Insecticidal Pilin Subunit from the Insect Pathogen Xenorhabdus nematophila

Abstract

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.

Bacteroides: the Good, the Bad, and the Nitty-Gritty

“…These vesicles appear in transmission electron microscopy as surface blebs and detached extracellular vesicles and have been shown to have a hemagglutinin function (185) and to contain sialidase activity that may be correlated to virulence (78). Similar outer membrane vesicles have been observed in Porphyromonas gingivalis, Pseudomonas fragi, and Xenorhabdus nematophilus, where it has been suggested that they may serve as a vehicle for toxins and attachment to host cells (80,109,132). Furthermore, similar vesicles in Pseudomonas aeruginosa have been shown to carry quinolone signaling molecules for intercellular signaling (153).…”

An Insecticidal GroEL Protein with Chitin Binding Activity from Xenorhabdus nematophila

“…The proteins were applied on the surface of artificial diet and allowed to percolate down. One neonatal larva (24 h old) of H. armigera was released on the surface of the diet in each well (19), and the plates were incubated at 25°C (16-h-day-length periods) with 80% relative humidity. Mortality and larval weight were recorded periodically over the entire larval period.…”

Role of Mrx Fimbriae of Xenorhabdus nematophila in Competitive Colonization of the Nematode Host

“…MrxA was shown to be associated with outer membrane vesicles (OMVs) that exhibited oral toxicity toward the lepidopteran Helicoverpa armigera (25). Purified MrxA exhibited cytotoxic properties and recombinant MrxA displayed oral toxicity and functioned as a pore-forming toxin on targeted insect hemocytes (5,26).…”