Proc. Natl. Acad. Sci. USA Vol. 93, pp. 3642-3646, April 1996 Biology

Rapid appearance and asymmetric distribution of glucose transporter SGTP4 at the apical surface of intramammalian-stage Schistosoma mansoni (tegument//schistosome) PATRICK J. SKELLY AND CHARLES B. SHOEMAKER Department of Tropical Public Health, Harvard School of Public Health, Boston, MA 02115 Communicated by Elkan Blout, Harvard Medical School, Boston, MA, December 20, 1995 (received for review June 4, 1995)

ABSTRACT Adult Schistosoma mansoni blood flukes re- in the various membranes of the tegument, including the side in the mesenteric veins of their vertebrate hosts, where -interactive surface membrane. As such, serum-exposed they absorb immense quantities of glucose through their schistosome GTPs may represent candidate vaccine targets tegument by facilitated diffusion. Previously, we obtained S. because they are probably essential for worm survival and mansoni cDNAs encoding facilitated-diffusion schistosome accessible to immune attack. glucose transporter 1 and 4 (SGTP1 and SGTP4) and We have previously obtained cDNA clones encoding schis- localized SGTP1 to the basal membranes of the tegument and tosome GTPs 1 and 4, (SGTP1 and SGTP4), and characterized the underlying muscle. In this study, we characterize the their function in Xenopus oocytes (8). Recently, we also expression and localization of SGTP4 during the schistosome showed that SGTP1 is located in the basal, but not the apical, life cycle. Antibodies specific to SGTP4 appear to stain only membranes of the tegument, as well as in muscle, suggesting the double-bilayer, apical membranes of the adult parasite that this functions to transport glucose from within the tegument, revealing an asymmetric distribution relative to the tegumental syncytium to the underlying tissues (9). Here we basal transporter SGTP1. On living worms, SGTP4 is avail- report that SGTP4 is detected in the host-interactive outer able to surface biotinylation, suggesting that it is exposed at tegument, asymmetrically distributed relative to SGTP1. Fur- the hose-parasite interface. SGTP4 is detected shortly after thermore, we demonstrate that SGTP4 appears during the the transformation of free-living, infectious cercariae into transformation of free-living cercariae into schistosomula, schistosomula and coincides with the appearance of the coincident with the development of the double-bilayer mem- double membrane. Within 15 min after transformation, anti- brane in the maturing schistosomula. Our results indicate that SGTP4 staining produces a bright, patchy distribution at the SGTP4 can serve as a unique surface membrane marker with surface of schistosomula, which becomes contiguous over the which to study the turnover and fate of this critical, host- entire surface of the schistosomula by 24 hr after transfor- interactive membrane and suggest that SGTP4 may provide an mation. SGTP4 is not detected in earlier developmental stages effective target for a schistosomiasis vaccine. (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 MATERIALS AND METHODS cycle and specifically localized within the host-interactive, Parasites. A schistosome life cycle was maintained with S. apical membranes of the tegument. mansoni (Puerto Rican strain)-infected Biomphalaria glabrata snails. Adult worms were obtained from C57 mice, 6-8 weeks Schistosoma mansoni is a parasitic platyhelminth that causes after infection with 50 cercariae each, by perfusing the portal the chronic, often debilitating disease, schistosomiasis. Adult blood vessels with 0.85% NaCl containing 0.75% sodium worms reside in the mesenteric veins of their mammalian hosts, citrate. For some experiments, male and female worms were where they are generally found, in copula, as male/female separated by teasing them apart in RPMI 1640 medium. pairs. The entire worm is surrounded by a continuous cyto- Cercariae and schistosomula were prepared as described (10). plasmic unit, or syncytium, called the tegument. The host- Schistosomula were cultured for various periods in RPMI 1640 interactive surface of the tegument is unusual in that it consists medium/20 mM Hepes, pH 7.5/gentamicin (100 ,tg/ml) in a of two tightly apposed, bilayer membranes that are highly 5% C02/95% air incubator. To obtain samples of sporocysts, invaginated. The internal, basal membrane of the tegument infected snail hepatopancreas tissue was recovered by dissec- consists of a normal (trilaminate) bilayer containing many tion. S. mansoni eggs were isolated from the livers of infected dilations. This bilayer extends periodically beneath the under- mice as described (11). lying muscle to enclose areas called "cell bodies," which Membrane Preparation and Gel Electrophoresis. Mem- contain nuclei and protein synthetic machinery (1-3). brane preparations of different life stages were prepared as Adult worms use large quantities of host glucose, reportedly described (12). Proteins were resolved by SDS/PAGE (13), consuming the equivalent of their dry weight every 5 h (4). and Western blot analysis was performed as detailed (14). Despite the possession of a functional gut, the adult parasites Generation of Anti-SGTP Antisera. DNA encoding the take up glucose across their external body surface or tegument largest predicted intracellular loop domain of SGTP1, from aa by facilitated diffusion (5-7). Little is known about the process 198 to 281, was amplified from SGTP1 cDNA by PCR. The by which schistosomes transport glucose from the host serum synthetic oligonucleotides lfx3 (5'-CCCGGATCCTCATTG- across the complex tegumental syncytium and into their bod- GTTACTTTACCG-3') and lfx4 (5'-CCGAATTCTGCAGC- ies. Precedent would suggest that one or more facilitated- TATTGTTGTAAAACTTGTA-3') were used with a cloned diffusion glucose transporter proteins (GTPs) are distributed SGTP1 cDNA as a template under reaction conditions de-

The publication costs of this article were defrayed in part by page charge Abbreviations: GTP, glucose transport protein; SGTP1 and -4, schis- payment. This article must therefore be hereby marked "advertisement" in tosome GTP 1 and 4, respectively; FITC, fluorescein isothiocyanate; accordance with 18 U.S.C. §1734 solely to indicate this fact. TRITC, tetramethylrhodamine B isothiocyanate. 3642 Downloaded by guest on September 29, 2021 Cell Biology: Skelly and Shoemaker Proc. Natl. Acad. Sci. USA 93 (1996) 3643 scribed in ref. 10. The 277-bp PCR product was digested with Biotinylation of Living Adult Worms. Living adult worms BamHI and PstI and ligated into the similarly digested vector were labeled with sulfosuccinimidobiotin (Pierce) as described pMAL-cRI (New England Biolabs). A maltose binding pro- (17). Surface membrane-enriched preparations were obtained tein/SGTP1 fusion protein was induced from the resulting by incubating the labeled worms in 0.1 M Tris-HCl, pH 7.4/12 expression plasmid, VA2-1, and purified by amylose affinity as mM sucrose/0.1% digitonin for 30 min at 4°C with gentle recommended by the manufacturer (New England Biolabs). shaking as described (18) following by centrifugation of the The purified fusion protein was used to generate antisera in a digitonin extract at 100,000 x g for 1 h at 4°C. The pellet was rabbit (East Acres Biologicals, Southbridge, MA). resuspended in 0.5 M NaCl/20 mM Tris-HCl, pH 8.0/0.1% A peptide (NH2-CQRPSRLTYKNEEPFYSDE-COOH) SDS/1% Triton-X 100/1 mM phenylmethylsulfonyl fluoride was synthesized (provided by Charles Dahl, Harvard Medical precipitation buffer. Streptavidin-agarose (25 tul of a 50% School) based on the sequence at the C terminus of SGTP4 slurry; Pierce) was added to -30 ,ug of the protein solution, corresponding to aa 487-505. The synthetic peptide was con- which was incubated overnight with rocking at 4°C. Beads were jugated separately to both bovine serum albumin and ovalbu- washed five times with the precipitation buffer, and bound min with m-maleimidobenzoyl-N-hydroxysuccinimide ester as protein was eluted by boiling the beads in SDS sample buffer described in ref. 15. Rabbits were immunized with the bovine before SDS/PAGE. serum albumin-coupled peptide as described (9). Antiserum against the control protein paramyosin (Pmy) was raised as described (16). RESULTS Antibody Purification. Anti-SGTP antibodies were purified SGTP4 and SGTP1 Expression During the Schistosome Life by affinity to their respective immunogens using either the Cycle. Antisera were generated against the large, predicted maltose binding protein/SGTP1 fusion protein or the SGTP4 internal loop of SGTP1 and against a peptide corresponding peptide conjugated to ovalbumin. About 1 mg of each was to the C terminus of SGTP4. The specific anti-SGTP1 and separately coupled to a 1-ml N-hydroxysuccinimide-activated anti-SGTP4 antibodies were purified from these antisera by HiTrap column (Pharmacia) as recommended by the manu- antigen-affinity chromatography. The resulting antibody prep- facturer. Two to 10 ml of undiluted serum was passed through arations specifically recognize the appropriate SGTP on West- the columns, which were washed extensively with phosphate- ern blots containing these proteins expressed by recombinant buffered saline (PBS), pH 7.4. Bound antibodies were eluted insect cells, but the preparations show no recognition for the in 0.1 M glycine (pH 2.5), immediately neutralized with other SGTP (not shown). Tris-HCl (pH 8.0), and finally dialyzed against PBS. Western blot analysis was used to compare the expressions Immunocytochemistry. Light microscope immunofluores- of SGTP4 and SGTP1 in schistosome eggs, sporocysts (within cent detection of SGTPs in whole schistosomula or adult worm the hepatopancreas tissue of infected snail hosts), cercariae, sections (fixed in acetone for 5 min) was done essentially as and adult male and female worms (Fig. 1A). SGTP1 protein described earlier for adult sections (9). Dual labeling was is detected within membrane extracts of all life cycle stages performed with biotinylated anti-SGTP4 and anti-SGTP1 tested. In contrast, SGTP4 is not found in eggs, sporocysts, and antibodies. To biotinylate anti-SGTP4 antibodies, 100 ,tg of cercariae, but is clearly seen in adult males and females. the purified antibodies was incubated in 0.1 M NaHCO3 with Because SGTP4 is detected only in the intramammalian stage 120 jtg biotin succinimide ester (Biosearch) in dimethyl sul- of the life cycle, it was of interest to establish the timing of its foxide for 4 h at room temperature, then dialyzed against initial expression. Western blot analysis shows that SGTP4 PBS/0.02% sodium azide overnight. Sections were first incu- rapidly appears within the first hour of transformation while bated with anti-SGTP1 antibodies (200 ,tg/ml in PBS), which the levels of SGTP1 (Fig. 1B) and other proteins (data not were detected with tetramethylrhodamine B isothiocyanate- shown) remain largely unchanged. conjugated donkey anti-rabbit IgG (H+L) F(ab')2s (Jackson Localization of SGTP4 in Adult Schistosomes. SGTP4 was ImmunoResearch). Next, biotinylated anti-SGTP4 antibodies localized within adult worms by indirect immunofluorescence (20 tLg/ml in PBS) were incubated with the sections and these on parasite sections, and the staining pattern was compared antibodies were detected using a 1:300 dilution of a strepta- with that obtained for SGTP1. As reported (9), anti-SGTP1 vidin-fluorescein isothiocyanate (FITC) conjugate (Pierce). antibodies stain the tegument and the underlying musculature Sections were washed three times in PBS/2% fetal calf serum (Fig. 2A). In contrast, bright staining with anti-SGTP4 anti- for 10 min each between steps. Sections were viewed using a bodies is limited to the tegument (Fig. 2B). The tegumental Nikon Diaphot and a Molecular Dynamics Sarastro 2000 staining for both SGTP1 and SGTP4 is consistently bright in confocal laser scanning microscope fitted with a 25-mW all examined sections, indicating that both transporters are argon-ion laser set at 495-nm or 552-nm excitation. Optical evenly distributed in females and males along their entire sections 0.5 ,im thick were collected with a x60 objective. lengths and on both the dorsal and ventral sides. A B

SGTPI SGTP4 SGTP1v SGTP4 FIG. 1. Developmental expression of F M S1 Su C E F M S1 Su C E 0 1 0 1 SGTP1 and SGTP4. Western blots contain 97 - 97 - schistosome membrane preparations resolved 68 - 68 - by SDS/15% PAGE. SGTP proteins are de- tected by use of purified anti-SGTP1 or anti- 43- 43 - SGTP4 antibodies. (A) SGTP1 and SGTP4 expression during the parasite life cycle. F, female; M, male; Si, hepatopancreas from 29 - infected snails; Su, hepatopancreas from un- infected control snails; C, cercariae; E, eggs. 29 - (B) SGTP1 and SGTP4 expression in newly transformed schistosomula. Schistosomula were harvested 0 h (0) or 1 h (1) after culture in RPMI 1640 medium. The arrows indicate 18 - 18- the SGTP proteins. The positions of molec- ular mass markers are indicated (kDa). Downloaded by guest on September 29, 2021 3644 Cell Biology: Skelly and Shoemaker Proc. Natl. Acad. Sci. USA 93 (1996) Pmy SGTP4 1 2 1 2

200 - 97- --.

68 -...... FIG. 3. SGTP4 is surface ex- 43 - .'ii-.l posed and available for biotiny- lation. Western blots contain teg- umental extracts of biotinylated worms resolved by SDS/10% 29 - PAGE. Proteins were detected with anti-paramyosin (Pmy) or anti-SGTP4 antibodies. Lanes contain either the total extract (lane 1) or the streptavidin- 18- bound portion of the extract (lane 2). The positions of molec- ular mass markers are indicated 14- (kDa). readily biotinylated when labeling is done with permeabilized worms (17). In contrast, biotinylated SGTP4 was easily de- tected in the extracts, indicating that a significant portion of this protein is exposed at the host-parasite interface (Fig. 3, SGTP4). Immunofluorescence Detection of SGTP4 During Schisto- somula Transformation. Expression and localization studies suggest that SGTP4 is exclusively localized within the rapidly induced, double-bilayer membranes, so it was of interest to FIG. 2. Immunolocalization of SGTP1 and SGTP4 in adult schis- analyze SGTP4 localization during the transformation process. tosomes. All sections are of adult S. mansoni labeled with the indicated Immediately after transformation, the schistosomula show a antibody and viewed by light fluorescence microscopy. For A-D, low internal fluorescence when stained with anti-SGTP4 an- detection was with FITC-conjugated anti-rabbit F(ab')2s. For C-E, tibodies (Fig. 4A, 0 h). Beginning after 15 min of incubation images were obtained by confocal microscopy and printed with and continuing for several hours, bright patches of fluores- artificial color. For E, SGTP1 was detected with anti-SGTP1 antibod- ies and TRITC-conjugated anti-rabbit F(ab')2s (red) while SGTP4 was detected with biotinylated anti-SGTP4 antibodies and a FITC- A B streptavidin conjugate (green). Differences in the tegumental staining for SGTP1 and SGTP4 become more apparent when the sections are exam- ined at higher magnification using confocal microscopy. The basally located SGTP1 (9) is seen beneath the tegumental surface, whereas SGTP4 is seen as a bright line of fluorescence at the surface of the section (Fig. 2 C and D). To confirm the distinct localization of the two transporters, double staining of worm sections was undertaken. SGTP1 was first stained with anti-SGTP1 antibodies followed by detection with TRITC- conjugated anti-rabbit immunoglobulin. SGTP4 was then stained with biotinylated anti-SGTP4 followed by FITC- labeled streptavidin for detection. Sections were simulta- neously scanned at 495 nm (for FITC detection) and at 552 nm (for TRITC detection). As seen in Fig. 2E, SGTP1 is detected as the inner, reddish band of fluorescence, and SGTP4 is seen as a clear green line that includes the external surface of the worm. This localization suggests that SGTP4 in present on the outer, apical membranes of the tegument, asymmetrically distributed relative to the basal localization of SGTP1. To test whether SGTP4 is surface exposed, living adult worms were labeled with sulfosuccinimidobiotin (17) and washed, and the apical membranes and tegumental cytosol were selectively extracted by a short treatment with digitonin (18). Western blot analysis indicated, as expected, that most of the SGTP4 but only a small fraction of the basal transporter FIG. 4. Immunofluorescent detection of SGTP4 in schistosomula. SGTP1 is present within the digitonin extracts not (A) Immunofluorescent detection of SGTP4 in optical sections (data the center of were shown). Surface extracts contained detectable levels of through schistosomula that cultured for 0 (0 h), 1 easily (1 h), or 24 h h) in RPMI 1640 medium. Immunolocalization the control nonsurface none is de- (24 (B) protein paramyosin yet of SGTP4 in an optical section at the surface of a schistosomulum tected in the biotinylated proteins (Fig. 3, Pmy), suggesting that cultured for 1 h in RPMI 1640 medium. The largely unstained anterior biotinylation of internal proteins did not occur. Paramyosin is of the parasite is at top. (Bar = 5 gm for A and B.) Downloaded by guest on September 29, 2021 Cell Biology: Skelly and Shoemaker Proc. Natl. Acad. Sci. USA 93 (1996) 3645 cence are visible on the surface of the schistosomula (Fig. 4A, basal membrane of the tegument, containing SGTP1, is a 1 h, and B). The patches often appear to exist in concentric typical bilayer membrane, the host-interactive outer surface of rows spaced 2-10 ,um apart and are estimated to number the tegument, containing SGTP4, is unique. Specifically, this 500-1000 on each schistosomulum. The anterior of the para- apical membrane consists of a double lipid bilayer that is site is unstained at 1 h of incubation (Fig. 4B). The surface produced only during the intramammalian stage of the schis- fluorescence coalesces to a bright, continuous band by 24 h tosome life cycle and is constantly exposed to the potentially after transformation (Fig. 4A, 24 h). This band extends around hostile environment of host serum. It seems likely that SGTP4 the entire parasite, including the previously unstained anterior. is specifically adapted to successfully accommodate the traf- ficking requirements needed to enter these host-exposed DISCUSSION apical membranes. Cercariae, the freshwater stage of S. mansoni that is infec- Adult schistosomes, residing in the vertebrate bloodstream, tious to vertebrates, are covered by a single bilayer membrane transport glucose across their syncytial tegument by facilitated that lacks SGTP4. The membrane is itself covered by an outer, diffusion (5-7, 19). To date, two different functional glucose dense, fibrillar coat called the . The formation of the transporters, SGTP1 and SGTP4, have been identified in double-bilayer membrane commences upon penetration of schistosomes and shown to have overall similarity in their cercariae into the vertebrate host and temporally correlates sequence (60%), substrate specificity, and kinetic properties with the appearance of SGTP4. The transformation process (8). However, the localizations of SGTP1 and SGTP4, as involves the migration of membranous structures from the revealed in this study, are quite distinct and indicate that these cell bodies beneath the muscle layers to the surface are involved in the of tegumental proteins centrally process glucose of the parasite, where they fuse to form patches of a multi- transport through the tegument. laminate outer membrane At the same the are in a (3). time, original SGTP1 and SGTP4 distributed distinctly asymmet- cercarial trilaminate membrane and the are cast off. ric manner in the tegument of adult worms. SGTP1 is confined glycocalyx to the basal membrane and its dilations whereas most Electron microscopy demonstrates that within 60 min after (9), a outer to with some transformation, significant portion of the tegumental SGTP4 immunolocalizes exterior SGTP1 expo- membrane is multilaminate The membranous sure at the host-parasite interface. In a separate study, im- already (3). munolocalization by electron microscopy has confirmed the structures continue to be synthesized by the underlying cell presence of SGTP4 in the tegument apical membrane (both bodies and transported to the surface until they eventually bilayers) but revealed no evidence of SGTP4 in the basal cover the entire developing schistosomula. membranes (20). This distribution suggests that SGTP4 trans- The appearance of anti-SGTP4 antibodies binding to the ports glucose from the glucose-rich host serum into the surface of the maturing schistosomula coincides with the tegument and that SGTP1 then transports a proportion of this appearance of the double-bilayer membrane. Although no glucose to the underlying tissues of the parasite. It is note- surface binding is detected in newly transformed schistoso- worthy that both of the S. mansoni glucose transporters are mula, within 15 min of cercarial transformation, a patchy found evenly distributed throughout the tegumental surfaces surface fluorescence is seen, spaced at intervals of 2-10 Jtm; (ventral and dorsal) of males and females, and this finding this fluorescence eventually covers the entire parasite. SGTP4 implies that both sexes have the ability to take up glucose protein is rapidly synthesized during this time and can easily be throughout the tegument. Neither of the two SGTPs is de- detected in schistosomula membrane preparation after 1 h. We tected in the adult gut or the caecum of schistosomula, sites propose that SGTP4 is present within, and specific to, the thought not to be involved in glucose transport (5). emerging double-bilayer membrane and that the patches seen Asymmetrically distributed glucose transporters have been in the early transforming parasites represent upwellings of observed in other biological systems in which sugar must cross membrane from single cell bodies. If so, this result implies that multiple membranes. In some cases, a single transporter is between 500 and 1000 cell bodies are space 2-10 gm apart in distributed in different concentrations at opposing cell sur- schistosomula, consistent with the reported estimate of a 5-10 faces. For example, the glucose transporter GLUT1 appears ,tm spacing between cell bodies in adults (26). The appearance asymmetrically distributed at the mammalian blood-brain of membrane is synchronous throughout the developing par- barrier (21), on the three membranes of the rat placenta asite because the patches detected with the anti-SGTP4 anti- separating the maternal and fetal bloodstreams (22), and on bodies simultaneously appear over the entire surface of the the plasma membranes of the eye epithelial cells (23). In these schistosomula (with the notable exception of the anterior of instances, it is speculated that different concentrations of the parasite). The rapid appearance of GTP at the surfaces of glucose transporter in the opposing membranes help to main the invading schistosomula may reflect the need for urgent a glucose gradient that promotes the movement of sugar across glucose uptake to replenish depleted sugar reserves and ensure the different membranes. More analogous to the schistosome the survival of the parasites within their new mammalian hosts. tegument, asymmetric distribution of two different glucose transporters is seen in intestinal and renal cells that are involved in absorbing glucose in the gut or reabsorbing glucose We thank Dr. John Caulfield and Dr. John Samuelson for their in the But these differ from that of helpful discussions during the experiments, Dr. Ed Pearce for sharing kidney (24, 25). examples unpublished data, and Hae Jin Kim for excellent technical assistance. SGTP1 and SGTP4 in that glucose is being absorbed from We thank Dr. Don Harn and Dr. Tao Liang-Feng for providing environments having a variable glucose concentration and is parasites and Dr. Rick Rogers, Bruce Elstein, and Jean Lai for their mediated by Na+/glucose cotransporters in an energy- expert help with confocal microscopy. These studies were funded, in dependent process capable of moving glucose against a con- part, by National Institutes of Health Grant AI28499, United Nations centration gradient. Development Programme/World Bank/World Health Organization It remains uncertain what purpose is served by maintaining Special Program for Research and Training in Tropical Diseases, and an asymmetric distribution of two different facilitated glucose the John and Catherine MacArthur Foundation. transporters in the schistosome tegument, particularly in light of the fact that SGTP1 and SGTP4 have similar apparent 1. Silk, M. H., Spence, I. M. & Gear, J. H. S. (1969) S. Afr. J. Med. substrate specificities and kinetic properties when expressed in Sci. 34, 1-10. Xenopus oocytes (8). However, because of the distinct nature 2. Smith, J. H., Reynolds, E. S. & von Lichtenberg, F. (1969) Am. of the schistosome tegument, two different transporters may J. Trop. Med. 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