Transcriptome Remodeling in Trypanosoma cruzi and Human Cells during Intracellular Infection.

Intracellular colonization and persistent infection by the kinetoplastid protozoan parasite, Trypanosoma cruzi, underlie the pathogenesis of human Chagas disease. To obtain global insights into the T. cruzi infective process, transcriptome dynamics were simultaneously captured in the parasite and host cells in an infection time course of human fibroblasts. Extensive remodeling of the T. cruzi transcriptome was observed during the early establishment of intracellular infection, coincident with a major developmental transition in the parasite. Contrasting this early response, few additional changes in steady state mRNA levels were detected once mature T. cruzi amastigotes were formed. Our findings suggest that transcriptome remodeling is required to establish a modified template to guide developmental transitions in the parasite, whereas homeostatic functions are regulated independently of transcriptomic changes, similar to that reported in related trypanosomatids. Despite complex mechanisms for regulation of phenotypic expression in T. cruzi, transcriptomic signatures derived from distinct developmental stages mirror known or projected characteristics of T. cruzi biology. Focusing on energy metabolism, we were able to validate predictions forecast in the mRNA expression profiles. We demonstrate measurable differences in the bioenergetic properties of the different mammalian-infective stages of T. cruzi and present additional findings that underscore the importance of mitochondrial electron transport in T. cruzi amastigote growth and survival. Consequences of T. cruzi colonization for the host include dynamic expression of immune response genes and cell cycle regulators with upregulation of host cholesterol and lipid synthesis pathways, which may serve to fuel intracellular T. cruzi growth. Thus, in addition to the biological inferences gained from gene ontology and functional enrichment analysis of differentially expressed genes in parasite and host, our comprehensive, high resolution transcriptomic dataset provides a substantially more detailed interpretation of T. cruzi infection biology and offers a basis for future drug and vaccine discovery efforts.

Violence and crime among male inpatients with severe mental illness: attempting to explain ethnic differences.

BACKGROUND: Studies report that in the U.K., among men with severe mental illness (SMI), those of black Caribbean ethnicity display increased risk of aggressive behaviour, criminal convictions, and schizophrenia. The study aimed to compare aggressive behaviour and criminal convictions among men with SMI of white British, black Caribbean and black African ethnicity, and to explore factors associated with differences across ethnicities. METHOD: Sample 1 included 1,104 male inpatients with SMI. Sample 2 included a representative sub-sample of 165 who completed interviews, and authorized access to medical and criminal files. Ethnicity was self-ascribed. RESULTS: Staff-rated violence prior to admission, self-reported aggressive behaviour, and convictions for non-violent and violent crimes differed among men with SMI of different ethnicities. Relative to men with SMI of white British ethnicity, those of black African ethnicity showed decreased risk of aggressive behaviour, and those of black Caribbean ethnicity showed elevated risk of convictions for non-violent, and marginally, for violent crimes. Relative to men with SMI of black African ethnicity, those of black Caribbean ethnicity showed elevated risk of aggressive behaviour and criminal convictions. Proportionately more of the men of both black African and black Caribbean ethnicity, than those of white British ethnicity, presented schizophrenia spectrum disorders. Multivariate analyses failed to identify factors that would explain differences in aggressive behaviour, and criminal convictions across ethnic groups. CONCLUSIONS: Differences in four different measures of aggressive and antisocial behaviour among men with SMI of different ethnicities were observed but factors associated with these differences were not found.

Suppressing glucose transporter gene expression in schistosomes impairs parasite feeding and decreases survival in the mammalian host.

Adult schistosomes live in the host's bloodstream where they import nutrients such as glucose across their body surface (the tegument). The parasite tegument is an unusual structure since it is enclosed not by the typical one but by two closely apposed lipid bilayers. Within the tegument two glucose importing proteins have been identified; these are schistosome glucose transporter (SGTP) 1 and 4. SGTP4 is present in the host interactive, apical tegumental membranes, while SGTP1 is found in the tegumental basal membrane (as well as in internal tissues). The SGTPs act by facilitated diffusion. To examine the importance of these proteins for the parasites, RNAi was employed to knock down expression of both SGTP genes in the schistosomula and adult worm life stages. Both qRT-PCR and western blotting analysis confirmed successful gene suppression. It was found that SGTP1 or SGTP4-suppressed parasites exhibit an impaired ability to import glucose compared to control worms. In addition, parasites with both SGTP1 and SGTP4 simultaneously suppressed showed a further reduction in capacity to import glucose compared to parasites with a single suppressed SGTP gene. Despite this debility, all suppressed parasites exhibited no phenotypic distinction compared to controls when cultured in rich medium. Following prolonged incubation in glucose-depleted medium however, significantly fewer SGTP-suppressed parasites survived. Finally, SGTP-suppressed parasites showed decreased viability in vivo following infection of experimental animals. These findings provide direct evidence for the importance of SGTP1 and SGTP4 for schistosomes in importing exogenous glucose and show that these proteins are important for normal parasite development in the mammalian host.

Imaging schistosomes in vivo.

Schistosomes are intravascular, parasitic helminths that cause a chronic, often debilitating disease afflicting over 200 million people in over 70 countries. Here we describe novel imaging methods that, for the first time, permit visualization of live schistosomes within their living hosts. The technology centers on fluorescent agent uptake and activation in the parasite's gut, and subsequent detection and signal quantitation using fluorescence molecular tomography (FMT). There is a strong positive correlation between the signal detected and parasite number. Schistosoma mansoni parasites of both sexes recovered from infected experimental animals exhibit vivid fluorescence throughout their intestines. Likewise, the remaining important human schistosome parasites, S. japonicum and S. hematobium, also exhibit gut fluorescence when recovered from infected animals. Imaging has been used to efficiently document the decline in parasite numbers in infected mice treated with the antischistosome drug praziquantel. This technology will provide a unique opportunity both to help rapidly identify much-needed, novel antischistosome therapies and to gain direct visual insight into the intravascular lives of the major schistosome parasites of humans.

Optimizing gene suppression in schistosomes using RNA interference.

Schistosomes are parasitic platyhelminths that constitute an important public health problem globally. Here we describe optimized protocols for effectively suppressing gene expression in the intra-mammalian life stages of Schistosoma mansoni using RNA interference (RNAi). RNAi is a mechanism by which gene-specific double stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. The gene encoding the cysteine protease cathepsin B (SmCB1 or Sm31) was targeted by exposing the parasites to dsRNA encoding part of the cathepsin B coding region. Suppression was measured using quantitative real time PCR. Electroporation as a mode of dsRNA delivery was substantially more efficient (100-1000-fold) than simply soaking the parasites in an equivalent dose. Soaking the parasites with dsRNA in the presence of different proprietary liposome preparations did not enhance gene suppression. In fact, all three reagents tested were variably toxic to the cultured schistosomes. Both long dsRNAs as well as synthetic short inhibitory RNAs (siRNAs) were effective at eliciting gene suppression. Different siRNAs exhibited variable efficiencies of suppression, perhaps reflecting differences in siRNA accessibility to the cathepsin B mRNA. Parasites cultured in vitro for 7 days or more following their emergence from the intermediate snail host were more susceptible to RNAi than those treated with dsRNA on the day of emergence (during the process of cercarial transformation into schistosomula). In addition, adult male and female parasites (49 days old) were susceptible to RNAi. Using fluorescein-labeled dsRNA to monitor the process, it was seen that in schistosomula (cultured for 7 days), electroporated dsRNA entered primarily through the mouth into the caecum while in young parasites (freshly emerged from snails) dsRNA appeared to enter primarily into the pre- and post-acetabular glands. The cathepsin B gene was significantly suppressed for up to 40 days after treatment suggesting that, as in some other organisms, the RNAi process can be amplified in schistosomes.

The tegument of the human parasitic worm Schistosoma mansoni as an excretory organ: the surface aquaporin SmAQP is a lactate transporter.

Adult schistosomes are intravascular parasites that metabolize imported glucose largely via glycolysis. How the parasites get rid of the large amounts of lactic acid this generates is unknown at the molecular level. Here, we report that worms whose aquaporin gene (SmAQP) has been suppressed using RNAi fail to rapidly acidify their culture medium and excrete less lactate compared to controls. Functional expression of SmAQP in Xenopus oocytes demonstrates that this protein can transport lactate following Michaelis-Menten kinetics with low apparent affinity (Km = 41+/-5. 8 mM) and with a low energy of activation (E(a) = 7.18+/-0.7 kcal/mol). Phloretin, a known inhibitor of lactate release from schistosomes, also inhibits lactate movement in SmAQP-expressing oocytes. In keeping with the substrate promiscuity of other aquaporins, SmAQP is shown here to be also capable of transporting water, mannitol, fructose and alanine but not glucose. Using immunofluorescent and immuno-EM, we confirm that SmAQP is localized in the tegument of adult worms. These findings extend the proposed functions of the schistosome tegument beyond its known capacity as an organ of nutrient uptake to include a role in metabolic waste excretion.

Protocols for gene silencing in schistosomes.

Schistosomes are parasitic platyhelminths that infect over 200 million people globally. In recent years there have been many advances in schistosome genomics and proteomics and in the development of molecular tools for use with these parasites. Among the more promising methodologies is RNA interference (RNAi) which is a mechanism by which gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. We aim to develop effective protocols utilizing RNAi for use in the intra-mammalian life stages of Schistosoma mansoni. In this work, the gene encoding alkaline phosphatase (SmAP) was targeted by exposing the parasites to dsRNA encoding part of the SmAP coding region. SmAP is known to be expressed in a variety of parasite tissues. We report that both long dsRNAs as well as synthetic short inhibitory RNAs (siRNAs) are effective at eliciting SmAP gene suppression in cultured schistosomula and in adult males and females. Electroporation as a mode of dsRNA delivery is more efficient than simply soaking the parasites in an equivalent dose. Relative SmAP RNA levels >90% lower than controls were routinely detected, when measured 2 days after treatment by electroporation, using quantitative real-time PCR. Commensurate with this decline in SmAP RNA, relative alkaline phosphatase enzyme activity levels >70% lower than controls were detected, 5 days after treatment. Protocols described here that result in the robust suppression of target genes in intravascular schistosomes may have wide applicability and promote functional schistosome genomics.

The flatworm spliced leader 3'-terminal AUG as a translation initiator methionine.

Spliced leader (SL) RNA trans-splicing contributes the 5' termini to mRNAs in a variety of eukaryotes. In contrast with some transsplicing metazoan groups (e.g. nematodes), flatworm spliced leaders are variable in both sequence and length in different flatworm taxa. However, an absolutely conserved and unique feature of all flatworm spliced leaders is the presence of a 3'-terminal AUG. We previously suggested that the Schistosoma mansoni spliced leader AUG might contribute a required translation initiator methionine to recipient mRNAs. Here we identified and examined trans-spliced cDNAs from a large set of newly available schistosome cDNAs. 28% of the trans-spliced cDNAs have the SL AUG in-frame with the major open reading frame of the mRNA. We identified over 40 cDNAs (40% of the SL AUG in-frame clones) that require the SL AUG as an initiator methionine to synthesize phylogenetically conserved N-terminal residues characteristic of orthologous proteins. RNA transfection experiments using several schistosome stages demonstrated that the flatworm SL AUG can serve as a translation initiator methionine in vivo. We also present in vivo translation studies of the schistosome initiator methionine context and the effect of the spliced leader AUG added upstream and out-of-frame with the main open reading of recipient mRNAs. Overall, our data have provided evidence that another function of flatworm spliced leader trans-splicing is to provide some recipient mRNAs with an initiator methionine for translation initiation.

Theileria parva candidate vaccine antigens recognized by immune bovine cytotoxic T lymphocytes.

East Coast fever, caused by the tick-borne intracellular apicomplexan parasite Theileria parva, is a highly fatal lymphoproliferative disease of cattle. The pathogenic schizont-induced lymphocyte transformation is a unique cancer-like condition that is reversible with parasite removal. Schizont-infected cell-directed CD8(+) cytotoxic T lymphocytes (CTL) constitute the dominant protective bovine immune response after a single exposure to infection. However, the schizont antigens targeted by T. parva-specific CTL are undefined. Here we show the identification of five candidate vaccine antigens that are the targets of MHC class I-restricted CD8(+) CTL from immune cattle. CD8(+) T cell responses to these antigens were boosted in T. parva-immune cattle resolving a challenge infection and, when used to immunize naïve cattle, induced CTL responses that significantly correlated with survival from a lethal parasite challenge. These data provide a basis for developing a CTL-targeted anti-East Coast fever subunit vaccine. In addition, orthologs of these antigens may be vaccine targets for other apicomplexan parasites.