Transcriptome analyses of inhibitor-treated Schistosome females provide evidence for cooperating Src-kinase and TGFbeta receptor pathways controlling mitosis and egshell formation

Schistosome parasites cause schistosomiasis, one of the most prevalent parasitemias worldwide affecting humans and animals. Constant pairing of schistosomes is essential for female sexual maturation and egg production, which causes pathogenesis. Female maturation involves signaling pathways controlling mitosis and differentiation within the gonads. In vitro studies had shown before that a Src-specific inhibitor, Herbimycin A (Herb A), and a TGFb receptor (TbR) inhibitor TRIKI) have physiological effects such as suppressed mitoses and egg production in paired females. As one Herb A target, the gonad-specifically expressed Src kinase SmTK3 was identified. Here, we comparatively analyzed the transcriptome profiles of Herb A- and TRIKI-treated females identifying transcriptional targets of Src-kinase and TbRI pathways. After demonstrating that TRIKI inhibits the schistosome TGFbreceptor SmTbRI by kinase assays in Xenopus oocytes, couples were treated with Herb A, TRIKI, or both inhibitors simultaneously in vitro. RNA was isolated from females for microarray hybridizations and transcription analyses. The obtained data were evaluated by Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA), but also by manual classification and intersection analyses. Finally, extensive qPCR experiments were done to verify differential transcription of candidate genes under inhibitor influence but also to functionally reinforce specific physiological effects. A number of genes found to be differentially regulated are associated with mitosis and differentiation. Among these were calcium-associated genes and eggshell-forming genes. In situ hybridization confirmed transcription of genes coding for the calcium sensor hippocalcin, the calcium transporter ORAI-1, and the calcium-binding protein calmodulin-4 in the reproductive system pointing to a role of calcium in parasite reproduction. Functional qPCR results confirmed an inhibitor-influenced, varying dependence of the transcriptional activities of Smp14, Smp48, fs800, a predicted eggshell precursor protein and SmTYR1. The results show that eggshell-formation is regulated by at least two pathways cooperatively operating in a balanced manner to control egg production

The Formin-Homology Protein SmDia Interacts with the Src Kinase SmTK and the GTPase SmRho1 in the Gonads of Schistosoma mansoni

BACKGROUND:Schistosomiasis (bilharzia) is a parasitic disease of worldwide significance affecting human and animals. As schistosome eggs are responsible for pathogenesis, the understanding of processes controlling gonad development might open new perspectives for intervention. The Src-like tyrosine-kinase SmTK3 of Schistosoma mansoni is expressed in the gonads, and its pharmacological inhibition reduces mitogenic activity and egg production in paired females in vitro. Since Src kinases are important signal transduction proteins it is of interest to unravel the signaling cascades SmTK3 is involved in to understand its cellular role in the gonads. METHODOLOGY AND RESULTS:Towards this end we established and screened a yeast two-hybrid (Y2H) cDNA library of adult S. mansoni with a bait construct encoding the SH3 (src homology) domain and unique site of SmTK3. Among the binding partners found was a diaphanous homolog (SmDia), which was characterized further. SmDia is a single-copy gene transcribed throughout development with a bias towards male transcription. Its deduced amino acid sequence reveals all diaphanous-characteristic functional domains. Binding studies with truncated SmDia clones identified SmTK3 interaction sites demonstrating that maximal binding efficiency depends on the N-terminal part of the FH1 (formin homology) domain and the inter-domain region of SmDia located upstream of FH1 in combination with the unique site and the SH3 domain of SmTK3, respectively. SmDia also directly interacted with the GTPase SmRho1 of S. mansoni. In situ hybridization experiments finally demonstrated that SmDia, SmRho1, and SmTK3 are transcribed in the gonads of both genders. CONCLUSION:These data provide first evidence for the existence of two cooperating pathways involving Rho and Src that bridge at SmDia probably organizing cytoskeletal events in the reproductive organs of a parasite, and beyond that in gonads of eukaryotes. Furthermore, the FH1 and inter domain region of SmDia have been discovered as binding sites for the SH3 and unique site domains of SmTK3, respectively

A Schistosome cAMP-Dependent Protein Kinase Catalytic Subunit Is Essential for Parasite Viability

Eukaryotes, protozoan, and helminth parasites make extensive use of protein kinases to control cellular functions, suggesting that protein kinases may represent novel targets for the development of anti-parasitic drugs. Because of their central role in intracellular signaling pathways, cyclic nucleotide–dependent kinases such as cAMP-dependent protein kinase (PKA) represent promising new targets for the treatment of parasitic infections and neoplastic disorders. However, the role of these kinases in schistosome biology has not been characterized and the genes encoding schistosome PKAs have not been identified. Here we provide biochemical evidence for the presence of a PKA signaling pathway in adult Schistosoma mansoni and show that PKA activity is required for parasite viability in vitro. We also provide the first full description of a gene that encodes a PKA catalytic subunit in S. mansoni, named SmPKA-C. Finally we demonstrate, through RNA interference, that SmPKA-C contributes to the PKA activity we detected biochemically and that inhibition of SmPKA-C expression in adult schistosomes results in parasite death. Together our data show that SmPKA-C is a critically important gene product and may represent an attractive therapeutic target for the treatment and control of schistosomiasis

Involvement of the Cytokine MIF in the Snail Host Immune Response to the Parasite Schistosoma mansoni

We have identified and characterized a Macrophage Migration Inhibitory Factor (MIF) family member in the Lophotrochozoan invertebrate, Biomphalaria glabrata, the snail intermediate host of the human blood fluke Schistosoma mansoni. In mammals, MIF is a widely expressed pleiotropic cytokine with potent pro-inflammatory properties that controls cell functions such as gene expression, proliferation or apoptosis. Here we show that the MIF protein from B. glabrata (BgMIF) is expressed in circulating immune defense cells (hemocytes) of the snail as well as in the B. glabrata embryonic (Bge) cell line that has hemocyte-like features. Recombinant BgMIF (rBgMIF) induced cell proliferation and inhibited NO-dependent p53-mediated apoptosis in Bge cells. Moreover, knock-down of BgMIF expression in Bge cells interfered with the in vitro encapsulation of S. mansoni sporocysts. Furthermore, the in vivo knock-down of BgMIF prevented the changes in circulating hemocyte populations that occur in response to an infection by S. mansoni miracidia and led to a significant increase in the parasite burden of the snails. These results provide the first functional evidence that a MIF ortholog is involved in an invertebrate immune response towards a parasitic infection and highlight the importance of cytokines in invertebrate-parasite interactions

Major prospects for exploring canine vector borne diseases and novel intervention methods using 'omic technologies

Canine vector-borne diseases (CVBDs) are of major socioeconomic importance worldwide. Although many studies have provided insights into CVBDs, there has been limited exploration of fundamental molecular aspects of most pathogens, their vectors, pathogen-host relationships and disease and drug resistance using advanced, 'omic technologies. The aim of the present article is to take a prospective view of the impact that next-generation, 'omics technologies could have, with an emphasis on describing the principles of transcriptomic/genomic sequencing as well as bioinformatic technologies and their implications in both fundamental and applied areas of CVBD research. Tackling key biological questions employing these technologies will provide a 'systems biology' context and could lead to radically new intervention and management strategies against CVBDs

Eukaryotic Protein Kinases (ePKs) of the Helminth Parasite Schistosoma mansoni

<p>Abstract</p> <p>Background</p> <p>Schistosomiasis remains an important parasitic disease and a major economic problem in many countries. The <it>Schistosoma mansoni </it>genome and predicted proteome sequences were recently published providing the opportunity to identify new drug candidates. Eukaryotic protein kinases (ePKs) play a central role in mediating signal transduction through complex networks and are considered druggable targets from the medical and chemical viewpoints. Our work aimed at analyzing the <it>S. mansoni </it>predicted proteome in order to identify and classify all ePKs of this parasite through combined computational approaches. Functional annotation was performed mainly to yield insights into the parasite signaling processes relevant to its complex lifestyle and to select some ePKs as potential drug targets.</p> <p>Results</p> <p>We have identified 252 ePKs, which corresponds to 1.9% of the <it>S. mansoni </it>predicted proteome, through sequence similarity searches using HMMs (Hidden Markov Models). Amino acid sequences corresponding to the conserved catalytic domain of ePKs were aligned by MAFFT and further used in distance-based phylogenetic analysis as implemented in PHYLIP. Our analysis also included the ePK homologs from six other eukaryotes. The results show that <it>S. mansoni </it>has proteins in all ePK groups. Most of them are clearly clustered with known ePKs in other eukaryotes according to the phylogenetic analysis. None of the ePKs are exclusively found in <it>S. mansoni </it>or belong to an expanded family in this parasite. Only 16 <it>S. mansoni </it>ePKs were experimentally studied, 12 proteins are predicted to be catalytically inactive and approximately 2% of the parasite ePKs remain unclassified. Some proteins were mentioned as good target for drug development since they have a predicted essential function for the parasite.</p> <p>Conclusions</p> <p>Our approach has improved the functional annotation of 40% of <it>S. mansoni </it>ePKs through combined similarity and phylogenetic-based approaches. As we continue this work, we will highlight the biochemical and physiological adaptations of <it>S. mansoni </it>in response to diverse environments during the parasite development, vector interaction, and host infection.</p

A Research Agenda for Helminth Diseases of Humans: Basic Research and Enabling Technologies to Support Control and Elimination of Helminthiases

Successful and sustainable intervention against human helminthiases depends on optimal utilisation of available control measures and development of new tools and strategies, as well as an understanding of the evolutionary implications of prolonged intervention on parasite populations and those of their hosts and vectors. This will depend largely on updated knowledge of relevant and fundamental parasite biology. There is a need, therefore, to exploit and apply new knowledge and techniques in order to make significant and novel gains in combating helminthiases and supporting the sustainability of current and successful mass drug administration (MDA) programmes. Among the fields of basic research that are likely to yield improved control tools, the Disease Reference Group on Helminth Infections (DRG4) has identified four broad areas that stand out as central to the development of the next generation of helminth control measures: 1) parasite genetics, genomics, and functional genomics; 2) parasite immunology; 3) (vertebrate) host–parasite interactions and immunopathology; and 4) (invertebrate) host–parasite interactions and transmission biology. The DRG4 was established in 2009 by the Special Programme for Research and Training in Tropical Diseases (TDR). The Group was given the mandate to undertake a comprehensive review of recent advances in helminthiases research in order to identify notable gaps and highlight priority areas. This paper summarises recent advances and discusses challenges in the investigation of the fundamental biology of those helminth parasites under the DRG4 Group's remit according to the identified priorities, and presents a research and development agenda for basic parasite research and enabling technologies that will help support control and elimination efforts against human helminthiases