Understanding the mechanisms of host-pathogen interaction can provide crucial information for successfully manipulating their relationships. Because of its genetic background and practical advantages over vertebrate model systems, the nematode Caenorhabditis elegans model has become an attractive host for studying microbial pathogenesis. Here we report a "Trojan horse" mechanism of bacterial pathogenesis against nematodes. We show that the bacterium Bacillus nematocida B16 lures nematodes by emitting potent volatile organic compounds that are much more attractive to worms than those from ordinary dietary bacteria. Seventeen B. nematocidaattractant volatile organic compounds are identified, and seven are individually confirmed to lure nematodes. Once the bacteria enter the intestine of nematodes, they secrete two proteases with broad substrate ranges but preferentially target essential intestinal proteins, leading to nematode death. This Trojan horse pattern of bacterium-nematode interaction enriches our understanding of microbial pathogenesis.Bacillus nematocida | Caenorhabditis elegans | chemotaxis | pathogen-host interaction | virulence protease M ost model organisms, such as the yeast Saccharomyces cerevisiae, the slime mold Dictyostelium discoideum, the mouseear cress plant Arabidopsis thaliana, the common fruit fly Drosophila melanogaster, and the nematode Caenorhabditis elegans, can be infected by microbes, including certain human-pathogenic bacteria (1). For several reasons, C. elegans is an attractive model organism to study host-pathogen interactions: It has simple growth requirements, a short generation time, a well-defined developmental process with invariant cell lineage sorting, a fully sequenced genome, and a suite of well-established genetic tools (2). Using C. elegans as a model, scientists in the last few years have identified a diversity of physical, chemical, and biochemical features involved in microbial pathogenesis (3). For example, Brevibacillus laterosporus secretes extracellular proteases that damage nematode cuticle, and Bacillus thuringiensis produces toxic crystal proteins that disrupt host cellular functions (4, 5). The common humanpathogenic bacterium Pseudomonas aeruginosa kills C. elegans with quorum-sensing controlled-virulence factors (6) and cyanide (7). Several other human pathogens such as the Gram-negative bacteria Burkholderia pseudomallei and Serratia marcescens and the Gram-positive bacteria Enterococcus faecalis, Streptococcus pyogenes, and Staphylococcus aureus also are reported to have nematoxic activities via a neuromuscular endotoxin, a cytolysin, two extracellular proteases (gelatinase and serine protease), and several other toxins (8-12). In S. aureus, several virulence determinants known to be important in mammalian pathogenesis, including the quorum-sensing global virulence regulatory system agr and the global virulence regulator sarA, the alternative sigma factor B, α-hemolysin, and the V8 serine protease, are all required for full pathogenicity against nematode...

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Lecanicillium psalliotae produced an extracellular protease (Ver112) which was purified to apparent homogeneity giving a single band on SDS-PAGE with a molecular mass of 32 kDa. The optimum activity of Ver112 was at pH 10 and 70 degrees C (over 5 min). The purified protease degraded a broad range of substrates including casein, gelatin, and nematode cuticle with 81% of a nematode (Panagrellus redivivus) being degraded after treating with Ver112 for 12 h. The protease was highly sensitive to PMSF (1 mM) indicating it to be a serine protease. The N-terminal amino acid residues of Ver112 shared a high degree of similarity with other cuticle-degrading proteases from nematophagous fungi which suggests a role in nematode infection.

A neutral protease (npr) (designated Bae16) toxic to nematodes was purified to homogeneity from the strain Bacillus nematocida. The purified protease showed a molecular mass of approximately 40 kDa and displayed optimal activity at 55 degrees C, pH 6.5. Bioassay experiments demonstrated that this purified protease could destroy the nematode cuticle and its hydrolytic substrates included gelatin and collagen. The gene encoding Bae16 was cloned, and the deduced amino acid sequence showed 94% sequence identity with npr gene from B. amyloliquefaciens, but had low similarity (13-43%) with the previously reported virulence serine proteases from fungi or bacteria, which reflected their differences. Recombinant mature Bae16 (rm-Bae16) was expressed in Escherichia coli BL21 using pET30 vector system, and its nematicidal activity confirmed that Bae16 could be involved in the infection process. Our present study revealed that the npr besides the known alkaline serine protease could serve as a potential virulence factor in the infection against nematodes, furthermore, the two proteases with different characteristics produced by the same strain co-ordinated efforts to kill nematodes. These data helped to understand the interaction between this bacterial pathogen and its host.