MicroRNAs Are Involved in the Regulation of Ovary Development in the Pathogenic Blood Fluke Schistosoma japonicum.

Schistosomes, blood flukes, are an important global public health concern. Paired adult female schistosomes produce large numbers of eggs that are primarily responsible for the disease pathology and critical for dissemination. Consequently, understanding schistosome sexual maturation and egg production may open novel perspectives for intervening with these processes to prevent clinical symptoms and to interrupt the life-cycle of these blood-flukes. microRNAs (miRNAs) are key regulators of many biological processes including development, cell proliferation, metabolism, and signal transduction. Here, we report on the identification of Schistosoma japonicum miRNAs using small RNA deep sequencing in the key stages of male-female pairing, gametogenesis, and egg production. We identified 38 miRNAs, including 10 previously unknown miRNAs. Eighteen of the miRNAs were differentially expressed between male and female schistosomes and during different stages of sexual maturation. We identified 30 potential target genes for 16 of the S. japonicum miRNAs using antibody-based pull-down assays and bioinformatic analyses. We further validated some of these target genes using either in vitro luciferase assays or in vivo miRNA suppression experiments. Notably, suppression of the female enriched miRNAs bantam and miR-31 led to morphological alteration of ovaries in female schistosomes. These findings uncover key roles for specific miRNAs in schistosome sexual maturation and egg production.

Comparative characterization of microRNAs in Schistosoma japonicum schistosomula from Wistar rats and BALB/c mice.

More than 40 kinds of mammals in China are known to be naturally infected with Schistosoma japonicum (S. japonicum) (Peng et al. Parasitol Res 106:967-76, 2010). Compared with permissive BALB/c mice, rats are less susceptible to S. japonicum infection and are considered to provide an unsuitable microenvironment for parasite growth and development. MicroRNAs (miRNAs), via the regulation of gene expression at the transcriptional and post-transcriptional levels, may be responsible for developmental differences between schistosomula in these two rodent hosts. Solexa deep-sequencing technology was used to identify differentially expressed miRNAs from schistosomula isolated from Wistar rats and BALB/c mice 10 days post-infection. The deep-sequencing analysis revealed that nearly 40 % of raw reads (10.37 and 10.84 million reads in schistosomula isolated from Wistar rats and BALB/c mice, respectively) can be mapped to selected mirs in miRBase or in species-specific genomes. Further analysis revealed that several miRNAs were differentially expressed in schistosomula isolated from these two rodents; 18 were downregulated (by <2-fold) and 23 were up-regulated (>2-fold) (expression levels in rats compare with those in mice). Additionally, three novel miRNAs were primarily predicted and identified. Among the 41 differentially expressed miRNAs, 4 miRNAs had been identified with specific functions in schistosome development or host-parasite interaction, such as sexual maturation (sja-miR-1, sja-miR-7-5p), embryo development (sja-miR-36-3p) in schistosome, and pathogenesis of schistosomiasis (sja-bantam). Then, the target genes were mapped, filtered, and correlated with a set of genes that were differentially expressed genes in schistosomula isolated from mice and rats, which we identified in a S. japonicum oligonucleotide microarray analysis in a previous study. Gene Ontology (GO) analysis of the predicted target genes of 13 differentially expressed miRNAs revealed that they were involved in some important biological pathways, such as metabolic processes, the regulation of protein catabolic processes, catalytic activity, oxidoreductase activity, and hydrolase activity. The study presented here includes the first identification of differentially expressed miRNAs between schistosomula in mice or rats. Therefore, we hypothesized that the differentially expressed miRNAs may affect the development, growth, and maturation of the schistosome in its life cycle. Our analysis suggested that some differentially expressed miRNAs may impact the survival and development of the parasite within a host. This study increases our understanding of schistosome development and host-parasite interactions.

Proteomic analysis of tegument-exposed proteins of female and male Schistosoma japonicum worms.

The interplay between sexes is a prerequisite for female growth, reproductive maturation, and egg production, and the basis of schistosome pathopoiesis and propagation. The tegument is in direct contact with the host environment and its surface membranes are particularly crucial for schistosome survival in the definitive host. In this study, a streptavidin-biotin affinity purification technique combined with LC-MS/MS was used to analyze putative tegument-exposed proteins in female and male adult Schistosoma japonicum worms. In total, 179 proteins were identified in females and 300 in males, including 119 proteins common to both sexes, and 60 female biased and 181 male biased proteins. Some (e.g., serpin and CD36-like class B scavenger receptor) were involved in host-schistosome interactions, while some (e.g., gynecophoral canal protein) were important in the interplay between sexes. Gene Ontology analysis revealed that proteins involved in protein glycosylation and lysosome were highly expressed in females, while proteins involved in intracellular signal transduction, regulation of actin filament polymerization, and proteasome core complex were highly expressed in males. These results might elucidate physiological differences between the sexes. Our study provides new insights into schistosome growth and sexual maturity in the final host and permits the screening of vaccine candidates or drug targets for schistosomiasis.

Proteomic analysis of schistosoma japonicum schistosomulum proteins that are differentially expressed among hosts differing in their susceptibility to the infection.

Schistosomiasis is a tropical, parasitic disease affecting humans and several animal species. The aim of this study was to identify proteins involved in the growth and survival of the parasitic forms inside a host. Schistosomula of Schistosoma japonicum were isolated from three different hosts: the susceptible BALB/c mice; the Wistar rats, which have a considerably lower susceptibility; and the resistant reed vole, Microtus fortis. Soluble proteins of the schistosomula collected from the above three hosts 10 days postinfection were subjected to two-dimensional difference gel electrophoresis. Comparative proteomic analyses revealed that 39, 21, and 25 protein spots were significantly differentially expressed between schistosomula from mice and rats, mice and reed voles, or rats and reed voles, respectively (ANCOVA, p < 0.05). Further, the protein spots were identified by liquid chromatography-tandem MS. Bioinformatics analysis showed that the differentially expressed proteins were essentially those involved in the metabolism of proteins, ribonucleotides, or carbohydrates, or in stress response or cellular movement. This study represents the first attempt at profiling S. japonicum living in different states and provides a basis for a better understanding of the molecular mechanisms in the development and survival of S. japonicum in different host environments.

Characterization of Schistosoma japonicum estrogen-related receptor beta like 1 and immunogenicity analysis of the recombinant protein.

The estrogen-related receptor beta like 1 (EsRRBL1) is a sex hormone receptor. Here, we describe the cloning and expression of the EsRRBL1 gene from Schistosoma japonicum (SjEsRRBL1). Quantitative real time PCR (qPCR) and Western blot analysis revealed that SjEsRRBL1 was highly expressed in 14-, 18-, 23- and 28-days-old schistosomes at the transcriptional and protein levels, when the schistosomes were undergoing early development of reproductive organs, male and female coupling, and egg-laying. qPCR also showed that schistosomula isolated from a S. japonicum-susceptible mouse host had 3- to 4-fold higher expression of SjEsRRBL1 than that from the S. japonicum non-permissive Microtus fortis host or the non-susceptible rat host. Moreover, SjEsRRBL1 expression was 2-fold higher in schistosomula from female mice than that from male mice. Western blot analysis revealed that rSjEsRRBL1 had good antigenicity. After immunization of BALB/c mice with recombinant (r)SjEsRRBL1, partial and significantly protective efficacy was observed in two independent trials (30.84% and 30.70% worm reduction; 35.39% and 35.61% liver eggs reduction), as compared with the blank control group. An enzyme-linked immunosorbent assay (ELISA) showed that mice vaccinated with rSjEsRRBL1 produced increased levels of specific IgG, IFN-γ and IL-4, but a reduced IgG1/IgG2a ratio, as compared to the adjuvant control group and the blank control group, suggesting that rSjEsRRBL1 vaccination could induce a mixed Th1/Th2 response. The results suggested that SjEsRRBL1 might be a critical regulator of schistosome development and represent a promising vaccine target for schistosomiasis.

Molecular cloning and characterization of a cyclophilin A homologue from Schistosoma japonicum.

Cyclophilins belong to a group of proteins that have peptidyl-prolyl cis-trans isomerase activity and have been identified in all cell types and all organisms studied. In both prokaryotes and eukaryotes, they have been characterized as functional chaperones and involved in cell signaling. In the present study, Sj cyclophilin A (CyPA) was cloned, characterized, and subcloned into a prokaryotic expression vector to produce soluble recombinant rSjCyPA protein. qPCR analysis revealed that SjCyPA was expressed at each schistosome developmental stage tested, but reached its highest levels at days 7 and 13. In addition, the gene was also found to be significantly downregulated in adult worms from Microtus fortis. The SjCyPA protein was located on the subtegumental musculature of Schistosoma japonicum as determined by immunohistochemical staining analysis. Direct administration of recombinant SjCyPA to mice induced partial protective efficacy against subsequent schistosome infection. Length and width of adult worms and expression of SjCyPA were significantly decreased in the immunized groups, at 42 days post-infection, indicating that immunization with recombinant SjCyPA may suppress the schistosomes development. rSjCyPA can also react with sera from S. japonicum-infected rabbits at different time points. The data presented here suggest that SjCyPA may be an important molecule in the schistosome life-cycle and may be useful as a therapeutic target to treat schistosomiasis infection or as a potential diagnostic antigen.

Schistosoma japonicum: cloning, expression and characterization of a gene encoding the α5-subunit of the proteasome.

The development of an effective vaccine against the schistosome is thought to be the most desirable means to control schistosomiasis, even though there is an effective means of chemotherapy with praziquantel. A full-length cDNA encoding the Schistosoma japonicum proteasome subunit alpha type 5 protein (SjPSMA5) was first isolated from 18-day-schistosomulum cDNAs. The cDNA had an open reading frame (ORF) of 747bp and encoded 248 amino acids. Real-time quantitative RT-PCR analysis revealed that SjPSMA5 is up-regulated in 18-day and 32-day schistosomes, and the level of expression in male is around fourfold higher than that in female worms at 42 days. The SjPSMA5 was subcloned into pET28a(+) and expressed as inclusion bodies in Escherichia coli BL21 (DE3) cells. Western blotting showed that the recombinant SjPSMA5 (rSjPSMA5) was immunogenic. After immunization of BALB/c mice with rSjPSMA5, reductions of 23.29% and 35.24% were obtained in the numbers of worms and eggs in the liver, respectively. The levels of specific IgG antibodies and CD(4)(+) cells were significantly higher (P<0.01) in the group vaccinated with rSjPSMA5 combined with Seppic 206 adjuvant than in the other groups, as detected by enzyme linked immunosorbent assay (ELISA) and flow cytometry. The study suggested that rSjPSMA5 induced partial immunoprotection against S. japonicum in BALB/c mice, and it could be a potential vaccine candidate against schistosomiasis.

Molecular characterizations of an inhibitor of apoptosis from Schistosoma japonicum.

Apoptosis is a normal process for regulating cellular death of many organisms. Here, we molecularly characterized an inhibitor of apoptosis from Schistosoma japonicum (SjIAP). The transcription of the SjIAP predominantly occurred at the developmental stages in a final host. Functional assay indicated that the SjIAP could inhibit caspase activity either in 293T cell or in schistosome lysates. Additionally, there were differently expressed profiles of the SjIAP in S. japonicum living in different hosts. Our preliminary results suggest that the SjIAP may play important roles in parasitic living and development as well as in the host-parasite interactions, and drug target of SjIAP might be a potential for controlling schistosomiasis.

Molecular cloning and characterization of a gene encoding methionine aminopeptidase 2 of Schistosoma japonicum.

Methionine aminopeptidase 2 (MAP2) is an essential enzyme that is involved in protein maturation. It plays a key role in the removal of the initiating methionine residue from nascent polypeptide chains. In the present study, a gene encoding methionine aminopeptidase 2 of Schistosoma japonicum (SjMAP2) was cloned and characterized. Real-time RT-PCR and Western blot analysis revealed that this was expressed in each testing developmental stage in S. japonicum, but more highly expressed at 42 days in male adult worm, suggesting this gene as male differentially expressed. The results also showed that the gene was differentially expressed in worms from three different host species. It was highly expressed in worms from the schistosome-susceptible mouse, expressed at a lower level in worms from the less susceptible rat, and at an even lower level in worms from the non-permissive host Microtus fortis. The expression of the gene was affected significantly when the hosts were treated with praziquantel: Expression was down-regulated by 92.17% and 49.01%, respectively, in treated male and female adult worms in comparison with untreated worms. An immuno-experiment in mice indicated that vaccination with recombinant SjMAP2 could induce partial protective efficacy against schistosome infection. The data presented here suggest that SjMAP2 is an important molecule in the development of the schistosome and that it may be a potential new drug target or vaccine candidate for schistosomiasis.

Molecular cloning and expression profiles of Argonaute proteins in Schistosoma japonicum.

Small non-coding RNAs including microRNAs and small interfering RNAs play important roles in many biological processes of many organisms. Argonaute proteins serve as a key component of the RNA-induced silencing complex for mediating miRNA/siRNA functions. In the present study, we systematically investigated Argonaute proteins in Schistosoma japonicum by using bioinformatics in combination with 5'- and 3'-Rapid Amplification of cDNA ends techniques and thus obtained three full-length cDNAs encoding Argonaute proteins, named as SjAgo1, SjAgo2, and SjAgo3, respectively. Additionally, SjAgo1/2/3 were differentially expressed in different developmental stages of schistosomes as determined by real-time RT-PCR and Western blot. Taken together, our preliminary results suggested that SjAgo1/2/3 may control gene expression during the life cycle of S. japonicum and therefore may regulate schistosome development and other biological processes.

[Evaluation on the immuno-protective efficacy of the recombinant antigen SjPGAM-SjEnol against Schistosoma japonicum in mice].

OBJECTIVE: To construct and express the recombinant plasmid pET32a-SjPGAM-SjEnol and evaluate its immuno-protective efficacy against the infection of Schistosoma japonicum in mice. METHODS: The peptides of SjPGAM and SjEnol containing the multivalent epitopes with higher binding capacity of human MHC II and mouse H2-dII but low homology with the host were analyzed and screened through bioinformatics. The corresponding nucleotide sequence of selected epitopes was spliced and the recombinant plasmid pET32a-SjPGAM-SjEnol was constructed and expressed in Escherichia coli BL21 cells. The antigenicity of the recombinant protein was detected by Western blotting and the protective effect induced with the recombinant was evaluated in mice. 55 BALB/c mice were randomly divided into 5 groups each with 11. Mice from groups A, B and C were injected with a mixture of recombinant protein (27 microg) pET32a-SjPGAM-SjEnol (A), pETL28a-SjPGAM (B) and pET28a-SjEnol (C) respectively together with 206 adjuvant, mice from groups D and E received adjuvant or PBS only, all injected for three times at two-week intervals. Mice were then challenged with 40 +/- 2 cercariae of S. japonicum at two weeks after the last vaccination, and sacrificed for perfusion by 6 weeks post infection. Adult worms were collected, the number of eggs in a gram of liver tissue was counted, and the rates of worm reduction and egg reduction were calculated. Serum samples were collected before vaccination, every one week after each inoculation and before sacrifice, and specific IgG was detected by ELISA. RESULTS: The sequences encoding the 96-147 aa of SjPGAM and 233-312 aa of SjEonl were chosen for constructing the recombinant plasmid, a cDNA fragment with the length of 447 bp was amplified by PCR. The recombinant plasmid was expressed in E. coli with a molecular weight of Mr 33,000. Western blotting revealed that the fusion protein was recognized by the rabbit serum specific to SjSWAP, and showed an adequate antigenicity. Vaccination experiment showed that when compared with those of the blank control, the worm reduction rate in group A was 39.7%, significantly higher than that of groups B (18.5%) and C (14.7%) (P < 0.05). The liver egg reduction rate in group A was 64.9%, also higher than that of groups B (47.5%, P < 0.05) and C (30.5%, P < 0.01). ELISA showed that the serum specific IgG in group A (2.372 +/- 0.268) was much higher than that of groups D (0.490 +/- 0.138) (P < 0.01 and E (0.220 +/- 0.088) (P < 0.01). CONCLUSION: The recombinant plasmid pET32a-SjPGAM-SjEnol has been constructed, and recombinant protein pET32a-SjPGAM-SjEnol induces higher immune-protection against S. japonicum than that of SjPGAM and SjEonl.

Common genetic heterogeneity of human interleukin-37 leads to functional variance.

Interleukin-37 (IL-37) is an inhibitory member of the IL-1 family of cytokines. We previously found that balanced selection maintains common variations of the human IL37 gene. However, the functional consequences of this selection have yet to be validated. Here, using cells expressing exogenous IL-37 variants, including IL-37 Ref and IL-37 Var1 and Var2, we found that the three variants of IL-37 exhibited different immunoregulatory potencies in response to immune stimulation. The protein level of IL-37 Var2 was found to be significantly less than that of IL-37 Ref or Var1, despite the comparable mRNA levels of all three variants. Further study showed that IL-37 Var2 was rapidly degraded by a proteasome-dependent mechanism mediated by enhanced polyubiquitination, leading to a transient upregulation of IL-37 Var2 after immune stimulation. Finally, when ectopically expressed in cells, human IL-37 Var2 exerted less inhibition on proinflammatory cytokine production than did other IL-37 variants. Conversely, purified extracellular IL-37 variant proteins demonstrated comparable inhibitory abilities in vitro. In conclusion, our study reveals that common genetic variants of IL37 lead to different immune-inhibitory potencies, primarily as a result of differences in IL-37 protein stability, suggesting the possible involvement of these variants in various human diseases.

Interleukin-37 gene variants segregated anciently coexist during hominid evolution.

IL37 is a member of IL-1 cytokine family but conveys anti-inflammatory functions. The biological characteristic and genetic heterogeneity of IL37 are not fully understood yet. Here using the whole-genome sequencing data from 1000 Genomes Project, we performed population and evolutionary genetic analysis of human IL37 gene. First, 2184 IL37 gene sequences from different human populations were retrieved. The IL37 protein sequences were inferred from the coding DNA sequences and multiple species alignment was made. Then, the phylogenetic tree of IL37 was built and dN/dS ratios were calculated for each evolutionary branch, the classic McDonald and Kreitman test was also performed. Next, we conducted intraspecific evolutionary genetic analysis and built the genealogy network of 116 unique IL37 haplotypes through median-joining network analysis. Finally, we compared IL37 sequences between the modern and archaic humans. Our results for the first time provide solid evidence that common IL37 variants other than NCBI reference sequence are present worldwide. Our data also supports that IL37 variants are shaped and maintained by selection instead of neutral evolution. We further identified that human IL37 variants consist of two major haplogroups and their presence in archaic humans corroborates its ancient origin in hominid evolution. In conclusion, these data indicate that common IL37 variants are maintained among human populations by selective force, suggesting their potential involvements in immune regulation and human diseases. In addition, the ancient history of IL37 variants reveals interesting insight into the complicated human evolutionary history.

Comparative analysis of microRNA in schistosomula isolated from non-permissive host and susceptible host.

The reed vole Microtus fortis is the only known mammal in which the schistosome is naturally prevented from maturing and schistosome infection does not cause significant pathogenesis. However, the mechanism behind this phenomenon remains unknown. In the present study, Solexa deep sequencing technology was used to carry out high-throughput sequencing and comparative analysis of microRNA (miRNA) between small RNA libraries isolated from 10 days oldschistosomula of M. fortis and BALB/c mice.In total, 10d schistosomula from M. fortis and BALB/c mice yielded 13.37 and 10.84 million reads, respectively, and nearly 39% and 40% of reads could be mapped to selected miRNAs in miRbase. Based on a bioinformatic analysis, we found that most of the miRNAs identified in Schistosoma japonicum were detected in our study. Further analysis revealed that 24 miRNAs were differentially expressed between the schistosomula from the two rodents, of which 21 were down-regulated and three were up-regulated in schistosomula from M. fortis. Also, six novel miRNAs were predicted and identified in this study. Target genes were mapped and filtered by correlating them with differentially expressed genes obtained from S. japonicum oligonucleotide microarray analyses performed in previous studies. miRNAs such as miR-10-3p, miR-10-5p, and miR-2b-5p may affect the growth, differentiation, and metabolism of worms via regulation of the expression of target genes such as enolase, aquaporin, TGF-beta-inducible nuclear protein, and paramyosin. Gene Ontology analysis of the predicted target genes of these six differentially expressed miRNAs revealed that some important biological pathways, such as metabolic processes,glycolysis, and catalytic activity, were involved. The results of this study highlight the function of miRNAs in the development and survival of the schistosome, and provide valuable information to increase our understanding of the regulatory function of miRNAs in schistosome development and host-parasite interactions in a differentially susceptible host environment.

MicroRNA expression profile in different tissues of BALB/c mice in the early phase of Schistosoma japonicum infection.

Schistosomiasis remains an important global public health problem that affects 200 million people in 76 countries. The molecular mechanisms of host-parasite interaction are complex, and in schistosome infection regulation of microRNA (miRNA) and the host micro-environment may be involved. In this study, an miRNA microarray was applied to investigate differences in miRNA expression in different tissues of mice before and 10 days post infection. In total, 220 miRNAs were detected in different tissues of the BALB/c mice before and after infection, including 8 miRNAs in liver, 8 in spleen and 28 in the lungs with up-regulated expression, and 3 miRNAs in liver, 5 in spleen and 28 in the lungs with down-regulated expression in mice 10 days post infection with schistosomes. The functions of these differentially expressed miRNAs are related mainly to the immune response, nutrient metabolism, cell differentiation, apoptosis, and signal pathways. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the differentially expressed miRNAs revealed that many important biological pathways are triggered by schistosome infection in BALB/c mice, such as the MAPK signaling pathway, insulin signaling pathway, Toll-like receptor signaling pathway and TGF-ß signaling pathway.The results reveal that miRNAs may be an important regulator of schistosome-host interaction in the early phase of Schistosoma japonicum infection. The data presented here provide valuable information to increase understanding of the regulatory function of the miRNAs in the host micro-environment, as well as the mechanism of host-parasite interactions. This may be helpful in the search for potential new drugs, and for biomarkers of early S. japonicum infection applicable in the future control of schistosomiasis.

Apoptosis phenomenon in the schistosomulum and adult worm life cycle stages of Schistosoma japonicum.

Apoptosis is an important aspect of a number of biological processes, from embryogenesis to the stress-injury response. It plays a central role in balancing cell proliferation and tissue remodeling activity in many organisms. In the present study, apoptosis in 14 days post infection schistosomula was evaluated using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assays and DAPI staining. Additionally, flow cytometry using the Annexin V-FITC/propidium iodide (PI) (Annexin V/PI) assay confirmed the percentage of early apoptotic, late apoptotic, and necrotic cells in 14 and 23 days post infection worms. Conserved Domain Database (CDD) BLAST analysis and alignment analysis of known schistosome proteins demonstrated the feasibility of detecting the activity of caspase-3 and -7 using the caspase-3/7 Glo analysis assay. Analysis of caspase-3 and -7 activities in schistosome demonstrated that both caspases were active in each developmental stage of Schistosoma japonicum, but was highest in the 14 days post infection schistosomula. Additionally, the caspase peptide inhibitor (Z-VAD-FMK) inhibited the caspase-3/7 activity at all developmental stages examined. Therefore, we hypothesized that two main signaling pathways are involved in apoptosis in S. japonicum, the caspase cascade and the mitochondrial-initiated pathway. We have constructed a model of these two pathways, including how they may interact and their biological outcomes. qRT-PCR analyses of the gene expression profiles of apoptosis-related genes supported our hypothesis of the relationship between the apoptotic pathway and parasite development. The data presented here demonstrates that apoptosis is an important biological process for the survival and development of the schistosome, and identifies potential novel therapeutic targets.

Comparison of the differential expression miRNAs in Wistar rats before and 10 days after S.japonicum infection.

BACKGROUND: When compared to the murine permissive host of Schistosoma japonicum, Wistar rats are less susceptible to Schistosoma japonicum infection, and are considered to provide a less suitable microenvironment for parasite growth and development. MicroRNAs (miRNAs), are a class of endogenous, non-coding small RNAs, that impose an additional, highly significant, level of gene regulation within eukaryotes. METHODS: To investigate the regulatory mechanisms provided by miRNA in the schistosome-infected rat model, we utilized a miRNA microarray to compare the progression of miRNA expression within different host tissues both before and 10 days after cercarial infection, in order to identify potential miRNAs with roles in responding to a schistosome infection. RESULTS: Among the analysed miRNAs, 16 within the liver, 61 within the spleen and 10 within the lung, were differentially expressed in infected Wistar rats. Further analysis of the differentially expressed miRNAs revealed that many important signal pathways are triggered after infection with S. japonicum in Wistar rats. These include the signal transduction mechanisms associated with the Wnt and MAPK signaling pathways, cellular differentiation, with a particular emphasis on adipocyte and erythroid differentiation. CONCLUSIONS: The results presented here include the identification of specific differentially expressed miRNAs within the liver, lungs and spleen of Wistar rats. These results highlighted the function of host miRNA regulation during an active schistosome infection. Our study provides a better understanding of the regulatory role of miRNA in schistosome infection, and host-parasite interactions in a non-permissive host environment.

Differential expression of microRNAs in the non-permissive schistosome host Microtus fortis under schistosome infection.

The reed vole Microtus fortis is the only mammal known in China in which the growth, development and maturation of schistosomes (Schistosoma japonicum) is prevented. It might be that the anti-schistosomiasis mechanisms of M. fortis associate with microRNA-mediated gene expression, given that the latter has been found to be involved in gene regulation in eukaryotes. In the present study, the difference between pathological changes in tissues of M. fortis and of mice (Mus musculus) post-schistosome infection were observed by using hematoxylin-eosin staining. In addition, microarray technique was applied to identify differentially expressed miRNAs in the same tissues before and post-infection to analyze the potential roles of miRNAs in schistosome infection in these two different types of host. Histological analyses showed that S. japonicum infection in M. fortis resulted in a more intensive inflammatory response and pathological change than in mice. The microarray analysis revealed that 162 miRNAs were expressed in both species, with 12 in liver, 32 in spleen and 34 in lung being differentially expressed in M. fortis. The functions of the differentially expressed miRNAs were mainly revolved in nutrient metabolism, immune regulation, etc. Further analysis revealed that important signaling pathways were triggered after infection by S. japonicum in M. fortis but not in the mice. These results provide new insights into the general mechanisms of regulation in the non-permissive schistosome host M. fortis that exploits potential miRNA regulatory networks. Such information will help improve current understanding of schistosome development and host-parasite interactions.

[Cloning, expression and immune function analysis of a gene encoding inositol monophosphate in Schistosoma japonicum].

OBJECTIVE: To clone and express a full-length cDNA encoding inositol monophosphate of Schistosoma japonicum (SjIM), and to access its immunoprotection in BALB/c mice for schistosomisis. METHODS: A full-length cDNA encoding the S. japonicum inositol monophosphate was isolated from 42 d schistosomes cDNAs. The expression profiles in different developmental stages were detected by real-time quantitative RT-PCR. The open reading frame (ORF) was subcloned into a pET28a(+) vector and transformed into BL21 and the recombinant protein was induced by IPTG. The immune characters of the purified recombinant protein were analyzed by Western blotting and immunoprotection in BALB/c mice. RESULTS: Bioinformatics analysis indicated that SjIM had an ORF of 834 base pairs that encoded 278 amino acids. Real-time quantitative RT-PCR analysis revealed that SjIM was upregulated in 35-day-old schistosomes, while the expression level in females was higher than that in male worms in 42nd day. Western blotting showed that the recombinant SjIM was immunogenic. An immunoprotection experiment in BALB/c mice showed that vaccination with recombinant SjIM could induce 48.76% and 41.29% reductions in the numbers of worms and eggs in the liver, respectively. CONCLUSIONS: The gene of SjIM is obtained from schistosomes cDNAs and the recombinant SjIM protein is induced successfully in E. coli. These aforementioned results demonstrate that the recombinant SjIM cand induce partial protection against schistosomiasis in BALB/c mice.

Differential gene expression in Schistosoma japonicum schistosomula from Wistar rats and BALB/c mice.

BACKGROUND: More than 46 species of mammals can be naturally infected with Schistosoma japonicum in the mainland of China. Mice are permissive and may act as the definitive host of the life cycle. In contrast, rats are less susceptible to S. japonicum infection, and are considered to provide an unsuitable micro-environment for parasite growth and development. Since little is known of what effects this micro-environment has on the parasite itself, we have in the present study utilised a S. japonicum oligonucleotide microarray to compare the gene expression differences of 10-day-old schistosomula maintained in Wistar rats with those maintained in BALB/c mice. RESULTS: In total 3,468 schistosome genes were found to be differentially expressed, of which the majority (3,335) were down-regulated (≤ 2 fold) and 133 were up-regulated (≥ 2 fold) in schistosomula from Wistar rats compared with those from BALB/c mice. Gene ontology (GO) analysis revealed that of the differentially expressed genes with already established functions or close homology to well characterized genes in another organisms, many are related to important biological functions or molecular processes. Among the genes that were down-regulated in schistosomula from Wistar rats, some were associated with metabolism, signal transduction and development. Of these genes related to metabolic processes, areas including translation, protein and amino acid phosphorylation, proteolysis, oxidoreductase activities, catalytic activities and hydrolase activities, were represented. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of differential expressed genes indicated that of the 328 genes that had a specific KEGG pathway annotation, 324 were down-regulated and were mainly associated with metabolism, growth, redox pathway, oxidative phosphorylation, the cell cycle, ubiquitin-mediated proteolysis, protein export and the MAPK (mitogen-activated protein kinases) signaling pathway. CONCLUSIONS: This work presents the first large scale gene expression study identifying the differences between schistosomula maintained in mice and those maintained in rats, and specifically highlights differential expression that may impact on the survival and development of the parasite within the definitive host. The research presented here provides valuable information for the better understanding of schistosome development and host-parasite interactions.