| It is reported that phagocytosis takes great effects in host defense mechanisms through the uptake and degradation of infectious pathogens and contributes to innate immune response. Regardless of different organisms, phagocytosis is of fundamental importance for innate immunity of animals. All phagocytic processes, driven by actin cytoskeleton, ultimately lead to the spatial and conformational changes of the actin cytoskeleton. In theory, the actin cytoskeleton may be involved in some or all of the steps. Therefore the actin cytoskeleton seems to have an important function during the stage of phagocytosis. The conformational changes of the actin cytoskeleton depend on some factors, such as Rho protein. It’s found that small G proteins including Rab6and Ran play important roles in phagocytosis. However, the biological system is so complicated that the molecular events in phagocytosis remain unknown. To address this issue, the molecular mechanism of Rab6and Ran in phagocytosis against virus infection is characterized in this study.At present, no cell line is available for over-expression of protein in shrimp. In order to establish a suitable approach for the over-expression of protein in shrimp, the recombinant GFP protein was purified, freeze-dried and injected to shrimp. The results showed that the injection of GFP is an efficient strategy for the delivery of target protein into shrimp. This method would be helpful for the characterizations of virus and host proteins in shrimp.In this study, it was unraveled that the Rab6protein was required for the formation of actin stress fibers by direct interaction with the actin protein during phagocytosis. The results showed that the depletion of Rab6by sequence-specific siRNA led to the significant decrease of phagocytic activity. In shrimp, actin fibers were distributed across the cells in the hemocytes of the control shrimp. In the hemocytes of shrimp treated with Rab6-siRNA, however, the actin fibers were distributed against the cells. In Drosophila melanogaster, the Rab6protein shared the same mechanism as that of Rab6in shrimp. The results indicated that the Rab6depletion in S2cells led to the lack of criss-crossing actin filaments through the cell by comparison with that of the control. In the cases of the expression of Rab6gene was silenced, over-expressed or rescued, the degree of actin conformation is changed. As known so far, the effective phagocytosis consists of distinct steps that include membrane invagination, coated vesicle formation, directed vesicle trafficking, formation of the phagocytic cup and engulfment of particles. Among these steps, the particle internalization and phagosomal maturation are two essential steps. The results in this study showed that the depletion of Rab6by sequence-specific siRNA led to the significant decrease of phagocytic activity. However, the RNAi assays indicated that the silencing of Rab5or LAMP gene expression had little effect on the phagocytosis. The sequence analysis of Rab6revealed that the Rab6protein was highly conserved in animals. The amino acid sequence encoded by shrimp Rab6shared88%and91%identites with those of fruit fly and human being, respectively. It could be speculated that the molecular events of phagocytosis mediated by the Rab6protein in invertebrates might be preserved in higher mammals during evolution. In this context, the Rab6-mediated regulatory mechanism of phagocytosis would be very helpful to enlarge our limited views about the the innate immune responses against pathogen infection in vertebrates.Widespread evidence indicates that the viral structural proteins play very important roles in virus infection to its host. However, the molecular mechanism concerning the protein-protein interaction between host and virus is limited. The previous study revealed that the host shrimp Rab6, tropomyosin, β-actin and the white spot syndrome virus (WSSV) envelope protein VP466formed a complex. In this study, it was shown that the viral VP466was a bifunctional protein, which could trigger two different pathways including the virus infection and the host antiviral phagocytosis in shrimp. In the VP466-Rab6-actin pathway, the interaction between the VP466and Rab6led to the enhancement of the Rab6GTPase activity. The increase of the Rab GTPase activity by VP466protein induced rearrangements of the actin cytoskeleton, resulting in the formation of actin stress fibers, which promoted the phagocytosis against virus. In the VP466-tropomyosin-actin pathway, the interaction between the viral VP466and host tropomyosin induced the conformation change of actin to facilitate the WSSV infection. The accumulating evidence indicates that the multiple functions of a protein widely exist in pathogens, but those in virus are limited. Therefore our findings revealed that a viral protein could be employed by virus or host to trigger different pathways, representing a novel molecular mechanism in the virus-host interaction in invertebrates.In our previous study, Ran was founded to be involved in regulating phagocytosis in shrimp. However, the mechanism of phagocytosis regulation by Ran remains unknown. In the present study, it was found that in Drosophila melanogaster, the Ran GTPase was up-regulated in phagocytosis, suggesting that the Ran might be implicated in the innate immune system against virus infection. It was evidenced that the depletion of Ran by RNAi caused a significant decrease of phagocytosis and the overexpression of Ran resulted in an obvious increase of phagocytic activity, suggesting that the Ran participated in the antiviral immunity by regulating phagocytosis. By comparison with the Rab5and LAMP, it was revealed that Ran took great effects on phagocytosis in both particle internalization and phagosomal maturation steps. The Ran protein could regulate the whole phagocytosis process. Therefore our study presented a novel finding on the Ran, which would be helpful to reveal the molecular events in phagocytosis. |
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