Spiroplasma eriocheiris Enters Drosophila Schneider 2 Cells and Relies on Clathrin-Mediated Endocytosis and Macropinocytosis.

Spiroplasma eriocheiris causes great economic losses in the crustacean aquaculture industry. However, the mechanism of S. eriocheiris infecting host cells has been poorly studied. We established a Spiroplasma-infected Drosophila Schneider 2 (S2) cell model and investigated its pathogenic mechanism. First, S. eriocheiris induced S2 cell apoptosis and necrosis, seriously decreased cell viability, and increased the production of intracellular reactive oxygen species. Further research showed that S. eriocheiris can invade S2 cells, and the number of copies of intracellular spiroplasmas is sharply increased by 12 h postinfection. In addition, S. eriocheiris can cause S2 cells to form typical inclusion bodies and exhibit large vacuoles. Second, S. eriocheiris is internalized into S2 cells and strongly inhibited through blocking clathrin-mediated endocytosis using chlorpromazine and dynasore. Inhibitors of macropinocytosis, protein kinase C and myosin II, cause a significant reduction in S. eriocheiris in S2 cells. In contrast, disruption of cellular cholesterol by methyl-ß-cyclodextrin and nystatin has no effect on S. eriocheiris infection. These results suggest that the entry of S. eriocheiris into S2 cells relies on clathrin-dependent endocytosis and macropinocytosis, but not via the caveola-mediated endocytic pathway. In addition, the intracellular numbers of S. eriocheiris are dramatically reduced after S2 cells are treated with cytoskeleton-depolymerizing agents, including nocodazole and cytochalasin B. Thus, cellular infection by S. eriocheiris is related to microtubules and actin filaments. This research successfully shows for the first time that S. eriocheiris can invade Drosophila S2 cells and provides a process for S. eriocheiris infection.

The function of serpin-2 from Eriocheir sinensis in Spiroplasma eriocheiris infection.

Serpin families classified serine protease inhibitors regulate various physiological processes. However, there is not study on the role of serpin in immune responses against Spiroplasma eriocheiris as a novel causative pathogen in the Chinese mitten crab, Eriocheir sinensis. In our study, quantitative real-time PCR (qRT-PCR) revealed that the mRNA transcripts of Esserpin-2 were ubiquitous in every tissue, relative higher expression in hepatopancreas, gill and hemocytes, while the intestine, muscle, heart and nerve showed relative lower expression. Followed by infection with S. eriocheiris, the transcripts of Esserpin-2 were significantly down-regulated from 1 d to 7 d. After double-stranded RNA injection, the transcripts of Esserpin-2 dramatically declined from 48 h to 96 h. The transcripts of proPO were found to be obviously increased after Esserpin-2 silenced, meanwhile, LGBP with no significant difference. The copy number of S. eriocheiris and subsequently the mortality of crabs in a silencing Esserpin-2 group were significantly less than control groups during infection. 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 cell death. These findings demonstrated that Esserpin-2 involved in the innate immune mechanism of E. sinensis in response to S. eriocheiris infection.

Identification and function analysis of ras-related nuclear protein from Macrobrachium rosenbergii involved in Spiroplasma eriocheiris infection.

A ras-related nuclear protein (Ran) protein was obtained from Macrobrachium rosenbergii, named MrRan. Phylogenetic analysis results showed that MrRan was clustered in one group together with other crustaceans. Tissue distribution analysis revealed that MrRan was expressed mainly in gill, intestine and stomach, and expressed weakly in muscle. The MrRan expression levels in gill and hemocyte of prawns were significantly up-regulated after challenged by Spiroplasma eriocheiris. The copy number of S. eriocheiris in MrRan dsRNA injection group was significantly less than control groups during infection. Meanwhile, silencing MrRan obviously increased the survival rate of prawns. The subcellular localization experiment suggested that recombinant MrRan was mainly located in the nucleus, and relatively weak in the cytoplasm. Finally, over-expression in Drosophila S2 cell indicated that MrRan could increase copies of S. eriocheiris and decrease of cell viability. The present study suggested that MrRan participated in regulating the phagocytosis of S. eriocheiris in M. rosenbergii.

Spiroplasma eriocheiris Invasion Into Macrobrachium rosenbergii Hemocytes Is Mediated by Pathogen Enolase and Host Lipopolysaccharide and ß-1, 3-Glucan Binding Protein.

Spiroplasma eriocheiris is a crustacean pathogen, without a cell wall, that causes enormous economic loss. Macrobrachium rosenbergii hemocytes are the major targets during S. eriocheiris infection. As wall-less bacteria, S. eriocheiris, its membrane protein should interact with host membrane protein directly and firstly when invaded in host cell. In this investigation, six potential hemocyte receptor proteins were identified firstly that mediate interaction between S. eriocheiris and M. rosenbergii. Among these proteins, lipopolysaccharide and ß-1, 3-glucan binding protein (MrLGBP) demonstrated to bind to S. eriocheiris using bacterial binding assays and confocal microscopy. Four spiroplasma ligand proteins for MrLGBP were isolated and identified. But, competitive assessment demonstrated that only enolase of S. eriocheiris (SeEnolase) could be a candidate ligand for MrLGBP. Subsequently, the interaction between MrLGBP and SeEnolase was confirmed by co-immunoprecipitation and co-localization in vitro. After the interaction between MrLGBP and SeEnolase was inhibited by antibody neutralization test, the virulence ability of S. eriocheiris was effectively reduced. The quantity of S. eriocheiris decreased in Drosophila S2 cells after overexpression of MrLGBP, compared with the controls. In addition, RNA interference (RNAi) knockdown of MrLGBP made M. rosenbergii more sensitive to S. eriocheiris infection. Further studies found that the immune genes, including MrLGBP and prophenoloxidase (MrproPO), MrRab7A, and Mrintegrin α1 were significantly up-regulated by SeEnolase stimulation. After SeEnolase pre-stimulation, the ability of M. rosenbergii resistance to S. eriocheiris was significantly improved. Collectively, this investigation demonstrated that MrLGBP and pathogen SeEnolase involved in mediating S. eriocheiris invasion into M. rosenbergii hemocytes.

Characterization of cathepsin D from Eriocheir sinensis involved in Spiroplasma eriocheiris infection.

Cathepsin D (catD) belongs to a lysosomal aspartic protease superfamily. The full-length catD cDNA from the Chinese mitten crab Eriocheir sinensis (EscatD) was 2748 bp and contained a 1158-bp ORF encoding a protein of 385 amino acids, including a signal peptide and two N-glycosylation sites. Phylogenetic analysis showed that EscatD was clustered into a single group, together with other catD for crustaceans. Quantitative real-time PCR revealed that EscatD was expressed mainly in the eyes, hemocytes, intestine and nerve and was expressed weakly in heart, muscle and gills. After challenge with Spiroplasma eriocheiris, the expression of EscatD was significantly up-regulated from 1 d to 9 d. The copy number of S. eriocheiris in a silencing EscatD group was significantly higher than those in the control groups during S. eriocheiris infection. Meanwhile, the survival rate of crabs decreased in an EscatD-dsRNA group. We further found that knockdown of EscatD by RNA interference resulted in a downward trend of expression levels of JNK, ERK, relish and p38 during the early stage, as well as a reduction in the expression of five antimicrobial peptides genes, namely, crusrin1, crustin2, ALF1, ALF2 and ALF3. 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 cell viability. The above results demonstrated that EscatD plays an important immune role in E. sinensis to S. eriocheiris challenge.