Functional Study Of The Type ? Secretion System In Entomopathogenic Nematode Symbiotic Bacteria

Entomopathogenic nematode symbiotic bacteria which form symbioses with the entomopathogenic nematodes are the Gram-negative bacteria.They are divided into Xenorhabdus spp.and Photorahbdus spp.In nature,these bacteria are mainly found in nematodes.A variety of virulence factors are secreted by bacteria to help nematodes kill insects.Studies have shown that the type III secretion system(T3SS)is the key secretion system for pathogenic bacteria to secrete virulence factors,and the functional mechanism of T3 SS in the symbiotic bacteria of insect pathogens has not been elucidated.In the previous research,the research team cloned the T3 SS synthetic gene cluster from Photorhabdus luminescens TT01 strain and recombined the conjugation and transfer elements into the recombinant plasmid,laying the foundation for heterologous expression of T3 SS in Xenorhabdus stockiae HN_xs01.In this study,a recombinant plasmid carrying the P.luminescens TT01 strain T3 SS synthetic gene cluster was transferred into the HN_xs01 strain by means of conjugative transfer.Whole genome scanning showed that the P.luminescens TT01 strain T3 SS synthetic gene cluster was successfully integrated into the chromosome of the HN_xs01 strain.Compared with the wild-type strain,the invasion ability of the recombinant strain HN_xs01-T3 SS on the midgut cell CF-203 was significantly enhanced.At the same time,bioassay experiments also showed that the recombinant strain HN_xs01-T3 SS had stronger injection toxicity to the fourth instar larva of cotton bollworm than the wild type strain HN_xs01.Using proteomics technology,we identified candidate type III effector proteins XopA and GogB analogs from the recombinant strain HN_xs01-T3 SS.Next,we identified candidate effector proteins XopA and GogB.First,the XopA and GogB prokaryotic expression vectors were constructed,and the XopA and Gog B prokaryotic expression vectors were successfully transferred into the wild-type strain HN_xs01 and the recombinant strain HN_xs01-T3 SS,respectively.Through cell incubation experiments,we found that the XopA protein in the recombinant strain HN_xs01-T3 SS can be efficiently secreted into CF-203 and HeLa cells,while the XopA protein in the wild type strain cannot be efficiently secreted into CF-203 and HeLa cells,confirming that XopA is T3 SS effector from HN_xs01 strain.In order to further study the mechanism of action of XopA protein,we constructed XopA eukaryotic expression vector and transfected it into HeLa cells.Western blot results showed that XopA protein was successfully expressed in HeLa cells;and the expression of XopA protein in cells would cause the cells to shrink and a large number of vacuoles appeared in the cells.MTT experiments and cell scratch experiments showed that XopA protein inhibited the proliferation and migration of HeLa cells.Then observed by transmission electron microscope: it was found that XopA protein caused the characteristics of apoptotic cells in HeLa cells such as cell shrinkage,a large number of vacuoles in the cell,and the nucleus was condensed and divided.At the same time,Hoechst33342 staining observation revealed that the nuclear characteristics of apoptotic cells,such as densely stained blocky blue fluorescence and nuclear fragmentation,further confirmed that XopA protein induced apoptosis in HeLa cells.By detecting the expression of apoptosis-related proteins in cells,it was found that XopA protein can cleave PARP protein and increase the activity of Caspase-3.Immunofluorescence results showed that XopA protein was localized on the cell membrane of HeLa cells,and XopA protein caused the cytoskeleton microtubules to disaggregate and the mitochondrial membrane potential decreased.Finally,co-immunoprecipitation and mass spectrometry were used to screen XopA-binding proteins for backbone proteins and heat shock protein HSP 90-?.In summary,in the functional study of T3 SS in HN_xs01,we found that T3 SS can enhance the pathogenicity of HN_xs01 and the invasion of eukaryotic cells.At the same time,we proved that XopA protein is an effector in HN_xs01,and explored the cytotoxicity of this protein on human tumor cells and the mechanism of its cytotoxicity,laying a theoretical foundation for the application research of this type of protein.