Identification and characterization of botulinum neurotoxin-like two-component toxins in Paeniclostridium ghonii

Abstract

Insecticidal bacterial proteins play key roles in insect-bacteria interactions and have been used as biopesticides. Here, we identify two insecticidal proteins in Paeniclostridium ghonii, designated PG-toxin 1 (PG1) and PG-toxin 2 (PG2), which are homologs of botulinum neurotoxins (BoNTs). Unlike BoNTs, PG1 and PG2 contain two separate proteins: One is the protease light chain (LC), and the other is the heavy chain containing the translocation domain and the receptor binding domain. Crystal and cryo-electron microscopy structures show a conserved BoNT-like architecture but without an interchain disulfide bond. Functional characterizations establish that the LCs of PG1 and PG2 cleave insect synaptosomal-associated protein 25 (SNAP25), but not human or rat SNAP25, and microinjection of PG1 and PG2 caused paralysis and death in Drosophila and Aedes mosquitoes. These findings identified unique two-component BoNT-like insecticidal proteins, revealing insights into the evolution of the BoNT family of toxins, and broadening our understanding of bacteria that can be used for biopest controls.

Fig. 1.. Identification and bioinformatical analysis of PG1 and PG2 in P. ghonii.

(A) Genomic neighborhoods surrounding PG1 (pg1-LC and pg1-HC) and PG2 (pg2-LC and pg2-HC) genes. The gene clusters containing representative BoNT and BoNT-like toxins are shown for comparison, including PMP1 (pmp1), BoNT/En (bont), BoNT/X, BoNT/A4 (a subtype of BoNT/A with its gene cluster containing orfX), and TeNT (tetX). Other neighboring genes encode ABC transporter ATP-binding protein (abc), radical SAM domain containing protein (radsam), ABC transporter ATP-binding protein/permease (abc-permease), filamentation induced by adenosine 3′,5′-monophosphate protein (fic), partitioning protein A (parA), insecticidal Cry8Ea1 toxin (cry8Ea1), RNA polymerase sigma factor D (rpoD), n-acetylmuramoyl-l-alanine amidase (amiD), metallophosphatase family protein (mpp), and BhlA/UviB family holin-like peptide (labeled “1”) aureocin-like type II bacteriocins (labeled “2” and “3”). Some duplicated labels between PG1 and PG2 were omitted to show adjacent labels clearer (mpp in PG1 and parA, rpoD, and amiD in PG2). (B) Schematic illustration of three-domain arrangement of PG1 and PG2 and their comparison to BoNT/A and PMP1. LC, light chain; HC, heavy chain; H_N, translocation domain; H_C, receptor binding domain. The conserved catalytic motif (HExxH) and the key motif in the translocation domain (PxxG) are marked. (C) Phylogenetic analysis of the LC (left) and HC (right) of BoNTs, BoNT-like toxins, PG1, and PG2. In both trees, PG-LC and PG-HC cluster with the BoNT/X/En/PMP1 lineage. Bootstrap values are indicated for all major clades. F7 and F5 are two subtypes of BoNT/F. HA/FA is the reported chimeric toxin BoNT/HA. (D) Phylogenetic clustering of P. ghonii genomes with the top 64 closest genomes in the NCBI database based on ANI. ANI values are shown on the right side of the tree and are averaged for the two P. ghonii strains. Strains encoding toxins were marked with a black circle.

Fig. 2.. LC/PG1 and LC/PG2 cleave insect SNAP25, but not human SNAP25.

(A) HA-tagged SNAP25 from the indicated species were coexpressed with the LCs of PG1, PG2, BoNT/A, E, or En in 293T cells via transient transfection. Cells lysates were analyzed by immunoblot detecting SNAP25 using an anti-HA antibody. Tubulin was detected as a loading control. (B) In vitro cleavage assays were carried out with the ratio of 5:1 (recombinantly purified SNAP25:LC). LC/PG1 and PG2 cleaved fly SNAP25 but did not cleave human SNAP25. (C and D) Fly SNAP25 was incubated with LC/PG1 or LC/PG2, respectively (PG1-pos and PG2-pos). As negative controls, equivalent samples were pretreated with trifluoroacetic acid and heat-inactivated before incubation (PG1-neg and PG2-neg). Peptides in the supernatants were extracted from each sample and subsequently analyzed with LC-MS/MS to determine molecular weight. Left panels showed eluted peptide peaks from the high-performance column over retention time (RT, x axis). The mass/charge ratio (m/z) for the cleaved peptides are listed from z = 1 to 5. Right panels showed MS/MS spectra of the deducted cleavage products from the left panels. The sequences are listed above the right panel, labeled with b and y fragmented ions for each positive sample of LC/PG1 (C) and LC/PG2 (D), respectively. (E) Sequence alignment of the indicated SNAP25 and fly SNAP24, with the cleavage sites of BoNT/A, E, PG1 and PG2 marked in red. (F) LC/PG1 did not cleave SNAP25 (E197K), while LC/PG2 did not cleave SNAP25 (R191E). R191E mutant also showed partial resistance to LC/PG1. (G) Crystal structure of LC/PG1 was resolved to 1.80-Å resolution (left). Zinc atom (red) and coordinated residues are shown. The right panel shows an overlay of LC/PG1 with LC/A [Protein Data Bank (PDB) ID: 4EJ5] (43) (LC/PG1, cyan color; LC/A, magenta color).

Fig. 3.. Cryo-EM structure of PG1 complex.

(A) Strep-II–tagged LC and His-tagged HC were coexpressed in E. coli using a pRSF-Duet vector. They were subjected to tandem purification, first using Ni–nitrilotriacetic acid column, followed with Strep tag column. Purified proteins were analyzed by SDS-PAGE and Coomassie Blue staining. LC and HC were copurified together for both PG1 and PG2. (B) Top: Representative cryo-EM 2D averages. Bottom: 3D reconstruction density map and atomic model of PG1 complex. Overlay of our built LC (cyan) and HC (blue) atom model into the cryo-EM density map shows local agreement of the refined model with the map. (C) The structure of PG1 complex was resolved to 3.3 Å by cryo-EM, and the front, back, and top views were presented. The three domains of PG1 complex are labeled in different colors: LC (cyan), H_N (green), and H_C (yellow). The N-terminal belt region of H_N is marked in magenta. A schematic illustration of PG1 domains is shown above the structures, with the residue numbers labeled. (D) Left: An overlay of LC/PG1 with LC of BoNT/A (PDB ID: 3V0C) (14). Right: An overlay of H_N/PG1 with the H_N of BoNT/A. PG1 in cyan and BoNT/A in gray. (E) Schematic diagrams of PG1 and BoNT/A structures show different orientations of belt region with respect to the rest of the molecule in the front (left) and back view (right). In contrast to BoNT/A, the belt region of PG1 only holds and interacts with LC of PG1 from the posterior side. (F) An overlay of H_C/PG1 with H_C of BoNT/A. PG1 in cyan and BoNT/A in gray. The H_C is composed of two subdomains, H_CN and H_CC.

Fig. 4.. LC-HC interfaces in PG1.

(A) Left: The close-up view of PG1 LC-belt region intermolecular β sheet (seat belt buckle structure) formed by the C-terminal region of LC/PG1 (cyan) and the N-terminal region of HC/PG1 (magenta). Right: The close-up view of BoNT/A (PDB ID: 3V0C) LC-HC belt region interface as well as the circled disulfide bond linkage (yellow) formed by cysteine residue C430 and cysteine residue C454. The PG1 LC (cyan), H_N (green), and H_C (beige) as well as the belt region (magenta) are shown. Interactions within the β sheets are shown as dashed yellow lines. (B) The close-up view of the second intermolecular three-stranded β sheet interface between the LC/PG1 and the belt region. The LC/PG1 (cyan), H_N (green), and H_C (yellow) as well as the belt region (magenta) are shown. Interactions between LC/PG1 and the belt region are shown as dashed yellow lines. (C) The close-up view of the interactions between the tip of the belt region with the LC/PG1. (D) The close-up view of PG1 LC-H_C interdomain interface of PG1 complex and interfacing residues in the front (left) and top (right) view. The PG1 LC (cyan), H_N (green), and H_C (yellow) as well as the belt region (magenta) are shown. The N306-T446 and E322-W439 interactions are shown as dashed yellow lines (left) and their interactions are circled and highlighted by red dashed lines viewed from the top (right). (E) Left: An overlay of the crystal structure of LC/PG1 (light gray) versus the cryo-EM structure of LC/PG1 (cyan) in the PG1 complex. Right: The close-up view of PG1 LC structural rearrangements induced by PG1 complex formation. The red arrows indicate a short β hairpin and the LC active site residues (H202, E203, and H206) and with E241 undergoing structural rearrangements upon complex formation.

Fig. 5.. PG1 and PG2 are toxic to flies and mosquitoes.

(A) Schematic illustrations of microinjection of toxins into adult D. melanogaster. (B) Survival curves of adult flies injected with 100 fmol of PG1. Injections of HC/PG1 and LC/PG1 were analyzed in parallel as controls. PG1 killed most flies within 48 hours (hr). (C) Survival curves of adult flies injected with 1 fmol of PG2. Injections of HC/PG2, LC/PG2 and inactivated PG2 mutant containing E206Q. PG2 killed all injected flies within 24 hours. (D and E) Survival curves for PG1 injected flies were recorded (D). The LD₅₀ was ~198 fmol for PG1 (E). (F and G) Survival curves for PG2 injected flies were recorded (F). The LD₅₀ was ~187 amol for PG2 (G). PG2 exhibited 1000-times higher potency than PG1. (H) Schematic illustration of PG1, PG2, and their mutants PG112, PG221, and PG2-SS. (I) SDS-PAGE analysis showed that the LC and HC were copurified in PG221 and PG112. (J) Flies were injected with PG1, PG2, PG221, and PG112 at the indicated doses (100, 1000, and 10,000 amol), and the percentage of survival flies after 24 hours were recorded and plotted. The toxicity level was PG2 (most toxic) > PG221 > PG112 > PG1 (least toxic). (K) SDS-PAGE analysis showing PG2 and PG2-SS under reducing (+2-ME, mercaptoethanol) versus nonreducing (−2-ME) conditions. PG2-SS appears to be a single band at ~140 kDa without the reducing agent, and it becomes two bands with the reducing agent, indicating that its LC and HC are linked by a disulfide bond. (L) PG2 and PG2-SS showed similar level of toxicity when injected into flies.