Identification and characterization of the receptor for the Bacillus sphaericus binary toxin in the malaria vector mosquito, Anopheles gambiae

The binary toxin (Bin) from Bacillus sphaericus exhibits a highly insecticidal activity against Culex and Anopheles mosquitoes. The cytotoxicity of Bin requires an interaction with a specific receptor present on the membrane of midgut epithelial cells in larvae. A direct correlation exists between binding affinity and toxicity. The toxin binds with high affinity to its receptor in its primary target, Culex pipiens, and displays a lower affinity to the receptor in Anopheles gambiae, which is less sensitive to Bin. Although the Bin receptor has previously been identified and named Cpm1 in C. pipiens, its structure in Anopheles remains unknown. In this study, we hypothesize that the Anopheles Bin receptor is an ortholog of Cpm1. By screening the Anopheles genomic database, we identified a candidate gene (Agm3) which is expressed primarily on the surface of midgut cells in larvae and which functions as a receptor for Bin. A Cpm1-like gene is also present in the Bin-refractory species Aedes aegypti. Overall, our results indicate that the three mosquito genes examined share a very similar organization and are strongly conserved at the amino acid level, in particular in the NH(2)-terminus, a region believed to contain the ligand binding site, suggesting that relatively few amino acids residues are critical for high affinity binding of the toxin.     

Monalysin, a novel ß-pore-forming toxin from the Drosophila pathogen Pseudomonas entomophila, contributes to host intestinal damage and lethality

Pseudomonas entomophila is an entomopathogenic bacterium that infects and kills Drosophila. P. entomophila pathogenicity is linked to its ability to cause irreversible damages to the Drosophila gut, preventing epithelium renewal and repair. Here we report the identification of a novel pore-forming toxin (PFT), Monalysin, which contributes to the virulence of P. entomophila against Drosophila. Our data show that Monalysin requires N-terminal cleavage to become fully active, forms oligomers in vitro, and induces pore-formation in artificial lipid membranes. The prediction of the secondary structure of the membrane-spanning domain indicates that Monalysin is a PFT of the ?-type. The expression of Monalysin is regulated by both the GacS/GacA two-component system and the Pvf regulator, two signaling systems that control P. entomophila pathogenicity. In addition, AprA, a metallo-protease secreted by P. entomophila, can induce the rapid cleavage of pro-Monalysin into its active form. Reduced cell death is observed upon infection with a mutant deficient in Monalysin production showing that Monalysin plays a role in P. entomophila ability to induce intestinal cell damages, which is consistent with its activity as a PFT. Our study together with the well-established action of Bacillus thuringiensis Cry toxins suggests that production of PFTs is a common strategy of entomopathogens to disrupt insect gut homeostasis.     

Bacillus sphaericus binary toxin elicits host cell autophagy as a response to intoxication

Bacillus sphaericus strains that produce the binary toxin (Bin) are highly toxic to Culex and Anopheles mosquitoes, and have been used since the late 1980s as a biopesticide for the control of these vectors of infectious disease agents. The Bin toxin produced by these strains targets mosquito larval midgut epithelial cells where it binds to Cpm1 (Culex pipiens maltase 1) a digestive enzyme, and causes severe intracellular damage, including a dramatic cytoplasmic vacuolation. The intoxication of mammalian epithelial MDCK cells engineered to express Cpm1 mimics the cytopathologies observed in mosquito enterocytes following Bin ingestion: pore formation and vacuolation. In this study we demonstrate that Bin-induced vacuolisation is a transient phenomenon that affects autolysosomes. In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells. Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment. Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.     

Monalysin,a Novel ?-Pore-Forming Toxin from the Drosophila Pathogen Pseudomonas entomophila,Contributes to Host Intestinal Damage and Lethality

Pseudomonas entomophila is an entomopathogenic bacterium that infects and kills Drosophila. P. entomophila pathogenicity is linked to its ability to cause irreversible damages to the Drosophila gut, preventing epithelium renewal and repair. Here we report the identification of a novel pore-forming toxin (PFT), Monalysin, which contributes to the virulence of P. entomophila against Drosophila. Our data show that Monalysin requires N-terminal cleavage to become fully active, forms oligomers in vitro, and induces pore-formation in artificial lipid membranes. The prediction of the secondary structure of the membrane-spanning domain indicates that Monalysin is a PFT of the ß-type. The expression of Monalysin is regulated by both the GacS/GacA two-component system and the Pvf regulator, two signaling systems that control P. entomophila pathogenicity. In addition, AprA, a metallo-protease secreted by P. entomophila, can induce the rapid cleavage of pro-Monalysin into its active form. Reduced cell death is observed upon infection with a mutant deficient in Monalysin production showing that Monalysin plays a role in P. entomophila ability to induce intestinal cell damages, which is consistent with its activity as a PFT. Our study together with the well-established action of Bacillus thuringiensis Cry toxins suggests that production of PFTs is a common strategy of entomopathogens to disrupt insect gut homeostasis.     

A secondary metabolite acting as a signalling molecule controls Pseudomonas entomophila virulence

Pseudomonas entomophila is an entomopathogenic bacterium that is lethal to Drosophila melanogaster within 1–2 days of ingestion of high doses. Flies orally infected with P. entomophila rapidly succumb despite the induction of both local and systemic immune responses. Recent studies suggest that its virulence relies on its ability to cause irreversible damages to the intestinal epithelium, in contrast to what is observed with milder pathogenic bacteria such as Erwinia carotovora carotovora Ecc15 or Pseudomonas aeruginosa PA14. The GacS/GacA two‐component system plays a key role in P. entomophila pathogenicity. Here, we report the identification of the pvf genes, whose products are involved in production of a secondary metabolite involved in P. entomophila virulence. A pvf mutant is impaired in its ability to persist within the gut, to trigger the fly immune responses and to inflict gut damages. The expression of several genes is affected in a pvf mutant, independently of the Gac system. Moreover, growing a pvf mutant in medium supplemented with supernatant extracts from either the wild‐type strain or a gacA mutant restore its pathogenicity. Collectively, our results indicate that we identified genes involved in the synthesis of a signalling molecule that controls P. entomophila virulence independently from the Gac system.     

X-ray and Cryo-electron Microscopy Structures of Monalysin Pore-forming Toxin Reveal Multimerization of the Pro-form

β-Barrel pore-forming toxins (β-PFT), a large family of bacterial toxins, are generally secreted as water-soluble monomers and can form oligomeric pores in membranes following proteolytic cleavage and interaction with cell surface receptors. Monalysin has been recently identified as a β-PFT that contributes to the virulence of Pseudomonas entomophila against Drosophila. It is secreted as a pro-protein that becomes active upon cleavage. Here we report the crystal and cryo-electron microscopy structure of the pro-form of Monalysin as well as the crystal structures of the cleaved form and of an inactive mutant lacking the membrane-spanning region. The overall structure of Monalysin displays an elongated shape, which resembles those of β-pore-forming toxins, such as Aerolysin, but is devoid of a receptor-binding domain. X-ray crystallography, cryo-electron microscopy, and light-scattering studies show that pro-Monalysin forms a stable doughnut-like 18-mer complex composed of two disk-shaped nonamers held together by N-terminal swapping of the pro-peptides. This observation is in contrast with the monomeric pro-form of the other β-PFTs that are receptor-dependent for membrane interaction. The membrane-spanning region of pro-Monalysin is fully buried in the center of the doughnut, suggesting that upon cleavage of pro-peptides, the two disk-shaped nonamers can, and have to, dissociate to leave the transmembrane segments free to deploy and lead to pore formation. In contrast with other toxins, the delivery of 18 subunits at once, nearby the cell surface, may be used to bypass the requirement of receptor-dependent concentration to reach the threshold for oligomerization into the pore-forming complex.