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.     

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.     

Fasciola hepatica: basal infolds and associated vacuoles of the tegument.

The tegument of Fasciola hepatica has been shown to contain long invaginations of its basal plasma membrane, hereafter called basal infolds. Associated with the membranes of the infolds, and with the apical and basal plasma membranes, is a Na⁺ K⁺ ATPase (EC 3.6.1.3). Furthermore, polymorphic masses of acid mucopolysaccharide lie close to or against the sides of the infolds and the basal plasma membrane and also fill cytoplasmic tubules which connect the tegument with the tegumental cells. Fixation and incubation of flukes in hypertonic and hypotonic media have shown that the infolds respond to changes in external osmolarity by collapsing in the former and swelling in the latter. This is not simply a passive response, however, since the infolds return to near normal configuration and morphology within 1 hr in either hypertonic or hypotonic media, even though the whole fluke may be shrunken or turgid depending on medium osmolarity. The tegument, therefore, has many of the characteristics of a transporting epithelium. A theory outlining the possible mode of operation of the tegument as a transporting epithelium is proposed on the basis of the present structural, chemical, and physiological findings combined with current ideas of the role and functioning of standing gradients, and forward and backward channel systems in other transporting epithelia.     

The cercarial tail in Proterometra macrostoma (Digenea: Azygiidae): permeability and fine structure of the tegument

Permeability of the cercarial tail in Proterometra macrostoma was examined in vitro with 1 mM 3H-glucose, which tails absorb by diffusion alone. Naturally emerged cercariae (bodies withdrawn into tails) were permeable, but they rapidly (3 min) equilibrated with glucose in the bathing medium and maintained steady state for 4 hr. Metabolism of absorbed glucose was not detectable until after 90 min, and radioactivity in bodies dissected from tails after 4 hr was negligible. On the basis of cercarial water content (90% of total weight) and absorbed isotope at steady state, the calculated volume of the equilibrating compartment was 4% of an intact cercaria. This value correlated well with that of the tegument (3-5%), which was 1-2 microm thick as seen by transmission electron microscopy. A continuous, electron-dense basal membrane/lamina separated the tegument from subtegument. We conclude that the glycocalyx and external plasma membrane are freely permeable, whereas the basal membrane is the barrier that effectively isolated the subtegument from exogenous glucose. The basal membrane also may be the primary structure that protects the subtegument and cercarial body from effects of osmotic stress.     

Ultrastructure of the Tegumental Basement Membrane of Fasciola hepatica(Trematoda)

The fine–structural characteristics of the basement membrane of the tegument of F. hepatica were examined following extraction fixations and tannic acid infiltration. The basement membrane was shown to consist of three layers: lamina lucida, lamina densa, and lamina reticularis. The lamina densa appeared amorphous and homogeneous with tannic acid impregnation. The lamina reticularis appeared as a dense network of 10–12 nm fibrils. Anchoring fibrils cross this layer and form loops. Along their length they contact hemidesmosomes of muscles, thus connecting muscle to muscle and to tegument. The tegument/basement membrane contact is enhanced by extensions of the lamina densa into infoldings of the tegumental basal membrane. Where tegumental spines reach the basement membrane, the contact is reinforced by hemidesmosomes that connect to anchoring fibrils reaching toward the underlying muscles. The basement membrane thus seems to be a complex structure integrating the distal tegumental layer with underlying tissues and transducing muscle contractions to the tegument and its spines.     

A role of trophoblastic cells in regulation of mouse blastocyst survival in vitro after microinjection and osmotic stress

We have evaluated the morphology of the mouse preimplantation embryos at developmental stages from morula to late blastocyst after two different impacts: microinjection of modified Witten’s medium and osmotic stress in physiological osmolarity (310 mOsM), in 5% glucose (560 mOsM) at high concentration of NaCl (614 mOsM). Results of our research showed that these stresses caused similar changes in embryo morphology: volume was reduced followed by its recovery in culture medium (osmolality was less than a physiological value, 260 mOsM). The ability of embryos to recover the volume and morphology up to the initial level depends on a stage of embryo development and consequently competence of TB cells. In this study it was revealed that a key role in regulation of volume homeostasis after microinjection and after short-time (30–60 min) osmotic stress belongs to TB cells. Both physical effects induce the further embryo development in vitro up to the formation of primary colonies of embryonic and trophoblastic cells. These data could be used to develop the morphological criteria for a prediction of blastocyst-stage embryonic implantation potential.     

Giardia intestinalis: influence of the composition of the osmolarity of the medium on in vitro excystation

The in vitro excystation of Giardia intestinalis was studied to make the osmolarity (from 50 to 500 mosmol/l) and the components of growth medium (MCI saline solution, MCII glucose solution, MCIII nutritive solution) varying. The percentage of excystation, the viability and the generation time were determined. Excystation was observed in the saline solution between 100 to 450 mosmol/l after cyst acid pepsin incubation. The trophozoite viability was increased by glucose addition (60 min in MCI; 300 min in MCII). Only a rich medium (MCIII) permitted a generation time from 225 to 425 mosmol/l.     

Fasciola hepatica: A light and electron microscope autoradiographic study of incorporation of monosaccharides into glycogen and glycoprotein

Incorporation of [³H]galactose and [³H]glucose into the parenchyma, tegument, testis, and muscle of Fasciola hepatica slices was studied by lightand electron-microscope autoradiography. “Accumulation” labeling periods of up to 60 min were used.Both monosaccharides were found to be readily incorporated into glycogen in the parenchymal cells and muscle and [³H]glucose entered the glycogen stores of spermatozoa.No evidence was found for the involvement of any particular cell organelle in glycogenesis, but the demonstration of high synthetic activity in parenchymal evaginations to the base of the surface syncytial tegument supports physiological evidence that glucose enters the fluke mainly across the tegument.Ethylene glycol-dehydrated preparations showed that [³H]galactose was incorporated into glycoprotein by Type I tegumental cells, and perhaps also by sperm morulae. The carbohydrate component seems to be added to the tegumental secretions in the vesicular-lamellar region of the Golgi complex.Following the longest periods of incubation, labeling was observed in the tubules connecting the tegumental cells and syncytium, but not in the surface syncytium itself.     

Schistosoma mansoni: analysis of cercarial transformation methods

Four methods of transforming cercariae to schistosomulae in vitro in ELAC buffer (pH 7.2, 37 C, 0-6 hr incubation) were compared in relation to biochemical and ultrastructural characteristics. The transformation methods used were chemical (3 mM linoleate), mechanical (centrifuge/vortex), mechanical/chemical, and heat (incubation at 37 C). Ultrastructural characteristics examined were based on the presence or absence of glycocalyx, heptalaminate membrane, cyton granules, and nuclear condition. Two EM fixation methods were used. Biochemical parameters assayed were loss of water tolerance (uptake of trypan blue dye), eicosanoid biosynthesis (PGE, LTB4, and 5-HETE), protein synthesis (leucine uptake), RNA synthesis (uracil and orotic acid uptake), and DNA synthesis (thymidine uptake). EM characteristics were remarkably similar for all transformation methods except heat incubation, with transformed cercariae evidencing the characteristics of schistosomulae (cyton granule migration, absence of glycocalyx and heptalaminate membrane); however, euchromatic nuclei could not be demonstrated using in vivo or in vitro transformation methods. Despite the ultrastructural similarities between transformation methods, biochemical data demonstrated that the resultant organisms were quite different. The chemical transformation method gave the highest rate of loss of water tolerance and eicosanoid production. RNA and protein synthesis were not correlated to ultrastructural changes and were highest in those organisms undergoing mechanical transformation methods, significantly higher than in those cercariae transformed by the chemical method. DNA synthesis was not demonstrated using any transformation method, although thymidine uptake did occur. Our data indicate substantial biochemical differences exist between morphologically similar organisms. Thus, experiments using any type of artificially transformed schistosomule must be interpreted with caution until additional biochemical and physiological studies on cercarial transformation are undertaken.     

Tegumental changes in adult Schistosoma mansoni harbored in mice treated with artemether

A detailed temporal examination was made of alterations induced by artemether in the tegument of adult Schistosoma mansoni worms using scanning electron microscopy (SEM). Mice infected with S. mansoni cercariae 42 days previously were treated intragastrically with artemether at a single dose of 400 mg/kg. Groups of 3 mice were killed at 24 hr, 72 hr, and 7 days after treatment; the worms were collected by perfusion and examined by SEM. Twenty-four hours after artemether treatment, focal damage to the tubercles on the tegumental surface of male worms was seen. In both male and female worms, there was focal swelling and fusion of tegumental ridges, and sometimes peeling. After 72 hr, the damage to the tegument had increased, especially in female worms, with extensive swelling, fusion, and peeling of the tegumental ridges. In the most severely damaged worms, host leukocytes were seen to be adhered to the damaged tegument. Damage to the oral sucker was also occasionally seen in both male and female worms. Seven days after treatment, the appearance of the tegument had returned to normal in some male and female worms, whereas others still showed apparent damage. The results demonstrate that artemether damages the tegument of adult S. mansoni, and the intensity of damage is more severe in female worms than in males.     

Schistosoma mansoni: cercaricidal effect of 2-tetradecenoic acid, a penetration stimulant

Schistosoma mansoni cercariae are stimulated by 2-tetradecenoic acid (TDA) to penetrate agar substrates. TDA simultaneously causes tegumental transformation similar to that seen when cercariae transform to schistosomula, reduces the Cercarienhüllen reaction in immune human serum, and reduces larval tolerance to water. TDA damages cercariae that fail to penetrate or have no opportunity to do so. This damage apparently stems from increased tegumental permeability to water. Preincubation in TDA for 60 min reduces the percutaneous infectivity of cercariae to mice by from 95% at 0.2 ppm to 100% at 0.7 ppm TDA, but does not reduce the infectivity of subcutaneously injected cercariae. The interference with percutaneous infection seems to be entirely due to osmotic damage. TDA does not induce premature secretion of the acetabular glands or block host-recognition chemoreceptors. TDA may be a promising cercaricide for schistosomiasis control. It is highly specific for schistosome cercariae and is effective at low concentrations (0.2 to 0.7 ppm). Both cercariae and TDA tend to collect in the upper few millimeters of standing water. It is unlikely that cercariae can evolve resistance to a chemical that triggers the host penetration mechanisms.     

Schistosoma mansoni: Tegumental damage as a consequence of lectin binding

Adult worms of Schistosoma mansoni exhibit gross tegumental damage following incubation in concanavalin A or Ricinus communis agglutinin. However, incubation with wheat germ agglutinin induces only minimal surface damage, while soybean agglutinin has no damaging effect upon the worms. Damage induced by Ricinus communis agglutinin or concanavalin A may be prevented by the addition of the appropriate competing sugar. In contrast, incubation of 3-hr artificially transformed schistosomula in concanavalin A and other lectins does not produce any disruption of the tegument. These results indicate that the surface membrane of the adult schistosome is readily disrupted by ligand binding and appears to be particularly sensitive and fragile. The membrane of the schistosomulum, however, is more resistant to the effects of lectin binding. Adult worms incubated in culture medium alone (ELAC or RPMI 1640) show background changes which seem to be related to the tonicity of the medium. Such results advocate that preliminary assessment of schistosome integrity be carried out prior to any experimental procedures which preclude the addition of serum to the basic incubation medium. Schistosomula do not exhibit comparable sensitivity.     

Schistosoma mansoni: increasing saline concentration signals cercariae to transform to schistosomula

Cercariae of S. mansoni shed the surface glycocalyx, form a double lipid bilayer on their surface, and transform to schistosomula when tails are removed and parasites are transferred from pond water to 300 mOsm phosphate-buffered saline. To determine whether the absolute concentration of saline or the relative change in saline concentration was the signal for surface transformation, cercariae were isolated from the snail hepatopancreas, sheared to remove the tails, and incubated in defined media for 3 hr at 37 degrees C. Surface transformation was assayed using the binding of the fluorescein-conjugated lectin concanavalin A to the schistosomular double unit membrane but not to the cercarial glycocalyx. An increase in salinity either from 18 mOsm (artificial pond water) to 120 mOsm (the snail osmolarity) or from 120 to 300 mOsm (the mammalian osmolarity) triggered transformation to schistosomula. Organisms constantly exposed to 120 mOsm or shifted from 120 mOsm to pond water did not transform their surfaces. The signal for transformation appeared to be increasing salinity rather than increasing osmolarity because cercarial bodies did not become schistosomula in 300 mOsm mannitol. Surface transformation was inhibited when cercariae were incubated with the acetylcholinesterase inhibitor eserine sulfate during a 10 min time when the osmolarity was raised. We conclude that increasing salinity rather than the absolute saline concentration is the signal for surface transformation and that eserine sulfate may inhibit the receipt of this signal.     

Functions of the tegument of schistosomes: clues from the proteome and lipidome

The tegumental outer-surface of schistosomes is a unique double membrane structure that is of crucial importance for modulation of the host response and parasite survival. Although several tegumental proteins had been identified by classical biochemical approaches, knowledge on the entire molecular composition of the tegument was limited. The Schistosoma mansoni genome project, together with recently developed proteomic and lipidomic techniques, allowed studies on detailed characterisation of the proteins and lipids of the tegumental membranes. These studies identified tegumental proteins and lipids that confirm the function of the tegument in nutrient uptake and immune evasion. However, these studies also demonstrated that compared to the complete worm, the tegument is enriched in lipids that are absent in the host. The tegument is also enriched in proteins that share no sequence similarity to any sequence present in databases of species other than schistosomes. These results suggest that the unique tegumental structures comprise multiple unique components that are likely to fulfil yet unknown functions. The tegumental proteome and lipidome, therefore, imply that many unknown molecular mechanisms are employed by schistosomes to survive within their host.     

Fasciola hepatica: Glycocalyx replacement in the juvenile as a possible mechanism for protection against host immunity

The response of newly excysted juvenile Fasciola hepatica to immune sheep serum under in vitro conditions was examined using indirect fluorescent antibody labeling and electron microscopy. Flukes acquired a continuous layer of host IgG over the surface during incubation in the presence of antiserum, but when transferred to a medium lacking antiserum they actively sloughed this layer and replaced the former glycocalyx, by a new antigenically similar surface coat. Electron microscope examination of juvenile flukes verified than an immune complex formed at and sloughed from the tegumental surface of those which were incubated in immune serum. T0 secretory bodies produced by the GER/Golgi system of the tegumental cells and stored in the metacercariae were discharged at the apical surface of the tegument, possibly in response to antibody binding. When cycloheximide was included with immune serum in the incubation medium the tegumental cells were unable to synthesize new T0 bodies to replace losses and the number of T0 bodies decreased so that the cytoplasm of the tegumental cells and surface syncytium became virtually devoid of T0 bodies within 48 hr.     

Isolation and characterization of glycosaminoglycans from Schistosoma mansoni

Tegumental tissues of paired adult Schistosoma mansoni were removed by treatment with Triton X-100 and recovered by centrifugation. The chloroform-methanol insoluble residues of this isolated tegumental fraction and of the denuded carcasses were analysed for glycosaminoglycan (GAG) and sialic acid contents. Treatment with GAG-specific enzymes followed by electrophoretic analysis showed that both the carcass and tegument contained heparin and/or heparan sulfate, chondroitin sulfate and hyaluronic acid. All these except hyaluronic acid were present in the tegumental fraction. Based on uronic acid content, about 73% of the total GAG was in the tegumental membrane, 15% in the tegmental matrix and the remaining 12% was in the carcass. The presence of heparin-like polysaccharide may present entrapment of the schistosoma by the hosts' blood-clotting process.     

Mouse embryos stressed by physiological levels of osmolarity become arrested in the late 2-cell stage before entry into M phase

Preimplantation mouse embryos of many strains become arrested at the 2-cell stage if the osmolarity of culture medium that normally supports development to blastocysts is raised to approximately that of their normal physiological environment in the oviduct. Arrest can be prevented if molecules that serve as "organic osmolytes" are present in the medium, because organic osmolytes, principally glycine, are accumulated by embryos to provide intracellular osmotic support and regulate cell volume. Medium with an osmolarity of 310 mOsM induced arrest of approximately 80% of CF1 mouse embryos at the 2-cell stage, in contrast to the approximately 100% that progressed beyond the 2-cell stage at 250 or 301 mOsM with glycine. The nature of this arrest induced by physiological levels of osmolarity is unknown. Arrest was reversible by transfer to lower-osmolarity medium at any point during the 2-cell stage, but not after embryos would normally have progressed to the 4-cell stage. Cessation of development likely was not due to apoptosis, as shown by lack of external annexin V binding, detectable cytochrome c release from mitochondria, or nuclear DNA fragmentation. Two-cell embryos cultured at 310 mOsM progressed through the S phase, and zygotic genome activation markers were expressed. However, most embryos failed to initiate the M phase, as evidenced by intact nuclei with decondensed chromosomes, low M-phase promoting factor activity, and an inactive form of CDK1, although a few blastomeres were arrested in metaphase. Thus, embryos become arrested late in the G(2) stage of the second embryonic cell cycle when stressed by physiological osmolarity in the absence of organic osmolytes.     

Osmotic stress,glucose transport capacity and consequences for glutamate overproduction in Corynebacterium glutamicum

Glucose uptake by Corynebacterium glutamicum is predominantly assured by a mannose phosphotransferase system (PTS) with a high affinity for glucose (Km=0.35 mM). Mutants selected for their resistance to 2-deoxyglucose (2DG) and lacking detectable PEP-dependent glucose-transporting activity, retained the capacity to grow on media in which glucose was the only carbon and energy source, albeit at significantly diminished rates, due to the presence of a low affinity (Ks=11 mM) non-PTS uptake system. During growth in media of different osmolarity, specific rates of glucose consumption and of growth of wild type cells were diminished. Cell samples from these cultures were shown to possess similar PTS activities when measured under standard conditions. However, when cells were resuspended in buffer solutions of different osmolarity measurable PTS activity was shown to be dependent upon osmolarity. This inhibition effect was sufficient to account for the decreased rates of both sugar uptake and growth observed in fermentation media of high osmolarity. The secondary glucose transporter was, however, not influenced by medium osmolarity. During industrial fermentation conditions with accumulation of glutamic acid and the corresponding increase in medium osmolarity, similar inhibition of the sugar transport capacity was observed. This phenomenon provokes a major process constraint since the decrease in specific rates leads to an increasing proportion of sugar catabolised for maintenance requirements with an associated decrease in product yields.     

Effect of osmotic pressure on neurogenesis in cultures of chich embryo spinal cords.

The osmotic pressure of the medium in stoppered, roller tube cultures increased by an average of 17 +/- 6 mOsM per kg of water during 3 days of incubation at 37 degrees C irrespective of the initial osmolality (280 to 340 mOsM) of the medium. The increase was apparently due to evaporation of water from the medium into the gas phase of the roller tube. This observation led us to study the effect of osmotic pressure on neuronal differentiation in cultures of chick embryo spinal cords. Spinal cords were excised from stage 16 to 19 (2.5 to 3 days of incubation) or stage 36 (10 days) chick embryos and cultured as fragments on collagen-coated cover slips in roller tubes at 37 degrees C for 21 days. The medium was adjusted to 283 +/- 3,300 +/- 3,323 +/- 3, or 342 +/- 3 mOsM per kg with saturated choline chloride solution or distilled water. The results indicate that the nature of the neuronal differentiation in vitro was not altered by the osmolality of the medium. The proportion of cultures containing neurons was influenced by osmolality. In the 300 +/- 3 mOsM medium, 75% of all the stage 36 cultures initiated contained neurons, and 52% of all the stage 16 to 19 cultures initiated contained neurons. In the other media the proportion of neuron-containing cultures was lower. Two conclusions were drawn. Neurogenesis in cultures of embryonic chick spinal cord fragments is sensitive to an increase in the initial osmotic pressure of the medium as small as 20 mOsM above the optimal 300 mOsM. As a result of the 17 mOsM increase which always occurred in the culture medium between feedings, the optimum osmolality for neuronal development is in fact a range, from 300 to 317 mOsM.     

A comparison of phlorizin and phloretin adsorption by the tapeworm Hymenolepis diminuta

Phloretin and phlorizin adsorb to the tegument surface of Hymenolepis diminuta, with KDs of 2.39 mM and 14.7 microM, respectively, and Vmaxs of 1446 and 12.54 nmoles/g tissue per 2 min, respectively. Phloretin adsorption is not inhibited by phlorizin or glucose. Glucose partially inhibits phlorizin adsorption. Phlorizin, but not phloretin, adsorption to isolated tegument brush border membrane preparations is partially inhibited by N-ethylmaleimide. No indications of phlorizin hydrolysis to phloretin during incubation with H. diminuta were obtained. The data are supportive of spacially separate and distinct binding sites for phloretin and phlorizin in the tegument brush border.