Characterization of Schistosoma japonicum CP1412 protein as a novel member of the ribonuclease T2 molecule family with immune regulatory function.
Ontology highlight
ABSTRACT: Schistosome infection typically induces a polarized Th2 type host immune response. As egg antigen molecules play key roles in this immunoregulatory process, clarifying their functions in schistosomiasis would facilitate the development of vaccine and immunotherapeutic methods. Schistosoma japonicum (Sj) CP1412 (GenBank: AY57074.1) has been identified as a new member of the RNase T2 family with immune regulatory functions.The expression plasmid Sj CP1412-pET28a was constructed and transformed into bacteria for production of recombinant Sj CP1412 protein (rSj CP1412) via IPTG induction. The RNase activity of Sj CP1412 was predicted by bioinformatic analysis and confirmed by digesting the yeast tRNA with rSj CP1412.C57BL/6j mice were immunized with rSj CP1412, and its immune regulatory effects in vivo and in vitro were investigated. Meanwhile, the relationship between the RNase activity of Sj CP1412 and its immune regulation was observed.Sj CP1412 was confirmed as a novel RNase T2 family protein with RNase activity. Immunoblotting and RT-PCR analyses demonstrated Sj CP1412 as a protein exclusively secreted/excreted from eggs, but not cercariae and adult worms. Stimulating RAW264.7 macrophages with rSj CP1412 raised the expression of CD206, Arg-1 and IL-10, which are related to M2 type macrophage differentiation. Stimulating dendritic cells (DCs) with rSjCP1412 failed to induce their maturation, and the recombinant protein also inhibited LPS-stimulated DC maturation. Depletion of Sj CP1412 from soluble egg antigen (SEA) impaired the ability of SEA to induce M2 type polarization of RAW264.7 macrophages. Immunizing mice with rSj CP1412 induced high antibody titers, increased serum IL-4 and TGF-? levels and splenic CD4?+?CD25?+?Foxp3 + T cells, downregulated serum IFN-? levels and alleviated the egg granuloma pathology of schistosome infection. In vitro stimulation by rSj CP1412 significantly increased CD4?+?CD25?+?Foxp3?+?T cell numbers in splenocytes of healthy mice. The rSj CP1412 protein with RNase activity inactivated by DEPC failed to induce M2 surface marker CD206 expression in RAW264.7 macrophages.The Sj CP1412 protein expressed specifically in S. japonicum eggs is a novel member of the RNase T2 family. Similar to Omega-1 of Schistosoma mansoni, the Sj CP1412 protein drives polarization of the host Th2 immune response, which is dependent on its RNase activity. These data provide new evidence towards understanding the immune regulatory role of RNase T2 family proteins during schistosome infection.
<h4>Background</h4>Schistosome infection typically induces a polarized Th2 type host immune response. As egg antigen molecules play key roles in this immunoregulatory process, clarifying their functions in schistosomiasis would facilitate the development of vaccine and immunotherapeutic methods. Schistosoma japonicum (Sj) CP1412 (GenBank: AY57074.1) has been identified as a new member of the RNase T2 family with immune regulatory functions.<h4>Methods</h4>The expression plasmid Sj CP1412-pET28a ...[more]
Project description:A. baumannii has the propensity to colonize abiotic surfaces and this is thought to mediate its transmission to susceptible patients. We found that disruption of A. baumannii ribonuclease T2 family protein (ATCC 17978 locus A1S_3026) severely diminishes the organism's ability to colonize abiotic surfaces. We used Affymetrix A. baumannii GeneChips (part number PMDACBA1) to compare the gene expression properties of wild type and isogenic ribonuclease T2 family protein mutant cells.
Project description:BACKGROUND: Schistosomal parasites can establish parasitization in a human host for decades; evasion of host immunorecognition including surface masking by acquisition of host serum components is one of the strategies explored by the parasites. Parasite molecules anchored on the membrane are the main elements in the interaction. Sjc23, a member of the tetraspanin (TSP) family of Schistosoma japonicum, was previously found to be highly immunogenic and regarded as a vaccine candidate against schistosomiasis. However, studies indicated that immunization with Sjc23 generated rapid antibody responses which were less protective than that with other antigens. The biological function of this membrane-anchored molecule has not been defined after decades of vaccination studies. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we explored affinity pull-down and peptide competition assays to investigate the potential binding between Sjc23 molecule and human non-immune IgG. We determined that Sjc23 could bind human non-immune IgG and the binding was through the interaction of the large extra-cellular domain (LED) of Sjc23 (named Sjc23-LED) with the Fc domain of human IgG. Sjc23 had no affinity to other immunoglobulin types. Affinity precipitation (pull-down assay) in the presence of overlapping peptides further pinpointed to a 9-amino acid motif within Sjc23-LED that mediated the binding to human IgG. CONCLUSION AND SIGNIFICANCE: S. japonicum parasites cloak themselves through interaction with human non-immune IgG, and a member of the tetraspanin family, Sjc23, mediated the acquisition of human IgG via the interaction of a motif of 9 amino acids with the Fc domain of the IgG molecule. The consequence of this interaction will likely benefit parasitism of S. japonicum by evasion of host immune recognition or immunoresponses. This is the first report that an epitope of schistosomal ligand and its immunoglobulin receptor are defined, which provides further evidence of immune evasion strategy adopted by S. japonicum.
Project description:Acinetobacter baumannii is an emerging bacterial pathogen of considerable medical concern. The organism's transmission and ability to cause disease has been associated with its propensity to colonize and form biofilms on abiotic surfaces in health care settings. To better understand the genetic determinants that affect biomaterial attachment, we performed a transposon mutagenesis analysis of abiotic surface-colonization using A. baumannii strain 98-37-09. Disruption of an RNase T2 family gene was found to limit the organism's ability to colonize polystyrene, polypropylene, glass, and stainless steel surfaces. DNA microarray analyses revealed that in comparison to wild type and complemented cells, the RNase T2 family mutant exhibited reduced expression of 29 genes, 15 of which are predicted to be associated with bacterial attachment and surface-associated motility. Motility assays confirmed that RNase T2 mutant displays a severe motility defect. Taken together, our results indicate that the RNase T2 family protein identified in this study is a positive regulator of A. baumannii's ability to colonize inanimate surfaces and motility. Moreover, the enzyme may be an effective target for the intervention of biomaterial colonization, and consequently limit the organism's transmission within the hospital setting.
Project description:A. baumannii has the propensity to colonize abiotic surfaces and this is thought to mediate its transmission to susceptible patients. We found that disruption of A. baumannii ribonuclease T2 family protein (ATCC 17978 locus A1S_3026) severely diminishes the organism's ability to colonize abiotic surfaces. We used Affymetrix A. baumannii GeneChips (part number PMDACBA1) to compare the gene expression properties of wild type and isogenic ribonuclease T2 family protein mutant cells. A. baumanni strain 98-37-09 (wild type) or isogenic ACJ7 (harboring a EZ-Tn5 insertion in A1S_3026) cells were grown to mid-exponential phase growth in Luria Burtani medium, total bacterial RNA was isolated and subjected to GeneChip hybridization and analysis. We sought to determine the regulatory effects of A1S_3026.
Project description:Schistosomiasis is a major human parasitic disease with a global impact. Schistosoma japonicum, the most difficult to control, can survive within host veins for decades. Mechanisms of immune evasion by the parasite, including antigenic variation and surface masking, have been implicated but not well defined. In this study, we defined the immunoglobulin-binding proteomes of S. japonicum using human IgG, IgM, and IgE as the molecular bait for affinity purification, followed by protein identification by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Several proteins situated at the tegument of S. japonicum were able to nonselectively bind to the Fc domain of host immunoglobulins, indicating a mechanism for the avoidance of host immune attachment and recognition. The profile of the immunoglobulin-binding proteomes provides further clues for immune evasion mechanisms adopted by S. japonicum.
Project description:Multi-organ failure caused by the inflammatory cytokine storm induced by severe infection is the major cause of death for sepsis. Sj-Cys is a cysteine protease inhibitor secreted by Schistosoma japonicum with strong immunomodulatory functions on host immune system. Our previous studies have shown that treatment with Sj-Cys recombinant protein (rSj-Cys) attenuated inflammation caused by sepsis. However, the immunological mechanism underlying the immunomodulation of Sj-Cys for regulating inflammatory diseases is not yet known. In this study, we investigated the effect of Sj-Cys on the macrophage M2 polarization and subsequent therapeutic effect on sepsis. The rSj-Cys was expressed in yeast Pichia pastoris. Incubation of mouse bone marrow-derived macrophages (BMDMs) with yeast-expressed rSj-Cys significantly activated the polarization of macrophages to M2 subtype characterized by the expression of F4/80+ CD206+ with the elated secretion of IL-10 and TGF-β. Adoptive transfer of rSj-Cys treated BMDMs to mice with sepsis induced by cecal ligation and puncture (CLP) significantly improved their survival rates and the systemic clinical manifestations of sepsis compared with mice receiving non-treated normal BMDMs. The therapeutic effect of Sj-Cys-induced M2 macrophages on sepsis was also reflected by the reduced pathological damages in organs of heart, lung, liver and kidney and reduced serological levels of tissue damage-related ALT, AST, BUN and Cr, associated with downregulated pro-inflammatory cytokines (IFN-gamma and IL-6) and upregulated regulatory anti-inflammatory cytokines (IL-10 and TGF-β). Our results demonstrated that Sj-Cys is a strong immunomodulatory protein with anti-inflammatory features through activating M2 macrophage polarization. The findings of this study suggested that Sj-Cys itself or Sj-Cys-induced M2 macrophages could be used as therapeutic agents in the treatment of sepsis or other inflammatory diseases.
Project description:The maturation of 5S ribosomal RNA in low G+C Gram-positive bacteria is catalyzed by a highly conserved, approximately 190 residue, enzyme, called ribonuclease M5 (RNase M5). Sequence alignment had predicted that the N-terminal half of RNase M5 would consist of a Toprim domain, a protein fold found in type IA and type II topoisomerases, DnaG-like primases, OLD family nucleases and RecR proteins [L. Aravind, D. D. Leipe and E. V. Koonin (1998) Nucleic Acids Res., 26, 4205-4213]. Here, we present structural modelling data and a mutational analysis of RNase M5 that confirms this hypothesis. The N-terminal half of RNase M5 can be fitted to the Toprim domain of the DnaG catalytic core. Mutation of amino acid residues highly conserved among RNase M5 enzymes and members of the Toprim domain family showed that alteration of residues critical for topoisomerase and primase activity also had a dramatic effect on the cleavage of 5S rRNA precursor by RNase M5 both in vivo and in vitro. This suggests that the mechanisms of double-stranded RNA cleavage by RNase M5 and double-stranded DNA cleavage by members of the topoisomerase family are related.
Project description:Schistosomiasis, a parasite infectious disease caused by Schistosoma japonicum, often leads to egg granuloma and fibrosis due to the inflammatory reaction triggered by egg antigens released in the host liver. This study focuses on the role of the egg antigens CP1412 protein of S. japonicum (SjCP1412) with RNase activity in promoting liver fibrosis. In this study, the recombinant egg ribonuclease SjCP1412, which had RNase activity, was successfully prepared. By analysing the serum of the population, it has been proven that the anti-SjCP1412 IgG in the serum of patients with advanced schistosomiasis was moderately correlated with liver fibrosis, and SjCP1412 may be an important antigen associated with liver fibrosis in schistosomiasis. In vitro, the rSjCP1412 protein induced the human liver cancer cell line Hep G2 and liver sinusoidal endothelial cells apoptosis and necrosis and the release of proinflammatory damage-associated molecular patterns (DAMPs). In mice infected with schistosomes, rSjCP1412 immunization or antibody neutralization of SjCP1412 activity significantly reduced cell apoptosis and necroptosis in liver tissue, thereby reducing inflammation and liver fibrosis. In summary, the SjCP1412 protein plays a crucial role in promoting liver fibrosis during schistosomiasis through mediating the liver cells apoptosis and necroptosis to release DAMPs inducing an inflammatory reaction. Blocking SjCP1412 activity could inhibit its proapoptotic and necrotic effects and alleviate hepatic fibrosis. These findings suggest that SjCP1412 may be served as a promising drug target for managing liver fibrosis in schistosomiasis japonica.
Project description:The 2',3'-cycling ribonuclease (RNase) genes are catalysts of RNA cleavage and include the RNase T2 gene family. RNase T2 genes perform important roles in plants and have been conserved in the genome of eukaryotic organisms. In this study we identified 21 EURNS genes in Eucommia ulmoides Oliver (E. ulmoides) and analyzed their structure, chromosomal location, phylogenetic tree, gene duplication, stress-related cis-elements, and expression patterns in different tissues. The length of 21 predicted EURNS proteins ranged from 143 to 374 amino acids (aa), their molecular weight (MW) ranged from 16.21 to 42.38 kDa, and their isoelectric point (PI) value ranged from 5.08 to 9.09. Two classifications (class I and class III) were obtained from the conserved domains analysis and phylogenetic tree. EURNS proteins contained a total of 15 motifs. Motif 1, motif 2, motif 3, and motif 7 were distributed in multiple sequences and were similar to the conserved domain of RNase T2. EURNS genes with similar structure and the predicted EURNS proteins with conserved motif compositions are in the same group in the phylogenetic tree. The results of RT-PCR and transcription data showed that EURNS genes have tissue-specific expression and exhibited obvious trends in different developmental stages. Gene duplication analysis results indicated that segment duplication may be the dominant duplication mode in this gene family. This study provides a theoretical basis for research on the RNase T2 gene family and lays a foundation for the further study of EURNS genes.
Project description:Schistosoma japonicum is a major infectious agent of schistosomiasis. It has been reported that large number of proteins excreted and secreted by S. japonicum during its life cycle are important for its infection and survival in definitive hosts. These proteins can be used as ideal candidates for vaccines or drug targets. In this work, we analyzed the protein sequences of S. japonicum and found that compared with other proteins in S. japonicum, excretory/secretory (ES) proteins are generally longer, more likely to be stable and enzyme, more likely to contain immune-related binding peptides and more likely to be involved in regulation and metabolism processes. Based on the sequence difference between ES and non-ES proteins, we trained a support vector machine (SVM) with much higher accuracy than existing approaches. Using this SVM, we identified 191 new ES proteins in S. japonicum, and further predicted 7 potential interactions between these ES proteins and human immune proteins. Our results are useful to understand the pathogenesis of schistosomiasis and can serve as a new resource for vaccine or drug targets discovery for anti-schistosome.
