Schistosoma mansoni miracidia transformed by particle

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Schistosoma	mansoni	miracidia	transformed
by	particle	bombardment	infect	Biomphalaria
glabrata	snails	and...
Article		in		Experimental	Parasitology	·	November	2003
DOI:	10.1016/j.exppara.2003.11.001	·	Source:	PubMed
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Experimental Parasitology 105 (2003) 174–178
www.elsevier.com/locate/yexpr
Research Brief
Schistosoma mansoni miracidia transformed by particle
bombardment infect Biomphalaria glabrata snails and develop
into transgenic sporocysts
Oliver Heyers,a Anna K. Walduck,b Paul J. Brindley,c Wilfrid Bleiß,a Richard Lucius,a
Tomislav Dorbic,d Burghardt Wittig,e and Bernd H. Kalinnaa,*
a Department of Molecular Parasitology, Institute for Biology, Humboldt University Berlin, 10115 Berlin, Germany
b Max Planck Institute for Infection Biology, 10117 Berlin, Germany
c Department of Tropical Medicine, Tulane University, Health Sciences Center, New Orleans, LA 70112, USA
d Centrum Somatische Gentherapie an der Freien Universit€at Berlin, Fachbereich Humanmedizin, UKBF and Geniotronic AG, Berlin, Germany
e Centrum Somatische Gentherapie an der Freien Universit€at Berlin, Fachbereich Humanmedizin, UKBF and Mologen Holding AG, Berlin, Germany
Received 5 June 2003; received in revised form 8 September 2003; accepted 4 November 2003
Abstract
Miracidia (and adults) of Schistosoma mansoni which had been subjected to particle bombardment with a plasmid DNA encoding enhanced green
fluorescent protein (EGFP) under control of the S. mansoni heat shock protein 70 (HSP70) promoter and termination elements were shown to express
the reporter gene. Bombarded miracidia were able to penetrate and establish in Biomphalaria glabrata the intermediate host snail. Gold particles
could be detected in the germ balls of parasites in paraffin-sections of snail tissue. The bombarded miracidia were able to develop normally and to
transform into mother sporocysts. Reporter gene activity could be determined at 10 days post-infection by RT-PCR in snail tissues, but not by
microscopy or Western blot which probably reflected sub-optimal expression levels of constructs. Our findings indicated that it is feasible to return
transgenic miracidia to the life cycle, a crucial step for the establishment of a transgenesis system for schistosomes.
� 2003 Elsevier Inc. All rights reserved.
Index Descriptors and Abbreviations: schistosome, miracidium, sporocyst, particle bombardment, gold particles, transgene, Biomphalaria glabrata;
germ ball; EGFP, enhanced green fluorescent protein; RT-PCR, reverse transcription-polymerase chain reaction; gDNA, genomic DNA; PBS,
phosphate buffered saline; TBS, Tris buffered saline; FCS, fetal calf serum; DEAE, diethylaminoethyldextran
Schistosomes are considered the most important of the human
helminths in terms of morbidity and mortality (Engels et al., 2002). A
tractable transfection system would represent a significant advance in
our ability to develop new interventions for control of schistosomiasis
(Boyle and Yoshino, 2003; Davis et al., 1999). Schistosoma japonicum
and Schistosoma mansoni sporocysts can be generated in vitro by
transforming miracidia (reviewed in Coustau and Yoshino, 2000), and
cultured sporocysts have been transiently transfected with pBluescript-based plasmids expressing green fluorescent protein (GFP) (Wippers-
teg et al., 2002a,b), demonstrating that it is possible in principle to
generate transgenic parasites. However, it is not clear yet whether a
transgenic line of schistosomes could be established from these tran-
siently transfected sporocysts. At least two important obstacles on the
path to the development of a reliable, tractable transgenesis system for
schistosomes now need to be surmounted: (1) development of appro-
priate constructs to achieve stable integration into the schistosome
* Corresponding author. Fax: +49-30-2093 6051.
E-mail address: bernd.kalinna@rz.hu-berlin.de (B.H. Kalinna).
0014-4894/$ - see front matter � 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.exppara.2003.11.001
genome and (2) a demonstration that the life cycle can be completed by
genetically modified parasites.
The life cycle of the schistosome includes two free-living, self-reli-
ant, and mobile infective stages. These are the miracidium, which seeks
out and directly infects the intermediate host snail, and the cercaria,
which accomplishes infection of the mammalian, definitive host by
direct penetration of host skin when a definitive host comes into
contact with water contaminated with cercariae. These are the only
two stages that could be reintroduced into the life cycle using a natural
route of infection, thereby obviating the inefficient and laborious tasks
of surgically implanting transformed sporocysts back into snails and/
or surgically implanting adult schistosomes into the portal system
blood vessels of mice (e.g., Cheever et al., 1994; Jourdane et al., 1985).
Here we subjected miracidia of S. mansoni to particle bombardment
and have been able to show that miracidia bombarded with DNA-
coated gold particles can directly and naturally infect the intermediate
host snail Biomphalaria glabrata and establish as sporocysts in a nat-
ural fashion. Further we were able to demonstrate transgene tran-
scription of enhanced GFP in both adult worms and snails infected
with transformed sporocysts.
mail to: bernd.kalinna@rz.hu-berlin.de
O. Heyers et al. / Experimental Parasitology 105 (2003) 174–178 175
Schistosoma mansoni (Puerto Rican strain) was propagated in the
laboratory as described (Copeland et al., 2003). Snails were maintained
at 26 �C in aerated water and fed with lettuce leaves, in a room with a
light/darkness cycle of 12 h each. At 8 weeks after S. mansoni infection,
mice were killed, adult worms were perfused from the mesenteric veins
with PBS, and the livers were removed. Adult worms were washed
three times in RPMI 1640 and maintained in vitro in RPMI
1640+ 10% FCS supplemented with penicillin/streptomycin at 37 �C in
5% CO2. The infected livers were forced through a plastic mesh to
release the schistosome eggs, after which the eggs were washed in cold
sterile 1.8% NaCl to remove host tissues and debris. The 1.8% NaCl
was replaced with sterile water to induce the eggs to hatch, miracidia
were collected from the water, and concentrated by centrifugation at
500 rpm. For particle bombardment (biolistics), approximately 500
miracidia or 20 male, adult worms were evenly spread onto a poly-
carbonate membrane (Transwell, Costar), water or medium was re-
moved and target schistosomes on the polycarbonate membranes were
positioned in a Biolistic PDS-1000/HE Particle Delivery System (Bio-
Rad Laboratories GmbH, M€unchen, Germany). Biolistic parameters
were 15 in. Hg of chamber vacuum, target distance of 3 cm (stage 1),
900 psi (miracidia) or 1800 psi (adult worms) particle acceleration
pressure, and 1.0lm gold microcarriers (Bio-Rad). Gold microcarriers
were prepared, and circular plasmid DNA was precipitated onto the
gold using methods recommended by Bio-Rad with the following
modification; 0.6mg of gold particles carrying �5lg of plasmid DNA
was used per bombardment.
To engineer plasmid constructs for these transient transfection
studies, a 532 bp fragment comprising the promoter region of the gene
encoding the 70 kDa heat shock protein (HSP70) of S. mansoni
(GenBank Accession No. L02415) (Neumann et al., 1992) was
amplified from S. mansoni genomic DNA using a 50 primer, 50-GCGC
GGAATTCGATTAGCACTAAG-30, and a BamHI-linked 30 primer,
50-GCGCGGGATTCTCCAAGATATGTTAAGAC-30. Genomic
DNA (gDNA) was isolated from adult schistosomes and from snails as
described (Copeland et al., 2003). The HSP70 30 end terminator region
was isolated by amplifying a 760-bp fragment from gDNA with the
XbaI-linked forward-primer, 50-CCCCCTCTAGACCTATAATTGT
TGTGATAAATTG-30, and a HindIII-linked reverse-primer, 50-CC
CCCAAGCTTAGAACCCTTCCACGGTGTG-30. To isolate an
EGFP-encoding fragment, pEGFP-N1 (Clontech) was digested with
BamHI and XbaI. The 720-bp fragment was purified by agarose elec-
trophoresis. The three different fragments were ligated into pUC19
digested with the appropriate restriction enzymes. A map of this
construct is presented in Fig. 1A. The construct was tested in COS7
cells and found to be functional in that �5% of the COS7 cells ex-
Fig. 1. (A) Schematic representation of the HSP70-GFP-HSP70 plasmid vec
The insert contains the promoter (50 HSP70), EGFP gene and terminator (HS
worm showing EGFP expression in the vicinity of the oral sucker 72 h after b
helium pressure of 1800 psi were used. The inset presents a scanning electron
region indicating the location shown in the fluorescence micrograph. A scale b
particle bombardment. Lanes 1, HSP70-promoter plasmid; 2, Marker; 3, pr
responding to the expected size of the EGFP RT-PCR product.
hibited EGFP fluorescence (not shown). To generate a control plas-
mid, pEGFP-N1 (Clontech) was modified by deletion of the CMV
promoter. Total RNA was extracted from snails or adult S. mansoni
worms using the RNeasy kit (Qiagen, Hilden, Germany). DNAse-
treated RNA was used as a template for oligo(dT) primed reverse
transcription. The resulting cDNA was used as the template in PCR
experiments using primers specific for the EGFP, 50-ATGG
TGAGCAAGGGCGAG-30 and 50-TTACTTGTACAGCTCGTC-30,
expected target size, 740 nt. One microliter of the first PCR was used as
template for a second round of nested PCR using primers 50-GC
AGTGCTTCAGCCGCTA-30 and 50-TAGTGGTTGTCGGGCAG
C-30, expected target size, 396 nt. Negative control reactions were
carried out using: (a) the non-transcribed DNAse-treated RNA and (b)
gDNA of uninfected snails as templates. DNA of plasmid pEGFP-N1
served as the positive control.
Adult worms were bombarded twice within a few minutes, culture
medium (above) was added, and the worms maintained in vitro for
24 h. Adult schistosomes were then heat shocked at 42 �C for 4 h and
cultured for an additional 24 h at 37 �C before examination for EGFP.
Larval (miracidia, sporocysts) schistosomes were not heat shocked
because the schistosome HSP70 promoter is constitutively active in
these developmental stages (Neumann et al., 1993). Fluorescence mi-
croscopy of bombarded worms was performed using a Zeiss Axioplan
fluorescence microscope (Oberkochen, Germany) with a blue excita-
tion filter set (No. 487909) and/or with a Leica TCS NT laser scanning,
confocal microscope, as described (Wippersteg et al., 2002a,b). Small
regions of EGFP fluorescence which co-localized with gold particles
could be distinguished from the relatively high background fluores-
cence only by using the laser scanning microscope. By contrast, green
(EGFP) fluorescence was not observed in untreated control worms,
worms that were bombarded with unlabelled gold, or with gold coated
with the promoter-less plasmid. Here only background fluorescence
was visible. EGFP fluorescence was not detectable by normal fluo-
rescence microscopy in any experiment (i.e., by using a Zeiss Axioplan
fluorescence microscope); it was detected only with the confocal mi-
croscope. This inability to detect fluorescence with normal fluorescence
microscopy has alsobeen noted by others (Wippersteg, personal
communication), and may reflect poor transcription/translation and/or
incorrect folding of the EGFP. Transfection rates varied between
0.5 and 1.0% in independent experiments, although prominent fluo-
rescence could only be detected in �0.1% of bombarded organisms.
The fluorescence pattern observed had no obvious specific distribution
but tended to be more often seen at the oral sucker (Fig. 1B) or at the
posterior end of the worm, in agreement with Wippersteg et al.
(2002a). In these cases, it was relatively straightforward to distinguish
tor used for transfection. The vector has a pUC19 plasmid backbone.
P70 30). Not drawn to scale. (B) Confocal microscope image of a male
ombardment. For this bombardment, 1.0lm gold microcarriers and a
micrograph of the anterior of an adult S. mansoni male, with the boxed
ar indicates the size. (C) EGFP mRNA expression in adult worms after
omoter-less plasmid control. The arrow indicates a 740 bp band cor-
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=nucleotide&cmd=search&term=L02415
176 O. Heyers et al. / Experimental Parasitology 105 (2003) 174–178
the EGFP fluorescence from background signals. To verify the ex-
pression of EGFP on the transcriptional level, an EGFP-specific RT-
PCR with RNA extracted from EGFP-expressing worms produced
amplicons of the expected size, 740 bp, confirming the transcription of
the EGFP in bombarded worms (Fig. 1C).
Infection of snails of �5mm diameter shell size with transformed
miracidia was accomplished by incubation of the snails with approx-
imately 150–250 transformed miracidia in 0.5ml of water for 3 h under
bright light. This high infection rate regimen was employed because we
anticipated that several only of the transfected sporocysts would de-
velop in each snail and because we did not know what percentage of
the bombarded miracidia would be capable of infection. Invasion of
snails by bombarded miracidia was monitored by microscopic obser-
vation. The snails survived this infection regimen, after which they
were maintained for several weeks. In some instances, bombarded
miracidia were cultured overnight in medium MEMSE-J+ 10% fetal
calf serum (FCS) for transformation in vitro into primary (mother)
sporocysts (Bayne et al., 1994; Jourdane et al., 1985).
To determine if miracidia could withstand particle bombardment,
miracidia were bombarded with unlabelled gold microcarriers at
900 psi. Subsequently, they were washed, transferred into MEMSE-
J+ 10% FCS, and cultured at 26 �C in air. About half the bombarded
miracidia survived, as adjudged by light microscopy for normal larval
movements (not shown). These miracidia transformed into mother
sporocysts within 24 h. Single gold particles or gold aggregates were
readily observed in �15% of these mother sporocysts. Further, mi-
croscopic examination of these sporocysts revealed the gold particles in
different focal planes (Figs. 2A–C), indicating that the microcarriers
Fig. 2. Transmission micrograph of in vitro generated mother sporocyst tha
subjected to particle bombardment with gold particles (not coated with DN
diameter). The same sporocyst is shown in the three panels in different foca
Fig. 3. Haematoxylin and eosin-stained paraffin section of B. glabrata snail
bardment with gold micro-carriers. (A) Overview section through the mouth
The box outlines the kidney region that is infected with sporocyst. This boxed
other key anatomical features are indicated by text. (B) Higher magnification
include developing mother sporocysts. The radula of the snail is prominent
features are indicated by text. (C) Higher power magnification of snail tissue
position of gold particles in close proximity to the germ ball cells in the spo
had penetrated into deeper tissue layers. Mother sporocysts containing
gold particles appeared to be otherwise intact and were indistin-
guishable in other ways from control, in vitro derived mother spo-
rocysts (not shown). Indeed, these sporocysts containing gold particles
were metabolically active as judged by the movement of the flame cells,
peristaltic movements, and depletion of culture media during the
course of the experiment. These findings indicated that many miracidia
survive the biolistics procedure and, second, that bombarded miracidia
can transform into mother sporocysts in vitro.
To examine whether transgenic parasites could survive in the in-
termediate host, miracidia which had been bombarded with gold
particles were used to infect B. glabrata snails. Snails were exposed to
bombarded miracidia and the progression and penetration of the
bombarded miracidia was monitored by light microscopy (Zeiss Axi-
oplan microscope). Most miracidia had penetrated the snails by 3 h.
Histological sections of the infected snails and of uninfected control
snails were prepared at 10–14 days after infection. Shells were removed
from the snails using forceps, the snails fixed in Bouin�s fluid, washed
in 70% ethanol, dehydrated in 100% ethanol, cleared with xylol, and
embedded in paraffin (Paraplast Plus, Sherwood Medical, St. Louis,
MO) (Lemos and Andrade, 2001; Romeis, 1989). Five micrometer
snail tissue sections were cut and then stained with haematoxylin and
eosin. Mother sporocysts could be observed in the tissues of the snails
(Fig. 3) and their structure and appearance were similar to that
described for normal sporocysts by Lemos and Andrade (2001). Germ
balls were clearly visible and gold particles (optically dense, 1–2lm
diameter) were located in the sporocyst tissues (Figs. 3A–C). The nu-
clei of the germ ball cells were large and euchromatic and hetero-
t has developed from a miracidium at 72 h after the miracidium was
A). Arrows indicate the position of gold particles (particles are 1.0lm
l planes. Scale bars indicate sizes.
2 weeks after infection with miracidia transformed by particle bom-
, mid-body, head–foot, and hepatopancreas/kidney region of the snail.
region is shown at higher magnifications in (B and C). The locations of
of snail tissues shown in (A); the regions in and around the box outline
in the upper left of the panel. The locations of other key anatomical
s from (B), with a close-up image of sporocysts. The arrows show the
rocysts. Scale bars are included at the top of each panel.
O. Heyers et al. / Experimental Parasitology 105 (2003) 174–178 177
chromatin was apparent at the nuclear membrane (Figs. 3B and C,
indicative of elevated metabolic activity in these cells. The prominent
nucleoli were also enlarged and showed lighter areas (Fig. 3C), indic-
ative of transcriptional activity (Smetana, 1974; Underwood, 1990). In
many cases, the gold particles co-localized with the germ ball cells
(Fig. 3C). This co-localization and the obvious integrity of the spo-
rocysts demonstrated that it is possible to transform miracidia by
particle bombardment and to use miracidia transformed in this way to
infect B. glabrata. Thus, these techniques represent useful tools for the
establishment of daughter lines of transformed schistosomes.
Next, we examined snails infected with miracidia that had been
bombarded with gold particles coated with plasmid constructs en-
coding the EGFP reporter under control of the schistosomal HSP70
gene regulatory regions, or with a control promoter-less plasmid. We
failed to observe EGFP in tissues of these snails by normal or confocal
fluorescence microscopy. Neither were we able to detect EGFP protein
by immunoblot analysis of snail tissues employing an anti-EGFP an-
tibody (not shown). However, when we examined the snail tissues for
transcription of mRNA encoding EGFP using an EGFP-specific nes-
ted RT-PCR with total RNA from infected snails, we detected am-
plicons of the expected size, 396 bp (Fig. 4). This confirmed that the
EGFP transgene introduced into miracidia by particle bombardment
was indeed transcribed in sporocysts developing in these snails.
To summarize,here we have demonstrated that miracidia of B.
glabrata can withstand and survive particle bombardment with gold
microcarriers carrying plasmid transgenes and that mother sporocysts
of S. mansoni were vital and developed in vitro from miracidia that
survived particle bombardment. In addition, we showed that gold
particles had entered into the miracidia at different depths. Indeed,
because the detection of single gold particles is difficult, we speculate
that the actual transformation efficiency may have been higher than we
observed. Also, we have demonstrated that bombarded miracidia were
able to infect B. glabrata snails. Numerous developing sporocysts were
evident within organs and tissues of the snails by about 2 weeks after
infection, and their morphology was indistinguishable from that de-
scribed by Lemos and Andrade (2001) for natural (non-transformed)
S. mansoni sporocysts in B. glabrata. Thus, we assume that sporocysts
which developed from bombarded miracidia were completely vital.
Also, many gold particles were visible and co-localized with germ ball
cells in these sporocysts. Since it has also been shown that organelles
Fig. 4. EGFP mRNA expression in tissues from a snail infected with
miracidia transformed by particle bombardment by gold microcarriers
coated with the S. mansoni HSP70 promoter-EGFP plasmid. Lanes 1
and 3: nested PCR with EGFP-specific primers; lanes 2 and 4: negative
control; no reverse transcription prior to PCR; lane 5: negative con-
trol; nested PCR with EGFP-specific primers using genomic DNA
from an uninfected snail as template; lane 6: positive control PCR with
plasmid DNA; lane 7: Marker. Lanes 1, 2 and 3, 4 correspond to two
independent experiments.
inside cells can be transfected by biolistic methods (Butow et al., 1996;
Daniell, 1993; Lorito et al., 1993; Rohou et al., 2001; Ruf et al., 1997),
it seems feasible that transfection of the sporocyst germ balls can be
achieved. Transfection of the totipotent cells that constitute the germ
balls of sporocysts will, theoretically, lead to the formation of trans-
genic daughter sporocysts. This, in turn, should facilitate the devel-
opment of a transgenic line of schistosomes.
The use of adult worms for transfection of schistosomes presents an
inherent problem in that adult worms cannot easily be introduced back
into the life cycle. Adult worms would have to be implanted into the
mesenteric veins of mice, which is technically difficult. Adult worms
are, however, useful for testing schistosomal expression plasmids.
Moreover, the use of mother sporocysts for transformation experi-
ments as described by Wippersteg et al. (2002a,b) may be less than
optimal. Although sporocysts can be generated in vitro from eggs and
methods for their long term culture and co-culture with a B. glabrata
embryo (bge) cells line have been developed (Bayne et al., 1994; Iva-
nchenko et al., 1999; Jourdane et al., 1985), implantation of in vitro
cultured sporocysts into the snails is difficult and survival rate of snails
and implanted sporocysts is low.
The coding region of the EGFP reporter used here contains mu-
tations which improve the intensity of green fluorescence. Furthermore
it contains other mutations which optimize protein folding and solu-
bility at 37 �C, apparently making it suitable for expression in adult
schistosomes, which are cultured at this temperature. Although EGFP
has been reported to have a 35-fold higher intensity than GFP (Heim
and Tsien, 1996), we were only able to detect EGFP fluorescence using
laser scanning microscopy. The expression pattern we observed was
comparable to that reported by Wippersteg et al. (2002a). After heat
shock, EGFP signals in adult worm tissues were seen preferentially in
tegumental areas, areas around the head sucker and at the posterior
end, and co-localized with gold particles in most cases. Expression of
EGFP in schistosomes is therefore possible but does not constitute a
significant improvement over the previous reports. Possibly the weak
EGFP signal results from low expression and/or incorrect folding of
EGFP in schistosomes. It is known, that the removal of a cryptic in-
tron in the GFP coding sequence is required to achieve prominent
fluorescence in transgenic Arabidopsis thaliana (Haseloff et al., 1997).
Introducing introns into the reporter sequence enhances GFP expres-
sion in Caenorhabditis elegans (Fire et al., 1990). Similar modifications
to constructs might enhance EGFP performance in schistosomes.
In infected snail tissues, EGFP could not be detected on the protein
level by immunoblot analysis, likely because the amount of EGFP
translated was below detectable levels in preparations of the extracts of
total snail proteins. On the other hand, EGFP transcripts in the snails
could be detected by nested RT-PCR, indicating that the reporter gene
was transcribed in these snails. Strong promoters that are constitu-
tively expressed in all schistosome developmental stages have not yet
been characterized and this remains as a serious impediment for de-
velopment of a tractable transgenesis system. The promoters used here
and by Wippersteg et al. (2002a,b) are developmentally regulated, and
may need specific, but as yet undefined, conditions for activity. This
could be an additional explanation for the poor expression of the
EGFP (or GFP) and the inability to detect fluorescence using con-
ventional fluorescence microscopy, which is straightforward in other
systems involving, for example, Drosophila melanogaster or C. elegans.
For future studies with transgenesis in schistosomes, the deployment of
additional reporters or promoters may improve expression levels of the
transgenes. Because numerous genomic sequences of schistosomes are
now becoming available, we can anticipate the introduction in the near
future of more appropriate endogenous gene promoters.
Finally, the findings reported here present proof of the principle
that miracidia can be transformed by biolistics procedures using gold
microparticles coated with transgene constructs, and that the trans-
formed miracidia retain the capacity to infect snails and to begin the
propagation of transgenic schistosomes. Yet simple plasmids located
episomally in the germ ball cell cytoplasm of transfected sporocysts
178 O. Heyers et al. / Experimental Parasitology 105 (2003) 174–178
will not likely or reliably lead to chromosomal integration of reporter
transgenes. In order to enhance the prospects for chromosomal inte-
gration of schistosome transgenes, we are currently investigating the
utility of mobile genetic elements including transposons, retrotrans-
posons, and pseudotyped retroviruses as transfection vectors. RNA
interference techniques in schistosomes (e.g., Skelly et al., 2003) may
also be compatible with biolistics procedures for transgenesis of
schistosomes.
Acknowledgments
We thank Grit Meusel for laboratory assistance, Anorte Marko for
preparing histological specimens and Ute Mackenstedt for interpre-
tation of snail histology. This study was supported by a grant from the
Deutsche Forschungsgemeinschaft (KA 866/2-1) to B.H.K. P.J.B. is a
recipient of a Burroughs Wellcome Fund scholar award in Molecular
Parasitology and is supported by an Infrastructure Grant from the
Ellison Medical Foundation (Award No. ID-IA-0037-02).
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https://www.researchgate.net/publication/7927756
	Schistosoma mansoni miracidia transformed by particle bombardment infect Biomphalaria glabrata snails and develop into transgenic sporocysts
	Acknowledgements
	References