Abstract
15-Acetyldeoxynivalenol (15-AcDON) is a low molecular weight sesquiterpenoid trichothecene mycotoxin associated with Fusarium ear rot of maize and Fusarium head blight of small grain cereals. The accumulation of mycotoxins such as deoxynivalenol (DON) and 15-AcDON within harvested grain is subject to stringent regulation as both toxins pose dietary health risks to humans and animals. These toxins inhibit peptidyltransferase activity, which in turn limits eukaryotic protein synthesis. To assess the ability of intracellular antibodies (intrabodies) to modulate mycotoxin-specific cytotoxocity, a gene encoding a camelid single domain antibody fragment (V(H)H) with specificity and affinity for 15-AcDON was expressed in the methylotropic yeast Pichia pastoris. Cytotoxicity and V(H)H immunomodulation were assessed by continuous measurement of cellular growth. At equivalent doses, 15-AcDON was significantly more toxic to wild-type P. pastoris than was DON. In turn, DON was orders of magnitude more toxic than 3-acetyldeoxynivalenol. Intracellular expression of a mycotoxin-specific V(H)H within P. pastoris conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging from 20 to 100 mug.ml(-1). We also documented a biochemical transformation of DON to 15-AcDON to account for the attenuation of DON cytotoxicity at 100 and 200 mug.ml(-1). The proof of concept established within this eukaryotic system suggests that in planta V(H)H expression may lead to enhanced tolerance to mycotoxins and thereby limit Fusarium infection of commercial agricultural crops.
Highlights
A toxin class commonly found within agricultural commodities infected by Fusarium are trichothecene mycotoxins
Regardless of size and structural composition, trichothecenes are potent inhibitors of eukaryotic protein synthesis with specific activity on ribosomal protein L3 within the 60 S subunit resulting in inhibition of peptidyltransferase activity [3, 4]
It was not possible to establish a dose-specific sensitivity to 3-AcDON relative to toxin-free DMSO media as cellular growth was not inhibited at the highest concentration (200 g1⁄7mlϪ1) (Table 1)
Summary
VHH Genes—NAT-267 VHH DNA sequence (GenBankTM EU676170.1) [18] was used to design a P. pastoris codon optimized version of the gene to which was added 3Ј HA and His epitope tags, as well as EcoRI- and XbaI-cloning sites at the respective 5Ј and 3Ј ends (GeneArtTM, Toronto, Ontario, Canada). Mean (n ϭ 3) A620 values for VHH and pPICZB (empty vector) transformants used in ribotoxin and control treatments, minus A 620 values of YPD media (blank) wells, were plotted against time (i.e. 25-min intervals). ⌬AUC values for each toxophore treatment were calculated based on addition of differences in cellular growth between VHH and control transformants at each 25-min time interval across the full time course of each assay. To decrease the bias of Western blot band intensities, VHH standard dilution series was prepared in P. pastoris cell lysate from pPICZB empty vector sample. 1:50 dilutions of NAT-267 or B-24 VHH samples were expressed as VHH mass per l of P. pastoris cell pellet (ng1⁄7lϪ1) based on the following: VHH quantity (nanogram) (from standard regression equation) divided by cell pellet lysis volume loaded into each well (microliter). Standards and samples were eluted at a flow rate of 1.0 ml1⁄7minϪ1 with a 20-min
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
