Intron length distribution
During annotation it was observed that many S. mansoni genes show a bias in their intron
sizes, with the size of introns markedly increasing from the 5’ end to the 3’ end. In particular,
small introns (<30 nt) are numerous at 5’ ends and introns of several kilobases are numerous
at 3’ ends of transcripts. To test this distribution, we extracted intron sequences from genes in
the S. mansoni genome annotation (v4). A large number of genes span gaps, preventing an
accurate determination of intron size. The analysis was, therefore, limited to 5,906 gene
models (5,890 introns; Supplementary Table 4), in which the start and stop codons for a gene
were present within a single contig. Despite limiting the analysis to approximately one-half
of the total predicted gene set, a size bias can clearly be seen (Figure 2, Supplementary Table
5) for introns at positions 1-6, relative to the 5’ or 3’ ends of each transcript. Because genes
with different sizes were compared directly alongside each other, the signal of skewed size
distribution is overtaken by noise after the first 5 or 6 introns.
The reason for the distribution remains to be determined but a recent study of human-
chimpanzee orthologue pairs found that more 5' introns are longer than 3' introns, because
they contain more regulatory elements1. Using version 53 of the Ensembl database2, we
extracted intron sizes from the C. elegans (release 190) and human genome (NCBI 36)
annotation (Supplementary Table 4) and confirmed that In both C. elegans and human, there
is a pronounced but opposite bias with considerably larger introns at the 5’ end of each
transcript (Supplementary Table 5).
Analysis of putative transcription-associated proteins (TAPs)
Schistosome transcription factor families were identified by searching against a customised
transcription factor database and clustered using the TRIBE-MCL algorithm. The clustering
analysis detected 362 protein families containing Schistosoma sequences, including 27
singletons, defined as clusters containing only a single member (Supplementary Fig. 2). The
335 S. mansoni TAP clusters were analysed with respect to the origin of the members from
the other 18 metazoan genomes queried with the HMMs (Supplementary Table 6). On
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average, 43% of the clusters contain chordate sequences, contrasting with nearly half that
number for nematodes (27%). Furthermore, the schistosome DNA-binding proteins appear to
display the greatest similarity with those of pufferfish (49% and 47% of the TAP families
have members originating from Tetraodon nigroviridis and Takifugu rubripes, respectively).
Evolution of distinct tissues
Schistosomes possess all the components of the Scribble, Par and Crumbs complexes3 that
interact directly with the cytoskeleton to specify where adherens and septate/tight junctions
form, thus controlling cell polarity (Supplementary Table 9). The presence of Claudin and
Zonula Occludens-1, critical proteins of the septate/tight junction, suggests that schistosome
epithelia are organised in a similar fashion to Drosophila while genes encoding other septate
junction proteins, e.g. Neurexin IV, Neuroglian, and Gliotactin4 accord with the ultrastructural
descriptions of septate structures in sensory nerve endings5. Cell-cell contact interactions
responsible for denoting tissue boundary lines may be specified by Notch signalling, and
homologues of the Notch-processing protease, Kuzbanian, are present, while the array of at
least ten Notch/Delta-like genes may be linked to the morphological diversity of distinct
phenotypes in the life cycle.
Schistosomes possess transcription factors (Supplementary Table 9), which, in insects
and vertebrates, mediate neurogenesis and act to pattern the nerve cord, supporting the ancient
origins of neural patterning6. These genes, well studied in vertebrates, act in concert with
Max-interacting protein upon neurogenin to influences Delta and promote lateral inhibition;
thus cells are selected from a pool of neural progenitors to become neurons. The use of
attractive or repulsive guidance cues in nervous system development, best characterised in C.
elegans7, directs nerves to their synaptic partners. In addition to Netrin and its membrane
receptor UNC-58, homologues of the antagonistic slit and roundabout proteins indicate that
schistosomes have all the tools needed to control axon growth cones and migration of neural
cells.
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Nuclear receptors
Nuclear receptors (NRs) are important transcriptional modulators in metazoans, which
regulate homeostasis, differentiation, metamorphosis and reproduction. There are 21 members
found in S. mansoni, more than half of them evolved from a second gene duplication, which is
independent from that of vertebrates. The data challenge the current theory of NR evolution9.
Two NRs are homologues of the thyroid hormone receptor that previously were thought to be
restricted to chordates10. Interestingly, a novel family of NR was identified in S. mansoni and
subsequently demonstrated to exist in other platyhelminths, mollusks and crustaceans9. In S.
mansoni there are three members of the family that each possesses two DNA-binding
domains (DBD) in tandem followed by a ligand-binding domain. The unique structure of
having two DBDs suggests a novel mechanism for regulation of target gene(s).
Redox biochemistry
Schistosome parasites reside in an aerobic environment and are vulnerable to damaging
reactive oxygen species, produced by their own aerobic respiration, as well as by the host
immune assault and schistosome redox enzymes have been shown to play a protective role11.
The redox biochemistry of S. mansoni is similar to but condensed relative to human pathways
(Supplementary Table 10 and Supplementary Fig. 3). Superoxide generated by the partial
reduction of molecular oxygen is converted to H2O2 by superoxide dismutase; H2O2 is
primarily reduced by peroxiredoxins, with electrons supplied by either thioredoxin or
glutathione12, which are reduced by a single, multifunctional protein thioredoxin glutathione
reductase, an essential protein and promising target for antischistosome drug development13.
Many redox proteins commonly found in other eukaryotes are apparently absent from the
genome (Supplementary Table 10). In addition, NADPH oxidase (superoxide generation) and
nitric oxide synthase (NO generation) are absent from the S. mansoni genome. Although there
are reports indicating the presence of nitric oxide synthase in worms14,15, diaphorase activity
in the parasite attributed to nitric oxide synthase could potentially be due to the activity of
ferredoxin NADP(H) oxidoreductase16 or perhaps to the putative cytochrome P450 reductase
(Smp_030760). NADPH oxidases are widespread in nature and are thought to have emerged
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before the divergence of fungi, plants and animals17; their apparent absence from the S.
mansoni genome is remarkable.
Lipid Metabolism
Lipid metabolism is an area in which previous biochemical studies have shown
schistosomes to be deficient but a genetic explanation for their inability to synthesise sterols
or free fatty acids (FFA) de novo was lacking18. The mevalonate pathway, a precursor to
steroid synthesis, is completely represented in the S. mansoni genome (Supplementary Table
11). However, there is scant evidence for the presence of enzymes that convert its products,
via squalene and lanosterol, to sterols19,20. Similarly, S. mansoni’s inability to synthesise FFA
is confirmed by loss of genes encoding both the cytosolic and mitochondrial fatty acid
synthase complexes. Only an acyl carrier protein, acetyl-CoA carboxylase and the
mitochondrial enzyme beta-ketoacyl synthase are present. As schistosomes can elongate
existing FFA derived from the host, these genes may still perform a function, acting in
concert with several homologues of yeast fatty acid elongation proteins. It should be noted
that the usually biochemically inactive beta oxidation pathway, for which S. mansoni
possesses a complete complement of requisite enzymes, might operate in reverse to perform a
similar function21.
As compensation, schistosomes do have the enzymes necessary to incorporate FFA or
sterols into, or derive them from, more complex lipid molecules. It appears that S. mansoni
depends upon a plethora of receptors and transport proteins dedicated to lipid uptake from the
host: at least three low density lipoprotein and two high density lipoprotein receptors for the
uptake of esterified-sterols, triacylglycerol (TAG) and phospholipids; at least four isoforms
of saposin, a protein involved intracellular lipid transfer and the hydrolysis of sphingolipids in
lysosomes; cholesterol and glycosphingolipid trafficking Niemann–Pick type C proteins;
several phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine transfer
proteins; and previously identified LDL receptor molecules8, complete the schistosome’s
extensive lipid carrying protein repertoire.
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Two isoforms of sterol esterase and several phospholipase homologues may provide
alterative sources of FFA, cholesterol or esterified intermediates such as lysophosphatidic
acid for membrane synthesis. In regard to esterified lipid precursors, it should be noted that
only one, glycerol-3-phosphate O-acyltransferase, a microsomal isoform22, appears capable of
producing lysophosphatidic acid from FFA and glycerol 3 phosphate, the mitochondrial
isoform is absent. TAG plays an uncertain role in the schistosome’s life cycle, despite
constituting 40% or more of the lipid content of adult worms18. They are slow to turn over, do
not contribute to the formation of other lipids such as phosphatidylcholine 18 and the apparent
inactivity of beta oxidation makes their use as an energy store doubtful21. Nevertheless, S.
mansoni possesses lipases capable of breaking TAG down, so the molecule may have
functions beyond preventing intracellular FFA concentrations from rising too high18.
Pathways responsible for synthesizing the phospholipid components of membranes are
well represented with two notable exceptions. Phosphatidylcholine can not be converted from
phosphatidylethanolamine but must be derived from diacylglycerol23 and, lacking inositol-3-
phosphate synthase, the parasite must depend on its host as source of inositol. Unlike
phosphatidylcholine, phosphatidylserine synthesis proceeds via a pathway similar to that
utilised by mammals. There is no evidence that it can be synthesised directly from
diacylglycerol.
The kinome and signalling
The TGF beta signalling pathway has been shown to play a potential role in host-
parasite interactions by transducing a signal from host TGF beta 1, via a parasite signalling
pathway, to regulate gene expression24 and reproductive development25. In schistosomes the
TGFβ pathway has been conserved and like Drosophila melanogaster and Caenorhabditis
elegans26, there are a limited number of members that are responsible for the diversity of
genes that are regulated. To date, two members of the schistosome TGFβ family of ligands,
two type II TGF- beta receptors, four type I TGF- beta receptors SmTβRI and three activin
receptor-like type I kinases, five members of the schistosome Smad family, and ten
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scaffolding/regulatory proteins and two target genes have been identified27 (Supplementary
Figure 5 and Supplementary Table 15). Both the TGF beta /Activin and BMP pathways are
represented by ligands and Smads. The type II and type I receptors for the BMP pathway have
not been identified.
In S. mansoni, few signalling molecules have been characterised, beyond those of the TGFβ
pathway. The study of the kinase complement (kinome) is of major importance for
understanding Schistosome physiology and provides insights into how to disrupt its
interactions with its host. The S. mansoni genome encodes 249 kinases, including 22 genes
with alternative splicing. A dendrogram using the kinase domain shows that the main
schistosome groups CAMK (calcium/calmodulin-dependent protein kinase), AGC, STE
(homologues of yeast sterile kinases) and CMGC are well separated (Supplementary Fig. 8),
although several kinase families fell outside of these clusters. All of the main CMGC families
are conserved between yeast, nematodes, mammals and S. mansoni, with the exception of the
RCK family, which is absent from S. mansoni and yeast. The S. mansoni MAPK include
representatives of ERK (extracellular signal–regulated kinase) and JNK (c-Jun NH2-terminal
kinase) types of MAPK, but appears to lack members of the p38 type (also not identified in S.
japonicum28). However, the presence of MAP kinases responsible for the activation of p38
(in humans and other species), suggests that this pathway (Supplementary Fig. 9) is active but
may have a different activator of the effector proteins, perhaps JNK. The second largest S.
mansoni kinase group, the AGC family, contains representatives of all family members
present in C. elegans and D. melanogaster. Interestingly, the most highly transcribed kinases
are 3 members of the AGC group that belongs to the GRK family of G protein signalling
regulators29 (Supplementary Table 16 and Supplementary Figure 8).
The least represented groups observed within the schistosome genome are the casein
kinase (CK1) and Receptor Guanylate Cyclase (RGC) families with only 7 and 3 members,
respectively. In contrast, CK1 is the largest group in C. elegans with 87 members and RGC
has 27 members. The CK1 and CMGC group members expressed in sperm or during
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spermatogenesis in C.elegans, are missing in S. mansoni, perhaps indicating distinct
regulatory mechanisms in this species.
The STE kinases, which function in the MAP kinase activation cascade, are also
comparatively abundant in the schistosome genome. In S. mansoni, this group includes 7
STE7 (MAPKK) kinases, 4 STE11 (MAPKKK), and 13 STE20 (MAPKKK) kinases.
Crosstalk occurs between protein kinases functioning at different levels of the MAPK
cascade. The large number of STE family kinases could translate into an enormous potential
for upstream signal specificity and diversity. Vertebrate EGF activates S. mansoni EGFR and
the downstream classical ERK pathway, indicating the conservation of EGFR function in S.
mansoni. Moreover, human EGF has been shown to increase protein and DNA synthesis as
well as protein phosphorylation in parasites, supporting the hypothesis that host EGF could
regulate schistosome development30.
The Degradome
ESTs from GenBank, dbEST, and the Wellcome Trust Sanger Institute
(ftp://ftp.sanger.ac.uk/pub/pathogens/Schistosoma/mansoni/ESTs/) were aligned to the S.
mansoni genome using BLAT (a minimum score 30 and 90% sequence similarity) to obtain
information related to life cycle stage and adult worm gender from the original library
description31. Of the 335 protease sequences, 80% have at least one matching EST
(Supplementary Table 18).
Twenty-one percent of the degradome comprises inactive protease homologues (IPHs),
i.e., proteases that contain mutations in one or more of the critical active site residues
(Supplementary Table 18). These IPHs are found in all five mechanistic classes, and some
have already been confirmed as being actively transcribed32,33. Though IPHs constitute a
significant proportion of the total degradome in other organisms (e.g., 15% in humans34), their
function(s) is unclear. They may regulate or inhibit, perhaps by a dominant-negative effect,
protease activity or titrate endogenous protein inhibitors, thereby increasing overall
proteolytic capacity34,35.
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Metabolic chokepoints
A total of 607 enzymatic reactions could be placed in pathways and 120 of these
enzymes were identified as chokepoints based on the capability to uniquely generate specific
products or utilise specific substrates (Supplementary Table 21). As validation of the
approach, the list of chokepoints includes many that are drug targets in S. mansoni or other
organisms. For instance, in the mevalonate pathway, almost all reactions are chokepoints. The
3-hydroxymethylglutaryl-coenzyme A reductase (HMG-CoA reductase) is well characterized:
its inhibition is accompanied by a cessation of egg production by the female parasite and a
reduced ability of the parasite to properly glycosylate proteins 36. That the enzyme is
significantly different from its mammalian orthologue and that its inhibitor mevinolin, has
anti-schistosomal activity37, provides further validation for HMG-CoA reductase as a target.
The chokepoint enzyme, L-DOPA decarboxylase, plays an important role in catecholamine
biosynthesis in S. mansoni, decarboxylating 5-hydroxytryptophan and L-DOPA 38. The
therapeutic potential of the enzyme is suggested by the drug Methyldopa, which is commonly
used to treat hypertension and inhibits L-DOPA decarboxylase in mammals, and which
inhibits the enzyme activity in schistosome extracts38. Glycolysis contains 4 chokepoint
reactions; one of which, phosphofructokinase, has been extensively studied 39,40and the
relationship established between its inhibition by trivalent organic antimonials and the
inhibition of schistosome growth in vitro41,42
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Supplementary Figure 1. Idiograms of S. mansoni chromosomes 1-7, W and Z. S. mansoni BAC clones were mapped to the karyotype of S. mansoni by FISH. The solid black areas are heterochromatin and the open areas are euchromatin. The BAC clones are identified by BAC numbers. Arrows identify the approximate location of the BAC on the chromosome. The scale is an arbritary measure of chromosome length and allows positioning of the BAC.
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Supplementary Figure 2. S. mansoni Transcription Associated Protein (TAP) families displayed as a 2D-hierarchical clustering. The rows are labelled with species names and the columns represent clusters which have schistosome sequences as members. If a cluster doesn't contain any sequences from the other genomes queried, the 'cell' in the matrix is yellow and orange if it does. The darker the shade of orange, the greater the number of sequences in the genome (after normalising for genome size).
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H2O2
GSH Biosynthesis
L-Glutamate + L-Cysteine
γ-Glu-Cys synthase
γ -L-glutamyl-L-cysteine
GSH Synthase
Glycine
GSH
Gut
Hemoglobindigestion
O2-
SODsec(Cu/Zn)
GPx/Prx?
H2O
Fe3+
OH•
Absent:GSH ReductaseTrx ReductaseMsrASulfiredoxinNADPH oxidaseNO synthaseCatalase (1.11.1.6)1-Cys PrxGST classes (a, k, p, s, z)GPx (1.11.1.9)Heme oxygenase
Cytoplasm
Mitochondria
Grx(S)2 → Grx(SH)2
2 GSH GSSGTGR
Protein(SH)2 → Protein(S)2
H2O
Trx(SH)2
2 GSH
GSH
O2-
SOD (Mn)
H2O
GSSG
TGR
Trx(SH)2
Trx(S)2
FDR
Mitochondrial e- transport
SOD (Cu/Zn)
O2-
H2O2
TGR
Trx(S)2
Prx
Prx-SO2
Sestrin
?
Prx
H2O2
H2O2
Other GSH functions
Other Trx functionsFe2+
Supplementary Figure 3. Enzymes and proteins involved in redox metabolism in the S. mansoni genome. Proteins are shown in red boxes; reactive oxygen species are shown in yellow. Abbreviations: Trx, thioredoxin; GSH, glutathione; Prx, peroxiredoxin; TGR, thioredoxin glutathione reductase; SOD, superoxide dismutase; GPx, GSH peroxidase; Grx, glutaredoxin; FDR, ferredoxin NADP(H) oxidoreductase.
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Supplementary Figure 4. The conserved cys-loop region of the putative cys-loop family ligand-gated ion channels from S. mansoni. The top ten sequences are nicotinic acetylcholine receptor subunits, and the bottom four sequences are from the non-nicotinic cys-loop groups, potentially GABA, glycine or acetylcholine-gated anion channels. The alignment of this segment was generated with ClustalX 2.0.9 with default parameters.
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NogginChordin
BMPDAN
THBS1
Decorin
LTBP1
TGFβ
Smurf1/2
RhoA
PP2A
P107
E2F4/5
DP1
Pitx2
ROCK1
p70S6Ka
SP1
IDSmad1
Smad2
SARA
Smad4
ERK1
Smad4
Rbx1
Cul1
SKP1
ActivinRI
TGFβRI
BMPRII
BMPRI
TGFβRII
ActivinRI
Smad2
Noggin
Smad1BSmad1
Smad1B
ERK2
p70S6Kb
SmCBP1
GCN5
SmCBP2
Growth factorRas/ MAPK
+p
+p
+u +u
-p
+p
+p
TAK1, MEKK1
DAXX/JUK MAPK signaling pathway
Ubiquitin mediated proteolysis
DNA
Transcription factors, co-activators and co-
repressors
ActivinRI
Smad3Smad3
Proteins/enzymes found in S. mansoni
Proteins/enzymes not found in S. mansoni but in humans
GCP
DNA
FKBP12
ActivinRII
Supplementary Figure 5. Schistosoma mansoni TGF-beta Signaling Pathway. Diagram showing the TGF-beta pathway of S. mansoni. The components of the pathway (identified in Supplementary Table 15) found in the schistosome genome are shown in blue. The white boxes depict the components present in the human TGF beta pathway but not present in the S. mansoni pathway.
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Supplementary Figure 6: Relative size of the S. mansoni kinome. A total of 249 kinases were identified in the predicted proteome of the parasite. For comparison, the % of the total predicted proteome that codes for kinases is shown for: Plasmodium falciparum, Homo sapiens, Trypanosoma cruzi, Trypanosoma brucei, Caernorhabditis elegans, Leishmania major, Mus musculus (see Supplementary Methods for data sources).
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Supplementary Figure 7. Distribution of kinase groups in S. mansoni and model organisms. S. mansoni kinases were classified according to KinBase (www.kinase.com/kinbase). A complete description of the kinome classification can be found in Supplementary Table 16. The group classification of various kinases (PTK- Protein Tyrosine Kinase; TKL- Tyrosine Kinase Like; AGC- cAMP-dependent protein kinase/protein kinase G/protein kinase C extended; CaMK -Calcium/Calmodulin regulated kinases; CMGC- Cyclin-dependent Kinases and other close relatives; CK1 - Cell Kinase I; STE- MAP Kinase cascade kinases; RGC- Receptor Guanylate Cyclases; and Other) of C. elegans, D. melanogaster, S. cerevisiae and H. sapiens is shown.
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Supplementary Figure 8. Dendrogram of the kinases of S. mansoni. To construct the phylogenetic tree of the kinase domains of the S. mansoni kinome, a phylogenetic tree was created using Neighbor joining and bootstrapping with 500 replicates in CLC Main Workbench (CLCbio V. 4.1.1, Denmark) and viewed in FigTree V1.2 (http://tree.bio.ed.ac.uk/software/figtree/). The major groups are labeled and color coded: Blue CMGC, orange STE, red AGC, green CAMK, pink TK, grey CK1, black RGC, light blue TKL yellow Other, light pink Unknown. The arrows point to the three most transcribed kinases according to EST evidence (Supplementary Table 16).
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NGF
BDNF
NT3/4
EGF
FGF
FDGF
TNF
IL1
PASL
TGFβ
Ras Raf1
G12 Gap1mNF1
Cdc42/Rac
CASP
TRAF2
DAXX
TRAF6
GADD45 MEKK4
TAO1/2
GLK
HGK
HPK1
TAB1
TAB2
Tpl2/Cot
MEKK1
LZK
MUKMLTK
ASK2
CBEB
Elk-1
Sap1ac-Myc
NFAT-2
NFAT-4
C-JUN
JunD
AFT-2
Elk-1
p53
Sap1a
GADD153
MAX
MEF2C
HSP27
Tau
STMN1
cPDA2
MNK1/2
RSK2
PPP3C
PRAK
MAPKAPK
MSK1/2Cdc25B
NLK
ERK5
PP2CA MKP
PTP
MKK3
MKK6
PP5
NIK
IKK
HSP72
Evil
MKK7JNK
JIP1/2
MKK4
ARRB
FLNA
CrkII
JIP3
p120GAP
PKCRasGRP
RasGRF CNasGEF
RafB
Mos
Rap1
MEK1
MEK2
MP1
ERK
PTP MKP
CREB
PP2CBECSIT
ASK1
TAK1
c-fosSRF
CACN
Trk
EGFRs
FGFRFDGFR
TNFR
IL1R
FAS
TGFβR
CD14
GRB2 SOS
PKA
NFkB
PAK
MST1/2
GCK
MLK3
MEKK2/3
MEK5 SmNR4A
p38
AKT
DNA
DNA
DNA
DNADNA
HerterotrimericG-protein
Ca2+
IP3cAMP
DAG
+p
+p +p
+p +p
+p
+p
+p +p
+p-p -p
+p
+p
+p
+p
+p
+p
+p
+p
+p
+p
+p
+p +p +p
+p
+p
+p
+p
-p
-p
-p-p -p
-p -p
+p
+p +p
+p+p
+p
-p-p
Classical MAP kinase pathway
JNK and p38 MAP kinase pathway
Serum, cyto toxic drugs, irradiation, heatshock, lipopolysaccharide and
other stress
LPS
DNA damage
ERK5 pathway
Serum, EGF, reactive oxygen species or srk
tryosinkinase downstream
MAPKKKK MAPKKK MAPKK MAPK Transcription factor
p53 signaling pathway Apoptosis
Proliferation, differentiation, inflammation Cell
cycle
Wnt signaling pathway
Proliferation, differentiation
Phosphatidylinositolsignaling system
+p Proliferation, inflammation anti-apoptosis
Proliferation, differentiation
Proteins/enzymes found in S. mansoni
Proteins/enzymes not found in S. mansoni but in human
Supplementary Figure 9. Schistosoma mansoni MAPK Signaling Pathway. Diagram showing the MAPK pathway of S. mansoni. The components of the pathway found in the schistosome genome are shown in blue. The white boxes depict the components present in the human MAPK pathway but not present in the S. mansoni pathway.
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