Schistosome apyrase SmATPDase1, but not SmATPDase2, hydrolyses exogenous ATP and ADP

Schistosomes are parasitic worms that can live in the bloodstream of their vertebrate hosts for many years. It has been proposed that the worms impinge on host purinergic signalling by degrading proinflammatory molecules like ATP as well as prothrombotic mediators like ADP. This capability may help explain the apparent refractoriness of the worms to both immune elimination and thrombus formation. Three distinct ectoenzymes, expressed at the host-exposed surface of the worm’s tegument, are proposed to be involved in the catabolism of ATP and ADP. These are alkaline phosphatase (SmAP), phosphodiesterase (SmNPP-5), and ATP diphosphohydrolase (SmATPDase1). It has recently been shown that only one of these enzymes—SmATPDase1—actually degrades exogenous ATP and ADP. However, a second ATP diphosphohydrolase homolog (SmATPDase2) is located in the tegument and has been reported to be released by the worms. It is possible that this enzyme too participates in the cleavage of exogenous nucleotide tri- and di-phosphates. To test this hypothesis, we employed RNA interference (RNAi) to suppress the expression of the schistosome SmATPDase1 and SmATPDase2 genes. We find that only SmATPDase1-suppressed parasites are significantly impaired in their ability to degrade exogenously added ATP or ADP. Suppression of SmATPDase2 does not appreciably affect the worms’ ability to catabolize ATP or ADP. Furthermore, we detect no evidence for the secretion or release of an ATP-hydrolyzing activity by cultured parasites. The results confirm the role of tegumental SmATPDase1, but not SmADTPDase2, in the degradation of the exogenous proinflammatory and prothrombotic nucleotides ATP and ADP by live intravascular stages of the parasite.     

Schistosome immunomodulators

Schistosomes are long lived, intravascular parasitic platyhelminths that infect >200 million people globally. The molecular mechanisms used by these blood flukes to dampen host immune responses are described in this review. Adult worms express a collection of host-interactive tegumental ectoenzymes that can cleave host signaling molecules such as the “alarmin” ATP (cleaved by SmATPDase1), the platelet activator ADP (SmATPDase1, SmNPP5), and can convert AMP into the anti-inflammatory mediator adenosine (SmAP). SmAP can additionally cleave the lipid immunomodulator sphingosine-1-phosphate and the proinflammatory anionic polymer, polyP. In addition, the worms release a barrage of proteins (e.g., SmCB1, SjHSP70, cyclophilin A) that can impinge on immune cell function. Parasite eggs also release their own immunoregulatory proteins (e.g., IPSE/α1, omega1, SmCKBP) as do invasive cercariae (e.g., Sm16, Sj16). Some schistosome glycans (e.g., LNFPIII, LNnT) and lipids (e.g., Lyso-PS, LPC), produced by several life stages, likewise affect immune cell responses. The parasites not only produce eicosanoids (e.g., PGE2, PGD2—that can be anti-inflammatory) but can also induce host cells to release these metabolites. Finally, the worms release extracellular vesicles (EVs) containing microRNAs, and these too have been shown to skew host cell metabolism. Thus, schistosomes employ an array of biomolecules—protein, lipid, glycan, nucleic acid, and more, to bend host biochemistry to their liking. Many of the listed molecules have been individually shown capable of inducing aspects of the polarized Th2 response seen following infection (with the generation of regulatory T cells (Tregs), regulatory B cells (Bregs) and anti-inflammatory, alternatively activated (M2) macrophages). Precisely how host cells integrate the impact of these myriad parasite products following natural infection is not known. Several of the schistosome immunomodulators described here are in development as novel therapeutics against autoimmune, inflammatory, and other, nonparasitic, diseases.     

Schistosoma mansoni: TGF-beta signaling pathways

Schistosome parasites have co-evolved an intricate relationship with their human and snail hosts as well as a novel interplay between the adult male and female parasites. We review the role of the TGF-beta signaling pathway in parasite development, host-parasite interactions and male-female interactions. The data to date support multiple roles for the TGF-beta signaling pathway throughout schistosome development, in particular, in the tegument which is at the interface with the host and between the male and female schistosome, development of vitelline cells in female worms whose genes and development are regulated by a stimulus from the male schistosome and embryogenesis of the egg. The human ligand TGF-beta1 has been demonstrated to regulate the expression of a schistosome target gene that encodes a gynecophoric canal protein in the schistosome worm itself. Studies on signaling in schistosomes opens a new era for investigation of host-parasite and male-female interactions.     

Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of infected host cells

Intracellular pathogens have evolved a wide array of mechanisms to invade and co-opt their host cells for intracellular survival. Apicomplexan parasites such as Toxoplasma gondii employ the action of unique secretory organelles named rhoptries for internalization of the parasite and formation of a specialized niche within the host cell. We demonstrate that Toxoplasma gondii also uses secretion from the rhoptries during invasion to deliver a parasite-derived protein phosphatase 2C (PP2C-hn) into the host cell and direct it to the host nucleus. Delivery to the host nucleus does not require completion of invasion, as evidenced by the fact that parasites blocked in the initial stages of invasion with cytochalasin D are able to target PP2C-hn to the host nucleus. We have disrupted the gene encoding PP2C-hn and shown that PP2C-hn-knockout parasites exhibit a mild growth defect that can be rescued by complementation with the wild-type gene. The delivery of parasite effector proteins via the rhoptries provides a novel mechanism for Toxoplasma to directly access the command center of its host cell during infection by the parasite.     

Conservation of CD4+ T cell-dependent developmental mechanisms in the blood fluke pathogens of humans

Schistosoma blood flukes are trematode parasites with a cosmopolitan distribution that infect over 200 million people globally. We previously showed that Schistosoma mansoni growth and development in the mammalian host is dependent on signals from host CD4+ T cells. To gain insight into the mechanisms that underlie this dependence, we sought to determine the evolutionary origins and limits of this aspect of the host-pathogen relationship. By infecting RAG-1-/- mice with a range of different schistosome species and strains, we tested several hypotheses concerning the time during Schistosoma evolution at which this dependence arose, and whether this dependence is specific to Schistosoma or is also found in other blood flukes. Our data indicate that the developmental dependence on CD4+ T cells previously described for S. mansoni is conserved in the evolutionarily basal species Schistosoma japonicum, suggesting this developmental adaptation arose early in Schistosoma evolution. We also demonstrate that the development of the more evolutionarily derived species Schistosoma haematobium and Schistosoma intercalatum are dependent on adaptive immune signals. Together, these data suggest that the blood fluke parasites of humans utilise common mechanisms to infect their hosts and to co-opt immune signals in the coordination of parasite development. Thus, exploitation of host-schistosome interactions to impair or prevent parasite development may represent a novel approach to combating all of the schistosome pathogens of humans.     

Vertebrate host protective immunity drives genetic diversity and antigenic polymorphism in Schistosoma mansoni

Schistosomes are gonochoric blood parasites with a complex life cycle responsible for a disease of considerable medical and veterinary importance in tropical and subtropical regions. Understanding the evolution of schistosome genetic diversity is clearly of fundamental importance to interpreting schistosomiasis epidemiology and disease transmission patterns of this parasite. In this article, we investigated the putative role of the host immune system in the selection of male genetic diversity. We demonstrated the link between genetic dissimilarity and the protective effect among male worms. We then compared the proteomes of three male clones with different genotypes and differing by their capacity to protect against reinfection. The identified differences correspond mainly to antigens known or supposed to be involved in the induction of protective immunity. These results underline the role played by host immune system in the selection of schistosome genetic diversity that is linked to antigenic diversity. We discuss the evolutionary consequences in the context of schistosome infection.     

Genomic organization of the Schistosoma mansoni aspartic protease gene, a platyhelminth orthologue of mammalian lysosomal cathepsin D

Schistosomes are considered the most important of the helminth parasites of humans in terms of morbidity and mortality. Schistosomes employ proteolytic enzymes to digest host hemoglobin from ingested human blood, including a cathepsin D-like, aspartic protease that is overexpressed in the gut of the adult female schistosome. Because of its key role in parasite nutrition, this enzyme represents a potential intervention target. To continue exploration of this potential, here we have determined the sequence, structure and genomic organization of the cathepsin D gene locus of Schistosoma mansoni. Using the cDNA encoding S. mansoni cathepsin D as a probe, we isolated several positive bacterial artificial chromosomes (BAC) from a BAC library that represents an approximately 8-fold coverage of the schistosome genome. Sequencing of BAC clone 25-J-24 revealed that the cathepsin D gene locus was approximately 13 kb in length, and included seven exons interrupted by six introns. The exons ranged in length from 49 to 294 bp, and the introns from 30 to 5025 bp. The genomic organization of schistosome cathepsin D was similar in sequence, structure and complexity to human cathepsin D, including to a greater or lesser extent the conservation of all six exon/intron boundaries of the schistosome gene. It was less similar to aspartic protease genes of the nematodes Caenorhabditis elegans and Haemonchus contortus, and dissimilar to those of plasmepsins from malarial parasites. Examination of the introns revealed the presence of endogenous mobile genetic elements including SR2, the ASL-associated retrotransposon, and the SINE-like element, SMalpha. Phylogenetically, schistosome cathepsin D appeared to be more closely related to mammalian cathepsin D than to other sub-families of eukaryotic aspartic proteases known from mammals. Taken together, these features indicated that schistosome cathepsin D is a platyhelminth orthologue of mammalian lysosomal cathepsin D.     

Secretion of an acid phosphatase provides a possible mechanism to acquire host nutrients by Plasmodium falciparum

As an intracellular proliferating parasite, Plasmodium falciparum exploits the human host to acquire nutrients. However, nutrients such as nucleotides and cofactors are mostly phosphorylated in the host cell cytosol and thus have to be dephosphorylated in order to be taken up by the parasite. Here we report the functional characterization of a unique secreted phosphatase in P. falciparum, which is expressed throughout the developmental stages in the red blood cell. We show that this enzyme, formerly described as anchoring glideosome‐associated protein 50 (GAP50), reveals a broad substrate profile with preference for di‐ and triphosphates at pH 5–7. Bioinformatic studies of the protein sequence identified an N‐terminal signal anchor (SA) as well as a C‐terminal transmembrane domain. By means of live microscopy of parasites transfected with GFP‐fusions of this secreted acid phosphatase (PfSAP), we demonstrate that PfSAP enters the secretory pathway en route to the parasite periphery – mediated by SA – and is subsequently engulfed into the food vacuole. We corroborate this with independent data where acid phosphatase activity is visualized in close proximity to hemozoin. The biochemical as well as the trafficking results support the proposed role of PfSAP in the acquisition of host nutrients by dephosphorylation.     

MalAvi: a public database of malaria parasites and related haemosporidians in avian hosts based on mitochondrial cytochrome b lineages

Research in avian blood parasites has seen a remarkable increase since the introduction of polymerase chain reaction-based methods for parasite identification. New data are revealing complex multihost-multiparasite systems which are difficult to understand without good knowledge of the host range and geographical distribution of the parasite lineages. However, such information is currently difficult to obtain from the literature, or from general repositories such as GenBank, mainly because (i) different research groups use different parasite lineage names, (ii) GenBank entries frequently refer only to the first host and locality at which each parasite was sampled, and (iii) different researchers use different gene fragments to identify parasite lineages. We propose a unified database of avian blood parasites of the genera Plasmodium, Haemoproteus and Leucocytozoon identified by a partial region of their cytochrome b sequences. The database uses a standardized nomenclature to remove synonymy, and concentrates all available information about each parasite in a public reference site, thereby facilitating access to all researchers. Initial data include a list of host species and localities, as well as genetic markers that can be used for phylogenetical analyses. The database is free to download and will be regularly updated by the authors. Prior to publication of new lineages, we encourage researchers to assign names to match the existing database. We anticipate that the value of the database as a source for determining host range and geographical distribution of the parasites will grow with its size and substantially enhance the understanding of this remarkably diverse group of parasites.     

Transport of nucleosides across the Plasmodium falciparum parasite plasma membrane has characteristics of PfENT1

Like all parasitic protozoa, the human malaria parasite Plasmodium falciparum lacks the enzymes required for de novo synthesis of purines and it is therefore reliant upon the salvage of these compounds from the external environment. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a nucleoside transporter that has been localized to the plasma membrane of the intraerythrocytic form of the parasite. In this study we have characterized the transport of purine and pyrimidine nucleosides across the plasma membrane of 'isolated' trophozoite-stage P. falciparum parasites and compared the transport characteristics of the parasite with those of PfENT1 expressed in Xenopus oocytes. The transport of nucleosides into the parasite: (i) was, in the case of adenosine, inosine and thymidine, very fast, equilibrating within a few seconds; (ii) was of low affinity [K(m) (adenosine) = 1.45 +/- 0.25 mM; K(m) (thymidine) = 1.11 +/- 0.09 mM]; and (iii) showed 'cross-competition' for adenosine, inosine and thymidine, but not cytidine. The kinetic characteristics of nucleoside transport in intact parasites matched very closely those of PfENT1 expressed in Xenopus oocytes [K(m) (adenosine) = 1.86 +/- 0.28 mM; K(m) (thymidine) = 1.33 +/- 0.17 mM]. Furthermore, PfENT1 transported adenosine, inosine and thymidine, with a cross-competition profile the same as that seen for isolated parasites. The data are consistent with PfENT1 serving as a major route for the uptake of nucleosides across the parasite plasma membrane.     

Cloning and functional expression of a gene encoding a P1 type nucleoside transporter from Trypanosoma brucei.

Nucleoside transporters are likely to play a central role in the biochemistry of the parasite Trypanosoma brucei, since these protozoa are unable to synthesize purines de novo and must salvage them from their hosts. Furthermore, nucleoside transporters have been implicated in the uptake of antiparasitic and experimental drugs in these and other parasites. We have cloned the gene for a T. brucei nucleoside transporter, TbNT2, and shown that this permease is related in sequence to mammalian equilibrative nucleoside transporters. Expression of the TbNT2 gene in Xenopus oocytes reveals that the permease transports adenosine, inosine, and guanosine and hence has the substrate specificity of the P1 type nucleoside transporters that have been previously characterized by uptake assays in intact parasites. TbNT2 mRNA is expressed in bloodstream form (mammalian host stage) parasites but not in procyclic form (insect stage) parasites, indicating that the gene is developmentally regulated during the parasite life cycle. Genomic Southern blots suggest that there are multiple genes related in sequence to TbNT2, implying the existence of a family of nucleoside transporter genes in these parasites.     

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.     

Gene flow and the coevolution of parasite range

Abstract The geographic range of many parasites is restricted relative to that of their hosts. We study possible evolutionary mechanisms for this observation using a simple model that couples coevolution and demography. The model assumes that the environment consists of two habitats connected by movement and that coevolution is governed by quantitative traits. Our results demonstrate that host gene flow is an important determinant of parasite geographic range. Fluctuations in the rate of host gene flow cause shifts in parasite population densities and associated range expansions or contractions. In extreme cases, changing the rate of host gene flow can lead to global extinction of the parasite. Through a process we term demographic compensation, these shifts in parasite density may occur with little or no change in parasite adaptation to the host. As a consequence, reciprocal adaptation between host and parasite can become uncoupled from the rate of host gene flow.     

Dispersal in a parasitic worm and its two hosts: consequence for local adaptation

Characterizing host and parasite population genetic structure and estimating gene flow among populations is essential for understanding coevolutionary interactions between hosts and parasites. We examined the population genetic structure of the trematode Schistosoma mansoni and its two host species (the definitive host Rattus rattus and the intermediate host Biomphalaria glabrata) using microsatellite markers. Parasites were sampled from rats. The study was conducted in five sites of the Guadeloupe Island, Lesser Antilles. Mollusks display a pattern of isolation by distance whereas such a pattern is not found neither in schistosomes nor in rats. The comparison of the distribution of genetic variability in S. mansoni and its two host species strongly suggests that migration of parasites is principally determined by that of the vertebrate host in the marshy focus of Guadeloupe. However, the comparison between genetic differentiation values in schistosomes and rats suggests that the efficacy of the schistosome rat-mediated dispersal between transmission sites is lower than expected given the prevalence, parasitic load and migration rate of rats among sites. This could notably suggest that rat migration rate could be negatively correlated to the age or the infection status of individuals. Models made about the evolution of local adaptation in function of the dispersal rates of hosts and parasites suggest that rats and mollusks should be locally adapted to their parasites.     

Differential Spatial Repositioning of Activated Genes in Biomphalaria glabrata Snails Infected with Schistosoma mansoni

Schistosomiasis is an infectious disease infecting mammals as the definitive host and fresh water snails as the intermediate host. Understanding the molecular and biochemical relationship between the causative schistosome parasite and its hosts will be key to understanding and ultimately treating and/or eradicating the disease. There is increasing evidence that pathogens that have co-evolved with their hosts can manipulate their hosts'' behaviour at various levels to augment an infection. Bacteria, for example, can induce beneficial chromatin remodelling of the host genome. We have previously shown in vitro that Biomphalaria glabrata embryonic cells co-cultured with schistosome miracidia display genes changing their nuclear location and becoming up-regulated. This also happens in vivo in live intact snails, where early exposure to miracidia also elicits non-random repositioning of genes. We reveal differences in the nuclear repositioning between the response of parasite susceptible snails as compared to resistant snails and with normal or live, attenuated parasites. Interestingly, the stress response gene heat shock protein (Hsp) 70 is only repositioned and then up-regulated in susceptible snails with the normal parasite. This movement and change in gene expression seems to be controlled by the parasite. Other differences in the behaviour of genes support the view that some genes are responding to tissue damage, for example the ferritin genes move and are up-regulated whether the snails are either susceptible or resistant and upon exposure to either normal or attenuated parasite. This is the first time host genome reorganisation has been seen in a parasitic host and only the second time for any pathogen. We believe that the parasite elicits a spatio-epigenetic reorganisation of the host genome to induce favourable gene expression for itself and this might represent a fundamental mechanism present in the human host infected with schistosome cercariae as well as in other host-pathogen relationships.     

Helminth parasite proteomics: from experimental models to human infections

Schistosomiasis is a major human helminth infection endemic in developing countries. Urogenital schistosomiasis, caused by S. haematobium, is the most prevalent human schistosome disease in sub-Saharan Africa. Currently control of schistosome infection is by treatment of infected people with the anthelmintic drug praziquantel, but there are calls for continued efforts to develop a vaccine against the parasites. In order for successful vaccine development, it is necessary to understand the biology and molecular characteristics of the parasite. Ultimately, there is need to understand the nature and dynamics of the relationship between the parasite and the natural host. Thus, my studies have focused on molecular characterization of different parasite stages and integrating this information with quantitative approaches to investigate the nature and development of protective immunity against schistosomes in humans. Proteomics has proved a powerful tool in these studies allowing the proteins expressed by the parasite to be characterized at a molecular and immunological level. In this review, the application of proteomic approaches to understanding the human-schistosome relationship as well as testing specific hypotheses on the nature and development of schistosome-specific immune responses is discussed. The contribution of these approaches to informing schistosome vaccine development is highlighted.