Luke Moertel

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.

Developmental gene expression profiles of the human pathogen Schistosoma japonicum

BackgroundThe schistosome blood flukes are complex trematodes and cause a chronic parasitic disease of significant public health importance worldwide, schistosomiasis. Their life cycle is characterised by distinct parasitic and free-living phases involving mammalian and snail hosts and freshwater. Microarray analysis was used to profile developmental gene expression in the Asian species, Schistosoma japonicum. Total RNAs were isolated from the three distinct environmental phases of the lifecycle – aquatic/snail (eggs, miracidia, sporocysts, cercariae), juvenile (lung schistosomula and paired but pre-egg laying adults) and adult (paired, mature males and egg-producing females, both examined separately). Advanced analyses including ANOVA, principal component analysis, and hierarchal clustering provided a global synopsis of gene expression relationships among the different developmental stages of the schistosome parasite.ResultsGene expression profiles were linked to the major environmental settings through which the developmental stages of the fluke have to adapt during the course of its life cycle. Gene ontologies of the differentially expressed genes revealed a wide range of functions and processes. In addition, stage-specific, differentially expressed genes were identified that were involved in numerous biological pathways and functions including calcium signalling, sphingolipid metabolism and parasite defence.ConclusionThe findings provide a comprehensive database of gene expression in an important human pathogen, including transcriptional changes in genes involved in evasion of the host immune response, nutrient acquisition, energy production, calcium signalling, sphingolipid metabolism, egg production and tegumental function during development. This resource should help facilitate the identification and prioritization of new anti-schistosome drug and vaccine targets for the control of schistosomiasis.

Transcriptional changes in Schistosoma mansoni during early schistosomula development and in the presence of erythrocytes.

Background Schistosomes cause more mortality and morbidity than any other human helminth, but control primarily relies on a single drug that kills adult worms. The newly transformed schistosomulum stage is susceptible to the immune response and is a target for vaccine development and rational drug design. Methodology/Principal Findings To identify genes which are up-regulated during the maturation of Schistosoma mansoni schistosomula in vitro, we cultured newly transformed parasites for 3 h or 5 days with and without erythrocytes and compared their transcriptional profiles using cDNA microarrays. The most apparent changes were in the up-regulation of genes between 3 h and 5 day schistosomula involved in blood feeding, tegument and cytoskeletal development, cell adhesion, and stress responses. The most highly up-regulated genes included a tegument tetraspanin Sm-tsp-3 (1,600-fold up-regulation), a protein kinase, a novel serine protease and serine protease inhibitor, and intestinal proteases belonging to distinct mechanistic classes. The inclusion of erythrocytes in the culture medium resulted in a general but less pronounced increase in transcriptional activity, with the highest up-regulation of genes involved in iron metabolism, proteolysis, and transport of fatty acids and sugars. Conclusions We have identified the genes that are up-regulated during the first 5 days of schistosomula development in vitro. Using a combination of gene silencing techniques and murine protection studies, some of these highly up-regulated transcripts can be targeted for future development of new vaccines and drugs.

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.

Comparative real-time PCR and enzyme analysis of selected gender-associated molecules in Schistosoma japonicum.

Schistosomes are complex parasitic helminths with discrete life-cycle stages, adapted for survival in their mammalian and snail hosts and the external aquatic environment. Recently, we described the fabrication and use of a microarray to investigate gender-specific transcription in Schistosoma japonicum. To address transcriptional differences, 8 gender-associated gene transcripts identified previously by the microarray analysis were selected for further study. First, differential transcription patterns were investigated in 4 developmental stages using real-time PCR. Subsequently, we undertook functional analysis of a subset of 4 transcripts encoding metabolic enzymes, so as to correlate gender-associated transcript levels with enzyme activity in protein extracts from adult worms. The 8 characterized molecules serve as a basis for further investigation of differential gene expression during the schistosome life-cycle and for studying the sexual dimorphism of adult worms. Continual refinement and annotation of the microarray used in the current study should support future work on these aspects.