Suppression of mRNAs Encoding Tegument Tetraspanins from Schistosoma mansoni Results in Impaired Tegument Turnover
et al. (2010) Suppression of mRNAs Encoding Tegument Tetraspanins from Schistosoma mansoni
Results in Impaired Tegument Turnover. PLoS Pathog 6(4): e1000840. doi:10.1371/journal.ppat.1000840
Suppression of mRNAs Encoding Tegument Tetraspanins from Schistosoma mansoni Results in Impaired Tegument Turnover
Mai H. Tran 0 1
Tori C. Freitas 0 1
Leanne Cooper 0 1
Soraya Gaze 0 1
Michelle L. Gatton 0 1
Malcolm K. Jones 0 1
Erica Lovas 0 1
Edward J. Pearce 0 1
Alex Loukas 0 1
Elisabetta Ullu, Yale University, United States of America
0 Current address: Trudeau Institute , Saranac Lake, New York , United States of America
1 1 Division of Infectious Diseases, Queensland Institute of Medical Research , Brisbane, Queensland , Australia , 2 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 3 School of Veterinary Sciences, The University of Queensland , Brisbane, Queensland , Australia
Schistosomes express a family of integral membrane proteins, called tetraspanins (TSPs), in the outer surface membranes of the tegument. Two of these tetraspanins, Sm-TSP-1 and Sm-TSP-2, confer protection as vaccines in mice, and individuals who are naturally resistant to S. mansoni infection mount a strong IgG response to Sm-TSP-2. To determine their functions in the tegument of S. mansoni we used RNA interference to silence expression of Sm-tsp-1 and Sm-tsp-2 mRNAs. Soaking of parasites in Sm-tsp dsRNAs resulted in 61% (p = 0.009) and 74% (p = 0.009) reductions in Sm-tsp-1 and Sm-tsp-2 transcription levels, respectively, in adult worms, and 67%-75% (p = 0.011) and 69%-89% (p = 0.004) reductions in Sm-tsp-1 and Sm-tsp-2 transcription levels, respectively, in schistosomula compared to worms treated with irrelevant control (luciferase) dsRNA. Ultrastructural morphology of adult worms treated in vitro with Sm-tsp-2 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls. Schistosomula exposed in vitro to Sm-tsp-2 dsRNA had a significantly thinner and more vacuolated tegument, and morphology consistent with a failure of tegumentary invaginations to close. Injection of mice with schistosomula that had been electroporated with Sm-tsp-1 and Sm-tsp-2 dsRNAs resulted in 61% (p = 0.005) and 83% (p = 0.002) reductions in the numbers of parasites recovered from the mesenteries four weeks later when compared to dsRNA-treated controls. These results imply that tetraspanins play important structural roles impacting tegument development, maturation or stability.
Funding: This work is supported by the Australian-American Fulbright Commission (MHT), Australian National Health and Medical Research Council (NHMRC)
grant #496600 to AL, National Institutes of Health (NIH) grants AI075266 and AI082548 to EJP and NIAID contract NO1-A1-30026. AL is supported by a senior
research fellowship from NHMRC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Schistosomes are parasitic trematodes that cause chronic
infection in over 207 million people in 76 developing tropical
countries. Schistosomiasis is generally associated with poverty,
poor water supply and inadequate sanitation . Infection rates
and intensities are high in early childhood, peak around 8 to 15
years and decrease in adulthood . Despite effective and
inexpensive widespread treatment with the anthelmintic drug
praziquantel for over 20 years, this parasitic disease still causes
more than 250,000 deaths per year and accounts for 1.7 to 4.5
million disability-adjusted life years (DALYs) lost annually .
Humans become infected with schistosomes when they are
exposed to free-living cercariae in fresh water. Cercariae penetrate
the skin, shed their tails and transform into schistosomula, which
reside in the dermis of the skin before entering the blood capillaries
to migrate through the vasculature to the portal venous system
where they mature into adult worms . The outer surface of
schistosomula and adult worms, the tegument, is a multinucleated
syncitium that contains tegumental cell bodies situated below the
muscular layers. During transformation from cercaria to
schistosomula, the outer surface of the tegument (the interface with the
host) is remodeled from a single membrane with a prominent
glycocalyx into an unusual double membrane (or
heptalaminate) structure . This double membrane is widely believed to
play an essential role in the ability of schistosomes to evade the
host immune system, a characteristic that allows them to live for
years within their hosts . The outer of the two surface
membranes also has the ability to adsorb host blood molecules,
masking its non-self status thereby contributing to immune evasion
and prolonged survival . We believe that tegumental proteins
are ideal targets for immunological and pharmacological
intervention . The generation of a large number of S. mansoni
expressed sequence tags  and the recently completed genome
sequence , in combination with advances in characterizing the
tegument proteome has led to the discovery of many tegument
Schistosomes, or blood flukes, reside in the blood vessels
surrounding the liver and bowel of their human hosts.
They infect 200 million people and kill many thousands
each year in developing countries. The parasites cover
themselves in a unique series of cell membranes called the
tegument. Molecules in the tegument membranes are a
major target for the development of new drugs and
vaccines against the parasite. Here we show that at least
one member of a family of tegument membrane proteins
called tetraspanins, Sm-TSP-2, is integral to the proper
formation of the tegument and subsequent survival of the
parasite in its human host, providing a potential
mechanism by which a vaccine based on Sm-TSP-2 protects
specific proteins . Among them are a group of membrane
proteins called tetraspanins, which are highly expressed in the
outer tegument membrane of adult schistosomes [12,13]. To date,
five tetraspanin cDNAs have been described from S. mansoni,
namely Sm-23 , Sm-tsp-1 and Sm-tsp-2 , Sm-tetraspanin-B
and Sm-tetraspanin-C .
Tetraspanins are a large superfamily of surface-associated
membrane proteins characterized by the conserved structure of
four hydrophobic transmembrane domains, a small and large
extracellular loop, an interconnecting intracellular loop, and
cytoplasmic amino- and carboxyl- termini . Tetraspanins
undergo post-translational modification in which palmitate is
bound to the membrane proximal cysteine residues and associates
with cholesterol-rich domains . This process enables
tetraspanins to play key roles in molecular organization of cell
membranes, interacting with one another and also specific partner
proteins such as integrins, MHC and co-stimulatory molecules to
form large signal transducing complexes termed
tetraspaninenriched microdomains (TEMs) . Tetraspanins are widely
distributed in many cell types but their physiological roles are
mostly unknown. Several lines of evidence have implicated
tetraspanins in the regulation of cell adhesion, differentiation,
motility, aggregation, cell signaling and sperm-egg fusion
[18,19,20,21]. They have been linked to various pathological
processes including lymphocyte activation , cancer ,
fertilization [23,24], and interactions between pathogens and host
cells such as HIV , HCV  and Plasmodium .
We previously identified two cDNAs, Sm-tsp-1 (Sm01494) and
Sm-tsp-2 (Sm12366), in adult S. mansoni using signal sequence
trapping , and showed that both of these tetraspanins were
expressed in the tegument of the adult parasite . Other authors
confirmed the surface expression of these tetraspanins using
various mass spectrometric approaches to characterize the
schistosome surface [11,29,30]. We expressed the large
extracellular loop of Sm-TSP-1 and Sm-TSP-2 in E. coli and used the
soluble recombinant proteins to immunize mice and then
challenged them with cercariae. Mice vaccinated with
recombinant Sm-TSP-1 and Sm-TSP-2 had significantly reduced adult
worm, liver egg and fecal egg burdens . Moreover, strong
IgG1 and IgG3 antibody responses against Sm-TSP-2 were
detected in sera of individuals deemed putatively resistant (PR)
to S. mansoni in comparison to sera from chronically infected
Despite their promise as vaccines against schistosomiasis, the
functions of Sm-TSP-1 and Sm-TSP-2 have not yet been
elucidated. We therefore employed RNA interference (RNAi) to
explore the roles of Sm-tsp-1 and tsp-2 in larval and adult S. mansoni.
RNAi has been utilized with S. mansoni to suppress endogenous
gene expression in schistosomula , adult worms , eggs 
and sporocysts . Here, we show that RNAi results in
reductions in expression of Sm-tsp-1 and tsp-2 mRNAs in
schistosomula and adult worms, and malformation of the
tegument in worms cultured in vitro. Moreover, silencing of tsp-1
and tsp-2 expression in schistosomula results in up to 90% fewer
worms maturing to adulthood when introduced into mice
compared with parasites exposed to control dsRNAs, highlighting
their essential roles in tegument biogenesis and maintenance and
further supporting the development of novel therapies targeting
these genes and their protein products.
Developmental expression of Sm-tsp-1 and Sm-tsp-2 in S.
Expression of Sm-tsp-1 and Sm-tsp-2 mRNAs in different stages
of the S. mansoni life cycle was determined relative to control
Sm-atubilin mRNA using qRT-PCR. Sm-tsp-1 and Sm-tsp-2 mRNAs
were detected in all stages of the schistosome life cycle with higher
levels identified in eggs, miracidia and cercariae than in 5-day old
schistosomula, males and female worms for tsp-1; a similar
expression profile was observed for tsp-2 but gene expression was
notably reduced in cercariae (Figure 1). Interestingly, the highest
level of Sm-tsp-1 expression was detected in cercariae whereas
Smtsp-2 expression was lowest in cercariae.
Expression of Sm-TSP-1 and Sm-TSP-2 in the tegument of
We previously demonstrated that Sm-TSP-1 and Sm-TSP-2 are
expressed on the tegument surface membrane of adult worms .
The tegument is fully formed by 3h after cercarial transformation
, so to determine whether these TSPs are expressed in the
tegument at this early stage after host entry and whether they are
accessible to antibodies on live parasites, we probed live newly
transformed schistosomula with antibodies against both proteins.
Both Sm-TSP-1 and Sm-TSP-2 were detected over the entire
surface tegument of live schistosomula when probed with mouse
anti-TSP-1 or -TSP-2 sera followed by FITC-labelled anti-mouse
IgG (Figure 2).
dsRNA-mediated knockdown of Sm-tsp expression in
Adult worms soaked for 7 days in Sm-tsp-1 dsRNA had a 61%
(p = 0.009) reduction in Sm-tsp-1 mRNA expression compared to
parasites soaked in control dsRNA (Figure 3A). A 74% (p = 0.009)
reduction in Sm-tsp-2 mRNA levels was detected in worms that
were cultured in media containing Sm-tsp-2 dsRNA compared to
parasites soaked in luciferase dsRNA (Figure 3B). Parasites were
visually monitored for motility on a daily basis but no differences
were detected between groups (not shown).
dsRNA-mediated knockdown of Sm-tsp expression in
Soaking of 3 h old schistosomula in Sm-tsp-1 dsRNA for 7, 14
and 21 days caused 75% (p,0.001), 67% (p = 0.019) and 69%
(p = 0.021) decreases in Sm-tsp-1 mRNA expression in comparison
to the control group (Figure 4A). Larval parasites incubated with
Sm-tsp-2 dsRNA for 7 days exhibited an 88% (p,0.001) decrease
in Sm-tsp-2 transcript levels compared to luciferase dsRNA treated
schistosomula (Figure 4B). RNAi knockdown was maintained with
reductions of 82% (p = 0.004) and 69% (p = 0.021) at days 14 and
21, respectively, compared to the control group. As observed in
adult worms, suppression of Sm-tsp RNAs resulted in no obvious
phenotypic differences compared to the luciferase dsRNA-treated
control group when examined by light microscopy. Cultures were
visually inspected using a light microscope on a daily basis and no
differences in early growth and development of schistosomula
(development of intestinal ceca or size of schistosomula)  were
apparent between test and control dsRNA treated groups.
Reduction in Sm-TSP2 protein expression in parasites
treated with Sm-tsp-2 dsRNA
To determine whether knockdown of Sm-tsp-2 RNA was evident
at the protein level, we performed Western blot analysis on dsRNA
treated adult (Figure 5A) and larval (Figure 5B) parasites. Parasites
were treated with Sm-tsp-2 or luciferase dsRNAs, lysed in 1% Triton
X-100 and immunoblotted with anti-Sm-TSP-2 or anti-Sm-Pmy
antibodies which target a sub-tegumental muscle protein,
paramyosin . Sm-TSP-2 protein expression was decreased in adult
worms treated with Sm-tsp-2 dsRNA compared to worms treated
with luciferase dsRNA for the four concentrations (2.0, 1.0, 0.5 and
0.25 mg) tested. In contrast, the Sm-Pmy protein expression levels
did not change in both test and control groups. The experiment
was repeated three times with similar results and a representative
image is shown (Figure 5A). Densitometry analysis was performed
on each band and the ratio of Sm-TSP-2 to Sm-Pmy at each
concentration was calculated. Analysis of whole worm lysates
(0.25 mg) by densitometry (not shown) revealed an average of 61%
(p = 0.027) reduction in Sm-TSP-2 expression in adult worms
treated with Sm-tsp-2 dsRNA compared to the control luciferase
group. For RNAi treated schistosomula, the amount of Sm-TSP-2
protein expressed by schistosomula after 7 days in culture with
Smtsp-2 dsRNA was reduced compared to parasites soaked in luciferase
dsRNA (Figure 5B). Densitometry analysis of lysates (2 mg, 1 mg
and 0.5 mg) showed an average decline of 36% (data not shown).
This decrease was lower than expected since suppression of
Sm-tsp2 mRNA was more pronounced in schistosomula than in adult
parasites. Adult and larval parasites soaked in Sm-tsp-1 dsRNA
demonstrated no obvious differences in protein expression to
luciferase dsRNA control worms by Western blotting analysis (data
Suppression of Sm-tsp-2 mRNA results in malformation of
the tegument when observed using transmission
Adult parasites and schistosomula treated with Sm-tsp-2 dsRNA
in vitro displayed modified tegument structure when visualized with
transmission electron microscopy (TEM) compared with luciferase
Figure 3. Suppression of Sm-tsp mRNAs in adult parasites by RNAi. Sm-tsp-1 (A) and Sm-tsp-2 (B) transcript levels relative to Sm-paramyosin
(mean6S.E.) in adult parasites soaked for 7 days with 1 mg/ml of Sm-tsp or luciferase control dsRNAs.
Figure 4. Suppression of Sm-tsp mRNAs in schistosomula by RNAi. Sm-tsp-1 (A) and Sm-tsp-2 (B) transcript levels relative to Sm-paramyosin
(mean6S.E.) in schistosomula soaked for 7, 14 and 21 days with 1 mg/ml of Sm-tsp or luciferase control dsRNAs.
dsRNA treated controls (Figure 6). The tegument of adult worms
incubated in vitro in Sm-tsp-2 dsRNA (Figure 6C,E) was more
highly vacuolated than luciferase dsRNA controls (Figure 6A), with
extensive and enlarged vacuoles throughout the surface layer. The
tegument of these parasites had less apparent cytoplasm and hence
fewer cytoplasmic inclusions and was frequently much thinner
than that of controls (Figure 6C,E). Schistosomula transformed
and cultured in vitro presented a tegument that resembled that of
larvae from natural or experimental infection (Figure 6B) .
The tegument in Sm-tsp-2 dsRNA treated schistosomula
(Figure 6D,F) was consistently thinner than those of luciferase
controls (P,0.001), measuring on average 0.378460.016 mm
compared with 0.584260.323 mm for luciferase controls
(Figure 6G). Volume density measures for invaginations and clear
vesicular compartments of the tegument showed higher volumes
for these compartments in Sm-tsp-2 treated schistosomula
(p = 0.014; Figure 6F). The morphology of the schistosomula
tegument was consistent with a failure to close invaginations of the
surface (Figure 6D,F). Adult worms and schistosomula soaked in
Sm-tsp-1 dsRNA showed no obvious differences to luciferase dsRNA
control worms when examined by transmission electron
microscopy (data not shown).
Suppression of Sm-tsp mRNAs in schistosomula affects
parasite survival in vivo
In the mammalian host, larval schistosomes migrate from the
skin through the lungs to the liver and then mature in the
mesenteric veins . In an effort to mimic in vivo conditions, 3 h
schistosomula were electroporated with 100 mg/ml of Sm-tsp-1,
Sm-tsp-2 or luciferase dsRNA and then injected intramuscularly
into female C57BL/6 mice. Four weeks later mice were perfused
to determine the number of parasites that reached maturity in
the mesenteries. Significantly fewer parasites were recovered
from the mesenteric veins compared to the luciferase control
group (see Figure 7A for results of three experiments). Mice
injected with schistosomula that were electroporated with
Smtsp-1 dsRNA yielded 48% (p = 0.045), 60% (p = 0.009) and 67%
(p = 0.019) reduction in the number of parasites recovered for
Experiments 1, 2 and 3, respectively in comparison to the
luciferase control group. Schistosomula pretreated with Sm-tsp-2
dsRNA and then injected into mice resulted in 70% (p = 0.039),
91% (p = 0.009) and 78% (p = 0.018) decreases in parasite
survival for Experiments 1, 2 and 3, respectively when compared
to the luciferase dsRNA group. The numbers of mature worms
harvested from the luciferase control group were very low, with
recovery ranging from 0.5-1.5%, however the data was
consistent between three experiments, with a reproducible and
significant reduction in worm recovery rates between tsp and
luciferase dsRNA treated parasites.
RNA was extracted from surviving worms that were perfused
from mice and transcript levels were analyzed by qRT-PCR.
Smtsp-1 expression was only slightly lower (17%) in worms recovered
from mice that were infected with Sm-tsp-1 dsRNA-treated
schistosomula compared to the control group. Likewise, Sm-tsp-2
expression was slightly reduced (15%) in worms recovered from
mice that were infected with Sm-tsp-2 dsRNA treated worms
compared to the luciferase control group (Figure 7B). However,
when the same batch of dsRNA electroporated schistosomula were
cultured in vitro for the same period of time (4 weeks), as opposed to
being injected into mice, significant knockdown of Sm-tsp-1 and
Sm-tsp-2 transcripts by 58% and 87%, respectively (Figure 7C),
was observed. These results illustrate that silencing of Sm-tsp-1 and
Sm-tsp-2 by either soaking or electroporation leads to suppression
of tetraspanin genes in schistosomes, and suppression is
maintained for at least 4 weeks in culture. The data also implies one of
three possible outcomes for Sm-tsp dsRNA treated schistosomula
that survived to adulthood after being transferred into mice; (1)
RNAi was not as effective in those individual schistosomula that
survived in mice as opposed to those that perished; (2) some of the
RNAi treated parasites received (or took up) less dsRNA, and
therefore the efficacy of gene suppression was variable between
individuals in a single electroporated batch; (3) it is also possible
that host developmental cues stimulate transcription.
Schistosomes express a family of tetraspanins in their tegument.
Sm23 was the first tetraspanin identified in S. mansoni , and is
of interest as a DNA vaccine antigen against schistosomiasis .
Its orthologue from S. japonicum, Sj23, protects water buffaloes
Figure 6. Ultrastructure of the tegument of parasites treated with Sm-tsp-2 dsRNA RNA observed using transmission electron
microscopy. A. Tegument of adult female treated with luciferase dsRNA. B. Tegument of schistosomulum incubated for 7 days with luciferase dsRNA.
C and E. Tegument of adult female incubated with Sm-tsp-2 dsRNA. The tegument is more highly vacuolated (C) and thinner (E) compared with
controls. D and F. Tegument of schistosomula incubated for 7 days with Sm-tsp-2 dsRNA. Digitate extensions (arrows) are more abundant on the
surface of the tegument. Abbreviations: Mus-muscles; teg-surface layer of tegument. The tegument of schistosomula were thinner, p,0.001 (G) and
more dense, p = 0.014 (H) in Sm-tsp-2 dsRNA treated schistosomula.
against challenge infection when administered as a DNA vaccine
. We identified two additional tetraspanins, Sm-tsp-1 and
Smtsp-2, which showed high levels of protection when administered to
mice as recombinant protein vaccines against S. mansoni [13,28].
However, despite the protective efficacy that these tetraspanins
afford, their functions in the parasite are unknown. To understand
the roles that these proteins play in the schistosome tegument, we
herein explored the effects of silencing the expression of Sm-tsp-1
and Sm-tsp-2 mRNAs in adult and larval S. mansoni.
RNAi has been used to suppress a number of schistosome genes
in an effort to determine their functions [40,41]. Soaking of S.
mansoni with dsRNA encoding the intestinal protease cathepsin B
(SmCB1), resulted in greater than 10-fold decrease in SmCB1
mRNA levels and significant growth inhibition compared to
parasites treated with control dsRNA . Suppression of the
mRNA encoding another intestinal protease, S. mansoni cathepsin
D (SmCD), in schistosomula by electroporation with dsRNA led to
reduction in RNA transcript levels, growth retardation in vitro and
Figure 7. Infection of mice with Sm-tsp dsRNA treated schistosomula. Schistosomula were electroporated with 100 mg/ml of Sm-tsp-1,
Smtsp-2 or luciferase dsRNAs, washed and counted. C57BL/6 female mice were immunized intramuscularly with 2,000 dsRNA treated schistosomula and
were perfused 4 weeks later to determine parasite numbers (A). Expression of Sm-tsp-1 and Sm-tsp-2 mRNA transcript levels of parasites harvested
from Experiment 1 (B). The Sm-tsp-1 and Sm-tsp-2 transcript levels of schistosomula that were electroporated and concurrently cultured in vitro for 4
weeks were also determined (C).
in vivo, and decreased cathepsin D enzymatic activity .
Silencing of the SmAQP gene encoding a water channel protein
by electroporating schistosomula with short interfering RNAs
suppressed mRNA and protein expression in the tegument, and
treated parasites cultured in vitro exhibited stunted growth and
lower viability . RNAi has been used to determine the
functional importance of tetraspanins in other organisms .
Suppression of tetraspanin-15 mRNA by feeding C. elegans with
dsRNA resulted in dissociation of the cuticle and degeneration of
the hypodermis, compromising epidermal integrity . RNAi
has also been used to determine the function of human
tetraspanins in various cell types . For example, the CD151
tetraspanin interacts with membrane proteins including the
laminin-binding integrin a3b1; when lung adenocarcinoma cells
were cultured on laminin-511 and then treated with CD151
siRNA, abnormal membrane protrusions on laminin-511 were
apparent and tyrosine phosphorylation dependent signalling was
reduced . These findings indicate a role for tetraspanins in the
maintenance of cell membrane biogenesis and structural integrity,
and support our observations on the compromised tegument
membrane formation in S. mansoni when tsp mRNA expression is
Numerous reports have documented molecular interactions
between tetraspanins and MHC, and involvement of human
tetraspanins in regulating T cell co-stimulation and peptide/MHC
presentation [48,49,50], indicating additional, non-structural roles.
Schistosomes acquire host MHC onto their surfaces ,
presenting the intriguing possibility that they function as a
receptor for host MHC. However, the majority of mammalian
tetraspanin binding partners identified to date are membrane
proteins rather than extracellular ligands ; moreover, our data
presented here implies that schistosome tetraspanins are pivotal for
proper tegument formation, even during in vitro culture in the
absence of immune cells, supporting a structural role in the
establishment and maintenance of the tegument. Indeed, the
tetraspanin CD9 complexes with numerous proteins including
Igcontaining proteins , a family of proteins which are also
present in the S. mansoni tegument membrane . Various
authors have described the contribution of tetraspanins, such as
CD9 and CD151, with members of the integrin family in
promoting cell-cell interactions and migration [53,54,55]. Mass
spectrometric analysis of the S. mansoni tegument revealed a
bintegrin subunit in the sub-tegumental layer . Suppression of
tetraspanin mRNA expression in schistosomes may affect lateral
interactions with integrins in the tegument, and the parasites
ability to migrate through the lungs to the liver and mesenteries
where they would mature. The binding partner(s) associated with
Sm-TSP-1 or Sm-TSP-2, or any of the other three S. mansoni
tegument tetraspanins, have yet to be identified. We have
produced monoclonal antibodies to Sm-TSP-2 and these
antibodies are being used to immunoprecipitate Sm-TSP-2 and its binding
partners in an effort to unravel the tegumental tetraspanin web.
To assess the viability of dsRNA treated parasites in vivo, we
injected tsp or luciferase dsRNA treated parasites into mice via the
intramuscular route . Recovery of adult worms from the
mesenteries 4 weeks later was very low but was in agreement with
other reports where newly transformed schistosomula were
electroporated with dsRNAs prior to intramuscular injection into
mice and subsequent recovery of adult worms from the
mesenteries . The natural route of S. mansoni infection is
through percutaneous penetration of cercariae; exposure of
laboratory mice to cercariae is generally performed via the
abdomen or tail. Intramuscular injection of mice with
schistosomula is not the natural infection route and consequently may
have contributed to the low recovery rates. Despite the low
recovery of adult parasites, we consistently over three experiments
recovered significantly fewer worms from the mice injected with tsp
dsRNA treated parasites. Moreover, tsp mRNA levels in those
parasites that were recovered from mice were higher than levels in
parasites cultured in vitro for the same time period after
electroporation with dsRNAs, indicating that the parasites that
survived in vivo had not succumbed to the effects of RNAi.
We envisage that interruption of Sm-TSP-1 and TSP-2
protein expression in the tegument of maturing schistosomula
results in impaired turnover of the tegument apical membrane
complex. Our observations from adults and schistosomula
treated with Sm-tsp-2 dsRNA would indicate that a likely role
for Sm-tsp-2 is in invagination and internalization of the surface
membrane, and perhaps the closure and internalization of
surface invaginations. This postulate is consistent with the
suggestion that TSP-2 binds other parasite sub-surface and
surface molecules in the tegument. The vaccine efficacy of
TSP-2 may thus result from impairment of the surface recycling
mechanisms in developing and adult schistosomes. While this
impaired surface turnover was not deleterious to in vitro
cultivated adult worms and schistosomula, the effect was
particularly marked in treated schistosomula transferred into
the host. In addition, schistosomes have the capacity to adsorb
host blood molecules that mask antigenic epitopes from the
hosts immune system . By affecting surface tegument
development and turnover, suppression of tsp expression (and
potential disruption of TEMs) may render the organism
susceptible to immune recognition and clearance.
Materials and Methods
All animals were maintained in accordance with the guidelines
of the Animal Ethics Committee (AEC) of Queensland Institute of
Medical Research and the Institutional Animal Care and Use
Committee (IACUC) of The University of Pennsylvania. All
studies and procedures were reviewed and approved by the AEC
and IACUC of Queensland Institute of Medical Research and
The University of Pennsylvania respectively.
The Puerto Rican strain of S. mansoni and Biomphalaria glabrata
snails were provided by the National Institutes of Allergy and
Infectious Diseases Schistosomiasis Resource Centre at the
Biomedical Research Institute (Rockville, Maryland, USA). B.
glabrata infected with miracidia were exposed to incandescent light
for 1h to obtain cercariae which were used to percutaneously
infect 68 week old C57BL/6 female mice (www.jax.org). After 8
weeks, adult parasites were recovered by hepatic-portal perfusion
and then washed three times with wash medium containing RPMI
1640, 1% antibiotic/antimycotic and 10 mM Hepes (www.
invitrogen.com) before experimentation.
To obtain schistosomula, cercariae were passed through a
22gauge emulsifying needle 25 times to mechanically shear the
cercarial tails from the bodies . The resulting schistosomula
were isolated from free tails by centrifugation through a 60%
percoll gradient , washed three times with washing medium
and incubated at 37uC under 5% CO2 atmosphere before
Immunofluorescent labelling of live schistosomula
Three hour schistosomula (n = 500) were blocked in blocking
buffer containing 1% goat serum in Dulbeccos Phosphate
Buffered Saline (DPBS) containing MgCl2 and CaCl2 (www.
invitrogen.com). Schistosomula were labelled with sera against
recombinant Sm-TSP-1, Sm-TSP-2 or control pre-vaccination sera
 diluted to 1:50 in blocking buffer for 1 h. Secondary goat
anti-mouse Ig-FITC (www.chemicon.com) was then introduced at
1:100 dilution in blocking buffer for 1 h followed by 4%
paraformaldehyde to fix the parasites. Incubations were carried
out at 4uC and parasites were washed in DPBS between
incubations. Approximately 200 schistosomula were examined
using a Leica MRIRB microscope and DC500 camera (www.leica.
Synthesis of dsRNAs
dsRNAs were prepared from DNA templates that were
amplified by PCR from S. mansoni paired adult worm cDNA
using primers flanked with T7 RNA polymerase promoter
sequence (underlined) at the 59 ends. A 523 bp fragment of the
Sm-tsp-1 coding DNA was generated using primers (forward:
59-TAATACGACTCACTATAGGGTTCGAAAGCTGCAATAGAAACA-39) and a 565 bp fragment of the
Smtsp-2 coding DNA was produced using primers (forward:
59-TAATACGACTCACTATAGGGGACCAATGCGAACAGAAACA-39). The GenBank accession numbers for
Smtsp-1 and Sm-tsp-2 are AF521093 and AF521091, respectively. The
PCR products were then utilized as templates for synthesis of dsRNAs
using the T7 Megascript kit (www.ambion.com), following the
manufacturers instructions. An irrelevant negative control, firefly
luciferase dsRNA derived from pGL3-basic (www.promega.com), was
prepared as described previously .
dsRNA delivery in schistosomes
Adult schistosomes were cultured in vitro in Medium 199 (www.
invitrogen.com) supplemented with 10% fetal calf serum (www.
gembio.com), 1% antibiotic/antimycotic and 10 mM Hepes at
37uC under 5% CO2 atmosphere. Five pairs of adult worms were
soaked in the presence of Sm-tsp-1, Sm-tsp-2 or luciferase dsRNAs at
1 mg/ml for 7 days with changes of media and dsRNAs every
second day. Schistosomula were maintained at 37uC with 5% CO2
in Medium 169  supplemented with 10% human AB serum
(www.gembio.com) and mouse whole blood. Larval parasites (3 h
old) were soaked in 1 mg/ml of Sm-tsp-1, Sm-tsp-2 or luciferase
dsRNAs and cultured in vitro at 37uC under 5% CO2 atmosphere
for 7, 14 and 21 days, with fresh changes of media, blood and
dsRNAs every second day. Adult and larval parasites were washed
in wash medium prior to RNA or protein extraction.
Infection of mice with dsRNA-treated schistosomula
Newly transformed schistosomula were incubated in wash
medium at 37uC with 5% CO2 for 3 h. Parasites were then
resuspended in 50 ml of wash medium with 100 mg/ml of Sm-tsp-1,
Sm-tsp-2 or luciferase dsRNAs and electroporated in a 4 mm cuvette
at 125 V for 20 ms using a square-wave BTX ECM 830
electroporator (www.btxonline.com). After three washes in wash
medium, schistosomula were counted and 2000 were injected
intramuscularly into each C57BL/6 female mouse (3 mice per
group) using a 23-gauge needle. Adult worms were perfused 28
days later to assess the number of worms that had matured and
reached the mesenteries.
Real-time quantitative RT-PCR
RNA was isolated from parasites using RNeasy Mini kit (www.
qiagen.com) and then treated with Turbo DNA-free endonuclease
(www.ambion.com) to remove contaminating genomic DNA. The
quantity of RNA was measured on a Nanodrop Spectrophotometer
(www.nanodrop.com) and 250 ng of total RNA, SuperScript II
reverse transcriptase (www.invitrogen.com) and oligo dT15 primer
(www.promega.com) were used to synthesize first strand cDNA.
The following primers were designed for real-time
qRTPCR; Sm-TSP-1 (forward: 59-TGGTTGTGCTTATTGGGTTG-39
and reverse: 59-TGATGTCTTGTGCCTCTGGT-39); Sm-TSP-2
(forward: 59-CGAAATTGAACCCCCACTAC-39 and revere:
59CATGCTCCAAACATCCCTAAA-39); Sm-Paramyosin (forward:
59-CGTGAAGGTCGTCGTATGGT-39 and reverse
59-GACGTTCAAATTTACGTGCTTG-39) and Sm-a-tubilin (forward:
59CCAGCAAAATCAGATGGTGAA-39 and reverse:
59-TTGACATCCTTGGGGACAAC-39). qRT-PCR was conducted in triplicate
and each reaction underwent 40 amplification cycles using an Applied
Biosystems 7500 real-time PCR system (www.appliedbiosystems.com)
with cDNA equivalent to 20 ng of total RNA, 50 nM of primers and
SYBR green PCR Master Mix (www.appliedbiosystems.com).
Dissociation curves were generated for each sample to verify the
amplification of a single PCR product. Sm-tsp transcript levels were
calculated relative to Sm-paramyosin in test and irrelevant dsRNA
treated parasites using the 22DDCt method , and data was
expressed as percent differences. For relative endogenous expression
of tsp mRNAs in schistosome life cycle stages, Sm-a-tubulin was used as
the endogenous standard. Sm-paramyosin was used as the housekeeping
gene for analyzing Sm-tsp expression in RNAi experiments.
Evaluation of protein expression
RNAi-treated adult parasites and schistosomula were harvested
after 7 days and then lysed with 1% Triton X-100 in Tris buffered
saline supplemented with complete protease inhibitor cocktail
EASYpacks (www.roche.com). Protein concentrations of lysates
were determined using a BCA protein assay kit (www.pierce.com),
and lysates were electrophoresed in 12% SDS-PAGE gels at
concentrations of 2, 1, 0.5 and 0.25 mg total protein per well.
Proteins were transferred to nitrocellulose membrane
(HybondECL, www.gehealthcare.com) and then probed with either
antiSm-TSP-2 (3H5/2) monoclonal antibody supernatants (L. Cooper,
M. Tran and A. Loukas, unpublished) diluted 1:1,000 followed by
anti-mouse Ig-HRP (www.chemicon.com) diluted 1:5,000.
Reactive proteins were detected by ECL (www.gehealthcare.com) as per
the manufacturers instructions. To assess equal protein loading,
nitrocellulose membranes were stripped after reacting with
antiTSP-2 antibodies and then re-probed with anti-paramyosin
(Sm4B1) monoclonal antibody supernatants  diluted at
1:1,000 followed by anti-mouse Ig-HRP. Experiments were
repeated three times and protein quantities in gel bands were
determined using Syngene Tools and software (www.syngene.com).
Adult parasites and schistosomula were soaked in 1 mg/ml of
Sm-tsp or luciferase dsRNAs for 7 days at 37uC under 5% CO2
atmosphere, washed three times in wash medium and then fixed in
3% glutaradehyde in 0.1M phosphate buffer at pH 7.4, followed
by fixation in potassium ferricyanide-reduced osmium tetroxide.
After fixation, parasites were dehydrated in acetone and
embedded in Epon Resin (ProSciTech). Ultrathin sections were
mounted onto copper grids, contrasted in uranyl acetate and lead
citrate and examined and photographed using a JEM 1011
transmission electron microscope operated at 80 kV and equipped
with a digital camera.
A morphometric approach was employed to quantify possible
changes to tegument structure in schistosomula treated with
Smtsp-2 relative to those treated with luciferase dsRNA. Point
counting stereology [61,62] was used to measure the volume of
tegument occupied by vacuolar compartments or tegument
invaginations in the tegument. Such regions were evident as
clear spaces in TEM sections. Twenty individual schistosomula
were selected at low magnification in the TEM. For each
parasite, the first region of tegument observed that fulfilled the
two criteria below was photographed at 610,000 magnification.
Criteria for selection were, firstly, that the region photographed
was from the lateral aspect of a parasite that was clearly longer
than wide and in which internal organs were present, and
secondly, that the region was not excessively spinous. Volume
density of vacuolar compartments of tegument were estimated
using grids generated by Image J analysis software (NIH
Besthesda), and were calculated as the number of points on the
grid intersecting a vacuolar space divided by the number of points
intersecting the tegument. This was measured across the entire
profile of the tegument in each electron micrograph, so that only
one measure was obtained for each schistosomulum. In addition
to the volume density measure, the thickness of the tegument was
measured at 10 different points using the line tool in Image J. For
each measure, a line was drawn digitally on each micrograph
from the basal membrane of the tegument to the apical
membrane. Regions where the tegument was excessively
invaginated, and those containing isolated spines and sensory
receptors were not measured. The 10 thickness measurements
were averaged for each schistosomulum.
All data are presented as the mean6standard error. Differences
between groups were assessed for statistical significance using
Student t-test (GraphPad Prism Software, www.graphpad.com). A
statistically significant difference for a particular comparison was
defined as p,0.050.
We thank Euihye Jung and Sarah E Galanti (University of Pennsylvania)
for technical assistance and advice, and Mary Duke (Queensland Institute
of Medical Research) and Fred Lewis (Biomedical Research Facility) for
their support in maintaining the schistosome life cycle. We also thank
Sujeevi Nawaratna for carrying out electron microscopy processes.
Conceived and designed the experiments: MHT TCF MKJ EJP AL.
Performed the experiments: MHT TCF LC SG EL. Analyzed the data:
MHT MLG MKJ EL. Contributed reagents/materials/analysis tools: SG
MLG MKJ EJP. Wrote the paper: MHT EJP AL.
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