Purification And Pathogenetic Mechanism Of Insecticidal Protein From Xenorhabdus Budapestensis D43

The entomopathogenic bacteria, symbiotically associate with different families ofnematodes, has been described to produce a range of metabolites that have insecticidal,antibacterial，anticancer activity, and so on. At present, these bacteria have been regardedas an important microbial resource and have huge development potential and goodapplication prospect. In particular, entomopathogenic bacteria can produce a range ofnovel toxin proteins that could be directly applied as protein insecticides or used in thetransgenic plants and microbes. Xenorhabdus budapestensis D43live in symbiosis withentomopathogenic nematode Steinernema ceratophorum, which was isolated fromnortheast China soils by using the insect bait entrapment method. In this study, we directlypurified new intracellular protein fractions from X. budapestensis D43that were screenedby insect bioassay. Subsequently, new insecticidal protein was studied in various aspectsinclude protein identification, gene cloning, bioinformatic analysis, recombinant proteinexpression and purification, and pathogenetic mechanism. The main results of this studycan be summarized as follows:1. In this report, we purified four protein fractions from the intracellular extract of X.budapestensis D43by preparative native PAGE. However, only one protein exhibitedinsecticidal activity against Galleria mellonella larvae by injection that was designatedHIP57. By injection, HIP57caused G. mellonella larval bodies to blacken and die with anLD50of206.81ng/larva. Especially, when we injected no less than490ng of HIP57perlarva, almost all G. mellonella larvae developed blackened body color within15min anddead after forty-eight hours.2. HIP57has a molecular weight (MW) of57379.7Da and a theoretical isoelectric point(pI) of4.77. The full-length1647bp sequence of hip57was obtained by sequencing and itssequence has been deposited in GenBank (accession no. JN863588), whereas its deducedamino acid sequence of548amino acids was deposited as AEU10771(GenBank accessionno.). HIP57protein is homologous to GroEL proteins from other bacteria. GroEL has beenaccepted as molecule chaperon, however, our research revealed that HIP57(GroEL)possesses another novel function as an insecticide in X. budapestensis D43.3. A bioinformatic analysis suggested that signal peptide is absent in HIP57protein, whichis stable and soluble in water. HIP57secondary structure prediction has been performed byfour different methods. At the same time, we predicted the accurate three-dimensional structure of HIP57with high accuracy by homology modeling. By multiple sequencealignment, we found that HIP57contained several specific amino acid residues, which didnot appear in any of the other four GroEL proteins from the family of mutualistic bacteriaat the same positions. A phylogenetic tree was constructed base on the homologues offorty-three GroEL proteins from the family of Enterobacteriaceae. In the phylogenetic tree,X. budapestensis D43were successfully classified into Xenorhabdus, which indicated thatGroEL have potential application in bacterial taxonomy and evolution.4. The recombinant HIP57, GFP and HIP57-GFP proteins were respectively expressed andpurified from transformed E. coli. Western blotting analysis show that these recombinantproteins could bind specifically to antibodies, respectively. Compared with the case ofoccurring melanism involve darkening of the whole body of wax moth larvae injected withnatural HIP57protein, recombinant HIP57protein did not show similar virulence.However, HIP57-GFP recombinant proteins was enable for the formation of black nodulein the haemocoel of larvae, but not lethal.5. G. mellonella larvae were injected with HIP57, resulting in extensive immune responsesinclude a significant increase in PO activity, a significant decline in hemocyte populationsand encapsulation response of hemocytes, compared to the controls. Our results illustratepronounced changes in the expression of four genes encoding hemolymph proteins,including ApoLp-III、Serpin、P27K and GST, were observed after HIP57exposure. Weinvestigated the hemolymph proteins that interact with HIP57protein usingco-immunoprecipitation. In vitro two interactions were arylphorin and β-1,3-glucanrecognition protein precursor, which were identified.In this research we unsuccessfully answer the problem that is if HIP57was secreted byentomopathogenic nematode-bacteria complex during infection host insect. However,HIP57show high lethal potency in the G. mellonella larva bioassay, thereby hypothesizingthat the bacteria could secret HIP57, which may play an important role inentomopathogenic nematode-bacteria complex infecting host insect. Even if the abovehypothesis is not confirmed, HIP57has research significance and application potency as aresult of excellent pathogenicity and novel insecticide mode. The results of my study forPh.D. degree are of greatly importance to elucidate the pathogenetic mechanism of HIP57,which will be useful for appropriate application of HIP57.