaryotic protein in bacteria. In our lab, we screened cDNA libraries for AMPs via expression systems (Kong et al, 2018;Wu et al, 2020), but this is also a time-and cost-consuming approach. In silico prediction is a time-and cost-effective approach for large-scale screening and detection of AMPs (Liu et al, 2017).…”

Two antimicrobial genes from Aegilops tauschii Cosson identified by the Bacillus subtilis expression system

“…As a pathogen indicator, B. subtilis cells can be used to screen for antimicrobial peptides. Although there is a limitation that some strong AMPs can kill B. subtilis cells too quickly to detect the desired genes, the B. subtilis screening strategy is able to isolate and clone for broad-spectrum antimicrobial peptides 54 . By screening cDNA libraries and observing autolyzed clones, it is feasible to rapidly identify resistance genes.…”

Isolation of Antimicrobial Genes from Oryza rufipogon Griff by Using a Bacillus subtilis Expression System with Potential Antimicrobial Activities

“…As with the previously mentioned method [ 27 , 43 ], initial screening and confirmation were carried out. The plasmid population was extracted and transferred into B. subtilis SCK6.…”

“…In our lab, using a B. subtilis expression system, we introduced a new method for isolating resistance genes [ 27 , 43 ]. This approach is efficient against ‘superbug’ bacteria and different plant microorganisms for antimicrobial gene isolation.…”