A single Photorhabdus gene, makes caterpillars floppy (mcf), allows Escherichia coli to persist within and kill insects

Abstract

Photorhabdus luminescens, a bacterium with alternate pathogenic and symbiotic phases of its lifestyle, represents a source of novel genes associated with both virulence and symbiosis. This entomopathogen lives in a "symbiosis of pathogens" with nematodes that invade insects. Thus the bacteria are symbiotic with entomopathogenic nematodes but become pathogenic on release from the nematode into the insect blood system. Within the insect, the bacteria need to both avoid the peptide- and cellular- (hemocyte) mediated immune response and also to kill the host, which then acts as a reservoir for bacterial and nematode reproduction. However, the mechanisms whereby Photorhabdus evades the insect immune system and kills the host are unclear. Here we show that a single large Photorhabdus gene, makes caterpillars floppy (mcf), is sufficient to allow Esherichia coli both to persist within and kill an insect. The predicted high molecular weight Mcf toxin has little similarity to other known protein sequences but carries a BH3 domain and triggers apoptosis in both insect hemocytes and the midgut epithelium.

Fig 1.

The mcf gene causes loss of insect body turgor and is encoded within a putative pathogenicity island. (A) Fifth instar M. sexta larvae 24 h after injection of 2 × 10⁶ E. coli cells containing H3 cosmid (left) and 2 × 10⁶ E. coli cells without H3 cosmid (right). Note the loss of body turgor, or floppy phenotype, in the caterpillar on the left. (B) Genomic location and insertional mutagenesis of the H3 cosmid. The 33 kb of the H3 cosmid predicts 12 ORFs (ORF numbers above open boxes). Transposon mutagenesis shows location of transposons removing floppy and lethal phenotypes (▴) or leaving both phenotypes intact (▵). The cluster of transposons that remove both phenotypes all lie within ORF nine, termed mcf. The relative location of a subclone containing only the mcf ORF is shown (E = EcoRI restriction sites). The shift in GC content (from 0.41 for the genome to 0.53 for the whole cosmid) across the region suggests that mcf lies within a pathogenicity island (see text). *, Probe used in the Southern analysis (see Fig. 2).

Fig 2.

The mcf gene predicts a novel toxin of high molecular weight. (A) Predicted amino acid sequence of the Mcf protein. The three shaded regions show regions of homology to other known proteins (see text). (B) Relative locations of the three regions of homology in relation to a Kyte-Doolittle hydrophilicity plot of the predicted protein. Note that the region homologous to the translocation domain of Toxin B is predominantly hydrophobic. (C) Match to the BH3 domain consensus sequence for Mcf toxins from two different Photorhabdus strains, W14 and K122. The consensus sequence for the BH3 motif is shown below. (D) SDS/PAGE gel of cytosolic fraction of Mcf expressing E. coli. An arrow indicates the presence of the Mcf protein (predicted weight of 324 kDa), which migrates at a higher molecular weight than the 174-kDa marker. (E) Southern blot of genomic DNA cut with EcoRI from four different Photorhabdus strains and E. coli probed with a fragment of mcf (see Fig. 1B for location of probe used). Note that the mcf DNA probe hybridizes at high stringency to all of the Photorhabdus strains but not E. coli (see text). Key to species and subspecies: P. luminescens subsp. akhurstii W14 (W14). P. luminescens subsp. laumondii TT 01(TT01), P. luminescens strain K122, which is probably subsp. temperata based on a description of its nematode host (K122), and P. asymbiotica ATCC43949 (P.a).

Fig 3.

GFP expressing E. coli carrying the mcf encoding subclone (pUC18-mcf) can persist within the insect model M. sexta. (A) Dissection of posterior of fifth instar larvae of M. sexta 16 h after injection of pUC18-mcf. Note the aggregations of GFP expressing bacteria within the fat body (Fb) and body wall (Bw) of the caterpillar. (B) High magnification of GFP-expressing bacterial clusters confirm that they consist of clumps of individual recombinant E. coli. (C and D) Parallel low magnification views of bacterial aggregations viewed under light and GFP-enhanced microscopy. Note how one of the bacterial aggregations (C) has been encapsulated by the insect hemocytes and brown melanin (arrow) deposited. A GFP enhanced view (D) of the same aggregations (arrows) shows the presence of GFP expressing and pUC18-mcf carrying E. coli. (Scale bar = 0.2 cm in A–E.) (E) Numbers of recoverable bacteria persisting over time in M. sexta. Note that E. coli carrying pUC18-mcf persist longer in an infected insect than E. coli carrying the pUC18 vector alone. E. coli carrying pUC18 alone were encapsulated as expected (data not shown).

Fig 4.

Hemocyte monolayers show that Mcf toxin acts on insect blood cells within 6 h by disrupting the cytoskeleton. (A and B) Hemocyte monolayers treated with cytosolic fractions of either pUC18 or pUC18-mcf carrying E. coli at the start of time-lapse differential interference contrast microscopy imaging. (C and D) The same preparations 6 h after treatment. Note that the plasmatocyte (P) indicated by an arrow (D) has begun to disintegrate by producing a series of rounded blebs. Note that the cell highlighted by an arrow is the same cell (B and D). Taken together with observations from TUNEL staining, the blebbing phenotype associated with Mcf-mediated hemocyte death is strongly supportive of programmed cell death. The full time lapse series of Mcf toxin action can be viewed at www.bath.ac.uk/bio-sci/quicktime.htm. (E and F) FITC-phalloidin staining of hemocyte monolayers treated either with cytochalasin D or Mcf. Note that treatment with both toxins disintegrates the actin cytoskeleton leaving a punctate staining pattern of residual actin. (Scale bar = 10 μm.)

Fig 5.

M. sexta infected with Mcf encoding E. coli show massive destruction of the midgut epithelium. (A) Light microscopy of a stained control section of M. sexta fourth instar midgut. Note the midgut epithelium (Me), which separates the lumen (L) of the gut from the insect hemocoel (H). (B) Section of midgut 16 h after infection with pUC18-mcf E. coli. The cells of the midgut have begun to bleb into the gut lumen (arrows). (C) Section of midgut 24 h after infection. The midgut epithelium has disintegrated leaving the space between the basal lamina (Bl) and the peritrophic membrane (Pm) packed with blebbed cells. (D) Detail of disintegrated epithelium showing that nuceli within the cellular blebs are pycnotic and often surrounded by vacuoles. (E) TUNEL staining of disintegrated epithelium suggesting that the cells are undergoing apoptosis. (Scale bar = 100 μm.)