Tegumental Phosphodiesterase SmNPP-5 Is a VirulenceFactor for Schistosomes�
Rita Bhardwaj, Greice Krautz-Peterson, Akram Da’dara, Saul Tzipori, and Patrick J. Skelly*Molecular Helminthology Laboratory, Division of Infectious Diseases, Department of Biomedical Sciences, Tufts University,
Cummings School of Veterinary Medicine, Grafton, Massachusetts 01536
Received 23 May 2011/Returned for modification 20 June 2011/Accepted 1 August 2011
The intravascular trematode Schistosoma mansoni is a causative agent of schistosomiasis, a disease thatconstitutes a major health problem globally. In this study we cloned and characterized the schistosometegumental phosphodiesterase SmNPP-5 and evaluated its role in parasite virulence. SmNPP-5 is a 52.5-kDaprotein whose gene is rapidly turned on in the intravascular parasitic life stages, following invasion of thedefinitive host. Highest expression is found in mated adult males. As revealed by immunofluorescence analysis,SmNPP-5 protein is found prominently in the dorsal surface of the tegument of males. Localization byimmuno-electron microscopy illustrates a unique pattern of immunogold-labeled SmNPP-5 within the tegu-ment; some immunogold particles are scattered throughout the tissue, but many are clustered in tight arrays.To determine the importance of the protein for the parasites, RNA interference (RNAi) was employed to knockdown expression of the SmNPP-5-encoding gene in schistosomula and adult worms. Both quantitative real-timePCR (qRT-PCR) and Western blotting confirmed successful and robust gene suppression. In addition, thesuppression and the ectolocalization of this enzyme in live parasites were evident because of a significantlyimpaired ability of the suppressed parasites to hydrolyze exogenously added phosphodiesterase substratep-nitrophenyl 5�-dTMP (p-Nph-5�-TMP). The effects of suppressing expression of the SmNPP-5 gene in vivowere tested by injecting parasites into mice. It was found that, unlike controls, parasites whose SmNPP-5 genewas demonstrably suppressed at the time of host infection were greatly impaired in their ability to establishinfection. These results demonstrate that SmNPP-5 is a virulence factor for schistosomes.
Blood-dwelling worms of the genus Schistosoma are thecausative agents of schistosomiasis, a tropical disease that isprevalent in Africa, South America, the Arabian Peninsula,China, the Philippines, and Indonesia (14). The disease infectsover 200 million people globally, and over 600 million live atrisk of infection (34). Schistosomiasis mansoni is characterizedby the presence of adult worms within the mesenteric venousplexus of an infected host. The disease is characterized clini-cally by chronic hepatic and intestinal fibrosis, portal hyper-tension, and anemia (14).
Adult schistosomes, living as male-female pairs, can survivefor many years in the vasculature and appear recalcitrant toelimination by the immune system. The worm surface likelyplays an important role in immune evasion, as it is a site ofintimate host-parasite interaction. This surface is structurallyunique and is multilaminate in appearance (21). In one modelof fine tegument morphology, the surface is interpreted as anapical plasma membrane that is covered by a laminate secre-tion called the membranocalyx (35). Specialized membranousbodies called multilamellar vesicles (MLVs) are synthesized incell bodies that lie beneath the peripheral muscle but connectto the tegument through thin cytoplasmic connections. TheseMLVs migrate from the cell bodies to the tegument properand have been reported to fuse with the tegumental apical
plasma membrane to contribute material to the overlyingmembranocalyx (35).
Using proteomics, the major protein components of thetegumental membranes of Schistosoma mansoni have beenidentified (6–9). These proteins belong to a variety of classes,including nutrient transporters, receptors, and enzymes andseveral proteins of unknown function. In these proteomic stud-ies, a putative phosphodiesterase was identified. The proteinwas not only greatly enriched in the tegument but was availablefor surface biotinylation of adult worms, highlighting its sur-face exposure (8). Its presence had been earlier implied by thedetection of phosphodiesterase activity in schistosome tegu-mental extracts (11). Recently, the cDNA encoding this en-zyme was cloned and characterized and its sequence was shownto exhibit greatest homology with sequences of members of thenucleotide pyrophosphatase-phosphosdiesterase 5 (NPP-5)family (26).
In this work we show that production of this protein (des-ignated SmNPP-5) is rapidly upregulated at the time of defin-itive host invasion. We confirm the surface localization ofSmNPP-5 and find that it displays a unique localization patternin the tegument as revealed by immuno-electron microscopy(immuno-EM). We show that suppressing SmNPP-5 gene ex-pression by the use of RNA interference (RNAi) impairs theability of larval schistosomes to establish infection in vivo,revealing this molecule to be important for parasite virulence.
MATERIALS AND METHODS
Parasites and mice. The Puerto Rican strain of Schistosoma mansoni was used.Schistosomula were prepared from cercariae that were released from infectedsnails, and those were cultured in Basch medium (lacking red blood cells [rbcs])
* Corresponding author. Mailing address: Molecular HelminthologyLaboratory, Division of Infectious Diseases, Department of Biomedi-cal Sciences, Tufts University, Cummings School of Veterinary Med-icine, Grafton, MA 01536. Phone: (508) 887-4348. Fax: (508) 839-7911.E-mail: [email protected].
at 37°C, in an atmosphere of 5% CO2, as described previously (2). These schis-tosomula were used in the RNAi work described below. Adult male and femaleparasites were recovered by perfusion from Swiss Webster mice that had beeninfected with 125 cercariae 7 weeks previously. Adult worms were maintained inBasch medium (lacking rbcs). Parasite eggs were isolated from infected mouseliver tissue (15). Miracidia were recovered from the eggs and transformed tosporocysts that were cultured for 24 h, as described previously (15). Snailsinfected with a single miracidium each were obtained from Fred Lewis, Biomed-ical Research Institute, Rockville, MD. Cercariae emerging from these snails areall of one sex, either male or female. Cercariae from individual snails were usedto infect mice; 7 weeks later, worms of a single sex were recovered from the miceby perfusion. These single-sex adult worms were used exclusively in developmen-tal expression analysis and not for any RNAi work.
Cloning SmNPP-5. Proteomic analysis of the S. mansoni tegument revealed aphosphodiesterase homolog (S. mansoni ORESTES database accession numberC606073.1) (7). BLAST interrogation of the S. mansoni genome (version 3) withthis sequence led to contig 0018606. Using this sequence as a guide, the predicted5�-most exon was extended in silico to a potential initiator methionine. Next,using oligonucleotides designed from the predicted 5� untranscribed region(5�UTR) just upstream of this methionine (PDE1f [5�-GTTATCGAAAAGCCAGTCGTAG-3�]) and the 3�UTR downstream of the last predicted exon(PDE3r [5�-TGGCAACAACAATTCATTCATTAG-3�]) together with adultparasite cDNA in a PCR, we amplified and then sequenced the completeSmNPP-5 coding DNA at the Tufts University Core Facility.
Anti-SmNPP-5 antibody production. NH2-TLKNKGAHGYDPDYK-COOH,a peptide comprising SmNPP-5 amino acid residues 354 to 369, was synthesizedby Genemed Synthesis, Inc., San Antonio, TX. A cysteine residue was added atthe amino terminus to facilitate conjugation of the peptide to bovine serumalbumin (BSA). Approximately 500 �g of the peptide-BSA conjugate mixed withFreund’s complete adjuvant was used to immunize two New Zealand Whiterabbits subcutaneously. The rabbits were given booster treatments of 100 �g ofpeptide alone in incomplete Freund’s adjuvant 20, 40, and 60 days later. Ten daysafter the last treatment, serum was recovered from both rabbits and pooled, andanti-SmNPP-5 antibodies were subjected to affinity purification using a peptide-ovalbumin conjugate and dialyzed against phosphate-buffered saline (PBS), aspreviously described (28).
SmNPP-5 gene expression analysis. The levels of expression of the SmNPP-5gene in different life stages of the parasite and in parasites treated with gene-specific small interfering RNAs (siRNAs) were measured by quantitative real-time PCR (qRT-PCR), using a custom TaqMan gene expression system (AppliedBiosystems, Carlsbad, CA). First, parasite samples were lysed by the addition of50 �l of cell disruption buffer (PARIS kit; Ambion, Austin, TX). Samples werehomogenized on ice using an RNase-free pestle for �1 min, and the parasitehomogenate was split into two halves. One half was used for isolating RNA andthe other for protein analysis. RNA was isolated from the parasite homogenateby the use of the PARIS kit per the manufacturer’s guidelines. Residual DNAwas removed by DNase digestion using a TurboDNA-free kit (Applied Biosys-tems). cDNA was synthesized using 1 �g of RNA, an oligo(dT)20 primer, andSuperScript III reverse transcriptase (Invitrogen, CA). The levels of expressionof the SmNPP-5 gene at different life stages were measured by qRT-PCR usingthe housekeeping gene encoding triose phosphate isomerase as the endogenouscontrol (17).
Primer sets and reporter probes labeled with FAM (6-carboxyfluorescein;Applied Biosystems, Carlsbad, CA) were used for qRT-PCR. To detectSmNPP-5 expression, the following primers and probe were used: primersSmPhosphod-F (5�-GGACGATTATTGCTGACAGAACGT-3�) and SmPhos-phod-R (5�-TGGAGACATCTCTTTGTAATCTGGATCA-3�) and probeSmPhosphod-M2 (5�-FAM-TTTATTTTTCAGGGTTATCCC-3�). Each qRT-PCR was performed using cDNA equivalent to 10 ng of total parasite RNA (ina final volume of 25 �l) according to the manufacturer’s PCR protocol foruniversal conditions. All samples were processed in triplicate and underwent 40amplification cycles on a StepOnePlus real-time PCR system instrument. Forquantifications, the ��Ct method was employed (20) and the gene encodingschistosome tubulin was used as the within-stage, endogenous control (13).
Western blot analysis. To monitor expression of the SmNPP-5 protein, para-site samples were first homogenized on ice in ice-cold cell disruption buffer andprotein content was measured using a BCA protein assay kit (Pierce Biotech-nology, Rockford, IL) according to the manufacturer’s instructions. Five micro-grams of protein from each sample was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions andblotted using a polyvinylidene difluoride (PVDF) membrane. The membranewas then incubated in a 5% skim milk–PBS solution containing 0.1% Tween 20(PBST) for 1 h at room temperature. Next, the membrane was probed overnight
at 4°C with affinity-purified rabbit anti-SmNPP-5 antibody at 1:200. After themembrane was washed twice in PBST, bound primary antibody was detectedusing horseradish peroxidase-labeled anti-rabbit IgG (GE Healthcare, NJ) (1:5,000) and a TMB membrane peroxidase system (from Kirkegaard and PerryLaboratories Inc., Gaithersburg, MD), following the instructions of the manu-facturers. The membrane was exposed to X-ray film and images were capturedusing a Kodak Image Station 2000RT system. To monitor protein loading perlane, a duplicate gel was stained with Coomassie brilliant blue.
Phosphodiesterase activities in live schistosomula and in schistosomulumextracts. To measure phosphodiesterase activity in live parasites, �500SmNPP-5-suppressed and control schistosomula (processed in triplicate)were incubated in assay buffer (50 mM Tris-HCl buffer [pH 8.9], 120 mMNaCl, 5.0 mM KCl, 60 mM glucose) containing 0.5 mM phosphodiesterasechromogenic substrate p-nitrophenyl 5�-dTMP (p-Nph-5�-TMP) (Sigma-Al-drich, MO), as described previously (26). Changes in optical density at 405nm were monitored continuously for �3 h.
To prepare schistosomulum extracts, parasites were harvested at 0, 2, and 4days after siRNA treatment, washed three times with PBS, and homogenized onice in ice-cold cell disruption buffer (Paris kit; Ambion) (30 �l). Protein contentwas measured using the BCA protein assay kit as described above. Schistoso-mulum extract (3 �g in 100 �l of phosphodiesterase assay buffer) was used in theenzyme assay, following the protocol described above.
SmNPP-5 immunolocalization. Adult worm sections 7 �m thick were obtainedusing a cryostat and fixed in cold acetone. Immunofluorescent detection ofSmNPP-5 was carried out using affinity-purified rabbit anti-SmNPP-5 antibodydiluted 1:25 and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen),essentially as described earlier (29). Control parasite sections were treated withsecondary antibody alone.
Immunogold labeling and electron microscopy. Freshly perfused adult para-sites were fixed overnight with 2% glutaraldehyde–0.1 M cacodylate buffer at4°C. The samples were then dehydrated in a graded ethanol series and theninfiltrated and embedded in LR White acrylic resin (London Resin Company).Ultramicrotomy was performed using a Leica Ultracut R ultramicrotome, andthe sections were collected on gold grids. Grids were immunolabeled in a two-step process according to the following procedure. The grids were conditioned inPBS three times for 5 min each time at room temperature, followed by blockingof nonspecific labeling for 30 min at room temperature using 5% nonfat drymilk–PBS. After rinsing, the grids were exposed to primary antibody diluted1:30 for 1 h at room temperature followed by washing in PBS, then incubatedwith secondary antibody diluted 1:30 (10-nm-diameter-gold-particle-labeledgoat anti-rabbit IgG [H&L; GE Healthcare]) for 1 h at room temperature,and finally rinsed thoroughly in water. Control parasite preparations weretreated with secondary antibody alone. Grids were exposed to osmium vaporand/or lightly stained with lead citrate to improve contrast and were examinedand photographed using a Philips CM 10 electron microscope at 80 kV.
RNA interference. Adult worms were treated either with a synthetic siRNAtargeting SmNPP-5 (SmNPP-5 siRNA1 [5�-TTGATGGATTTCGTTATGATTACTTTG-3�]) or with control siRNAs. Control siRNAs were used in two forms.The first siRNA control type targets unrelated schistosome genes by the use ofschistosome siRNA control 1 (5�-AAACAGCATACACCAATTTATTTGGCT-3�), which targets the SmATPdase 1 gene (GenBank accession numberAY323529), and schistosome siRNA control 2 (5�-AAGAAATCAGCAGATGAGAGATTTAAT-3�), which targets the SmAP gene (GenBank accession num-ber EU040139). The second siRNA control type targets no sequence in theschistosome genome (5�-CTTCCTCTCTTTCTCTCCCTTGTGA-3�). Deliveryof siRNAs to the parasites was performed by electroporation as described pre-viously, using 10 �g of each siRNA (18, 22). Gene suppression was assessedposttreatment as described above by comparing mRNA levels (using qRT-PCR)and protein levels (by Western blotting and enzyme activity measurements) intarget versus control groups.
Infection of mice with siRNA-treated schistosomula. One-day-old culturedschistosomula were electroporated with either SmNPP-5, or control or nosiRNA. Some parasites were then used to infect female Swiss Webster mice byadding �1,000 schistosomula to 100 �l of RPMI medium without phenol red andinjecting the inoculum into the thigh muscle of the animals using a 1-ml tuber-culin syringe and a 25-gauge, 1-in needle (19). The remaining parasites werecultured in vitro to allow an assessment of gene suppression levels at differenttime points. Three different infection protocols were followed. In protocol 1,parasites were used to infect mice immediately after RNAi treatment (day 0) andworms were recovered by vascular perfusion 28 days later. In protocol 2, para-sites were maintained in culture for 2 days prior to being washed and countedand used to infect mice. Those worms were recovered 14 days after infection. Inprotocol 3, parasites were maintained in culture for 4 days before being washed
and counted and used to infect mice. These worms were recovered 28 days afterinfection. All recovered worms were counted and examined under a light micro-scope; subsequently, their SmNPP-5 gene expression levels were determined byqRT-PCR, as described earlier. To compare parasite sizes, images of the wormswere captured using an inverted microscope (TH4-100; Olympus, Tokyo, Japan)equipped with a Retiga 1300 camera (Q Imaging, Surrey, British Columbia,Canada), and the area occupied by each individual parasite was measured usingImageJ 1.41 software (U.S. National Institutes of Health, Bethesda, MD).
Statistical analysis. For data generated by qRT-PCR and for the determina-tions of enzyme activity in worm extracts, one-way analysis of variance(ANOVA) and Tukey’s honestly significant difference test were used for post hocanalysis. To analyze the live worm enzyme activity data, two-way repeated-measurement ANOVA was used. To assess worm recovery and worm size data,one-way ANOVA, Student’s t test, or the Mann-Whitney test was used, asappropriate. In all cases, P values � 0.05 were considered significant.
Nucleotide sequence accession number. The GenBank accession number forSmNPP-5 is EU769293.
RESULTS
SmNPP-5 sequence analysis. The cloned SmNPP-5 cDNApotentially encodes a 458-amino-acid protein with a molecularweight (MW) of 52,563 and a pI of 6.28. This sequence issimilar to that of a cDNA independently cloned by Rofatto etal. (26) but extends the 5� end by 9 amino acids. This encom-passes the 27-amino-acid, N-terminal potential signal peptidesequence 1MYCIETMQKMIILLLICFFPYIERIYA27 (perSignalP 3.0 software [http://www.cbs.dtu.dk/services/SignalP-3.0/]). The sequence identity of SmNPP-5 to animal homologs(e.g., humans [ENPP5, GenBank accession no. AAQ88878]and in the sea anemone Nematostella vectenis [GenBank ac-cession no. XP001641023]) is 29 to 35%; the sequence identityis somewhat lower (24%) to a Saccharomyces cerevisiae enzyme(GenBank accession no. NP009955). In SmNPP-5, the pro-posed catalytic site 84TLTFPSH90 is conserved, as are aminoacids reported to be important in metal binding (D45, D207,H211, D255, H256, and H362). The protein has a single predictedtransmembrane domain at the carboxyl terminus (436LSIIFIIKFIILSIFMV452). The 355TLKNKGAHGYDPDYK369 pep-tide was used to generate anti-SmNPP-5 antibodies.
SmNPP-5 developmental expression. The developmental ex-pression of SmNPP-5 was examined in several schistosome lifestages by qRT-PCR, and the results are shown in Fig. 1A. Ofthe various life stages tested, the relative gene expression ofSmNPP-5 was negligible in eggs, cercariae, and sporocysts. Inessence, the gene appears to be turned on following invasionof the definitive host, with relatively high expression in theintramammalian life stages (schistosomula and adults) andhighest expression in the adult male parasites. Females fromsingle-sex infections (Fig. 1A, lane F�) expressed largeramounts of SmNPP-5 than females from a mixed infection(Fig. 1A, lane F).
Concurrent findings were observed at the protein level, asdetermined by Western blotting and shown in Fig. 1B.SmNPP-5 was detected at about its predicted size (�55 kDa)(arrow, Fig. 1B) in extracts of 14-day cultures of schistosomula(Som14) and all adult male and female worms (from bothmixed and single-sex infections). SmNPP-5 protein was notdetected in egg and cercarial (Cer) extracts, and levels werebarely detectable in freshly transformed schistosomula (Som0).The strip of a Coomassie brilliant blue-stained duplicate gel(Fig. 1B, bottom panel) demonstrates that protein was presentin all lanes.
Localization of SmNPP-5 in adult tissues. Figure 2A showsthe immunolocalization pattern of SmNPP-5 in a section of anadult schistosome pair. It is clear that the protein is promi-nently expressed in the tegument of males. The most intensestaining is seen in the dorsal surface (arrow, Fig. 2A). Local-ization of SmNPP-5 by immunogold electron microscopy (Fig.2B and C) confirmed that the protein is distributed on the hostinteractive tegumental membrane. Two distinctive patterns ofimmunogold particle localization within the tegument—scat-tered and clustered—are discernible. Scattered individual im-munogold particles are seen distributed widely throughout thesection. In addition, clusters of from 5 to 20 or more immu-nogold particles are also widely apparent (arrowheads, Fig. 2Band C). The inset shows a cluster at higher magnification(white arrow, Fig. 2C). The top left inset (Fig. 2C, black arrow)is interpreted to represent a cluster dispersed at the parasitesurface. Parasites treated with secondary antibody alone dem-onstrated no tissue staining (data not shown).
SmNPP-5 gene suppression. SmNPP-5 gene expression wastargeted for suppression in adult parasites in vitro by introduc-tion of a specific siRNA via electroporation. Figure 3A showsthe robust (�95%) suppression of SmNPP-5, measured byqRT-PCR, 8 days after treatment. Western blotting demon-strated that the siRNA treatment also resulted in a substantialdiminution in SmNPP-5 protein production (Fig. 3B). Notethat, in this instance, SmNPP-5 resolves as two bands: a dom-inant lower band of about the expected size of SmNPP-5 (�55kDa) (arrow, Fig. 3B) and an upper, fainter band of lowerelectrophoretic mobility (arrowhead, Fig. 3B). That fainterband likely represents a posttranslationally modified (perhaps
FIG. 1. Developmental expression of SmNPP-5. (A) SmNPP-5gene expression determined by qRT PCR. The following developmen-tal stages were examined: egg, cercaria (Cer), sporocyst (Spo), 7-day-cultured schistosomulum (Som), adult male (M), adult female (F),adult male from a single-sex infection (M�), and adult female from asingle-sex infection (F�). (B) SmNPP-5 protein expression determinedby Western analysis. Protein extracts from egg, cercaria (Cer), freshlytransformed schistosomulum (Som0), 14-day-cultured schistosomulum(Som14), adult male (M), adult female (F), adult male from a single-sexinfection (M�), and adult female from a single-sex infection (F�) wereprobed with anti-SmNPP-5 antibody. The arrow indicates the positionof the SmNPP-5 protein (�55 kDa). The bottom panel shows a strip ofthe polyacrylamide gel stained with Coomassie brilliant blue to illus-trate the presence of parasite protein in each lane.
gylcosylated) SmNPP-5 variant. Notably, lower levels of bothforms of the SmNPP-5 protein were detected in extracts fromSmNPP-5 siRNA-treated parasites (left lane, Fig. 3B) versuscontrols (middle and right lanes, Fig. 3B) at 8 days posttreat-ment. The lower panel in Fig. 3B shows a fragment of a Coo-massie brilliant blue-stained polyacrylamide gel, distant fromthe location of SmNPP-5, to illustrate that all lanes containedroughly equivalent amounts of parasite protein. The robustsuppression of SmNPP-5 did not result in any detectablechange in schistosomulum or adult parasite morphology orbehavior. However, live schistosomula whose SmNPP-5 ex-pression was suppressed by RNAi treatment, unlike controls(treated with the irrelevant control siRNA or left untreated),had a significantly diminished ability to cleave the exogenouslyadded, synthetic phosphodiesterase substrate p-Nph-5�-TMP(P � 0.05) (Fig. 3C).
Effect of SmNPP-5 gene suppression on parasites in vivo. Toinvestigate whether RNAi-mediated gene silencing ofSmNPP-5 had any impact on the parasites in vivo, we infectedmice with 1-day-old SmNPP-5-suppressed or control schisto-somula. Using protocol 1, mice were infected at �1 h afterRNAi treatment. At 28 days postinfection, worm burdens werecompared across the groups; the data are shown in Fig. 4A. Inthis experiment, no significant differences in worm burdenwere found in the SmNPP-5-suppressed group compared toeither control group (Fig. 4A). Furthermore, no phenotypicdifferences between the worms from each group were ob-served. SmNPP-5 gene expression analysis was undertaken us-ing the worms recovered from the infected mice and the par-asites that had been maintained in vitro for 7 or 28 days.SmNPP-5-suppressed parasites cultured for 7 days exhibitedclose to 100% suppression of SmNPP-5 (Fig. 4B, left panel).Even after 28 days in culture, the mRNA levels in theSmNPP-5 siRNA-treated worms were still markedly (�90%)suppressed (Fig. 4B, middle panel). In contrast, parasites re-
covered from the vertebrate host were no longer suppressed;SmNPP-5 transcript levels had returned to control levels (Fig.4B, right panel).
Using an alternative RNAi treatment and infection protocol(protocol 2), schistosomula were maintained in culture for 2days after RNAi treatment before we introduced them intomice. Those mice were perfused 14 days later to recover anyworms present. A total of 125 parasites were recovered fromthe control group versus 72 parasites from the SmNPP-5knockdown group. Since the numbers of individual worms permouse were not counted, no statistical analysis can be per-formed on those data. However, the sizes of the parasites fromboth groups of mice were compared (Fig. 5A). The mean sizeof the parasites from the SmNPP-5-suppressed group was sig-nificantly less than that of parasites from the control group(P � 0.05). SmNPP-5 gene expression was compared in theworms recovered from the infected mice versus parasites thathad been maintained in vitro for the same time period (14days). As Fig. 5B shows, parasites maintained for 14 days invitro exhibited almost 100% target gene suppression (Fig. 5B,left pair of bars) whereas SmNPP-5-suppressed parasites re-covered from the infected mice now exhibited �75% suppres-sion (Fig. 5B, right pair of bars). This amount is still substantialcompared to controls but is significantly less than that seen inthe parasites maintained in culture for the entire experiment(P � 0.05).
Using a final RNAi treatment and infection method (proto-col 3), schistosomula were cultured for longer (4 days) afterRNAi treatment before introducing them into mice, whichwere perfused 28 days later. Data from two experiments per-formed using this protocol yielded essentially the same results.As shown in Fig. 6A and B, using protocol 3, there was asignificant reduction in worm burden in the SmNPP-5-sup-pressed group compared to the burden seen with a controlgroup treated with an irrelevant siRNA (Fig. 6A; 1 � 0.8
FIG. 2. Immunolocalization of SmNPP-5 in adult parasites. (A) Cross-section through a male-female couple, showing strong tegumentalimmunofluorescent staining with anti-SmNPP-5 antibody (arrow). (B and C) Electron micrographs of the adult tegument, showing immunogoldlabeling of SmNPP-5. Arrowheads indicate clustered localizations of gold particles in the tegument. Higher-magnification images highlight clusterswithin the tegument (white arrow, inset in panel C) and at the surface (black arrow, upper left in panel C). Numbers above scale bars representmicrometers. Immunogold particles are 10 nm in diameter.
worms versus 20 � 6 worms [P � 0.05]) or to that seen with acontrol group that had received no siRNA treatment (Fig. 6B;0.4 � 0.2 worms versus 6 � 3 worms [P � 0.05]). We detectedno significant difference in the mean size of the small numberof SmNPP-5 siRNA-treated parasites recovered from miceversus the mean size of control parasites. Next, the expressionlevels of SmNPP-5 in the recovered worms were compared. Asnoted above, the SmNPP-5-suppressed parasites that had beencultured for 28 days still exhibited profound (�95%) genesuppression (Fig. 6C, left pair of bars). In contrast, the smallnumber of parasites recovered from infected mice after 28 dayswere no longer suppressed; SmNPP-5 transcript levels hadeffectively returned to control levels (Fig. 6C, right pair ofbars).
In final experiments, we sought to determine whether the 3different RNAi protocols used in this work had any bearing onthe level of target enzyme activity at the time of infection.Therefore, phosphodiesterase activity assays were carried outusing parasite extracts generated at 0, 2, and 4 days post-RNAitreatment; these data are shown in Fig. 7. It is clear that activityincreased in the control parasite group (gray bars) after day 0,with day 2 and 4 parasites exhibiting approximately twice theactivity of their day 0 counterparts. This finding corroboratesthe Western blot studies described above (Fig. 1B), which
demonstrated SmNPP-5 expression increasing as cercariaetransformed and schistosomula matured. Figure 7 (left panel)demonstrates that enzyme activity in the day 0 suppressed-parasite extract did not differ significantly from the corre-sponding control value. In contrast, at day 2 (Fig. 7, middlepanel), the controls exhibited about twice the activity of theirsuppressed counterparts (P � 0.05). It is clear that SmNPP-5gene suppression prevented the surge in activity seen in thecontrol day 2 schistosomula. In a similar vein, extracts of day 4SmNPP-5-suppressed schistosomula exhibited low enzyme ac-tivity compared to the day 4 controls (P � 0.05) (Fig. 7, rightpanel). The suppressed day 4 worms exhibit only �30% of theenzyme activity of controls.
DISCUSSION
Schistosomes are globally successful intravascular parasites.Our laboratory seeks to understand how the molecules ex-pressed at the host-interactive surface contribute to their suc-cess (27). Among the enzyme activities detected at the schis-tosome surface is that of phosphodiesterase, which was firstdescribed in a study of the schistosome tegument publishedover 30 years ago (11). A cDNA encoding such an enzyme wasrecently cloned and designated SmNPP-5 (26). We have inde-
FIG. 3. RNAi suppression of SmNPP-5. (A) Relative SmNPP-5 gene expression levels (means � standard errors [SE]) in adult parasites 8 daysafter electroporation with SmNPP-5 (white bar) or control 1 or control 2 siRNAs (gray bars). The asterisk signifies a statistically significantdifference between the SmNPP-5-suppressed group and either the control 1 or control 2 group (P � 0.05). (B) SmNPP-5 protein expressionanalysis. SmNPP-5 protein was detected in adult worm extracts obtained 8 days following treatment with the indicated siRNA (top panel).SmNPP-5 resolves here as two bands, a prominent band at �55 kDa (arrow) and a minor band of lower relative mobility (arrowhead). Both formsof SmNPP-5 are diminished in extracts of SmNPP-suppressed parasites, as shown in the left lane. The positions of migration of molecular massmarkers are shown at right (kilodaltons). The lower panel shows a strip of the gel stained with Coomassie brilliant blue to illustrate roughlyequivalent protein loadings in each lane. (C) SmNPP-5 enzyme activity in live schistosomula. Changes in optical density at 405 nm (OD405) withtime in solution containing live schistosomula incubated with chromogenic phosphodiesterase substrate p-Nph-5�-TMP 8 days after treatment withSmNPP-5 or with irrelevant siRNA (control) or in the absence of siRNA treatment. The asterisk signifies a statistically significant differencebetween the SmNPP-5-suppressed group and either control group from the 30-min time point onward (P � 0.05).
pendently cloned this cDNA, and here we confirm and extendprevious observations. Sequence analysis catalogs SmNPP-5 asa type 1 transmembrane protein; it is predicted to be a single-pass transmembrane protein, with its N terminus, and themajority of the protein, being external to the cell.
Expression of the SmNPP-5 gene accompanies cercarialtransformation, which coincides with vertebrate host invasion.This suggests that the protein performs a function for theintravascular worms. Maximal SmNPP-5 expression is seen inthe mature adults—particularly males from a mixed-sex infec-tion. Upon comparing SmNPP-5 gene expression results de-termined for adult parasites from single-sex infections, wefound that females from a single-sex infection expressed �3times more SmNPP-5 than females from a mixed infection.Mated females reduced their expression of SmNPP-5, whereasmales increased theirs. This suggests that the male “takes over”this function upon mating, perhaps permitting the mated fe-male to divert the resources necessary to express SmNPP-5 toother tasks, notably expression of egg-laying genes. The sexualdimorphism of schistosome adults suggests that adult malesand females have separate and distinctive functions in vivo, amajor role for males being, e.g., to transport the female, andfor females (lying in the male’s gynecophoric canal) being, e.g.,to produce and release eggs (3). However, the precise functionof SmNPP-5 is not known and the advantage to the schisto-some couple for the male to begin to monopolize this activityis unclear.
In adult schistosome sections, the tegument stains stronglywith anti-SmNPP-5 antibodies, demonstrating that the proteinlocalizes there, including at the host-parasite interface. Thistegumental localization of SmNPP-5 has been reported byother research groups using proteomic approaches (7, 33) andimmunofluorescence localization (26); also, as noted earlier,phosphodiesterese activity in tegument-enriched fractions of
FIG. 4. Schistosome recovery from infected mice following SmNPP-5 suppression. In protocol 1, parasites were injected on day 0 after RNAitreatment and recovered at day 28. (A) Mice were injected intramuscularly (i.m.) with schistosomula on day 0 after treatment with SmNPP-5 orcontrol (Control) or no (None) siRNA. Mice were perfused 28 days later, and worm burdens were measured. Each dot represents the worm burdenfrom a single mouse, and the lines indicate the means for each group. (B) Expression of SmNPP-5 (means � SE) in schistosomula at different timesafter treatment with SmNPP-5 siRNA (white bars) or after treatment with control or no siRNA (gray bars). Parasites were maintained in culturefor 7 days after treatment (left panel) or for 28 days after treatment (middle panel) or were recovered from infected mice 28 days after treatment(right panel). Each asterisk signifies a statistically significant difference between the SmNPP-5-suppressed group and either control group (P �0.05).
FIG. 5. Schistosome recovery from infected mice followingSmNPP-5 suppression. In protocol 2, parasites were injected on day 2after RNAi treatment and recovered at day 14. (A) Schistosomulawere injected i.m. into mice on day 2 after treatment with SmNPP-5 orcontrol siRNA, and mice were perfused 14 days later. Parasite size wasmeasured using ImageJ 1.41 software. Each dot represents the size ofan individual parasite recovered from the mice in the given group. Thelines indicate the medians for each group. (B) Expression of SmNPP-5(means � SE) in schistosomula 14 days after treatment with SmNPP-5(white bars) or control (gray bars) siRNAs. Parasites either were main-tained in culture (left pair of bars) or were recovered from infectedmice (right pair of bars). Each asterisk signifies a statistically significantdifference between the SmNPP-5-suppressed group and the controlgroup (P � 0.05).
adult worms was previously reported (10, 11, 23, 25). Thelocalization of SmNPP-5 as determined using immunogoldelectron microscopy and reported here confirms and extendsthose data. We noted two patterns of immunogold particlelocalization—clustered and scattered—in the tegument. Whilescattered immunogold particles have been seen in other im-munolocalization experiments involving tegumental molecules(4, 16, 17), the report of the clustered pattern presented hereis, to our knowledge, unique. We speculate that the clusteredparticles overlay membranous bodies within the tegumentcalled multilaminate vesicles (MLV). These structures are dif-ficult to visualize in sections chemically prepared for im-muno-EM analysis. MLVs have been proposed to play animportant role in surface membrane formation and turnover(27). In one model of tegument morphology, the surface (mul-tilaminate in appearance) is interpreted as an apical plasmamembrane that is covered by a laminate secretion called themembranocalyx (35). MLVs have been reported to fuse with
the apical plasma membrane to contribute material to theoverlying membranocalyx (35). If, as we propose, SmNPP-5enzymes are clustered within MLVs, much of the enzymewould be delivered into the membranocalyx to participate in-timately in host-parasite interactions. Immunogold labeling iscertainly apparent at the host-parasite interface, and SmNPP-5can clearly access exogenous substrate. Suppressing expressionof SmNPP-5 by the use of RNAi greatly impedes the ability oflive worms to cleave a synthetic phosphodiesterase substrateadded to the medium. However, the idea of placing SmNPP-5in the membranocalyx is at odds with recent work involving theidentification of schistosome tegumental proteins that can beremoved from live parasites by external application of theprotease trypsin (9). In that work, a number of tegumentalproteins, perhaps located in the membranocalyx, are accessedby trypsin, but not SmNPP-5 (9). Thus, the definitive localiza-tion of SmNPP-5 within the tegumental membranes requiresfurther experimental analysis.
To investigate the importance of SmNPP-5 for schistosomes,gene expression was suppressed using RNAi. IntroducingsiRNAs targeting SmNPP-5 by electroporation resulted in veryrobust (�95%, at the RNA level) gene suppression in adultparasites and schistosomula. Robust suppression at the proteinlevel, as determined by Western blotting and by comparativeenzyme activity assays, was also observed. Despite this finding,suppressing SmNPP-5 gene expression did not cause any visi-ble morphological or behavioral change in the SmNPP-5-sup-pressed versus control parasites even after prolonged culturefor up to 28 days. This was not because the RNAi effect woreoff in the cultured parasites; even after 28 days in vitro,SmNPP-5 gene expression levels were �95% lower than thecontrol value. This suggests that SmNPP-5 does not play animportant role for schistosomes in vitro.
In order to test the effects of knocking down expression ofthe SmNPP-5 gene in vivo, we first followed a protocol in whichschistosomula were used to infect mice immediately aftersiRNA treatment (12, 19, 24, 32). Using this method (which wedesignate “protocol 1”), no differences in worm burden in thetest versus control groups were found after 28 days and no
FIG. 6. Schistosome recovery from infected mice following SmNPP-5 suppression. In protocol 3, parasites were injected on day 4 after RNAitreatment and recovered at day 28. Note that panels A and B show data from replicate infection experiments in which the control groups differed.(A) Schistosomula were injected i.m. into mice on day 4 after treatment with SmNPP-5 or control siRNA. Mice were perfused 28 days later, andworm burdens were measured. Each dot represents the worm burden from a single mouse, and the lines indicate the means for each group.(B) Schistosomula were injected i.m. into mice on day 4 after treatment with SmNPP-5 or in the absence of siRNA treatment (None). Mice wereperfused 28 days later, and worm burdens were measured. Each dot represents the worm burden from a single mouse, and the lines indicate themeans for each group. (C) Expression of SmNPP-5 (means � SE) in parasites 28 days after treatment with SmNPP-5 (white bars) or in the absenceof siRNA treatment (gray bars). Parasites either were maintained in culture (left pair of bars) or were recovered from infected mice (right pairof bars). The asterisk signifies a statistically significant difference between the SmNPP-5-suppressed group and the control group (P � 0.05).
FIG. 7. Relative percent Nph-5�-TMP (phosphodiesterase sub-strate) cleavage activity (means � SE) in schistosomulum extracts atdifferent time points (day 0, day 2, and day 4) after treatment withSmNPP-5 (white bars) or control (gray bars) siRNAs. Each asterisksignifies a statistically significant difference between the SmNPP-5-suppressed group and the control group (P � 0.05).
obvious morphological differences between the recoveredworms from the different groups were seen. Furthermore, thelevels of expression of the SmNPP-5 gene in the worms recov-ered from infected mice were all similarly high. In other words,the SmNPP-5-suppressed parasites overcame the suppressionin vivo, in contrast to their counterparts that were maintainedin culture. We speculate that the rapid growth and metabolicvigor of schistosomes in an infected animal, compared with thegenerally observed stunting of the parasites maintained in cul-ture, may contribute to the ability of the parasites within miceto overcome gene suppression. In support of this notion, it waspreviously reported that rapid cell multiplication can quicklydilute an RNAi effect (1). Since suppressing SmNPP-5 had noimpact on schistosomes introduced into mice, this may meanthat (as is the case in vitro) the gene plays no essential role forthe worms in vivo. However, we reasoned that, as a result of theuse of protocol 1, the parasites may have been introduced intothe mice before gene suppression was well established and thatthis allowed the worms that were growing in vivo to overcomeany incipient RNAi effect. We inferred, therefore, that main-taining the suppressed parasites in vitro for several days wouldallow mRNA and protein levels to decline and permit us tobetter gauge the real importance of SmNPP-5.
Using a new procedure (protocol 2), RNAi-treated parasiteswere first cultured for 2 days after treatment before they wereintroduced into mice. The mice were perfused 14 days later. Incontrast to protocol 1, this protocol led to a clear and demon-strable gene suppression effect on the recovered parasites. Thetest group showed a reduced total number of parasites com-pared to the control group, and worms that were recoveredfrom the test group were generally smaller in size than thosefrom the control group. Thus, impeding SmNPP-5 function inthis case impaired parasite development. In addition, expres-sion of the SmNPP-5 gene in worms recovered from the testgroup remained low (�75% lower than the expression levelseen with control parasites). Unlike the SmNPP-5-suppressedworms recovered following implementation of protocol 1 asdescribed above, here the SmNPP-5-suppressed worms hadnot effectively overcome the RNAi effect at the 14-day timepoint.
Our final gene suppression and infection method (protocol3) differed from the earlier version in that parasites were cul-tured for longer (4 days) prior to infection and were recoveredfrom mice 28 days later. Using this protocol, we saw the mostdrastic differences between the different groups in worm re-covery results. Very few parasites were recovered from theSmNPP-5-suppressed group (a mean of �10-fold fewer thanwere recovered from the control groups). In other words, whenprotocol 3 was implemented, SmNPP-5-suppressed schistoso-mula were significantly impaired in their ability to establishinfection. This outcome demonstrates that SmNPP-5 is an im-portant virulence determinant for schistosomes.
To test the hypothesis underpinning these infection experi-ments, namely, that retaining parasites in culture after RNAitreatment significantly diminishes SmNPP-5 enzyme activitycompared to that seen with controls, we set out to establish thetime dependency of suppression in schistosomula. Studies wereundertaken to determine the levels of phosphodiesterase sub-strate cleavage activity in extracts of control and test parasitesat day 0, day 2, and day 4 after siRNA exposure. The results
validate the hypothesis. As expected, there were no significantdifferences in enzyme activity in extracts of control or RNAi-treated parasites that were tested immediately after siRNAexposure. In contrast, at day 2 (and more so at day 4), thedifferences in enzyme activity between suppressed and controlparasites were stark. Therefore, when suppressed parasitesthat have been maintained in culture are later injected intomice, they display substantially less enzyme activity than con-trols. We argue that this enzymatic impairment is responsiblefor the decreased ability of the suppressed worms to establisha vigorous infection.
As noted earlier, following implementation of protocol 3,the level of SmNPP-5 suppression remained substantial in cul-tured parasites even after a month in vitro. In contrast, inequivalent parasites recovered from infected mice 4 weeksafter RNAi treatment, the SmNPP-5 gene was no longer sup-pressed. The few parasites that survived in vivo had SmNPP-5mRNA levels that had returned to control levels. A possibleexplanation for this observation is that RNAi is variably effec-tive in different parasites and/or that different individuals in thetreated parasite population received different amounts ofsiRNA. Those in which SmNPP-5 knockdown was least effec-tive, or that received less double-stranded RNA (dsRNA),would have survived because expression of their SmNPP-5gene was minimally impaired. Another possibility is that someworms are more metabolically robust in vivo and that this leadsto a shorter half-life of the dsRNA and/or its downstreameffectors.
What is the function of SmNPP-5, an essential gene productfor infecting schistosomes? We proposed earlier that tegumen-tal phosphodiesterase may participate with other schistosometegumental phosphatase homologs in the catabolism of extra-cellular nucleotides (5). However, sequence analysis of thistegumental enzyme shows that it clearly belongs to the pyro-phosphatase/phosphosdiesterase-5 (NPP-5) family (26), andmembers of this family have not been reported to metabolizenucleotides (30, 31). In agreement with this sequence analysis,in our unpublished work, we found that SmNPP-5-suppressedparasites, though impaired in their ability to cleave the syn-thetic substrate p-nitrophenyl 5�-dTMP, are not impaired intheir ability to degrade exogenous ATP, ADP, or AMP (datanot shown). No natural substrate of any member of the NPP-5family has been identified to date (30, 31); thus, the function ofSmNPP-5 for schistosomes has not yet been determined.
Only by maintaining the suppressed parasites in vitro prior tousing the parasites to infect mice was the importance ofSmNPP-5 made clear. However, we and other groups haveshown an impact of gene suppression in schistosomes by in-troducing parasites immediately after RNAi treatment (i.e.,using protocol 1) (12, 19, 24). This apparent discrepancy maybe due to differences in the ability of parasites to recover fromsuppression of different gene targets and/or due to the highersusceptibility of schistosomula to a diminution of a particulartarget gene product during the initial phase of infection.
In summary, we report here the characterization of the S.mansoni tegumental enzyme SmNPP-5. The SmNPP-5 gene israpidly upregulated following host invasion. The protein ex-hibits an intriguing clustered distribution in the tegument asrevealed by immuno-EM localization. Suppressing SmNPP-5gene expression impairs the ability of living schistosomes to
cleave exogenous phosphodiesterase substrate but exerts noovert morphological effect on the worms. This illustrates thatSmNPP-5 is not essential for schistosomes in culture. In con-trast, parasites whose SmNPP-5 gene is demonstrably sup-pressed at the time of host infection are greatly impaired intheir ability to establish infection. This demonstrates thatSmNPP-5 is a virulence factor for schistosomes. While almostcertainly involved in some intimate aspect of host parasiterelations, the natural substrate of this surface enzyme and itsprecise molecular function await discovery.
ACKNOWLEDGMENTS
This work was supported by grant AI-056273 from the NationalInstitutes of Health—National Institute of Allergy and Infectious Dis-eases (NIH-NIAID).
Schistosome-infected snails were provided by the Biomedical Re-search Institute through NIH-NIAID contract HHSN272201000009I.We thank Chuck Shoemaker for helpful discussions, Phyllis Mann forassistance with statistical analysis, and John Nunneri for help withelectron microscopy.
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