Bosheng Zhao

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, expression of a glycoprotein hormone receptor homolog in the amphioxus Branchiostoma belcheri with implications for origin of vertebrate GpHRs

Amphioxus is phylogenetically located at the most primitive position of the chordate clade. Its endocrine system has been extensively studied; however, key information about the glycoprotein hormone (GpH) and its receptor (GpHR) in the endocrine system remained to be elucidated. In this study, a GpHR homologous gene, BbGpHR-like, has been identified in the amphioxus Branchiostoma belcheri. It contains a 1377bp open reading frame that corresponds to a deduced protein of 458 amino acid residues with a predicted molecular mass of approximately 50kDa. Phylogenetic analysis shows that BbGpHR-like is located at the position between vertebrate and invertebrate, indicating amphioxus B. belcheri is in an evolutionary transitional state between vertebrate and invertebrate in the GpHR lineage. BbGpHR-like has been successfully expressed in Pichia pastoris. Western blot analysis confirmed that the receptor produced a cross-immunoreactivity with human thyrotropin receptor (TSHR), the luteinizing hormone receptor (LHR) and the follicle-stimulating hormone receptor (FSHR). In situ hybridization and immunohistochemical analyses revealed that the BbGpHR-like transcripts and proteins were strongly distributed in the ovaries, testes and/or endostyle. These findings suggest that BbGpHR-like possibly plays an ancient and fundamental role in the control of thyroid hormone synthesis and gametogenesis in chordates. Presumably, the amphioxus BbGpHR-like represents the ancestral form of the GpHR gene prior to its split to the vertebrate paralogs gonadotropin receptor and thyrotropin receptor.

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.

Cloning, characterization and expression of tyrosinase-like gene in amphioxus Branchiostoma japonicum

Prophenoloxidase (tyrosinase) widely distributed in invertebrates and vertebrates, and plays a crucial role in the innate immune. In the present study, the full-length cDNA of a tyrosinase-like (designated AmphiTYR) was cloned from amphioxus Branchiostoma japonicum by PCR techniques. The full-length cDNA of AmphiTYR is 2314 bp, and its predicted open-reading frame codes for a protein of 544 amino acids with a predicted molecular mass of approximately 60.9 kDa and an isoelectric point of 5.65. It has a conserved putative copper-binding domain with six histidines in tyrosinase proteins. Six potential N-linked glycosylation sites and 14 conserved cysteine residues were also predicted to be present in B. japonicum tyrosinase. Homology analysis revealed that AmphiTYR was higher similar to vertebrates tyrosinases (32.5-40.5%) than to invertebrates phenoloxidase (6.4-25.4%). In the adult, AmphiTYR mRNA was expressed in the muscle, epidermis, notochord, ovary, hepatic caecum, pharynx and gill, but not in the neural tube, intestines and testis. During the different development stages from unfertilized egg to larvae of amphioxus, AmphiTYR expressed during all the amphioxus development. These results indicated that AmphiTYR gene not only play a pivotal role in innate immune but also play an important role during embryonic development of cephalochordate amphioxus.

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.

Sirt7 regulates circadian phase coherence of hepatic circadian clock via a body temperature/Hsp70-Sirt7-Cry1 axis

The biological clock is generated in the hypothalamic suprachiasmatic nucleus (SCN), which synchronizes peripheral oscillators to coordinate physiological and behavioral activities throughout the body. Disturbance of circadian phase coherence between the central and peripheral could disrupt rhythms and thus cause diseases and aging. Here, we identified hepatic Sirt7 as an early element responsive to light, which ensures the phase coherence in mouse liver. Loss of Sirt7 leads to advanced liver circadian phase; restricted feeding in daytime entrains hepatic clock more rapidly in Sirt7-/- mice compared to wild-types. Molecularly, a light-driven body temperature (BT) oscillation induces rhythmic expression of Hsp70, which binds to and promotes the ubiquitination and proteasomal degradation of Sirt7. Sirt7 rhythmically deacetylates Cry1 on K565/579 and promotes Fbxl3-mediated degradation, thus coupling hepatic clock to the central pacemaker. Together, our data identify a novel BT/Hsp70-Sirt7-Cry1 axis, which transmits biological timing cues from the central to the peripheral and ensures circadian phase coherence in livers.