Xindong Xu

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.

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.

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.

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.

Protein array identification of protein markers for serodiagnosis of Mycobacterium tuberculosis infection

The lack of effective and accurate diagnostic tools contributes to the high prevalence of tuberculosis (TB) worldwide. The current serodiagnostics for TB are inadequate mainly due to lack of TB-specific antigens with highly accurate diagnosis. In the current study, we aimed to identify novel diagnostic antigens using glutathione S-transferase (GST)-fusion protein technique. We determined the reactivity of these recombinant proteins arrayed in solution and on GSH-immobilized microplates with TB patient sera. Of 409 TB proteins produced, ninety-two yielded seropositive reactions, fourteen including eight novel proteins showed strong immunoreactivity. Further, six were selected and constructed as a multiple-antigen combination set through analysis of various combinations. A comparative study of the multiple-antigen combination set and a commercially available kit revealed that the combination set showed 66.3% (95% CI 60.5–71.8) sensitivity, which was significantly higher than that of the commercial kit [31.6% (95% CI 26.3–37.3)]. The specificity of both methods was similar at 89.6% (95% CI 83.3–95.4) and 90.6% (95% CI 83.0–95.6), respectively. This study provides a set of novel diagnostic protein markers with great potential for the development of novel diagnostic tools for active TB.

Having a pair: the key to immune evasion for the diploid pathogen Schistosoma japonicum.

Schistosomes, unlike malaria parasites, are in their diploid stage when targeted by the human immune system. Diploids can be either homozygous or heterozygous. The difference has profound significance for developing immunity and yet has not previously been addressed. We examined the implications of zygosity on immunity to a diploid pathogen, Schistosoma japonicum and showed that the diploid state, and its associated heterozygous advantage, significantly affects the outcome of attack by the immune system and the accumulation of antigenic diversity in the parasite population. We demonstrate here that diploidy provides a novel means of immune evasion for diploid pathogens.

Influence of HLA-DRB1 Alleles on Antibody Responses to PfCP-2.9-Immunized and Naturally Infected Individuals

IntroductionThe Plasmodium falciparum chimeric protein, PfCP-2.9, which consists of apical membrane antigen (AMA)-1(III) and merozoite surface protein (MSP)1–19, is a promising asexual-stage malaria vaccine currently being evaluated in clinical trials. This study attempts to investigate the potential association between human leukocyte antigen (HLA)-DRB1 genotype and antibody response against PfCP-2.9 in healthy population and malaria patients.Materials and methodsWe investigated the HLA-DRB1 alleles in 40 participants from phase I trial and 86 malaria patients from southern China by polymerase chain reaction with allele sequence-specific primers. The antibody and cellular response against PfCP-2.9 or its components were measured by enzyme-linked immunosorbent assay and T lymphocyte proliferation assay.ResultsIn clinical subjects, the anti-PfCP-2.9 antibody response was likely suppressed by HLA-DR6 alleles, which was consistent with the T lymphocyte proliferation assay. Nevertheless, HLA-DR7 positively correlated with antibody responses in naturally infected individuals while DR8 correlated with weaker antibody responses for all the three recombinant proteins. Moreover, parasitemia was significantly lower in samples with higher antibody levels against PfCP-2.9 or rMSP1–19, but not for rAMA-1(III).ConclusionThese data suggest that antibody responses against PfCP-2.9, AMA-1(III), or MSP1–19 elicited by vaccine formulation or natural infection are controlled by different HLA-II alleles. Moreover, the antibody response to MSP1–19 contributed more to protection immunity than AMA-1(III).

Protective immunity against Schistosoma japonicum infection can be provided by IgG antibodies towards periodate-sensitive or periodate-resistant glycans

BackgroundIt has been well accepted that glycans present in schistosomes are highly antigenic. However, it is not clear what kind of worm glycans can affect the infected host to mount IgG responses and whether mounted anti-glycan IgG responses are protective.MethodsThe contribution of antigenicity by glycans was measured by using competitive ELISA assay in sera from infected mice and humans. Monoclonal antibodies towards soluble Schistosoma japonicum egg antigens (SjEA) were generated from SjEA immunizated mice. The expression of glycans on surfaces of cercaria or young worm and their distributions were examined by immunofluorescence assay. The protective roles of glycans-specific mAbs were assayed by determination of the worm and egg burden in infected mice.ResultsBoth periodate-resistant glycans and periodate-sensitive glycans are antigenic in schistosome infections. When monoclonal antibodies against either periodate-sensitive or periodate-resistant glycans were administered prior to schistosome infections in mice, both kinds of anti-glycan antibodies were found to successfully provide protective immunity to infected mice.ConclusionsBoth periodate-resistant and periodate-sensitive glycans are antigenic, and dominant anti-glycan IgG responses can play important roles in protective immunity in schistosome infected hosts.

Identification and characterization of six novel tetraspanins from Schistosoma japonicum.

BackgroundTetraspanins (TSPs), also known as members of the trans-membrane 4 super-family (TM4SF), comprise an assemblage of surface antigens reported in eukaryotic organisms. In the work presented here, six novel TSP proteins from the human blood fluke Schistosoma japonicum (S. japonicum) were produced and analyzed through a combination of bioinformatics and experimental approaches.ResultsSix novel TSP proteins of Schistosoma japonicum (designated as Sj-TSP-#1~6) contained four trans-membrane regions and one large extracellular loop (LEL) with a conserved CCG motif. Size of the proteins varied from 227 to 291 amino acid residues. All the six proteins were produced in E.coli and immune sera to each protein were prepared. Analysis of transcription profiles of the proteins by RT-PCR showed that Sj-TSP-#4 was transcribed only in the egg stage while transcription of the Sj-TSP-#2 was detected in female worms but not in males. The similar results were obtained by Western blot. Immunolocalization of the TSP proteins by immunofluorescence assay showed that the Sj-TSP-#2, Sj-TSP-#5 and Sj-TSP-#6 were located in the tegument of worms.ConclusionsThis study provided six novel TSP members of S. japonicum including their sequences and recombinant proteins. Availability of the novel proteins and information on their expression profile and location provided a basis for further investigation of the TSP proteins for their biological functions and as vaccine candidates.