A Novel Secreted Protein Toxin from the Insect Pathogenic Bacterium Xenorhabdus nematophila

Brown, Susan E.; Cao, Anh T.; Hines, Eric R.; Akhurst, Raymond J.; East, Peter D.

doi:10.1074/jbc.m309859200

Cited by 49 publications

(37 citation statements)

References 45 publications

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“…Several factors in X. nematophila that are directly responsible for the death of insects or assist in the pathogenic process have been described (6,14,16,26). Outer membrane proteins of the pathogenic bacteria perform several critical functions in the host environment, such as recognition and interaction with the target host cells (3), transport of effector proteins (22), and secretion of virulence factors and antibacterial proteins (31).…”

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Insecticidal Pilin Subunit from the Insect Pathogen Xenorhabdus nematophila

et al. 2004

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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.

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Insecticidal Pilin Subunit from the Insect Pathogen Xenorhabdus nematophila

et al. 2004

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“…High-molecular-weight genomic DNA was isolated from P. luminescens strain V16 (31). The DNA was partially digested with Sau3AI and used to create a cosmid library, as described previously (9). This library was screened by hybridization using the previously discovered A24tox gene from X. nematophila strain A24 as a probe (9).…”

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confidence: 99%

“…We recently described a novel toxin, A24tox, from X. nematophila strain A24. This 42-kDa toxin is a secreted protein that is lethal to the larvae of insects such as Galleria mellonella and Helicoverpa armigera when it is injected at doses of 30 to 40 ng/g larvae (9).…”

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“…Injection of the Tca complex of P. luminescens strain W14 or ingestion by Manduca sexta larvae damaged the midgut cells, resulting in shedding of the midgut epithelium into the gut lumen, followed by lysis of the epithelium (5). Injection of A24tox into lepidopteran larvae caused the larvae to cease feeding almost immediately, and preliminary histological studies indicated that the main site of action for the toxin was the insect midgut (9). Many other insecticidal toxins are known to act on the insect gut, including the well studied ␦-endotoxins from Bacillus thuringiensis and cholesterol oxidase from Streptomyces (5).…”

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

Brown

Cao

Dobson

et al. 2006

Appl Environ Microbiol

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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.

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“…X. nematophila is adapted to two diverse environmental niches, the nematode gut and the insect larval body, in which it produces potent toxins to kill its larval prey (6,7,24,29).…”

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The Cytotoxic Fimbrial Structural Subunit of Xenorhabdus nematophila Is a Pore-Forming Toxin

et al. 2006

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We have purified a fimbrial shaft protein (MrxA) of Xenorhabdus nematophila. The soluble monomeric protein lysed larval hemocytes of Helicoverpa armigera. Osmotic protection of the cells with polyethylene glycol suggested that the 17-kDa MrxA subunit makes pores in the target cell membrane. The internal diameter of the pores was estimated to be >2.9 nm. Electron microscopy confirmed the formation of pores by the fimbrial subunit. MrxA protein oligomerized in the presence of liposomes. Electrophysiological studies demonstrated that MrxA formed large, voltage-gated passive-diffusion channels in lipid bilayers.

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A Novel Secreted Protein Toxin from the Insect Pathogenic Bacterium Xenorhabdus nematophila

Cited by 49 publications

References 45 publications

Insecticidal Pilin Subunit from the Insect Pathogen Xenorhabdus nematophila

Insecticidal Pilin Subunit from the Insect Pathogen Xenorhabdus nematophila

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

The Cytotoxic Fimbrial Structural Subunit of Xenorhabdus nematophila Is a Pore-Forming Toxin

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