Isolation Of The Entomopathogenic Nematode Symbiotic Bacteria And Studies On Its Activity And Tumor Targeting

Entomopathogenic nematode symbiotic bacteria which form mutualistic symbioses with the entomopathogenic nematodes (EPN) are the Gram-negative bacteria of family Enterobacteriaceae. They colonize the intestine of the nematodes and are divided into two genuses——Xenorhabdus sp. and Photorahbdus sp., living together with Steinernema and Heterorhabditis respectively. These bacteria show multiple broad-spectrum and high efficient bioactivities such as insecticidal, antimicrobial and antitumor activities and produce irreversible phenotypic variants, from primary form (phase I) to secondary form (phase II), when they are cultured in vitro. This research has completed the isolation and identification of entomopathogenic nematode symbiotic bacteria, studied on the multiple bioactivities and cultural characteristics of isolated strains, provided an insight into this strain as an antitumor targeting bacterium agent and it’s mode of action of antitumor, and obtained the small molecular secondary metabolites with high-efficiency antitumor activity.Using the "Galleria bait method",3 strains of entomopathogenic nematode symbiotic bacteria were isolated from soil sample of Yuelu Mountain (City of Changsha, Hunan Province, China) and De Mountain (City of Changde, Hunan Province, China). All of them were identified as Xenorhabdus sp. by a series of methods including colonial morphology, microscopy, comparison of protein patterns and the homologous analysis of 16S rRNA nucleotides. These strains were named as X. stockiae HN_xs01, X. stockiae HN_ds01 and X. stockiae HN_dsO2 and their 16S rRNA gene sequences were deposited in the GenBank database under accession number HQ840745.1, JQ219853.1 and JQ219854.1 respectively. Based on the preliminary bioactivities test, X. stockiae HN xs01 was chosen for studies on cultural characteristics as growth curve, changes of pH value, optimal growth temperature, optimal pH value and antibiotics resistance.The insecticidal, antimicrobial and antitumor activities were analyzed by contrast between primary form and secondary form of X. stockiae HN xs01. Results showed that two forms of this strain have apparent inhibitory effect on Enterobacter aerogenes, Bacillus subtillis, Lysinibacillus sp., Staphylococcus aureus, Salmonella typhimurium and Escherichia coli and primary form has better bacteriostasis effect. Two forms have no significant insecticidal activity on agriculture pests as Galleria mellonella L. and Helicoverpa armigera, and have high cytotoxicity to tumor cells as murine melanoma cell B16, human cervical carcinoma cell Hela, human epithelial carcinoma A431 and human chronic myelogenous leukemia A562. In the cell medium,2.78% of the 72 h fermentation culture supernatant of X. stockiae HN_xs01 can amount to the semi-suppressive dose to B16.In my thesis, entomopathogenic nematode symbiotic bacteria——Xenorhabdus sp. was used as an antitumor targeting bacterium agent for the first time and good effect was achieved. Mice bearing tumors were injected with X. stockiae HN_xs01 by intratumoral injection and intravenous injection, and growth of the mice and their tumors were observed. Results showed that X. stockiae HN xs01 with certain amount can grow and reproduce in tumor and significantly restrain the tumor growth and inhibit the pulmonary metastasis. In addition, X. stockiae HN_xs01 showed great tumor targeting with little toxic and side effects to organism. This strain can be removed from the liver, kidney and spleen by immune system of mice. Using HE staining, 3 modes of action of antitumor in vivo, that is anti-microangiogenesis, induction of inflammation of the tumor and production of antitumor activity secondary metabolites of this strain were concluded preliminarily.Four small molecule secondary metabolites, XS560, XS674, XS787 and XS845, with efficient antitumor activity, were purified and identified from 72 h fermentation culture supernatant of X. stockiae HN_xs01 by using adsorption chromatography, centrifugation, filtration, extraction. HPLC. LC-MS/MS and NMR. XS845 is a macrolide. XS560, XS674 and XS787 are Phenylacetamide derivatives. XS787 has the strongest antitumor activity and the semi-suppressive dose to human chronic myelogenous leukemia K562 is 1.5 μg/mL.