Influence Of XeGroEL Protein From Xenorhabdus Ehlersii On Immune Responses In Galleria Mellonella

The bacteria Xenorhabdus spp. are entomopathogenic symbionts that can produce several toxicproteins that interfere the immune system of insects, which facilitate the successful infection ofentomopathogenic nematode Steinernema. We purified a toxic protein with injectable activity fromXenorhabdus ehlersii, and the toxic protein can interferet the cellular and humoral immune response inGalleria mellonella；we cloned the gene and expressed it in Escherichia coli. We determined the effectof XeGroEL on proteome and gene transcriptal level by comparative proteomic analyses andRealtime-qPCR. Our results help to understand anti-microbial immune responses in G. mellonella,providing a meaningful hypothesis for the interaction between nematode-symbiotic bacteria and host.The main results are as summarized as follows:1. A toxic protein with injectable activity was purified and identified.A toxic protein from X. ehlersii was purified by ammonium sulfate precipitation, sequential HiTrap QHP, HiTrap Butyl FF and RESOURCE Q chromatography, and identified as a GroEL homology with ahighest matching against a GroEL from X. nematophila by LC-MS/MS on HDMS High DefinitionMass Spectrometry system. The apparent molecular weight and isoelectric point were estimated as58kDa and5.0by2-D PAGE. G. mellonella larva injected with XeGroEL presented melanization after10min, and dead in48h. An LD50of0.76±0.08μg/larva was calculated.2. Effect of XeGroEL on hemocytes immune and PO activity in G. mellonella was determined.After injecting XeGroEL into the G. mellonella hemolymph, the total hemocyte number decreased,especially the number of plasmatocytes, and a minor reduction in hemocyte numbers was observed byincubation in vitro, however plasmatocytes were greatly diminished in both treatments. XeGroEL canweaken the encapsulation of G. mellonella hemocytes by injection or incubation in vitro, whilepromoted the phagocytosis of G. mellonella hemocytes in vitro. XeGroEL increased the PO activity inhemolymph,and the effect was more intensive in vivo.3. XeGroEL gene was cloned and then expressed in E. coli, and the polyclonal antibody againstrecombinant XeGroEL was prepared.According to the MS result, degenerate primers were designed for PCR, XeGroEL with1647bpencoding548amino acid residues is a ubiquitous house keeping geng in bacteria, phylogenetic analysisshowed that XeGroEL is a valuable molecular marker in evolution analysis of enterobacteriaceae;sequence anlignment showed that GroEL of symbiotics had lower conservation than that of free-livingbacteria, and the multifunctionality of GroEL in symbiotics may be related to several unconservativeposition. Alignment of XeGroEL and XnGroEL indicates that substitution at17positions may attributedto the different activity. In addition, we expressed the XeGroEL ligating to pET28a vector, andpolyclonal antibody was prepared using purified recombinant XeGroEL.4. The changes of proteome and transcriptal level in XeGroEL-challenged G. mellonella weredeteimined.we demonstrated that XeGroEL can promote the expression of some immune-related factors acting as an effective immunogen by comparative proteomic analyses and Realtime-qPCR. The expressionlevel of PGRPs (PGRP-LB, PGRP-A and PGRP-B), antimicrobial peptides (anionic antimicrobialpeptide2, gallerimycin, cecropin, gloverin), antidotic proteins (ferritin and GST),27kDa hemolymphprotein and apolipophorin-3in XeGroEL-challenged larvae were all increased. Additionally,co-immunoprecipitation presented2hemolymph proteins binding with XeGroEL, which provided thebasis for further interaction study.