Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth

Abstract

Two recently sequenced genomes of the insect-pathogenic bacterium Photorhabdus and a large Serratia entomophila plasmid, pADAP, have phage-related loci containing putative toxin effector genes, designated the "Photorhabdus virulence cassettes" (PVCs). In S. entomophila, the single plasmid PVC confers antifeeding activity on larvae of a beetle. Here, we show that recombinant Escherichia coli expressing PVC-containing cosmids from Photorhabdus has injectable insecticidal activity against larvae of the wax moth. Electron microscopy showed that the structure of the PVC products is similar to the structure of the antibacterial R-type pyocins. However, unlike these bacteriocins, the PVC products of Photorhabdus have no demonstrable antibacterial activity. Instead, injection of Photorhabdus PVC products destroys insect hemocytes, which undergo dramatic actin cytoskeleton condensation. Comparison of the genomic organizations of several PVCs showed that they have a conserved phage-like structure with a variable number of putative anti-insect effectors encoded at one end. Expression of these putative effectors directly inside cultured cells showed that they are capable of rearranging the actin cytoskeleton. Together, these data show that the PVCs are functional homologs of the S. entomophila antifeeding genes and encode physical structures that resemble bacteriocins. This raises the interesting hypothesis that the PVC products are bacteriocin-like but that they have been modified to attack eukaryotic host cells.

FIG. 1.

PVC loci of P. asymbiotica. (Top panel) Conserved PVC unit consisting of 13 to 15 open reading frames encoding predicted proteins with high levels of similarity to different phage components. Note the variable number of putative effector proteins encoded at one end of each PVC locus. These effectors are either unnamed or bear the name of the protein to which they exhibit the highest level of similarity in a BlastX search (see text). (Bottom panel) 2D gels of E. coli proteins prepared from recombinant bacteria with (left gel) and without (right gel) the PaPVCpnf cosmid. The circled proteins are unique to PaPVCpnf-expressing E. coli, and three of the most abundant of these proteins were confirmed to be PVC-encoded proteins by picking the spots from the gel, analyzing them by MALDI-TOF analysis, and subsequently matching predicted tryptic digests of all PVC-encoded proteins. Orf 1, Orf 2, and Orf 3 correspond to the first three open reading frames in the PaPVCpnf unit.

FIG. 2.

Transmission electron micrographs of PVC products, showing their similarity to R-type pyocins. (A) Relaxed PVC product, showing only the outer sheath (arrow). (B) Contracted PVC product, showing extrusion of the inner needle (white arrow) from outer sheath (black arrow).

FIG. 3.

Mortality of Galleria larvae into which E. coli expressing individual PVC-containing cosmids was injected. (A) Percentage of larvae showing blackening at 24 h after injection of either whole PVC-expressing E. coli cultures (left) or culture supernatant of PaPVCpnf alone (right). Note that all the PVC-expressing cosmids from P. asymbiotica (PaPVC) caused blackening in 80 to 100% of the larvae, whereas those from P. luminescens (PlPVC) exhibited a phenotype in only 10 to 40% of the larvae. (B) Larvae into which only supernatants from E. coli expressing PaPVCpnf was injected showed rapid blackening and death within 30 min. (C) Control larvae into which a heated supernatant from E. coli expressing PaPVCpnf was injected showed no effects.

FIG. 4.

Decrease in the number of recoverable hemocytes associated with injection of PVC-expressing E. coli and effect on the actin cytoskeleton. (A) Histogram comparing the number of hemocytes recovered 30 min after injection of heat-treated PaPVCpnf supernatants (Control mean) to the number recovered after injection of native supernatant. Note the dramatic decrease in the number of recoverable hemocytes following injection of the native supernatant from PVC-expressing E. coli. (B) Control hemocytes stained with TRITC-phalloidin. Note the numerous actin-rich projections in healthy Galleria hemocytes. (C) Hemocytes recovered from Galleria larvae into which E. coli carrying a cosmid expressing PaPVCpnf was injected. Note the marked condensation of the cell body and the remnant bundle of condensed actin in the remaining dead hemocytes.

FIG. 5.

Expression of PVC effectors in mammalian tissue culture cells. (A) NIH-3T3 cells transfected with the pRK5myc (red) vector alone and cotranfected with a second construct expressing actin-EGFP (green) had a normal actin cytoskeleton. Nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI) (blue). (B) Cells transfected with pRK5myc expressing the Mcf-like effector Plu1690 contained only fragments of successfully transfected cells which showed actin condensation. (C) Cells transfected with pRK5myc expressing the Pnf-like effector had contracted cell bodies and condensed actin. (D) Cells transfected with pRK5myc expressing the SepC-like effector had remarkable actin-rich cabling, a phenotype never seen in cells from the pRK5myc transfected controls.

FIG. 6.

Transposon mutagenesis of PaPVCpnf. The positions of insertions in the PVC that either disrupt toxicity (solid triangles) or maintain toxicity (open triangles) are indicated. Note that insertions that disrupt toxicity are all clustered in the center of the PVC unit in a region that contains the gene encoding the putative Lop-T-like effector.