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Virulence factors of schistosomes

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DOI: 10.1016/j.micinf.2012.09.001 Source: PubMed

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Microbes and Infection 14 (2012) 1442e1450

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Virulence factors of schistosomes

R. Alan Wilson*
Centre for Immunology & Infection, Department of Biology, University of York, Heslington, York YO10 5DD, UK

Received 17 April 2012; accepted 3 September 2012

Available online 11 September 2012

Abstract

This review considers whether the products of schistosomes in the mammalian host can be considered as virulence factors. These include: the
cercarial secretions used in infection, those of the migrating schistosomulum, surface-exposed proteins of adult worms in the portal system and
their gut vomitus in the context of immune evasion, secretions of the egg facilitating its escape from gut tissues and micro-exon gene products.
2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Schistosoma mansoni; Tegument; Host physiology; MEGs

1. Introduction with sequencing of the genomes and transcriptomes of the

The concept of virulence factors first came to prominence
in the 1970s, applied almost exclusively to pathogenic
bacteria. The term encompasses microbial products, generally
macromolecules, which facilitate establishment and mainte-
nance of an infection, contributing to its pathogenicity. They
include cell surface components essential for infection and
evasion of the immune response, as well as secreted toxins that
are directly linked to virulence. The purpose of this article is to
evaluate whether the virulence factor concept is applicable to
macroparasitic helminths, specifically the blood dwelling
schistosomes of humans. It has only been used overtly with the
helminth parasite Schistosoma mansoni in a single paper
published in 2011 [1]. The task is not straightforward because,
apart from scale, there is a major difference between microbes
and helminths. Once a microbe has established it undergoes
replication, usually rapid, until it is checked by the immune
system or the host is overwhelmed. Conversely, helminths do
not multiply in the mammalian host, each invasion being
a singular event. Thus, the parasite burden accumulates and
may be terminated acutely or persist chronically according to
helminth species. The recent advances in proteomics, coupled
* Tel.: 44 1904 328600; fax: 44 1904 328505.
E-mail address: raw3@york.ac.uk.
major schistosome species have made it possible, for the first
time, to define the major constituents of the schistoso-
meemammalian host interface, and the secretions of the larval
and adult stages associated with the host tissues and blood-
stream [2]. Can these constituents be termed or represented as
virulence factors?
2. Schistosomes and the mammalian host
The schistosomes parasitic in humans are threadlike and
about 1 cm long, the adults living as paired males and females
in the hepatic portal vasculature (S. mansoni and Schistosoma
japonicum) or the vessels of the bladder wall (Schistosoma
haematobium). The eggs laid by the females in the tissues of
the gut or bladder wall are the main pathogenic agents. In the
intestinal infections they may dislodge and travel downstream
to embolise in the liver; in the case of S. haematobium they
have a direct effect on the tissues of the urogenital tract. The
adult worms are remarkable for living, apparently unprotected,
in the host bloodstream for decades so must clearly deploy
sophisticated means of immune evasion. The infective agents
are microscopic cercaria larvae, shed from fresh water snail
intermediate hosts, which directly penetrate the skin to reach
dermal blood vessels and begin migration to the portal system.
The dramatic physiological transition from fresh water to the
human body is accompanied by loss of the cercarial tail and

1286-4579/$ - see front matter 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.micinf.2012.09.001
 Author's personal copy
R.A. Wilson / Microbes and Infection 14 (2012) 1442e1450
metamorphosis into the schistosomulum. Coincident with this,
the parasite replaces its surface membranes and becomes
invulnerable to killing by antibody-dependent killing mecha-
nisms (ADCC) [3]. The cercaria possesses five pairs of
acetabular glands to assist skin penetration, and a minute head
gland that facilitates parasite entry into a blood vessel. It is
apparent that the schistosome eggs, deposited in host tissues,
have their own system for secreting proteins required for exit
from the host. Production of factors essential for onward
transmission is not an aspect of virulence that has been applied
to microbes but it seems apposite for schistosomes.
3. Host invasion
We are on safe ground in suggesting that the secretions
used by cercariae to penetrate host skin are directly analogous
to bacterial virulence factors. However, while the work on
schistosomes has focused largely on proteases, with little
information on adhesion molecules, the situation is reversed in
bacteria with the emphasis placed on adhesion proteins.
Additionally, bacteria secrete a whole range of exotoxins;
schistosomes may produce the equivalent, but data on their
function is lacking at present. The three pairs of cercarial post-
acetabular glands were shown decades ago to produce mucins
[4] that enabled the cercaria to make firm adhesions to the
stratum corneum of the skin, preparatory to penetration.
However, no attempt to date appears to have been made to
characterise their molecular composition. Instead, researchers
have focused on the soluble proteins packaged in vesicles in
the two pairs of pre-acetabular glands.
The soluble serine proteases of S. mansoni cercariae have
been well characterised over the last 20 years. They are
generally referred to as elastases [5] but this has provoked
controversy because the acetabular gland cells where they are
synthesised have disappeared before the vast majority of
schistosomula enter the dermis where elastin fibres are situ-
ated [6]. At least three of these serine proteases have been
biochemically characterised from the acetabular gland secre-
tions [7] and their transcripts identified in the precursor germ
balls of the daughter sporocyst stage in the snail host [8]. (The
latest version (v5) of the S. mansoni genome lists seven
homologues but gives no information on the stage where
each is expressed.) Since the majority of S. mansoni parasites
do not enter the dermis for at least 48 h after penetration,
the substrate of the characterised serine proteases is likely
to be the proteins of the stratum corneum and the epidermis
where the incoming cercarial body creates a penetration tun-
nel (http://www.york.ac.uk/cii/media-library/cercariae/). The
cercarial tail and the thick protective glycocalyx are shed as
the parasite enters the epidermis.
A proteomic analysis of the soluble secretions released by
the schistosome cercariae during artificial transformation
in vitro [9] identified a hitherto unsuspected major component
as a metalloprotease. This is now classified in the S. mansoni
genome database as an invadolysin (Smp_090100). Whilst
tandem mass spectrometry pinpointed only a single protein, v5
of the S. mansoni genome lists seven homologues [10].
1443
Furthermore, a microarray analysis of the infection process
indicates that five of them were the most highly-upregulated
metalloprotease genes in the germ ball stage, when cercarial
proteins are being synthesised [8]. Given the role of
mammalian metalloproteases in degradation of tissues
matrices, a thorough investigation of these enzymes in the
context of skin invasion is warranted. Why up to seven elas-
tases and five metalloproteases, representing 34% and 13%
respectively of the material released from the pre-acetabular
glands, are needed for skin invasion is unclear, unless they
have very fine distinctions in substrate specificity. It has been
suggested that expressing multiple copies of genes for abun-
dant secretory proteins might itself be part of an evasion
strategy [8]. It is noteworthy that skin penetration by cercariae
of Trichobilharzia regenti (a bird schistosome) and S. japo-
nicum appears to be facilitated by a cysteine peptidase,
cathepsin B2, so there may be some latitude in the enzymes
involved in establishment among different schistosomes
[11,12]. Finally, experiments in vitro suggest that the serine
proteases secreted by cercariae can cleave host immunoglob-
ulins such as IgE [13] and interfere with complement-
mediated lysis [14]; such properties deployed in vivo could
well enhance parasite establishment in the host.
It is possible that the acetabular gland secretions also
contain the schistosome equivalents of bacterial toxins. Cur-
wen et al. [9], identified three proteins with SCP (sperm coat
protein) domains that have since been classified as part of
a 28-member family of VAL (venom allergen like) proteins,
approximately two thirds of which are secreted by different
stages. The three constituents of the cercarial secretions are
SmVAL4, SmVAL10 and SmVAL18, collectively representing
3% of released protein. Similar proteins have been identified
in other helminth secretions, e.g. Heligmosomoides polygyrus
[15]. The problem is that no clear function has yet emerged for
any VAL in any helminth species. The same is true of another
enigmatic constituent of the cercarial secretions, Sm16
(SmSLP/SPO-1), originally ascribed anti-inflammatory prop-
erties [16], and more recently a caspase-dependent apoptotic
function [17]. Its presence in extruded material may simply
reflect the fact that acetabular glands release their contents by
holocrine secretion. Indeed, the localisation of Sm16 tran-
scripts to numerous internal tissues of the cercaria by
WISH [18], plus the absence of a signal peptide suggests that
it has an internal function in the newly penetrated parasite.
Potentially, if it is pro-apoptotic, this could be in the
tissue remodelling that accompanies transformation to the
schistosomulum.
The final constituents of cercarial secretions that merit
attention as virulence factors are the glycans. Both the proteins
in soluble cercarial secretions and those in the soluble egg
secretions are heavily glycosylated [19,20]. A plethora of both
N- and O-linked glycan structures have been identified by
mass spectrometry and many proved to be shared between the
two transmission stages, suggesting a commonality of function
by the stages entering and leaving the host [19,20]. Moreover,
these glycans are highly immunogenic [21,22], but there is no
evidence that the responses invoked are protective. This has
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1444 R.A. Wilson / Microbes and Infection 14 (2012) 1442e1450
led to the suggestion that they are part of the immune evasion
mechanism, serving as a smoke screen to deflect the antibodies
away from vulnerable and functional peptide epitopes [21,23]
or in the matadors cloak variant of the hypothesis [20], actu-
ally luring leucocytes away from the incoming larva so that it
can escape attack.
The molecular definition of secreted factors released by
invading schistosomes is ahead of the research to delineate
their functions. Nevertheless, evidence is accumulating for
their interaction with the dermal immune response, although
the identity of the specific factors involved has yet to be
elucidated. Since the process of schistosome infection, per se,
does not appear to elicit protective immunity, it is reasonable
to assume that observed effects are parasite-, not host-
protective. The secretions from CFDA-SE (carboxy-
fluorescein diacetate-succinimidyl ester) labelled cercariae
released into the skin during penetration are initially taken up
by neutrophils [24]. By 24 h dermal macrophages and
dendritic cells are labelled, and both of these cells are
observed in the skin-draining lymph nodes at 48 h, perhaps
a reflection of their transit time from the skin. In vitro,
macrophages are slower to take up the secretions than
dendritic cells but produce more (suppressive) IL-10 [24].
Ligands for the mannose receptor (CD206), presumably
glycans, are abundant in the secretions [25]. In this context it
is notable that schistosomes, unlike the mammalian hosts, do
not terminate their sugar chains with sialic acid, lacking the
necessary transferase enzyme. Furthermore, in mannose
receptor-null mice, after exposure to cercariae, fewer leuco-
cytes in the skin had taken up parasite antigen but there was
enhanced proliferation of the skin-draining lymph node cells
secreting interferon gamma. Thus it appears that the cercarial
secretions down-modulate the production of pro-inflammatory
cytokines in the skin. This conclusion is reinforced by the
observation that multiple exposures of mice to cercariae
(before the onset of egg production) induce hypo-
responsiveness of CD4 T cells in the skin-draining lymph
nodes [26]. The crucial cells are alternatively activated
Fig. 1. A, Whole mount in situ hybridisation (WISH) of the MEG-3.2 gene in a day-3 schistosomulum. The dark staining area at the anterior reveals a high level of
expression in the head gland. The lighter areas are tegument cell bodies. B, A mature egg with the sub-shell envelope syncytium highlighted by fluorescent dextran;
an unstained miracidium (m) is visible in the centre. C, In situ hybridisation to detect MEG-3.2 expression performed on liver sections containing mature eggs. The
mRNA revealed by the blue stain is localised to the sub-shell envelope.
macrophages and functionally compromised MHCIIhi cells
that are unable to present antigen.
4. Intravascular migration
Much less is known about the factors secreted by the
schistosomulum after it has completed its transformation in
the skin, shedding its cercarial plasma membrane and
replacing it with a new surface in an apparently seamless
process. (Whether that shed material represents a source of
signals to the host skin has not been investigated.) The
parasites use of the head gland to gain access to a blood vessel
was documented in ultrastructural studies [27], but its minute
size means that little material is available for analysis.
Biosynthetic labelling with 35S-methionine has revealed that
skin and lung stage schistosomula release a dominant protein
of wMr 20 kDa into the external environment [28]. In 2010 it
was identified by proteomics as a mixture of two proteins,
MEG-3.1 and MEG-3.2, encoded by micro-exon genes ([29]
see Egg escape). Furthermore, whole mount in situ hybrid-
isation (WISH) revealed that expression of the MEG-3.2 gene
was localised to the head gland [29], (the only transcript so far
identified from that tissue) and to a lesser extent the tegument
cells bodies (Fig. 1A; Parker-Manuel, pers. comm.). This
indicates that the MEG-3 proteins are likely to be released
across the tegument surface as well as from the head gland and
makes them candidate constituents of the enigmatic homoge-
neous secretory bodies, uniquely found in the lung stage [30].
For information on other likely secreted or exposed proteins of
migrating lung schistosomula we have to rely on gene
expression patterns revealed by cDNA sequencing or micro-
array analyses, which have identified known tegument
constituents of the adult worm [31].
5. The life of adult worms in the bloodstream
In most hosts once migrating schistosomes have reached
the hepatic portal blood vessels and begun to feed on
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R.A. Wilson / Microbes and Infection 14 (2012) 1442e1450
erythrocytes, they appear to be invulnerable to immune attack;
the exceptions are the laboratory rat and the rhesus macaque,
which eliminate pre-adult and adult worms, respectively [32].
5.1. Products of the schistosome gut
The schistosome gut is tidal, with food entering and waste
leaving via the mouth. Essentially it is an elongate tube
(in some places bifurcated) comprising a uniform layer of
epithelium, sometimes referred to as the gastrodermis. This
tube is invested by smooth muscle fibres responsible for the
peristalsis that propels the contents along the lumen. The
females of the species are more voracious than the males,
consuming eight times as much blood [33]. Haemoglobin from
ingested erythrocytes is the major protein source but plasma
proteins, especially albumin, are also important [34]. A black
haematin/haemozoin pigment, the residue of haemoglobin
digestion is periodically voided into the portal blood and then
filtered out by the resident macrophages of the liver. As
a detoxification product it is highly likely that the haemozoin
is relatively inert. Indeed the version from S. japonicum failed
to exert any significant effects on the development and activ-
ities of murine myeloid dendritic cells [35]. Other major
products of the gut vomitus are the circulating anodic and
cathodic polysaccharide antigens, which were shown to
comprise the glycocalyx lining the gut [36] and subsequently
developed as the basis for diagnostic tests [37]. No evidence
has been presented for effects on host physiology other than
the formation of immune complexes with antibodies that
facilitates antigen clearance. Finally, as a result of earlier
biochemical studies and recent proteomic analyses we now
have information on the proteins that are secreted into the
worm gut, and ultimately voided into the bloodstream [34,38].
These come in three categories, hydrolytic enzymes, carrier
proteins, and protease inhibitors, many of which are associated
with lysosomal activity. The enzymes include Cathepsins B1
(2 isoforms), C and K/S, a Pro-X carboxylpeptidase and
a Glucan 1,4 beta-glucosidase. The transporters include
Ferritins, Saposins, NPC-like cholesterol binding protein, and
Calumenin, a Ca transporter. The protease inhibitors identified
are a serpin and the schistosome homologue of alpha-2-
macroglubulin.
Since adult intestinal schistosomes inhabit the hepatic
portal venules of the gut wall, the contents of their guts that are
voided into the bloodstream will be carried downstream to the
liver. The role of the liver as an organ of the immune system
[39], particularly in the context of tolerance generation [40], is
now well established. Therefore, it seems surprising that there
is little information on the interaction of any of the protein
constituents of gut vomitus with hepatic antigen presenting
cells. Of course, this might be due to the difficulty of stimu-
lating worms in vitro to open their mouths and regurgitate their
gut contents [34]. However, in vivo the gut vomitus must flow
past the fixed antigen presenting cells of the sinusoids. Hae-
mozoin pigment is very evident in the resident Kupffer cells of
the sinusoids and its presence implies that other vomitus
constituents will also be endocytosed; an investigation of the
1445
induction of tolerance to schistosome antigens in the liver is
clearly warranted.
5.2. The tegument and immune evasion
The schistosome interface with the bloodstream, termed
a tegument, is a syncytial layer of cytoplasm, connected to cell
bodies beneath the musculature. The unusual appearance of its
surface in juvenile and adult schistosomes, when stabilised by
uranyl acetate, was described w40 years ago as a heptalami-
nate membrane [30], and this term is still used by some
researchers. However, the structure was interpreted as
a normal plasma membrane overlain by a laminate secretion,
the membranocalyx, which originated in multilaminate vesi-
cles present within the cytosol of the tegument syncytium [41].
It was suggested that the secreted layer lacked macromolec-
ular components and so acted as a barrier to prevent antibodies
from binding to vulnerable functional proteins in the plasma
membrane. This hypothesis was based on two of observations.
Firstly, binding of anti-worm antibodies to the tegument
surface of live worms cannot easily be demonstrated, so
something must be getting in the way. Secondly, without
uranyl acetate treatment the processing of worms for electron
microscopy with organic solvents extracts most of the vesicle
contents, revealing their predominantly lipid composition, and
the membranocalyx disappears. However, a small residue
remained at the centre of each empty vesicle, which stained
positively for glycans [41]. Furthermore, incubation of live
worms with the lectin Concavalin A froze vesicles in the act
of fusion with the plasma membrane, revealing the presence of
exposed mannose residues in the membranocalyx before
secretion [42]. Recent proteomic analyses indicate that the
hypothesis of the membranocalyx as an immunologically inert
barrier might need to be modified.
Clearly, the tegument surface is the principal location
where evasion of the host immune response occurs. Its prop-
erties and composition have been comprehensively reviewed
in recent years [43e45] and proteomics has contributed much
of the new knowledge about the surface layers [2]. However,
some misguided conclusions have been reached by researchers
who confused the secretome with the necrotome (the
contaminating cytosolic proteins released by damaged or
dying parasites). The most exposed proteins have been tagged
by biotinylation of live worms [46,47] while GPI-anchored
proteins have been detached by incubation with phosphatidyl
inositol phospholipase C (PiPLC). In the context of virulence
factors there are three aspects to be considered. Firstly, the
actual composition of the membranocalyx lipid barrier, and of
the endogenous glycans associated with lipids or proteins, is
completely unexplored. Secondly, the proteins exposed on the
surface may function in a parasite-protective role. Thirdly, any
proteins and/or glycan constituents of the membranocalyx,
released when it turns over into the bloodstream, could serve
as modifiers of host responses or physiology.
A small number of enzymes exposed on the outer leaflet of
the tegument plasma membrane have been implicated as
potential virulence factors due to their ability to modify the
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1446 R.A. Wilson / Microbes and Infection 14 (2012) 1442e1450
immediate environment of the parasite in the blood vessel
(reviewed by Bhardwaj & Skelly [48]). The demonstration of
ATP-diphosphohydrolase activity externally on the tegument
led to the suggestion that the enzyme could regulate the
concentration of purine nucleotides around the parasites and
hence enable them to escape the host haemostasis by pre-
venting ADP-induced platelet activation [49]. The GPI-
anchored ADP-ribosyl cyclase enzyme also on the external
surface, whilst apparently unable to generate cyclic adenosine
diphosphate ribose, may catabolize extracellular NAD() to
prevent its use by host enzymes that facilitate immune
responses [50]. It has also been suggested that the adenosine
generated from adenosine monophosphate by tegumental
alkaline phosphatase acts as a localised immunosuppressant
[51]. Lastly, the tegumental phosphodiesterase, SmNPP-5, has
actually been directly implicated as a virulence factor by the
use of RNAi treatment to suppress gene expression in para-
sites, followed by demonstration of their greatly reduced
ability to establish after administration to mice [1].
Although many tegument surface constituents have been
identified, the difficulty is to place them geographically within
the complex surface structure. Currently, six proteins: Sm200,
Sm29, Sm13, Annexin Smp_077720, Tetraspanin-TSP2, and
Low Molecular Weight Protein (LMWP) are the prime candi-
dates for location in the secreted membranocalyx [2]; since the
membranocalyx turns over into the external environment [42],
this means they will be released into the bloodstream. Inde-
pendent evidence for this fate comes in part from short term
culture experiments to collect vomitus (and inevitably any true
surface secretions) [34] that identified Sm200. In addition, this
protein has been reported from the lipid fraction of host serum,
providing confirmation of its release [52]. What the function of
these proteins might be is a matter of conjecture, but it seems
likely that the annexin and tetraspanin play a structural role; that
leaves Sm200, Sm29, Sm13 and LMWP as potential virulence
factors interacting with the host.
The apparent invulnerability of the adult worm to antibody
binding and immune attack was noted above. It therefore came
as a surprise that compositional analysis of the tegument
surface complex identified the presence of host antibodies of
IgM, IgG1 and IgG3 classes [53,54]. Furthermore the C3 and
C4 components of the complement cascade were also identi-
fied but not those of the membrane attack complex C5eC9.
This implies that complement fixation is initiated but then
inhibited at an early stage. Possible candidates for this role are
the two GPI-anchored schistosome homologues of host CD59
(Smp_019350, Smp_105220), that can be detached from live
worms by PiPLC treatment [31]. The human equivalent
protects self-cells by binding to the membrane attack complex.
Counter to this suggestion, a quantitative estimate of the
amounts of C3 and IgG at the schistosome surface using the
QconCAT technique [55] found much less C3, indicating that
the complement pathway was blocked earlier than attack
complex formation at the level of C3b convertase. One further
protein could play a role in immune evasion at the surface,
namely the schistosome homologue (Smp_194920) of the host
T cell immunomodulatory protein. In the host it has
a protective effect in graft-v-host disease [56]; does it modify
the interaction of host leucocytes with the tegument surface?
6. Egg escape from host tissues
The schistosome egg is a composite structure comprising
w30 vitelline cells and a fertilised ovum packaged within an
elastic tanned protein shell, laid by a female in an undeveloped
state in a venule. The ovum develops into a ciliated mira-
cidium whilst the egg remains in the vessel using nutrients
from the vitelline cells but also, since it has been estimated to
increase in mass by 3.33, acquiring them from the host as
well [57]. During development 3e4 cells detach from the
embryonic mass and move to the periphery where they fuse to
form the syncytial sub-shell envelope (Fig. 1B), which
differentiates into a protein synthesising and exporting tissue
[57]. However, secretion only becomes appreciable when the
miracidium is mature, clearly a device to prevent premature
expulsion of the partially developed egg. Release of secretions
is the trigger that initiates egg migration across the tissues to
the gut (or bladder) lumen, and voiding in excreta. The role of
these secretions from live eggs is thus crucial for parasite
transmission and should not be confused with the products of
moribund or dead eggs (not considered here) that contribute to
granulomatous pathology that is the hallmark of the disease.
Definition of the proteins secreted by viable mature eggs to
facilitate escape from host tissues has proved controversial. The
first report detailed a large number of intracellular proteins
identified by tandem mass spectrometry and postulated their
release via a non-classical pathway [58]. A later report
described a very different situation with a relatively simple
mixture secreted from eggs that were judged to be >96% viable
[59]. The secretions were dominated by previously described
IPSE (Interleukin-4 inducing principle of schistosome eggs
[60];) and Omega-1 [61], plus a few minor spots When the
secretions were depleted of the heavily glycosylated IPSE and
Omega-1 using Aleuria aurantia agglutinin the minor spots
were greatly enriched and many could be identified by mass
spectrometry [29]. The bulk of the minor components were
encoded by micro-exon genes, five from the MEG-2 and three
from the MEG-3 family, with at least 10 variants of the former
and 12 of the latter. IPSE was demonstrated by immunocyto-
chemistry to originate in the sub-shell envelope [60] whilst
MEG-3.2 gene was revealed by WISH to be transcribed in the
same tissue (Fig. 1C). As a footnote, the recently characterised
Sm-kappa antigenic glycoprotein [62] is associated with the
miracidium and not found in the egg secretions [59,62],
providing strong evidence that the sub-shell envelope is the true
interface between the living egg and the host tissues. The glycan
composition of the egg secretions has been characterised,
revealing a wide variety of N- and O-linked structures, many of
them shared with the secretions of the invasive cercaria [20].
What are the functions of these egg secretions? The precise
way in which the egg traverses host tissues remains a conun-
drum. Protease activity of MW 14 and 31 kDa was reported on
casein protease substrate gels of fractionated, unreduced eggs
secretions [57] but no known proteases have been linked to
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R.A. Wilson / Microbes and Infection 14 (2012) 1442e1450 1447

these activities by later proteomic analysis [59]. Omega-1 has
ribonuclease activity implying that it could by cytotoxic, but
which host cells are the target? Egg excretion in immuno-
compromised mice is inhibited and can be restored by
immune serum or the adoptive transfer of lymphoid cells [63].
A proportion of eggs laid in the gut tissues may become the
centre of a granulomatous response, but the majority reach the
gut lumen. Furthermore, when eggs mature, they attract leu-
cocytes to their vicinity (Fig. 2). Add to this the fact that egg
glycans are highly immunogenic [21,22] but may serve as
a smoke screen rather than mediating protective responses
[2,23]. Thus, we can speculate that the eggs actively recruit the
host immune response via their secretions, to aid their passage
through the tissues. Escape of the mature egg from the blood
vessel is the first step (see MEG section below). Does the
extensive glycosylation of IPSE and Omega-1, the major
egg secretions, made them sticky to ensure their retention in
the eggs vicinity? Are they the agents that attract the leuco-
cytic focus, potentially acting via Toll-like, C-type lectin or
mannose cell surface receptors? Host matrix metal-
loproteinases, a feature of macrophage-mediated inflammation
capable of degrading all kinds of extracellular matrix proteins,
could then provide the hydrolytic capacity. This, coupled with
the motive power provided by smooth muscle peristalsis,
would facilitate egg transit across the gut tissues.
A useful corollary to this biological process that may be
beneficial to the schistosome, is the now well-attested capacity
of the schistosome egg to drive T helper cell polarisation in
a Th2 direction [64]. Indeed, two groups have reported that
Omega-1 is the factor responsible for polarising the immune
response in that direction [65,66]. However, if we are evalu-
ating outcomes that benefit the schistosome, then there is
a contradictory element because of the claims that protective
immunity to schistosomes in humans is correlated with Th2
responses [67,68]. An equally intriguing consequence of
immune polarisation via Th2 or T-regulatory pathways is the
demonstration, in animal models at least, of the prevention of
autoimmunity (reviewed by [69]). We must presume that this
protection is a bystander effect rather than of benefit to the
schistosome.
7. Micro-exon encoded proteins
The final element in this appraisal of schistosome virulence
factors is the recent discovery of micro-exon-encoded secre-
tory proteins, collectively referred to as MEGs [29,70].
Currently, >70 MEGs are known, classified into 18 distinct
families with 1e23 members. They appear unique to schis-
tosomes in that approximately 80% of the protein coding
sequence is made up of small exons ranging from 6 to 36 bp,
the commonest frequency being 21 bp. Microarray experi-
ments have shown that the majority of MEGs are up-regulated
in life cycle stages associated with establishment in the
mammalian host after skin penetration [8]. Furthermore, RT-
PCR of transcripts has revealed numerous variants generated
by exon skipping and, as we have seen for both larval and egg
secretions, these are translated to provide a pool of variant
proteins. Unfortunately none of the MEG proteins has any
homology to proteins with known functions in other organ-
isms, and the only motif encoded is a signal sequence. Lastly,
WISH and immunochemistry have been used to localise
a small number of MEGs to specific structures: MEG-3.2,
already mentioned; MEG-4.1 to the oesophageal gland [71],
and MEG-14 and MEG-4.2 to the primordia of the developing
gut in the Day 3 schistosomulum [29]. The puzzle is what
advantage the parasite gets from breaking down its coding
regions into small modules, generating a range of transcripts
by exon skipping and then translating them into variant
proteins? There must be an energy cost to this as not all of the
variants can be functional. Is it worthwhile because it assists

Fig. 2. Multiphoton microscopy of schistosome eggs (yellow auto-fluorescence) located in venules stained with FITC-labelled Lysopersicon esculentum lectin
(green). VaDS Red B6 mice, which have CD2, CD3 red T cells were infected with schistosome cercariae 5e7 weeks earlier. The serosal collagen layer appears
blue. A, An immature egg in a venule; the red T cells are randomly distributed throughout the sub-mucosa. B, A mature egg, still within a venule, but surrounded
by an aggregate of T cells. This is a prelude to egg entry into the mucosal tissues and exit to the gut lumen. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
 Author's personal copy

1448 R.A. Wilson / Microbes and Infection 14 (2012) 1442e1450

Table 1

immune evasion, and if so how is that achieved? Until specific 8. Conclusions

functions have been established for one or more MEG
proteins this will be hard to authenticate. The MEG-3 family Schistosomes do not replicate in the mammalian host and
is a case in point. The proteins are secreted by the migrating lack the genetic plasticity of microbes, so thus far purposeful
skin and intravascular schistosomulum, and the mature egg products that make a schistosome infection more virulent have
also located in a blood vessel. The obvious inference is that not been defined. This means that the term virulence factor
the MEG-3 proteins interact with circulating cells, or the cannot have precisely the same meaning as for microbes.
vascular endothelium, most likely the latter. In the case of the Nevertheless, it is clear that schistosomes deliberately expose
larva, secretion from the head may first facilitate entry into proteins and glycoproteins on their surface or release them
a venule, then from the tegument, its travel through the into their host environment. Ironically the constituents of
vascular beds of internal organs to reach the portal vein. For moribund or dead eggs are the major contributors to the
the egg, secretion of MEG-3 proteins may be associated with pathogenicity of schistosomiasis, but it is hard to see how they
escape from the portal venule where it was laid, into the benefit the parasite since their release is incidental to trans-
tissues e the reverse of what the larva achieves, but still mission; hence their omission from this review. That still
involving the breach of a vessel wall. The longest isoform of leaves a significant list of candidate virulence factors
MEG-3.2 possesses 16 cysteine residues that can be aligned in (Table 2). A major task is to identify the function of such
two groups of four pairs, each separated by three amino acids candidates at the parasiteehost interface, given that significant
(Table 1). amounts of native protein are difficult or impossible to obtain
It is thus tempting to conclude that the mature protein is from the parasites themselves. RNA interference, (reviewed by
a dimer capable of cross-linking surface receptors on the [72]) is one avenue of investigation. Expression of proteins
vascular endothelium to achieve a biological effect. This like IPSE and their testing for biological effects in vitro or
should be an eminently testable proposition, provided a small in vivo is another way forward (e.g., [60]). The secreted
protein with 16 cysteines can be produced in a correctly folded proteins with multiple cysteine bonds such as those encoded
and functional form, not a trivial task. by most MEGs, may need expression in a eukaryotic system to
achieve correct folding, before testing for function. With skill,
good fortune and access to funds such studies could yield
a major dividend for our understanding of schistosomeehost
Table 2
Selected potential virulence factors. interactions.
Genome v4/v5 Genome v4/v5
Cercarial secretions Membranocalyx proteins Acknowledgements
Elastasea Smp_119130 Sm200 Smp_017730
Elastase Smp_112090 Sm29 Smp_072190 Thanks are due to Dr Adrian Mountford for commenting on
Invadolysinb Smp_090100 Sm13 Smp_195190 the manuscript, to Dr Sophie Parker-Manuel and Mr Henrique
VAL4 Smp_002070 LMWP Smp_194860
Roffato for use of their WISH images of MEG-3.2 expression
VAL18 Smp_001890 Egg secretions
VAL10 Smp_002060 IPSE Smp_112110 in the lung schistosomulum and mature egg, to Miss Hayley
Sm16 Smp_113760 Omega-1c Smp_193860 Tyrer and Mrs Jo Marrison for the multiphoton microscope
Schistosomula secretions MEG-2.1 Smp_181510 images of eggs in the intestinal wall, and Dr Mark Coles for
MEG-3.1 Smp_138080 MEG-2.2 Smp_159810 the gift of VaDS Red/B6 mice.
MEG-3.2 Smp_138070 MEG-2.5 Smp_180320
Exposed tegument enzymes MEG-2.6 Smp_180310
ATP-diphosphohydrolase 1 Smp_042020 MEG-2.8 Smp_180340 References
Phosphodiesterase Smp_153390 MEG-3.1 Smp_138080
SmNPP-5
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