Txp40, a ubiquitous insecticidal toxin protein from Xenorhabdus and Photorhabdus bacteria

Abstract

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.

FIG. 1.

Schematic diagram showing the regions where amino acid insertions and deletions occur in the Txp40 toxin. The diagram was constructed from the alignment of the amino acid sequences of toxins from 23 species of Xenorhabdus and Photorhabdus. aa, amino acids.

FIG. 2.

Inhibition of cell growth observed for various doses of recombinant Txp40_A24 protein in the in vitro cytotoxicity assay with A. aegypti, S2, Sf21, Sf9, and SP2 cells. Inhibition is expressed as a percentage relative to the growth of the control cells, which were treated with MBP. For each cell type, the inhibition with 0, 0.2, 2, 20, 200, and 2,000 ng/ml of Txp40_A24 is shown. As there was no significant difference between the growth of the toxin-treated cells and the growth of the MBP-treated cells at doses of 0.002 and 0.02 ng/ml, the data for these concentrations were omitted for clarity.

FIG. 3.

Histological images of toxin-treated H. armigera larvae, showing the effect of recombinant toxin on the midgut. All images (magnification, ×90) are images of longitudinal sections through the anterior region of the midguts of larvae inoculated with 100 ng of either MBP or Txp40_A24 toxin. BM, basement membrane; E, midgut epithelium; L, midgut lumen; PM, peritrophic matrix. The open arrowheads indicate spaces between cells of the gut epithelium, the solid arrowheads indicate breakdown of the basement membrane, the asterisk indicates cells sloughed into the midgut lumen, and the multiplication signs indicate rafts of unidentified material in the midgut lumen. (a, c, and e) Larvae treated with MBP. (b, d, and f) Larvae treated with Txp40_A24. Tissues were fixed at zero time (a and b) and at 12 h (c and d) and 18 h (e and f) after toxin injection.

FIG. 4.

Histological images of toxin-treated H. armigera larvae, showing the effect of recombinant toxin on the peritrophic matrix. All images are images of longitudinal sections through the anterior region of the midguts of larvae inoculated with 100 ng of either MBP or Txp40_A24 toxin. Tissues were fixed 12 h after injection of the toxin, and autofluorescence was monitored after sections were treated with citric acid and heat. BM, basement membrane; E, midgut epithelium; L, midgut lumen; PM, peritrophic matrix. The multiplication signs indicate rafts of unidentified material in the midgut lumen. (a and c) Larvae following MBP treatment. (b and d) Larvae following Txp40_A24 treatment. (a and b) Magnification, ×370. (c and d) Magnification, ×1,100.

FIG. 5.

Histological images of toxin-treated H. armigera larvae, showing the effect of recombinant toxin on the midgut and the fat body. All images are images of longitudinal sections through the anterior region of the midguts of larvae inoculated with either MBP or Txp40_A24 toxin. BM, basement membrane; E, midgut epithelium; L, midgut lumen; FB, fat body; PM, peritrophic matrix. The open arrowheads indicate spaces between cells of the gut epithelium, the solid arrowheads indicate breakdown of the basement membrane, the asterisks indicate cells sloughed into the midgut lumen, the multiplication sign indicates rafts of unidentified material in the midgut lumen, and N indicates fat body nuclei that show signs of damage. (a and c) Larvae following treatment with 100 ng MBP. (b) Larvae following treatment with 100 ng Txp40_A24. (d) Larvae after treatment with 10 ng Txp40_A24. Tissues were fixed at 18 h after injection of toxin. (a and b) Magnification, ×230. (c and d) Magnification, ×370.