Hongkuan Deng

De Novo Transcriptome Analysis Provides Insights into Immune Related Genes and the RIG-I-Like Receptor Signaling Pathway in the Freshwater Planarian (Dugesia japonica)

Background The freshwater planarian Dugesia japonica (D. japonica) possesses extraordinary ability to regenerate lost organs or body parts. Interestingly, in the process of regeneration, there is little wound infection, suggesting that D. japonica has a formidable innate immune system. The importance of immune system prompted us to search for immune-related genes and RIG-I-like receptor signaling pathways. Results Transcriptome sequencing of D. japonica was performed on an IlluminaHiSeq2000 platform. A total of 27,180 transcripts were obtained by Trinity assembler. CEGMA analysis and mapping of all trimmed reads back to the assembly result showed that our transcriptome assembly covered most of the whole transcriptome. 23,888 out of 27,180 transcripts contained ORF (open reading fragment), and were highly similar to those in Schistosoma mansoni using BLASTX analysis. 8,079 transcripts (29.7%) and 8,668 (31.9%) were annotated by Blast2GO and KEGG respectively. A DYNLRB-like gene was cloned to verify its roles in the immune response. Finally, the expression patterns of 4 genes (RIG-I, TRAF3, TRAF6, P38) in the RIG-I-like receptor signaling pathway were detected, and the results showed they are very likely to be involved in planarian immune response. Conclusion RNA-Seq analysis based on the next-generation sequencing technology was an efficient approach to discover critical genes and to understand their corresponding biological functions. Through GO and KEGG analysis, several critical and conserved signaling pathways and genes related to RIG-I-like receptor signaling pathway were identified. Four candidate genes were selected to identify their expression dynamics in the process of pathogen stimulation. These annotated transcripts of D. japonica provide a useful resource for subsequent investigation of other important pathways.

The role of a novel C-type lectin-like protein from planarian in innate immunity and regeneration

ABSTRACT Planarian, a representative of platyhelminthes, has strong regeneration ability and less complicated innate immune system. However, planarian immune system remains poorly understood. In this paper, a novel C‐type lectin‐like protein, namely, DjCTL was identified and characterized in Dugesia japonica. DjCTL was mainly expressed in the pharyngeal and epidermis and up‐regulated upon the induction of lipopolysaccharide (LPS), peptidoglycan (PGN), Gram‐positive and Gram‐negative bacteria indicating that DjCTL may be involved in the immune responses. Recombination DjCTL protein agglomerated rabbit red blood cells and interacted with LPS, PGN, mannose and galactose as well as both Gram‐positive and Gram‐negative bacteria, but it can only cause the agglutination of Gram‐negative bacteria. Importantly, in the early periods of regeneration, DjCTL had a significantly high expression and was mainly expressed in early blastemas. RNA interference of DjCTL by dsRNA‐DjCTL led to a slow wound healing during regeneration. These findings suggest that DjCTL participates in the innate immune response and plays an important role in early stages of regeneration. HighlightsA C‐type lectin‐like gene, DjCTL, was cloned from Planarian, Dugesia japonica.DjCTL involves in the innate immune response as a pattern recognition receptor.DjCTL plays an important role in early stages of regeneration.

Identification and characterization of a phospholipid scramblase encoded by planarian Dugesia japonica

Phospholipid scramblases (PLSCRs) are the conserved calcium-binding, type II transmembrane proteins synthesized in all eukaryotic organisms. In mammals, these proteins play essential roles in various physiological processes, especially in the immune responses. However, the existence of PLSCRs and their biological functions in planarian are still unknown at present. In this study, a new member of PLSCRs was identified in planarian Dugesia japonica (D. japonica), named DjPLSCR. The sequence analysis revealed that it contains an opening reading frame consisting of 726bp encoding a putative protein of 241 amino acids with a predicted molecular mass of ~28.7kDa and an isoelectric point of 6.21. Whole-mount in situ hybridization showed that mRNAs of DjPLSCR are predominantly expressed in adult and regenerative pharynx which is an important organ of immune system in planarians. Importantly, we found that the transcription level of DjPLSCR was significantly upregulated when planarians were stimulated with the pathogen-associated molecular patterns [polyinosinic-polycytidylic acid, lipopolysaccharide, peptidoglycan and β-glucan], suggesting that DjPLSCR is involved in the immune response upon pathogen invasion. Our findings provide the first experimental insights into the characteristics and potential functions of PLSCR in planarians.

14-3-3 α and 14-3-3 ζ contribute to immune responses in planarian Dugesia japonica

14-3-3 proteins are a family of highly conserved acidic proteins that regulate cellular processes. They act as a kind of important signaling molecules taking part in many crucial decisions throughout the development process. We have isolated and characterized two members of the 14-3-3 family, namely, Dj14-3-3 α and Dj14-3-3 ζ in the planarian Dugesia japonica. The Dj14-3-3 α and ζ genes encode polypeptides of 260 and 255 amino acids respectively. We have proved that the Dj14-3-3 α and ζ genes were especially expressed in the pharynx in adult and regenerating planarians by in situ hybridization and they were not involved in regeneration process. Besides, Dj14-3-3 α and ζ genes can compensate each other in planarians by RNA interference. The Dj14-3-3 α and ζ were significantly up-regulated expression when planarians were stimulated with the pathogen-associated molecular patterns including lipopolysaccharide (LPS), peptidoglycan (PGN), β-Glu and Poly (I:C), indicating that the Dj14-3-3 α and ζ may be involved in the immune responses.

Identification and characterization of a novel multifunctional placenta specific protein 8 in Dugesia japonica

Placenta specific protein 8 (Plac8) has been well studied in vertebrates, yet little is known in invertebrates. In this study, a novel Plac8 from the planarian Dugesia japonica was identified and its functions in immune responses and development were characterized. Our results show that Djplac8 was expressed in the pharynx, epidermis and intestine of intact adult planarian. The expression of DjPlac8 increased significantly upon lipopolysaccharide (LPS) challenge, and inhibited the growth of the Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), suggesting the role of Plac8 in immune response. Spatial and temporal expression and distribution of DjPlac8 mRNA in regenerated planarians indicates that DjPlac8 was mainly expressed in the pharynx. In situ hybridization also revealed the elevated expression of the DjPlac8 gene in the embryonic pharynx, germ band and parenchyma cells, indicating an important role in embryonic development of D. japonica. When DjPlac8 was deactivated by RNA interference-mediated knockdown, the head of planarians underwent abnormal development. In summary, we identified DjPlac8 as a novel multifunctional protein that plays essential roles in immune response and development of planarians.

Identification and functional analysis of invasion associated locus B (IalB) in Bartonella species

Bartonellosis is caused by the genus Bartonella. Bartonella is widely distributed in the ruminants, cats, dogs, rodents and other mammals including humans. At least 13 species or subspecies of Bartonella are zoonotic, and each species appears to be highly adapted to one or a limited number of reservoir animals in which it is asymptomatic, while it can be transmitted to humans in which a variety of clinical manifestations can be caused. It was reported that Bartonella henselae infection rate among domestic cats was high in nature, making it one of the leading, important, and easily neglected zoonotic diseases. The aims of this study were to identify the expression, localization, immunogenicity and functional mechanism of Bartonella virulence factor IalB. We found that recombinant IalB protein could react with the serum from infected reservoir hosts and anti-IalB polyclonal antibodies could react with different Bartonella species by western blot analysis. According to these results, we proposed that IalB protein and anti-IalB antibodies would be good candidates for diagnosis of Bartonella infection by antigen-based anti-IalB antibodies or antibody-based IalB antigen capture immunoassay, respectively. We also found that IalB had a putative 22-amino-acid signal sequence and little IalB was localized to the outer membrane of Bartonella birtlesii by electron microscopy assay. Incubation with anti-IalB polyclonal antibodies resulted in inhibition of the invasion of mouse erythrocytes by B. birtlesii. According to these results, we propose that IalB could be a secreted protein that facilitates Bartonella entry into erythrocytes. In conclusion, these results improve our understanding of IalB as a candidate for immunodiagnosis and how IalB affects Bartonella-erythrocyte entry.

Identification and characterization of a TNF receptor-associated factor in Dugesia japonica

The tumor necrosis factor (TNF) superfamily consists of a wide variety of inflammatory cytokine, including cell-bound and secreted proteins. These TNFs function through binding and activation of the TNF receptors for modulating TNF-associated intracellular signals. A set of mammalian TNF receptor-associated factors (TRAFs) that have emerged as the major signal transducers for the TNF receptor superfamily, play an important role in both adaptive and innate immunity. However, the existence of TRAFs and their biological functions in planarian are still unknown. In this study, a new member of TRAFs, DjTRAF2, was identified in planarian Dugesia japonica. Phylogenetic analysis revealed that DjTRAF2 could be a new member of the invertebrate TRAF2 family. Sequence analysis showed that the open reading frame of DjTRAF2 had 1353 bp in length and encoded a putative protein of 450 amino acids with a predicted molecular mass of ~51.8 kDa and an isoelectric point of 7.052. Whole-mount in situ hybridization showed that DjTRAF2 was predominantly expressed in adult and regenerative pharynx, which is an important immune organ of planarian. Quantitative real-time PCR revealed that the transcriptional level of DjTRAF2 was significantly up-regulated after induced by pathogen-associated molecular patterns (polyinosinic-polycytidylic acid, lipopolysaccharide, peptidoglycan and β-glucan), suggesting that DjTRAF2 is involved in the immune response against pathogen invasion. Collectively, these results demonstrated that DjTRAF2 might play important roles in the innate immunity of planarian.

DjRlc is required for the intestinal regeneration in planarian Dugesia japonica

The myosin regulatory light chain (RLC) proteins play an important role in cellular processes, especially in muscle contraction. The planarian intestine is a fascinating system for studying the organogenesis during regeneration. In this paper, A homolog gene of Rlc, DjRlc, was identified and characterized in Dugesia japonica. The DjRlc sequence analysis revealed that it contains an opening reading frame encoding a putative protein of 175 amino acids with functionally domains that are highly conserved, including an EF-Hand motif and Ca2+ binding sites. Whole mount in situ hybridization showed that DjRlc is predominantly expressed in the intestine of intact and regenerating planarians. The cross sections of planarians revealed that the DjRlc distributes in the muscle of intact planarians. Knockdown of RNA interference of DjRlc by dsRNA-DjRlc affected the intestinal morphology, causing distinct defects in branching morphogenesis. These finding suggest that DjRlc is required for intestinal regeneration.

The inhibition of GSK-3β promotes the production of reactive oxygen species via β-catenin/C/EBPα signaling in the spleen of zebrafish (Danio rerio)

&NA; In this study, the mechanism that the inhibition of glycogen synthase kinase‐3&bgr; (GSK‐3&bgr;) promotes the production of reactive oxygen species (ROS) via &bgr;‐catenin/CCAAT/enhancer binding protein &agr; (C/EBP&agr;) signaling was investigated in the spleen of zebrafish (Danio rerio). The results demonstrated that the inhibition of GSK‐3&bgr; induced the mRNA expression of &bgr;‐catenin and C/EBP&agr; by lithium (Li) treatments or GSK‐3&bgr; RNA interference. The levels of hydrogen peroxide (H2O2), superoxide anion (O2.‐), and hydroxy radical (·OH) as well as the activity of superoxide dismutase (SOD) were increased, while the activities of catalase (CAT) and glutathione peroxidase (GSH‐PX) were decreased in the spleen and ZF4 cells of zebrafish by Li+ treatments. In addition, GSK‐3&bgr; RNA interference increased ROS levels and decreased the activities of CAT and GSH‐PX in the spleen. The fluorescence intensity of ROS was increased but the mitochondrial membrane potential (MMP) was decreased by Li+ treatments in ZF4 cells labeled with 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) and Rhodamine‐123, respectively. The results of present study indicated that the inhibition of GSK‐3&bgr; promoted the ROS production via &bgr;‐catenin/C/EBP&agr; signaling in the spleen of zebrafish, and the balance between ROS and antioxidants could be destroyed by the GSK‐3&bgr;/&bgr;‐catenin/C/EBP&agr; signaling. The results may be a valuable contribution to understanding the modulatory mechanism of GSK‐3&bgr;/&bgr;‐catenin/C/EBP&agr; signaling on the antioxidant system in fish species. HighlightsThe inhibition of GSK‐3&bgr; promotes the production of ROS.The inhibition of GSK‐3&bgr; inhibits the activities of GSH‐PX and CAT.The inhibition of GSK‐3&bgr; induces the &bgr;‐catenin/CEBP/&agr; signaling.The inhibition of GSK‐3&bgr; promotes ROS production via &bgr;‐catenin/CEBP/&agr; signaling.

Identification and characterization of an atypical RIG-I encoded by planarian Dugesia japonica and its essential role in the immune response

ABSTRACT Retinoic acid‐inducible gene I (RIG‐I), an RNA sensor with a conserved structure, activates the host interferon (IFN) system to produce IFNs and cytokines for eliminating pathogens upon recognizing PAMPs. However, the biological functions and the mechanism by which RIG‐I regulates the innate immunity response in invertebrates are still unknown at present. Here we identified an atypical RIG‐I in planarian Dugesia japonica. Sequence analysis, 3D structure modeling and phylogenetic analysis showed that this atypical protein was clustered into a single clade at the base of the tree in invertebrates, suggesting that DjRIG‐I is an ancient and unique protein of the RIG‐I‐like receptors (RLRs). In situ hybridization analysis revealed that the DjRIG‐I mRNAs were predominantly expressed in the pharynx and head of the adult and regenerative planarians. Stimulation with PAMPs induced the over‐expression of DjRIG‐I in planarians. The molecular simulation demonstrated that DjRIG‐I formed a large hole‐structure for the docking of dsRNAs, and the pull‐down assay confirmed the interaction between DjRIG‐I and viral analog poly(I:C). Importantly, some representative antiviral/antibacterial genes in the RIG‐I‐mediated IFN and P38 signaling pathway, TBK1, IRF‐3, Mx, and P38, were significantly upregulated in planarians stimulated with PAMPs. Interference of the DjRIG‐I expression by RNAi, inhibited the PAMPs‐induced over‐expression, suggesting that DjRIG‐I is a key player for downstream signaling events. These results indicate that DjRIG‐I triggered the intracellular signaling cascades independent of the classical CARD domains and played an essential role in the virus/bacteria‐induced innate immunity of planarian. HighlightsAn atypical RIG‐I‐like gene (DjRIG‐I) is isolated and characterized from planarian Dugesia japonica.DjRIG‐I mRNAs are significantly distributed throughout the body of the intact adult and regenerating planarians.Stimulation with PAMPs induced the over‐expression of DjRIG‐I, which can also interact with viral analog poly(I:C).DjRIG‐I participates in the virus‐ and bacteria‐induced innate immunity of planarian.