ABSTRACT
Antibodies are a useful tool for assistance to map the binding epitopes in Bacillus thuringiensis Cry toxins and their receptors, and even determine how receptors promote toxicity. In this work, a monoclonal antibody (mAb-1D2) was produced by the hybridoma cell line raised against Cry2Aa toxins, with a half inhibition concentration (IC50) of 9.16 µg/mL. The affinity constant of two recombinant toxin-binding fragments derived from Helicoverpa armigera and Plutella xylostella cadherin-like protein (HaCad-TBR or PxCad-TBR) to Cry2Aa toxin was measured to be 1.21 µM and 1.24 µM, respectively. Competitive ELISA showed that mAb-1D2 competed with HaCad-TBR or PxCad-TBR binding to Cry2Aa. Meanwhile, the toxicity of the Cry2Aa toxin to the H. armigera and P. xylostella larvae were greatly reduced when the toxin was mixed with mAb-1D2, which indicated that cadherin may play an important functional role in the toxicity of Cry2Aa. After transforming mAb-1D2 to a single-chain variable fragment (scFv), the hot spot residues of Cry2Aa with 1D2-scFv, PxCad-TBR, and HaCad-TBR were analyzed by molecular docking. It was demonstrated that the hot spot residues of Cry2Aa involving with 1D2-scFv interaction were mainly in Domain II, and some residues in Domain I. Moreover, mAb-1D2 and the two cadherin fragments shared the common hot spot residues on Cry2Aa, which could explain mAb-1D2 inhibited Cry2Aa binding with cadherin fragments. This monoclonal antibody could be a useful tool for identifying the binding epitopes between Cry2Aa and cadherin, and even assist to analyze the roles of cadherin in Cry2Aa toxicity.
Subject(s)
Bacillus thuringiensis , Moths , Animals , Bacillus thuringiensis/metabolism , Endotoxins/toxicity , Endotoxins/metabolism , Cadherins/chemistry , Cadherins/metabolism , Antibodies, Monoclonal , Epitopes/analysis , Epitopes/chemistry , Epitopes/metabolism , Molecular Docking Simulation , Bacillus thuringiensis Toxins/metabolism , Larva , Hemolysin Proteins/toxicity , Hemolysin Proteins/chemistry , Hemolysin Proteins/metabolism , Bacterial Proteins/chemistryABSTRACT
In this study, we investigated the effects of a symbiotic bacterium and phosphate (PO4(3-)) nutrition on the toxicity and metabolism of arsenate (As(V)) in Dunaliella salina. The bacterium was identified as Alteromonas macleodii based on analysis of its 16S rRNA gene sequence. When no As(V) was added, A. macleodii significantly enhanced the growth of D. salina, irrespective of PO4(3-) nutrition levels, but this effect was reversed after As(V)+PO4(3-) treatment (1.12mgL(-1)) for 3 days. Arsenic (As) absorption by the non-axenic D. salina was significantly higher than that by its axenic counterpart during incubation with 1.12mgL(-1) PO4(3-). However, when the culture was treated with 0.112mgL(-1) PO4(3-), As(V) reduction and its subsequent arsenite (As(III)) excretion by non-axenic D. salina were remarkably enhanced, which, in turn, contributed to lower As absorption in non-axenic algal cells from days 7 to 9. Moreover, dimethylarsinic acid was synthesized by D. salina alone, and the rates of its production and excretion were accelerated when the PO4(3-) concentration was 0.112mgL(-1). Our data demonstrate that A. macleodii strongly affected As toxicity, uptake, and speciation in D. salina, and these impacts were mediated by PO4(3-) in the cultures.
