Jean-François Charles

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens.

Photorhabdus luminescens is a symbiont of nematodes and a broad-spectrum insect pathogen. The complete genome sequence of strain TT01 is 5,688,987 base pairs (bp) long and contains 4,839 predicted protein-coding genes. Strikingly, it encodes a large number of adhesins, toxins, hemolysins, proteases and lipases, and contains a wide array of antibiotic synthesizing genes. These proteins are likely to play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver, making P. luminescens a promising model for the study of symbiosis and host-pathogen interactions. Comparison with the genomes of related bacteria reveals the acquisition of virulence factors by extensive horizontal transfer and provides clues about the evolution of an insect pathogen. Moreover, newly identified insecticidal proteins may be effective alternatives for the control of insect pests.

The receptor of Bacillus sphaericus binary toxin in Culex pipiens (Diptera: Culicidae) midgut: molecular cloning and expression ☆

Culex pipiens larval midgut is the primary target of the binary toxin (Bin) present in parasporal inclusions of Bacillus sphaericus. Cpm1, a 60-kDa protein purified from brush border membranes, has been proposed as the receptor of the Bin toxin in the midgut epithelial cells of mosquitoes. We have cloned and characterized the corresponding cDNA from midgut of Culex pipiens larvae. The open reading frame predicted a 580 amino-acid protein with a putative signal peptide at the N-terminus and a putative GPI-anchoring signal at the C-terminus. The amino acid sequence of the cloned Cpm1 exhibited 39-43% identities with insect maltases (alpha-glucosidases and alpha-amylases). Recombinant Cpm1 expressed in E. coli specifically bound to the Bin toxin and had a significant alpha-glucosidase activity but no alpha-amylase activity. These results support the view that Cpm1 is an alpha-glucosidase expressed in Culex midgut where it constitutes the receptor for the Bin toxin. To date, this is the first component involved in the mosquitocidal activity of the Bacillus sphaericus Bin toxin to be characterized. Its identification provides a key step to elucidate the mode of action of the Bin toxin and the mechanisms of resistance developed against it by some mosquito strains.

Identification of the receptor for Bacillus sphaericus crystal toxin in the brush border membrane of the mosquito Culex pipiens (Diptera: Culicidae).

The binary toxin (Bin) from Bacillus sphaericus crystals specifically binds to soluble midgut brush border membrane proteins from Culex pipiens larvae. A single 60 kDa midgut membrane protein is identified as the binding protein. This protein is anchored in the mosquito midgut membrane via a glycosyl-phosphatidylinositol (GPI) anchor, and is partially released by phosphatidylinositol specific-phospholipase C (PI-PLC). Fractionation of soluble proteins by anion exchange chromatography indicates that the binding protein does not co-elute with leucine aminopeptidase activity. After partial purification, the sequences of internal amino acid fragments of the 60 kDa protein were determined. The peptide sequences were compared with data in GenBank, and showed a very high degree of similarity with enzymes belonging to the alpha-amylase family. Further enzymatic investigation showed that the receptor of the Bin toxin in C. pipiens larval midgut may be an alpha-glucosidase.

Bacteriological larvicides of dipteran disease vectors

The apparent success in vector control observed between 1950 and 1970 was followed by worldwide resistance to organosynthetic insecticides wherever they were used intensively. Insect resistance to one or more categories of insecticides has limited the effectiveness of these compounds, and their non-selective mode of action adversely affects non-target organisms. This scenario highlights the need for selective agents in integrated vector control programs. This article gives an overview of the main fundamental and applied research topics on entomopathogenic bacteria in relation to their role in vector control.

Growth, sporulation and larvicidal activity of Bacillus sphaericus

SummaryA new medium (MBS) for optimal sporulation of Bacillus sphaericus was defined. With the two main mosquito pathogenic strains grown in this medium, 1593-4 and 2297, highest cell and spore yields were obtained, concomitantly with an highest larvicidal activity against Culex pipiens. Study of both strains asporulated mutants showed a decrease in larvicidal power. After plasmid curing treatments, toxicity of strain 1593-4 did not decrease, neither toxic parasporal inclusion bodies of strain 2297 disappear.

Ultrastructural midgut events in culicidae larvae fed with Bacillus sphaericus 2297 spore/crystal complex

Ingestion of Bacillus sphaericus 2297 spore/crystal complex by Culicidae larvae Anopheles stephensi, Culex pipiens subsp. pipiens and Aedes aegypti was rapidly followed by a dissolution of the protein crystalline inclusions inside the anterior stomach of the three species. During the first day of intoxication, B. sphaericus spores germinated within the midgut lumen, and were in a vegetative stage between 36-48 h after ingestion when the larvae began to die. Ultrastructural observations focused on larval midgut showed alterations which differed according to the mosquito species, being localized mainly in the gastric caeca and posterior stomach. With the bacterial concentration used, neither general cell swelling nor complete breakdown of the midgut epithelium was recorded before larval death. In A. stephensi larval midgut epithelium large low-electron-density areas appeared, rough endoplasmic reticula formed numerous concentrical structures and mitochondria swelled. Large vacuoles (of unknown origin) appeared early on in the C. pipiens midgut cells, and rough endoplasmic reticula broke into small vesicles. Midgut epithelial cells of A. aegypti showed mitochondria swelling except in the anterior stomach, and a vacuolisation of smooth reticula: these aspects remained unchanged until the larvae died.

Loss of the membrane anchor of the target receptor is a mechanism of bioinsecticide resistance

The mosquitocidal activity of Bacillus sphaericus is because of a binary toxin (Bin), which binds to Culex pipiens maltase 1 (Cpm1), an α-glucosidase present in the midgut of Culex pipiens larvae. In this work, we studied the molecular basis of the resistance to Bin developed by a strain (GEO) of C. pipiens. Immunohistochemical and in situ hybridization experiments showed that Cpm1 was undetectable in the midgut of GEO larvae, although the gene was correctly transcribed. The sequence of the cpm1GEO cDNA differs from the sequence we previously reported for a susceptible strain (cpm1IP) by seven mutations: six missense mutations and a mutation leading to the premature termination of translation. When produced in insect cells, Cpm1IP was attached to the membrane by a glycosylphosphatidylinositol (GPI). In contrast, the premature termination of translation of Cpm1GEO resulted in the targeting of the protein to the extracellular compartment because of truncation of the GPI-anchoring site. The interaction between Bin and Cpm1GEO and the enzyme activity of the receptor were not affected. Thus, Bin is not toxic to GEO larvae because it cannot interact with the midgut cell membrane, even though its receptor site is unaffected. This mechanism contrasts with other known resistance mechanisms in which point mutations decrease the affinity of binding between the receptor and the toxin.

Mosquitocidal bacterial toxins: diversity, mode of action and resistance phenomena.

Bacteria active against dipteran larvae (mosquitoes and black flies) include a wide variety of Bacillus thuringiensis and B. sphaericus strains, as well as isolates of Brevibacillus laterosporus and Clostridium bifermentans. All display different spectra and levels of activity correlated with the nature of the toxins, mainly produced during the sporulation process. This paper describes the structure and mode of action of the main mosquitocidal toxins, in relationship with their potential use in mosquito and/or black fly larvae control. Investigations with laboratory and field colonies of mosquitoes that have become highly resistant to the B. sphaericus Bin toxin have shown that several mechanisms of resistance are involved, some affecting the toxin/receptor binding step, others unknown.

Permeabilization of Model Lipid Membranes by Bacillus sphaericus Mosquitocidal Binary Toxin and its Individual Components

Abstract. The high larvicidal effect of Bacillus sphaericus (Bs), a mosquito control agent, originates from the presence of a binary toxin (Bs Bin) composed of two proteins (BinA and BinB) that work together to lyse gut cells of susceptible larvae. We demonstrate for the first time that the binary toxin and its individual components permeabilize receptor-free large unilamellar phospholipid vesicles (LUVs) and planar lipid bilayers (PLBs) by a mechanism of pore formation. Calcein-release experiments showed that LUV permeabilization was optimally achieved at alkaline pH and in the presence of acidic lipids. BinA was more efficient than BinB, BinB facilitated the BinA effect, and their stoichiometric mixture was more effective than the full Bin toxin. In PLBs, BinA formed voltage-dependent channels of ≈100–200 pS with long open times and a high open probability. Larger channels (≥400 pS) were also observed. BinB, which inserted less easily, formed smaller channels (≤100 pS) with shorter mean open times. Channels observed after sequential addition of the two components, or formed by their 1:1 mixture (w/w), displayed BinA-like activity. Bs Bin toxin was less efficient at forming channels than the BinA/BinB mixture, with channels displaying the BinA channel behavior. Our data support the concept of BinA being principally responsible for pore formation in lipid membranes with BinB, the binding component of the toxin, playing a role in promoting channel activity.

Transposon-mediated resistance to Bacillus sphaericus in a field-evolved population of Culex pipiens (Diptera: Culicidae)

The binary toxin is the major active component of Bacillus sphaericus, a microbial larvicide used for controlling some vector mosquito‐borne diseases. B. sphaericus resistance has been reported in many part of the world, leading to a growing concern for the usefulness of this environmental friendly insecticide. Here we characterize a novel mechanism of resistance to the binary toxin in a natural population of the West Nile virus vector, Culex pipiens. We show that the insertion of a transposable element‐like DNA into the coding sequence of the midgut toxin receptor induces a new mRNA splicing event, unmasking cryptic donor and acceptor sites located in the host gene. The creation of the new intron causes the expression of an altered membrane protein, which is incapable of interacting with the toxin, thus providing the host mosquito with an advantageous phenotype. As a large portion of insect genomes is composed of transposable elements or transposable elements‐related sequences, this new mechanism may be of general importance to appreciate their significance as potent agents for insect resistance to the microbial insecticides.