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.

Conversion at large intergenic regions of mitochondrial DNA in Saccharomyces cerevisiae.

Saccharomyces cerevisiae mitochondrial DNA deletion mutants have been used to examine whether base-biased intergenic regions of the genome influence mitochondrial biogenesis. One strain (delta 5.0) lacks a 5-kilobase (kb) segment extending from the proline tRNA gene to the small rRNA gene that includes ori1, while a second strain (delta 3.7) is missing a 3.7-kb region between the genes for ATPase subunit 6 and glutamic acid tRNA that encompasses ori7 plus ori2. Growth of these strains on both fermentable and nonfermentable substrates does not differ from growth of the wild-type strain, indicating that the deletable regions of the genome do not play a direct role in the expression of mitochondrial genes. Examination of whether the 5- or 3.7-kb regions influence mitochondrial DNA transmission was undertaken by crossing strains and examining mitochondrial genotypes in zygotic colonies. In a cross between strain delta 5.0, harboring three active ori elements (ori2, ori3, and ori5), and strain delta 3.7, containing only two active ori elements (ori3 and ori5), there is a preferential recovery of the genome containing two active ori elements (37% of progeny) over that containing three active elements (20%). This unexpected result, suggesting that active ori elements do not influence transmission of respiratory-competent genomes, is interpreted to reflect a preferential conversion of the delta 5.0 genome to the wild type (41% of progeny). Supporting evidence for conversion over biased transmission is shown by preferential recovery of a nonparental genome in the progeny of a heterozygous cross in which both parental molecules can be identified by size polymorphisms.

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%.

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.

Expression of Schistosoma mansoni genes involved in anaerobic and oxidative glucose metabolism during the cercaria to adult transformation.

Schistosomes switch rapidly from the use of stored glycogen to a reliance on host glucose during the transformation from free-living cercariae to parasitic schistosomula. We have cloned a set of cDNAs encoding proteins involved in glucose metabolism to allow us to examine the expression of these genes during this transformation. We first obtained and characterized Schistosoma mansoni cDNA clones encoding the tricarboxylic acid cycle enzyme, mitochondrial malate dehydrogenase (SMDH) and the mitochondrial encoded electron transport protein, cytochrome oxidase subunit 1 (SCOX1). Northern blots were then prepared using mRNA isolated from whole cercariae, cercarial tails, schistosomula, adult males and adult females. The Northern blots were successively hybridized with a variety of probes including those for SMDH, SCOX, the glycolytic enzymes, hexokinase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase and several control probes. Probes were additionally hybridized to mRNA dot blots and the signals were quantified using storage phosphor technology. These studies reveal that transcripts encoding these metabolic enzymes are localized at much higher levels in cercarial tails than in whole cercariae or transformed schistosomula, and support the notion of a dominant aerobic metabolism in tails. Male and female adult worms express each of the mRNAs at roughly equal levels. Adults express the metabolic mRNAs, including those involved in oxidative glucose metabolism, at relatively high levels suggesting that adult schistosomes retain a significant capacity to produce energy through aerobic metabolism.

Characterization of the Schistosoma mansoni gene encoding the glycolytic enzyme, triosephosphate isomerase.

The complete gene encoding Schistosoma mansoni triosephosphate isomerase (TPI) was isolated from a lambda phage genomic library on 2 overlapping clones. These genomic clones have been characterized by restriction mapping and DNA sequencing of the 5' flanking region, the exons, the intron boundaries and the polyadenylation addition site. S. mansoni TPI is encoded by 6 exons spanning a region of about 12 kb. The 5 introns are located at positions precisely analogous to those of mammalian TPI genes but one of the 6 mammalian TPI introns is missing in S. mansoni. We find no evidence of spliced leader involvement in TPI gene expression. The gene is preceded by at least 4 tandem copies of a 2.5-kb repetitive sequence. While the 12-kb size for the S. mansoni TPI gene is much larger than the 3-4 kb typical of mammalian TPI genes, the 42-bp first intron is unusually short. The transcription initiation site for the S. mansoni TPI gene is heterogeneous. Genomic Southern blot analysis suggests that TPI is expressed from a single copy gene.

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.

Failure of schistosomiasis to significantly decrease testosterone levels in Brazilian men.

Schistosomes can decrease the reproductive potential or castrate both invertebrate (snail) hosts as well as vertebrate (mouse, rat, and hamster) hosts. To determine if host castration occurs in human males, we examined testosterone levels in the sera of 38 Brazilian males, 16-35 years of age, who had Schistosoma mansoni infections. We found that individuals with intestinal schistosomiasis exhibited serum testosterone levels similar to those of noninfected controls. Four subjects with severe hepatosplenic disease also exhibited testosterone levels within the normal range. We did observe a negative correlation between parasite load (as predicted by fecal egg count) and testosterone levels but could not dissociate this relationship from the effect of age on either parameter. Therefore, in contrast to rodent models, host castration does not appear to be a usual side effect of human schistosomiasis.

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.

A molecular genetic study of the variations in metabolic function during schistosome development.

During their complex life cycle schistosomes alternate between the use of stored glycogen and reliance on host glucose to provide for their energy needs. In addition, there is dramatic variation between the relative contribution of aerobic versus anaerobic glucose metabolism during development. We have cloned a set of representative cDNAs that encode proteins involved in glucose uptake, glycolysis, Kreb's cycle and oxidative phosphorylation. The different cDNAs were used as probes to examine the expression of glucose metabolism genes during the schistosome life cycle. Steady state mRNA levels from whole cercariae, isolated cercarial tails, schistosomula and adult worms were analysed on Northern blots and dot blots which were quantified using storage phosphor technology. These studies reveal: (1) Transcripts encoding glycogen metabolic enzymes are expressed to much higher levels in cercarial tails than whole cercariae or schistosomula while the opposite pattern is found for glucose transporters and hexokinase transcripts; (2) Schistosomula contain low levels of transcripts encoding respiratory enzymes but regain the capacity for aerobic glucose metabolism as they mature to adulthood; (3) Male and female adults contain similar levels of the different transcripts involved in glucose metabolism.

Distribution of mitochondrial intron sequences among 21 yeast species.

The mitochondrial and nuclear genomes of 21 yeast species belonging to 12 genera have been tested for the presence of sequences similar to seven S. cerevisiae mitochondrial introns (Se cox1.1,2,3,4,5c, Sc cob.4 and Sc LSU.1) and one K. lactis mitochondrial intron (K1 cox1.2). Some introns, (Sc cox1.4, Sc cob.4, Sc LSU.1 and Kl cox1.2-all group I type), are widely distributed and are found in species with either basidiomycete or ascomycete affinities. This distribution is suggestive of recent sequence transfer between species. The remaining S. cerevisiae introns cross react with an additional species but with no set pattern. Pulsed field gel electrophoretic studies confirm that none of the tested mitochondrial introns cross react with nuclear DNA. These introns are, therefore, mitochondria-specific. Seven strains of K. lactis exhibit striking variability in intron content. In contrast to all mitochondrial introns tested, two introns of nuclear genes (the K. lactis actin gene and the S. cerevisiae RP29B gene) are not detected beyond their source species.

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.

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.

Polymorphisms in tandemly repeated sequences of Saccharomyces cerevisiae mitochondrial DNA.

A spontaneously arising mitochondrial DNA (mtDNA) variant of Saccharomyces cerevisiae has been formed by two extra copies of a 14-bp sequence (TTAATTAAATTATC) being added to a tandem repeat of this unit. Similar polymorphisms in tandemly repeated sequences have been found in a comparison between mtDNAs from our strain and others. In 5850 bp of intergenic mtDNA sequence, polymorphisms in tandemly repeated sequences of three or more base pairs occur approximately every 400-500 bp whereas differences in 1-2 bp occur approximately every 60 bp. Some polymorphisms are associated with optional G + C-rich sequences (GC clusters). Two such optional GC clusters and one A + T repeat polymorphism have been discovered in the tRNA synthesis locus. In addition, the variable presence of large open reading frames are documented and mechanisms for generating intergenic sequence diversity in S. cerevisiae mtDNA are discussed.

Rolling circle replication of DNA in yeast mitochondria.

The conformation of mitochondrial DNA (mtDNA) from yeasts has been examined by pulsed field gel electrophoresis and electron microscopy. The majority of mtDNA from Candida (Torulopsis) glabrata (mtDNA unit size, 19 kb) exists as linear molecules ranging in size from 50 to 150 kb or 2-7 genome units. A small proportion of mtDNA is present as supercoiled or relaxed circular molecules. Additional components, detected by electron microscopy, are circular molecules with either single- or double-stranded tails (lariats). The presence of lariats, together with the observation that the majority of mtDNA is linear and 2-7 genome units in length, suggests that replication occurs by a rolling circle mechanism. Replication of mtDNA in other yeasts is thought to occur by the same mechanism. For Saccharomyces cerevisiae, the majority of mtDNA is linear and of heterogeneous length. Furthermore, linear DNA is the chief component of a plasmid, pMK2, when it is located in the mitochondrion of baker's yeast, although only circular DNA is detected when this plasmid occurs in the nucleus. The implications of long linear mtDNA for hypotheses concerning the ploidy paradox and the mechanism of the petite mutation are discussed.

Sequence rearrangements at the ori 7 region of Saccharomyces cerevisiae mitochondrial DNA.

Three ori elements (ori 2, ori 5, and ori 7) have been sequenced in Saccharomyces cerevisiae strain Dip 2 and compared to the equivalent ori elements of a second strain (B). Both ori 2 and ori 5 exhibit 98% base matching between strains Dip 2 and B. In contrast, the third ori element (ori 7) exhibits extensive sequence rearrangements whereby a segment located downstream in the consensus strain occurs within the ori structure in Dip 2. This represents a novel polymorphic form of the yeast mitochondrial genome.

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.

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.