In Vivo and In Vitro Interactions between Exopolysaccharides from Bacillus thuringensis HD270 and Vip3Aa11 Protein

Abstract

Bacillus thuringiensis (Bt) secretes the nutritional insecticidal protein Vip3Aa11, which exhibits high toxicity against the fall armyworm (Spodoptera frugiperda). The Bt HD270 extracellular polysaccharide (EPS) enhances the toxicity of Vip3Aa11 protoxin against S. frugiperda by enhancing the attachment of brush border membrane vesicles (BBMVs). However, how EPS-HD270 interacts with Vip3Aa11 protoxin in vivo and the effect of EPS-HD270 on the toxicity of activated Vip3Aa11 toxin are not yet clear. Our results indicated that there is an interaction between mannose, a monosaccharide that composes EPS-HD270, and Vip3Aa11 protoxin, with a dissociation constant of Kd = 16.75 ± 0.95 mmol/L. When EPS-HD270 and Vip3Aa11 protoxin were simultaneously fed to third-instar larvae, laser confocal microscopy observations revealed the co-localization of the two compounds near the midgut wall, which aggravated the damage to BBMVs. EPS-HD270 did not have a synergistic insecticidal effect on the activated Vip3Aa11 protein against S. frugiperda. The activated Vip3Aa11 toxin demonstrated a significantly reduced binding capacity (548.73 ± 82.87 nmol/L) towards EPS-HD270 in comparison to the protoxin (34.96 ± 9.00 nmol/L). Furthermore, this activation diminished the affinity of EPS-HD270 for BBMVs. This study provides important evidence for further elucidating the synergistic insecticidal mechanism between extracellular polysaccharides and Vip3Aa11 protein both in vivo and in vitro.

Keywords: Bacillus thuringiensis; Spodoptera frugiperda; Vip3Aa11; extracellular polysaccharides.

Figure 1

Purification results of EPS-HD270. (A) The purification result of EPS-HD270 by anion exchange. (B) The purification result of EPS-HD270 by gel filtration chromatography.

Figure 2

Analysis of the purification and activation results of Vip3Aa11 protoxin by SDS-PAGE. M: Marker 26614; (A) 1: Vip3Aa11 soluble expression component; 2: Vip3Aa11 purification process flow through the liquid; 3: binding buffer; 4: elution buffer; and 5: Vip3Aa11 purification result. (B) Y: Vip3Aa11 protoxin; H: the Vip3Aa11 trypsin activation result; and C: the purification result of the activated protein using a Q column and a desalting column.

Figure 3

Laser confocal observation of midgut injury and fluorescence signal localization in S. frugiperda induced by Vip3Aa11 prototoxin and EPS-HD270; (A) PBS buffer feeding control group; (D) EPS-HD270 feeding treatment group; (B,E) Vip3Aa11 protoxin was fed in the treatment group (the white box is the damaged part of intestinal wall cells); (C,F) the mixture of Vip3Aa11 protoxin and EPS-HD270 was fed in the treatment group (the yellow box line shows the damaged part of intestinal wall cells and the co-localization part of fluorescent signal; the yellow arrows represent regions where fluorescence signal of EPS co-localizes with the Vip3Aa11 protein); (G–I) the fluorescence images of the mixture of Vip3Aa11 protoxin and EPS-HD270 under different excitation light; (G) the green fluorescence of ConA stained EPS-HD270; (H) the orange fluorescence of Alexa Fluor 555-labeled Vip3Aa11 protoxin; and (I) the merged mixed images of each channel.

Figure 4

Determination of Vip3Aa11 and carbohydrate binding by isothermal titration calorimetry. ITC reaction curves of Vip3Aa11 protoxin with mannose (A), galactose (B), glucose (C), and glucosamine (D).

Figure 5

Effects of EPS-HD270 on insecticidal activity of Vip3Aa11 protoxin (A) and the activated Vip3Aa11 toxin (B) against S. frugiperda (7 days). The presented data represent the mean ± standard deviation derived from three separate experiments. Statistical significance is indicated by asterisks (** p < 0.01 and * p < 0.05).

Figure 6

ELISA analyzed the interaction between EPS-HD270 and Vip3Aa11 protoxin (A) and the activated Vip3Aa11 toxin (B).

Figure 7

Influence of EPS-HD270 on the interaction of the activated Vip3Aa11 toxin with BBMVs of S. frugiperda. M: Marker 26616. (A) The binding analysis of Vip3Aa11 toxin and BBMVs, and CK⁺ shows the saturated bound Vip3Aa11 protoxin and BBMVs. (B) The impact of EPS-HD270 on the interaction between the activated Vip3Aa11 toxin and BBMVs. Furthermore, 1:0, 1:10, 1:50 and 1:100 are the mass ratios of activated Vip3Aa11 toxin to EPS-HD270.