The use of imaging to detect schistosomes and diagnose schistosomiasis.

Several imaging modalities have been employed to examine schistosomes and monitor schistosome-induced pathology. Ultrasound is a noninvasive imaging method that has long been used in the laboratory and in the field to evaluate pathological changes, notably fibrosis, that arise as a consequence of the host response to schistosome eggs lodging in a variety of tissues. Ultrasonography has been widely used to monitor changes in the extent of fibrosis and in spleen/liver enlargement following chemotherapeutic treatment for schistosomiasis. Imaging methods to monitor schistosomes themselves in vivo (as opposed to detecting schistosome-induced pathology) include positron emission tomography and fluorescence molecular tomography. Both approaches rely on schistosome uptake of tracers that are introduced into infected animals and that can be detected externally. These methods have been used to successfully detect schistosomes in vivo and to monitor their elimination following chemotherapeutic treatment. Direct monitoring of live schistosomes in vivo has been achieved using intravital microscopy, when the infected tissues of anaesthetized animals are exposed. Finally, schistosome eggs have been visualized by confocal laser scanning microscopy in infected mice as well as in a human patient with schistosomiasis hematobium. Further advances in imaging technologies seem likely to provide greater insight into disease progression and into the biology of schistosomes in the most relevant setting-within a live animal.

Metabolite movement across the schistosome surface.

Intravascular schistosome parasites are covered by an unusual double lipid bilayer. Nutrients, such as glucose and amino acids, as well as other metabolites, are known to be transported across this surface via specific transporter proteins. For instance, the glucose transporter protein SGTP4 is found in the host-interactive tegumental membranes. A second glucose transporter, SGTP1, localizes to the tegumental basal membrane (and internal tissues). Following expression in Xenopus oocytes, SGTP1 and SGTP4 both function as facilitated-diffusion sugar transporters. Suppressing the expression of SGTP1 and SGTP4 in juvenile schistosomes using RNA interference (RNAi) impairs the parasite's ability to import glucose and severely decreases worm viability. Amino acids can also be imported into schistosomes across their surface and an amino acid transporter (SPRM1lc) has been localized in the parasite surface membranes (as well as internally). In Xenopus oocytes, SPRM1lc can import the basic amino acids arginine, lysine and histidine as well as leucine, phenylalanine, methionine and glutamine. To function, this protein requires the assistance of a heavy-chain partner (SPRM1hc) which acts as a chaperone. Water is transported across the tegument of schistosomes via the aquaporin protein SmAQP. Suppressing SmAQP gene expression makes the parasites less able to osmoregulate and decreases their viability. In addition, SmAQP-suppressed adult parasites have been shown to be impaired in their ability to excrete lactate. Analysis of tegumental transporter proteins, as described in this report, is designed to generate a comprehensive understanding of the role of such proteins in promoting parasite survival by controlling the movement of metabolites into and out of the worms.

Schistosoma mansoni proteases Sm31 (cathepsin B) and Sm32 (legumain) are expressed in the cecum and protonephridia of cercariae.

Adult Schistosoma mansoni parasites live in the bloodstream of their vertebrate hosts where they consume red blood cells. Hemoglobin, released from the ingested red blood cells, is degraded by a variety of parasite proteases, including Sm31 (cathepsin B) and Sm32 (schistosome legumain). In this study the localization pattern of the Sm31 and Sm32 enzymes in cercariae (the infectious life cycle stage) was examined. Antibodies generated against recombinant Sm31 and Sm32 recognize their respective proteins in Western blots of soluble parasite extracts. Highest levels are seen in adult female extracts, whereas the level of both proteins is below detection in cercarial extracts. However, in fixed, whole cercariae, both proteins are seen in the cecum and protonephridia. In the cecum, the staining pattern has a granular appearance, suggesting that the proteins are packaged in vesicles. In the protonephridial system, Sm31 and Sm32 are detected in all 8 flame cells in the cercarial body and in both flame cells in the cercarial tail. The distribution of the 2 proteins differs in the flame cells. Examination of immunostained cercariae using laser scanning confocal microscopy shows that whereas Sm31 is located in the tubule cell, Sm32 is found in both the tubule cell and its adjoining cap cell. These findings suggest that the proteins are involved in the proposed excretory and osmoregulatory roles of flame cells.

Cloning and characterization of a muscle isoform of a Na,K-ATPase alpha subunit (SNaK1) from Schistosoma mansoni.

A cDNA encoding a Na,K-ATPase alpha subunit homologue, designated SNaK1, was isolated from an adult cDNA library of Schistosoma mansoni. The 3.8 kb DNA contained a 3021 bp open reading frame potentially encoding a 1,007 amino acid protein that had an Mr of 111,817 and a pl of 5.48. Homology searches for SNaK1 revealed approximately 70% sequence identity with a variety of Na, K-ATPases from evolutionarily diverse organisms. SNaK1 is predicted to contain 10 transmembrane regions typical of this protein family as well as other conserved domains, such as the phosphorylation site and ATP binding domain. Antibodies raised against an amino terminal peptide detected the protein in membrane preparations of eggs, cercariae and adult males and females, suggesting a general role for SNaK1. The mobility of the protein differed in various life-stages suggestive of post-transcriptional or post-translational modification. Immunolocalization of SNaK1 in sections of adult worms using epifluorescence and electron microscopy, revealed antibody labelling in the subtegumental and peripheral layers. Strong staining was discernible in the peripheral muscle band indicating that SNaK1 plays a central role in muscle contraction in adult parasites and may be the primary target of ouabain action. Staining was also detected in the secretory bodies in sections of ducts in this region and over the RER of the presumed gastrodermis. Immunogold labelling was also localized over neuronal vesicles in axons associated with the peripheral muscle layer.

Immunization with plasmid DNA encoding the integral membrane protein, Sm23, elicits a protective immune response against schistosome infection in mice.

Schistosomes are helminth parasites infecting at least 200 million people worldwide. In this study, we evaluated the feasibility of using a nucleic acid vaccine to induce protective immune responses to the Schistosoma mansoni integral membrane protein Sm23. C57BL/6 mice were immunized by intramuscular injection in three separate vaccination trials. ELISA and Western Blot analyses indicated that mice immunized with a DNA plasmid construct encoding Sm23 (Sm23-pcDNA) generated specific IgG for Sm23, while sera from mice immunized with the control pcDNA plasmid did not. The vaccine elicited IgG(2a), and IgG(1) antibody isotypes. We also tested the adjuvant activity of IL-12 and IL-4 on humoral responses to Sm23. Co-immunization with plasmid encoding IL-12 did not affect the level of anti-Sm23 IgG(2a), but did reduce the IgG(1) level. In contrast, co-injection with a plasmid encoding IL-4 significantly reduced the level of anti-Sm23 IgG(2a), while the level of IgG(1) was largely unchanged. Importantly, the Sm23-pcDNA vaccine provided statistically significant levels of protection against challenge infection (21-44%, P<0.001-0.02). Co-administration of plasmids encoding either IL-12 or IL-4 did not significantly enhance this protective effect.

Schistosoma mansoni: molecular characterization of a tegumental Ca-ATPase (SMA3).

A cDNA encoding a Ca-ATPase homologue, designated SMA3, was isolated from an adult cDNA library of Schistosoma mansoni. The full-length cloned DNA contains a 3105-bp open reading frame that potentially encodes a 1035-amino-acid protein with a M(r) of 113,729 and a pl of 6.48. Homology searches for SMA3 reveal high sequence identity with a variety of Ca-ATPases from evolutionarily diverse organisms. SMA3 is predicted to contain 10 transmembrane regions typical of this protein family as well as other conserved domains, such as the phosphorylation site and FITC binding domain. The greatest sequence identity (40-50%) is found to those Ca-ATPases belonging to the secretory pathway subclass. Identification of the 5' end of the SMA3 cDNA by RACE analysis reveals the presence of a 36-base spliced leader RNA, suggesting that the SMA3 pre-mRNA is processed by trans-splicing. Northern analysis reveals a single dominant transcript of 5 kb in adult RNA preparations. Antibodies raised against an amino terminal peptide detect the protein in the adult tegument, suggesting that SMA3 functions to help control Ca homeostasis within the tegument and may play a role in signal transduction at the host parasite interface.

The Schistosoma mansoni host-interactive tegument forms from vesicle eruptions of a cyton network.

During trans-dermal invasion of the vertebrate host, larval schistosomes (cercariae) transform into schistosomula and become enveloped by a double lipid bilayered, tegumental membrane. The glucose transporter protein SGTP4 is found exclusively in these host-interactive tegumental membranes and in membranous vesicles proposed to be their precursor. In this study, we monitored the appearance and migration of this tegumental marker protein during larval transformation to test the current model of tegumental membrane biosynthesis in parasitic blood flukes. Only minutes after transformation was initiated, SGTP4 began accumulating in a previously unrecognized, bilaterally symmetrical, 'cyton network' beneath the peripheral muscle. Approximately 30 min after the initiation of transformation the marker protein was seen in tubules connecting the network to the surface and erupting onto the surface in discrete patches. After I h the patches were regularly arrayed over the schistosomula body and began to cover the anterior organ. By 3 h the staining has largely resolved into a contiguous layer of fluorescence covering most of the worm surface. These findings confirm earlier suggestions, based on electron microscopy, that the parasite's surface tegumental membranes are derived from the migration of membranous vesicles produced within cytons and reveal a new subtegumental architecture interconnecting the cytons.

Induction cues for tegument formation during the transformation of Schistosoma mansoni cercariae.

Adult schistosomes are parasitic blood flukes that have a continuous double lipid bilayered membrane surrounding the entire worm. This tegumental membrane is synthesised during invasion of the vertebrate host by free-swimming infectious forms called cercariae. As cercariae invade their final hosts they lose their tails and encounter a changing environment that includes altered temperature, sugar concentration and osmolarity. We have identified a glucose transporter protein designated SGTP4 that is found exclusively in the outer adult tegument and on membranous vesicles within the tegumental cytoplasm. By using immunofluorescence analysis to monitor the appearance and distribution of SGTP4 we can track the process of new tegumental membrane formation and examine the cues that trigger this developmental pathway. Cercariae in water do not transform their tegument while those incubated in rich medium do so rapidly. We have examined which of the many constituents of rich medium are responsible for triggering this transformation. Incubation in a solution of moderate osmolarity (120 mOsM PBS) is sufficient by itself to trigger tegument transformation, albeit at a slower rate relative to incubation in rich medium. Adding either glucose (to 100 mM) to the solution or increasing the temperature of incubation (from 22 degrees C to 37 degrees C) further increased the rate of tegument biogenesis. The introduction of glucose together with an increase in the incubation temperature further accelerated the process, suggesting that these factors act synergistically to promote transformation rates. The critical nature of osmolarity in inducing the process is highlighted by the fact that transformation proceeds as efficiently in 360 mOsM alone as it does in rich medium. While the fatty acids linolenic acid (cis-9, cis-12, cis-15-octadecatrienoic acid at 1 mM) and capric acid (Decanoic acid, at 0.1 mM) have both been proposed to stimulate tegumental transformation, we show that neither promotes the morphogenesis of a normal schistosomulum tegument. The schistosomicide praziquantel (to 1 mM) has no detectable effect on new tegument formation.

A cDNA encoding a nuclear hormone receptor of the steroid/thyroid hormone-receptor superfamily from the human parasitic nematode Strongyloides stercoralis.

A cDNA encoding a nuclear hormone receptor of the steroid/thyroid receptor superfamily was obtained from third-stage larvae(L3) of the parasitic roundworm Strongyloides stercoralis. A recombinant clone was isolated via screening of an S. stercoralis cDNA library with a polymerase chain reaction (PCR)-generated probe. The insert of 2,583 bp contained the complete coding sequence of the receptor homologue. The conceptually translated amino acid sequence of this open reading frame encodes a 753-amino-acid-residue protein with an apparent molecular weight of 83.6 kDa and a predicted isoelectric point (pI) of 8.52. The coding sequence is 69% AT and the noncoding sequence is 72% AT, reflecting a characteristic A/T codon bias of S. stercoralis. In this report the amino acid sequence of the S. stercoralis nuclear hormone receptor of the steroid/thyroid receptor superfamily is compared with that of nuclear hormones of Caenorhabditis elegans, human orphan nuclear receptors, and insect ecdysone receptors. The potential role of steroids in the induction of hyperinfection syndrome is also discussed.

Studies on phospholipid turnover argue against sloughing of tegumental membranes in adult Schistosoma mansoni.

The tegumental membrane complex of Schistosoma mansoni is the site of interaction between the parasite and the host. The tegument is involved in uptake of many nutrients, but also plays a crucial role in the evasion of the actions of the host immune system. Essential for the success of this evasion is maintaining the integrity of the tegumental membranes. The rate of turnover of phospholipids was investigated by pulse-labelling worms cultured in vitro, followed by additional incubation in the presence of unlabelled substrates. Tegumental membranes were isolated, characterized using antibodies against specific tegumental proteins, and analysed. It was demonstrated that the most prominent fatty acid found in tegumental phospholipids, palmitate, incorporated rapidly into the phospholipid fraction during a 30 min pulse labelling. In a subsequent 20 h chase with unlabelled substrates, the incorporated radioactivity was lost again from the tegumental membrane complex. This high turnover of palmitate was found to be limited to phosphatidylcholine (PC) only. The turnover was due to deacylation/reacylation, and not to the sloughing of membranes as is the case in schistosomula. It is speculated that this rapid turnover of PC in the tegument of adult schistosomes plays a new and important role in the immune evasion by the parasite.

SPRM1lc, a heterodimeric amino acid permease light chain of the human parasitic platyhelminth, Schistosoma mansoni.

The Schistosoma mansoni protein, SPRM1lc, is a light chain member of a new family of heterodimeric amino acid permeases. These proteins require covalent association with a type II glycoprotein (like h4F2hc) for functional surface localization when expressed in Xenopus oocytes. We previously reported that, when co-expressed with h4F2hc, the transport properties of SPRM1lc resemble system y and y+ while its human homologue, E16, functions as an L-type permease. Here we extend the functional characterization of SPRM1lc in oocytes and show by competitor studies that its amino acid transport capacity is similar to that of whole adult schistosomes. We demonstrate by Northern and Western analysis that SPRM1lc is expressed within both larval and adult schistosomes. In all stages, SPRM1lc is associated into a high molecular weight complex that can be disrupted by reducing agents, consistent with the hypothesis that a significant fraction of the endogenous SPRM1lc is linked by a disulphide bond to an uncharacterized schistosome amino acid transporter heavy chain. Immunofluorescence localization detects SPRM1lc in miracidia, daughter sporocysts and adult worms. Confocal microscopy demonstrates that SPRM1lc is found in the apical membrane of the syncytial, double-lipid bilayer tegument which surrounds adult worms. Aqueous biotinylation studies on living worms show that SPRM1lc is exposed on the host-interactive surface of this tegumental membrane. Host exposed, functionally important surface proteins such as SPRM1lc could form the basis of an effective schistosomiasis vaccine. These studies are the first to describe a helminth amino acid transporter, and the first to characterize an invertebrate heterodimeric amino acid transporter.

Functional heterodimeric amino acid transporters lacking cysteine residues involved in disulfide bond.

The protein mediating system L amino acid transport, AmAT-L, is a disulfide-linked heterodimer of a permease-related light chain (AmAT-L-lc) and the type II glycoprotein 4F2hc/ CD98. The Schistosoma mansoni protein SPRM1 also heterodimerizes with h4F2hc, inducing amino acid transport with different specificity. In this study, we show that the disulfide bond is formed by heavy chain C109 with a Cys residue located in the second putative extracellular loop of the multi-transmembrane domain light chain (C164 and C137 for XAmAT-L-lc and SPRM1, respectively). The non-covalent interaction of Cys-mutant subunits is not sufficient to allow coimmunoprecipitation, but cell surface expression of the light chains is maintained to a large extent. The non-covalently linked transporters display the same transport characteristics as disulfide bound heterodimers, but the maximal transport rates are reduced by 30-80%.

Amino-acid transport by heterodimers of 4F2hc/CD98 and members of a permease family.

Amino-acid transport across cellular plasma membranes depends on several parallel-functioning (co-)transporters and exchangers. The widespread transport system L accounts for a sodium-independent exchange of large, neutral amino acids, whereas the system y(+)L exchanges positively charged amino acids and/or neutral amino acids together with sodium. The molecular nature of these transporters remains unknown, although expression of the human cell-surface glycoprotein 4F2 heavy chain (h4F2hc; CD98 in the mouse) is known to induce low levels of L- and/or y(+)L-type transport. This glycoprotein is found in activated lymphocytes, together with an uncharacterized, disulphide-linked lipophilic light chain with an apparent relative molecular mass of 40,000 (M(r) 40K). Here we identify the permease-related protein E16 as the first light chain of h4F2hc and show that the resulting heterodimeric complex mediates L-type amino-acid transport. The homologous protein from Schistosoma mansoni, SPRM1, also associates covalently with coexpressed h4F2hc glycoprotein, although it induces amino-acid transport of different substrate specificity. The coexpression of h4F2hc is required for surface expression of these permease-related light chains, which belong to a new family of amino-acid transporters that form heterodimers with cell-surface glycoproteins.

Molecular and functional characterization and tissue localization of 2 glucose transporter homologues (TGTP1 and TGTP2) from the tapeworm Taenia solium.

Tapeworms absorb and consume large quantities of glucose through their syncytial tegument, storing the excess as glycogen. Although some studies on the metabolism of glucose in several tapeworms are available, the proteins that mediate its uptake and distribution in their tissue have not been identified. We describe the isolation and characterization of cDNA clones encoding 2 facilitated diffusion glucose transporters (TGTP1 and TGTP2) from Taenia solium, the causal agent of human and porcine cysticercosis. Radio-isotope labelled hexose uptake mediated by TGTP1 expressed in Xenopus oocytes is inhibited by the natural stereoisomers D-glucose and D-mannose but not by L-glucose. Transport by TGTP1 is sensitive to classical inhibitors of facilitated diffusion such as phloretin and cytochalasin B, and insensitive to ouabain. TGTP2 did not function in Xenopus oocytes. Localization studies using specific anti-TGTP1 and anti-TGTP2 antibodies show that TGTP1 is abundant in a number of structures underlying the tegument in adult parasites and larvae, whereas TGTP2 appears to be localized only on the tegumentary surface of the larvae and is not detected in adults.

Glucose Transport and Metabolism in Mammalian-stage Schistosomes.

Adult schistosomes transport nutrients from the host bloodstream across their outer body covering or tegument. The tegument is a cytologically unusual structure; it is a syncytium bounded externally by two lipid bilayer membranes. In this review, Patrick Skelly, Louis Tielens and Chuck Shoemaker reconsider our understanding of how glucose enters schistosomes across this unusual outer covering in the light of recent papers characterizing glucose transport proteins and glucose metabolism pathways in these parasites.

The Schistosoma mansoni epidermal growth factor receptor homologue, SER, has tyrosine kinase activity and is localized in adult muscle.

DNA encoding the full-length Schistosoma mansoni epidermal growth factor receptor, SER, was obtained by combining partial cDNA clones. Three different anti-SER antibody preparations, specific either to the SER amino-terminus or the predicted ligand binding domain were generated with recombinant or synthetic peptides and purified by antigen affinity. Recombinant SER was expressed within insect cells using the baculovirus expression system and detected by each of the anti-SER antibodies as well as anti-phosphotyrosine antibodies. By in vitro phosphorylation of immunoprecipitated recombinant SER, the protein has been shown to be capable of tyrosine autophosphorylation but this activity is not affected by human epidermal growth factor. Native SER is detected as a 170 kDa protein in Western blots of S. mansoni adult worm membrane preparations. Adult worm sections, labeled with anti-SER antibodies, localize SER predominantly to the muscle of adult male and female worms. These results confirm a place for SER in the EGFR family of tyrosine kinases and strongly suggest that it participates in schistosome signal transduction, perhaps related to muscle development or function.

Rapid appearance and asymmetric distribution of glucose transporter SGTP4 at the apical surface of intramammalian-stage Schistosoma mansoni.

Adult Schistosoma mansoni blood flukes reside in the mesenteric veins of their vertebrate hosts, where they absorb immense quantities of glucose through their tegument by facilitated diffusion. Previously, we obtained S. mansoni cDNAs encoding facilitated-diffusion schistosome glucose transporter proteins 1 and 4 (SGTP1 and SGTP4) and localized SGTP1 to the basal membranes of the tegument and the underlying muscle. In this study, we characterize the expression and localization of SGTP4 during the schistosome life cycle. Antibodies specific to SGTP4 appear to stain only the double-bilayer, apical membranes of the adult parasite tegument, revealing an asymmetric distribution relative to the basal transporter SGTP1. On living worms, SGTP4 is available to surface biotinylation, suggesting that it is exposed at the hose-parasite interface. SGTP4 is detected shortly after the transformation of free-living, infectious cercariae into schistosomula and coincides with the appearance of the double membrane. Within 15 min after transformation, anti-SGTP4 staining produces a bright, patchy distribution at the surface of schistosomula, which becomes contiguous over the entire surface of the schistosomula by 24 hr after transformation. SGTP4 is not detected in earlier developmental stages (eggs, sporocysts, and cercariae) that do not possess the specialized double membrane. Thus, SGTP4 appears to be expressed only in the mammalian stages of the parasite's life cycle and specifically localized within the host-interactive, apical membranes of the tegument.

Schistosoma mansoni: the glucose transport protein SGTP4 is present in tegumental multilamellar bodies, discoid bodies, and the surface lipid bilayers.

Schistosomes metabolize large quantities of glucose obtained from the host serum by facilitated diffusion through the tegument. Here we have used rabbit antibodies affinity purified against the carboxyl terminus of a facilitated glucose transporter, SGTP4, to localize the antigen in both schistosomula and adults. By ultrastructural immunocytochemical analysis, SGTP4 was localized to both lipid bilayers that cover the tegumental surface of adults and schistosomula. In the inner bilayer of adults, SGTP4 was apparently oriented with the carboxyl terminus on the internal side of the bilayer. SGTP4 was also present in the discoid and multilamellar bodies in adults and the membranous bodies in schistosomula. Finally, the affinity purified antibodies against SGTP4 bound nonspecifically to the head glands and postacetabular glands in schistosomula. The localization of the antigen in the two surface lipid bilayers suggests that SGTP4 may be responsible for transporting glucose from mammalian host serum into the tegument.

Immunolocalization of a Schistosoma mansoni facilitated diffusion glucose transporter to the basal, but not the apical, membranes of the surface syncytium.

Adult parasites of Schistosoma mansoni reside within vertebrate mesenteric veins where they consume immense quantities of host glucose after transporting the sugar through their surface syncytium or tegument. Previously we obtained cDNA clones encoding two functional facilitated diffusion glucose transporter proteins expressed by S. mansoni adult worms (Skelly et al. 1994). Antibodies specific for one transporter (SGTP1) have been generated against an extrafacial and an internal domain of the protein and used to localize the protein by light and electron microscopy. By light microscopy both antibodies stain a linear structure approximately 1-5 microns from the surface of the tegument of adult male and female schistosomes. Electron microscopic examination of frozen thin sections show binding of the antibodies to membranes in the base of the tegument and not to the membranes covering the outer surface or their invaginations. Analysis of the gold distribution suggests that the extrafacial domain is disposed toward the interstitial space beneath the tegument and the internal domain faces the syncytial plasm. The localization suggests that SGTP1 may function to transport free glucose from within the tegument and into the interstitial fluids that bathe the internal organs of these parasites.