Characterization of a cytotoxic pilin subunit of Xenorhabdus nematophila

Xenorhabdus nematophila is an insect pathogenic bacterium, known to produce protein toxins that kill the larval host. We have described a cytotoxic pilin subunit of X. nematophila, which is expressed on the cell surface and also secreted in the extracellular medium associated with outer membrane vesicles. A 17kDa pilin subunit was isolated and purified from X. nematophila cell surface. The protein showed cytotoxicity to larval hemocytes of Helicoverpa armigera in an in vitro assay, causing agglutination of the cells, and releasing cytoplasmic enzyme lactate dehydrogenase in the medium. The pilin protein was able to bind to the surface of larval hemocytes. The binding and cytotoxicity of the purified 17kDa protein to hemocytes was inhibited by antiserum raised against the pilin protein. The study demonstrates for the first time a cytotoxic structural subunit of pilin from an entomopathogenic bacterium X. nematophila that is excreted in the extracellular medium with outer membrane vesicles.     

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.     

A novel secreted protein toxin from the insect pathogenic bacterium Xenorhabdus nematophila

The bacterium Xenorhabdus nematophila is an insect pathogen that produces several proteins that enable it to kill insects. Screening of a cosmid library constructed from X. nematophila strain A24 identified a gene that encoded a novel protein that was toxic to insects. The 42-kDa protein encoded by the toxin gene was expressed and purified from a recombinant system, and was shown to kill the larvae of insects such as Galleria mellonella and Helicoverpa armigera when injected at doses of around 30-40 ng/g larvae. Sequencing and bioinformatic analysis suggested that the toxin was a novel protein, and that it was likely to be part of a genomic island involved in pathogenicity. When the native bacteria were grown under laboratory conditions, a soluble form of the 42-kDa toxin was secreted only by bacteria in the phase II state. Preliminary histological analysis of larvae injected with recombinant protein suggested that the toxin primarily acted on the midgut of the insect. Finally, some of the common strategies used by the bacterial pathogens of insects, animals, and plants are discussed.     

The hmsHFRS operon of Xenorhabdus nematophila is required for biofilm attachment to Caenorhabditis elegans

The bacterium Xenorhabdus nematophila is an insect pathogen and an obligate symbiont of the nematode Steinernema carpocapsae. X. nematophila makes a biofilm that adheres to the head of the model nematode Caenorhabditis elegans, a capability X. nematophila shares with the biofilms made by Yersinia pestis and Yersinia pseudotuberculosis. As in Yersinia spp., the X. nematophila biofilm requires a 4-gene operon, hmsHFRS. Also like its Yersinia counterparts, the X. nematophila biofilm is bound by the lectin wheat germ agglutinin, suggesting that beta-linked N-acetyl-D-glucosamine or N-acetylneuraminic acid is a component of the extracellular matrix. C. elegans mutants with aberrant surfaces that do not permit Yersinia biofilm attachment also are resistant to X. nematophila biofilms. An X. nematophila hmsH mutant that failed to make biofilms on C. elegans had no detectable defect in symbiotic association with S. carpocapsae, nor was virulence reduced against the insect Manduca sexta.     

Xenorhabdus nematophila发酵动力学研究

在分批发酵中,研究了Xenorhabdus nematophila YL001的生长、基质消耗及抗菌物质产生的特性.基于Logistic方程和Luedeking-Piret方程,得到了描述分批发酵过程的动力学模型及模型参数,同时对实验数据与模型进行了验证比较.模型计算值与实验数据拟合良好,模型基本反映了Xenorhabdus nematophila YL001分批发酵过程的动力学特征.分批发酵中细胞生长与产物合成属于偶联型.     

The thermostable direct hemolysin of Vibrio parahaemolyticus is a pore-forming toxin.

The hemolytic mechanism of thermostable direct hemolysin (TDH), a possible virulence factor of Vibrio parahaemolyticus, was studied. We demonstrated that TDH acts as a "pore-forming toxin" in temperature-dependent and -independent steps. The first temperature-dependent step requires only about 1-2 min incubation at 37 degrees C and makes a "pore" with a functional diameter of approximately 2 nm. The pore size was deduced from the molecular diameter of the colloidal inhibitory polysaccharides. The formation of the pores on TDH-treated erythrocyte membranes was also demonstrated by electron microscopic examination. The second step, which is a temperature-independent lytic step, causes the erythrocytes to swell owing to a colloidal osmotic influx of water via the "pores" into cells, resulting in erythrocyte lysis (or rupture) owing to increased intracellular pressure.     

Identification and functional characterization of a Xenorhabdus nematophila oligopeptide permease

The bacterium Xenorhabdus nematophila is a mutualist of Steinernema carpocapsae nematodes and a pathogen of insects. Presently, it is not known what nutrients the bacterium uses to thrive in these host environments. In other symbiotic bacteria, oligopeptide permeases have been shown to be important in host interactions, and we therefore sought to determine if oligopeptide uptake is essential for growth or symbiotic functions of X. nematophila in laboratory or host environments. We identified an X. nematophila oligopeptide permease (opp) operon of two sequential oppA genes, predicted to encode oligopeptide-binding proteins, and putative permease-encoding genes oppB, oppC, oppD, and oppF. Peptide-feeding studies indicated that this opp operon encodes a functional oligopeptide permease. We constructed strains with mutations in oppA(1), oppA(2), or oppB and examined the ability of each mutant strain to grow in a peptide-rich laboratory medium and to interact with the two hosts. We found that the opp mutant strains had altered growth phenotypes in the laboratory medium and in hemolymph isolated from larval insects. However, the opp mutant strains were capable of initiating and maintaining both mutualistic and pathogenic host interactions. These data demonstrate that the opp genes allow X. nematophila to utilize peptides as a nutrient source but that this function is not essential for the existence of X. nematophila in either of its host niches. To our knowledge, this study represents the first experimental analysis of the role of oligopeptide transport in mediating a mutualistic invertebrate-bacterium interaction.     

Identification and Functional Characterization of a Xenorhabdus nematophila Oligopeptide Permease

The bacterium Xenorhabdus nematophila is a mutualist of Steinernema carpocapsae nematodes and a pathogen of insects. Presently, it is not known what nutrients the bacterium uses to thrive in these host environments. In other symbiotic bacteria, oligopeptide permeases have been shown to be important in host interactions, and we therefore sought to determine if oligopeptide uptake is essential for growth or symbiotic functions of X. nematophila in laboratory or host environments. We identified an X. nematophila oligopeptide permease (opp) operon of two sequential oppA genes, predicted to encode oligopeptide-binding proteins, and putative permease-encoding genes oppB, oppC, oppD, and oppF. Peptide-feeding studies indicated that this opp operon encodes a functional oligopeptide permease. We constructed strains with mutations in oppA₁, oppA₂, or oppB and examined the ability of each mutant strain to grow in a peptide-rich laboratory medium and to interact with the two hosts. We found that the opp mutant strains had altered growth phenotypes in the laboratory medium and in hemolymph isolated from larval insects. However, the opp mutant strains were capable of initiating and maintaining both mutualistic and pathogenic host interactions. These data demonstrate that the opp genes allow X. nematophila to utilize peptides as a nutrient source but that this function is not essential for the existence of X. nematophila in either of its host niches. To our knowledge, this study represents the first experimental analysis of the role of oligopeptide transport in mediating a mutualistic invertebrate-bacterium interaction.     

Epsilon toxin: a fascinating pore-forming toxin

Epsilon toxin (ETX) is produced by strains of Clostridium perfringens classified as type B or type D. ETX belongs to the heptameric β-pore-forming toxins including aerolysin and Clostridium septicum alpha toxin, which are characterized by the formation of a pore through the plasma membrane of eukaryotic cells consisting in a β-barrel of 14 amphipatic β strands. By contrast to aerolysin and C. septicum alpha toxin, ETX is a much more potent toxin and is responsible for enterotoxemia in animals, mainly sheep. ETX induces perivascular edema in various tissues and accumulates in particular in the kidneys and brain, where it causes edema and necrotic lesions. ETX is able to pass through the blood-brain barrier and stimulate the release of glutamate, which accounts for the symptoms of nervous excitation observed in animal enterotoxemia. At the cellular level, ETX causes rapid swelling followed by cell death involving necrosis. The precise mode of action of ETX remains to be determined. ETX is a powerful toxin, however, it also represents a unique tool with which to vehicle drugs into the central nervous system or target glutamatergic neurons.     

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.     

A toxin protein from Xenorhabdus nematophila var. pekingensis and insecticidal activity against larvae of Helicoverpa armigera

Xenorhabdus nematophila var. pekingensis, which is highly virulent for many insects, is a symbiotic bacterium of Steinernema carpocapsae isolated from Beijing soil in China. Previous studies demonstrated that the bacterium had high antifeedant activity against larvae of Helicoverpa armigera, Plutella xylostella and Spodoptera exigua. Herein, we report the purification, molecular cloning and antifeedant activity of an intracellular toxic protein from the bacterium. The purified protein displayed a single band and a relative molecular weight of over 212 kDa determined by SDS-PAGE. We designated the protein as XnAFP2. Peptide segments were obtained by MALDI-TOF and covered 40% of the amino acid sequence of a toxin protein from X. nematophilus PMFI1296. The full cDNA sequence encoding for XnAFP2 (Genbank accession number FJ222606) was amplified from X. nematophlia var. pekingensis and consists of 7575 bp. The gene showed homology with up to 99% identity to the A2 gene from X. nematophila strain BP (GenBank accession number AY282763) and 92% identity to the insecticidal toxin xptA2 gene from X. nematophila PMFI 1296 (GenBank accession number AJ308438). The protein caused a rapid cessation in feeding and reduction in larval weight of H. armigera. When fed to third instar larvae of H. armigera in an artificial diet at 6.0 µg/g (w/w) toxin protein, growth reduction reached 97.9%. The insecticidal protein greatly decreased fourth instar larval weight, lengthened larval stage, and reduced pupation and emergence rates. The antifeedant rate in choice and no-choice leaf disk tests against fifth instar larvae was 78.4 and 87.6% in 24 h, respectively.     

Friend and foe: the two faces of Xenorhabdus nematophila

Comparisons of mutualistic and pathogenic relationships are necessary to decipher the common language of microorganism-host interactions, as well as the subtle differences in dialect that distinguish types of symbiosis. One avenue towards making such comparisons is to study a single organism that speaks both dialects, such as the gamma-proteobacterium Xenorhabdus nematophila. X. nematophila inhabits and influences the lives of two host animals, helping one to reproduce optimally while killing the other.     

Enhanced antibiotic activity of Xenorhabdus nematophila by medium optimization

Nutrition had highly influence on the antibiotic production by Xenorhabdus nematophila YL001. Glucose and peptone were identified as the best carbon and nitrogen sources that significantly affected antibiotic production using one-factor-at-a-time approach. Response surface methodology was applied to optimize the medium constituents (Glucose, peptone and minerals) for antibiotic production by X. nematophila YL001. Higher antibiotic activity (328.9 U/ml) was obtained after optimizing medium components. The optimal levels of medium components were (g/l): glucose 6.13, peptone 21.29, MgSO(4).7H(2)O 1.50, (NH(4))(2)SO(4) 2.46, KH(2)PO(4) 0.86, K(2)HPO(4) 1.11 and Na(2)SO(4) 1.72. An overall 16% and 35% increase in antibiotic activity were obtained as compared with mean observed response (283.7U/ml) at zero level of all variables and YSG medium.     

The Xenorhabdus nematophila nilABC genes confer the ability of Xenorhabdus spp. to colonize Steinernema carpocapsae nematodes

Members of the Steinernema genus of nematodes are colonized mutualistically by members of the Xenorhabdus genus of bacteria. In nature, Steinernema carpocapsae nematodes are always found in association with Xenorhabdus nematophila bacteria. Thus, this interaction, like many microbe-host associations, appears to be species specific. X. nematophila requires the nilA, nilB, and nilC genes to colonize S. carpocapsae. In this work, we showed that of all the Xenorhabdus species examined, only X. nematophila has the nilA, nilB, and nilC genes. By exposing S. carpocapsae to other Xenorhabdus spp., we established that only X. nematophila is able to colonize S. carpocapsae; therefore, the S. carpocapsae-X. nematophila interaction is species specific. Further, we showed that introduction of the nilA, nilB, and nilC genes into other Xenorhabdus species enables them to colonize the same S. carpocapsae host tissue that is normally colonized by X. nematophila. Finally, sequence analysis supported the idea that the nil genes were horizontally acquired. Our findings indicate that a single genetic locus determines host specificity in this bacteria-animal mutualism and that host range expansion can occur through the acquisition of a small genetic element.     

嗜线虫致病杆菌CB6菌株培养特性的初步研究

研究了嗜线虫致病杆菌(Xenorhabdus nematophila)CB6菌株的菌体增殖规律及对主要营养成分的利用规律。结果表明,该菌延缓期、对数生长期、稳定期、衰亡期分别为0~6h、6~18h、18~48h和48h以后。培养18h时,糖和蛋白质的含量达到最低并保持稳定;氨基氮含量在6h时最低,以后逐渐升高,36h达到最高。药效实验表明,该菌培养42h时其菌悬液杀虫活性最高。     

Analysis of the PixA inclusion body protein of Xenorhabdus nematophila

The symbiotic pathogenic bacterium Xenorhabdus nematophila produces two distinct intracellular inclusion bodies. The pixA gene, which encodes the 185-residue methionine-rich PixA inclusion body protein, was analyzed in the present study. The pixA gene was optimally expressed under stationary-phase conditions but its expression did not require RpoS. Analysis of a pixA mutant strain showed that PixA was not required for virulence towards the insect host or for colonization of or survival within the nematode host, and was not essential for nematode reproduction. The pixA gene was not present in the genome of Xenorhabdus bovienii, which also produces proteinaceous inclusions, indicating that PixA is specifically produced in X. nematophila.     

Trade-offs shape the evolution of the vector-borne insect pathogen Xenorhabdus nematophila

Our current understanding on how pathogens evolve relies on the hypothesis that pathogens' transmission is traded off against host exploitation. In this study, we surveyed the possibility that trade-offs determine the evolution of the bacterial insect pathogen, Xenorhabdus nematophila. This bacterium rapidly kills the hosts it infects and is transmitted from host cadavers to new insects by a nematode vector, Steinernema carpocapsae. In order to detect trade-offs in this biological system, we produced 20 bacterial lineages using an experimental evolution protocol. These lineages differ, among other things, in their virulence towards the insect host. We found that nematode parasitic success increases with bacteria virulence, but their survival during dispersal decreases with the number of bacteria they carry. Other bacterial traits, such as production of the haemolytic protein XaxAB, have a strong impact on nematode reproduction. We then combined the result of our measurements with an estimate of bacteria fitness, which was divided into a parasitic component and a dispersal component. Contrary to what was expected in the trade-off hypothesis, we found no significant negative correlation between the two components of bacteria fitness. Still, we found that bacteria fitness is maximized when nematodes carry an intermediate number of cells. Our results therefore demonstrate the existence of a trade-off in X. nematophila, which is caused, in part, by the reduction in survival this bacterium causes to its nematode vectors.