Gene expression patterns during adaptation of a helminth parasite to different environmental niches
Genome Biology
eJ2Vto0ola0llulys7.meea8r,cIshsue 4, Article R65 Re Gene expression patterns during adaptation of a helminth parasite to different environmental niches
Emmitt R Jolly 1
Chen-Shan Chin 1
Steve Miller 1
Mahmoud M Bahgat 0
KC Lim 1
Joseph DeRisi 1
James H McKerrow 1
0 Theraputic Chemistry Department, Infectious Diseases and Immunology Laboratory, the Road to Nobel Project, the National Research Center , Dokki, 12311 Cairo , Egypt
1 California Institute for Quantitative Biomedical Research (QB3) of the University of California, San Francisco , 4th Street, San Francisco, CA 94158 USA
Background: Schistosome bloodflukes are complex trematodes responsible for 200 million cases of schistosomiasis worldwide. Their life cycle is characterized by a series of remarkable morphological and biochemical transitions between an invertebrate host, an aquatic environment, and a mammalian host. We report a global transcriptional analysis of how this parasite alters gene regulation to adapt to three distinct environments. Results: Utilizing a genomic microarray made of 12,000 45-50-mer oligonucleotides based on expressed sequence tags, three different developmental stages of the schistosome parasite were analyzed by pair-wise comparisons of transcript hybridization signals. This analysis resulted in the identification of 1,154 developmentally enriched transcripts. Conclusion: This study expands the repertoire of schistosome genes analyzed for stage-specific expression to over 70% of the predicted genome. Among the new associations identified are the roles of robust protein synthesis and programmed cell death in development of cercariae in the sporocyst stages, the relative paucity of cercarial gene expression outside of energy production, and the remarkable diversity of adult gene expression programs that reflect adaptation to the host bloodstream and an average lifespan that may approach 10 years.
Background
Schistosomiasis is a chronic debilitating parasitic disease
affecting some 200 million people across 74 countries within
Africa, Asia, the Middle East and South America. In terms of
public health and socio-economic impact, it ranks second
only to malaria among parasitic diseases [1,2]. The causative
agents of schistosomiasis are schistosome bloodflukes,
multicellular trematodes whose life cycle is characterized by a
series of striking morphological and biochemical transitions
between an intermediate host snail in an aquatic
environment, two free-swimming aquatic larval forms, and a
warmblooded mammalian host (Figure 1). As such, the schistosome
represents an ideal but challenging biological system in which
to identify programs of gene regulation that have evolved to
facilitate adaptation of metazoa to different biological
microenvironments.
Molluscan Host
Sporocyst transcripts
function primarily in
protein synthesis and J
metabolism, and
differentiation
JE
Joly
(f) Sporocyst
(e) Miracidium
Free-swimming
aquatic stages
(a) Cercaria
Transcripts from cercariae function primarily
in energy metabolism and motility
(b) Schistosomulum
(c) Adult
Adult transcripts function in egg
production, acquisition of nutrients,
host adaptation and evasion of the
host immune response
(d) Egg
TFhigeusrcehi1stosome life cycle is complex, with morphologically distinct stages occupying several ecological niches
The schistosome life cycle is complex, with morphologically distinct stages occupying several ecological niches. Infective cercariae (a) swim in fresh water
to find and infect a mammalian host. After host invasion, cercariae transform into schistosomula (b) and adapt to survival in the host bloodstream. The
schistosomula mature into adult male or female schistosomes (c), which pair and produce eggs (d). Eggs are excreted from the host. In fresh water, the
eggs hatch into miracidia (e), which infect a snail host and develop into sporocysts (f). Daughter sporocysts generate infectious cercariae, completing the
life cycle.
In the intermediate host aquatic snail, sac-like clusters of
differentiating larvae called daughter sporocysts give rise to
large numbers of free-swimming aquatic cercariae. Cercariae
navigate their environment by a side to side motion of their
tails and find the mammalian host through chemical and
environmental signals [3-6]. Upon entering the mammalian
host, the tail is detached, and an early vascular stage called
the schistosomulum rapidly readjusts to the new host
environment. During this time, the schistosomulum undergoes
changes in basic metabolism and surface properties [7]. The
schistosomulum navigates the vascular system, including
transiting through at least two capillary plexuses. Between 14
and 28 days, the organisms undergo rapid growth and
differentiation to adult male and female forms in specific regions of
the host vasculature. Male and female worms form
matingpairs and the female produces hundreds to thousands of eggs
per day, depending on the species of schistosome. The eggs, in
turn, pass out of the host via feces or urine, and hatch in fresh
water to yet another morphologically distinct form, the
miracidium. This larval form moves through fresh water by means
of numerous cilia, and invades the soft parts of the
intermediate host snail, transforming into the mother sporocyst. The
cycle is complete as the mother sporocyst produces daughter
sporocysts, which serve as the developmental stratum for
production of cercariae.
Efforts to reduce schistosome prevalence have included
combinations of sanitation, health education, snail control, better
diagnosis and chemotherapy [8]. Control of parasitemia has
relied primarily on praziquantel, an effective
chemotherapeutic drug that has been in use for over 20 years. Recurring
morbidity and evidence of emerging resistance to praziquantel in
endemic countries emphasizes the need for both an effective
vaccine and new chemotherapy [9-11]. Several potential
vaccine candidates have been identified [12,13], but to date none
is sufficiently effective for practical use. Given the complexity
of the schistosome lifecycle and its ability to evade the
immune system, a more comprehensive approach to
understanding the molecular biology and biochemistry of
schistosomes is essential to rationally identify and develop potential
vaccine candidates or new drugs.
The approximately 270 Mb genome of Schistosoma mansoni
[14] is organized into 8 chromosomes [15], including the
heterologous female pair WZ [16]. It is estimated to have 30%
GC content [17]. Currently, the S. mansoni genome is thought
to contain some 14,000 predicted genes [18,19]. Until
recently, there have been relatively few attempts to analyze
the functional genomics of schistosomes due to limited
genomic sequence information. Identification of genes
expressed in a sex-specific manner by adult worms was made
using either a 576, 7,335, or 4,608 oligonucleotide cDNA
based microarray system [20-22] and an analysis of genes
associated with pairing of adult male and female worms was
addressed [23]. The transition between miracidium and
mother sporocyst was also analyzed using the same 7,335
component oligonucleotide array noted above [24].
Comparisons of adult transcripts of two schistosome species, and
comparison of lung stage versus experimentally produced
schistosomula, were carried out using a larger array [7,25]. A
comparative study between the S. japonicum proteome and
transcriptome was also undertaken [26].
Now that the genome is nearing completion, we present a
comprehensive analysis of gene expression during three of
the major stages of S. mansoni development: daughter
sporocyst, cercaria, and adult. These stages represent key
transitions from intermediate host snail to aquatic environment to
mammalian host. We designed a 12,000 oligonucleotide
microarray chip made of 45-50-mer oligonucleotides and we
analyzed expression of a majority of the predicted 14,000 S.
mansoni genes under very stringent conditions. While the
present annotation of the genome does not distinguish gene
insert orientation, we found that over 9,700 of the
oligonucleotides printed yielded clear transcriptional signals in at least
one parasite stage. We have examined pair-wise differential
expression at each stage and identify those genes whose RNA
expression profile is conserved or differentially expressed
across developmental stages. Noteworthy gene clusters that
support previous hypotheses or provide new insights into the
responses of the parasite to environmental transitions during
the lifecycle are identified. Highly expressed transcripts in
sporocysts include those involved in general protein synthesis
and quality control, consistent with the function of sporocysts
in production of large numbers of cercariae. Cercarial
transcripts are dominated by genes involved in mitochondrial
function, supporting the energy production necessary for
cercarial swimming. Adult worms express a diverse pattern of
transcripts necessary for egg production, energy metabolism,
immune evasion and physiological maintenance of a
relatively long-lived organism.
Results
The 12,000 element schistosome array
The microarray used in this study contained 12,000
individual 45-50-mer oligonucleotides based on 11,998 tentative
consensus sequences (TCs), as documented by the
Schistosoma mansoni Genome Index maintained at The Institute for
Genomic Research (TIGR) [27]. TCs are created by virtual
assemblage of full or partial cDNA sequences into transcripts.
Spliced forms of genes are listed separately. Of the 12,912
sequences provided by TIGR, 11,998 TCs were chosen based
on the maximum size of the TC available. Thus, this
microarray contains a majority of known sequences available in the S.
mansoni genome. There have been concerns raised about the
orientation of inserts in the current annotation of the TIGR
assemblage; however, we found that >9,700 of the 12,000
oligonucleotides printed gave some transcript signals in one or
another parasite stage by type II analysis (see Materials and
methods) and, as detailed below, many key gene programs
could be identified and correlated with environmental
transitions.
The microarray chip is specific for S. mansoni transcripts
Extraction of RNA from schistosome daughter sporocysts
involves excision of the entire snail hepatopancreas, and
colysis of the snail and schistosome material. To control for any
background hybridization of snail-specific material (to our S.
mansoni cDNA-based 12,000-oligonucleotide microarray),
we hybridized hepatopancreas RNA from infected snails
containing daughter sporocysts versus uninfected snail
hepatopancreas RNA across four different chips. With the exception
of two genes, we found that intermediate host snail RNA was
not detected (data not shown). Even for these two genes
(TC8129, similar to beta-1 4-galactosyltransferase, and
TC6896, similar to Unknown), hybridization signals were not
seen on every run and were detected only by type II analysis.
In summary, no significant contribution of snail RNA to the
hybridization analysis was found. Daughter sporocysts
contain cercariae at various stages of differentiation.
Nevertheless, the transcriptome profile of this stage in the snail
differed significantly from that of mature aquatic cercariae.
We also found that microarray samples and chips were
reproducible and correlative. There was an average Pierson
correlation coefficient (log ratio of medians) of 0.7 for biological
replicates and 0.96 for technical replicates. Dye switch
experiments showed that data differences were not due to
dye-labeling bias (Additional data file 1).
S. mansoni transcriptome overview
This clustering analysis includes 431 genes analyzed by
pairwise comparison with at Examination of all developmental
stages showed a transcript pattern specific to each. Figure 2
(Table 1) is a clustering analysis of the major transcripts
identified in which duplicates of sporocyst (Cy5) were compared
to cercariae (Cy3). For example, cluster 1 represents
transcripts that are upregulated in the daughter sporocyst stage
but not significantly in adults or cercariae. Cluster 2 shows
transcripts up-regulated in both sporocysts and adults
relative to cercariae. Clusters 3 and 4 show transcripts enriched
in cercariae relative to adults. Clusters 5 and 6 show
transcripts that are upregulated in adult worms but are either
relatively low or not enriched in cercariae or sporocysts. least a
R65.4 Genome Biology 2007,
Spo V Cercs
(a)
(b)
Adults V Cercs
(c)
(d)
Cluster 1
Cluster 2
Cluster 3
Cluster 4
Cluster 5
Cluster 6
SFmciaghjiuosrtoegseo2nmeecltursatnesrcsripts are developmentally regulated and divided into six
Schistosome transcripts are developmentally regulated and divided into six
major gene clusters. Genes are differentially expressed in sporocyst/
cercarial pair-wise comparisons (columns (a) and (b), 'Spo v Cercs') or
adult/cercarial pair-wise comparisons (columns (c) and (d), 'Adults v
Cercs'). Genes are highly enriched (three-fold) in sporocysts relative to
cercariae (clusters 1-3), in cercariae relative to adults (cluster 3 and 4),
and in adults relative to cercariae (clusters 2, 5, and 6).
3-fold difference in transcript level between developmental
stages. Overall, we identified enrichment of 1,154 genes
during the schistosome developmental life cycle, the largest
proportion of which is expressed in adult worms (type II
analysis). Of the 1,154 genes, 406 represent genes of unknown
function (Figure 3).
Validation of microarray data by real time PCR
To test the validity of the microarray results, we performed a
quantitative analysis study on the expression levels of several
parasite genes by real-time PCR. Among the transcripts
analyzed were actin, cathepsin B, cytochrome C, eggshell protein
precursor, Sm23 and cathepsin L. Gene-specific primers were
designed to validate the gene expression profile for sporocyst,
cercariae, and adult developmental stages (Additional data
file 2). To ascertain gene expression transitions between
cercariae and adults, we also analyzed transcript levels in
24hour schistosomula, the stage of schistosomal development
between cercarial penetration and adult worms. We found
Sporocysts
158 (29)
47 (8)
78 (38)
71 (12)
16 (2)
1,154 Gene transcripts
sVFceihgniusntrodesiao3gmraemdeovfeklnoopwmnengtene transcripts enriched (1.5-fold) during
Venn diagram of known gene transcripts enriched (1.5-fold) during
schistosome development. The number of unknown genes associated with
each section is in parentheses.
R65.6 Genome Biology 2007,
Some of the major transcripts from clustering analysis of the microarray data
Transcripts enriched in specific schistosome stages are organized into six major clusters. For an updated list of the latest version of TC sequences
see Additional data file 8.
that the expression of genes analyzed by RT-PCR correlates
with our microarray analysis in 10 of 11 cases (Figure 4).
elastase [
32
]. In general, fewer transcripts are found in
cercariae relative to other stages.
Genes expressed in daughter sporocysts reflect production of cercariae
The daughter sporocyst stage resides in the intermediate host
snail. The key biological function of this stage is to support the
differentiation and development of large numbers of
cercariae, the aquatic larval stage that will initiate invasion of the
mammalian host. The function of this stage in generating
cercariae is underscored by the fact that many of the most highly
expressed transcripts are gene products that function in
general protein synthesis (40S and 60S ribosomal subunits,
elongation factor) and post-translational protein folding and
fidelity (chaperones, ubiquitin). Furthermore, many of the
proteins that will be utilized by cercariae in the initial stages
of host skin invasion are also produced in this stage, including
cercarial elastase (aka cercarial protease), cercarial muscle
proteins (actin, dynein light chain isoforms), and calcium
binding proteins that are also abundant in the proteome of
cercarial secretions [28].
As would be expected from the large-scale differentiation of
cercariae within the daughter sporocyst, transcription factors
such as the Y-box binding protein and stathmin, a phosphoryl
protein involved in vertebrate growth and regulation of
differentiation [29] are highly expressed. Sporocysts express
a cathepsin L-like cysteine protease homolog (TC9217) that is
not significantly enriched in adults (Figure 4f). In adults a
cathepsin L is gut localized and functions in digestion [30].
The lack of significant enrichment of other gut-specific genes
in sporocysts, such as those encoding cathepsins B, B1, and C,
and the unique profile of the cathepsin-like protease
homolog, suggests that this protease isoform may function
differently in the daughter sporocyst, possibly in the
generation of cercarial progeny. In Caenorhabditis elegans, a
cathepsin L is essential for embryogenesis and development [31].
Genes expressed in cercariae reflect energy production and motility
Cercariae are a relatively short-lived 'transitional' stage that
are released from snails into fresh water and must swim to
find, and ultimately invade, a mammalian host. Cercariae will
not survive if they fail to enter the mammalian host before
energy sources are exhausted. Consistent with this concept of
the biological function of cercariae, transcripts that are
upregulated are primarily factors necessary to sustain
swimming behavior and invasion (Figure 2, clusters 3 and 4). The
highest expressed transcripts are genes involved in
mitochondrial function or energy metabolism. These include NADH
dehydrogenase and its various subunits, cytochrome C and its
homologues, and ATP/ADP carrier proteins. Other
transcripts highly expressed in cercariae include structural and
motility genes like actin and fibrillin and transcripts coding
for a protease that plays a role in host invasion, cercarial
Genes expressed in adult worms
Compared to sporocysts and cercariae, the most abundant
transcripts in adult worms (male and female worm pairs) are
significantly more diverse. This reflects the fact that adult
worms must evade the mammalian host immune system,
maintain motility, acquire and metabolize a variety of
nutrients, form mating pairs, and produce large numbers of eggs.
In parallel with these functions, highly abundant transcripts
in adult worms include a group of genes involved in protein
degradation in the gut (cathepsin B, cathepsin L), egg
production, and oxidative stress responses [
33-36
]. Genes involved
in gonadal differentiation for both male and female worms, as
well as genes coding for proteins for egg-associated proteins
are readily identified. These include the anti-mullerian
hormone receptor for males, and members of the RXR family and
Smad family, which are cell signaling pathways previously
associated with function of female gonads [
37,38
]. Highly
expressed egg transcripts include eggshell protein precursor,
major egg antigen, and several homologues of these proteins.
Perhaps most striking is the abundance of transcripts in adult
worms from genes coding for surface proteins, including
nutrient transporters such as the glucose transporter and a
number of surface proteins of unknown function previously
identified in research projects aimed at subunit vaccine
development. These include Sm23 [
39-45
], integral membrane
protein 25 [
46
], Sm14 [
47,48
], and 26 and 28 glutathione
Stransferases [
49-52
].
Discussion
The stage-specific transcriptome of S. mansoni provides an
informatics foundation for the study of parasite gene
regulation and a correlative for proteomic studies. This
microarray study validates and extends observations made
with a 7,335-oligonucleotide array from previously available
expressed sequence tags (ESTs) [
53
], and correlates well with
comparative analyses between the transcriptome and
proteome of adult Schistosoma japonicum and S. mansoni [26].
With 12,000 oligonucleotides, a more complete picture of
gene programs that mark transitions between key
schistosome stages in distinct environmental niches is now
apparent. The entire schistosome genome is estimated to have
14,000 functional genes. While it has been noted that the
TIGR annotation to date has not distinguished the
orientation of the inserts sequenced, we found that 9,700 of the
12,000 genes analyzed on this array gave clear positive
signals for at least one stage. More importantly, many genes
could be functionally annotated and gene programs
correlating with environmental transitions of the helminth parasite
discovered or validated.
Actin
S C L A
(sRFAtieag)agulertsi:ems4peoPrCoRcyastnsal(ySs)i,scoefrcsearleiacet t(Cra)n,ssccrhipisttsoascormousslafo(Lu)r, adnedvealoduplmtewnotramls
Real time PCR analysis of select transcripts across four developmental
stages: sporocysts (S), cercariae (C), schistosomula (L), and adult worms
(A). The upward arrow indicates transcript level where fewer RT cycles
are indicative of an increase in transcripts relative to a cytochrome c
internal control. The microarray ratio of sporocyst to cercaria (SvC) and
adult to cercaria (AvC) is designated 'fold change' and is in the same
format as Figure 2. Cathepsin L numbers are from type II analysis.
One of the most remarkable aspects of the schistosome
parasite is the dramatic morphological change that takes place
between life cycle stages (Figure 1). These morphological
changes parallel transition of the parasite between three
markedly different environments. The daughter sporocyst
stage is the late developmental stage in the intermediate host
snail - a mollusk of fresh water habitats. Motile cercariae
emerge from the snail and must navigate an aquatic
environment to find the mammalian host. Following entry into the
skin of the mammalian host, the parasite must now adapt to
a different osmotic environment and a warm-blooded host.
Finally, within the mammalian host, two distinct sexual
stages develop and eggs are subsequently produced.
Array analysis comparing daughter sporocysts to the cercarial
stage highlighted gene programs responsible for supporting
larval development. The sporocyst stage is a very efficient
protein synthesis factory. This correlates with the need for
mass production of cercariae and the availability of nutrients
from an intimate relationship with the snail host. In keeping
with the complex morphological differentiation of cercariae
with distinct body parts and 'organs' from an embryonic cell
mass, daughter sporocyst transcripts included genes involved
not only in general protein synthesis such as ribosomal and
heat shock genes, but also transcripts annotated to function
in programmed cell death (TC18024) and ubiquitination, a
process key to morphological differentiation.
In Figure 2, cluster 2, daughter sporocysts share with adult
worms a previously unrecognized requirement for
transcriptional upregulation of antioxidant genes such as
glutathioneS-transferase, thioredoxin, and thioredoxin peroxidase. This
may reflect oxidative stress generated within the immediate
snail host, or storage of these proteins in developing
cercariae, so that they are available in the earliest stages of
mammalian host invasion. Utilizing a 7,335 oligonucleotide array,
Vermeire et al. [24] documented gene expression patterns
between miracidia, the aquatic stage that invades the snail
host, and the mother sporocyst stage, which is the initial stage
following invasion of the snail host. As is the case for daughter
sporocysts analyzed in our study, they also found
upregulation of several genes involved in protein synthesis, the redox
pathway, and proteolysis. This suggests that these gene
programs are initiated in mother sporocysts following entry of
miracidia into the snail, and sustained throughout the
daughter sporocyst stage until mature cercariae leave the snail.
Transition to the cercarial stage is marked by a reduction in
transcript level of a large number of genes as seen in clusters
1 and 2 of Figure 2. We found 116 genes whose abundance is
reduced in cercariae by 3-fold relative to sporocysts (type II
analysis where sporocyst intensity units are set >3,000). In
comparison to other stages, the cercariae are less
transcriptionally active. Fewer than 7 genes are upregulated more than
2-fold relative to adults and only 34 are upregulated when
compared pair-wise to adult transcripts.
The cercariae express a cluster of genes consistent with the
energy required to move rapidly through water in search of a
mammalian host. These include genes functioning in ATP
production and utilization, presumably for muscle function
and swimming behavior. Consistent with previous Northern
blot analyses and biochemical studies, much of the repertoire
of proteins that cercariae use to invade the skin of their host,
as well as structural proteins, have already been produced
during cercarial development in the daughter sporocyst stage
within the nutrient rich intermediate host snail (Figure 2a-d).
Cercariae do express caspases and related cell death
programs, which may be required for morphological remodeling
during transition into the schistosomulum stage. Cercariae
detach their motile tail and surface glycocalyx shortly after
entry into the mammalian host. Large gland structures
(acetabular glands) producing invasive proteases involute
within 48-72 hours of invasion.
Residence of adult schistosomes in the mammalian host
bloodstream is supported by expression of gene families that
respond to oxidative stress and genes involved in adjustment
and adaptation to a new osmotic environment (aquaporin)
[
54-56
]. Adult worms have a major requirement to digest
blood-proteins and acquire other nutrients (cathepsin B,
glucose transporter) [
33,35,57
]. Schistosomes differentiate into
male and female worms and expression of an anti-mullerian
hormone receptor [
58,59
] is likely related to sexual
differentiation. Female parasites produce hundreds to thousands of
eggs per day as reflected in the upregulation of egg shell
proteins. Recent analysis of gene expression in earlier
intravascular stages can now be compared with the adult (versus
cercariae) transcriptome presented here. Using a cDNA
array, Dillon et al. [60] equated seven-day cultured
schistosomula with lung schistosomula and compared gene
expression across life stages. Chai et al. [7] utilized schistosomula
directly obtained from the lungs of infected mice, and
compared transcripts expressed to adult worms, cercariae,
and newly transformed schistosomula. By and large, the
genes we found expressed in adult parasites versus cercariae
were similar to those observed in the comparison of lung
worms versus cercariae. These included nutrient acquisition
genes, such as those encoding the glucose transporter and the
proteolytic cathepsins. The genes down-regulated in adult
worms relative to lung schistosomula include several we
found expressed in cercariae or daughter sporocysts,
including those encoding the anti-inflammatory protein Sm16 and
paramyosin.
The intravascular stages of schistosome parasites have a
complex and highly adapted relationship with the mammalian
host [
61,62
]. To support this relationship, intravascular
stages upregulate surface proteins or receptors, some of
which have homology to mammalian receptors and/or factors
involved in signaling cascades (Additional data file 6). These
include the anti-Mullerian hormone type II receptor [63] and
the thyroid receptor interacting protein [
64
].
Developmental regulation of cell number and type by
programmed cell death appears to be an important function in all
stages of schistosome development analyzed, as it is in the
nematode C. elegans [
65
]. Homologues for genes involved in
programmed cell death include TC11294 and the DAP-1
homolog TC18024 (type II analysis).
Since the initial stages of the genome analysis of
schistosomes, it has been clear that transposon-like sequences are
common. Efforts to exploit these elements for genetic
manipulation are ongoing [
66,67
]. It is noteworthy that one
transposon (TC17720) is more abundantly expressed in the
sporocyst than in any other developmental stage. It is also
expressed in adults, albeit at low levels, as corroborated in the
study by Gobert et al. [25]. There is also another retroelement
(TC7011) highly enriched in adult worms.
This expanded array data set, focusing on life cycle stage
transitions, should aid in current attempts to develop transfection
and gene knockout studies for schistosomes by identifying
those genes that are stage-specific versus others shared
among different developmental states. For example, the gene
encoding cytochrome C is expressed at comparable levels in
all stages studied. In contrast, genes such as those encoding
the cercarial elastase in the sporocyst stage, or eggshell
components in the adult female stage, represent potential models
for identifying and characterizing key spatial and temporal
promoter elements, and ultimately molecular mechanisms of
gene regulation. Fitzpatrick et al. and Moertel et al. also have
studied gender-specific gene transcription in S. mansoni
using a 7,335 oligonucleotide array [
53
] and a 22,575
combined S. japonicum and S. mansoni array [
68
]. The results
reported here for genes that can be attributed to either male
or female worms largely validate these studies.
Analysis of the stage-specific transcriptional program of
schistosomes also helps to validate and underscore
differences in the levels of gene products noted in proteomic
studies of schistosome life cycle stages. The levels of many gene
products identified by proteomic analysis parallel
transcription levels in this microarray analysis. In contrast to
protozoan parasites like Leishmania [
69
], this may indicate that
less post-transcriptional regulation operates in schistosomes.
Having transcriptome data for three major stages, combined
with proteomic data, should now facilitate a more focused
analysis to determine to what extent RNA stability or other
post-transcriptional mechanisms play a role in schistosome
gene regulation. It was noted from proteome analysis that
many genes are expressed across stages and these were,
therefore, seen as less attractive vaccine targets [
26,70
]. The
identification of several stage-specific patterns of expression
in this present study should help to redirect efforts aimed at
finding the most logical candidates for a subunit vaccine, and
also identify new targets to explore for drug therapy.
Conclusion
We show that the daughter sporocyst stage in the
intermediate host snail functions primarily to support the development
of invasive larvae (cercariae) by up-regulating expression of
genes involved in protein synthesis, cellular differentiation,
and programmed cell death. Many of the major structural and
functional components of cercariae, utilized to later invade
the mammalian host, are expressed and packaged prior to
larval release from the snail. The aquatic cercariae themselves
are less transcriptionally active than other stages, with an
emphasis on production of proteins involved in energy
metabolism and motility. This is in keeping with the function
of these larvae in swimming from snail to mammalian host.
Finally, adult parasites, which have adapted to survival in the
mammalian host bloodstream, have a complex
transcriptional program that supports adaptation to a new host
temperature and chemical environment, evasion of the host
immune response, acquisition of nutrients, and production of
eggs for transmission to a new host.
Materials and methods
All experiments were performed with S. mansoni of the
NMRI Puerto Rican strain maintained routinely through
Biomphalaria glabrata snails and Syrian golden hamsters as
previously detailed [
71
].
Schistosome stage collection
Daughter sporocysts were collected by dissection of whole
hepatopancreas from six-week old infected B. glabrata
snails, during maturation of daughter sporocysts and prior to
cercarial release. Uninfected snail hepatopancreas was also
collected as a control. Cercariae were collected in distilled
water from infected B. glabrata snails using the light
induction method as previously described [
71,72
]. Following
exposure to light for 2 hours, 50-60 snails shed about
200300 cercariae/snail. Several collections were pooled and used
for RNA extraction. Twenty-four hour schistosomula were
mechanically transformed as previously described [73]. Adult
worms were recovered from the mesenteric veins by portal
perfusion as previously described [
74
].
RNA extractions
Total RNA was extracted from all biological samples for
Combimatrix microarray analysis. Multiple uninfected and
infected hepatopancreas, cercariae, and adult worms were
homogenized in 1 ml Trizol (Invitrogen, Carlsbad, CA, USA)
and RNA was extracted using the standard Invitrogen
protocol. RNA was eluted in deionized water, quantified, and
checked for RNA quality by UV spectrometry and agarose gel
analysis. Total RNA (15 μg) for individual stages (coupled to
Cy3 or Cy5) was hybridized to Combimatrix custom array
microarray chips, as described in detail below (Combimatrix
Corporation, Mukilteo, WA, USA).
S. mansoni DNA oligonucleotide probe design
All probes were designed using the Combimatrix software
CombiMatrix Automated Probe-design Suite (PDS), based on
11,997 TC groups from sequences at TIGR. TCs represent a
virtual assemblage of ESTs. Of the 12,717 TIGR TC groups,
11,998 were used for the 12,000 spot array based on size of the
TC source cDNA. Those with the smallest TC source cDNA
sequences were excluded. The 12,000 sequences were loaded
into the Combimatrix Custom Array content probe array
system for probe design, one of which was spotted in triplicate
using different oligonucleotide designs constructed by hand.
The probe design system took each TC sequence and designed
45-50-mer probes to be unique to each gene with predictable
thermodynamic behaviors.
Microarray hybridization and data analysis
Combimatrix Custom Array microarray chip hybridizations
were stringently performed in duplicate or triplicate.
Hybridizations (3× SSC, 0.025 M HEPES, 0.28 μg/μl polyA, 0.05%
SDS) occurred at 63°C for 18 hours to maintain specific
binding efficacy. DNA microarray chips were scanned using an
Axon4000B scanner at 5 μ resolution and 100% laser power
and the images were analyzed with GenPix Pro 4 (Molecular
Devices, Sunnyvale, CA, USA). Microarray data were stored
in the NOMAD microarray database [
78
] and normalized by
a global normalization using unflagged features with a
regression correlation coefficient ≥0.75 and median intensity value
>0 as previously described [
75
]. Data were clustered using
Cluster 3.0 [
76
] and visualized in Java TreeView version
1.0.11 [
77
]. Clustering was done as below: all data were log
transformed and filtered for the presence of 100% of genes
and a standard deviation value of log2 1.5 or 3-fold gene
expression, followed by correlation uncentered clustering of
genes and arrays by complete linkage analysis. A second and
less stringent analysis was repeated using the same
methodology, except clustering was done with a standard deviation
value of log2 0.6, or 1.5-fold gene expression (Additional data
file 3). Each Combimatrix array has 12,500 spots; 148 are
empty controls (no oligonucleotides) and 352 represent
various small oligonucleotide controls. For a less sophisticated
analysis of gene expression, on or off, each channel (635 and
532) was treated independently. The data were reanalyzed
and normalized as described above. The average median
value of the 148 empty spots for each chip was used as
background and subtracted from the initial intensity unit value to
produce a 'normalized value'. Spots from the 'normalized
value' (IU) were retained as expressed genes. We refer to this
type of analysis as type II analysis. To compare all three
stages, the average median of all cercariae median values was
used as a standard (Additional data file 4). Transcripts with
an IU >2,000 are defined as enhanced.
RT-PCR
A total of 1 μg parasite RNA from each stage was used to
prepare double stranded cDNA using SuperScript™ II reverse
transcriptase (Invitrogen) in the presence of oligo dT.
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