Fig 1.
Bacillus thuringiensis Vpb4Da2 crystal structure.
(A) Crystal structure of Vpb4Da2 structure and domains labeled D1 to D6. Predicted pore-forming loop is in pale green and calcium ions are orange spheres. (B) Topology diagrams of Vpb4Da2 domain 4 (left) and domain 6 (center) versus PA domain 4 from PDB ID Acc1 (right). (C) Vpb4Da2 domain 4 (left) versus Cry3Bb1 domain 3 from PDB ID 1ji6 (center) and Clostridium thermocellum xylanase carbohydrate binding module from PDB ID 1gmm (right). (D) Vpb4Da2 domains 5 (left) and 6 (center) versus PA domain 4 (right).
Fig 2.
Structural conservation of Vpb4Da2 domains and its evolutionary relationship to Bacterial_exotoxin_B homologs.
(A) Mapping amino acid sequence conservations and variabilities from multiple sequence alignments of Vpb4Da2 homologs onto the surface structure of Vpb4Da2. The degree of conservation is color-coded (high conservation = blue; low conservation = red). Higher sequence variability occurs within domains 4–6, whereas more sequence conservation is found in domains 1–3. (B) Phylogenetic tree of relative relationships between Bacterial_exotoxin_B homologs. Tip annotations show protein and global percent identity relative to PA precursor (AAA22637.1). Sequences used in this analysis include Vpb4Da2 (AZJ95709.1), Vpb4C.6693 (this study), Vpb4Aa1 (AEB52299.1), Vpb4Ba1 (OUB778819.1), CDTb (AUA37847.1), Iota toxin component Ib (CAA51960.1), C2 toxin component-II (BAA32537.1), Vpb1Ac1 (AEH05932.1), Vpb1Ba1 (AAR40886.1), Vpb1Ca1 (AAO86514.1), Vpb1Da1 (CAI40767.1), and Protective antigen precursor (AAA22637.1).
Fig 3.
Structural comparison of Vpb4Da2 domains to corresponding domains of the Protective Antigen (PA).
(A) Unique to Vpb4Da2, when compared to PA (PDB ID Acc1), are additional domains 5–6 found at the carboxyl-terminus. Moreover, Vpb4Da2 domain 4 displays a distinct topology. (B) Domains 1–3 of both proteins exhibit similar structural organization. The receptor binding interface and pore forming loop of PA are indicated.
Fig 4.
Vpb4Da2 processing by proteases occurs within domain 1.
(A) Schematic representation of Vpb4Da2 domains and protease cleavage sites. Yellow and white arrowheads are gut fluid and trypsin cleavage sites, respectively. Cleavage sites at the amino terminal end, within domain 1 are indicated by clear vertical lines. (B) Surface rendition of Vpb4Da2 structure showing proteolytic cleavage sites (yellow spheres). (C) Analytical size-exclusion chromatography (SEC) profiles of the full-length (dark blue trace) and the trypsin-processed (light blue trace) Vpb4Da2. Full-length (105.9 kDa) and processed (85.3 kDa and 19.7 kDa complex) protein samples exhibit approximately the same retention time when ran separately through an SEC column. The calibration curve is shown in the inset and the experimentally determined molecular weights of full-length and processed Vpb4Da2 are indicted by dark blue and light blue squares, respectively. (D) The SDS-PAGE profile of the elution peaks from the full-length Vpb4Da2 (lane 1) and processed (lane 2) samples, along with the molecular size of the eluted protein bands, are shown. The SDS-PAGE profile of the elution peak at approximately 42 min designated “Peptides” in (C) and derived from processed Vpb4Da2 ran through the SEC column is shown in lane 3. No apparent peptides of molecular weights higher than 10 kDa were observed (lane 3), suggesting that peptides are very small breakdown products of the proteolytic reaction.
Fig 5.
Schematic representation of Vpb4Da2 and Vpb4C.6693 domain-swap and truncation variants.
Domains and putative pore-forming loop (L) are labeled and color coded.
Table 1.
Insecticidal activity of Vpb4Da2, Vpb4C.6693, and corresponding chimeras and deletions.
Fig 6.
Competition bioassay evaluation using the pore-forming disabled variant.
(A) Structural representation (PyMOL™2.0.2) of the Vpb4Da2 pore-forming loop within domain 2 (pale green) and amino acid substitutions (orange spheres) representing the disabled insecticidal protein (DIP) variant. Domains D1-D5 are also represented. (B) Mass action in vivo competition bioassay. Wild-type Vpb4Da2 was competed with increasing challenge ratio of the Vpb4Da2-DIP variant. Bars represent the mean percent larval mortality with standard error. Mean values with same letter are not statistically different (One Way ANOVA Student-Newman-Keuls’ test, α = 0.05). (C) Multiple sequence alignment of the Vpb4Da2 pore-forming loop and selected β−PFPs from the Bacterial_exotoxin_B family using CLCBio™ version 7.6.4. Numbers on sequences are relative to Vpb4Da2. The degree of sequence conservation is represented by a green background gradient.
Fig 7.
Vpb4Da2 forms an oligomer at acidic pH in the presence of the BBM from WCR (Full SDS-PAGE figures are depicted in supporting information).
(A) Trypsin-processed Vpb4Da2 (tryptic core) forms a reversible oligomer in the presence of WCR BBM as shown in the gel following treatment with a homo-bifunctional cross-linker (BS3). BS3 titration at 0, 1, 2, and 5 mM are in lanes 1, 2, 3, and 4, respectively. Reactions were carried out at pH 6.0 and at room temperature. The oligomerized Vpb4Da2 tryptic core is indicated by the asterisk at the entrance of gel wells. (B) The Vpb4Da2 tryptic core forms a BBM bound oligomer in the absence of crosslinker at pH 6.0 and 37°C. The Vpb4Da2 tryptic core without BBM is shown as a monomer (lane 1). An SDS-resistant oligomer, asterisk (lane 2), is formed in the presence of WCR BBM. (C) The trypsin-processed Vpb4Da2 disabled insecticidal protein (DIP) variant does not form an SDS-resistant oligomer in the presence of WCR BBM. The full-length Vpb4Da2 and the processed DIP variant are in lanes 1 and 2, respectively. All reactions were at pH 6 and at 37°C.
Fig 8.
Effects of Vpb4Da2 on WCR larvae development and dissected gut morphology.
(A) Vpb4Da2 larvicidal activity on WCR measured by increasing protein concentration. Data points represent mean mortality (± SD), not corrected for buffer control mortality (5.56%). (B) Larval growth inhibition and gut morphology evaluation from 24 h to 72 h following exposure to Vpb4Da2. Larval growth was inhibited as early as 24 h. Dissected midguts from treated and untreated larvae are also shown. (C) Bar graph representation of growth inhibition. Data represent mean larval length (± SD). Asterisks indicate statistical significance at p = 0.05 (*) and p = 0.01(**) using the student-t test (n = 5).
Fig 9.
Western corn rootworm midgut morphology after feeding with Vpb4Da2.
Vpb4Da2 staining by Alexa-fluor-488 conjugated secondary antibody is in green. Nuclei staining by DAPI is in blue. (A) Longitudinal section of the midgut after 6 h exposure to Vpb4Da2. (B) Cross section after 48 h exposure to Vpb4Da2. (C) Cross section after 96 h exposure to Vpb4Da2. (A) and (B) White arrows indicate blebbing of the epithelial cells. (A), (B) and (C) White arrowheads designate a complete or near complete loss of the apical microvilli layer. (D), (E), and (F) Are untreated controls where black arrowheads indicate the intact apical microvilli layer. Scale bars are all 50 μm.