Transcriptional Changes in Schistosoma mansoni during Early Schistosomula Development and in the Presence of Erythrocytes
et al. (2010) Transcriptional Changes in Schistosoma mansoni during Early Schistosomula
Development and in the Presence of Erythrocytes. PLoS Negl Trop Dis 4(2): e600. doi:10.1371/journal.pntd.0000600
Transcriptional Changes in Schistosoma mansoni during Early Schistosomula Development and in the Presence of Erythrocytes
Geoffrey N. Gobert 0
Mai H. Tran 0
Luke Moertel 0
Jason Mulvenna 0
Malcolm K. Jones 0
Donald P. 0
Alex Loukas 0
Elodie Ghedin, University of Pittsburgh, United States of America
0 1 Division of Infectious Diseases, Queensland Institute of Medical Research , Herston, Queensland , Australia , 2 School of Veterinary Sciences, The University of Queensland , Brisbane, Queensland , Australia
Background: Schistosomes cause more mortality and morbidity than any other human helminth, but control primarily relies on a single drug that kills adult worms. The newly transformed schistosomulum stage is susceptible to the immune response and is a target for vaccine development and rational drug design. Methodology/Principal Findings: To identify genes which are up-regulated during the maturation of Schistosoma mansoni schistosomula in vitro, we cultured newly transformed parasites for 3 h or 5 days with and without erythrocytes and compared their transcriptional profiles using cDNA microarrays. The most apparent changes were in the up-regulation of genes between 3 h and 5 day schistosomula involved in blood feeding, tegument and cytoskeletal development, cell adhesion, and stress responses. The most highly up-regulated genes included a tegument tetraspanin Sm-tsp-3 (1,600-fold up-regulation), a protein kinase, a novel serine protease and serine protease inhibitor, and intestinal proteases belonging to distinct mechanistic classes. The inclusion of erythrocytes in the culture medium resulted in a general but less pronounced increase in transcriptional activity, with the highest up-regulation of genes involved in iron metabolism, proteolysis, and transport of fatty acids and sugars. Conclusions: We have identified the genes that are up-regulated during the first 5 days of schistosomula development in vitro. Using a combination of gene silencing techniques and murine protection studies, some of these highly up-regulated transcripts can be targeted for future development of new vaccines and drugs.
Funding: This work was supported by grants 496600 and 290247 from the National Health and Medical Research Council, Australia (NHMRC). AL is supported by
a senior research fellowship from NHMRC. MT was supported by a U.S./Australia Fulbright award. 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.
The schistosome tegument, an unique double lipid bilayered
syncitium that covers the external surface of the intra-mammalian
developmental stages, represents the point of interaction between
the parasite and mammalian host tissues. This structure is pivotal
for parasite survival within the host and is therefore a primary
target of anthelmintic drugs  and vaccines [2,3]. In similar
fashion, the intestine, or gastrodermis of schistosomes is a source of
secreted proteins and another point of interaction with host tissues
(i.e. blood). The Schistosoma mansoni genome sequence has recently
been reported  and the secreted proteome (secretome) has also
been characterised with a major focus on the proteins present in
the tegument and excretory/secretory (ES) products [4,5,6,7,8,9].
While the schistosome gastrodermal proteome has not yet been
explored, we recently described tissue-specific gene profiling for
adult S. japonicum and characterised the transcriptome of
gastrodermal cells using a combination of laser microdissection
microscopy followed by cDNA microarray analysis . Despite
the progress made in characterising the mRNA and protein
compositions of cells at the host-parasite interface, it is only now
with the recent application of gene silencing technologies to the
study of schistosomes, that we are understanding the functions of
these proteins and how they enable schistosomes to exist as
In terms of vaccine development, the newly transformed
schistosomulum is widely viewed as the most susceptible stage to
antibody-mediated damage [2,3,12,13,14]. After cercariae
transform into schistosomula, parasites undergo changes in their surface
protein composition . The schistosomula surface is dynamic,
with some proteins appearing and others disappearing  as the
parasites mature during their migration to the lungs. Once the
parasites reach the lungs they are refractory to antibody-mediated
damage  and cloak themselves in host blood group antigens
 and other proteins involved in immune responses [19,20].
Obtaining sufficient quantities of schistosomula directly from
lung tissue for most research purposes is time consuming and
involves working with mammalian hosts. As a result, many
Schistosome blood flukes cause more mortality and
morbidity than any other human worm infection, but
current control methods primarily rely on a single drug.
There is a desperate need for new approaches to control
this parasite, including vaccines. People become infected
when the free-swimming larva, the cercaria, enters
through the skin and becomes the schistosomulum.
Schistosomula are susceptible to immune responses
during their first few days in the host before they become
adult parasites. We characterised the genes that these
newly transformed parasites switch on when they enter
the host to identify molecules that are critical for survival
in the human host. Some of these highly up-regulated
genes can be targeted for future development of new
vaccines and drugs.
researchers mechanically transform cercariae and culture them in
serum-containing medium . This in vitro strategy also confers
uniformity in parasite maturation, which is critical for short
culturing periods and cannot be achieved in vivo due to the
variation in the time required for individual parasites to penetrate
host skin and enter the vasculature. Erythrocytes are not usually
included in the culture medium yet the parasites are bathed in
blood in vivo. Moreover, schistosomula acquire erythrocyte 
and other cellular  proteins onto their teguments, and this is
thought to aid in evasion of the immune response. Despite their
vascular existence, the effect of erythrocytes on gene expression of
in vitro cultured parasites has not been addressed until now.
We sought to identify the transcriptional changes in genes
encoding surface and secreted proteins during the first 5 days of in
vitro culture of schistosomula in the presence or absence of
erythrocytes. Some of these surface exposed proteins are proving
to be efficacious vaccines [23,24], yet the expression profiles of
only some of these genes have been explored, and have involved
using arrays covering only ,3,000 genes . Here we show that
the major transcriptional changes which occur during this 5 day
time period involve a wide range of biological functions but
prominent processes include tegument maturation, cellular
development and organisation, gut function/nutrient acquisition
and stress responses. The data provide a framework by which to
select targets for vaccine and drug design based on genes that are
critical for the development of S. mansoni larvae during their first
few days in the mammalian host.
Materials and Methods
Culture of S. mansoni and total RNA isolation
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). To
obtain cercariae, B. glabrata snails infected with miracidia were
exposed to incandescent light for 1 h and then washed twice in
RPMI 1640, 1% antibiotic/antimycotic and 10 mM Hepes
(Invitrogen). Cercariae were passed through a 22-gauge
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 and
washed three times with wash medium before experimentation
. Schistosomula were cultured at 37uC in modified Baschs
medium (containing 10% Fetal Calf Serum) under 5% CO2
atmosphere [21,28], either in the presence or absence of
erythrocytes, for 3 h or 5 days. Parasites were washed in RPMI
and stored in Trizol at 280uC until the total RNA was isolated
following manufacturers instructions. RNA quality, integrity
checks and concentration were assessed using Nanodrop
ND1000 spectrophotometer and Agilent 2100 Bioanalyzer . Due
to the limited availability of schistosomula material, a single
biological sample was used.
The design and construction of the schistosome microarray has
been previously reported . The microarray comprises of
19,222 target sequences printed twice from two independent
probe designs, including 12,166 probes derived from S. mansoni
contiguous sequences (contigs) and 7,056 probes derived from S.
japonicum contigs. The putative genes designated from S. mansoni
contiguous sequences were the primary but not exclusive focus of
the analysis, and the genes derived from S. japonicum contigs were
also fully considered. Further details of the microarray design and
the normalised data from this study are presented in Tables S1
Microarray hybridisation and feature extraction
The methods used in microarray hybridisation and feature
extraction have been previously reported  and followed the
manufacturers instructions (One-Color Microarray-Based Gene
Expression Analysis Protocol; Version 5.5, February 2007 Agilent).
For each sample 300 ng of total RNA was used to synthesise
fluorophore-labelled cRNA using Cyanine 3-CTP (Agilent
Technologies One Color Microarray Kit). Samples were purified
using the Qiagen RNeasy kit. Cyanine-labeled cRNA samples
were examined at A260 and A550 using a ND-1000
spectrophotometer to determine yield, concentration, amplification efficiency
and abundance of cyanine fluorophore. CY3c (1.65 mg aliquot)
was incubated with the fragmentation mix (Agilent Technologies)
for 30 min at 60uC. Samples were then combined with 26 Gene
Expression Hybridisation Buffer HI-RPM, mixed and applied to a
gasket slide pre-positioned in a hybridisation chamber (Agilent
Technologies), placed in a hybridisation oven and incubated for
17 h at 65uC. Each hybridisation was performed in duplicate as a
technical replicate. After hybridisation, microarray slides were
washed using the standard protocol (Agilent Technologies) and
scanned on an Agilent microarray scanner at 550 nm. The tag
image format files (tiff) produced by the scanner were loaded into
the image analysis program Feature Extraction 18.104.22.168 (Agilent
Technologies) to establish standardised data for statistical analysis.
All microarray slides were checked for background evenness by
viewing the tiff image on Feature Extraction.
Feature extracted data were analysed using GENESPRING
software, version 7.3.1 (Agilent Technologies/Silicon Genetics).
Duplicated microarray data (technical replicates) were normalised
using the GeneSpring normalisation scenario for Agilent FE
onecolor which included Data Transformation: Set measurements
less than 5.0 to 5.0, Per Chip: Normalise to 50th percentile and
Per Gene: Normalise to median. Samples were also normalised
to individual samples depending on the comparison to be made.
To compare developmental effects of the transformation from
cercariae to schistosomula, 3 h schistosomula with erythrocytes
and 5 day schistosomula with erythrocytes were normalised to
expression levels of cercariae. To determine the effects of culturing
schistosomula with erythrocytes, 3 h schistosomula with
erythrocytes were normalised to 3 h schistosomula without erythrocytes;
similarly 5 day schistosomula with erythrocytes were normalised to
5 day schistosomula without erythrocytes. Data sets were further
analysed using published procedures [30,31] consisting of methods
related to one-colour experiments and utilised the signal intensity
(gProcessedSignal) values determined using Agilents Feature
Extraction software including aspects of signal/noise ratio, spot
morphology and homogeneity. ProcessedSignal represents signal
after localised background subtraction and includes corrections for
surface trends. Features were deemed Absent when the processed
signal intensity was less than two-fold the value of the processed
signal error value. Features were deemed Marginal when the
measured intensity was at a saturated value or if there was a
substantial amount of variation in the signal intensity within the
pixels of a particular feature. Features that were not Absent or
Marginal were deemed Present. Data points were included only if
Present and contigs were retained if all data points were Present.
Secretory signal sequence analysis
Transcripts that were at least two-fold up-regulated in a
particular experiment were searched against a database consisting
of the full protein datasets from the S. japonicum  and S. mansoni
 genome sequencing projects using BLASTX. Protein hits with
an identity greater than 95% were used as the protein translation
of the EST and analysed using local versions of SignalP 3.0 
for secretory signal sequences and TMpred (http://www.ch.
embnet.org/software/TMPRED_form.html) for transmembrane
domains. Transcripts that had no high scoring identities in the
predicted protein database were searched against the NCBI
nonredundant protein database using BLASTX. If the top scoring
protein had a bit score greater than 50 the reading frame of the
blast translation was used to translate the EST into protein
sequence. The protein sequence was then analysed using SignalP
Real time PCR
Gene expression patterns of a subset of genes were validated
using real time PCR. Genes were chosen to span a range of
biological functions and life cycle expression patterns, with a focus
on including genes that encode known secreted/membrane
proteins of interest as vaccines and drug targets, such as
tetraspanins and intestinal proteases. Complementary DNA was
synthesised from total RNA using a QuantiTect whole
transcriptome kit (Qiagen). All cDNA samples were synthesised from
aliquots of the same total RNA and used for the microarray
hybridisations at a concentration of 50 ng/ml quantified using a
Nanodrop ND-1000 spectrophotometer. Subsequently, 1 ml
aliquots were combined with 10 ml of SYBR Green, 3 ml of water
and 2 ml (5 pmol) of the forward and reverse primers in a 0.1 ml
tube. All reactions were performed on a Rotor-Gene 3000 real
time thermal cycler (Corbett) and analysed using Rotor Gene 6
Software (Corbett). In order to minimise indiscriminate binding of
double-stranded DNA, which can produce readings in the no
template controls, separate reverse transcription and PCR steps
were included. Primer sets used are described in Table S3. The
housekeeping gene TC15682 (DNA segregation ATPase)  was
used for primary normalisation for all experiments. This
housekeeping gene was selected from the initial microarray data
since it was consistently unchanged throughout all of the
comparisons made. Each experiment was performed in duplicate
and the confidence threshold (CT) of the second set was
normalised to the first set before evaluation. The analysis of
correlation between microarray and quantitative PCR was
performed in Graphpad Prism Version 5 (Graphpad Software
Inc.) and was based on a previously published analysis . To
correlate results from microarray and quantitative PCR platforms,
we first determined whether the data were distributed normally.
This involved the use of both the DAgostino & Pearson omnibus
normality test and Shapiro-Wilk normality test. Both tests
indicated that the data were not normally distributed; thus a
Spearman correlation (Rho) was employed. All methods used an
alpha value of 0.05.
Supplementary Information (RAW Data) has been submitted at
GEO- Gene Expression Omnibus, (http://www.ncbi.nlm.nih.
gov/geo/) with accession numbers for the platform GPL7160,
and series GSE18335.
Two questions were addressed using the microarray analysis of
S. mansoni schistosomula cultured for 3 h and 5 days.
1. Which genes undergo changes in expression during the transition from cercariae to 3 h schistosomula to 5 day schistosomula?
2. What effect does the inclusion of erythrocytes have on gene expression during culture?
Genes modulated during the transformation of cercariae
Three hour and 5 day schistosomula were cultured in the
presence of erythrocytes and the subsequent microarray data were
normalised to the gene expression of cercariae. Data were filtered
for each replicated data point for each of the 38,444 probes
(19,222 contigs) on the microarray. Data points were filtered to
preserve signals that were flagged during the feature extraction
process as Present in all hybridisations; this resulted in the retention
of 13,466 probes (7,764 contigs). A final cut-off was applied to the
microarray data generating lists of genes that were $2-fold up- or
down- regulated (relative to cercariae) for both 3 h and 5 day
schistosomula. The number of genes (contigs) that were $2-fold
up-regulated in the 3 h and/or 5 day schistosomula were 1,608 at
3 hours and 3,600 at 5 days, with 1,270 genes maintaining
upregulation at both time points (Figure 1A). Fewer genes were
down-regulated $2-fold, with 1,183 at 3 hrs and 1,343 at 5 days;
831 genes remained down-regulated at both time points.
The differential fold-change occurring between 3 h and 5 day
cultured schistosomula was determined by plotting the signal
intensity of the two time points and applying a .2 or ,0.5 cut-off
to represent a 2-fold up- or down- regulation. A larger number of
genes were up-regulated during the transition from 3 h to 5 days
(2,327 genes) compared with those that were down-regulated
during this time period (439 genes) (Figure 1B). Examples of
differentially expressed genes with novel annotations as well as the
expression levels of genes encoding surface proteins of recently
described vaccine antigens are presented in Table 1. Genes of
particular interest that were highly up-regulated during the
transition from cercariae to 3 h and 5 day schistosomula included
the tegumental tetraspanin Sm-TSP-3 which was up-regulated
almost 1,600-fold between cercariae and 5 day schistosomula.
Other genes encoding tegument proteins that were upregulated
included the Sm22.6 tegument associated antigen (137-fold
upregulated at day 5) and a protein with homologues from the
tegument of other flukes  and with low sequence identity to
protein kinases (TC7982, 68-fold increased expression at day 5).
Genes encoding two additional tegument proteins were rapidly
upregulated at 3h (annexin 7-fold and cytosol aminopeptidase
14fold) and then further up-regulated by day 5 (annexin 21-fold and
aminopeptidase 45-fold). A gene with sequence similarity to
fasciclin 1 was up-regulated 100-fold in day 5 schistosomula
(TC14173); the full-length protein (Smp_141680) was obtained
from S. mansoni GeneDB (http://www.genedb.org/genedb/
smansoni/) and contained two predicted transmembrane domains.
Genes encoding intestinal proteases with known or suspected roles
in digestion of the blood meal were highly upregulated by day 5
and included cathepsin B (65-fold), cathepsin L (37-fold), cathepsin
D (13-fold) and cathepsin C (11-fold). The third most highly
upregulated gene in 5 day schistosomula was a S01 family serine
protease (TC16843; 108150-fold), although its anatomical
location, and therefore potential function, has not been
determined. Some of the genes that were up-regulated very quickly
after transformation (by 3 h), included cathepsin B (10-fold),
cathepsin L (2-fold) and cathepsin D (2-fold). At a more global
level, the transformation between cercariae and 3 h/5 day
schistosomula resulted in up-regulation of genes encoding a wide
range of gene ontology (GO) categories including stress effectors
(HSP70 and mitochondrial dicarboxylate transporter), enzymes
involved in digestion of the blood meal (proteases) and iron storage
(ferritin 2 (somal ferritin); upregulated 135-fold) (Figure 2). Other
noteworthy genes included transmembrane transporters of zinc
that were elevated in both 3 h and 5 day schistosomula (TC7518
ZNT4_RAT Zinc transporter 4, 3-fold at 3 h and 9-fold at 5 day;
TC18440 solute carrier family 30 zinc transporter member 6,
2fold at 3 h and 4-fold at 5 days).
Changes in gene expression of schistosomula in the
presence of erythrocytes
Schistosomula cultured for 3 h or 5 days were maintained in the
presence or absence of erythrocytes and the subsequent
microarray data were normalised to the gene expression of parasites
cultured without erythrocytes. Data were filtered for each
replicated data point for each of the 38,444 probes (19,222
contigs) on the microarray. Data points were filtered to preserve
signals that were flagged during the feature extraction process as
Present in all hybridisations; this resulted in the retention of 13,140
probes (7,599 contigs) (Figure 3A). A final cut-off was applied to
the microarray data generating lists of genes that were $2-fold
upor down- regulated (relative to culture conditions without
erythrocytes) for both 3 h and 5 day cultured schistosomula. A
total of 788 genes at 3 h and 2,479 at 5 days were $2-fold
upregulated; 399 genes were up-regulated at both time points
(Figure 3B). A small number of genes (388) were down-regulated in
3 h schistosomula. A small proportion of genes that were
upregulated at 5 days were down-regulated initially at 3 h (317
genes). Examples of novel genes and gene whose expression was
up-regulated during the development of schistosomula from the
cercarial stage are presented in Table 1, and further examples of
novel up-regulated genes due to the presence of erythrocyte either
at 3 h or 5 day schistosomula are presented in Table 2. The
presence or absence of erythrocytes had little to no effect on the
expression of genes encoding intestinal proteases, and of the genes
encoding exposed tegument proteins, only TSP-4 and alkaline
phosphatase III were up-regulated (2-fold) in the presence of
erythrocytes. However, the inclusion of erythrocytes induced more
obvious expression changes in genes involved in development and,
to a lesser extent, the stress response (Table 1). These included
genes encoding proteins involved in iron storage (ferritin-2, 20-fold
increase), divalent metal transporter, heme-binding protein,
proteolytic enzymes (serine protease), a fatty acid receptor,
glycogen formation (glycogenin) and other components potentially
involved in cellular differentiation/morphology. An overview of
the differentially expressed genes in the presence/absence of
erythrocytes is presented as GO categories in Figure 4.
Validation of mRNA expression using quantitative PCR
To validate the microarray transcription data, mRNA
expression profiles for 15 genes from different functional categories were
assessed using quantitative real time PCR. Two independent
experiments were carried out for this validation. The relative
differential gene expression obtained by microarray analysis and
by quantitative PCR was similar for the majority of data points for
the 15 genes assessed (Figure 5). There was a significant
correlation of 0.9208 between the two data sets (Spearmans
Rho, P,0.0001, n = 34).
Normalised to Cercariae
Normalised to no RBCs
epidermal growth factor receptor
Paramyosin. [Blood fluke], complete
Normalised to Cercariae
Normalised to no RBCs
Microarray data are presented as fold changes relative to the period of culture (cercariae for the developmental changes) or culture condition (presence of erythrocytes).
Genes are presented under broad themes based on gene function. F = Gene failed filtering in that particular comparison.
Up-regulation of genes encoding for secreted proteins
Genes that were upregulated $2-fold in any of the comparisons
were assessed for the presence of signal peptides/anchors and
transmembrane domains, suggestive of an extracellular location
and therefore potentially involved in host-parasite interactions.
Eight percent of genes that were up-regulated in 3 h old
schistosomula (compared with cercariae) encoded for secreted
proteins; all of these had 20-fold or less increases in expression
except for contig 680_298, which was upregulated 3,447-fold in
the absence of erythrocytes and an additional 742-fold in the
presence of erythrocytes (Figure 6). Other genes up-regulated in
3 h parasites that encoded secreted proteins of interest included
TC6882_676, a serine protease inhibitor that was up-regulated
2fold and then an additional 144-fold in 5 day old parasites. More
than 9% of genes that were up-regulated in 5 day parasites
(compared to 3 hr parasites) encoded for secreted proteins;
foldchanges in expression were generally higher than those seen in 3 h
parasites, with the top ten ranging from 1,598-fold (TC18051_669
tetraspanin Sm-TSP-4) to 65-fold (TC13586_1222 cathepsin B)
(Figure 6). Four of these top 10 most highly upregulated secreted
proteins shared no identity with any proteins of known function.
The complete list of genes encoding for secreted proteins that
underwent $2-fold increased expression in any of the
experimental groups is provided in Table S4.
Table 2. Examples of genes that were up-regulated either in schistosomula of S. mansoni cultured for 3 hours or 5 days in the
presence of erythrocytes.
Developmental Effects Culturing Conditions
Normalised to Cercariae Normalised to no RBCs
TC13678 similar to low-density lipoprotein receptor [Chiloscyllium plagiosum], partial (3%)/
SJCHGC03880 vitellogenin receptor
TC10050 SJCHGC04667 protein [Schistosoma japonicum], hypothetical protein
extracellular serine-threonine rich protein
mucin-associated surface protein (MASP), putative [Trypanosoma cruzi]
TC13651 similar to seven transmembrane helix receptor [Homo sapiens], partial (5%)
TC10651 SJCHGC03199 protein [Schistosoma japonicum], similar to lipopolysaccharide-induced TNF
similar to mucin 5 [Homo sapiens], partial (2%)
weakly similar to Cullin homolog 3 (CUL-3). [Human], partial (30%)/SJCHGC07208
TC13461 homologue to PAXILLIN-LIKE PROTEIN [Dictyostelium discoideum], partial (4%)/mucin 17
TC16784 glucan 1 4-beta-glucosidase [Xanthomonas axonopodis pv. citri str. 306], partial (1%)/
CG33300-PA [Drosophila melanogaster]
TC15511 activin receptor type
TC16880 similar to MAP kinase [Strongylocentrotus purpuratus], partial (85%)/mitogen-activated
protein kinase 3
TC10495 homologue to cathepsin B1 isotype 1 [Schistosoma mansoni], partial (27%)
TC16719 epidermal growth factor receptor [Schistosoma mansoni], partial (4%)
weakly similar to unnamed protein product [Homo sapiens], partial (9%)/SJCHGC05056 epsin 2 1.4
Microarray data are presented as fold changes relative to schistosomula cultured for the same period in the absence of erythrocytes.
In this study we have presented a comprehensive analysis of the
transcriptional changes that are associated with two distinct and
critical phases of the early maturation of the intra-mammalian
stages of S. mansoni. We investigated the developmental changes
that occur in vitro (1) in the first few hours after transformation as
the cercarial glycocalyx is shed and the parasite adapts to an
intramammalian environment, and (2) as schistosomula mature in vitro
over a 5 day period. This developmental window corresponds to
the in vivo phase of parasite migration from the skin (3 h) into the
vasculature en route to the lungs (5 days). In vitro culture exposes
the parasite to host serum (and erythrocytes) but does not entirely
mimic intra-mammalian development due to the absence of tissue
barriers to penetrate, a vascular system to navigate and a complete
immune system to avoid. We also examined the effects on gene
transcription when erythrocytes were present in the culture
medium. For both analyses, we placed emphasis on genes that
encode proteins at the host-parasite interface, namely exposed
tegument proteins and key proteins involved in nutrient
The development of newly transformed S. mansoni
schistosomula over the first 57 days as they enter the vasculature and
progress to the lungs represents what many believe to be a critical
window of opportunity for vaccine-mediated protection
[3,37,38]. At this stage the parasite presents a distinct suite of
proteins on its tegument and has not yet become fully cloaked in
host-derived molecules . Moreover, juvenile schistosomula
are more susceptible to antibody-dependent cellular cytotoxicity
 than are older schistosomula and adult worms. We therefore
reasoned that genes that are highly expressed at this stage,
particularly those encoding secreted and membrane proteins, are
worthy targets for the development of vaccines and new drugs.
Recent microarray based studies of S. mansoni  and S.
japonicum  have profiled the gene expression patterns of a wide
range of lifecycle stages. However, one aspect of the parasite
lifecycle that has not been examined in detail for either species is
the transformation of the cercarial stage to the skin
schistosomula, and the maturation of these parasites into lung
schistosomula. Dillon and co-workers  compared cDNAs
from 2 day and 7 day old cultured schistosomula with a control
pool of mixed lifecycle stage cDNAs. They utilised a cDNA
microarray consisting of 3,088 unique contigs and identified
broad categories of differentially expressed genes in the
schistosomulum stage, including energy metabolism, cytoskeletal
organisation, protease activity and chromosome remodelling.
Our study differs from that of Dillons in a number of ways; (1) we
focused on different time points of cercarial transformation and
maturation, 3 hours and 5 days post-transformation, since these
times better approximate the skin and lung schistosomula stages
of S. mansoni; (2) we compared the gene expression profiles of
each developmental stage to the stage preceding it, i.e.
schistosomula to cercariae instead of using a pooled multi-stage
cDNA preparation for determining baseline expression; and (3)
we utilized an array consisting of 12,166 S. mansoni unique
Our results indicate that the transcriptional changes that
accompany the development of cercariae to 3 hour and then to
5 day schistosomula are mostly found in the up-regulation of genes
associated with many different molecular functions, but
particularly catalytic activity and binding. Considerably fewer genes were
down-regulated in the first 3 hours after transformation, most
likely representing genes that are important for the free-swimming
cercariae and are no longer required by the intra-mammalian
stages. Jolly and co-workers found that the most highly
upregulated genes in cercariae (compared to other life cycle stages)
were mitochondrial in nature representing the increased energy
requirements needed for motility during this stage . This
observation was paralleled in our study where a number of
mitochondrial genes such as NADH subunit 2 and NADH subunit
5 (see Table S2) were down regulated (up to 25-fold) at 3 h
posttransformation compared with the cercarial stage. We observed
that most probes to actin or actin related genes and fibrillin 2 were
upregulated in the schistosomula relative to cercariae. The
differential expression of structural genes such as actin may reflect
the increased levels of tegument matrix generation and turnover as
well as smooth muscle formation in newly transformed and
growing parasites [42,43].
Figure 6. Percentage of genes encoding for secreted/membrane proteins that underwent $2-fold increased expression in each
category. All genes that were up-regulated were screened for the presence of a signal peptide or anchor using SignalP; those ORFS with a signal
peptide/anchor were then further screened for transmembrane (TM) domains using TMPred. The tables show the ten most highly upregulated genes
in each category.
We were particularly interested in the expression of genes
encoding exposed proteins on the tegument of the parasite.
Tetraspanins are four transmembrane spanning proteins
represented by at least 5 distinct members in the tegument membranes
of the adult parasite [2,5,6,44]. Sm-TSP-1 and TSP-2 are
efficacious vaccines ; tsp-1 mRNA was up-regulated almost
4-fold in 5 day schistosomula but tsp-2 expression levels did not
change. However, one of the most highly up-regulated gene on the
entire microarray (TC18051), undergoing 1,598-fold
up-regulation between 3 h and 5 day cultured parasites, was another
tegument tetraspanin  that we have termed Sm-tsp-3 . This
considerable up-regulation of tsp-3 was confirmed by quantitative
PCR. Genes encoding other tegument proteins that were
significantly up-regulated during this developmental period
included Sm22.6, an unknown protein, and annexin. Sm22.6 is
an inhibitor of human thrombin , and its up-regulation upon
entry into the host vasculature likely represents an important
survival strategy. Recombinant Sm22.6 confers partial protection
as a vaccine in murine studies  but it is a major target of IgE in
infected people , so its utility as a vaccine is likely limited. The
third most highly up-regulated gene in 5 day schistosomula was a
S01 family serine protease (150-fold), sharing identity with
cercarial elastases that digest connective tissue proteins [48,49].
Unlike the cercarial elastases where mRNA expression is highest in
sporocysts and is switched off in cercariae and intra-mammalian
stages , this new serine protease likely plays a distinct role due
to its up-regulation in maturing schistosomula. We also detected
100-fold up-regulation in 5 day schistosomula of a gene encoding
for a homologue of fasciclin 1, a family of GPI- anchored proteins
that mediate cell adhesion through an interaction with alpha3/
beta1 integrin .
As schistosomula mature their gastrodermis forms and they begin
to ingest blood as a source of nutrition. The digestive process begins
with haemolysis where erythrocytes are ingested and lysed by the
action of a haemolysin(s) within the oesophagus and intestine .
Saposin-like proteins (SAPLIPs) are candidate pore-forming
haemolysins in the schistosome gut , and we identified a SAPLIP
that was highly up-regulated in 5 day schistosomula (TC_14899,
25fold up-regulated). Two other SAPLIP-encoding genes, TC10647
and TC10646, were also up-regulated (5- and 3-fold respectively) in
5 day schistosomula, suggesting a role for this protein in blood
feeding, either via haemolysis or transport of lipids. An anion sugar
transporter, distinct from the well characterised glucose transporter
family from the S. mansoni tegument , was up-regulated 25-fold
in 5 day schistosomula, although its site of expression (gut, tegument
or elsewhere) has not yet been determined. Other intestinal genes
that were up-regulated between 3 h and 5 day schistosomula were
the digestive proteases. The role of cysteine and aspartic proteases in
digestion of haemoglobin and serum proteins for nutritional support
is well recognised [55,56], and the increase in transcription of these
genes as schistosomula begin to feed on blood supports their roles in
Erythrocytes provide the parasite with an important nutritional
source. However, other host factors also have an impact on
parasite development; for example, the presence of insulin in the
culture medium induced the increased expression in S. japonicum of
many genes related to sexual reproduction and protein translation
in general . In this current study, serum (which contains
insulin) was present throughout during culture, but other
components, such as glycoproteins or glycolipids, derived directly
from the erythrocytes may have impacted on gene expression. The
up-regulation of a low-density lipoprotein receptor in the presence
of erythrocytes may be influential in the development of the female
reproductive system since this protein is also a putative vitellogenin
receptor (Table 1). Lipids are a component of the female
reproductive tract in schistosomes, as indicated previously by the
localisation of a fatty acid-binding protein within lipid droplets of
vitelline cells . The most commonly encountered transcripts
that were up-regulated in the presence of erythrocytes were of
retroviral/retrotransposon origin. This is not surprising, as we
have previously reported the up-regulation of retrotransposons in
geographical isolates of schistosomes , and their up-regulation
here in the presence of erythrocytes may be in response to
Other genes that were up-regulated in 5 day schistosomula in
the presence of erythrocytes included at least three distinct
mucins that were .3-fold up-regulated, and an activin
receptor, which have been described from secretions of
schistosome eggs and cercariae  and the tegument .
Glycosidases were also upregulated, an example being glucan 1
4-beta-glucosidase (TC16784). This was 2.8 fold up-regulated
in 5 day schistosomula in the presence of erythrocytes
compared to their erythrocyte-free cultured counterparts,
further implying a general increase in expression of genes
involved in acquisition and metabolism of erythrocyte proteins,
lipids and glycans. Only one of the digestive proteases showed
greater than 2-fold increased expression in the presence of
erythrocytes (TC10495, homologue to cathepsin B1), implying
that their up-regulated expression is predominantly
independent of erythrocytes (and therefore haemoglobin), but instead is
dependent on serum proteins. Selected examples of the
most highly up-regulated (non-retroviral) genes in the
presence of erythrocytes in 5 day schistosomula are provided
in Table 2.
The transformation from cercaria to schistosomulum requires
the adaption of the parasite to radically different environments
and subsequent large scale cellular differentiation and growth.
This stressful process is reflected by the up-regulation of multiple
stress related genes , including numerous heat shock proteins
and minichromosomal maintenance complex component 2, all of
which were up-regulated at 3 h and were maintained at elevated
expression levels through day 5, relative to cercariae.
Another major feature of the maturing schistosomula is the
extensive musculature that begins to form beneath the tegument
. This increase in muscle tissue was reflected in our study by
increased expression levels of a number of muscle related genes
including paramyosin, myosin light chain kinase and myosin light
polypeptide 5 regulatory sequence (See Table S2). Paramyosin is
of particular importance to intra-mammalian stages due to its dual
function as a structural element of smooth muscle and its
immunomodulatory function in schistosomula [63,64,65].
The tegument of S. mansoni schistosomula is the major target of
the immune response of a population of putatively resistant (PR)
individuals in S. mansoni endemic areas of Brazil , and in
schistosomiasis resistant rats . By identifying genes that are
highly upregulated during this developmental process, particularly
those that are accessible to antibodies such as the exposed
tegument and gut proteins, a new suite of vaccine antigens can be
produced and screened with sera from resistant hosts. We envisage
this study as an initial screen for potential vaccine antigens and
drug targets, to be followed by murine protection studies and
functional analysis where approaches such as RNA interference
could be used to verify the consequences of silencing these genes
under different in vitro and in vivo conditions.
Table S1 Complete lists of contiguous sequences listed in the
custom designed schistosome microarray manufactured by Agilent
Technologies used in this study. Column A (ProbeID): Unique
identifier of probe on the microarray. Column B (Sequence):
Nucleotide sequence of the 60mer probe. Column C (EST
Sequence): Complete nucleotide sequence of the assembled EST
contig. Column D (TargetID): Contig designation for either S.
japonicum (Contig) or S. mansoni (TC). Column E (Accessions):
Genbank accession number corresponding to the EST sequences.
Column F (Description-Nucleotide): BLASTn annotation result
based on nucleotide sequence. Column G (GeneSymbols):
Designation of primary or secondary probe design to the
corresponding contig. Column H (Protein Homology): BLASTX
annotation result based on protein sequence. Column G (Gene
Ontology): Gene Ontology number and description.
Found at: doi:10.1371/journal.pntd.0000600.s001 (8.96 MB ZIP)
Table S2 Complete list of differentially expressed genes, shown
on separate sheets of a Microsoft Excel File.
Found at: doi:10.1371/journal.pntd.0000600.s002 (7.41 MB XLS)
Table S3 Primer sets for real time PCR validation of a subset of
genes that were differentially expressed in schistosomula under
different culture conditions and/or relative to the cercarial stage.
Found at: doi:10.1371/journal.pntd.0000600.s003 (0.03 MB XLS)
Table S4 Genes encoding for secreted/membrane proteins that
underwent $2-fold increased expression in each category. All
genes that were up-regulated were screened for the presence of a
signal peptide or anchor using SignalP; those ORFS with a signal
peptide/anchor were then further screened for transmembrane
(TM) domains using TMPred.
Found at: doi:10.1371/journal.pntd.0000600.s004 (0.15 MB XLS)
We thank Mary Duke for maintenance of the S. mansoni life cycle.
Conceived and designed the experiments: GNG MHT AL. Performed the
experiments: GNG MHT LM. Analyzed the data: GNG JM AL.
Contributed reagents/materials/analysis tools: GNG LM JM MKJ
DPM. Wrote the paper: GNG MHT AL. Proofed the paper: DPM.
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