| Xenorhabdus sp. is motile gram-negative, rod-shaped, facultatively anaerobic bacteria, which is insect pathogen bacteria symbiotically associated with entomopathogenic nematode Steinernema carpocapsae sp.. This bacteria can produce a variety of toxins in insect intestines and interfere with the host’s immune response, eventually kill the host insect.Our previous work has separated X. stockiae HN_xs01. To discover its toxicity, we constructed the X. stockiae Fosmid genome library. It consisted of 12950 clones, which was preserved in 80 tubes. We randomly selected 24 clones and used Not I to digest DNA. The result showed that the inserted fragments size may be 30-45 kb, and no-load condition does not exist. The library covers 7.2 times of the pathogenic bacterium genome, and obtained probability of any single copy DNA sequence is 99.9%. Fosmid stability assays indicated that the library was stable during propagation in the fosmid system. In order to facilitate the follow-up screenning work, to reduce the workload, to rapid positioning objective monoclonal, we constructed secondary pools to our second screen work.When using the midgut cell lines CF203/205(from Choristoneura fumiferan), IPLB-Sf9(from pupal ovarian tissue of Spdoptera frugiperda) and a variety of tumor cells as target cells, FS2 cloning tube was found has a broad spectrum of cell toxicity. Nextly, we built the secondary pools of FS2 to confirm that the clone FS2C1 was the positive one to CF203/2.5. Through the sequencing and homology analysis, we found FS2C1 clone contained the srfABC homologous cassette and it was tentatively named srfABC. In order to study the role of SrfABC toxins in the host colonization and virulence, the srfABC was deleted by the Red/ET recombination. Compared with the wild type FS2C1, the mutant lost its cellular toxicity, which showed that SrfABC toxin are the main cytotoxin factor of FS2C1.In this study, we constructed the srfABC expression vector and delected vector of C components, then extract total proteins to cell toxicity experiment analysis. The result showed that SrfABC plays an important role in cell toxicity, but C component does not contribute to the cytotoxcity. When cell was treated by SrfABC toxin in low concentration, the intact cytoskeleton, nuclear shrinkage and mitochondria disappeared of CF203/2.5 cell were observed by confocal laser microscopy. moreover, cytoskeleton depolymerized, nuclear shrinkage and mitochondria disappeared in high concentration toxin. We deduce that the potent target of srfABC may be mitochondria. DNA Ladder multiplying degradation experiment indicated cell apoptosis. In addition, the cell toxicity mechanism of srfABC remains to be study.In this study, we also constructed SrfA, SrfB, SrfC were heterologously expressed in E.coli by IPTG induction respectively. When incubated these proteins with midgut cell lines CF203/2.5 from C. fumiferana, we found that SrfA exhibited obvious cytotoxicity in low concentration, but SrfB and SrfC exhibited inhibition cell proliferation only in high concentration. We deduce that SrfA component could be the enzyme active component, and SrfB and SrfC components may be the auxiliary components in the complex.This study laid a foundation for further study on the mechanism of SrfABC toxin, contributed to insure SrfABC toxin resistance to host defense in pathogenic bacteria strains, toxicity and the important role of colonization in the host. Our research also provide a theory basis for pest control by pathogenic bacteria, and offer new idea to basic research, disease therapy and biological control. |
PDF Full Text Request