Weiqing Pan

Identification and Characterization of Novel MicroRNAs from Schistosoma japonicum

Background Schistosomiasis japonica remains a major public health problem in China. Its pathogen, Schistosoma japonicum has a complex life cycle and a unique repertoire of genes expressed at different life cycle stages. Exploring schistosome gene regulation will yield the best prospects for new drug targets and vaccine candidates. MicroRNAs (miRNAs) are a highly conserved class of noncoding RNA that control many biological processes by sequence-specific inhibition of gene expression. Although a large number of miRNAs have been identified from plants to mammals, it remains no experimental proof whether schistosome exist miRNAs. Methodology and Results We have identified novel miRNAs from Schistosoma japonicum by cloning and sequencing a small (18–26 nt) RNA cDNA library from the adult worms. Five novel miRNAs were identified from 227 cloned RNA sequences and verified by Northern blot. Alignments of the miRNAs with corresponding family members indicated that four of them belong to a metazoan miRNA family: let-7, miR-71, bantam and miR-125. The fifth potentially new (non conserved) miRNA appears to belong to a previously undescribed family in the genus Schistosome. The novel miRNAs were designated as sja-let-7, sja-miR-71, sja-bantam, sja-miR-125 and sja-miR-new1, respectively. Expression of sja-let-7, sja-miR-71 and sja-bantam were analyzed in six stages of the life cycle, i.e. egg, miracidium, sporocyst, cercaria, schistosomulum, and adult worm, by a modified stem-loop reverse transcribed polymerase chain reaction (RT-PCR) method developed in our laboratory. The expression patterns of these miRNAs were highly stage-specific. In particular, sja-miR-71 and sja-bantam expression reach their peaks in the cercaria stage and then drop quickly to the nadirs in the schistosomulum stage, following penetration of cercaria into a mammalian host. Conclusions Authentic miRNAs were identified for the first time in S. japonicum, including a new schistosome family member. The different expression patterns of the novel miRNAs over the life stages of S. japonicum suggest that they may mediate important roles in Schistosome growth and development.

An “In-Depth” Description of the Small Non-coding RNA Population of Schistosoma japonicum Schistosomulum

Parasitic flatworms of the genus Schistosoma are the causative agents of schistosomiasis, which afflicts more than 200 million people yearly in tropical regions of South America, Asia and Africa. A promising approach to the control of this and many other diseases involves the application of our understanding of small non-coding RNA function to the design of safe and effective means of treatment. In a previous study, we identified five conserved miRNAs from the adult stage of Schistosoma japonicum. Here, we applied Illumina Solexa high-throughput sequencing methods (deep sequencing) to investigate the small RNAs expressed in S. japonicum schistosomulum (3 weeks post-infection). This has allowed us to examine over four million sequence reads including both frequently and infrequently represented members of the RNA population. Thus we have identified 20 conserved miRNA families that have orthologs in well-studied model organisms and 16 miRNA that appear to be specific to Schistosoma. We have also observed minor amounts of heterogeneity in both 3′ and 5′ terminal positions of some miRNA as well as RNA fragments resulting from the processing of miRNA precursor. An investigation of the genomic arrangement of the 36 identified miRNA revealed that seven were tightly linked in two clusters. We also identified members of the small RNA population whose structure indicates that they are part of an endogenously derived RNA silencing pathway, as evidenced by their extensive complementarities with retrotransposon and retrovirus-related Pol polyprotein from transposon.

A Critical Role of Perinuclear Filamentous Actin in Spatial Repositioning and Mutually Exclusive Expression of Virulence Genes in Malaria Parasites

Many microbial pathogens, including the malaria parasite Plasmodium falciparum, vary surface protein expression to evade host immune responses. P. falciparium antigenic variation is linked to var gene family-encoded clonally variant surface protein expression. Mututally exclusive var gene expression is partially controlled by spatial positioning; silent genes are retained at distinct perinuclear sites and relocated to transcriptionally active locations for monoallelic expression. We show that var introns can control this process and that var intron addition relocalizes episomes from a random to a perinuclear position. This var intron-regulated nuclear tethering and repositioning is linked to an 18 bp nuclear protein-binding element that recruits an actin protein complex. Pharmacologically induced F-actin formation, which is restricted to the nuclear periphery, repositions intron-carrying episomes and var genes and disrupts mutually exclusive var gene expression. Thus, actin polymerization relocates var genes from a repressive to an active perinuclear compartment, which is crucial for P. falciparium phenotypic variation and pathogenesis.

Serodiagnosis of Schistosoma japonicum infection: genome-wide identification of a protein marker, and assessment of its diagnostic validity in a field study in China

BACKGROUND Schistosomiasis remains a highly prevalent and serious parasitic disease. A major factor preventing its effective management is the scarcity of effective diagnostic tools. We did a genome-wide identification of diagnostic protein markers for schistosome infection and assessed their diagnostic validity in a field study. METHODS We predicted putative secreted proteins of Schistosoma japonicum (SjSPs) and expressed them as glutathione S-transferase (GST)-fusion proteins. The fusion proteins were arrayed on glutathione (GSH)-immobilised microplates and screened with serum samples from patients with schistosomiasis diagnosed by the Kato-Katz method. We further assessed an identified protein marker for sensitivity and specificity, first in infected serum samples collected from Jiangxi and Hunan Provinces, China, and then through a field study, done in two villages located in a high schistosomiasis-endemic area of the southeast of China. FINDINGS Of 204 recombinant proteins, 35 yielded seropositive reactions, eight showed strong immunoreactivity, and only one (SjSP-13) reacted to the entire panel of 14 archived samples. The reactivity of SjSP-13 to 476 serum samples showed 90·4% (95% CI 86·5-93·5) sensitivity and 98·9% (95% CI 95·9-99·9) specificity. Of 1371 residents enrolled in a field study from Dec 6, 2010, to June 23, 2011, only 74 individuals were identified as being egg-positive, whereas 465 were diagnosed as positive by the SjSP-13-based ELISA kit (rSP13-ELISA). Of the 394 individuals found egg-negative but rSP13-ELISA-positive, 363 (92·4%) were confirmed to be positive for schistosome infection by PCR detection of S japonicum SjR2 retrotransposon. INTERPRETATION The application of this sensitive, specific, and affordable rSP13-ELISA method should help reduce schistosomiasis transmission through targeted treatment of individuals, particularly with low intensity infections, and therefore support schistosomiasis control and elimination strategies. FUNDING National 973 project in China.

No miRNA were found in Plasmodium and the ones identified in erythrocytes could not be correlated with infection

BackgroundThe transcriptional regulation of Plasmodium during its complex life cycle requires sequential activation and/or repression of different genetic programmes. MicroRNAs (miRNAs) are a highly conserved class of non-coding RNAs that are important in regulating diverse cellular functions by sequence-specific inhibition of gene expression. What is know about double-stranded RNA-mediated gene silencing (RNAi) and posttranscriptional gene silencing (PTGS) in Plasmodium parasites entice us to speculate whether miRNAs can also function in Plasmodium-infected RBCs.ResultsOf 132 small RNA sequences, no Plasmodium-specific miRNAs have been found. However, a human miRNA, miR-451, was highly expressed, comprising approximately one third of the total identified miRNAs. Further analysis of miR-451 expression and malaria infection showed no association between the accumulation of miR-451 in Plasmodium falciparum-iRBCs, the life cycle stage of P. falciparum in the erythrocyte, or of P. berghei in mice. Moreover, treatment with an antisense oligonucleotide to miR-451 had no significant effect on the growth of the erythrocytic-stage P. falciparum.MethodsShort RNAs from a mixed-stage of P. falciparum-iRBC were separated in a denaturing polyacrylamide gel and cloned into T vectors to create a cDNA library. Individual clones were then sequenced and further analysed by bioinformatics prediction to discover probable miRNAs in P. falciparum-iRBC. The association between miR-451 expression and the parasite were analysed by Northern blotting and antisense oligonucleotide (ASO) of miR-451.ConclusionThese results contribute to eliminate the probability of miRNAs in P. falciparum. The absence of miRNA in P. falciparum could be correlated with absence of argonaute/dicer genes. In addition, the miR-451 accumulation in Plasmodium-infected RBCs is independent of parasite infection. Its accumulation might be only the residual of erythroid differentiation or a component to maintain the normal function of mature RBCs.

A Schistosoma japonicum chimeric protein with a novel adjuvant induced a polarized Th1 immune response and protection against liver egg burdens.

BackgroundSchitosomiasis japonica is still a significant public health problem in China. A protective vaccine for human or animal use represents an important strategy for long-term control of this disease. Due to the complex life cycle of schistosomes, different vaccine design approaches may be necessary, including polyvalent subunit vaccines. In this study, we constructed four chimeric proteins (designated SjGP-1~4) via fusion of Sj26GST and four individual paramyosin fragments. We tested these four proteins as vaccine candidates, and investigated the effect of deviating immune response on protection roles in mice.MethodsThe immunogencity and protection efficacy of chimeric proteins were evaluated in mice. Next, the chimeric protein SjGP-3 was selected and formulated in various adjuvants, including CFA, ISA 206, IMS 1312 and ISA 70M. The titers of antigen-specific IgG, IgE and IgG subclass were measured. The effect of adjuvant on cytokine production and percentages of CD3+CD8-IFN-γ+ cells and CD3+CD8-IL-4+ cells were analyzed at different time points. Worm burdens and liver egg counts in different adjuvant groups were counted to evaluate the protection efficacy against cercarial challenge.ResultsImmunization of mice with chimeric proteins provided various levels of protection. Among the four proteins, SjGP-3 induced the highest level of protection, and showed enhanced protective efficacy compared with its individual component Sj26GST. Because of this, SjGP-3 was further formulated in various adjuvants to investigate the effect of adjuvant on immune deviation. The results revealed that SjGP-3 formulated in veterinary adjuvant ISA 70M induced a lasting polarized Th1 immune response, whereas the other adjuvants, including CFA, ISA 206 and IMS 1312, generated a moderate mixed Th1/Th2 response after immunization but all except for IMS 1312 shifted to Th2 response after onset of eggs. More importantly, the SjGP-3/70M formulation induced a significant reduction in liver egg deposition at 47.0–50.3% and the number of liver eggs per female at 34.5–37.2% but less effect on worm burdens at only 17.3–23.1%, whereas no effect of the formulations with other adjuvants on the number of liver eggs per female was observed.ConclusionConstruction of polyvalent subunit vaccine was capable to enhance immunogenicity and protection efficacy against schistosomiasis. There was correlation of the polarized Th1 response with reduction of liver egg burdens, supporting the immune deviation strategy for schistosomiasis japonica vaccine development.

Host serum miR-223 is a potential new biomarker for Schistosoma japonicum infection and the response to chemotherapy

BackgroundNumerous studies have shown that aberrant microRNA (miRNA) expression is associated with the pathogenesis and progression of various human diseases. Hence, serum miRNAs are considered to be potential biomarkers for the diagnosis of human diseases. This study examined whether several miRNAs known to be commonly deregulated in liver diseases are deregulated in the serum of hosts with hepatic schistosomiasis, and thus whether they could serve as potential markers for detection of schistosome infection and evaluation of the effectiveness of chemotherapy.MethodsWe analyzed the serum levels of six selected candidate miRNA molecules (miR-146b, miR-122, miR-223, miR-199a-5p, miR-199a-3p, miR-34a) from mice, rabbits, buffalos and humans infected with Schistosoma japonicum using qPCR. We evaluated liver pathology by determining the hydroxyproline content in liver tissues. Primary resident liver cells were isolated to quantify the expression level of deregulated miRNAs. Bioinformatics analyses were also conducted to assess the potential function of miR-223.ResultsUsing a mouse model of Schistosoma japonicum infection, we found that the expression level of serum miR-223 was significantly elevated after infection, but returned to near normal levels after the treatment with praziquantel (PZQ). Importantly, the level of serum miR-223 reflected the extent of liver pathology post-infection. We validated the elevated level of the circulating miR-223 in serum samples of other host species including rabbits, buffalos and humans. In addition, our results showed that miR-223 was primarily located in the Kupffer cells, but its expression levels were significantly up-regulated in hepatocytes, hepatic stellate cells and Kupffer cells after infection. Bioinformatics analyses revealed a potential functional role of miR-223 in transcription regulator activity, transcription factor activity and DNA binding.ConclusionsThis study suggested that the circulating miR-223 could serve as a potential new biomarker for the detection of schistosome infection and the assessment of the response to chemotherapy.

Analysis and characterization of the genes encoding the Dicer and Argonaute proteins of Schistosoma japonicum

BackgroundThe Dicer and Argonaute(AGO) proteins within the small RNA regulatory pathways (SRRPs) play an indispensable role in regulation of gene expression. In this study, we analyzed two genes, Dicer and Argonaute, from Schistosoma japonicum, along with their expression through a combination of bioinformatics and experimental approaches.ResultsOur results indicate that one Dicer and four Argonaute genes exist in Schistosoma japonicum, termed SjDicer and SjAGO1, 2, 3, and 4, respectively. SjDicer encodes 2590 amino acid residues that contains 5 conserved domains, including one amino-terminal helicase domain, one PAZ (Piwi-Argonaut-Zwille) domain, two RNAse III domains, and one dsRNA-binding domain. SjAGO1, 2, and 3 encode 1009, 945, and 904 amino acid residues, respectively, all of which contain PAZ and PIWI domains. In addition, we analyzed the expression profiles of SjDicer and SjAGO1 genes by qRT-PCR in eggs, miracidium, cercariae, schistosomula, and adult worms. Results showed consistent expression of both SjDicer and SjAGO1 in different stages; however, their expression levels were stage-dependent, with the highest being in the miracidium stage.ConclusionsThis study provided the sequence of the Dicer and Ago genes of S. japonicum and their expression profiles which are essential for further investigation of functions of miRNA in Schistosoma japonicum.

From In Vivo to In Vitro: Dynamic Analysis of Plasmodium falciparum var Gene Expression Patterns of Patient Isolates during Adaptation to Culture

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var gene family, plays a crucial role in disease virulence through its involvement in binding to various host cellular receptors during infection. Growing evidence suggests that differential expression of the various var subgroups may be involved in parasite virulence. To further explore this issue, we have collected isolates from symptomatic patients in south China-Myanmar border, and characterized their sequence diversity and transcription profiles over time of var gene family, and cytoadherence properties from the time of their initial collection and extending through a two month period of adaptation to culture. Initially, we established a highly diverse, DBLα (4 cysteines) subtype-enriched, but unique local repertoire of var-DBL1α sequences by cDNA cloning and sequencing. Next we observed a rapid transcriptional decline of upsA- and upsB-subtype var genes at ring stage through qRT-PCR assays, and a switching event from initial ICAM-I binding to the CD36-binding activity during the first week of adaptive cultivation in vitro. Moreover, predominant transcription of upsA var genes was observed to be correlated with those isolates that showed a higher parasitemia at the time of collection and the ICAM-1-binding phenotype in culture. Taken together, these data indicate that the initial stage of adaptive process in vitro significantly influences the transcription of virulence-related var subtypes and expression of PfEMP1 variants. Further, the specific upregulation of the upsA var genes is likely linked to the rapid propagation of the parasite during natural infection due to the A-type PfEMP1 variant-mediated growth advantages.

Transcriptome Bioinformatical Analysis of Vertebrate Stages of Schistosoma japonicum Reveals Alternative Splicing Events

Alternative splicing is a molecular process that contributes greatly to the diversification of proteome and to gene functions. Understanding the mechanisms of stage-specific alternative splicing can provide a better understanding of the development of eukaryotes and the functions of different genes. Schistosoma japonicum is an infectious blood-dwelling trematode with a complex lifecycle that causes the tropical disease schistosomiasis. In this study, we analyzed the transcriptome of Schistosoma japonicum to discover alternative splicing events in this parasite, by applying RNA-seq to cDNA library of adults and schistosomula. Results were validated by RT-PCR and sequencing. We found 11,623 alternative splicing events among 7,099 protein encoding genes and average proportion of alternative splicing events per gene was 42.14%. We showed that exon skip is the most common type of alternative splicing events as found in high eukaryotes, whereas intron retention is the least common alternative splicing type. According to intron boundary analysis, the parasite possesses same intron boundaries as other organisms, namely the classic “GT-AG” rule. And in alternative spliced introns or exons, this rule is less strict. And we have attempted to detect alternative splicing events in genes encoding proteins with signal peptides and transmembrane helices, suggesting that alternative splicing could change subcellular locations of specific gene products. Our results indicate that alternative splicing is prevalent in this parasitic worm, and that the worm is close to its hosts. The revealed secretome involved in alternative splicing implies new perspective into understanding interaction between the parasite and its host.