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Discovery of novel Schistosoma japonicum antigens using a targeted protein microarray approach.

ABSTRACT: BACKGROUND: Novel vaccine candidates against Schistosoma japonicum are required, and antigens present in the vulnerable larval developmental stage are attractive targets. Post-genomic technologies are now available which can contribute to such antigen discovery. METHODS: A schistosome-specific protein microarray was probed using the local antibody response against migrating larvae. Antigens were assessed for their novelty and predicted larval expression and host-exposed features. One antigen was further characterised and its sequence and structure were analysed in silico. Real-time polymerase chain reaction was used to analyse transcript expression throughout development, and immunoblotting and enzyme-linked immunosorbent assays employed to determine antigen recognition by antibody samples. RESULTS: Several known and novel antigens were discovered, two of which showed up-regulated transcription in schistosomula. One novel antigen, termed S. japonicum Ly-6-like protein 1 (Sj-L6L-1), was further characterised and shown to share structural and sequence features with the Ly-6 protein family. It was found to be present in the worm tegument and expressed in both the larval and adult worms, but was found to be antigenic only in the lungs that the larvae migrate to and traverse. CONCLUSIONS: This study represents a novel approach to vaccine antigen discovery and may contribute to schistosome vaccine development against this important group of human and veterinary pathogens.

SUBMITTER: McWilliam HE

PROVIDER: S-EPMC4080988 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Discovery of novel Schistosoma japonicum antigens using a targeted protein microarray approach.

McWilliam Hamish E G HE Driguez Patrick P Piedrafita David D McManus Donald P DP Meeusen Els N T EN

Parasites & vectors 20140625

<h4>Background</h4>Novel vaccine candidates against Schistosoma japonicum are required, and antigens present in the vulnerable larval developmental stage are attractive targets. Post-genomic technologies are now available which can contribute to such antigen discovery.<h4>Methods</h4>A schistosome-specific protein microarray was probed using the local antibody response against migrating larvae. Antigens were assessed for their novelty and predicted larval expression and host-exposed features. On ...[more]

PMID: 24964958

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Project description:BackgroundSchistosomiasis is a chronic debilitating parasitic disease that afflicts more than 200 million individuals worldwide. Long-term administration of chemotherapy with the single available drug, praziquantel, has led to growing concerns about drug resistance. The PSD-95/Dlg/ZO-1 (PDZ) domain is an important module found in many scaffolding proteins, which has been recognized as promising targets for the development of novel drugs. However, the parasite-derived PDZ domains and their associated functions are still largely unknown.Methodology/principal findingsThe gene encoding the Schistosoma japonicum Scribble protein (SjScrib) was identified by homologous search with the S. mansoni Scrib sequence. By screening an arbitrary peptide library in yeast two-hybrid (Y2H) assays, we identified and confirmed the ligand binding specificity for each of the four PDZ domains of SjScrib. Both SjScrib-PDZ1 and SjScrib-PDZ3 recognize type I C-terminal PDZ-domain binding motifs (PBMs), which can be deduced as consensus sequences of -[Φ][x][E][TS][x][ILF] and -[x][RKx][ETS][T][WΦ][ILV], respectively. SjScrib-PDZ2 prefers stringent type II C-terminal PBMs, which significantly differs from that of its human ortholog. SjScrib-PDZ4 binds to typical II C-terminal PBMs with a consensus sequence -[x][FW][x][LI][x][LIV], in which the aromatic residue Phe is predominantly selected at position -4. The irregular and unconventional internal ligand binding specificities for the PDZ domains of SjScrib were confirmed by point mutations of the key amino acids within the ligand binding motifs. We also compared the differences in ligand specificities between SjScrib-PDZs and hScrib-PDZs, and explored the structural basis for the ligand binding properties of SjScrib-PDZs.Conclusions/significanceIn this study, we characterized and confirmed the ligand binding specificities of all four PDZ domains of SjScrib for the first time. We denoted the differential ligand binding specificities between SjScrib-PDZs and hScrib-PDZs as well as the structural basis for these properties. This work may provide a fundamental basis for the rational design of novel anti-schistosomal drugs.

Project description:BackgroundDespite successful control efforts in China over the past 60 years, zoonotic schistosomiasis caused by Schistosoma japonicum remains a threat with transmission ongoing and the risk of localised resurgences prompting calls for a novel integrated control strategy, with an anti-schistosome vaccine as a core element. Anti-schistosome vaccine development and immunisation attempts in non-human mammalian host species, intended to interrupt transmission, and utilising various antigen targets, have yielded mixed success, with some studies highlighting variation in schistosome antigen coding genes (ACGs) as possible confounders of vaccine efficacy. Thus, robust selection of target ACGs, including assessment of their genetic diversity and antigenic variability, is paramount. Tetraspanins (TSPs), a family of tegument-surface antigens in schistosomes, interact directly with the host's immune system and are promising vaccine candidates. Here, for the first time to our knowledge, diversity in S. japonicum TSPs (SjTSPs) and the impact of diversifying selection and sequence variation on immunogenicity in these protiens were evaluated.MethodsSjTSP sequences, representing parasite populations from seven provinces across China, were gathered by baiting published short-read NGS data and were analysed using in silico methods to measure sequence variation and selection pressures and predict the impact of selection on variation in antigen protein structure, function and antigenic propensity.ResultsHere, 27 SjTSPs were identified across three subfamilies, highlighting the diversity of TSPs in S. japonicum. Considerable variation was demonstrated for several SjTSPs between geographical regions/provinces, revealing that episodic, diversifying positive selection pressures promote amino acid variation/variability in the large extracellular loop (LEL) domain of certain SjTSPs. Accumulating polymorphisms in the LEL domain of SjTSP-2, -8 and -23 led to altered structural, functional and antibody binding characteristics, which are predicted to impact antibody recognition and possibly blunt the host's ability to respond to infection. Such changes, therefore, appear to represent a mechanism utilised by S. japonicum to evade the host's immune system.ConclusionWhilst the genetic and antigenic geographic variability observed amongst certain SjTSPs could present challenges to vaccine development, here we demonstrate conservation amongst SjTSP-1, -13 and -14, revealing their likely improved utility as efficacious vaccine candidates. Importantly, our data highlight that robust evaluation of vaccine target variability in natural parasite populations should be a prerequisite for anti-schistosome vaccine development.

Project description:BackgroundSchistosomiasis 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 resultsWe 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.ConclusionsAuthentic 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.

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Discovery of novel Schistosoma japonicum antigens using a targeted protein microarray approach.

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Discovery of novel Schistosoma japonicum antigens using a targeted protein microarray approach.

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