Dataset Information

A New Membrane Lipid Raft Gene SpFLT-1 Facilitating the Endocytosis of Vibrio alginolyticus in the Crab Scylla paramamosain.

ABSTRACT: Pathogens can enter their host cells by way of endocytosis in which the membrane lipid raft gene flotillins are probably involved in the invasion process and this is an important way to cause infection. In this study, a new gene SpFLT-1 was identified in Scylla paramamosain, which shared high identity with the flotillin-1 of other species. The SpFLT-1 gene was widely distributed in tissues and showed the highest level of mRNA transcripts in the hemocytes. This gene might be a maternal gene based on the evident results that it was highly expressed in maternal ovaries and in the early developmental stages of the zygote and early embryo stage whereas it gradually decreased in zoea 1. SpFLT-1 positively responded to the challenge of Vibrio alginolyticus with a significantly increased level of mRNA expression in the hemocytes and gills at 3 hours post infection (hpi). The SpFLT-1 protein was detected densely in the same fraction layer where the Vibrio protein was most present in the hemocytes and gills at 3 hpi. Furthermore, it was found that the expression of SpFLT-1 decreased to the base level following disappearance of the Vibrio protein at 6 hpi in the gills. Silencing SpFLT-1 inhibited the endocytosis rate of V. alginolyticus but overexpression of the gene could facilitate bacterial entry into the epithelioma papulosum cyprinid cells. Our study indicated that SpFLT-1 may act as a key protein involved in the process of bacterial infection and this sheds light on clarifying the pathogenesis of pathogens infecting S. paramamosain.

SUBMITTER: Chen F

PROVIDER: S-EPMC4506021 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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A New Membrane Lipid Raft Gene SpFLT-1 Facilitating the Endocytosis of Vibrio alginolyticus in the Crab Scylla paramamosain.

Chen Fangyi F Bo Jun J Ma Xiaowan X Dong Lixia L Shan Zhongguo Z Cui Qian Q Chen Huiyun H Wang Kejian K

PloS one 20150717 7

Pathogens can enter their host cells by way of endocytosis in which the membrane lipid raft gene flotillins are probably involved in the invasion process and this is an important way to cause infection. In this study, a new gene SpFLT-1 was identified in Scylla paramamosain, which shared high identity with the flotillin-1 of other species. The SpFLT-1 gene was widely distributed in tissues and showed the highest level of mRNA transcripts in the hemocytes. This gene might be a maternal gene based ...[more]

PMID: 26186350

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Project description:In this study, we investigated the hemocytes' immune response to white spot syndrome virus (WSSV) or Vibrio alginolyticus infection at the protein level. The differential proteomes from crab hemocytes infected with WSSV or V. alginolyticus were analyzed using the isobaric tags for relative and absolute quantitation approach immediately after infection. Using this approach, we identified 1,799 proteins by their by LC-MS/MS spectra and sequencing data. These included 157 upregulated proteins and 164 downregulated proteins after WSSV infection. Similarly, 243 proteins were determined to be differentially expressed during V. alginolyticus infection, of these, 121 were upregulated and 122 were downregulated after infection. Interestingly, among these differentially expressed proteins, 106 were up- or downregulated significantly in both WSSV and V. alginolyticus infection. Six genes, ?-actin, myosin-9, anti-lipopolysaccharide factor isoform 4, anti-lipopolysaccharide factor 4, transketolase-like protein 2-like isoform 1, and sarcoplasmic calcium-binding protein 1 were chosen for further study. The expression of these genes all showed a trend of upregulation at 24?h post-WSSV or V. alginolyticus infection except for myosin-9 in response to WSSV. To confirm the protective effects of the six genes, crabs were injected with specific dsRNAs before WSSV or V. alginolyticus challenge. The results showed that the knockdown of these genes led to an increase in the morbidity and mortality (P?<?0.01) rate, and a decrease in infection time in WSSV-infected crabs. During the first 84?h, knockdown of these genes also led to an increase in the morbidity rates in V. alginolyticus -infected crabs, and results of four genes showed a higher mortality rate than that of the control after they were knocked down. This is the first report of the proteome response in crab hemocytes during WSSV or V. alginolyticus infection. These findings will contribute to our understanding of the immune response to WSSV and V. alginolyticus infection in crabs.

Project description:BackgroundInfection of bacterial Vibrio parahaemolyticus is common in mud crab farms. However, the mechanisms of the crab's response to pathogenic V. parahaemolyticus infection are not fully understood. MicroRNAs (miRNAs) are a class of small noncoding RNAs that function as regulators of gene expression and play essential roles in various biological processes. To understand the underlying mechanisms of the molecular immune response of the crab to the pathogens, high-throughput Illumina/Solexa deep sequencing technology was used to investigate the expression profiles of miRNAs in S. paramamosain under V. parahaemolyticus infection.Methodology/principal findingsTwo mixed RNA pools of 7 tissues (intestine, heart, liver, gill, brain, muscle and blood) were obtained from V. parahaemolyticus infected crabs and the control groups, respectively. By aligning the sequencing data with known miRNAs, we characterized 421 miRNA families, and 133 conserved miRNA families in mud crab S. paramamosain were either identical or very similar to existing miRNAs in miRBase. Stem-loop qRT-PCRs were used to scan the expression levels of four randomly chosen differentially expressed miRNAs and tissue distribution. Eight novel potential miRNAs were confirmed by qRT-PCR analysis and the precursors of these novel miRNAs were verified by PCR amplification, cloning and sequencing in S. paramamosain. 161 miRNAs (106 of which up-regulated and 55 down-regulated) were significantly differentially expressed during the challenge and the potential targets of these differentially expressed miRNAs were predicted. Furthermore, we demonstrated evolutionary conservation of mud crab miRNAs in the animal evolution process.Conclusions/significanceIn this study, a large number of miRNAs were identified in S. paramamosain when challenged with V. parahaemolyticus, some of which were differentially expressed. The results show that miRNAs might play some important roles in regulating gene expression in mud crab under V. parahaemolyticus infection, providing a basis for further investigation of miRNA-modulating networks in innate immunity of mud crab.

Project description:"Immune priming" plays a vital part in the immune system of invertebrates, protecting against recurrent infections by pathogens, and can provide some ideas for the prevention and treatment of invertebrate diseases. Many invertebrates have been demonstrated recently to have immune priming, but the relevant mechanisms are not known. Expression of immune system-related genes in the hemocytes and hepatopancreas of the mud crab (Scylla paramamosain) before and after repeated stimulation with Vibrio parahaemolyticus were analyzed by real-time fluorescence quantitative polymerase chain reaction. Some molecules that may participate in the immune priming of S. paramamosain were screened out, and their possible roles in immune priming were interpreted. Crabs injected first with heat-killed V. parahaemolyticus (HkVp group) or physiologic (0.9%) saline (PS group) were rechallenged at 168 h with live V. parahaemolyticus (HkVp+Vp group and PS+Vp group, respectively). The log-rank test shows a significant difference in survival rate between the HkVp+Vp group and the other groups after the ICH (p < 0.05). Expression of genes involved in the toll-like receptor (TLR) signaling pathway and some antimicrobial peptide genes were detected. By, respectively, comparing gene quantification at different time points in hemocytes and the hepatopancreas, the molecules that may play a part in the early stage of the immune priming of S. paramamosain in the hemocytes are found to be down syndrome cell adhesion molecule (Dscam), Hyastatin, Cactus, Arasin, antilipopolysaccharide factor 3 (ALF3), ALF4, ALF5, and ALF6 as well as later acting molecules, such as Crustin, Dorsal, Pelle, and myeloid differentiation factor 88 (MyD88). The molecules that functioned throughout the entire period are TLR and Spaetzle. In the hepatopancreas, the molecules that may play a part in the early stages of immune priming are Dscam, Hyastatin, Arasin, ALF6, Pelle, Spaetzle, Dorsal and, in the later stage, ALF4. The molecules that functioned throughout the entire period are TLR, Crustin, Cactus, MyD88, ALF3, and ALF5. In summary, the immune function of S. paramamosain is enhanced after it receives the same repetitive stimulation by V. parahaemolyticus, indicating immune priming in S. paramamosain. Our study enriches research on immune priming in invertebrates and lays the foundation for further studies revealing the molecular mechanism of immune priming in crabs.

Dataset Information

A New Membrane Lipid Raft Gene SpFLT-1 Facilitating the Endocytosis of Vibrio alginolyticus in the Crab Scylla paramamosain.

Publications

A New Membrane Lipid Raft Gene SpFLT-1 Facilitating the Endocytosis of Vibrio alginolyticus in the Crab Scylla paramamosain.

Similar Datasets

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