The Study On The Enzyme Active Components And Mechanism Of SrfABC Complex Toxin

Xenorhabdus sp.is a symbiotic species of insect pathogens with Steinernema sp.which secretes a variety of insecticidal,bacteriostatic and anti-tumor active metabolites that interfere with host immune response,leading to the death of the insect hosts.In the previous study,we constructed the Fosmid genomic library of X.stockiae HN_xs01strain,and screened out the complex toxin SrfABC,which was obviously toxic to insect cells and tumor cells,and found that there were interactions between the three proteins.In this study,the activity of each component of SrfABC complex toxin and its mechanism of action were studied,which laid the foundation for the function of this toxin in the pathogenic and antitumor activities of Xenorhabdus.The GST-tagged vector was used to express SrfA,SrfB and SrfC proteins and assayed the cytotoxicity to Helicoverpa armigera by bioassay,and the three were the most toxic.The midgut cell lines CF-203/2.5 and human cervical cancer cell line HeLa are used as targets.It was found by microscope and MTT that SrfA,SrfB and SrfC proteins exhibited cytotoxicity against CF-203/2.5 and HeLa,and the toxicity is the greatest when the three are mixed.It was found by Hoechst/PI double staining and flow cytometryn show that SrfA,SrfB and SrfC proteins can destroy cell membrane integrity and cause apoptosis,and the three cells have the greatest damage to cell membrane integrity in CF203/2.5 cells.And all three proteins can block the cell cycle in the G2/M phase.Next,we investigated the localization of the components of the SrfABC toxin in cells.The results of Immunofluorescence showed that SrfA,SrfB and SrfC proteins were concentrated in the cytoplasm region in CF203/2.5 cells alone or mixed;SrfA protein was concentrated in the cytoplasm,SrfB protein was concentrated in the nucleus,SrfC protein was distributed in the cytoplasm when it alone,and is transferred from cytoplasm to nucleus during mixing in HeLa cells.Western blot showed that SrfA,SrfB,and SrfC proteins were able to enter insect cells and tumor cells with independently and high efficiently.In order to study the mechanism of action of the components of SrfABC toxin,we transfected the expression vectors of mammalian cells and insect cells of SrfA,SrfB and SrfC into tumor cell HeLa and insect cell Sf9,respectively.The results showed that SrfA,SrfB and SrfC proteins are successfully expressed in HeLa cells.The binding proteins were Skeletal protein mainly by immunoprecipitation and mass spectrometry.Western blot showed that an extra band appeared in HeLa cells transfected with SrfA protein,and its molecular weight was larger than the target band.It was analyzed that the expression of SrfA protein in tumor cells was post-translationally modified,and it was treated by site-directed mutagenesis and glycosidase.Its modification was initially excluded as glycosylation.This study explored the cytotoxicity of SrfABC toxins on human tumor cells and insect cells,and identified the location of each component in cells.The enzyme active components of the ternary compound toxin were preliminarily analyzed,and the mechanism of the toxicity of SrfABC toxoid in cells was preliminarily grasped.This study provide a theoretical basis for the study of the toxins in pathogenic microorganisms against host defense,virulence and host colonization.