Purification And Pathogenetic Mechanism Of Haemocoel Insecticidal Toxin From Xenorhabdus Nematophila

Xenorhabdus nematophila is insect pathogen bacteria symbiotically associated with entomopathogenic nematode Steinernema carpocapsae, which is motile gram-negative bacteria belonging to the family of Enterobacteriaceae. The infective juvenile nematodes can carry and release the bacteria into the haemocoel of host-insect. The bacteria produce a number of toxins to overcome immune response of insect hosts and kill their hosts. To explore the lethal mechanisms of the bacteria, we isolated and characterized two novel haemocoel insecticidal toxins by using methods of salting out and native-PAGE from the intracellular proteins of X. nematophila HB310. The purified proteins were named Tp40 and Tp60 respectively. The SDS-PAGE spectrum of Tp40 and Tp60 were all showed a single band which were about 42 kDa and 60 kDa respectively. The injectable hemocoelic potency (LD₅₀) of Tp40 and Tp60 at 48 h were 68.54 ng/larva and 181.5 ng/larva respectively against fifth-instar Galleria mellonella larvae.Western blotting with an antibody that was highly specific to the known Txp40 indicated that Tp40 was homologous with Txp40 and only existed inside cells. The determined nucleotide sequences of tp40 gene have been deposited in GenBank (accession number GenBank: EU095326). The size of the open reading frame of tp40 was 1107 bp, encoding a peptide of 368 amino acid residues, with a theoretical predicted molecular weight 41.5 kDa and an isoelectricpoint (IP) 8.66. The Tp40 shared 85%～99% homology of nucleotide sequences and 70%～99% amino acids with those of 13 other symbiotic bacteria with entomopathognic nematodes. Tp40 showed high injectable virulent for G. mellonella larvae. The larvae injected with the dose of more than (70±0.02) ng/larva of Tp40 rapidly died in 20 min. The larvae started to exhibit an exciting symptom, and quickly moved right away after Tp40-injection. However the larvae move more and slower after several minutes, the Tp40-injected larvae raised its head and chest, the mouth kept opening and closing rhythmically. After 10 min, the larvae showed the symptom of strong spasm that the feet and mouth quivered and the body twitched, finally died. The dead larvae turned soft but no phenomenon of dehydration, and the color of body did not change. After death for 6 h, the chest and head of the dead larva became black. The activity of the carboxylesterase (CarE) and the acetylcholinesterase (AchE) of G. mellonella larvae injected with the LD₅₀ dose of Tp40 were obviously higher than that of PBS-control (P < 0.05), respectively. However, the polyphenoloxidase (PO) activity of the Tp40-injected larvae was markedly lower than that of the control (P < 0.05).Tp60 was digested in-gel by trypsin and identified as GroEL protein by matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS). Following Tp60-injection, a rapid, extensive hemolymph melanization occurred going with obvious changes of the polyphenoloxidase (PO) activity in G. mellonella larvae from high to be low, the carboxylesterase (CarE) activity increased obviously. However the acetylcholinesterase (AchE) activity did change distinctly.The study on the histopathology of the midgut of G. mellonella indicated that Tp40 and Tp60 all could destroyed the epithelium and the peritrophic membrane of the midgut.These results suggested that Tp40 and Tp60 were likely to be associated with the immunosuppressant of X. nematophila. The midgut tissues of the host may be the acting target of Tp40 and Tp60.