A novel Schistosoma japonicum endonuclease homologous to DNase II
Hou et al. BMC Genomics
A novel Schistosoma japonicum endonuclease homologous to DNase II
Nan Hou 1
Xianyu Piao 1
Pengfei Cai 1
Chuang Wu 1
Shuai Liu 1
Yan Xiao 1
Qijun Chen 0 1
0 Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin University , Xi An Da Lu 5333, Changchun , People's Republic of China
1 MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College , Dong Dan San Tiao 9, Beijing , People's Republic of China
Background: Recent advances in studies of the Schistosoma japonicum genome have opened new avenues for the elucidation of parasite biology and the identification of novel targets for vaccines, drug development and early diagnostic tools. Results: In this study, we surveyed the S. japonicum genome database for genes encoding nucleases. A total of 130 nucleases of 3 classes were found. Transcriptional analysis of these genes using a genomic DNA microarray revealed that the majority of the nucleases were differentially expressed in parasites of different developmental stages or different genders, whereas no obvious transcriptional variation was detected in parasites from different hosts. Further analysis of the putative DNases of S. japonicum revealed a novel DNase II homologue (Sjda) that contained a highly conserved catalytic domain. A recombinant Sjda-GST protein efficiently hydrolysed genomic DNA in the absence of divalent iron. Western-blot and immunofluorescence assays showed that Sjda was mainly expressed on the teguments of female adult parasites and induced early humoral immune responses in infected mice. Conclusions: A novel DNase II homologue, Sjda, was identified in S. japonicum. Sjda was mainly distributed on the teguments of adult female parasites and possessed a typical divalent iron-independent DNA catalytic activity. This protein may play an important role in the host-parasite interaction.
Schistosoma japonicum; Nuclease; DNase II homologue; Host-parasite interaction
Schistosomiasis is one of the most serious parasitic
diseases, infecting over 200 million people in 76 tropical
and subtropical countries . The pathogenesis of
schistosomiasis is mainly caused by egg-induced granuloma
formation and subsequent fibrosis. However, tools for
the early diagnosis and abrogation of the
pathophysiological effects of the parasite, especially the eggs, are still
lacking. Treatment of schistosomiasis has relied on a
single drug, praziquantel; however, strains of S. mansoni
that are resistant to praziquantel have emerged .
Understanding the parasite biology and the mechanism of
the hostparasite interaction are critical for the
development of an effective vaccine and anti-parasite drugs ,
which are urgently needed for schistosomiasis control.
Recent advances in research on the S. japonicum
genome, transcriptome, and proteome have provided
information that contributes to the understanding of parasite
biology and the hostparasite interplay [4-7]. Nucleases,
including DNases, RNases, topoisomerases, recombinases,
ribozymes, and RNA splicing enzymes, have diverse
functions ranging from DNA replication,
recombination and repair and RNA maturation and processing
to nutrient regeneration and cell death in various
species . Recent evidence has highlighted a novel role of
nucleases, especially DNases, in pathogen evasion of
host defence mechanisms. Nucleases of bacteria, such
as Streptococcus [9-11], Staphylococcus aureus  and
Aeromonas hydrophila , have been suggested to act
as virulence factors in resistance to host neutrophil
extracellular traps (NET), while members of the DNase
II family in the roundworm Trichinella spiralis have
been found to be secreted into circulation to counteract
host innate immune responses . Thus, disrupting
the functions of vital nucleases will not only hinder the
homeostasis and development of parasites but also be
beneficial to the host immune system and allow
parasite control. However, no nucleases have been identified
and characterised in the Schistosoma genus to date,
except for a dicer enzyme in S. mansoni . In this
study, we first surveyed the S. japonicum genome for
genes encoding potential nucleases and then analysed
the transcription of these genes during different
developmental stages using a DNA microarray. Numerous
genes encoding potentially important nucleases were
identified, and a novel DNase II predominantly
expressed during the mature parasite stage was
Identification and characterisation of putative nuclease
sequences of S. japonicum
Using known nucleases from Brugia malayi,
Caenorhabditis briggsae, Caenorhabditis elegans, Hydra vulgaris,
Nematostella vectensis, Schistosoma mansoni and
Trichoplax adhaerens as bait sequences, 238 homologous
proteins encoded in the S. japonicum genome were
identified. Of these, 130 proteins were found to contain
domains with potential nuclease activities. SignalP4.0 was
also used to predict signal peptides. Potential proteins
with a D score of greater than 0.45 were considered to
have an N-terminal signal peptide sequence. Among the
130 nucleases, 12 were predicted to have signal peptide
sequences. The proteins were also characterised
according to their substrates, enzymatic properties, and
divalent cation dependencies. The catalytic activities of 24
nucleases were potentially divalent cation-dependent
(Table 1). The potential nucleases were grouped into
three classes as follows: 4.6% were classified as DNases,
60.8% as RNases, and the remainder (34.6%) possessed
the potential for hydrolysing both DNA and RNA
Transcriptional analysis of genes encoding putative
nucleases of S. japonicum
The transcriptional characteristics of the putative
nucleases of S. japonicum were investigated using a
targetspecific microarray. The expression of the nucleases
greatly differed among the parasites at different
developmental stages (Figure 1A) and between those of different
genders (Figure 1B), whereas few differences were
observed among adult worms of the same gender isolated
from different hosts (Figure 1B). There were a total of
22 genes that were distinctly expressed at particular
developmental stages (Figure 2). A total of 19 genes were
differentially expressed in the male or female adults from
various hosts, including1 DNase, 6 nucleases and 7
RNases, which were up-regulated in the female adults, and
3 nucleases and 2 RNases, which were up-regulated in
the male adults (Figure 2B).
The transcription of the 6 putative DNases was further
analysed. Among them, only the expression of
AAW25360.1 was significantly up-regulated in the
schistosomula and paired adults compared with egg and
cercariae (Figure 3A). These results were confirmed by
QPCR (data not shown). A phylogenetic comparison
showed that these DNases were clearly divided into 5
major groups, including DNase I, DNase II, exoDNase III,
ATP-dependent exoDNase and Tat DNase (Figure 3B).
AAW25360.1 was separate from the other DNases
(Figure 3B). The similarity of AAW25360.1 with
known DNase II sequences from other organisms was
analysed (Figure 4A). AAW25360.1 shared 70.3%,
25.4%, 27.5%, and 25.4% identity with the DNase II
proteins of S. mansoni, human, mouse and C. elegans,
respectively. The full-length cDNA of AAW25360.1
contained an open reading frame of 1,134 bp encoding
378 amino acids with a molecular mass of 43,607 Daltons,
and it was termed Sjda (S. japonicum DNase). Sjda
contained conserved amino acid residues, especially in
the catalytic domain, and had a composition typical of
DNaseII nucleases (Figure 4B and data not shown). It
also had a classical peptide sequence at the N-terminus.
Characterisation of DNase activity of Sjda
To confirm the endonuclease activity of Sjda, a
recombinant Sjda-GST protein and Sjda-His were generated
(Figure 5A and B) and tested for DNA hydrolysis
activity. Sjda-GST could efficiently digest genomic DNA
from human, bovine, rabbit and murine sources, and
its catalytic activity was independent of any divalent
cation (Figure 6A, B). Sjda could also digest the
genomic DNA of S. japonicum (Figure 6C). We next
determined the optimum pH for its activity and found that
Table 1 The characteristics of the putative nuclease sequences of S. japonicum
Figure 1 Heat maps of transcription of all putative nucleases in S. japonicum parasites of different stages, genders or hosts. A.
Transcription of all putative schistosome nucleases from parasites at different developmental stages. The cercariae (C) were obtained from
infected Oncomelania hupensis, and the eggs (E), hepatic schistosomula (S) and paired adult worms (A) were obtained from S. japonicum-infected
BABL/c mice. B. Transcription of all putative nucleases from parasites of different genders and hosts. Male and female adults were obtained from S.
japonicum-infected mammals, including BABL/c mice (1), C57BL/6 mice (2), rabbits (3) and buffaloes (4). The transcriptional data is based on a genomic
DNA microarray dataset.
Figure 2 Heat maps of nuclease genes with distinct transcription profiles from parasites of different stages or genders. A. Genes
encoding putative nucleases with distinct transcriptional patterns in parasites of different developmental stages. The parasites were obtained as
described in Figure 1. B. Genes encoding putative nucleases with distinct transcriptional patterns in adult parasites of different genders. A 2-fold
difference was consistent with differential expression (p < 0.05) throughout the life cycle or between the female and male adults.
Figure 3 Transcriptional characteristics and phylogenetic classification of putative schistosomal DNases. A. Quantitative comparison of
the transcriptional data of the putative DNases in parasites of different developmental stages (E: egg; C: cercariae; S: schistosomulum; M: male;
and F: female) based on a DNA microarray dataset. *** indicates p < 0.001. B. Phylogenetic tree constructed with amino acid sequences of the
putative DNases. A scale of 0.2 is shown below the tree. The frequency of each sequence is shown in brackets before the name.
Sjda-GST activity occurred over abroad pH range and was
highly active at acidic pH levels (Figure 6D). A further
assay with supercoiled plasmids as substrates showed that
Sjda-GST-digested products were not only short
fragments but also fragments that were slightly smaller than
the linear DNA produced by XhoI (Figure 6E), indicating
that it may also be able to produce nicks along DNA
chains apart from double-strand digestion.
Sjda was mainly identified on surface membranes of
female adult worms
To confirm Sjda expression, a Westernblot assay was
first carried out to detect this protein in parasites at
different developmental stages. A thick protein band
for Sjda was observed in the female adults, and a dim
band was detected in the schistosomula, while no band
was observed in the eggs, cercariae or male adults
(Figure 7A). These results indicated that Sjda was
mainly expressed in the female parasites.
Immunofluorescence further confirmed that this protein was
mainly localised to the surfaces of the female parasites
Dynamics of Sjda-specific antibodies in infected hosts
Sjda-specific IgG levels in the sera of infected mice on
days 0, 7, 14, 28, 42 post-infection were detected by
Western blotting. The Sjda-His protein was specifically
recognised by IgG in some infected mice as early as day
7 post-infection. This immune reactivity increased
gradually over time, peaking on day 42 (Additional files 1
and 2). The serum positive rates of Sjda-specific IgG on
days 0, 7, 14, 28, 42 were 0, 50 14.1, 77.4 8.4, 91.7
11.8, and 100 0%, respectively (Additional file 1).
Figure 4 (See legend on next page.)
(See figure on previous page.)
Figure 4 Comparison of amino acid sequence of Sjda with DNaseII molecules from various species. A. Phylogenetic tree constructed with
amino acid sequences of Sjda and 10 DNase II molecules from other species. The protein name includes the species name and NCBI protein
bank ID. A scale of 0.2 is shown below the tree. The frequency for each sequence is shown in brackets before the name. B. Amino acid sequence
alignments of Sjda and S. mansoni, human, mouse and C. elegans DNase II homologues. The catalytic domains are enclosed inboxes. * indicates
conserved cysteine residues. Residues that are present in all sequences are highlighted with dark-grey shading. Residues that are conserved in at
least three sequences are highlighted in grey. The dots indicate missing residues.
Nucleases, particularly DNases, have recently been
found to play novel roles in pathogen invasion in
evading host defence mechanisms. For example, the M1
serotype strain of Group A Streptococcus (GAS) has
been found to express a potent DNase that is both
necessary and sufficient to promote GAS neutrophil
resistance and virulence in a murine model of necrotising
fasciitis . The membrane-attached surface-exposed
DNA-entry nuclease EndA of Streptococcus pneumoniae
has been shown to play an important role during the
establishment of invasive infections by degrading
extracellular chromatin and thereby overcoming the innate
immune responses in mammals . S. pyogenes
nuclease A promotes bacterial survival in whole human blood
and in neutrophil killing assays . The expression of
nucleases by S. aureus has also been found to lead to
increased bacterial dissemination in mice . In addition
to those found in bacteria, a multitude of DNase family
members have been found in T. spiralis. These DNases
are believed to enhance the degradation of DNA
released from host phagocytes, which are involved in the
down-regulation of host inflammatory processes . In
this study, we performed a genome-wide investigation to
identify the nucleases of S. japonicum with potential
functions in host-parasite interactions.
We identified 130 nucleases of 3functional classes,
which all contained conserved domains of classical
nucleases (Table 1 and Additional file 3). The majority of
these nucleases were found to be differentially expressed
in eggs, cercariae or flukes (including schistosomula and
adults), but only a few genes were differentially
expressed between the schistosomulum and adult stages.
This finding indicated that the schistosomula and adults
have similar nucleicacid metabolic pathways.
Furthermore, the expression levels of the nuclease genes in
adult worms of the same gender from different hosts
were also similar, indicating that the parasites have
evolved a capacity for adaptation to different host
environments (Figures 1 and 2). Additionally, the expression
levels of nucleases were greatly increased in the female
parasites compared with the male parasites (Figure 1B),
suggesting that they are critical components that are
much more metabolically active in female compared
with male parasites. However, it cannot be ruled out that
some nucleases actively participate in the host-parasite
Among all 130 putative nucleases, six genes encoding
putative DNases were identified in the S. japonicum
genome. These DNases only accounted for 4.6% of all of the
putative nucleases. AAW25360.1 (Sjda), with a sequence
characteristic of DNase II, was the only DNase that
showed significantly increased expression in the adult
parasites (Figure 3A). Since the identification of human
DNase II in 1998, murine , porcine  and bovine
 DNase II genes have been sequentially identified.
Other putative genes encoding proteins homologous to
DNase II have recently been observed in the genomes of
many species, including Gallus gallus (chicken), Fugu
rubripes (puffer fish), Xenopus laevis (frog), Anopheles
Figure 5 Generation of recombinant Sjda proteins. A. Detection of purified GST-tagged recombinant Sjda protein. The molecular weight of
Sjda-GST is 69 kDa. B. Detection of purified His-tagged recombinant Sjda protein. The molecular weight of Sjda-His is 43.5 kDa. Lane M, protein
molecular weight markers; Lane 1, purified proteins were analysed by SDS-PAGE and stained with Coomassie brilliant blue; Lane 2, Western blotting of
the recombinant protein with specific mouse anti-Sjda antibodies; and Lane 3, Western blotting of the recombinant protein with anti-GST-tag mAb (A)
or anti-His-tag mAb (B).
Figure 6 DNase activity of the recombinant protein Sjda-GST. Agarose gel electrophoresis was carried out to analyse the catalytic products
of the DNase. A. The left panels show the DNA digested by 40 g Sjda-GST or GST proteins at the indicated times. The right panels show the
digested DNA according to the indicated amount of Sjda-GST or GST proteins with an incubation time of 15 min. All assays were carried out with
200 ng human genomic DNA in a final volume of 25lPBS (pH7.0) at 37C. B. The DNA from different hosts digested by Sjda-GST or GST. H, human; C,
calf; R, rabbit; and M, C57BL/6 mouse. C. S. japonicum DNA digested by Sjda-GST or GST. D. Human DNA digested by Sjda-GST in PBS of different pH
levels, ranging from 4.0 to 7.5. E. Supercoiled plasmid DNA digested by Sjda-GST, GST or XhlI.
gambiae (mosquito), Drosophila, and the slime mould
Dictyostelium discoideum, in addition to parasitic
organisms, such as Trichinella spiralis, Trichomonas vaginalis,
and the bacterium Burkholderia pseudomallei .
Analysis of these DNase II genes and their homologues has
revealed a striking conservation of amino acid residues
surrounding the catalytic site of the molecule . The
high conservation of amino acid residues in the catalytic
domain of Sjda in the DNase II family (Figure 4B)
strongly indicates that its function is similar to those of
other DNase II members.
The catalytic activity of the recombinant Sjda-GST
protein was divalent cation-independent, and its
preference for an acidic environment led to its characterisation
as an endogenous DNase II (Figure 6A-D). However, it
seemed to be able to introduce nicks along one strand of
the DNA, similar to human DNase I  (Figure 6E).
Sjda was mainly up-regulated in the schistosomula and
adult parasites, but not in the cercariae or eggs,
suggesting that this molecule may play a role in the adaptation
of parasites to the host circulation (Figure 7A). The
expression of Sjda was mainly localised to the surfaces of
the adult female parasites, but it was not detected in the
gut, further supporting this hypothesis (Figure 7B).
Interestingly, Sjda was not expressed in the male
parasites (Figure 7B). Previous studies have shown that the
teguments of male and female parasites are structurally
different, and the surfaces of male schistosomes are
coated with an increased number of sugar components
compared to those of female parasites . These
findings indicate that male and female parasites possess
different arsenals for the resistance of host immune
Furthermore, although Sjda was primarily found on
the surfaces of the female parasites, it cannot be ruled
out that it is secreted by the parasite. It has been
reported that Trichinella spiralis constantly secretes
DNase II into the surrounding environment in the
host and affects the host immune system [22,23]. We
predicted the localisation of a signal peptide sequence
with 19 amino acids to the N-terminus of Sjda. This
finding strongly indicates that it is secreted by the
parasite. Furthermore, the detection of Sjda-specific
antibodies in the infected host serum as early as one
week after infection also suggested that it was
recognised by the host immune system and played a role in
the host-parasite interaction.
In this study, we discovered a novel DNase II homologue,
Sjda, with a DNA hydrolysis function. This protein was
found to be mainly expressed on the tegument of female
Figure 7 Detection of Sjda protein in parasites of S. japonicum. A. Detection of Sjda protein in parasites at different developmental stages with
specific anti-Sjda antibodies by Western blotting. A specific band is present only for the proteins extracted from the female parasite. B. Detection of the
Sjda protein on the surfaces of adult worms by immunofluorescence. A specific signal (in red colour) was observed only on the teguments of the female
parasite. The lower panels depict magnifications of the upper panels. , female adult; and , male adult.
adult parasites, and it also stimulated early humoral
immune responses after infection. Sjda is likely to play an
important role in the hostparasite interaction. These
findings provide a novel starting point for the discovery
of novel targets for drug and vaccine development
All procedures performed on the animals in this study
were conducted according to the animal husbandry
guidelines of the Chinese Academy of Medical Sciences.
The human peripheral blood sample was donated by a
healthy volunteer and anonymised. Written informed
consent was obtained from this volunteer for the
publication of this study and any accompanying images. A
copy of the written consent is available for review by the
Editor of this journal. All procedures performed on
human samples were carried out in accordance with the
tenets of the Declaration of Helsinki. This research was
reviewed and approved by the Experimental Animal
Committee and the Ethical Committee of the Chinese
Academy of Medical Sciences.
Parasites and animals
Parasite-infected Oncomelania hupensis was purchased
from the Hunan Institute of Parasitic Diseases, Yueyang,
China. The freshly released cercariae were harvested
immediately. Special pathogen-free (SPF) C57BL/6 mice
(males, 6 weeks old) and New Zealand white rabbits
(both from the Vital River Laboratory Animal Technology
Co. Ltd., Beijing, China) were percutaneously infected with
S. japonicum cercariae (20 parasites per mouse or 1,000 to
1,500 per rabbit). Sera from the infected animals were
collected on days 0, 7, 14, 28 and 42. Hepatic
schistosomula were isolated from the animals at 2 weeks
postinfection, while mixed adult worms were obtained at
6 weeks post-infection by portal perfusion and manual
separation under a light microscope. Eggs were purified
from the liver tissues as previously described .
Adult worms from infected buffaloes were provided by
the Hunan Institute of Parasitic Diseases.
Total RNA isolation and quality control
Parasites of all stages were soaked in RNAlater
solution (Ambion, CA, USA) overnight and stored at
80C. Total RNA was extracted using an RNeasy
Mini Kit (QIAGEN GmbH, Hilden, Germany), and
the contaminating genomic DNA was completely removed
from the RNA samples with a TURBO DNA-freeKit
(Ambion). RNA quantification and quality control was
conducted with a Nanodrop ND-1000 spectrophotometer
(Thermo Fisher Scientific, Wilmington, DE, USA) and
denaturing agarose gel electrophoresis.
Microarray construction, hybridisation and validation
The sequences used for the design and construction of
the microarray were obtained from three databases and
included putative transcriptome data for S. japonicum
(12,657 sequences)  and S. mansoni (13,197
sequences)  and UniGene data from NCBI for S.
japonicum (10,809 sequences). Putative transcriptome data
were also predicted in-house (15,685 sequences) .
Redundant sequences were eliminated according to a
coverage 90% and identity 80% using cd-hit-v4.3
software (http://bioinformatics.ljcrf.edu/cd-hi/). A total of
21,861 target sequences (20,194 sequences derived from
S. japonicum and 1,667 sequences derived from S.
mansoni) were determined, and 131,092 experimental probes
with 3 or 4 unique 60-mer oligonucleotide probes per
sequence corresponding to both strands of the genomic
DNA were designed. The microarray was manufactured
by Roche NimbleGen (Basel, Switzerland), and
microarray hybridisation was carried out at the core facility of
the Capital BioCorporation (Beijing, China). The
procedure for microarray design and hybridisation was
performed as described previously . An expression
dataset of target genes was generated based on the
fluorescence intensity output from the Roche NimbleGen
platform. The expression data of the genes encoding
nucleases were retrieved and deeply analysed using Microsoft
Excel and GraphPad Prism 5, and heat maps were
generated using ClustlX 3.0 and Treeview. The expression levels
of the genes at each developmental stage (including the
cercariae, schistosomula, male adult, and female adult)
were obtained by comparing them with those at the egg
stage. Expression levels 2-fold (p < 0.05) were regarded
as significantly up-regulated. The microarray data were
further validated by two independent QPCR assays .
The expression levels of six putative DNase genes
(AAW25685.1, Sjp_0035270, AAW25360.1, AAW26473.1,
CAX69654.1, and CAX72944.1) were also analysed in this
study (data not shown). Quantification of the expression
of each gene was performed by normalisation against the
expression of the housekeeping gene, NADPH. Statistical
analyses were conducted using GraphPad Prism 5 or
Prediction of nucleases encoded in S. japonicum genome
For the prediction of potential S. Japonicum nucleases,
nuclease protein sequences of 7 species (Brugia malayi,
Caenorhabditis briggsae, Caenorhabditis elegans, Hydra
vulgaris, Nematostella vectensis, Schistosoma mansoni,
and Trichoplax adhaerens) were selected from the
KEGG database. These 7 species are the most closely
related to flatworms according to the KEGG Organisms
html). We constructed an S. japonicum protein database
including information from the following three sources:
S. japonicum proteins from NCBI (25319 proteins), S.
japonicum proteins from CHGC (13469 proteins) and
proteins predicted from the 21861 genes described above
by Augustus software. Protein sequences of S. japonicum
were compared with reference nuclease sequences using
BLASTP with an E-value < 1e-9. Potential nuclease
sequences of S. japonicum were further analysed for
conserved motifs and domains using the Conserved
Domain Database (CDD) v3.08 of NCBI [29,30]. The CDD
content includes NCBI-curated domains, as well as domain
models imported from a number of external source
databases, including Pfamv.22.0 [31,32], SMART v.5.0 ,
COG , PRK , and TIGRFAM v.13 . Additionally,
proteins that were shorter than 100 residues or with lengths
of less than 80% of the nuclease core sequences were not
retained, and the remaining sequences were subjected to
Sequence comparison and phylogenetic analysis of Sjda
with homologous genes from other species
The homologous sequences of S. japonicum AAW25360.1
encoding a putative DNase (Sjda) were retrieved from
NCBI by performing a BLAST search (version 2.2.26)
based on bidirectional best-hit (BBH) identification, an
E-value <1e-10 and an identity >30%. Ten sequences from
animals, parasites and humans were obtained and
subjected to phylogenetic analysis. Multiple alignments of the
selected sequences were performed with DNAMAN for
sequence comparison or with MUSCLE algorithm
implemented in CLC Sequence DNA Workbench 6.6.2 software
(CLC bio). A phylogenetic tree was constructed using the
maximum likelihood approach implemented in PhyML3.0
. Bootstrap values, expressed as a percentage of 100
replicates, were given at branching points.
Plasmid construction and recombinant protein generation
Recombinant Sjda-GST and GST proteins of S. japonicum
were generated for confirmation of the DNase activity of
Sjda. A recombinant Sjda-His protein was generated to
detect antibodies in the mouse sera. Briefly, the gene
fragment encoding Sjda was amplified from S. japonicum
cDNA using a high-fidelity Phusion DNA polymerase
(FinnzymesOy, Finland). PCR was performed with the
following primers, which carried an EcoR I and SalI
restriction site, respectively: forward 5-GAG GAA TTC
ATG CTC GTA TTT CTG GGA C-3 and reverse
CTT GTC GAC TTA GGC ATA CAT AAT AAA AAC
ATA TTG-3. The amplified product was purified using a
DNA Gel Extraction Kit (Axygen, CA, USA) and cloned
into pGEX-4 T-1 and pET-32a expression vectors. The
recombinant plasmids were transformed into DH5 (DE3)
Escherichia coli, and positive clones were selected for
sequencing. The correct recombinant plasmids were then
transformed into Rosetta E. coli for protein expression.
The recombinant proteins Sjda-GST and GST were
purified with Glutathione Sepharose (GE Healthcare, Uppsala,
Sweden), and Sjda-His was purified with Ni-NTA Agarose
(QIAGEN) according to the manufacturers instructions.
All proteins were analysed with a 12% SDS-PAGE gel and
Western blotting with monoclonal antibodies against the
His-tag or GST-tag (all from Cell Signaling Technology,
Detection of DNase activity of Sjda
Genomic DNA from humans, rabbits, mice, and calves,
bacterial plasmid DNA (pGEX-6p-1) and Xhol-digested
linear plasmid DNA were used as substrates for the
assessment of DNase activity. Human whole blood, New
Zealand white rabbit ear tissue and C57BL/6 mouse tail
tissue were used for the extraction of genomic DNA
with a TIANamp Genomic DNA Kit (Tiangen Biotech.,
Beijing, China) according to the manufacturers
instructions. Calf genomic DNA was purchased from Sigma
Aldrich (St. Louis, MO, USA). The recombinant Sjda
protein and 200 ng genomic DNA were mixed in 25 l
PBS with different pH values, prepared on ice and
incubated at 37C from 5 min to 1 h. The reactions were
terminated by incubation at 70C for 20 min. A 10-l
aliquot of each reaction mixture was analysed by 0.6%
agarose gel electrophoresis.
Detection of Sjda protein in S. japonicum parasites
To confirm the expression of the Sjda protein in the
parasites, a monoclonal antibody to Sjda was generated.
Briefly, the amino acid sequence of Sjda was analysed for
antigenic determinants, hydrophilicity and secondary
structures using Geneious, and a 237-bp gene fragment
(from amino acid 25 to 108) was chosen and cloned into
pET-30a plasmids to generate a recombinant protein.
The purified recombinant protein was used to immunise
BABL/c mice (females, 6 weeks old). Splenocytes from
the immunised mice were fused into SP2/O cells. The
positive fusion cells were selected to generate a
monoclonal antibody against Sjda. The specificity of this antibody
was proven by Western blotting.
Parasites at all stages stored at 80C were
homogenised by grinding in liquidnitrogen followed by
incubation with lysis buffer (8 M urea, 4% CHAPs, 1% DTT,
1% EDTA, 10 mM Tris, and 35 g/ml PMSF) for 30 min
on ice. The mixture was centrifuged at 12,000 rpm for
30 min at 4C, and the protein concentrations were
quantified with a BCA kit (Pierce, Rockford, IL, USA) in
accordance with the manufacturers directions. The
extracted proteins were denatured by boiling in
SDSPAGE buffer, separated on 12% SDS-PAGE gels, and
transferred to polyvinylidenedifluoride membranes
(Millipore, Bedford, MA, USA). The blots were blocked
with a blocking buffer containing 5% skim milk for 1 h
at room temperature and incubated in the same buffer
with an anti-Sjda monoclonal antibody (2 mg/ml,
1:1,000 dilution) or mouse IgG control overnight at 4C.
After washing, detection was accomplished by
incubation with an IRDye 800 CW conjugated goat anti-mouse
IgG (H + L) antibody (Li-COR Biosciences, Lincoln, NE,
USA), using Odyssey (Li-COR).
Localisation of the Sjda protein in adult worms was
carried out with an immunofluorescence assay using
anti-Sjda monoclonal antibodies according to the
standard protocol with minor modifications. Briefly, parasites
were fixed in 4% formaldehyde and then permeabilised
for 2 h with 1% SDS in PBS followed by treatment with
proteinase K (2 g/ml) for 10 min at room temperature.
After permeabilisation, the parasites were re-fixed for
10 min in 4% formaldehyde and rinsed with PBSTx (PBS
plus 0.3% Triton X-100). The parasites were incubated
in blocking solution (5% horse serum, 0.3% Triton
X-100, and 0.05% Tween 20 in PBS) for 2 h at room
temperature. An anti-Sjda monoclonal antibody (2 mg/ml)
diluted 1:500 in blocking solution was incubated with the
parasites at 4C overnight. Parasites incubated with
normal mouse IgG (Calbiochem, Darmstadt, Germany)
were included as controls. After being washed four
times (for 30 min each), the parasites were further
incubated with Alexa Flour 555 donkey anti-mouse IgG
(H + L) and DAPI (all from Invitrogen, OR, USA) at
4C overnight. Fluorescence was visualised with a TCS
SP5 confocal microscope (Leica Microsystems, Wetzlar,
Detection of anti-Sjda antibodies in host sera by Western
Mouse sera were collected on days 0, 7, 14, 28 and 42
post-infection and stored at 80C. Recombinant Sjda-His
was resolved in 12% SDS-PAGE gels and transferred to
polyvinylidenedifluoride membranes. Sera from infected
mice were used as primary antibodies (diluted 1:200), and
sera from uninfected mice were used as a control.
AntiSjda antibodies in the mouse sera were detected by
Western blotting as described above.
Additional file 1: Dynamics of Sjda-specific antibodies in host sera.
A. Sjda-specific antibodies in the sera of S. japonicum-infected mice on0,
7, 14, 28 and 42 days post-infection were detected by Western blotting.
Sera collected at each time point were pooled. Lane M, protein molecular
weight markers. B. Percentages of sera with specific anti-Sjda antibodies at
the indicated time points post-infection detected by Western blotting. Three
independent experiments were carried out, with 57 mice in each group.
Additional file 2: WB images of the recognition of the Sjda-His
protein by antibodies in each S. japonicum-infected mouse at the
indicated infection time points.
NH carried out bioinformatics analysis, recombinant protein generation, and
immunoassay and drafted the manuscript. XP carried out the detection of
DNase activity. PC and SL performed bioinformatics analysis. CW and YX
participated in the immunofluorescence assay. QC conceived and designed
the experiment and wrote the paper. All authors read and approved the final
This study was supported by the National Natural Science Foundation of
China (Grant No.81270026), the National S & T Major Program (Grant No.
2012ZX10004-220), the Program for Changjiang Scholars and Innovative
Research Team in University (Grant No. IRT13007) and an intramural grant
from the Institute of Pathogen Biology CAMS (Grant No. 2014IPB104). We
thank Zhenglin Du very much for his help with bioinformatics analysis and
1. Steinmann P , Keiser J , Bos R , Tanner M , Utzinger J. Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk . Lancet Infect Dis . 2006 ; 6 ( 7 ): 411 - 25 .
2. Ismail M , Botros S , Metwally A , William S , Farghally A , Tao LF , et al. Resistance to praziquantel: direct evidence from Schistosoma mansoni isolated from Egyptian villagers . Am J Trop Med Hyg . 1999 ; 60 ( 6 ): 932 - 5 .
3. McManus DP . Prospects for development of a transmission blocking vaccine against Schistosoma japonicum . Parasite Immunol . 2005 ; 27 ( 7-8 ): 297 - 308 .
4. Liu F , Lu J , Hu W , Wang SY , Cui SJ , Chi M , et al. New perspectives on host-parasite interplay by comparative transcriptomic and proteomic analyses of Schistosoma japonicum . PLoS Pathog . 2006 ; 2 ( 4 ): e29 .
5. Hu W , Yan Q , Shen DK , Liu F , Zhu ZD , Song HD , et al. Evolutionary and biomedical implications of a Schistosoma japonicum complementary DNA resource . Nat Genet . 2003 ; 35 ( 2 ): 139 - 47 .
6. Liu F , Chen P , Cui SJ , Wang ZQ , Han ZG . SjTPdb: integrated transcriptome and proteome database and analysis platform for Schistosoma japonicum . BMC Genomics . 2008 ; 9 : 304 .
7. Ghildiyal M , Zamore PD . Small silencing RNAs: an expanding universe . Nat Rev Genet . 2009 ; 10 ( 2 ): 94 - 108 .
8. Yang W. Nucleases : diversity of structure, function and mechanism . Q Rev Biophys . 2010 ; 44 ( 1 ): 1 - 93 .
9. Buchanan JT , Simpson AJ , Aziz RK , Liu GY , Kristian SA , Kotb M , et al. DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps . Curr Biol . 2006 ; 16 ( 4 ): 396 - 400 .
10. Midon M , Schafer P , Pingoud A , Ghosh M , Moon AF , Cuneo MJ , et al. Mutational and biochemical analysis of the DNA-entry nuclease EndA from Streptococcus pneumoniae . Nucleic Acids Res . 2010 ; 39 ( 2 ): 623 - 34 .
11. Chang A , Khemlani A , Kang H , Proft T. Functional analysis of Streptococcus pyogenes nuclease A (SpnA), a novel group A streptococcal virulence factor . Mol Microbiol . 2011 ; 79 ( 6 ): 1629 - 42 .
12. Tseng CW , Kyme PA , Arruda A , Ramanujan VK , Tawackoli W , Liu GY . Innate immune dysfunctions in aged mice facilitate the systemic dissemination of methicillin-resistant S . aureus. PLoS One . 2012 ; 7 ( 7 ): e41454 .
13. Brogden G , von Kockritz-Blickwede M , Adamek M , Reuner F , Jung-Schroers V , Naim HY , et al. Beta-Glucan protects neutrophil extracellular traps against degradation by Aeromonas hydrophila in carp (Cyprinus carpio) . Fish Shellfish Immunol . 2012 ; 33 ( 4 ): 1060 - 4 .
14. Liu MF , Wu XP , Wang XL , Yu YL , Wang WF , Chen QJ , et al. The functions of Deoxyribonuclease II in immunity and development . DNA Cell Biol . 2008 ; 27 ( 5 ): 223 - 8 .
15. Krautz-Peterson G , Skelly PJ . Schistosoma mansoni: the dicer gene and its expression . Exp Parasitol . 2008 ; 118 ( 1 ): 122 - 8 .
16. Baker KP , Baron WF , Henzel WJ , Spencer SA . Molecular cloning and characterization of human and murine DNase II . Gene. 1998 ; 215 ( 2 ): 281 - 9 .
17. Shiokawa D , Tanuma S. Cloning of cDNAs encoding porcine and human DNase II. Biochem Biophys Res Commun . 1998 ; 247 ( 3 ): 864 - 9 .
18. Krieser RJ , Eastman A. The cloning and expression of human deoxyribonuclease II: a possible role in apoptosis . J Biol Chem . 1998 ; 273 ( 47 ): 30909 - 14 .
19. Evans CJ , Aguilera RJ . DNase II: genes, enzymes and function . Gene . 2003 ; 322 : 1 - 15 .
20. MacLea KS , Krieser RJ , Eastman A. A family history of deoxyribonuclease II: surprises from Trichinella spiralis and Burkholderia pseudomallei . Gene . 2003 ; 305 ( 1 ): 1 - 12 .
21. Zhang M , Hong Y , Han Y , Han H , Peng J , Qiu C , et al. Proteomic analysis of tegument-exposed proteins of female and male Schistosoma japonicum worms . J Proteome Res . 2013 ; 12 ( 11 ): 5260 - 70 .
22. Cui J , Liu RD , Wang L , Zhang X , Jiang P , Liu MY , et al. Proteomic analysis of surface proteins of Trichinella spiralis muscle larvae by two-dimensional gel electrophoresis and mass spectrometry . Parasit Vectors . 2013 ; 6 : 355 .
23. Wang L , Cui J , Hu DD , Liu RD , Wang ZQ . Identification of early diagnostic antigens from major excretory-secretory proteins of Trichinella spiralis muscle larvae using immunoproteomics . Parasit Vectors . 2014 ; 7 : 40 .
24. Dalton JP , Day SR , Drew AC , Brindley PJ . A method for the isolation of schistosome eggs and miracidia free of contaminating host tissues . Parasitology . 1997 ; 115 (Pt 1): 29 - 32 .
25. Schistosoma japonicum Genome Sequencing and Functional Analysis Consortium. The Schistosoma japonicum genome reveals features of host-parasite interplay . Nature . 2009 ; 460 ( 7253 ): 345 - 51 .
26. Berriman M , Haas BJ , LoVerde PT , Wilson RA , Dillon GP , Cerqueira GC , et al. The genome of the blood fluke Schistosoma mansoni . Nature . 2009 ; 460 ( 7253 ): 352 - 8 .
27. Piao X , Cai P , Liu S , Hou N , Hao L , Yang F , et al. Global expression analysis revealed novel gender-specific gene expression features in the blood fluke parasite Schistosoma japonicum . PLoS One . 2011 ; 6 ( 4 ): e18267 .
28. Liu S , Cai P , Hou N , Piao X , Wang H , Hung T , et al. Genome-wide identification and characterization of a panel of house-keeping genes in Schistosoma japonicum . Mol Biochem Parasitol . 2012 ; 182 ( 1-2 ): 75 - 82 .
29. Marchler-Bauer A , Anderson JB , Cherukuri PF , DeWeese-Scott C , Geer LY , Gwadz M , et al. CDD: a conserved domain database for protein classification . Nucleic Acids Res . 2005 ; 33(Database issue):D192-6.
30. Marchler-Bauer A , Panchenko AR , Shoemaker BA , Thiessen PA , Geer LY , Bryant SH. CDD: a database of conserved domain alignments with links to domain three-dimensional structure . Nucleic Acids Res . 2002 ; 30 ( 1 ): 281 - 3 .
31. Punta M , Coggill PC , Eberhardt RY , Mistry J , Tate J , Boursnell C , et al. The Pfam protein families database . Nucleic Acids Res . 2011 ; 40 (Database issue): D290 - 301 .
32. Finn RD , Mistry J , Schuster-Bockler B , Griffiths-Jones S , Hollich V , Lassmann T , et al. Pfam: clans, web tools and services . Nucleic Acids Res . 2006 ; 34 (Database issue): D247 - 51 .
33. Letunic I , Copley RR , Pils B , Pinkert S , Schultz J , Bork P. SMART 5: domains in the context of genomes and networks . Nucleic Acids Res . 2006 ; 34 (Database issue): D257 - 60 .
34. Tatusov RL , Fedorova ND , Jackson JD , Jacobs AR , Kiryutin B , Koonin EV , et al. The COG database: an updated version includes eukaryotes . BMC Bioinformatics . 2003 ; 4 : 41 .
35. Altschul SF , Gish W , Miller W , Myers EW , Lipman DJ . Basic local alignment search tool . J Mol Biol . 1990 ; 215 ( 3 ): 403 - 10 .
36. Haft DH , Selengut JD , White O. The TIGRFAMs database of protein families . Nucleic Acids Res . 2003 ; 31 ( 1 ): 371 - 3 .
37. Guindon S , Gascuel O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood . Syst Biol . 2003 ; 52 ( 5 ): 696 - 704 .