Sujeevi Nawaratna

Exposed proteins of the Schistosoma japonicum tegument.

The ability of the mammalian blood fluke Schistosoma japonicum to survive in the inhospitable environment of the mammalian bloodstream can be attributed, at least in part, to its host-exposed outer surface, called the tegument. The tegument is a dynamic organ and is involved in nutrition, immune evasion and modulation, excretion, osmoregulation and signal transduction. Given its importance for parasite survival, proteins exposed to the host at the surface of the tegument are ideal targets for the development of vaccines and drugs. By biotinylating live adult worms and using a combination of OFFGEL electrophoresis and tandem mass spectrometry 54 proteins were identified as putatively host-exposed in S. japonicum. These included glucose transport proteins, an amino permease, a leucine aminopeptidase and a range of transporters, heat shock proteins and novel immune-active proteins. Members of the tetraspanin protein family and a homologue of Sm 29, a tegument membrane protein from Schistosoma mansoni, both effective vaccine antigens in S. mansoni, were also identified. The fate of labelled surface proteins was monitored over time using electron microscopy and revealed that biotinylated proteins were rapidly internalised from the surface of the tegument and trafficked into the cytoplasmic bridges that connect the distal cytoplasm of the tegument to the underlying cell bodies. The results reported herein dramatically increase the number of S. japonicum proteins known to be exposed to the host and, hence, those of interest as therapeutic targets. The ability of the parasite to rapidly internalise proteins at its surface has implications for the development of vaccines and may explain how these parasites are able to avoid the host immune system for long periods of time.

The secreted and surface proteomes of the adult stage of the carcinogenic human liver fluke Opisthorchis viverrini

Infection with the human liver fluke, Opisthorchis viverrini, is a serious public health problem in Thailand, Laos and nearby locations in Southeast Asia. Both experimental and epidemiological evidence strongly implicate liver fluke infection in the etiology of one of the liver cancer subtypes, cholangiocarcinoma (CCA). To identify parasite proteins critical for liver fluke survival and the etiology of CCA, OFFGEL electrophoresis and multiple reaction monitoring were employed to characterize 300 parasite proteins from the O. viverrini excretory/secretory products and, utilizing selective labeling and sequential solubilization, from the host‐exposed tegument. The excretory/secretory included a complex mixture of proteins that have been associated with cancers, including proteases of different mechanistic classes and orthologues of mammalian growth factors and anti‐apoptotic proteins. Also identified was a cysteine protease inhibitor which, in other helminth pathogens, induces nitric oxide production by macrophages, and, hence may contribute to malignant transformation of inflamed cells. More than 160 tegumental proteins were identified using sequential solubilization of isolated teguments, and a subset of these was localized to the surface membrane of the tegument by labeling living flukes with biotin and confirming surface localization with fluorescence microscopy. These included annexins, which are potential immuno‐modulators, and orthologues of the schistosomiasis vaccine antigens Sm29 and tetraspanin‐2. Novel roles in pathogenesis were suggested for the tegument–host interface since more than ten surface proteins had no homologues in the public databases. The O. viverrini proteins identified here provide an extensive catalogue of novel leads for research on the pathogenesis of opisthorchiasis and the development of novel interventions for this disease and CCA, as well as providing a scaffold for sequencing the genome of this fluke.

Tissue specific profiling of females of Schistosoma japonicum by integrated laser microdissection microscopy and microarray analysis.

Background The functions of many schistosome gene products remain to be characterized. A major step towards elucidating function of these genes would be in defining their sites of expression. This goal is rendered difficult to achieve by the generally small size of the parasites and the lack of a body cavity, which precludes analysis of transcriptional profiles of the tissues in isolation. Methodology/Principal Findings Here, we describe a combined laser microdissection microscopy (LMM) and microarray analysis approach to expedite tissue specific profiling and gene atlasing for tissues of adult female Schistosoma japonicum. This approach helps to solve the gene characterization “bottle-neck” brought about by acoelomy and the size of these parasites. Complementary RNA obtained after isolation from gastrodermis (parasite gut mucosa), vitelline glands and ovary by LMM were subjected to microarray analyses, resulting in identification of 147 genes upregulated in the gastrodermis, 4,149 genes in the ovary and 2,553 in the vitellaria. Conclusions This work will help to shed light on the molecular pathobiology of this debilitating human parasite and aid in the discovery of new targets for the development of anti-schistosome vaccines and drugs.

Gene Atlasing of Digestive and Reproductive Tissues in Schistosoma mansoni

Background While considerable genomic and transcriptomic data are available for Schistosoma mansoni, many of its genes lack significant annotation. A transcriptomic study of individual tissues and organs of schistosomes could play an important role in functional annotation of the unknown genes, particularly by providing rapid localisation data and thus giving insight into the potential roles of these molecules in parasite development, reproduction and homeostasis, and in the complex host-parasite interaction. Methodology/Principal Findings Quantification of gene expression in tissues of S. mansoni was achieved by a combination of laser microdissection microscopy (LMM) and oligonucleotide microarray analysis. We compared the gene expression profile of the adult female gastrodermis and male and female reproductive tissues with whole worm controls. The results revealed a total of 393 genes (contigs) that were up-regulated two-fold or more in the gastrodermis, 4,450 in the ovary, 384 in the vitelline tissues of female parasites, and 2,171 in the testes. We have also supplemented these data with the identification of highly expressed genes in different regions of manually dissected male and female S. mansoni. Though relatively crude, this dissection strategy provides low resolution localisation data for critical regions of the adult parasites that are not amenable to LMM isolation. Conclusions This is the first detailed transcriptomic study of the reproductive tissues and gastrodermis of S. mansoni. The results obtained will help direct future research on the functional aspects of these tissues, expediting the characterisation of currently unannotated gene products of S. mansoni and the discovery of new drug and vaccine targets.

Increased iron stores correlate with worse disease outcomes in a mouse model of schistosomiasis infection.

Schistosomiasis is a significant parasitic infection creating disease burden throughout many of the worlds developing nations. Iron deficiency anemia is also a significant health burden resulting from both nutritional deficit as well as parasitic infection in these countries. In this study we investigated the relationships between the disease outcomes of Schistosoma japonicum infection and iron homeostasis. We aimed to determine if host iron status has an effect on schistosome maturation or egg production, and to investigate the response of iron regulatory genes to chronic schistosomiasis infection. Wild-type C57BL/6 and Transferrin Receptor 2 null mice were infected with S. japonicum, and sacrificed at the onset of chronic disease. Transferrin Receptor 2 null mice are a model of type 3 hereditary hemochromatosis and develop significant iron overload providing increased iron stores at the onset of infection. The infectivity of schistosomes and egg production was assessed along with the subsequent development of granulomas and fibrosis. The response of the iron regulatory gene Hepcidin to infection and the changes in iron status were assessed by real-time PCR and Western blotting. Our results show that Hepcidin levels responded to the changing iron status of the animals, but were not significantly influenced by the inflammatory response. We also show that with increased iron availability at the time of infection there was greater development of fibrosis around granulomas. In conclusion, our studies indicate that chronic inflammation may not be the primary cause of the anemia seen in schistosomiasis, and suggest that increased availability of iron, such as through iron supplementation, may actually lead to increased disease severity.

Suppression of the Insulin Receptors in Adult Schistosoma japonicum Impacts on Parasite Growth and Development: Further Evidence of Vaccine Potential.

To further investigate the importance of insulin signaling in the growth, development, sexual maturation and egg production of adult schistosomes, we have focused attention on the insulin receptors (SjIRs) of Schistosoma japonicum, which we have previously cloned and partially characterised. We now show, by Biolayer Interferometry, that human insulin can bind the L1 subdomain (insulin binding domain) of recombinant (r)SjIR1 and rSjIR2 (designated SjLD1 and SjLD2) produced using the Drosophila S2 protein expression system. We have then used RNA interference (RNAi) to knock down the expression of the SjIRs in adult S. japonicum in vitro and show that, in addition to their reduced transcription, the transcript levels of other important downstream genes within the insulin pathway, associated with glucose metabolism and schistosome fecundity, were also impacted substantially. Further, a significant decrease in glucose uptake was observed in the SjIR-knockdown worms compared with luciferase controls. In vaccine/challenge experiments, we found that rSjLD1 and rSjLD2 depressed female growth, intestinal granuloma density and faecal egg production in S. japonicum in mice presented with a low dose challenge infection. These data re-emphasize the potential of the SjIRs as veterinary transmission blocking vaccine candidates against zoonotic schistosomiasis japonica in China and the Philippines.

Transcriptional profiling of the oesophageal gland region of male worms of Schistosoma mansoni

The intestinal tract of schistosomes opens at the mouth and leads into the foregut or oesophageal region that is lined with syncytium continuous with the apical cytoplasm of the tegument. The oesophagus is surrounded by a specialised gland, the oesophageal gland. This gland releases materials into the lumen of the oesophagus and the region is thought to initiate the lysis of erythrocytes and neutralisation of immune effectors of the host. The oesophageal region is present in the early invasive schistosomulum, a stage potentially targetable by anti-schistosome vaccines. We used a 44k oligonucleotide microarray to identify highly up-regulated genes in microdissected frozen sections of the oesophageal gland of male worms of S. mansoni. We show that 122 genes were up-regulated 2-fold or higher in the oesophageal gland compared with a whole male worm tissue control. The enriched genes included several associated with lipid metabolism and transmembrane transport as well as some micro-exon genes. Since the oesophageal gland is important in the initiation of digestion and the fact that it develops early after invasion of the mammalian host, further study of selected highly up-regulated functionally important genes in this tissue may reveal new anti-schistosome intervention targets for schistosomiasis control.

Characterising granuloma regression and liver recovery in a murine model of schistosomiasis japonica

For hepatic schistosomiasis the egg-induced granulomatous response and the development of extensive fibrosis are the main pathologies. We used a Schistosoma japonicum-infected mouse model to characterise the multi-cellular pathways associated with the recovery from hepatic fibrosis following clearance of the infection with the anti-schistosomal drug, praziquantel. In the recovering liver splenomegaly, granuloma density and liver fibrosis were all reduced. Inflammatory cell infiltration into the liver was evident, and the numbers of neutrophils, eosinophils and macrophages were significantly decreased. Transcriptomic analysis revealed the up-regulation of fatty acid metabolism genes and the identification of Peroxisome proliferator activated receptor alpha as the upstream regulator of liver recovery. The aryl hydrocarbon receptor signalling pathway which regulates xenobiotic metabolism was also differentially up-regulated. These findings provide a better understanding of the mechanisms associated with the regression of hepatic schistosomiasis.

An Ex Vivo Model for Studying Hepatic Schistosomiasis and the Effect of Released Protein from Dying Eggs

Background We report the use of an ex vivo precision cut liver slice (PCLS) mouse model for studying hepatic schistosomiasis. In this system, liver tissue is unfixed, unfrozen, and alive for maintenance in culture and subsequent molecular analysis. Methods and Findings Using thick naive mouse liver tissue and sterile culture conditions, the addition of soluble egg antigen (SEA) derived from Schistosoma japonicum eggs, followed 4, 24 and 48hrs time points. Tissue was collected for transcriptional analysis and supernatants collected to quantitate liver enzymes, cytokines and chemokines. No significant hepatotoxicity was demonstrated by supernatant liver enzymes due to the presence of SEA. A proinflammatory response was observed both at the transcriptional level and at the protein level by cytokine and chemokine bead assay. Key genes observed elevated transcription in response to the addition of SEA included: IL1-α and IL1-β, IL6, all associated with inflammation. The recruitment of antigen presenting cells was reflected in increases in transcription of CD40, CCL4 and CSF1. Indications of tissue remodeling were seen in elevated gene expression of various Matrix MetalloProteinases (MMP3, 9, 10, 13) and delayed increases in TIMP1. Collagen deposition was significantly reduced in the presence of SEA as shown in COL1A1 expression by qPCR after 24hrs culture. Cytokine and chemokine analysis of the culture supernatants confirmed the elevation of proteins including IL6, CCL3, CCL4 and CXCL5. Conclusions This ex vivo model system for the synchronised delivery of parasite antigen to liver tissue provides an insight into the early phase of hepatic schistosomiasis, corresponding with the release of soluble proteins from dying schistosome eggs.

Lysosome-associated membrane glycoprotein (LAMP)--preliminary study on a hidden antigen target for vaccination against schistosomiasis.

Our previously reported gene atlasing of schistosome tissues revealed transcripts that were highly enriched in the digestive tract of Schistosoma mansoni. From these, we selected two candidates, Sm-LAMP and Sm-NPC2 for testing as vaccine targets. The two molecules were selected on the basis of relatively high expression in the gastrodermis, their potentially important biological function, divergence from homologous molecules of the host and possible apical membrane expression in the gastrodermis. Bacterially expressed recombinant peptides corresponding to regions excluding trans-membrane domains of the selected vaccine targets were used in blinded vaccine trials in CBA mice using alum-CpG as adjuvant. Vaccine trials using the recombinant insoluble Sm-LAMP protein showed 16–25% significant reduction in total worm burden. Faecal egg count reduction was 52% and 60% in two trials, respectively, with similar results for the solubly expressed protein. Liver egg burden was reduced significantly (20% and 38%) with an insoluble recombinant Sm-LAMP in two trials, but not with the soluble recombinant form. Parasite fecundity was not affected by either Sm-LAMP protein preparations in the trials. It is concluded that Sm-LAMP may provide limited protection towards S. mansoni infections but could be used in combination with other vaccine candidates, to provide more comprehensive protection.