Txp40, a ubiquitous insecticidal toxin protein from Xenorhabdus and Photorhabdus bacteria

Xenorhabdus and Photorhabdus are gram-negative bacteria that produce a range of proteins that are toxic to insects. We recently identified a novel 42-kDa protein from Xenorhabdus nematophila that was lethal to the larvae of insects such as Galleria mellonella and Helicoverpa armigera when it was injected at doses of 30 to 40 ng/g larvae. In the present work, the toxin gene txp40 was identified in another 59 strains of Xenorhabdus and Photorhabdus, indicating that it is both highly conserved and widespread among these bacteria. Recombinant toxin protein was shown to be active against a variety of insect species by direct injection into the larvae of the lepidopteran species G. mellonella, H. armigera, and Plodia interpunctella and the dipteran species Lucilia cuprina. The protein exhibited significant cytotoxicity against two dipteran cell lines and two lepidopteran cell lines but not against a mammalian cell line. Histological data from H. armigera larvae into which the toxin was injected suggested that the primary site of action of the toxin is the midgut, although some damage to the fat body was also observed.     

Txp40, a Ubiquitous Insecticidal Toxin Protein from Xenorhabdus and Photorhabdus Bacteria

Xenorhabdus and Photorhabdus are gram-negative bacteria that produce a range of proteins that are toxic to insects. We recently identified a novel 42-kDa protein from Xenorhabdus nematophila that was lethal to the larvae of insects such as Galleria mellonella and Helicoverpa armigera when it was injected at doses of 30 to 40 ng/g larvae. In the present work, the toxin gene txp40 was identified in another 59 strains of Xenorhabdus and Photorhabdus, indicating that it is both highly conserved and widespread among these bacteria. Recombinant toxin protein was shown to be active against a variety of insect species by direct injection into the larvae of the lepidopteran species G. mellonella, H. armigera, and Plodia interpunctella and the dipteran species Lucilia cuprina. The protein exhibited significant cytotoxicity against two dipteran cell lines and two lepidopteran cell lines but not against a mammalian cell line. Histological data from H. armigera larvae into which the toxin was injected suggested that the primary site of action of the toxin is the midgut, although some damage to the fat body was also observed.     

嗜线虫致病杆菌血腔毒素Tp40的纯化和特性分析

[目的]嗜线虫致病杆菌是一种昆虫病原线虫共生菌,它能够产生多种杀虫毒素.本研究旨在从嗜线虫致病杆菌Xenorhabdus nematophila HB310菌株的细胞内纯化新的杀虫蛋白毒素,并对其进行基因克隆和序列分析.[方法]应用盐析和制备型非变性凝胶电泳等方法纯化蛋白,再通过对5龄大蜡螟幼虫血腔注射进行活性筛选.对获得的目的蛋白与已知蛋白进行同源分析,克隆出该目的蛋白的基因序列,从而进行相应的基因和氨基酸序列分析.[结果]本研究纯化的Tp40蛋白对大蜡螟LD50为68.54 ng/头,其SDS-PAGE电泳图谱只显示出一条分子量约为42 kDa的多肽.Western印迹分析表明Tp40与已知的Txp40为同源蛋白,并且仅存在于细胞内.编码该蛋白的基因开放读码框全长1107bp(GenBank登录号:EU095326),编码368个氨基酸残基,预测分子量为41.5 kDa,等电点为8.66,与GenBank中的其余13株昆虫病原线虫共生菌所包含的相似基因核苷酸序列及推导的氨基酸序列比较,同源性分别为85%～99%和70%～99%.[结论]Tp40蛋白具有很高的血腔杀虫活性,其基因序列具有较强的保守性,是昆虫病原线虫共生菌复合体杀虫过程中的一种关键因子.     

Insecticidal pilin subunit from the insect pathogen Xenorhabdus nematophila

Xenorhabdus nematophila is an insect pathogen and produces protein toxins which kill the larval host. Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of X. nematophila. Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of X. nematophila isolated from that protein complex. The gene was amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient. The protein oligomerized during in vitro refolding, forming multimers. Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation. The recombinant protein was cytotoxic to cultured Helicoverpa armigera larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase. The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives. The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein. The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest, H. armigera, causing extensive damage to the midgut epithelial membrane. To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium.     

Stages of infection during the tripartite interaction between Xenorhabdus nematophila, its nematode vector, and insect hosts

Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.     

Insecticidal toxin complex proteins from Xenorhabdus nematophilus: structure and pore formation

Toxin complexes from Xenorhabdus and Photorhabdus spp. bacteria represent novel insecticidal proteins. We purified a native toxin complex (toxin complex 1) from Xenorhabdus nematophilus. The toxin complex is composed of three different proteins, XptA2, XptB1, and XptC1, representing products from class A, B, and C toxin complex genes, respectively. We showed that recombinant XptA2 and co-produced recombinant XptB1 and XptC1 bind together with a 4:1:1 stoichiometry. XptA2 forms a tetramer of ～1,120 kDa that bound to solubilized insect brush border membranes and induced pore formation in black lipid membranes. Co-expressed XptB1 and XptC1 form a tight 1:1 binary complex where XptC1 is C-terminally truncated, resulting in a 77-kDa protein. The ～30-kDa C-terminally cleaved portion of XptC1 apparently only loosely associates with this binary complex. XptA2 had only modest oral toxicity against lepidopteran insects but as a complex with co-produced XptB1 and XptC1 had high levels of insecticidal activity. Addition of co-expressed class B (TcdB2) and class C (TccC3) proteins from Photorhabdus luminescens to the Xenorhabdus XptA2 protein resulted in formation of a hybrid toxin complex protein with the same 4:1:1 stoichiometry as the native Xenorhabdus toxin complex 1. This hybrid toxin complex, like the native toxin complex, was highly active against insects.     

Bacterial metabolites of an entomopathogenic bacterium, Xenorhabdus nematophila, inhibit a catalytic activity of phenoloxidase of the diamondback moth, Plutella xylostella

A monoterpenoid compound, benzylideneacetone (BZA), is identified from bacterial metabolites synthesized by an entomopathogenic bacterium, Xenorhabdus nematophila. It inhibits phospholipase A2 of target insects to shut down biosynthesis of various eicosanoids, which play significant roles in insect immunity. This study discovered another novel activity of BZA that directly inhibited phenoloxidase (PO) activity required for immune-associated melanization. When it was injected into larvae of Plutella xylostella, it suppressed PO activity in the plasma by inhibiting its activation from inactive proPO. However, BZA did not influence on gene expression of PO, which was analyzed by RT-PCR using gene-specific primers designed from a partial cDNA sequence of PO of the P. xylostella identified in this study. To test a direct inhibitory activity of BZA against PO, the activated PO of P. xylostella was prepared from the hemolymph collected from the larvae challenged by bacteria. When the activated PO was incubated in vitro with BZA, it was inhibited in a dose-dependent manner. The inhibition of PO by BZA was recovered by addition of increasing amounts of substrate, L-3,4-dihydroxyphenylalanine. Three other known bacterial metabolites containing a benzene propane core structure synthesized by X. nematophila also inhibited the PO enzyme activity. However, modification of the core structure by hydroxylation of BZA lost its strong inhibitory activity against the activated PO.     

A Recombinant Truncated Cry1Ca Protein Is Toxic to Lepidopteran Insects and Forms Large Cuboidal Crystals in Insect Cells

A truncated version of the cry1Ca gene from Bacillus thuringiensis was introduced into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) under the control of two promoters. A recombinant virus (vSyncry1c) was isolated and used to infect insect cells in culture and insect larvae. Structural and ultrastructural analysis of insects infected with vSyncry1C showed the formation of large cuboidal crystals inside the cytoplasm of insect cells in culture and in insect cadavers late in infection. Infected insect cell extracts were analyzed by SDS-PAGE and Western blot and showed the presence of a 65-kDa polypeptide probably corresponding to the protease processed form of the toxin. Bioassays using purified recombinant toxin crystals showed a CL₅₀ of 19.49 ng/ml for 2^nd instar A. gemmatalis larvae and 114.1 ng/ml for S. frugiperda.     

The xaxAB genes encoding a new apoptotic toxin from the insect pathogen Xenorhabdus nematophila are present in plant and human pathogens

Xenorhabdus nematophila, a member of the Enterobacteriaceae, kills many species of insects by strongly depressing the immune system and colonizing the entire body. A peptide cytotoxin has been purified from X. nematophila broth growth, and the cytolytic effect on insect immunocytes and hemolytic effect on mammalian red blood cells of this toxin have been described (Ribeiro, C., Vignes, M., and Brehélin, M. (2003) J. Biol. Chem. 278, 3030-3039). We show here that this toxin, Xenorhabdus alpha-xenorhabdolysin (Xax), triggers apoptosis in both insect and mammalian cells. We also report the cloning and sequencing of two genes, xaxAB, encoding this toxin in X. nematophila. The expression of both genes in recombinant Escherichia coli led to the production of active cytotoxin/hemolysin. However, hemolytic activity was observed only if the two peptides were added in the appropriate order. Furthermore, we report here that inactivation of xaxAB genes in X. nematophila abolished the major cytotoxic activity present in broth growth, called C1. We also show that these genes are present in various entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus, in Pseudomonas entomophila, in the human pathogens Yersinia enterocolitica and Proteus mirabilis, and in the plant pathogen Pseudomonas syringae. This toxin cannot be classified in any known family of cytotoxins on the basis of amino acid sequences, locus organization, and activity features. It is, therefore, probably the prototype of a new family of binary toxins.     

Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated strains of Pseudomonas fluorescens

Pseudomonas fluorescens CHA0 and the related strain Pf-5 are well-characterized representatives of rhizosphere bacteria that have the capacity to protect crop plants from fungal root diseases, mainly by releasing a variety of exoproducts that are toxic to plant pathogenic fungi. Here, we report that the two plant-beneficial pseudomonads also exhibit potent insecticidal activity. Anti-insect activity is linked to a novel genomic locus encoding a large protein toxin termed Fit (for P.   f luorescens i nsecticidal t oxin) that is related to the insect toxin Mcf ( M akes c aterpillars f loppy) of the entomopathogen Photorhabdus luminescens , a mutualist of insect-invading nematodes. When injected into the haemocoel, even low doses of P. fluorescens CHA0 or Pf-5 killed larvae of the tobacco hornworm Manduca sexta and the greater wax moth Galleria mellonella . In contrast, mutants of CHA0 or Pf-5 with deletions in the Fit toxin gene were significantly less virulent to the larvae. When expressed from an inducible promoter in a non-toxic Escherichia coli host, the Fit toxin gene was sufficient to render the bacterium toxic to both insect hosts. Our findings establish the Fit gene products of P. fluorescens CHA0 and Pf-5 as potent insect toxins that define previously unappreciated anti-insect properties of these plant-colonizing bacteria.     

Interaction of the bacteria Xenorhabdus nematophila (Enterobactericeae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasiocampidae)

Malacosoma disstria larvae are a pest of deciduous trees. Little is known on the interaction of bacteria with the immediate hemocytic antimicrobial responses of these insects. Incubating dead Xenorhabdus nematophila and Bacillus subtilis with a mixture of serum-free granular cells and plasmatocytes in vitro revealed differential bacterial-hemocyte adhesion and differential discharge of lysozyme and phenoloxidase but not total protein. Although active phenoloxidase adhered equally to both bacterial species, X. nematophila limited enzyme activation whereas B. subtilis enhanced activation. Serum with active phenoloxidase (as opposed to tropolone-inhibited phenoloxidase) and purified insect lysozyme increased bacterial-hemocyte adhesion of both bacterial species. An apolipophorin-III-like protein when incubated with hemocytes, limited their responses to glass slides and bacterial adhesion. However, initial binding of the protein to both bacteria increased granular cell levels with bacteria while lowering the plasmatocyte levels with adhering procaryotes. The protein also increased lysozyme and phenoloxidase activities. Although B. subtilis in vivo elicited a nodulation-based decline in total hemocyte counts and did not affect hemocyte viability, dead X. nematophila elevated hemocyte counts and damaged the hemocytes as lipopolysaccharide levels increased and X. nematophila emerged into the hemolymph. Apolipophorin-III-like protein once bound to the bacteria slowed their removal from the hemolymph.     

Fate of Bacillus thuringiensis strains in different insect larvae

In favorable conditions Bacillus thuringiensis spores germinate and vegetative cells multiply, whereas in unfavorable conditions Bacillus thuringiensis sporulates and produces insecticidal crystal proteins. The development of B. thuringiensis strains was investigated in the larvae of insects belonging to the orders Lepidoptera and Diptera. Bacillus thuringiensis strains able to kill the insects did not always multiply in cadavers. Strains with no specificity to kill the insect sometimes multiplied when the insects were killed mechanically. These results indicate that some insect larvae represent an environment that favors the germination of B. thuringiensis spores and the multiplication of vegetative cells; however, there was no correlation between the toxin specificity and the specificity of the host.     

First report of detection of the putative receptor of Bacillus thuringiensis toxin Vip3Aa from black cutworm (Agrotis ipsilon)

Black cutworm (BCW) Agrotis ipsilon, an economically important lepidopteran insect, has attracted a great attention. Bacillus thuringiensis (Bt) is spore forming soil bacteria and is an excellent environment-friendly approach for the control of phytophagous and disease-transmitting insects. In fact, bio-pesticide formulations and insect resistant transgenic plants based on the bacterium Bt delta-endotoxin have attracted worldwide attention as a safer alternative to harmful chemical pesticides. The major objective of the current study was to understand the mechanism of interaction of Bt toxin with its receptor molecule(s). The investigation involved the isolation, identification, and characterization of a putative receptor – vip3Aa. In addition, the kinetics of vip toxin binding to its receptor molecule was also studied. The present data suggest that Vip3Aa toxin bound specifically with high affinity to a 48-kDa protein present at the brush border membrane vesicles (BBMV) prepared from the midgut epithelial cells of BCW larvae.     

Insecticidal effects of Buthus occitanus tunetanus BotIT6 toxin expressed in Escherichia coli and baculovirus/insect cells

BotIT6 is a neurotoxin polypeptide derived from the venom of the scorpion Buthus occitanus tunetanus (Bot). Its mature form is composed of 62 amino acids. BotIT6 has been reported to be the most potent toxin from Bot venom that has a strict selectivity for insects. Such toxin may have potential as a potent animal-harmless tool against insects. Using RT-PCR, we isolated and sequenced a cDNA encoding 62 amino acid residues corresponding to the known amino acid sequence of BotIT6. We have expressed a recombinant active form of BotIT6 in significantly high amounts in Escherichia coli. We have also engineered the cDNA into the Autographa californica Nuclear Polyhedrosis Virus (AcMNPV) genome and expressed the protein under control of the polyhedrin promoter. Supernatants of AcIT6-virus infected Sf9 insect cells exhibit a typical intoxication effect when injected to Spodoptera littoralis larvae. Moreover, injection of the recombinant virus showed enhanced insecticidal potency against S. littoralis larvae compared with wild type AcMNPV.     

The tc genes of Photorhabdus: a growing family

The toxin complex (tc) genes of Photorhabdus encode insecticidal, high molecular weight Tc toxins. These toxins have been suggested as useful alternatives to those derived from Bacillus thuringiensis for expression in insect-resistant transgenic plants. Although Photorhabdus luminescens is symbiotic with nematodes that kill insects, tc genes have recently been described from other insect-associated bacteria such as Serratia entomophila, an insect pathogen, and Yersinia pestis, the causative agent of bubonic plague, which has a flea vector. Here, recent advances in our understanding of the tc gene family are reviewed in view of their potential development as insect-control agents.     

Purification and functional characterization of insecticidal sphingomyelinase C produced by Bacillus cereus.

Bacillus cereus isolated from the larvae of Myrmeleon bore was found to secrete proteins that paralyze and kill German cockroaches, Blattela germanica, when injected. One of these active proteins was purified from the culture broth of B. cereus using anion-exchange and gel-filtration chromatography. The purified toxin, with a molecular mass of 34 kDa, was identified as sphingomyelinase C (EC 3.1.4.12) on the basis of its N-terminal and internal amino-acid sequences. A recombinant sphingomyelinase C expressed in Escherichia coli was as potent as the native protein in killing the cockroaches. Site-directed mutagenesis (His151Ala) that inactivated the sphingomyelinase activity also abolished the insecticidal activity, suggesting that the rapid insect toxicity of sphingomyelinase C results from its phospholipid-degrading activity.     

Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis delta-endotoxin by protein blot analysis.

Binding sites for insecticidal toxins of Bacillus thuringiensis are located in the brush border membranes of insect midguts. Two approaches were used to investigate the interactions of B. thuringiensis subsp. kurstaki HD-73 CryIA(c) toxin with brush border membrane vesicles from sensitive and naturally resistant insects: 125I-toxin-vesicle binding assays and protein blots probed with 125I-CryIA(c) toxin. In bioassays, Manduca sexta and Heliothis virescens larvae were highly sensitive, Helicoverpa zea larvae were moderately sensitive, and Spodoptera frugiperda larvae were resistant to CryIA(c) toxin. Studies of binding of 125I-CryIA(c) toxin to brush border membrane vesicles from the larval midguts revealed that all insects tested had high-affinity, saturable binding sites. Significantly, S. frugiperda larvae bind but are not killed by CryIA(c) toxin. Labeled CryIA(c) toxin incubated with protein blots identifies a major binding molecule of 120 kDa for M. sexta and 148 kDa for S. frugiperda. H. virescens and H. zea are more complex, containing 155-, 120-, 103-, 90-, and 63-kDa proteins as putative toxin-binding molecules. H. virescens also contains a minor toxin-binding protein of 81 kDa. These experiments provide information that can be applied toward a more detailed characterization of B. thuringiensis toxin-binding proteins.