Isolation And Identification Of A Surface Protein(SP84) Of Nosema Bombycis Associated With Spore Invasion

The microsporidia are obligatory, intracellular parasites found in members of all animal phyla, and are most important pathogens of insects, fish and human, as well as promising protozoa for use in pest control. Spore germination is a prerequisite process during invasion, and certain spore surface protein is related to the spore germination and invasion mechanism. The studies on the microsporidian surface proteins crucial to invasion are important for controlling the microsporidian infection and utilization. In our experiment, a monoclonal antibodies (Mab) 3C2, against an exospore protein of the microsporidium Nosema bombycis (N. bombycis) was prepared, and its effects on microsporidial germination and reproduction in vitro were studied and we identified a surface protein (SP84) involved in invasion The antigen was isolated and identified by two-dimensional gel electrophoresis and MS-MS.The present results provided some crucial information to reveal the mechanism of microsporidian invasion.1.Preparation of monoclonal antibodies (Mab) against exospore of the microsporidium Nosema bombycis (N. bombycis).Seven Mabs 1A6,3B1,3C1,3C2,3C3,3C4,3F1 were developed against N. bombycis after infection, cell fusion, clone and ascites production. The reactivity of antibodies with mature and immature N. bombycis and also another microsporidium (Endoreticulatus-like microsporidium) were assessed by ELISA. It showed that only 1A6 strongly cross-reacted with the Endoreticulatus-like microsporidium, while the others did not cross-react with this microsporidium. The Mabs were capable of differentiating between N. bombycis and Endoreticulatus-like microsporadium (Esp) which showed lower infectivity to Bombyx mori. The method of indirect ELISA based on the Mabs was sensitive and was used for identifying the surface proteins associated with invasion and also for detecting N. bombycis in sericulture crops and silkworm seed inspection. 2. Pretreatment of spores with Mab 3C2 inhibited N. bombycis germination in vitro.There was considerable reduction in the number of germinated spores as monitored spectrophotometrically. When the spores were pre-incubated for 24 h with Mab 3C2 and 1A6 at different concentrations compared with spores pre-incubated with an irrelevant isotype Mab with same concentrations (p<0.05).3C4 showed a similar effect (p<0.05). Other Mabs did not strongly affect the germination of spores (p<0.05).3. Pre-exposure of spores to Mab 3C2 inhibited N. bombycis infecting Bombyx mori cells in vitro.Mab 3C2, which produced the strongest inhibition of N. bombycis spore germination was selected for this study in which 1 X 106 spores were cultured with 1 mg/mL of either Mab 3C2 or isotype control Mab. Pretreatment of spores with Mab 3C2 for 1 h reduced the amount of infected Bombyx mori cells up to 48.6%(72 h post inoculation) compared with isotype control Mab 3E12 (p<0.05); To determine whether the effect of continuous exposure of N. bombycis to anti-exospore Mab 3C2 on microsporidial reproduction, N. bombycis/BmN cell cultures were maintained in media containing either Mab 3C2 or control isotype Mab at different concentrations. Cultures were fixed and stained with DAPI after inoculation. The number of infected cells was significantly lower in Mab 3C2-treated cultures compared to control isotype Mab-treated cultures (p<0.05) from 12 to 96 h post-culture.4. Analysis of antigen by SDS-PAGE and 2D-Electrophoresis.Immunoblotting revealed a 84 kDa protein corresponding to pI (7.2) on the 2D gel.The SDS-PAGE profiles of N. bombycis exhibited 11 distinct proteins of 84 kDa,59 kDa,55 kDa,46 kDa,37 kDa,34 kDa,31 kDa,26 kDa,17 kDa,14 kDa,12 kDa.The protein profile of 2D-PAGE using 120μg of spore protein, most of proteins were small molecules with neutral or acidic isoelectric point (<18kDa, pI 5-7). The antigen which recognized by Mab 3C2 was a 84 kDa protein corresponding to pI 7.2 and revealed it was low abundant.5. Monoclonal antibody-based immunogold electron microscopy and indirect immuno-fluorescence test (IFAT).The IFAT of N. bombycis with the Mab 3C2 detected bright green fluorescence; but the IFAT of N. bombycis extracted spore wall proteins by SDS with the monoclonal antibody 3C2 did not show fluorescence. The immunogold reaction showed that Mab 3C2 bound exclusively to the exospore. The reaction was specific and Mab 3C2 demonstrated a generalized localization to antigens-not clear. There was no immunogold reaction in the control sample using Mab 3E12 (data not shown).6. Co-Immunoprecipitation (Co-IP) revealed there was protein-protein interactions between SP84 and other spore proteins.SP84 was identified by MS-MS as gi|19074407|ref|NP₅85913.1| hypothetical protein ECU06₁560 [Encephalitozoon cuniculi GB-M1], the protein P34 was identified as tubulin alpha chain, it must be confirmed because of the lower protein cover percent. The studies on microsporidian protein SP84 is useful in analyzing crucial information to elucidate the mechanism of microsporidian invasion.