Screening And Function Of Mutant Proteins In The Blood Cells Of Macrobrachium Rosenbergii Infected By

The freshwater prawn Macrobrachium rosenbergii, also known as Malaysia prawns, long arm of Freshwater prawns, one of the largest freshwater individuals, is currently the major one of the three freshwater shrimps in China. Since 1976, when M. rosenbergii was originally introduced into China, its farming area and production expanded rapidly. However, with its constant expansion of the farming scale and intensive degree, diseases caused by bacteria or viruses are becoming more and more serious, which also include a novel pathogen Spiroplasma eriocheiris. Early researches showed that, S. eriocheiris entered into crab body through the gills or body surface, invaded its target cell-semi-granular cells, propagated in hemocytes, then be carried to connective tissue by infected hemocytes, invaded into neural and muscle organization. The diseased crab was weakness and did not eat any food at early stage of infection and then tremored at later stage of infection. How does the special pathogen adhered and infected the host target cells. Which proteins play important roles between pathogen-host interactions. Firstly, we identified the different expression proteins with iTRAQ technology. The prawns was infected by spiroplasma, then total proteins of hemocytes was extracted at the seven days of infection. We try to study the possible mechanism of S. eriocheiris infection and host defense through proteomics. Secondly, the candidate receptor proteins in hemocytes of M. rosenbergii were screened by Far-western blotting experiment. Then, bacterial binding activity and confocal laser experiment were conducted, receptor functon was analyzed by specific double-stranded RNA. Finally, ligands of the spiroplasmas were identified using Far-western blotting, hemocytes of M. rosenbergii was cultured in vitro, the function of ligand protein was investigated through competitive experiments. Results of this paper play important roles in studying the mechanism of infection and host immunity, this study includes the following six aspects:1. Identify different hemocytes proteins of M. rosenbergii with iTRAQ during spiroplasma infectionThe changes for number and kinds of M. rosenbergii hemocytes proteins during S. eriocheiris infection were studied by iTRAQ, which was dynamic, overall and quantitative. The identified proteins, with clear function, were related with binding, catalysis, structure and transfer function. Using a 1.5-fold change in expression as a physiologically significant benchmark,69 differentially expressed proteins were reliably quantified by iTRAQ analysis. Up-regulated proteins included 6 immune related protein,14 physiological protein,25 intracellular proteins,4 unknown or hypothetical proteins. Down-regulated proteins include 4 immune related protein,3 cytoskeletal proteins,11 physiological protein,2 unknown or hypothetical proteins. Seven proteins were selected to conduct qRT-PCR, and the fluctuation was basically consistent with the iTRAQ.2. Far-western blotting experiment to screening receptors of M. rosenbergii hemocytesM. rosenbergii hemocyte proteins was separated by SDS-PAGE, then transferred onto PVDF membranes, incubated with formalin-fixed spiroplasmas, the anti-spiroplasma rabbit polyclonal antibody, and the secondary antibody. This study identified six receptors which may be related with the infection of S. eriocheiris. According to molecular function, the receptor protein is divided into three groups: cytoskeleton, the innate immune system and signal transduction. The cytoskeleton group include Beta-Actin, Beta-Tubulin and Alpha-Tubulin protein. The innate immune system group include the LGBP and proPO protein. Signal transduction factor group include Ran protein.3. Expression of Beta-Actin protein and receptor function analysisIn this research, the bacteria combination experiment and confocal laser experiment were conducted to verify the reliability of receptor protein. In this part, receptor function verification of Beta-Actin was investigated with in vitro bacterial binding assay and confocal laser scanning experiment. For bacterial binding assay, the purified recombinant Beta-Actin protein and S. eriocheiris were incubated for 2 h at room temperature, then washed with PBS, and finally treated with 8 M urea. Western blot result showed that Beta-Actin can be combined on the surface of S. eriocheiris. Confocal laser scanning results showed that S. eriocheiris labeled with green fluorescence could attached with Actin protein labeled with red fluorescent, and yield yellow fluorescence, the interaction between Beta-Actin and S. eriocheiris was further confirmed.4. Expression of LGBP protein and receptor function analysisLGBP can recognize and bind with lipopolysaccharide and β-1,3-glucan to participate in the identification and immune response of gram-negative bacteria and fungi, but there is no report on that LGBP could bind with bacteria lack of cell wall. In this study, bacteria binding experiment was conducted in vitro, and we established that LGBP protein can be combined with S. eriocheiris. In confocal laser scanning experiment, LGBP protein was marked by red fluorescent and the results showed that 5. eriocheiris labeled with green fluorescence could attached onto the LGBP protein. Then, LGBP specific double-stranded RNA was synthesized to analyze its influence on S. eriocheiris copy number and survival rate of M. rosenbergii. The results showed that, S. eriocheiris copy number in LGBP dsRNA injection group was significantly less than the GFP dsRNA and PBS injection group at third days of S. eriocheiris challenge experiment. Prawn survival statistics showed that silencing LGBP increased the sensitivity of the host to S. eriocheiris infection, mortality is higher than that of two control group. The above results suggest that LGBP as an extracellular receptor protein is involved in the process of S. eriocheiris adhesion, but in addition to LGBP, there must be other receptor protein.5. Full length cloning of Ran protein and receptor function analysisPrevious studies showed that the small GTPase protein Ran of shrimp or drosophila was involved in virus phagocytosis. This study further investigated the interaction between Ran and S. eriocheiris, and elucidate the molecular regulating mechanisms of Ran protein on phagocytosis. Firstly, design degenerate primers of Ran gene using mass spectrometry peptide sequences, then amplify its full length by RACE technology. Then, S. eriocheiris binding activity was confirmed by bacteria binding experiment in vitro. Finally, Ran specific double strand RNA was synthesized to analyze its influence on S. eriocheiris copy number and survival rate of M. rosenbergii. The results showed that, S. eriocheiris copy number in Ran dsRNA injection group was significantly less than the GFP dsRNA and PBS injection group during challenge experiment. Prawn survival statistics showed that silencing Ran increased prawns’survival rate. We speculated that Ran protein as an intracellular receptor was involved in the phagocytic process of S. eriocheiris, and Ran may be involved in the process of foreign body ingestion.6. Far-western blotting experiment to screening ligand of spiroplasma and their function analysisIn this research, we identified the candidate ligand for Beta-Actin was transketolase, and the candidate ligands for LGBP included enolase, transketolase, acetaldehyde dehydrogenase, DNA directed RNA polymerase subunit Beta. M. rosenbergii hemocytes was cultured in vitro, and purified recombinant ligand protein was incubated prior S. eriocheiris infection. Results showed that enolase protein can effectively inhibit infection of spiroplasma, and hemocytes survival rate increased 20 percentage in this group. Transketolase and acetaldehyde dehydrogenase protein had no or little significant on hemocytes survival rate. Western blot showed that the enolase protein was distributed both in membrane and cytosolic component. We hypothesized that enolase is involved in the adhesion process of S. eriocheiris to M. rosenbergii hemocytes, and promote S. eriocheiris adhesion and colonization by combining with LGBP protein.