A Photorhabdus Natural Product Inhibits Insect Juvenile Hormone Epoxide Hydrolase

Simple urea compounds (“phurealipids”) have been identified from the entomopathogenic bacterium Photorhabdus luminescens, and their biosynthesis was elucidated. Very similar analogues of these compounds have been previously developed as inhibitors of juvenile hormone epoxide hydrolase (JHEH), a key enzyme in insect development and growth. Phurealipids also inhibit JHEH, and therefore phurealipids might contribute to bacterial virulence.

larvae that were infected with a suspension of P. luminescens TT01 cells. For this, an overnight culture of TT01 was washed with LB and adjusted to an optical density of OD 600 1.0 and each larva was injected by syringe in the midabdominal proleg with 5 µl of cell suspension. Aseptic LB medium was used as a control. All infected larvae were dead after 48 hours, while 95% of the control group survived. Each sample was snap-frozen in liquid nitrogen. The frozen larvae were ground to a crude powder using a pestle and mortar. The powder was extracted for 1 hour with 50 mL of a methanol/acetone (1:1) solvent mixture, filtered and evaporated. The crude extract was re-dissolved in 3 mL of methanol and diluted 1:10 before HPLC-MS analysis. Production kinetics were analysed using triplicates in LB broth. P. luminescens TT01 was cultivated in 1 L Erlenmeyer flasks containing 100 mL LB using the described cultivation conditions. For each time point 4 mL of culture broth was extracted with the same solvent mixture, filtered and the solvents removed under reduced pressure. The crude extract was then dissolved in 1 mL methanol and without further dilution submitted to HPLC-MS analysis.

Construction of the carbamoyl transferase and the methyl transferase mutants.
Carbamoyl transferase and methyl transferases encoded in the genome of P. luminescens TT01 [13] were identified using their homology to known enzymes from E. coli and by comparison between all Xenorhabdus [14,15] and Photorhabdus [13,16] genomes currently available. The genes plu2076 and plu2237 were only present in P. luminescens and encode a carbamoyl transferase and a methyl transferase, respectively. They were disrupted by plasmid integration as described previously. Briefly, the suicide vector pDS132 [17] or pCKcipB (a pDS132 derivative with an additional BglII recognition site) both carrying a chloramphenicol resistance gene (cat) were used. An internal fragment of plu2237 (pliB) was amplified with λpir phage lysogen). Subsequently this construct was conjugated into a rifampcin-resistant TT01 strain as previously described [1] .
The plasmid donor strain and the acceptor strain were grown to an OD 600 of 0. then injected into first day 5 th instar Manduca sexta larvae, which were reared as previously described [18] . Briefly, larvae were maintained individually at 25˚C under a photoperiod of 17 hours light: 7 hours dark and fed on an artificial diet based on wheat germ. Unless otherwise stated, larvae 1 day after ecdysis to the 5th instar were used for all experiments.
For the two strains approximately 100, 1000 and 10000 viable cells (confirmed by overnight plating) were injected into a cohort of 12 animals each, which were then monitored for mortality (movement and turgidity) by regular physical stimulus over 3 days and again at 7 days.
To induce immune responses in G. mellonella larvae were injected with the Data were analyzed as described previously [22] . Statistics. Four datasets with 6 PBS control embryos, 6 DMSO control embryos and 12 embryos for each compound/concentration were recorded. Embryos that completed dorsal closure properly were scored as completing the development. Significance was determined by the two-sided student's t-test.

Chemical synthesis of glycine amides and phurealipids.
If not noted differently, the chemicals were purchased in the highest purity available.
General synthetic procedure I. 1 eq of the corresponding isocyanides were dissolved in acetone (4 mL/mmol) and 2 eq of the amine were added. The reaction mixture was stirred overnight and the forming precipitate recrystallized.
General synthetic procedure II. 1 eq of the free acid was dissolved in benzene (45 mL/mmol) and 6 eq of triethylamine and 3 eq of diphenylphosphoryl azide were added. After refluxing for approximately 1h the reaction mixture was cooled to room temperature, acetone (30 mL/mmol) and ca. 10 eq of methylamine (40% in water) or ammonium hydroxide (28 -30% in water) were added and the mixture stirred at room temperature overnight. The solvents were removed under reduced pressure and the precipitate purified with normal phase liquid chromatography or recrystallization.  The iso-branched fatty acid was purchased with Larodan AB (Limhamn, Sweden).
Recrystallization from acetone yielded the white crystalline urea (