A Trojan horse mechanism of bacterial pathogenesis against nematodes

Abstract

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. nematocida-attractant 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.

Fig. 1.

PA and NA of four strains of B. nematocida against C. elegans. PA of culture filtrate of strain B16 using 0.2 M casein as the substrate was 7.9 U/mL (SD 0.3) = 100%. Means ± SD of three replicates were plotted.

Fig. 2.

Microscopic examination of B. nematocida strain B16 target sites. (A) Both the intestine and cuticle of nematodes were intact when treated with E. coli. (B) Structures of pharynx, muscle, and intestine were disorganized when treated with B. nematocida strain B16. (C) Nematodes in the E. coli-treated control group had smooth undisturbed surfaces with a healthy cuticle structure that included the regular striae and lateral lines. (D) Nematodes infected with B. nematocida strain B16 showed a lightly exfoliated cuticle. (E) The cross-section of an untreated, healthy nematode showed a highly ordered and compact intestinal structure. (F) The cross-section of a nematode infected with B. nematocida strain B16 showed numerous defects including fusion, vesiculation, and loosening of various organs. (G) Low-magnification TEM of the midgut of the control nematode showed ordered, densely arrayed, and normal-looking microvilli. (H) Microvilli in strain B16-infected nematodes appeared destroyed with significant membrane-tethering defects. Arrows indicate healthy (G) and damaged (H) and microvilli.

Fig. 3.

Intestinal damage in C. elegans by the two proteases when infected by strain B19. In each panel, subpanels A, B, C, and D represent, respectively, nematodes observed under visible light, Bace16-GFP fluorescence, Bae16-DsRed fluorescence, and an overlay of the two fluorescent signals. (A) Live C. elegans 2 h after microinjection. (B) Dying C. elegans 5 h after microinjection. (C) Dead C. elegans 8 h after microinjection. (D) Decomposed C. elegans 24 h after microinjection.

Fig. 4.

Assays of AA and NA in soil. (A) The scheme for AA assays in soil (details are given in SI Materials and Methods). (B) Results of assays of AA in soil. (C) Activity levels in soil at points A, B, and C as determined by distances from the bacterial inoculum. (D) Assays for different NA levels in soil. (E) Assay of the nematode-immobilizing ability of B. nematocida strain B19 in natural soil.