The Establishment And Application Of Spiroplasma Infection Of Drosophila S2 Cell Model

Spiroplasma is a type of microrganism with a spiral shape,motility and no cell wall.Its host is widely distributed.Among them,Spiroplasma eriocheiris was discovered from Eriocheir sinensis with the tremor disease(TD)and a new type of spiroplasma pathogen found in aquatic crustaceans for the first time.Previous studies have shown that Spiroplasma eriocheiris entered into crustaceans' body through the gills or skin,and and propagated in hemocytes(target cells).And the pathogen was brought to the connective tissues of crustaceans' organs along with blood circulation.After infection,crustaceans became inactive,did not have any food at early stage of infection and then emerged tremor symptoms at late stage of infection.S.eriocheiris can not only infect aquatic crustaceans in the natural environment,but also infect chicken embryos and suckling mice,leading to cause cataract in mice.In order to effectively control the disease caused by S.eriocheiris,it is necessary not only to study the biological characteristics of pathogenic bacteria,but also to study the behavior and mechanism of target cells infected by the bacteria at the cellular level.Drosophila S2 cells are used as a model to research the behave of spiroplasma,due to the lack of a crustacean cell line.Therefore,this paper established a model of S.eriocheiris infected Drosophila S2 cells and used this model to study the function of Esserpin-2 and EscatD in vitro.1.The model of Spiroplasma eriocheiris infected Drosophila S2 cellIn this study,a cell infected model was established by S.eriocheiris strain TDA-040725-5T infected invertebrate Drosophila S2 cells in vitro.The morphology of S2 cells was observed by inverted fluorescence microscopy.It was found that the proliferation of S2 cells gradually decreased along with the prolongation of infection time.Flow cytometry was used to detect the apoptosis of S2 cells infected by S.eriocheiris for 48 h.And it was found that S2 cells' apoptosis was significantly increased.The detection of reactive oxygen species showed that the fluorescence value of S.eriocheiris was highest after infection S2 cells for 9 h,indicating that S2 cells produced an immune response to S.eriocheiris.Immunofluorescence technique was utilized to observe the distribution of S.eriocheiris in S2 cells.It was found that S.eriocheiris infected S2 cells with mainly a single individual.Transmission electron microscopy(TEM)was used to show the S2 cells infected by S.eriocheiris.It was found that S.eriocheiris entered the cells and formed many inclusion bodies.And the infection of S.eriocheiris can cause vacuolization of S2 cells.When S2 cells were pretreated with drugs to blocked clathrin-mediated endocytosis,including chlorpromazine and Dynasore,S.eriocheiris infected S2 cells was strongly inhibited.Inhibition of key regulators of macropinocytosis,such as protein kinase C(PKC)and myosin II,significantly reduced S.eriocheiris infection.In contrast,disruption of cellular cholesterol by methyl-?-cyclodextrin and nystatin had no effect on bacterium infection.These results suggested that S.eriocheiris entered S2 cells by the clathrin-dependent endocytosis and macropinocytosis but not via caveolae-mediated endocytic pathway.In addition,intracellular S.eriocheiris motility was dramatically attenuated by cytoskeleton-depolymerizing agents including nocodazole and cytochalasin B,indicating that the movement of S.eriocheiris in cells depended on actin filaments or microtubules.These results for the first time has successfully shown that S.eriocheiris could invade Drosophila S2 cells thus providing an ideal model for exploring the behavior of S.eriocheiris in living cells.And it laid the foundation to study the mechanism of invertebrate cells infected by S.eriocheiris in the future.2.Functional study of Esserpin-2 in S2 cellsThe function of Esserpin-2 in vitro was studied using the model of S2 cells.The subcellular localization experiment suggested that recombinant Esserpin-2 was mainly located in the cytoplasm.Finally,over-expression assay in Drosophila S2 cells indicated that Esserpin-2 could increase copies of S.eriocheiris and result in S2 cell death.3.Functional study of EscatD in S2 cellsThe model of S2 cells was used to study the function of EscatD in vitro.The subcellular localization experiment suggested that recombinant EscatD was mainly located in the cytoplasm.The over-expression in Drosophila S2 cells indicated that EscatD could decrease the copy number of S.eriocheiris and increase S2 cell viability.These results indicated that the function of Esserpin-2 and EscatD in vitro could be elucidated by the model of S2 cells.And the S2 cells' model could be used to explore the behavior of S.eriocheiris in living cells.Furthermore,it helped us better understand the underlying mechanisms about the interaction between S.eriocheiris and the host.