Genome sequencing of C. bifermentans strains

To identify mosquitocidal components, we sequenced genomes of two Pb mosquitocidal strains Pbm and Pb paraiba (Pbp)6, which show higher selectivity to Anopheles than Aedes mosquitoes (Supplementary Table 1), and the non-mosquitocidal Pb, Pbm, Pbp, and Pb genomes have similar chromosome sizes and belong to the group of extremely low GC clostridia, with 28% content (Supplementary Table 2, Supplementary Fig. 1A).

Eight extra scaffolds from Pbm sequencing data did not match chromosomic sequences. PCR amplification from these scaffolds’ ends confirmed their circularity, and identified them as the Pbm plasmids. Similarly, PCR confirmed the presence of five Pbp and two Pb plasmids. Notably, the mosquitocidal strains share four plasmids, which were not present in non-mosquitocidal Pb (Table 1).

Table 1 Presence of plasmids (marked with X) in mosquitocidal and non-mosquitocidal Pb strains Full size table

Pbm toxicity is linked to a plasmid with two toxin loci

Loss of function Pbm mutants were generated by γ-irradiation. A mutant, Pbm∆109, which completely lost toxicity against Aedes and Anopheles larvae, was sequenced. Data showed that this non-toxic mutant lost four Pbm plasmids, which are also present in Pbp (Table 1). A 109 kb plasmid was analyzed (Fig. 1a, Supplementary Data 1) and contained cry16A/17A and hemolysin-like genes in a cry operon, previously characterized4,5. We found a second toxin locus (ptox) (Fig. 1a) flanked by insertion sequences and transposon elements. This locus encodes a protein, which we named paraclostridial mosquitocidal protein 1 (PMP1), with high similarity to CNTs, a group that includes the tetanus neurotoxin (TeNT) and botulinum neurotoxins (BoNTs). Recently, similar putative BoNT genes were reported in non-clostridial species from Weissella (BoNT-Wo) and Enterococcus (BoNT-En)7,8,9. The ptox locus has genes, which encode for non-toxic non-hemagglutinin (NTNH), OrfX1, OrfX2, OrfX3, PMP1, and P47 proteins, and a putative metallophosphatase family protein (MPP) (Fig. 1b).

Fig. 1 Analysis of ptox locus and paraclostridial mosquitocidal protein 1 (PMP1) sequences. a Map of the 109 kb megaplasmid in Paraclostridium bifermentans subsp. malaysia (Pbm) and Pb paraiba (Pbp). b Configuration of clostridial neurotoxin loci in different Clostridium and non-Clostridium strains and in Pbm. c Phylogenetic split network covering all botulinum neurotoxin (BoNT) serotypes, subtypes, mosaic toxins, and tetanus neurotoxin (TeNT). PMP1 is in bold. The scale bar represents the split support for the edges47. d Schematic drawing of the three domains of PMP1 showing the conserved elements found in clostridial neurotoxins: HELXH motif in the light chain (LC), the cysteines that form disulfide bond between LC and heavy chain (HC) and the tandem SxWY motif in the HC Full size image

BoNTs characterized to date mainly affect mammals and avian populations to various degrees, with BoNTs A, B, and E the main agents of human botulism, whereas BoNTs C and D are preponderant in cattle10. BoNTs intoxicate mainly by ingestion, thus they resist extreme pH and gut proteolysis to reach the bloodstream and then the nerve terminals11. Following receptor binding, the toxin is endocytosed and the acidic vesicular pH causes a conformational change that mediates translocation of the light chain (LC) within the neuron cytosol where LC, a zinc endopeptidase, cleaves its target SNARE proteins12. In the gut, BoNTs travel as high-molecular-weight complexes with associated protein components, like NTNH and the HA proteins, which stabilize the toxin13 and promotes crossing of the host intestinal barrier14,15,16. The function of the OrfX proteins remains unknown; however, recent structural information suggests they may be involved in lipid interactions17,18.

Pbm NTNH, OrfX1–3, PMP1, and P47 proteins have 35–57% amino acid identity to Clostridium proteins. PMP1’s closest relative is BoNT/X from C. botulinum strain 111 (36% identity)19, followed by BoNT/En, the Enterococcus BoNT-like protein7,9 (34% identity) (Fig. 1c, Supplementary Fig. 1B). PMP1 presents the conserved SxWY motif in the binding domain (H C ), which in BoNTs is involved in ganglioside receptor binding (Fig. 1d, Supplemenatry Fig. 1C), as well as the conserved disulfide bond that links the toxin heavy and light chains, and is essential for toxicity20. The zinc-coordinating motif HExxH, which confers the LC its metalloprotease activity is also conserved (Fig. 1d, Supplementary Fig. 1D).

The ptox locus has a gene organization with an OrfX1–3 gene cluster located between NTNH and PMP1 under the same promoter (Fig. 1b). This configuration, which differs from other CNT loci, suggests that the horizontal gene transfer to Pbm or Pbp likely occurred from an ancestral bacterium as speculated for the Enterococcus BoNT-like cluster20.

Pmp operon proteins show oral toxicity to Anopheles larvae

PMP1 was immunodetected as a ~140 kDa protein in Pbm cultures (Supplementary Fig. 2A). High molecular complexes from Pbm were concentrated21 and the sample, which contained PMP1 and Cry16A (Supplementary Fig. 2B), was separated by native PAGE, subjected to analysis by ultra-performance liquid chromatography-tandem mass spectrometer (UPLC/MS/MS), and compared with a similar extracted fraction from the Pbm∆109 mutant (Supplementary Fig. 2C, 1st lane, Fig. 2a). All proteins from the cry and ptox loci were detected in the extracted Pbm sample (Supplementary Table 3), but as expected, absent in Pbm∆109. Since proteins from the cry locus are inactive against Anopheles mosquitoes5, our data provide strong evidence that proteins encoded by the ptox locus are responsible for the larvicidal activity to Anopheles.

Fig. 2 Paraclostridial mosquitocidal protein 1 (PMP1) and its associated proteins are toxic to mosquitoes. a Native PAGE of Paraclostridium bifermentans subsp. malaysia (Pbm), P. bifermentans (Pb), and Pbm∆109 extracted fractions. Lanes were split into E1 and E2 for tandem mass spectrometry (MS/MS) analyses. Greater abundance of OrfX1–3 in E2 may indicate that they are not strongly associated to a high molecular weight complex. b Schematic of constructs expressing proteins in the pmp operon (left panel) and their corresponding mortality to Anopheles and Aedes mosquito larvae (right panel) (Supplementary Note 1) (n = 15/assay, see Methods). The lethal concentration 50 (LC50) values of the pmp operon is 0.42 and 0.48 ml in two replicates. c Toxicity of PMP1 and inactive PMP1 E209Q mutant to larvae by injection in a dose–mortality plot. d Aedes 3rd instar larvae (n = 15) show reduced movement after injection with PMP1, but not when injected with water or inactive PMP1 E209Q mutant (t test, p value ≤0.05). Median in bold, box edges are 25th and 75th percentiles and vertical lines are min and max values. e Adult mosquitoes and flies (percent) stopped flying after 24 h of injection with PMP1. f Pre-incubation with 1,10-phenanthroline decreases PMP1 toxicity by injection (t test, p value ≤0.05). Error bars represent ± s.d. of three replicates. Source data are provided as a Source Data file Full size image

To verify that the pmp1 operon encodes the anopheline active toxin, we expressed these proteins in different combinations in B. thuringiensis 4Q7 strain (Fig. 2b). The Bt cultures expressing either PMP1 or NTNH protein alone had no toxicity to Anopheles coluzzi. However, cultures expressing both NTNH and PMP1 proteins showed 33% mortality, whereas the one expressing the full operon (Supplementary Fig. 2C, 2nd lane) had 70% mortality (Fig. 2b). Hence, the NTNH protein likely protects the PMP1 toxin from degradation in the gut, a mechanism common among BoNT complexes22. The OrfX1–3 proteins could also act similarly and/or facilitate PMP1 toxin absorption in the gut, since increased activity is observed in their presence than in their absence (Fig. 2b). Importantly, our data show a role in toxicity for the OrfX proteins. None of the constructs was significantly toxic to Ae. aegypti. Thus, the selectivity observed in Pbm to Aedes is likely produced by the cry operon alone5.

The toxicity of Bt expressing the pmp operon is lower than that of wild-type Pbm. Potentially other proteins in the megaplasmid may contribute to the anopheline toxicity. Alternatively, differences in toxin stability and/or differences in toxin availability in Pbm and Bt contribute to the lower toxicity of the latter.

PMP1 is toxic to mosquito larvae in vivo

To evaluate if PMP1 alone is toxic when the gut barrier is by passed, we injected recombinant PMP1 into mosquito larvae (Fig. 2c). Strikingly, injected PMP1 was toxic to both Aedes and Anopheles mosquitoes with an LD 50 of 14 pg (98 amol) and 6.5 pg (44.5 amol) per larva, respectively. The toxicity observed is 10–100 times greater than that of spider toxins that were also injected23.

Aedes larvae injected with the LC 99 (54 pg/larva) fully recovered from the injection, but at 3 h showed significant slowing of motion (Fig. 2d, Supplementary Movie 1), consistent with the paralysis associated with CNTs’ intoxication. PMP1 was also toxic to adult mosquitoes by injection, since a dose-dependent impairment in their ability to fly was observed (Fig. 2e). Pre-incubation of the toxin with the metalloprotease inhibitor 1,10-phenanthroline before injection decreased PMP1 toxicity (Fig. 2f). Further, the mutation E209Q in the metalloprotease active site (H E xxH motif) abolished activity (Fig. 2c), which confirms that PMP1 is a metalloprotease and this activity is essential for toxicity.

Since PMP1 is toxic to both mosquito species by injection, we determined if its specificity extends to other diptera or mammals once the gut barrier is bypassed. Although Pbm culture was not toxic by feeding to Drosophila larvae and adults (Supplementary Table 1), recombinant PMP1 was toxic to adult flies by injection (Fig. 2e). Further, PMP1 shows no toxicity to mice by the Digit Abduction Score assay and by intraperitoneal injections.

PMP1 cleaves mosquito syntaxin

LC metalloprotease activity is specific for one of the three neuronal SNARE proteins in mammals and their cleavage prevents neuro-exocytosis. In particular, VAMP-2/n-synaptobrevin is the target of TeNT and BoNTs B, D, F, G, X19,24,25,26, BoNT/Wo, and BoNT/En9,27, while syntaxin 1 is the target of BoNT/C and SNAP-25 is cleaved by BoNTs A, C, and E and BoNT/En9,28,29. To determine if PMP1 can cleave one of these SNARE protein homologs in mosquitoes, we incubated recombinant Anopheles gambiae syntaxin1A, n-synaptobrevin, and SNAP-25 with PMP1 LC. Only the C terminus of mosquito syntaxin was cleaved by PMP1 LC but not by the catalytically inactive PMP1 LC E209Q mutant (Fig. 3a). Interestingly, PMP1 LC was unable to cleave the recombinant human syntaxin1A (Fig. 3a), the C terminus of which is identical in mouse, and hence consistent with the lack of toxicity of PMP1 to mice.

Fig. 3 Paraclostridial mosquitocidal protein 1 (PMP1) light chain (LC) cleaves mosquito syntaxin. a Representation of the recombinant tagged SNARE proteins used in PMP1 LC cleavage assays (upper panel). Immunodetection of SNARE proteins and syntaxin mutants performed in the absence or in the presence of PMP1 LC, and PMP1 catalytically inactives E209Q mutant. b Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of His-syntaxin cleavage assay. The fragment of 4.5 kDa band released after incubation with PMP1 LC is indicated and was analyzed by ultra-performance liquid chromatography-tandem mass spectrometer (UPLC/MS/MS). c Alignment of the C-termini of syntaxins in different species. Amino acids that differ from human syntaxin are in red. The position of PMP1 cleavage site and the mutations introduced in syntaxin and tested in cleavage assays are indicated with arrows. Source data are provided as a Source Data file Full size image

To determine the PMP1 cleavage site, a peptide of ~4.5 kDa released from syntaxin cleavage (Fig. 3b) was purified and analyzed by UPLC-MS/MS and the peptide HAMDYVQTATQDTKK was detected (Supplementary Fig. 3B). Since Anopheles syntaxin1A C terminus is rich in positive charges, making it difficult for MS/MS detection, a syntaxin mutant (syxΔ2myc), where the charged region was deleted and an additional myc tag added, was created and similarly analyzed (Supplementary Fig. 3A). MS/MS identified peptides from the C terminus of syntaxin from H255 (Supplementary Fig. 3B). Thus, PMP1 LC cleaves syntaxin between E254 and H255 releasing a peptide that matches the observed size (Fig. 3b).

PMP1 does not cleave human syntaxin despite the cleavage site being conserved (Fig. 3c). However, a region closer to the C terminus shows sequence variation between human and mosquito syntaxins that could potentially influence substrate recognition. Indeed, CNTs are known to have an extensive substrate length requirement that involve multiple exosites away from the catalytic pocket30. To test this hypothesis, we generated Anopheles syntaxin single or double mutants where we changed amino acids in this region to the corresponding residues in human syntaxin (Fig. 3c) and incubated with PMP1 LC. These mutants cleaved less efficiently than Anopheles syntaxin, and L271V completely abolished cleavage (Fig. 3a). The presence of non-polar amino acids, such as valine in human syntaxin C terminus, likely disrupts the interaction with PMP1 LC and prevents cleavage.

Canonical CNT GBS is not critical for PMP1 mosquito toxicity

CNTs bind to the presynaptic membrane of peripheral nerve terminals with high specificity31. Polysialogangliosides recruit and concentrate CNTs, while specific proteins such as associated vesicle protein 2 (SV2) or synaptotagmin have been described as cell entry mediators12. A conserved ganglioside-binding site (GBS), which contains the SxWY motif has been identified in the C terminus of BoNT/A, B, E, F, G, X and TeNT H C , where the tryptophan is essential in maintaining hydrophobic interactions with the ganglioside sugar and is involved in toxicity32. Notably, PMP1 also shares a SGWY motif in the corresponding site, and a tandem repeat in front (Fig. 1d, Supplementary Fig. 1C).

To analyze if the SxWY motif is involved in toxicity and to determine the presence of other cell recognition regions, recombinant PMP1 H C was crystallized (residues 825–1260) and its structure determined at 1.95 Å resolution (Table 2). PMP1 H C has a fold similar to other CNTs33 and a well-conserved secondary structure (Fig. 4a, Supplementary Fig. 4A, B) despite having <30% sequence identity. PMP1 H C includes two subdomains, a N-terminal lectin-like fold (H CN ) consisting primarily of 15 β-strands arranged in a jelly-roll fold and a C-terminal β-trefoil fold mostly composed of seven pairs of β-strands linked by loops (H CC ) (Fig. 4a). The H CC is the main region associated with cell recognition, and in PMP1, presents the most structural variation compared to other CNTs. One striking feature of PMP1 H CC is its general hydrophobicity, with an array of a dozen aromatic residues exposed on its surface (Fig. 4b) and its lack of clear binding pockets (Supplementary Fig. 4C). The SxWY motif (S1229, W1231, and Y1232) is in a shallow pocket flanked on one side by a short lysine-rich α-helix (1242–1246) that is unique to PMP1 and may prevent the binding of gangliosides with branched carbohydrate head groups (Fig. 4b, Supplementary Fig. 4C).

Table 2 Data collection and refinement statistics (molecular replacement) Full size table

Fig. 4 Difference in the H CC domain of paraclostridial mosquitocidal protein 1 (PMP1) contributes to its selective toxicity. a Superimposition of the PMP1 H C crystal structure (cyan) with the full-length botulinum neurotoxin (BoNT)/B (blue, PDB 1EPW). The domains are labeled as LC (catalytic light chain), H N (translocation domain), and the H C (binding domain) with its two subdomains (H CN and H CC ). b Crystal structure of PMP1 H C. The aromatic residues exposed on PMP1 H CC surface, the mutations tested for toxicity (purple sticks), the loop 1 SWYG motif (orange sticks), and the conserved ganglioside-binding motif (red sticks) are labeled. The E1099-K1168 salt bridge is indicated as a dashed line. c Toxicity of PMP1 H CC mutants to A. aegypti 4th instar larvae by injection (t test, p value ≤0.05). Error bars represent ± s.d. of at least three replicates. Source data are provided as a Source Data file Full size image

Of particular interest are the three extended loops found almost in parallel in the H CC subdomain. Extended loops 1094–1099 (loop 1), 1164–1172 (loop 2), and 1202–1208 (loop 3) (Fig. 4b) present bulky hydrophobic side chains. Loops 1 and 2 come close to each other with E1099 making a salt bridge with K1168 (Fig. 4b), at a location where BoNT/B and TeNT bind synaptotagmin and disialyllactose or a tri-peptide, respectively34 (Supplementary Fig. 4C), suggesting a different cell recognition mechanism for PMP1. A fourth loop composed of residues 1213–1222 (loop 4) was disordered, but seems to occupy a different position compared to the equivalent loops in BoNT/B and TeNT that are more protuberant. This loop was recently shown to play an essential role in lipid bilayer insertion for BoNT/B, DC, and G19,35.

The toxicity of PMP1 H C mutants was tested by injecting an LC 99 dose into A. aegypti larvae (Fig. 4c). Remarkably, W1231A and W1224A mutations of the tandem SxWY motifs did not decrease toxicity (Fig. 4c). The mutation of S1229, which disrupts ganglioside binding in BoNTs36, did not affect toxicity either (Fig. 4c). Mutation of an exposed F1202 in loop 3 did not decrease toxicity, but other mutations on loops 1 to 3 decreased toxicity to different extents. We observed a slight reduction in the quadruple mutants of loops 2 and 3 (K1168A/I1169A/K1170A/E1171A and M1165A/Y1166D/M1204A/Y1205D) and a strong reduction to 8% mortality in the quadruple mutant of loop 1 (S1095A/W1096A/Y1097A/G1098A) (Fig. 4c). Interestingly, mutation of Trp alone (W1096A) in loop 1 strongly decreased the toxicity to 30%. In addition, the mutant affecting a surface-exposed tyrosine hydrophobic patch (Y1100D/Y1101D/Y1173A/Y1227A) also nearly abolished toxicity (Fig. 4c, Supplementary Fig. 4).