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.

Keywords: Bacillus subtilis; Isatis indigotica; antimicrobial peptide; gene expression system; membrane-interrupting peptides; plant resistance gene.

Figure 1

Damaging effect of foreign proteins on host Bacillus subtilis cells. The engineered B. subtilis strains harboring IiR515, IiR915, and empty vector were separately spotted onto Luria-Bertani (LB) plates and incubated at 37 °C. (a) IiR515 after 12 h of incubation and (b) after 36 h of incubation; (c) IiR915 after 12 h of incubation and (d) after 36 h of incubation. The empty vector-transformed B. subtilis strain (WB800-e) was used as a control. Spots on the right represent the test clones with damaging effects and spots on the left represent the control.

Figure 2

Cell membrane disruptions of transformed B. subtilis cells. Bacillus pellets were fixed with glutaraldehyde after washing with PBS buffer (phosphate buffer saline). Finally, the samples were lyophilized and then gold coated. (a) Cellular disruption of IiR515, IiR915, and WB800-e (empty vector-transformed B. subtilis WB800) under the scanning electron microscope. Test bacteria were washed and resuspended in PBS buffer at 1 × 10⁹ CFU/mL and stained with PI (40 mg/mL). Confocal images under (b) ordinary light and (c) fluorescence. (d) Percentages of fluorescent events (relative value of PI staining) in gate shown in the region on the right. The x-axis shows the relative fluorescence intensity and the y-axis shows the side scatter light.

Figure 3

Fluorescence intensity and nucleic acid release of B. subtilis cells. The fluidity and electrical potential of the cytoplasmic membrane of B. subtilis were measured. Excitation and emission wavelengths for (a) The DPH were 365 nm and 425 nm and for (b) DiSC₃(5) were 622 nm and 670 nm, respectively. The WB800-e strain was used as a control. The detection of released DNA and RNA was also performed. The empty vector was used as a blank control. (c) The concentration of DNA and (d) RNA in shaking media at different time intervals. Data are the mean values from three individual experiments. Vertical bars represent the SD (Standard deviation). For significance analysis, t-tests were performed; * p < 0.05, ** p < 0.01.

Figure 4

Analysis of potential antimicrobial peptides (AMPs) against pathogens. For antimicrobial activity assays, the B. subtilis WB800-e strain was used as a control. (a) Inhibition of Gram-positive and Gram-negative bacteria in response to IiR515 and IiR915 compared to WB800-e. (b) Percent inhibition of IiR515 and IiR915 against Botrytis cinerea compared to WB800-e. Temperature curves for (c) Clavibacter fangii and (d) Xanthomonas oryzae. Data are the mean values from three individual experiments. Vertical bars represent the SD. For significance analysis, t-tests were performed; * p < 0.05, ** p < 0.01.

Figure 5

Western blot analysis of fusion peptides.

Figure 6

Percent inhibition of Phytophthora capsici and Botrytis cinerea on Nicotiana benthamiana leaves. Images of P. capsici disease inhibition (a) under normal light and (b) ultraviolet light (365 nm). (c) Percent inhibition compared to the control. (d) Images of B. cinerea inhibition and (e) percent inhibition compared with the control. The y-axis shows the logarithmic values of the bacterial population (log CFU/10 g of soil). The inhibitory potential of (f) the IiR515 strain and (g) the IiR915 strain against C. fangii. Inhibitory potential of (h) the IiR515 strain and (i) the IiR915 strain against C. michiganensis compared to the control WB800-e strain. Data are the mean values from three individual experiments. Vertical bars represent the SD. For significance analysis, t-tests were performed; * p < 0.05, ** p < 0.01.