Insect Control: Biological and Synthetic Agents - Index of

320 9: Insecticidal Toxins from Photorhabdus and Xenorhabdus
are proapoptotic (Budd, 2001), leading to the working
hypothesis that Mcf itself may also promote apoptosis.
Interestingly, if proved, this would be the first
BH3 domain found outside of eukaryotes.
The observation that E. coli expressing Mcf alone
can survive in the presence of the insect immune
system is unexpected. Normally, E. coli carrying a
pUC18 plasmid are cleared from the insect, presumably
via the action of both antibacterial peptides
and the insect phagocytes or hemocytes. In
contrast, E. coli carrying pUC18-mcf and a second
green fluorescent protein (GFP) expressing plasmid
persist longer in the hemolymph. Dissection of infected
animals shows that the Mcf and GFP expressing
bacteria can colonize tissues in the insect and
that the hemocytes fail to encapsulate the bacteria in
melanin. Therefore, it was speculated that this failure
to encapsulate Mcf expressing bacteria was due
to hemocytes, dying via Mcf induced apoptosis. To
investigate whether Mcf can promote apoptosis in
hemocytes, the hemocyte monolayers were exposed
to recombinant Mcf. The results show that exposed
hemocytes, disintegrate rapidly, within 6 h, by producing
numerous characteristic blebs. The residual
actin cytoskeleton also shows a characteristic punctate
pattern of staining. This proapoptotic effect on
hemocytes helps to explain how recombinant E. coli
can persist in the insect but fails to explain how the
insects lose body turgor and die.
To examine the cause of the floppy phenotype, the
histopathology of insects infected with E. coli
expressing Mcf was investigated. Given that both
the Malpighian tubules and the midgut are responsible
for osmoregulation, destruction of either of these
organs could result in a rapid loss of body turgor.
Sections of infected caterpillars showed that the
Malpighian tubules were intact at the time of death
but cells of the midgut epithelium were severely
affected as early as 12 h post injection. Both goblet
and columnar cells shed circular blebs, often including
the nucleus, into the midgut lumen. Nuclei within
the dying cells appear pycnotic and stain terminal
deoxynucleotidyl transferase-mediated duTP end
labeling (TUNEL) positive, suggesting that they are
apoptotic. These observations are consistent with
Mcf having a proapoptotic function and with its
primary site of action being the insect midgut. However,
some caution is necessary in interpreting these
results as insects ingesting B. thuringiensis Cry of Vip
toxins also show a similar histopathology of the
midgut. More specific experiments to show that
Mcf is proapoptotic are therefore needed.
Comparison of the genomic location of mcf1-like
genes between Photorhabdus strains confirms two
important points (Figure 7). First, mcf1-like genes
appear to be present in all strains of Photorhabdus,
as would be expected if Mcf1 is a dominant toxin.
Second, like the tc toxins, the location of mcf1 differs
between genomes and is often associated with
either a Phe tRNA or integrase genes. In fact in
strain TT01, mcf1 is adjacent to a Phe tRNA on
one side and the downstream of the tccC locus,
suggesting that in this case both a tc and mcf locus
may be found together on the same pathogenicity
Figure 7 Comparison of mcf1 containing loci from four different Photorhabdus strains. Note that mcf1 lies alongside a Phe tRNA in
both W14 and TT01; however, W14 also carries palBA. In the other two strains sequenced, K122 and ATCC43949, mcf1 appears to
occupy different genomic locations suggesting that the toxin is mobile within the Photorhabdus genome.