A Neurotoxin That Specifically Targets Anopheles Mosquitoes
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ARTICLE https://doi.org/10.1038/s41467-019-10732-w OPEN A neurotoxin that specifically targets Anopheles mosquitoes Estefania Contreras1, Geoffrey Masuyer 2, Nadia Qureshi1, Swati Chawla1, Harpal S. Dhillon1, Han Lim Lee 3, Jianwu Chen1, Pål Stenmark2,4 & Sarjeet S. Gill 1 Clostridial neurotoxins, including tetanus and botulinum neurotoxins, generally target ver- tebrates. We show here that this family of toxins has a much broader host spectrum, by 1234567890():,; identifying PMP1, a clostridial-like neurotoxin that selectively targets anopheline mosquitoes. Isolation of PMP1 from Paraclostridium bifermentans strains collected in anopheline endemic areas on two continents indicates it is widely distributed. The toxin likely evolved from an ancestral form that targets the nervous system of similar organisms, using a common mechanism that disrupts SNARE-mediated exocytosis. It cleaves the mosquito syntaxin and employs a unique receptor recognition strategy. Our research has an important impact on the study of the evolution of clostridial neurotoxins and provides the basis for the use of P. bifermentans strains and PMP1 as innovative, environmentally friendly approaches to reduce malaria through anopheline control. 1 Department of Molecular, Cell and Systems Biology, University of California, Riverside, CA 92521, USA. 2 Department of Biochemistry and Biophysics, Stockholm University, 106 91 Stockholm, Sweden. 3 Unit of Medical Entomology, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia. 4 Department of Experimental Medical Science, Lund University, Lund 22100, Sweden. Correspondence and requests for materials should be addressed to P.S. (email: [email protected]) or to S.S.G. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:2869 | https://doi.org/10.1038/s41467-019-10732-w | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10732-w fi alaria continues to impact populations with signi cant Table 1 Presence of plasmids (marked with X) in deaths worldwide. Various approaches used to attenu- Pb M ’ mosquitocidal and non-mosquitocidal strains ate this disease s incidence include the control of Ano- pheles mosquito species that vector plasmodium transmission. Pb malaysia Pb paraiba Pb PbmΔ109 Presently, chemical insecticides are the main stay of such vector (toxic) (toxic) (non-toxic) (non-toxic) control programs, although the development of resistance has Genome → size (kb) ~3.9 ~3.9 ~3.6 ~3.7 impacted their use. Biological approaches used in vector control Plasmid↓ size (kb) 1.84 X X can be effective, as evidenced by successful intervention of 1.96 X X X Onchocerca parasitic worm transmission in West Africa using 3.6 X X Bacillus thuringiensis israelensis (Bti)1. In its over two-decade use 4XX controlling Simulium blackflies, no resistance was observed to Bti, 7.2 X X 14.8 X X X X but this biological is not effective against anophelines. 35.8 X X With the aim to identify additional biologicals, Paraclostridium 109 X X bifermentans subsp. malaysia (Pbm) was isolated from a man- grove swamp soil in Malaysia and this strain has high mosqui- 7–9 tocidal activity primarily to Anopheles2, even though the P. from Weissella (BoNT-Wo) and Enterococcus (BoNT-En) . The bifermentans (Pb) type strain is not mosquitocidal. Pbm is ptox locus has genes, which encode for non-toxic non-hemag- innocuous to mammals, fish, and non-target invertebrates3. glutinin (NTNH), OrfX1, OrfX2, OrfX3, PMP1, and P47 proteins, Unfortunately, the lack of knowledge about the toxins involved in and a putative metallophosphatase family protein (MPP) the mosquitocidal activity prevents its utilization as a (Fig. 1b). bioinsecticide. BoNTs characterized to date mainly affect mammals and avian Previous attempts to characterize Pbm toxic components led to populations to various degrees, with BoNTs A, B, and E the main fi agents of human botulism, whereas BoNTs C and D are the identi cation of Cry16 and Cry17, which have similarity to B. 10 thuringiensis Cry toxins and two proteins with low amino acid preponderant in cattle . BoNTs intoxicate mainly by ingestion, 4 thus they resist extreme pH and gut proteolysis to reach the similarity to Aspergillus fumigatus hemolysins . These proteins, as 11 a complex, are orally toxic to Aedes mosquito larvae but not to bloodstream and then the nerve terminals . Following receptor Anopheles, even though the Pbm strain is more toxic to binding, the toxin is endocytosed and the acidic vesicular pH Anopheles5. causes a conformational change that mediates translocation of the light chain (LC) within the neuron cytosol where LC, a zinc Here we analyze by comparative genomics Pb mosquitocidal 12 and non-mosquitocidal strains and show that the active compo- endopeptidase, cleaves its target SNARE proteins . In the gut, nent in Pbm responsible for Anopheles toxicity is a complex that BoNTs travel as high-molecular-weight complexes with asso- ciated protein components, like NTNH and the HA proteins, contains a clostridial neurotoxin (CNT) that has high selectivity 13 to anopheline mosquitoes, acquired through a megaplasmid. which stabilize the toxin and promotes crossing of the host intestinal barrier14–16. The function of the OrfX proteins remains unknown; however, recent structural information suggests they Results may be involved in lipid interactions17,18. Genome sequencing of C. bifermentans strains. To identify Pbm NTNH, OrfX1–3, PMP1, and P47 proteins have 35–57% mosquitocidal components, we sequenced genomes of two Pb amino acid identity to Clostridium proteins. PMP1’s closest mosquitocidal strains Pbm and Pb paraiba (Pbp)6, which show relative is BoNT/X from C. botulinum strain 111 (36% identity)19, higher selectivity to Anopheles than Aedes mosquitoes (Supple- followed by BoNT/En, the Enterococcus BoNT-like protein7,9 mentary Table 1), and the non-mosquitocidal Pb, Pbm, Pbp, and (34% identity) (Fig. 1c, Supplementary Fig. 1B). PMP1 presents Pb genomes have similar chromosome sizes and belong to the the conserved SxWY motif in the binding domain (HC), which in group of extremely low GC clostridia, with 28% content (Sup- BoNTs is involved in ganglioside receptor binding (Fig. 1d, plementary Table 2, Supplementary Fig. 1A). Supplemenatry Fig. 1C), as well as the conserved disulfide bond Eight extra scaffolds from Pbm sequencing data did not match that links the toxin heavy and light chains, and is essential for chromosomic sequences. PCR amplification from these scaffolds’ toxicity20. The zinc-coordinating motif HExxH, which confers the ends confirmed their circularity, and identified them as the Pbm LC its metalloprotease activity is also conserved (Fig. 1d, plasmids. Similarly, PCR confirmed the presence of five Pbp and Supplementary Fig. 1D). two Pb plasmids. Notably, the mosquitocidal strains share four The ptox locus has a gene organization with an OrfX1–3 gene plasmids, which were not present in non-mosquitocidal Pb cluster located between NTNH and PMP1 under the same (Table 1). promoter (Fig. 1b). This configuration, which differs from other CNT loci, suggests that the horizontal gene transfer to Pbm or Pbm Pbp likely occurred from an ancestral bacterium as speculated for toxicity is linked to a plasmid with two toxin loci. Loss of 20 function Pbm mutants were generated by γ-irradiation. A mutant, the Enterococcus BoNT-like cluster . PbmΔ109, which completely lost toxicity against Aedes and Anopheles larvae, was sequenced. Data showed that this non-toxic Pmp operon proteins show oral toxicity to Anopheles larvae. mutant lost four Pbm plasmids, which are also present in Pbp PMP1 was immunodetected as a ~140 kDa protein in Pbm cul- (Table 1). A 109 kb plasmid was analyzed (Fig. 1a, Supplementary tures (Supplementary Fig. 2A). High molecular complexes from Data 1) and contained cry16A/17A and hemolysin-like genes in a Pbm were concentrated21 and the sample, which contained PMP1 cry operon, previously characterized4,5. We found a second toxin and Cry16A (Supplementary Fig. 2B), was separated by native locus (ptox) (Fig. 1a) flanked by insertion sequences and trans- PAGE, subjected to analysis by ultra-performance liquid poson elements. This locus encodes a protein, which we named chromatography-tandem mass spectrometer (UPLC/MS/MS), paraclostridial mosquitocidal protein 1 (PMP1), with high simi- and compared with a similar extracted fraction from the larity to CNTs, a group that includes the tetanus neurotoxin PbmΔ109 mutant (Supplementary Fig. 2C, 1st lane, Fig. 2a). All (TeNT) and botulinum neurotoxins (BoNTs). Recently, similar proteins from the cry and ptox loci were detected in the extracted putative BoNT genes were reported in non-clostridial species Pbm sample (Supplementary Table 3), but as expected, absent in 2 NATURE COMMUNICATIONS | (2019) 10:2869 | https://doi.org/10.1038/s41467-019-10732-w | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10732-w ARTICLE a 0 b 100,000 10,000 Inner circle: GC bias 2nd circle (from the center): G+C content Positive values Negative value Strain/subtype 90,000 20,000 3rd circle Toxin containing operons. Ptox is arrowed orfX3 orfX2 orfX1 p47 ntnh bont/e 4th circle C. botulinum Beluga/E1 Predicted genes in forward strand th ha70 ha17 ha33 botR p47 ntnh bont/b1 5 circle C. botulinum Okra/B1 80,000 30,000 Predicted genes on the reverse strand Outer circle: genes encoded by the plasmid in both strands orfX1 orfX2 orfX3 p47 ntnhbont/x orfX2b Regulatory C. botulinum 111/X Toxin loci Conserved hypothetical orfX1orfX2 orfX3 p47 ntnh Bont/en 70,000 40,000 Unknown E. faecium/En Transposon related, mobile elements Cell wall associated, surface associated; IS′ p47 ntnh orfX1 orfX2 orfX3 pmp1 mpp fla′ Miscellaneous metabolic genes 60,000 50,000 Pbm and Pbp/PMP1 c 0.1 pmp operon (11.6 Kb) BoNT/A ptox locus (15.7 Kb) BoNT/F5A (H) BoNT/G d C395–C406 832 1260 BoNT/B LC HN HC BoNT/F HELIH Tetanus HC toxin BoNT/E BoNT/En BoNT/X PMP1 BoNT/C-D BoNT/Wo Fig.