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Laser capture microdissection in combination with mass spectrometry: Approach to characterization of tissue-specific proteomes of Eudiplozoon nipponicum (Monogenea, Polyopisthocotylea)
research-article
Author(s):
Pavel Roudnický
1
,
* ,
David Potěšil
2 ,
Zbyněk Zdráhal
2
,
3 ,
Milan Gelnar
1 ,
Martin Kašný
1
Publication date (Electronic):
17 June 2020
Journal:
PLoS ONE
Publisher:
Public Library of Science
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Abstract
Eudiplozoon nipponicum (Goto, 1891) is a hematophagous monogenean ectoparasite which inhabits the gills of the common carp ( Cyprinus carpio). Heavy infestation can lead to anemia and in conjunction with secondary bacterial infections cause poor health and eventual death of the host. This study is based on an innovative approach to protein localization which has never been used in parasitology before. Using laser capture microdissection, we dissected particular areas of the parasite body without contaminating the samples by surrounding tissue and in combination with analysis by mass spectrometry obtained tissue-specific proteomes of tegument, intestine, and parenchyma of our model organism, E. nipponicum. We successfully verified the presence of certain functional proteins (e.g. cathepsin L) in tissues where their presence was expected (intestine) and confirmed that there were no traces of these proteins in other tissues (tegument and parenchyma). Additionally, we identified a total of 2,059 proteins, including 72 peptidases and 33 peptidase inhibitors. As expected, the greatest variety was found in the intestine and the lowest variety in the parenchyma. Our results are significant on two levels. Firstly, we demonstrated that one can localize all proteins in one analysis and without using laboratory animals (antibodies for immunolocalization of single proteins). Secondly, this study offers the first complex proteomic data on not only the E. nipponicum but within the whole class of Monogenea, which was from this point of view until recently neglected.
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A Systematically Improved High Quality Genome and Transcriptome of the Human Blood Fluke Schistosoma mansoni
Anna V Protasio, Isheng J. Tsai, Anne Babbage … (2012)
Introduction Schistosoma spp. are platyhelminth (flatworm) parasites responsible for schistosomiasis, a tropical disease endemic in sub-tropical regions of Africa, Brazil, Central America, regions of China and Southeast Asia, which causes serious morbidity, mortality and economic loss. An estimated 779 million people are at risk of infection and more than 200 million are infected [1]. The paired adult males and females of S. mansoni reside in the hepatic portal vasculature, each female depositing 200–300 eggs per day near the intestinal wall. These eggs either pass into the gut lumen to be voided in the faeces and continue the life cycle or pass up the mesenteric veins and lodge in the liver, where they can cause serious pathology including granulomatous inflammation response and fibrosis. On contact with fresh water, free-living motile miracidia hatch from the eggs to infect aquatic snails (Biomphalaria spp.), where parasites undergo two rounds of asexual multiplication and are released as infective cercariae into water. Cercariae infect the human host, by penetrating unbroken skin, and transform into schistosomula. After several days the parasites exit the cutaneous tissue via blood (or lymphatic) vessels and travel first to the lungs and onward into the systemic vasculature. They may make multiple circuits before arriving in the hepatic portal system; only then do they start to feed on blood, mature and pair up, the whole process taking approximately five weeks [2]. Two Schistosoma draft genomes (S. mansoni and S. japonicum) were recently published [3], [4] and represent the only described genomes amongst parasitic flatworms to date. Their assemblies were generated by conventional capillary sequencing but are highly fragmented (S. mansoni, 19,022 scaffolds; S. japonicum, 25,048 scaffolds) and severely compromise gene prediction, as well as comparative and functional genomics analyses. The transcriptome has similarly only been partially characterised by large-scale expressed sequence tag (EST) sequencing and low-resolution cDNA-based microarrays. Second-generation sequencing technologies provide new opportunities to characterise both genomes and transcriptomes in depth. In addition to whole genome de novo sequencing [5], [6] and genome improvement [7], massively parallel cDNA sequencing (RNA-seq) can identify transcriptionally active regions at base-pair resolution [8]–[11] and accurately define the exon coordinates of genes [12]. In addition, the quantitative nature and high dynamic range of RNA-seq allows gene expression to be scrutinised [11], [13], [14] in a more sensitive and accurate way than other previous high-throughput methods [15], [16]. In this study we systematically improved the draft genome of S. mansoni, using a combination of traditional Sanger capillary sequencing, second generation DNA sequencing from clonal parasites and reanalysis of existing genetic markers [17]. This allowed us to assemble 81% of the genome sequence into chromosomes. We have also used RNA-seq data from several life-cycle stages to refine the structures of 45% of existing genes as well as to identify new genes and alternatively spliced transcripts. In addition to cis splicing, our data highlight extensive trans-splicing and provide clear evidence that the latter can be used to resolve polycistronic transcripts. With RNA-seq we profiled the parasite's transcriptome during its transformation from the free-living, human-infectious cercariae to the early stages of infection and in the mature adult. As the infective form transforms into a mammalian-adapted parasite, the relative abundance of transcripts shifts markedly during a 24-hour period, from those involved in glycolysis, translation and transcription to those required for complex developmental and signalling pathways. The improved sequence and new transcriptome data are available to the community in a user-friendly and easy to query format via both the GeneDB (www.genedb.org) and SchistoDB (www.schistodb.net) databases. These data demonstrate that revisiting a previously published draft genome, to upgrade its quality, is an option that should not just be reserved for model organisms. Materials and Methods The full description of materials and methods is presented in Supplementary Materials (Text S1). A synopsis of the methods used in this paper is presented below. Parasite material, library preparation and sequencing S. mansoni clonal DNA was obtained from single miracidium infections of Biomphalaria snails. Male and female adults (NMRI strain, Puerto Rican origin) were obtained from infected C57Bl/6 mice. DNA extraction was performed and sequencing libraries were prepared as previously described [18]. Eight and lanes were sequenced for the male samples and one lane for the female sample, both as 108-base paired reads. For RNA-seq samples, total RNA samples were obtained from cercariae, 3 hours and 24 hours post-infection schistosomula, and 7-week old mixed sex adult worms. Schistosomula samples were obtained using mechanical transformation [19]. RNA-seq libraries were prepared using a modified version of the protocol described in [8] and sequenced as 76-base paired reads. All samples were sequenced using the Illumina Genome Analyzer IIx platform. Raw sequence data were submitted to public data repositories; DNA reads were submitted to ENA http://www.ebi.ac.uk/ena/ under accession number ERP000385 and RNA-seq reads were submitted to ArrayExpress http://www.ebi.ac.uk/arrayexpress/ under accession number E-MTAB-451). Generating a new assembly and transferring previous gene annotation The Arachne assembler (version 3.2, [20]) was used to produce a new assembly using the existing capillary reads from the previously published draft assembly [3], supplemented with an additional ∼90,000 fosmid and BAC end sequences. FISH-mapped BACs [3] were also end-sequenced generating 438 reads that were incorporated into the assembly. Illumina reads were used to close gaps with the IMAGE pipeline [7]. The sequences of 243 published linkage markers [17] of S. mansoni were retrieved and used as anchors within the assembly by incorporating them as faux capillary reads. Scaffolds containing these reads were ordered, orientated and merged into chromosomes. Except where indicated, all analyses reported in the present study refer to a frozen dataset taken at this stage of the assembly process (S. mansoni genome v5.0, available at http://www.sanger.ac.uk/resources/downloads/helminths/schistosoma-mansoni.html). All comparisons were made against the previously published draft genome (v4.0). As part of the active finishing process, we randomly checked ∼20% (2,062) of the gaps automatically closed by IMAGE and found 90% of these could be verified by visual inspection. Contigs containing telomeric repeat sequences (TTAGGG) [21] were extended by oligo-walking pUC clones until a unique sequence was identified. Where the unique sequence was linked to a known marker, the telomere could be placed onto a chromosome. All manual improvement changes were included in a subsequent snapshot of the data (v6.0). To transfer the existing annotation to the latest reference we used RATT [22] (with the old assembly split into four parts and using options –q and –r) to define regions with synteny between both assemblies and transform the annotation coordinates onto the new assembly. The annotated genome sequence was submitted to EMBL http://www.ebi.ac.uk/embl/ under the accession numbers HE601624 to HE601631 (nuclear chromosomes); HE601612 (mitochondrial genome); and CABG01000001 to CABG01000876 (unassigned scaffolds). Gene finding using RNA-seq Each lane of RNA-seq reads was independently aligned to the genome using TopHat [23] and the resulting alignment files used as the input for the gene finder Cufflinks [12]. Transcript fragments with less than 10× average read depth coverage and fewer than 50 codons were excluded from subsequent analyses. JIGSAW [24] was used to combine existing models with Cufflinks' transcript fragments. The final set of gene models can be accessed through GeneDB http://www.genedb.org/Homepage/Smansoni and SchistoDB http://www.schistodb.net. Trans-splicing and polycistronic transcription RNA-seq read pairs that contained the splice leader (SL) sequence [25] were used to find trans-splicing sites; where a gene was found within 500 bases from a trans-splice site its transcript was tagged as putative trans-spliced. By looking for genes whose 3′ end was located within 2,000 bp upstream of a putative trans-spliced acceptor site, putative polycistronic units were identified. RT-qPCR was performed to validate both trans-spliced and polycistronic transcripts. Quantification of RNA-seq and differential expression RNA-seq reads were aligned to the reference genome using SSAHA2 [26]. A minimum mapping score 10 was applied to filter aligned reads. Reads per gene and RPKMs (reads per Kilobase per million mapped reads [8]) were calculated using only coding regions coordinates. We also estimated the background signal for non-coding regions (RPKM background RPKM) in pair wise comparisons (adjusted p-value 100 kb) and a further 114 unplaced scaffolds (∼1.1 Mb) that were W-specific. Repeats comprise 90% of the latter, and include previously identified female-specific repeat [32] as well as 0.1 Mb of previously uncharacterised female-specific sequences. These scaffolds usually have female reads mapped many fold higher than the average coverage of the assembly, for example scaffold 1570 has 26 times higher coverage than the average, suggesting that the heterochromatin portion of the W chromosome have been collapsed into these scaffolds. Based on the differences between the genome-wide assembly coverage and the coverage of these scaffolds, we estimate these heterochromatin portions of the W chromosome to comprise ∼3.3 Mb collapsed into the 1 Mb of consensus. Interestingly, the W-specific scaffolds appear to contain no coding genes whereas the Z-specific portion of Z/W sequence contains 782 genes, ∼95% of which exist as single-copies within the assembly. The mitochondrial genome Amongst the unassembled reads there were 5,647 that originated from mitochondrial DNA. An independent assembly of these reads using CAP3 [33] generated a single contig of 21 kb (to which 15 scaffolds from the previous genome assembly could be aligned). The first 14 kb of the contig was 99.9% identical to the published coding portion of the S. mansoni mitochondrial genome [34]. Based on restriction fragment analysis, a long non-coding region that is repetitive and highly variable between individuals has previously been partially characterised [35]. In our data, the additional 9 kb non-coding portion of the mitochondria genome is now complete and comprises known 62 bp repeats [35], plus additional 558 bp repeats and long tracts of low complexity sequence. Improvements to gene models using RNA-seq We obtained total RNA from four time points of the life cycle of S. mansoni: 1) free-living mammalian-infectious cercariae, mechanically transformed schistosomula at 2) three hours and 3) twenty hours post infection, and 4) seven-week old mixed-sex adults recovered from hamster host. The 183 million 76-base RNA-seq read pairs were mapped to the new reference genome using SSAHA2 alignment tool [26]. An average 70% of the RNA-seq reads generated in each sequenced library aligned as proper pairs to the genome (Table 2), an improvement over the previous version of the genome. Less than 6% of reads mapped to the mitochondrial genome in each sample; the lowest (0.5%) corresponding to the schistosomula stages. 10.1371/journal.pntd.0001455.t002 Table 2 Summary of RNA-seq mapping. Cerc 3 h Som 24 h Som Adult Total read pairs sequenced (out of 183,590,080) 69,498,003 53,041,873 50,528,949 10,521,255 Properly mapped read pairsa (%) 70.7 68.6 69.8 72.3 Additional properly mapped read pairs in new assemblyb (%) 2.0 0.2 0.4 2.8 Pairs mapped to repeats (%) 23.8 14.0 16.2 19.7 Pairs mapped to different scaffolds (%) 0.2 2.1 3.0 0.3 One mate mapped or mapped in wrong orientation (%) 4.3 12.2 9.7 6.1 Unmapped (%) 1.0 3.2 1.4 1.6 Proportion of reads mapped to mitochondria 5.1 0.6 0.4 3.7 Number of RNA-seq reads mapped using SSAHA2 to the genome from libraries prepared from cercariae (Cerc); 3-hour post-infection schistosomula (3 h Som); 24-hour post-infection schistosomula (24 h Som); and mixed male and female adult worms (Adult). a reads mapped within expected distance apart and in the correct orientation. b reads that were properly mapped to the new assembly but not in the previous. The majority (91%) of the 11,799 gene models from the previous version of the genome could unambiguously be transferred onto the new assembly. Splitting gene models from the previous assembly increased the gene count by 307; however, the coalescence of genes previously located on multiple different scaffolds caused some redundancy (an example is shown in Figure 2), removal of which reduced the number of transferred genes to 10,123. Of the 1,065 genes that could not be transferred to the new assembly, at least 83% were presumed to represent incorrect annotations due to a lack of sequence similarity and their short lengths, 1- or 2-exon structures (Figure S4) or a lack of start or stop codons. 10.1371/journal.pntd.0001455.g002 Figure 2 Removal of assembly redundancies produces a more reliable set of gene models. Gene models were migrated from previous version using RATT [22]. Repeats and sequencing errors in the old assembly resulted in ambiguities and sequences being represented more than once. In the new version, many scaffolds coalesced into one region and hence the gene models contained in them overlap each other. In this example, four supercontigs from the previous version collapsed on an unplaced region of Chromosome 3 in the new assembly. The smaller gene models are now obsolete as they were clearly incomplete annotations and their coding region are part of the exons of the larger gene model. RNA-seq data has been used to refine and improve gene model predictions in various organisms [10], [36], [37]. In the first draft of the S. mansoni genome, gene models were generated using a combination of ab initio gene predictions and EST evidence [38], with only a few hundred manually curated genes. To systematically upgrade the quality of annotations, we aligned pooled RNA-seq reads using TopHat [23], which allows gaps in the read-to-reference alignment at putative splice sites. Using the upgraded genome sequence 30% more RNA-seq reads with putative splice junctions aligned, highlighting putative new genes or structural refinements that could be made to existing genes. Cufflinks [12] was used to aid the refinement of gene structures by creating transcript “fragments” with sharply defined exon boundaries [23]. Using transcript fragments with at least 10 reads coverage at each base we found 78% of previous gene models had evidence of transcriptional activity within the sampled life cycle stages. Of these models, 3,604 (45%) were modified to include new exons derived from RNA-seq data, hence generating alternative gene predictions (Table 3). Using the transcript data as a guide, 236 genes were merged and 26 split into two or more gene models. 10.1371/journal.pntd.0001455.t003 Table 3 Fate of gene models. Number Total gene models in old genome version a 11,719 Not transferred 1,088 Deleted models 545 Split or merged models 731 Models with additional exons 3,438 Models that have been automatically replaced 1,116 New genes 504 Genes in new version b 10,852 The criteria for including genes into each category are described in the main text. a Version 4.0. b Version 5.0. To assess the accuracy of gene models, we calculated two metrics: the proportion of intron-exon junctions found in previous models that matched to the same intron-exon junction in a transcript fragment, and the proportion of the coding sequence in previous models that overlapped with the transcript fragments. Figure 3A is a heatmap showing these two metrics; existing models are clustered around top right of the plot, which indicates that RNA-seq evidence-based transcript fragments are similar to the existing models. Sixteen percent of gene models were perfectly reproduced by the transcript fragments (Figure 3B), while 90% of gene models with transcriptional evidence have at least 70% of the coding region covered by the transcript fragments. 10.1371/journal.pntd.0001455.g003 Figure 3 Improvement of gene annotation using RNA-seq. (A) Heatmap displaying comparisons between previous gene models and transcript fragments generated from Cufflinks. For each model, the extent of coding region that overlaps with a Cufflinks' model and the proportion of correctly predicted exon boundaries was calculated and categorised into bins of 70–100%. Models in this plot were excluded with less than 70% of their exon boundaries or coding regions predicted. (B), (C) and (D) Example scenarios of Cufflinks' models compared with previous gene models where (B) the Cufflinks prediction is identical to the 1,239 existing models; (C) Cufflinks fails to identify small introns; (D) Cufflinks removes incorrect introns present in the previous gene model, probably due to the improved assembly which, by correcting gaps, produced a longer single exon while the reading frame is preserved. In the new dataset, only 53% of gene models have at least 70% of their exon boundaries preserved. There are two main reasons for this low specificity in predicting exon boundaries. First, Cufflinks was unable to successfully predict the small introns typically observed in the 5′ end of many S. mansoni genes (Figure 3C and [3]). Consistently, when the first four exons of the old gene models were excluded, we found that transcript fragments could perfectly predict 90% of exon boundaries. Second, sequencing errors in the previous assembly resulted in introns being falsely incorporated into gene models during prediction to compensate for apparent frameshifts. These “intron” sequences are no longer necessary to preserve the reading frame and were identified as part of exons by Cufflinks in the new assembly (Figure 3D). For the two reasons above, we used JIGSAW [39] to combine existing models with those produced from RNA-seq data, resulting in 1,264 exon coordinates being changed. We identified 1,370 transcripts corresponding to putative full length coding sequences but which did not overlap with existing gene models. To check whether they indeed represented novel genes, we first screened them against known repeats and transposable elements. The 36 previously published transposable element sequences in S. mansoni matched 866 of the transcribed fragments, the longest of which (5,061 bp) was 99% identical to the coding portion of the LTR retrotransposon Saci-1 [40]. Of the remaining 504 complete transcript fragments we found sequence similarity for 231 in the NCBI nr protein database, mostly to other genes already annotated in S. mansoni (presumably representing gene duplications or members of multi-gene families) or S. japonicum. However, seven out of the remaining 273 full-length transcript fragments did show at least one conserved domain: a putative Tpx-1/SCP related allergen, a rhodopsin-like GPCR domain, a DNA-protein interaction domain, a epidermal growth factor-like (EGF-like) domain, and a polypeptide encoding a fascicline-like domain (FAS1) domain), and two transcripts with ArsR transcriptional regulator sequences. The new transcript fragments were on average shorter (261 bp) and exhibited unusual codon usage (Wilcoxon rank sum test, p<0.01, Figure S5) compared with a typical schistosome gene. Although we cannot rule out at this stage that the small set of atypical genes are non-coding RNA species, they are included in the total number of putative protein coding genes, which stands at 10,852. Trans-splicing Both cis and trans-splicing are used to produce mature transcripts in S. mansoni. By filtering for RNA-seq reads containing the spliced leader (SL) sequence [25], strongly supported trans-splicing events could be mapped on a genome-wide scale and highlighted 1,178 (∼11%) genes (an example is shown in Figure 4A), a figure in close agreement with a previous prediction [41]. For validation, we randomly chose ten putative trans-spliced gene models and could verify the existence of their trans-spliced transcripts by RT-PCR (Figure 4B, Table S1). In many cases, mapping information suggests a second trans-splicing acceptor site, usually within 20–50 bases from the primary acceptor site, indicating that alternative splicing operates at the trans as well as cis levels. Using Gene Ontology enrichment [30], we could find no particular functions or processes enriched within the trans-spliced genes, agreeing with the previous report [41]. 10.1371/journal.pntd.0001455.g004 Figure 4 RNA-seq reveals trans-spliced transcripts. (A) Schematic view of the 5′ end of trans-spliced gene Smp_176420. Shaded coverage plots represent non-normalized RNA-seq reads still containing the spliced-leader (SL) sequence (green – unclipped reads) and reads previously found to contain the SL sequence (orange - clipped). In the latter, the SL sequence was removed prior to aligning the reads to the genome; which improved the reads mapability (lower in the unclipped reads than in the orange reads). (B) RT-PCR validation of 10 putative trans-spliced genes with SL1 as forward primer and a gene-specific reverse primer. Smp_024110.1, previously described as trans-spliced [41], was included as a positive control (indicated with ‘+’) while Smp_045200.1 was included as a negative control (‘−’). All PCRs but one (Smp_176590.1) show bands corresponding to expected PCR product size. (C) Schematic view of the putative polycistron Smp_079750-Smp_079760. PCR1 represents the amplicon obtained from the unprocessed polycistronic transcript containing the intergenic region while PCR2 the trans-spliced form of Smp_079760. (D) RT-PCR validation of 5 putative polycistrons and a positive control (Smp_024110-Smp_024120; lane 9) previously reported in [45]. Each putative polycistron was subjected to two PCRs that correspond to PCR1 (e.g lane 1) and PCR2 (e.g lane 2) in panel C. Polycistronic transcripts originate from a single promoter but are later processed to generate two or more individual mRNAs. This type of transcriptional regulation is characteristic of trypanosomatids [42] and is present in C. elegans [43] and other organisms [44]. It has been suggested [45] that the S. mansoni Ubiquinol-cytochrome-c-reductase (UbCRBP) and phosphopyruvate hydratase (Smp_024120 and Smp_024110 respectively) genes might be transcribed as a polycistronic unit and that trans-splicing of the phosphopyruvate hydratase might resolve the polycistron into individual transcripts. In our study we provide strong evidence that this is indeed the case. One of the characteristics of polycistronic transcripts is a short intergenic distance (<200 bp) between individual “monocistrons”. We identified a total of 46 trans-splicing acceptor sites that fall between gene models that have a maximum intergenic distance of 200 bp, and 115 cases (Figure 4C, Table S2) where the intergenic regions expands up to 2 kb (maximum reported for C. elegans). We validated four of these polycistrons by RT-PCR (Figure 4D, Table S1) and Sanger sequencing (data not shown). Unlike C. elegans, which uses a second spliced leader (SL2) to resolve polycistrons [43], S. mansoni seems to use the same SL for both polycistronic- and non-polycistronic- trans-spliced transcripts. The role of polycistrons in schistosome gene expression remains to be determined but no pattern could be discerned between the ascribed functions of genes within each polycistron. Transcriptome analysis and differentially expressed genes In order to profile the transcriptional landscape of the parasite establishing in the mammalian host, the RNA-seq data from four key time points in the parasite's life cycle were analysed independently. Consistent with RNA-seq experiments elsewhere [16], we found good reproducibility between biological replicates, indicated by high correlation coefficients (average Pearson correlation of log RPKM values, across five pairs of biological replicates, was 0.95; Figure S6). A total of 9,535 (88%) genes were expressed (above an empirically determined background RPKM cut-off of 2 – Text S1 and Figure S7) in at least one surveyed time point and the remaining 12% were regarded as genes with expression too low to be detected or expressed during life stages not surveyed in this study (e.g. intra-molluscan stages) and therefore were excluded from further analysis. Of the excluded genes, 65% are annotated as hypothetical proteins (higher than the genome-wide figure of 44%). To gain better insight into the resolution of the RNA-seq approach in S. mansoni, we compared our results with a few example genes that have been described to undergo pronounce changes in their expression along the parasite's life cycle: an 8 kDa calcium binding protein, associated with tegument remodelling during cercariae transformation into schistosomula [46], [47]; a heat shock protein 70 (HSP70), active in schistosomula after penetration through mammalian host skin [48]–[50]; and the tegument antigen Sm22.6 [51], associated with resistance to re-infection in adult patients of endemic areas [52]. Our RNA-seq results broadly agree (Figure 5) with relative gene expression measurements obtained through other approaches. We also investigated how well the RNA-seq data correlate with previous microarray studies [53], [54]. Comparing normalised intensity values of the array features against the RNA-seq read depth for each microarray probe location in the genome (Figure S8) suggests that these data broadly correlate (Pearson's correlation of the log values 0.67). 10.1371/journal.pntd.0001455.g005 Figure 5 Comparison of expression of genes previously identified to be developmentally regulated. Barplots represent relative normalized reads (from RNA-seq data) for 3 transcripts, asterisks represent comparisons where differential expression is significant (adjusted p-value<0.01). Relative expression reported in the literature [46], [49], [51] is shown at the bottom (+++, high expression, ++ medium expression, + some expression, − not expressed, NA no information available). C = cercariae, 3S = 3-hour schistosomula, 24S = 24-hour schistosomula, A = adult. A total of 2,194 genes had detectable expression in at least one stage but not another and were therefore differentially expressed. We also used a pair-wise approach to analyse genes differentially expressed between the following life cycle stages: cercariae vs. 3-hour schistosomula, 3-hour schistosomula vs 24-hour schistosomula, and 24-hour schistosomula vs. adult. A total of 3,396 non-redundant transcripts (excluding alternative spliced forms) were differentially expressed (adjusted p-value<0.01) within the three pair wise comparisons (Table 4 and Table S3). An example showing differential expression between cercariae and 3-hour post-infection schistosomula is presented in Figure 6. To obtain a broad overview of the biological changes occurring at the gene expression level, we used Gene Ontology term enrichment to identify annotated functions and processes that were overrepresented in genes that were statistically (adjusted p-value<0.01) up- or down- regulated. Aerobic energy metabolism pathways were down regulated in schistosomules compared to cercariae and antioxidant enzymes were overrepresented in transcripts from adults. Three-hour post-infection schistosomula showed enrichment of transcripts involved in transcriptional regulation, G-protein coupled receptor (GPCR) and Wnt signalling pathways, cell adhesion and a considerable number of genes involved in potassium/sodium transport (Table S4). Most of the categories enriched at 3 hours post transformation persist through to 24 hours (e.g. GPCR signalling pathways). A total of 165 proteins are found to be associated with GPCR signalling pathways (annotated via GO). Of these, 30 and 18 were up regulated in 3 and 24 hours post-infection schistosomula, respectively, compared with cercariae. 10.1371/journal.pntd.0001455.g006 Figure 6 Detection of differentially expressed genes. The plot (left) shows the log fold change (y-axis) vs. log relative concentration (x-axis) for the cercariae – 3-hour schistosomula comparison. A total of 1,518 genes are differentially expressed between these two life cycles stages (adjusted p-value<0.01). On the right, example coverage plots for differentially and non-differentially expressed genes. Of particular interest, genes up regulated in the 3-hour schistosomula stage are enriched in G-protein coupled receptors and integrins, suggesting that signalling is a key process in this life-cycle transition. 10.1371/journal.pntd.0001455.t004 Table 4 Number of differentially expressed genes. Stage comparison Up regulated Down regulated Total Cercariae - 3 hour schistosomula 1,002 516 1,518 3 hour schistosomula - 24 hour schistosomula 433 595 1,028 24 hour schistosomula - adult 1,141 935 2,076 Figures refer to those genes with significant differential expression (adjusted p-value<0.01). NB the v5.0 assembly contains 10,852 genes. In order to investigate major processes occurring individually in each life cycle stage, we studied genes with expression above the 95 percentile in cercariae, 24-hour schistosomula and adults (Figure 7). Across the life cycle stages studied, some core cellular processes are consistently highly expressed, including glycolytic enzymes and protein translation but other broad changes are also apparent. Free-living cercariae utilise internal glycogen stores; accordingly genes involved in glycolysis and the tricarboxylic acid cycle (TCA) are highly expressed. After penetrating the skin and transforming into obligate endoparasites, the schistosomula switch to anaerobic metabolism [55], [56] before aerobic metabolism partly resumes in the adult. These events are also reflected in the transcriptome. At the schistosomulum stage there is a switch to high expression of L-lactate dehydrogenase, while TCA cycle transcription markedly decreases. As noted above, the cercariae and adult samples have relatively high contributions from the mitochondrial transcriptome (Figure S9) reflecting the high energy-demands of these two stages. 10.1371/journal.pntd.0001455.g007 Figure 7 Genes with expression above the 95 percentile different in cercariae and intra-mammalian stages. Venn diagram represents the distribution of genes above 95 percentile of expression in 3 different life cycle stages of the parasite. Examples of the genes/processes found within these groups are discussed in the main text. Other genes highly expressed in the schistosomula are involved in protein re-folding and chaperone function: 5 heat shock proteins (Smp_008545, Smp_035200, Smp_062420, Smp_072330, HSP70/Smp_106930) are among the top 50 most expressed genes at this stage and may reflect a response to the rapid temperature rise between fresh-water (∼28°C), in which the cercariae are found, and the warmer mammalian host (∼37°C). Within the host, schistosomes are exposed to potentially damaging reactive oxygen species produced during metabolism. Consistent with previous work [57] we found that antioxidant enzymes - particularly the peroxiredoxins (Prx1, Smp_059480 and Prx2, Smp_158110) - are highly expressed in adults, 24 hours after transformation and for Prx1, as early as 3 hours after transformation. Our results highlight the advantages of RNA-seq transcriptome profiling, especially its ability to dramatically improve the gene annotation alongside accurately recording changes in gene expression. Discussion In 2009 a draft genome of S. mansoni was published and provided a major resource for gene discovery and data mining. Our motivation for this study was to take S. mansoni's genome to the next level, to systematically upgrade its draft sequence so that gene structures can be more accurately predicted and the genomic context of genes can be better explored. Although systematic manual finishing has occurred for some parasite genomes, it is not an economically viable option for most non-model organisms. The genome of S. mansoni is approximately 10 times larger that the genomes of protozoan parasites and is set in the context of a field that attracts less funding. Although additional “traditional” targeted, long-range capillary sequence was introduced, more than 40,000 gaps were closed simply by re-sequencing at deep coverage, from a low-polymorphic population of adult worms. Further substantial changes were made from re-evaluating existing genetic marker information. As a result, the genome is measurably more accurate and its continuity has been transformed; 81% of the data is now assembled into chromosomes. We have also upgraded the annotation using deep coverage RNA-seq. Compared with the 2009 draft genome, the net change in the gene content is that there are now ∼900 fewer genes. However, 500 genes are new and more than 1600 low confidence or erroneous predictions have been removed. Across the genome, more than one third of genes now have new sequences. The value of the genome resource will therefore be tangibly improved: data mining approaches to identify genes will be more sensitive and trawling through kilobases of sequence for missing exons will be come less common. Our results also highlight the major benefit of using RNA-seq for transcriptome profiling - its ability to dramatically improve the gene annotation, whilst accurately recording changes in gene expression. We see major expected changes, for example, the well described metabolic switch on host penetration, plus some previously overlooked ones, such as a battery of receptors up regulated at the onset of infection in the mammalian host. Our data also define with high resolution some of the important building blocks of the schistosome transcriptome – long transcripts, cis and trans-splicing, and for the first time, clear evidence of the trans-splicing being used to resolve polycistrons. By increasing the quality of the genome, we have increased the utility of our RNA-seq data and taken it well beyond the levels attainable by previous microarray approaches. Although only a broad view of gene expression changes are presented herein, the resolution of our analyses reflects the functional annotation that has been previously ascribed. The true value of these data will arise from their use within the context of genome databases such as GeneDB and SchistoDB to query the behaviour of specific genes or groups of genes. The quality of a genome directly influences the uses to which it can be put and with many more, low-cost, draft-genome sequencing projects underway, the requirement for higher quality reference material, is increasing. Chain et al. 2009 recently defined several levels or standards for genome assemblies [58]. In the present study, we have taken an existing draft genome and demonstrated that in relatively modest period of time it can be upgraded to annotation-directed grade using second generation sequencing technology without the need for extensive manual finishing. The much improved genome assembly and gene structures, along with the expression data, are available at GeneDB and SchistoDB and will be an excellent resource not only for the helminth research community but also for in depth comparative genomics studies across metazoa. Supporting Information Figure S1 The frequency and length of newly inserted sequences at gaps. (PDF) Click here for additional data file. Figure S2 The S. mansoni v5.0 genome assembly superimposed over a genetic linkage map [17] . The numbers on the left of chromosomes are map distances in centimorgans, and the identifiers on the right of each chromosome denote contigs and scaffolds of assembly v5.0 (e.g. 6569_28 is contig 6569, which is assembled into scaffold 28). Lines connecting chromosomes indicate where an assembly scaffold contains contigs from two different chromosomes. There are multiple possible reasons for such occurrences, including repetitive sequences, assembly errors. All assembly ambiguities of this kind have been manually inspected and cannot be resolved using the current data. (PDF) Click here for additional data file. Figure S3 Analysis of male and female specific sequences. Sequence data from both Z and W chromosomes assembled together but was resolved by aligning male (blue) and female (red) genome sequence reads. The arrowheads indicate Z-specific genetic linkage markers. (PDF) Click here for additional data file. Figure S4 Plot showing (A) transcript length and (B) number of exons for the three different categories of gene models transfered using the Rapid Annnotation Transfer Tool (RATT). Outliers were not drawn in the boxplot. (PDF) Click here for additional data file. Figure S5 Codon usage of the (manually) curated genes and the 466 novel genes. (PDF) Click here for additional data file. Figure S6 Correlation between replicate experiments. Biological replicates are evaluated by calculating the Pearson's correlation for each pair of samples. (PDF) Click here for additional data file. Figure S7 Cumulative distribution of RNA-seq coverage (expressed as RPKM values, see Methods) for exons, introns, intergenic sequences and untranslated regions. (PDF) Click here for additional data file. Figure S8 Correlation of RNA-seq data and microarray data. The scatter plots show the coverage (Log2-transformed) of reads per probe location compared with normalized microarray intensities (Log2-transformed) from (A) Fitzpatrick et al. 2009 [54] and (B) Parker-Manuel et al. 2011 [53]. The graphs was generated using the smoothScatter function from the R software package [31]. (PDF) Click here for additional data file. Figure S9 Relative gene expression levels for mitochondrial genes. C = cercariae; 3S = 3 hour schistosomula; 24S = 24 hour schistosomula; A = adult. (PDF) Click here for additional data file. Table S1 Primers used for validation of trans-spliced (top) and polycistronic (bottom) transcripts. (XLS) Click here for additional data file. Table S2 Putative polycistrons with a maximum intergenic distance of 200 bp and 2000 bp. (XLS) Click here for additional data file. Table S3 Differentially expressed genes in the cercariae vs. 3 hr schistosomula comparison, 3 hr vs. 24 hr schistosomula comparison and 24 hr schistosomula vs. adult comparison. Only significantly differentially expressed transcripts (adjusted p.value<0.01 – BH correction) are listed. (XLS) Click here for additional data file. Table S4 Gene Ontology (Biological Processes) enrichment for differentially expressed genes in the cercariae vs. 3 hr schistosomula comparison, 3 hr vs. 24 hr schistosomula comparison and 24 hr schistosomula vs. adult comparison. The top 20 hits are shown. (XLS) Click here for additional data file. Text S1 Supplementary Materials and Methods. (DOC) Click here for additional data file.
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New Perspectives on Host-Parasite Interplay by Comparative Transcriptomic and Proteomic Analyses of Schistosoma japonicum
FENG LIU-SMITH, Jiong Lu, Wei-wei Hu … (2006)
Introduction Schistosomiasis remains one of the most prevalent and serious of the parasitic diseases, with an estimated 200 million people infected in 76 countries and territories, located predominantly in tropical and subtropical regions. The disease is caused by three major schistosome species, Schistosoma japonicum, Schistosoma mansoni, and Schistosoma haematobium [1]. Schistosomes have a complex life cycle with specific, differential gene expression for adaptation to their intermediate, snail, and definitive mammalian host environments. Eggs deposited by the adult female schistosomes embolize in the liver, intestines, and other sites and represent the key contributor to the pathology and morbidity associated with schistosomiasis. The highly adapted relationship between schistosomes and their mammalian hosts appears to involve parasite exploitation of host endocrine and immune signals [2–4]. Evasion strategies that underpin avoidance of the host immune system, allowing schistosomes to survive for years despite strong host immunological responses, have long confounded and intrigued investigators intent on controlling these parasites through development of an effective vaccine. A comprehensive deciphering of the schistosome genome, transcriptome, and proteome has become increasingly central for understanding the complex parasite-host interplay and for delivering candidate drug and vaccine targets [5,6]. Although many tens of thousands of expressed sequence tags (ESTs) derived from S. japonicum and S. mansoni were recently released to GenBank by us and others [7,8], and whereas ESTs are useful for cataloging expressed genes, these ESTs are not the ideal format of genetic information to study gene function because many provide only partial sequence coverage of the matching gene. Accordingly, genome-scale collections of the full-length cDNAs with potential coding sequences (CDSs) of expressed genes have become important for the analysis of the structure and function of the estimated 14,000–20,000 genes of the schistosome parasite [8,9]. Furthermore, global proteomics analyses offer a unique means for determining not only protein identification for genome annotation but also for subcellular localization of these proteins. In this present report, we describe the isolation of ~ 8,420 potential protein-coding RNAs from S. japonicum. Moreover, in tandem with nucleotide sequence analysis of the CDSs, we undertook high-throughput proteomics analyses, involving high resolution in-line two-dimensional-nano-liquid chromatography (2D-nano-LC) and tandem mass spectrometry (MS/MS) analysis to characterize gene and protein expression within different developmental stages of the schistosome (cercariae, hepatic schistosomula, adults, eggs, and miracidia), as well as in tegumental preparations and the eggshell. We interrogated the protein and peptide datasets deduced from these CDSs (Figure S1). We anticipate that the findings from these comparative genomic, transcriptomic, and proteomic analyses should lead to a more profound understanding of schistosome biology, the host-parasite relationship, molecular mechanisms of immunopathology of schistosomiasis, and the development of innovative intervention strategies. Results The Transcriptome of S. japonicum Previously, we reported an initial analysis of the transcriptomes of adult and egg stages of S. japonicum from 43,707 5′ ESTs that we assigned to 13,131 gene clusters, of which 2,706 clusters were similar to known proteins deposited in GenBank (BLASTP cutoff of E value 10−20), and of which 611 genes containing complete CDSs were isolated [7]. To isolate more full-length cDNAs with complete or partial CDSs of S. japonicum genes, we constructed new cDNA libraries with longer inserts (mean size ~ 2 kb) from mixed-sex adult worms and hepatic schistosomula. Long-read sequencing of both 5′ and 3′ ends was subsequently performed on clones randomly selected from the new cDNA libraries. Representative clones of assembled sequences with potential CDSs were also sequenced. A total of 98,770 raw ESTs derived from 79,639 clones, which were composed of 55,063 new ESTs and the 43,707 ESTs reported previously [7], were quality-trimmed using Phred 20 after removing repetitive, mitochondrial, ambiguous, and vector sequences (Table S1). The available 84,449 clean ESTs, including 67,383 (79.9%) 5′ ESTs and 17,026 (20.1%) new 3′ ESTs, were assembled into 14,962 clusters with an average length of 783 base pairs (bp) (Table S1) (10,518 contigs and 4,444 singletons), where the length of gene clusters was extended and the quality was significantly improved as compared to our earlier report [7]. To independently evaluate the cluster redundancy and gene coverage of this expanded transcriptomic dataset, we collected 213 S. japonicum mRNAs deposited in GenBank prior to the large-scale EST sequencing project (before December, 2002). After removing retrotransposon and ribosomal sequences, the remaining 172 CDSs, of which 105 encode complete CDSs and 67 have partial coding sequences, may represent 126 distinct genes. Of these 126 genes, 110 (87.3%) could be found in the new expanded S. japonicum EST dataset, whereas 92 (73%) were represented by 159 of the new 8,420 clusters with potential CDSs, where an average of 1.73 clusters was assigned into a gene. This analysis indicated that the majority of S. japonicum genes may be present in the expanded transcriptome dataset. Of these 14,962 clusters, 8,420 (56.3%) appeared to have potential CDSs according to similarity, protein identification, or length (hypothetical genes encoding at least 100 amino acids) (Figure S1). Specifically, of these 8,420 clusters with CDSs, 3,077 (36.5%) were included with apparently complete CDSs, whereas 3,695 (43.9%) were considered to have partial CDSs lacking the start or stop codons, although these CDSs exhibited identity to known proteins from other organisms (BLASTP cutoff of E value 10−20). The remaining 1,648 (19.6%) clusters were considered to be genes encoding hypothetical proteins of unknown function. The average length of the 3,077 genes with entire CDSs, including the 611 genes reported previously [7], was 1,024 bp; within these genes, the average length of the CDS was 606 bp, encoding 202 deduced amino acid residues (Figure S2). The majority (2,112; 68.6%) of these entire CDSs could encode proteins with lengths ranging from 100 to 300 amino acid residues. To characterize the transcriptomic information, we further analyzed the guanine-cytosine (GC) content of those clusters with or without CDSs (Figure 1A). The GC content of all 14,962 clusters ranged broadly between 10% and 55% with a significant peak at ~ 32%, whereas the prominent peak of GC content of the clusters with CDSs, in particular protein-coding regions, was apparent at ~ 37% GC. By contrast, the GC content of the 3′ UTRs was obviously reduced, with a peak at only ~ 27%. Furthermore, 419 (8.9%) and 583 (13.6%) of 4,725 complete and hypothetical CDSs were predicted as secretory and membrane proteins, respectively (Figure 1B and Table S2). To explore the biological characteristics of S. japonicum, the 5,077 CDSs with identity to known proteins (BLASTP E value of 10−10), were further categorized based on Gene Ontology (GO) (see Materials and Methods for details and http://function.chgc.sh.cn/sj-proteome/index.htm). We assigned 873 (17.2%) to 12 main molecular functional categories and 829 (16.3%) to 15 main biological process categories. Similarly, among 2,984 CDSs encoding InterPro protein domains (Tables S2 and S3), 842 (28.2%) and 894 (30%) were assigned into molecular functional and biological process categories, respectively (Figure S3 and Table S4). A Proteomic View of the S. japonicum Life Cycle We employed 2D-nano-LC, one-dimensional PAGE, and MS/MS in a shotgun proteomics approach to further profile the protein expression of various developmental cycle stages of S. japonicum (including cercariae, hepatic schistosomula [whole worms and their tegument], adults [mixed-sex, females, males, and their teguments], eggs [intact eggs and empty eggshells], and miracidia). This was accomplished by interrogation of the combined human, mouse, and rabbit mammalian host protein and peptide databases and comparison with S. japonicum, which we had assembled from the newly obtained transcriptomic data (Figure S1 and Protocol S1), as previously described [10]. More than 420,000 highly accurate MS/MS spectra, generated from more than 400 protein sub-fractions from all of the discrete samples, were searched against the combined host-parasite protein database using probability-based scoring. The resulting findings indicated that a total of 26,484 distinct peptides with significant probability scores could be confidently assigned into 3,260 unique proteins that accounted for 38.7% of the 8,420 CDSs. Of these unique proteins, 1,181, 1,154, 1,375, 1,441, and 918 were identified from cercariae, hepatic schistosomula, adults, eggs, and miracidia, respectively (Figure 2A and Table S2). There were 337, 258, 444, 473, and 237 proteins that were identified in one or another of these developmental stages only; suggesting that, despite the fact that the GO classification displays little discrimination among the different developmental stages (Table S2), certain proteins are stage-enriched or are expressed in response to different environmental stimuli. However, some proteins identified in only one stage by the proteomics analysis had ESTs in more than one stage, as did some gender-enriched proteins. This apparent inconsistency may reflect the incompleteness of the proteomic dataset due to limitations in sensitivity of the proteomic technologies that we employed. Also, some transcripts may be relatively stable and might persist through several stages but be translated in a much shorter window, contributing to the discrepancy between the proteomic and transcriptomic data. Only those proteins with more than 3-fold differences throughout the life cycle or between the female and male worms based on quantitative proteomics [11], and which were consistent with the transcriptome data, are highlighted in Figures 3 and S4. Interestingly, sex-enriched expression was far more dramatic than stage-enriched expression. Herein, the correlation between transcriptomic and proteomic data (p < 0.05) (based on EST copy numbers using tools available at http://www.igs.cnrs-mrs.fr/~audic/significance.html) [12] among the three developmental stages, including hepatic schistosomula, adults, and eggs, or between female and male worms, was considered to be significant. For cercariae and miracidia, we only show some proteins with more than 3-fold differences throughout the life cycle based on quantitative proteomics, since only a small number of ESTs were available for these two larval stages that were insufficient to statistic analysis. Putative deoxyribodipyrimidine photo-lyase (DNA photolyase) was found only in cercariae (the mammalian-infective, larval form), implying that the free-living cercariae might catalyze the light-dependent monomerization of cyclobutyl pyrimidine dimers as a photo-reactivating enzyme, upon exposure to ultraviolet radiation [13]. The stage-specific protein SPO-1, which is preferentially expressed in schistosome sporocysts within their snail hosts [14], and putative sex-determining region Y protein (SRY), which is a testis-determining factor [15], were all expressed in cercariae, indicating that this stage undergoes significant development associated with male sex determination (Table S2). In addition, proteins similar to a number of well-known receptors, including vasopressin-activated calcium-mobilizing receptor (cullin 5), dioxin receptor, and tri-spanning orphan receptor were expressed in cercariae (although not only in this stage), suggesting that these larvae may have evolved specific molecular processes for detecting their mammalian hosts. However, an ortholog of S. mansoni cercarial elastase, a major enzyme involved in skin penetration, has not been identified to date from S. japonicum [16]. A number of proteins with identity to homologs, known in other species to be associated with neural development, including ubiquitin-protein ligase NEDD4-like, prion protein interacting protein, karyopherin (which facilitates nuclear import), budding uninhibited by benzimidazoles1 (a kinase involved in spindle checkpoint function), and Su (var) 3–9 (the major heterochromatin-specific HMTase) were highly expressed in hepatic schistosomula. Their presence may reflect the complex physiology of these immature adult worms, which are adapting to dramatic environmental changes as they migrate from the liver to the mesenteric veins of the intestines, which is the preferred site of the adult stage. The hepatic schistosomula also expressed numerous enzymes associated with digestion of hemoglobin [17,18], which reflects the nutritional dependence of this stage (and mature adult worms) for ingested host red blood cells. Of the 1,375 proteins identified from adult worms, 491, 574, and 723 were identified from females, males, and mixed adults, respectively; of these, 444 proteins were identified only in adults (Figure 2A and Table S2). In addition to cytoskeleton and motor proteins, chaperones, extracellular matrix molecules, and enzymes associated with digestion of haemoglobin, a number of proteins similar to known proteins associated with developmental and sexual maturation, including forebrain embryonic zinc-finger like (Fezl), histone H2A (gonadal), and eggshell protein were identified, and they may play key roles in schistosome growth and sexual maturation (Figures 3A and S4, Table S2). Putative ribophorin II, extracellular superoxide dismutase, and female-specific 800 protein appeared to be preferentially expressed in adult females; whereas gynecophoral canal protein, F-box only protein 9, and amidase were preferentially expressed in males (Figures 3B and S4, Table S2). The gynecophoral canal protein has been shown previously to be localized to the gynecophoral canal of S. mansoni male worms [19]. It is noteworthy that putative C1-tetrahydrofolate synthase was also identified in adults and hepatic schistosomula, suggesting that folic acid and its derivatives could be critical for growth, development, and differentiation, as well as for normal cellular function. However, other folic acid pathway elements, e.g., dihydrofolate reductase, which has been characterized in flatworms [20,21], were not found in this study. Schistosome eggs are directly responsible for granuloma formation in the liver and are the major cause of pathology in schistosomiasis. It is noteworthy that calcium influx could be important for eggs because several Ca2+-associated polypeptides, including high voltage-activated calcium channel (beta subunit 2) and calcium/calmodulin-dependent protein kinase II (delta isoform 3) were found only in eggs [22]. Additional proteins that could be involved in development, including twister, nocturnin (a circadian clock-regulated gene), craniofacial development protein 1, and transducin-like enhancer of split 3 (TLE3) were highly expressed in eggs. Furthermore, many molecules involved in mitosis, including microtubule-associated protein and regulator of G-protein signaling 2, were also expressed in eggs; this suggests that a small proportion of miracidia would still be maturing within eggs newly laid by female adults, although the majority of eggs deposited in the liver would already be fully developed and quiescent. Of 918 proteins identified from newly hatched miracidia, 412 (44.7%) were also located in eggs, while 237 were found only in miracidia (Figure 2A and 2B). Along with several motor proteins, some receptor-like proteins, and related proteins involved in neural development, including Notch receptor, GABA receptor, dioxin receptor, and acetylcholine receptor (alpha-3 chain) were expressed in this developmental stage (Table S2). This implies that the free-living, motile miracidium can accept external signaling molecules from the snail intermediate host through receptors linked to the miracidial nervous system, in addition to being able to respond to internally produced (self) signals; although supporting functional evidence is currently not yet available. Comparison of the S. japonicum Transcriptome and Proteome To further investigate the relationship between the transcriptome and proteome of S. japonicum, we compared the available ESTs and proteins identified in the various developmental stages examined in this study. Of 18,579 ESTs representing 3,540 potential CDSs derived from hepatic schistosomula, more than half, 679 (58.8%) of 1,154 proteins identified, were consistent with the transcriptomic data for this developmental stage (Figure 2C). Similarly, 41.7%, 54.9%, and 61.5% of the proteins identified in egg and male and female worms were consistent with the transcripts, respectively. In the adult stage samples, 1,193 (86.8%) of 1,375 appeared to have transcripts in 6,699 CDSs assigned from 52,742 ESTs from adult stages, exhibiting the highest overlap between the proteomic and transcriptomic data for the developmental stages investigated here, suggesting that a large amount of transcriptomic data could be helpful to the annotation of proteomic resource in addition to the protein identification. The lowest level (3.5%) of concordance between the transcriptomic and proteomic datasets was obtained with the miracidium, and this finding probably reflects the small number of ESTs available for this stage. Nonetheless, the overall findings indicated that tandem proteomic and transcriptomic approaches will provide distinct, yet complementary, views in profiling gene expression in discrete schistosome developmental stages, in like fashion to the situation reported for Plasmodium [23]. Tegument and Eggshell Proteins The surface tegument that covers the schistosomulum and adult stages of schistosomes contributes centrally to host-parasite interactions, being critical for nutrient uptake, parasite growth, and development and as a protective barrier against host immune responses. In light of their importance for parasite survival, tegumental proteins are recognized as prime candidate targets for chemotherapy and immunotherapy of schistosomiasis [24]. We prepared the tegumental samples according to an established method [25] and identified 373 tegumental proteins—134 from adult females, 58 from adult males, 156 from mixed-sex adults, and 159 from hepatic schistosomula (Figure 2D and Table S2). It is noteworthy that 85 tegument proteins were only found in mixed-sex adults, which might reflect heterogeneity in protein extraction and peptide detection with the mass spectrometer for different tegumental batch samples. Among the tegument proteins in our preparations, several cytoskeleton and motor proteins (actins, tubulins, paramyosin, tropomyosin, myosin, and dynein light chain 1) [23], 22.6 kDa tegument membrane-associated antigen [26], tegumental antigen Sm20 [27], glutathione-S-transferase [28], nitric oxide synthase 1 (NOS1) [29], leucine aminopeptidase [30], 14–3–3 proteins [31], and SnaK [32], as well as a cathepsin B-like cysteine protease precursor [33] and 21.7 kDa antigen [34] have been characterized previously as tegumental proteins. Many chaperones, including heat-shock proteins (60, 70, 86, and 90 kDa) and chaperonins, were also identified in the tegument samples. Further, it is possible other extracellular matrix proteins, transporters, and membrane proteins that were located in the tegumental protein assemblage, including collagen (type I, alpha 3), annexins, osteonectin (SPARC), and presenilin may have roles in the host-parasite interplay at the schistosome surface. Several enzymes involved in redox homeostasis, such as antioxidative thioredoxin peroxidases and manganese superoxide dismutase were situated in the tegument, implying that this layer could provide protection against therapeutic drugs, environmental toxins, and products of oxidative stress through detoxification pathways. Interestingly, we did not detect glutathione-S-transferase among the tegumental proteins in our samples. Components of Ca2+ ion signaling pathways, including calpain, calreticulin, and calcineurin A were expressed in the tegument, suggesting that these pathways could play an important role in the maintenance of tegumental functions, and thus the key components in the pathways might be considered as putative drug targets [35,36]. Furthermore, to evaluate the proteomic data, an immunofluorescence assay was employed to confirm the localization of immunophilin (FK506-binding protein 50) identified as a tegumental protein, using a monoclonal antibody prepared against recombinant S. japonicum immunophilin. The assay indicated that immunophilin was limitedly localized in the subtegumental region (Figure 2E), directly supportive of the proteomics findings. The schistosome eggshell is sclero-proteinaceous in nature and is lined internally by a vitelline membrane within which the miracidium develops [37]. A mechanism for eggshell production in S. mansoni has been proposed [22]. In the present study, the eggshell-containing samples were collected for proteomic identification soon after the miracidia had hatched from the eggs. Of 520 proteins found in the eggshell preparations, 258 and 218 were also located in samples from intact eggs and from miracidia, respectively (Figure 2B). Several proteins with similarity to known eggshell proteins, including p48 eggshell protein [38] and thioredoxin peroxidase [39], as well as previously characterized egg proteins, such as p40 major egg antigen, 21.7 kDa, and SM22.6 antigens (A12) were identified in the eggshell sample. Additionally, many motor proteins and chaperones were found in the eggshell. It is noteworthy that some enzymes or proteins involved in redox homeostasis were also found in the eggshell-containing sample, and this suggests that like the tegument, the eggshell could provide a protective biochemical barrier to oxidative stress. It is generally accepted that antigens released by the miracidium within the egg are responsible for the onset of granuloma formation around the egg, leading to disease [40]. Interestingly, immunity-associated and cell adhesion-related proteins, such as endoplasmin (gp96), immunophilin (FK506-binding protein 50), HLA-B associated transcript 1, and platelet glycoprotein IIIa (GPIIIa) were identified in the eggshell sample. These antigens may be contributing to the molecular mechanisms associated with granuloma formation. For example, endoplasmin (gp96), a known inflammatory mediator, not only promotes CD8+ and CD4+ T cell effector functions, as a specific co-stimulatory molecule [41,42], but also activates dendritic cells, neutrophils, or monocytes and promotes phagocytosis [43]. Like the tegument, the eggshell also contains proteins involved in calcium flux pathways, as well as signaling molecules such as 14–3–3 proteins. Genetic Polymorphisms Genetic variation in schistosome populations can be expected to contribute to differences in infectivity, development in intermediate and definitive hosts, drug sensitivity, pathogenicity, and immunogenicity. In the present study, about 13,000 cercariae from naturally infected snail populations from Anhui Province, China were employed to experimentally infect laboratory mice in order to obtain the schistosome samples that we investigated in the transcriptomic analyses. Within 5,267 contigs with at least four ESTs (the minimum required for redundancy-based single nucleotide polymorphism [SNP] detection) [44], we could identify 7,286 SNP sites, including 6,038 in the cluster with CDS and the reminding 1,248 in non-protein coding clusters, with a redundancy of two or more ESTs in 1,812 (21.5%) contigs with an average SNP density of 1/288 bp, according to stringent criteria [44,45] (Tables S5 and S6). Of these 1,812 contigs, 1,496 contained potential CDSs, whereas 316 did not. Interestingly, of 6,038 SNP sites occurring in the 1,496 genes with CDSs, 3,673 were localized in protein-encoding regions of 1,121 genes with a SNP density of 1/244 bp; 521 were found in the 5′ UTRs of 270 contigs with a density of 1/133 bp and 1,844 in the 3′ UTRs of 625 contigs with a density of 1/158 bp, indicating that the protein-encoding regions of the S. japonicum genome display lower SNP density compared to those of UTRs. In these SNPs, the transition of C-T/T-C and A-G/G-A was found in 33% and 37% of SNPs, respectively, whereas transversions led by SNPs accounted for 30% (Figure 4A). Moreover, 2,272 (61.8%) of 3,824 SNPs in 3,673 sites could only induce synonymous substitutions in coding regions, whereas 1,552 (40.6%) nonsynonymous SNPs may lead to protein variations in 601 CDSs. Furthermore, a small number of the SNPs may abrogate or introduce stop codons to cause an extension or truncation. To evaluate the potential significance of the genes with potential nonsynonymous SNPs within S. japonicum, and to begin to investigate evolutionary pressures acting on schistosome genes, we first calculated the ratio of nonsynonymous to synonymous substitutions (dN/dS) between 1,514 orthologs found in S. japonicum and S. mansoni. In general, it appeared that the orthologous gene pairs were under purifying selection pressure due to low average dN/dS ratios (0.149) (Figure 4B). Interestingly, of the 601 CDSs with nonsynonymous SNPs, 185 (93.0%) of 199 with detectable orthologs in S. mansoni had a higher than average dN/dS ratio value (Table S6), including putative ribosome-associated protein P40 (3.125), protein disulfide isomerase (0.694), 21.7 kDa antigen (0.524), and immunophilin (0.457). This may endow the schistosome population with the potential for adaptation to environmental niches under diverse selection pressures, including host immune responses. Moreover, 335 of the 601 CDSs detected by the proteomics analysis, including 72 tegument-localized proteins such as paramyosin, fimbrin, and prosaposin, as well as 89 eggshell proteins such as antigen SM22.6 (A12), calcium-binding protein Sj66, G protein alpha subunit, and flavoprotein (Fp) may display antigenic polymorphisms due to the candidate SNPs. The SNPs appear to represent a capacity of the schistosome population to parry, modify, or attenuate host immunological responses during infection of the mammalian host. To estimate the potential SNPs representing either polymorphisms between chromosomal homologs or polymorphisms between individuals, we performed genomic DNA sequencing on PCR-amplified products from 30 individual worms for three genes with one or two potential SNP sites each, a total of four SNP sites. This revealed that the homogeneous 62 (83.8%) of all available 74 sequences exhibited differences at all four sites between individuals, with the remaining 12 (16.2%) sequences showing heterogeneous peaks at three sites within single individuals, suggesting that a small proportion of this polymorphism data as “background” variation could be due to differences between both alleles of the same gene. Some genes exhibited complex genetic variation (Figures 5A and S5). For example, putative SM22.6 antigen (A12) was selected for PCR-sequencing verification using genomic DNAs isolated from field samples of S. japonicum from five provinces of southern China: Jiangxi, Hunan, Hubei, Sichuan, and Anhui. The resulting sequences revealed that in addition to those found by the EST strategy, multiple SNPs in putative SM22.6 antigen (A12) were identified by the DNA sequencing (Figure 5A). This suggests that certain genes might include complex genetic variants involved in immune evasion and natural selection. Indels (insertion/deletion length variations) represent another source of genetic polymorphism distinct from SNPs. There were 2,806 indel sites, including 2,225 in 948 clusters with CDSs, and 581 in 263 clusters without CDSs, apparent in 1,211 clusters with at least four ESTs. There were 1,078 (38.4%) indel sites in the coding regions of 560 genes with CDSs (Tables S5 and S6). The length of indels was primarily in the range of one to three nucleotides, with the majority, 2,459 (78.1%) of all 3,147 indels, exhibiting an insertion/deletion of just a single nucleotide. Significantly, some proteins deduced from the known genes with indels were localized on tegument or eggshell-containing samples. For example, the motor proteins (e.g., paramyosin), enzymes (e.g., peptidylprolyl isomerase), membrane proteins (e.g., 21.7 kDa antigen), and others (e.g., 14–3–3 epsilon and Hsp60) showed significantly higher indel frequencies than other genes, again suggesting that polymorphisms could confound effective host responses targeting these antigens. In addition, legumain (antigen Sj32), phosphomannose isomerase (type I), and cytochrome c oxidase (subunit 1) exhibited indel genetic polymorphisms. Previous reports have demonstrated microsatellite polymorphisms in field and laboratory populations of S. mansoni [46] and S. japonicum [47]. In the present report, among a total of 14,962 consensus sequences, we identified 1,026 repeat motifs, in which there were 345 (33.1%) with at least ten di-nucleotide repeats, and 625 (60.1%), 70 (6.7%), and two (0.2%) with at least five repeats for tri-, tetra-, and penta-nucleotide repeats, respectively. The 444 microsatellite repeats were found in 411 clusters with CDSs of which 174 (39.1%) were localized in the protein-encoding regions, while 50 (11.2%) and 220 (49.5%) were found in 5′ and 3′ UTRs, respectively (Tables S5 and S6). This higher frequency of microsatellites localized within UTRs could contribute to the pronounced regulatory effects on protein translation and stabilization of mRNAs. The dinucleotide motif (TA)n was found commonly in 313 (91.5%) of 342 clusters with di-nucleotide repeats, whereas (CA)n and (GA)n repeats were found in 23 and nine clusters, respectively. Most of the microsatellite repeats (159 of 174, 91.4%) localized in protein-encoding regions were tri-nucleotide repeats, whereas these accounted for 146 (54.1%) of 270 microsatellites in UTRs, implying that some proteins might be prone to accumulate polymorphisms involving tri-nucleotide repeat microsatellites. Furthermore, some microsatellite repeats, including (TAA)n, (CAT)n, (CAT)n, (CAA)n, (TAG)n, (CAA)n, (TAA)n, (TGG)n, and (GAA)n were common within the protein-encoding regions (Figure 4C). The stretches of polymers of Asn (encoded by AAT), Asp (GAT), Ser (TCA), Gln (CAA), Thr (ACT, ACA), Ile (ATA), Pro (CCA), and Glu (GAA, encoded by specific microsatellite repeats) could have important molecular functions (Table S6). Moreover, 59 homopolymers with more than ten tandem amino acids were here found in 54 proteins, a low frequency as compared to that of Dictyostelium discoideum [48] and Plasmodium falciparum [49]. The stretches of polymers of Asn and Ser, as the most common homopolymers, occurred in 19 and 15 S. japonicum proteins, respectively, somewhat different from the situation in the genome of D. discoideum and P. falciparum, where poly N and Q or poly N and K are the predominant motifs, respectively [48,49]. Furthermore, I33 were the longest homopolymers encoded by microsatellite DNA repeats in the 8,420 S. japonicum CDSs (Table S6), where the DNA sequence composed of (TAA)33 tri-nucleotide repeats can encode the ATA/(I)33 homopolymer. Of 174 CDSs with microsatellite repeats, 52 gene products were identified by our proteomics analyses, including receptor kinase I-interacting protein (SIP), which appears to be localized on the tegument or eggshell at the host-parasite interface. The proteomic data from the MS/MS spectra were further employed to identify the translated variants due to the nonsynonymous SNPs, indels, and microsatellites. Five peptide variants due to the nonsynonymous SNPs were identified by the MS/MS spectra, where both wild-type and a peptide variant of SJCHGC01743 protein were found to match perfectly with the proteomics data (Figure 5B and Table S6). This strongly suggests that the genetic polymorphisms by SNPs indeed may result in variant translated products. Conserved Proteins The 8,420 CDSs were further compared with the protein datasets derived from model organisms with sequenced genomes. Of translated CDSs, 38% or 62% were similar to mammalian proteins at BLASTP Expectation (E) values with less than 10−20 or 10−5, respectively (Figure 6A). Of these CDSs, 30%–37% showed significant similarity with proteins from fishes (Tetraodon nigroviridis and Takifugu rubripes), insects (Drosophila melanogaster and Anopheles gambiae), and nematodes (Caenorhabditis elegans and Caenorhabditis briggsae), at BLASTP E values of 10−20. Furthermore, only about 10% of the S. japonicum CDSs shared significant sequence similarity with proteins examined for the four Apicomplexan parasitic protozoa, P. falciparum (strain 3D7), Plasmodium yoelii nigeriensis (17XNL), Cryptosporidium parvum, and Cryptosporidium hominis, respectively; overall, a total of 1,092 (13%) S. japonicum CDSs were similar to those of these protozoan parasites. Less than 1,213 (14.4%) S. japonicum CDSs shared sequence similarity with yeast (Saccharomyces cerevisiae) proteins at an E cutoff value of 10−20 (Figure 6A). To explore potential molecular mechanisms of the schistosome-mammalian host interplay, 1,336 CDSs with high similarity (E < 10−50) with mammalian genes were analyzed more extensively. Significantly, mammalian-like receptor and related proteins, including insulin receptor protein kinase RTK-2, receptor tyrosine phosphatase (gamma and delta), purinergic receptor P2X (ligand-gated ion channel, 4), dioxin receptor, vasopressin-activated calcium-mobilizing receptor, and feline leukemia virus (subtype-B) receptor were identified by the transcriptomic and proteomic approaches, implying that the parasite can accept certain hormone and cytokine signals from the mammalian host in addition to endogenous schistosome signals. However, functional evidence is needed to further support this hypothesis. Interestingly, among 820 (61.4%) of 1,336 highly conserved proteins identified with confidence by our proteomics approaches, 174 and 217 were potentially localized to the tegument and eggshell, respectively, i.e., at the host-parasite interface (Table S2). These included numerous cytoskeleton and motor-associated proteins, chaperones, extra cellular matrix molecules, as well as enzymes involved in redox homeostasis, which could be involved with evasion of immune responses by antigenic mimicry, a strategy that has long been predicted (along with others including host antigen masking) to account for the chronic nature of schistosome infection [50]. Additionally, other immune-associated molecules including immunophilin, cyclophilin B, endoplasmin (gp96), and HLA-B associated transcript 1 may be contributing to the immune evasion and immune-dependent growth of the parasite by modulating the innate and adaptive immune systems of the mammalian host. Schistosome-Specific Proteins Phylum Platyhelminthes- and genus Schistosoma-specific genes are potential targets for vaccines, drugs, and diagnostic reagents for schistosomiasis. We first analyzed all 8,420 potential CDSs by comparing them with the known genes of all other organisms except flatworms. This analysis revealed that 40%–68% of the S. japonicum CDSs had similarity with known genes at cutoff E values of 10−20 to 10−5, respectively, and indicated that the remaining clusters represented a resource for identifying potential candidate flatworm-specific and schistosome-specific genes. The remaining 32% −60% CDSs (at cutoff E values of 10−20 to 10−5) were then compared with the 223,321 public nucleotide entries deposited in GenBank for species belonging to the Phylum Platyhelminthes (except for S. japonicum), which included 12,621 entries for cestodes, 747 for monogeneans, 198,809 for digeneans, and 11,144 for turbellarians. The comparisons revealed that 50%–59% CDSs had significant similarity with the nucleotide entries at a cutoff of tBLASTN E value of 10−20, implying that these genes may be Phylum Platyhelminthes-specific (Figure 6B). Furthermore, to identify schistosome-specific genes, the candidate Platyhelminthes-specific CDSs with high similarity with nucleotide entries for the Phylum Platyhelminthes (tBLASTN < 10−20), were compared with 195,620 nucleotide entries only for the genus Schistosoma (except S. japonicum), including 195,414 from S. mansoni. These comparisons revealed that 50%–58% were similar to the available Schistosoma transcriptomic data at tBLASTN E value of 10−20 (Figure 6B). However, it should be pointed out that 61%–75% of all 8,420 S. japonicum CDSs were similar to the available S. mansoni transcriptomic data at tBLASTN, with expectation values of 10−20–10−5, respectively (Figure 6C). The S. japonicum genes share significantly closer identity with the genes of S. mansoni than with other organisms, which suggests that most schistosome genes share pair-wise orthologs between S. japonicum and S. mansoni. Therefore, 1,323 CDSs representing stringent genus Schistosoma-specific genes warrant consideration as candidate targets for new interventions. Moreover, 402 (30.1%) of 1,323 gene products were confidently identified by our proteomics approaches, where 111 and 102 were found in cercariae and hepatic schistosomula, respectively; while 26 and 50 proteins, including Sj-Ts4 and MF3 appeared to be tegument and eggshell proteins, respectively (Table S2). Discussion Using a transcriptomics approach, it has been estimated that S. mansoni has a complement of ~ 14,000 genes [8]. The 14,962 gene clusters, generated from a new suite of 84,449 high-quality ESTs from egg, larval, and adult developmental stages of S. japonicum appeared to represent 8,420 potential CDS-encoding proteins, which accordingly probably represent 60%–70% of all S. japonicum proteins (if we assume that the gene number of S. japonicum and S. mansoni is about the same). Moreover, 3,260 proteins, accounting for 38.7% of 8,420 potential CDSs, were confidently identified throughout the parasite life cycle using high-throughput proteomics approaches, implying that the S. japonicum transcriptomic dataset is a relatively reliable resource of genetic information. Notably, these 3,260 proteins identified by our proteomics approaches represent a more than 100-fold increase in the number of schistosome proteins so far reported using similar approaches [51,52]. Furthermore, by describing the presence of numerous SNPs and indels in many S. japonicum genes, we have revealed extensive genetic polymorphism in this parasite, which should resolve the long-standing debate over the extent of genetic heterogeneity in populations of the Oriental schistosome [53,54]. Most genes of S. japonicum and S. mansoni appear to share pair-wise orthologs because 5,161 (61.3%) of the new 8,420 S. japonicum gene sets were similar to the S. mansoni EST data with limited transcriptomic information at a tBLASTN E value of 10−20. It can be expected, as more sequence data become available, that additional comparative genomic analysis between both these two species will provide more pair-wise orthologs, including single-copy genes and gene families with many paralogs. Phylum Platyhelminthes- and Schistosoma-specific genes can be considered to be potential candidates for new drugs and vaccines, in like fashion to the situation with parasites from the phylum Nematoda [55]. Our comparative genomic analysis revealed that at least half of the CDSs, without similarity to known genes from all organisms other than platyhelminths, could be considered as Schistosoma genus-conserved genes across the genus due to orthologs known from the S. mansoni transcriptome. The highly co-evolved relationship of schistosomes and their hosts appears to include exploitation of host endocrine and immune signals, although the molecular mechanisms involved in the host-parasite interplay remain poorly understood [2–4]. Characterization of these key genes and their cognate proteins related to the parasite-host interplay should lead to a better understanding of this intriguing biological phenomenon. Together with the potential for accepting the mammalian-derived hormones, cytokines, chemokines, and immune cells that facilitate parasite growth, development, and maturation, various schistosome motor proteins and chaperones may play key roles in avoidance of immunological attack and maintenance of parasitism and parasite survival through antigen mimicry strategies. The presence of other protein groups at the interface, including anti-oxidant enzymes and protease inhibitors, supports the notion that they play roles in facilitating parasite evasion of host immunological responses [56,57]. In addition, immunity-related molecules with strong similarity to host proteins, including immunophilin, cyclophilin B, endoplasmin (gp96), and HLA-B associated transcript 1 were localized on the schistosome tegument or eggshell. These proteins can be expected to contribute to the immunopathology and chronicity associated with schistosomiasis, by facilitating escape from host immunosurveillance mechanisms through molecular mimicry, antigen presentation, and immune modification or immune inhibition. A recent, timely reassessment of schistosomiasis-related disability [58], combined with new information on the global prevalence of schistosome infection [1], indicates that the true public health burden of schistosomiasis is substantially greater than previously appreciated. The abundance of new gene and protein sequences of S. japonicum reported here should lead to a more fundamental understanding of the biology of this important human parasite and the molecular mechanisms underpinning the pathology of schistosomiasis. Furthermore, we anticipate that this new information will contribute significantly to the elucidation of complete sequences for the schistosome genome, proteome, and transcriptome, which in turn can be expected to provide new insights for the development of novel interventions leading to improved treatment and control of schistosomiasis. Materials and Methods Schistosome materials. A field-collected isolate of S. japonicum from Anhui Province, China was used in all of the transcriptome and proteome investigations. To evaluate whether genetic polymorphisms occur in natural, geographically discrete populations of S. japonicum, additional Chinese field isolates were collected from Jiangxi, Hunan, Hubei, and Sichuan Provinces. Cercariae of S. japonicum were shed from naturally infected Oncomelania hupensis hupensis snails collected in the field from these provinces. In addition, O. hupensis hupensis were infected in the laboratory with miracidia hatched from eggs. Each rabbit and mouse was experimentally infected percutaneously with 1,000 and 100 cercariae, respectively. Developing unpaired hepatic schistosomula were isolated from livers of experimentally infected mice at 14 d post-cercarial challenge, and adult worms and eggs were obtained from the mesenteric veins and liver of infected mice or rabbits, respectively, at 42–45 d post-infection. Adult parasites were manually separated into male and female worms with the aid of a microscope. Eggs, miracidia, cercariae, hepatic schistosomula, male, female, and mixed-sex adults were washed thoroughly in PBS to remove host cell debris, and then stored at −80 °C for up to 6 mo. To obtain eggshells after miracidia had been freshly hatched, eggs collected from infected livers were incubated in distilled water under a bright light for 6 h at room temperature [59]. After the miracidia were removed, the remaining eggshell-containing pellet was collected by centrifugation, and after washing three times with PBS, examined by light microscopy to ensure it contained empty eggshells only. Schistosome tegumental preparations were isolated from hepatic schistosomula, mixed-sex, and separated male and female adult worms for the proteomic survey using an optimized Triton X-100 detergent-based technique [25]. cDNA libraries and DNA sequencing. We isolated poly (A)+ mRNA from total RNA on oligo-dT sepharose (Qiagen, Valencia, California, United States), after extracting total RNA from the frozen schistosome life-cycle stages using TRIzol Reagent (GIBCO-BRL, San Diego, California, United States). We employed the poly (A)+ RNAs from hepatic schistosomula, mixed-sexed adults, and miracidia to construct new cDNA libraries with long inserts (larger than 2 kb) in the directional phage vector Uni-ZAP XR, using oligo dT priming (Stratagene, La Jolla, California, United States). Long-read DNA sequencing was carried out on an ABI 3730 DNA sequencer (ABI, Columbia, Maryland, United States) on clones selected randomly from the new cDNA libraries and the representative clones derived from the assembled clusters with potential CDSs. All EST sequences were quality-trimmed through Phred 20 prior to assembling the data. Phred was employed as a base-calling program to evaluate the quality of raw EST sequences by assigning an error probability to each base. A Phred score of 20 for a given peak in the sequence chromatogram indicates that a base is incorrectly called one time in every 100 bases, and so, in general, a Phred 20 means that the sequence is reliable. cDNA assembly and ORF prediction. The cDNA assembly procedure and prediction and annotation of CDSs were carried out according to the stringent criteria described in the Protocol S1. Analysis of statistical significance of gene expression was performed using tools available at http://www.igs.cnrs-mrs.fr/~audic/significance.html [12]. Comparative genomic analysis. Comparative genomic analysis was performed using BLAST programs based on public nucleotide and protein databases via the public access Web sites: GenBank at National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov) for gene, protein, and EST entries; Ensembl (http://www.ensembl.org) for human, Rattus norvegicus, Mus musculus, T. rubripes, A. gambiae, D. melanogaster, C. briggsae, and C. elegans; http://www.genoscope.cns.fr/externe/tetraodon for T. nigroviridis; http://www.plasmodb.org for P. yoelii nigeriensis (17XNL) and P. falciparum (3D7); http://www.hominis.mic.vcu.edu for C. hominis; and GenBank and http://bioinfo.iq.usp.br/schisto for S. mansoni genes and ESTs. We also prepared a local copy of the S. mansoni dataset on our Web server under the link http://function.chgc.sh.cn/sj-proteome/download/download.php. Conserved protein domains or motifs and families were identified by interrogation of the InterPro protein domain database version 7.0 (http://www.ebi.ac.uk/interpro) using S. japonicum protein sequences deduced from putative CDSs as queries. The CDSs that were similar to known genes and domains were further assigned into different molecular functions and biological processes based on GO (http://www.geneontology.org). Signal peptide and transmembrane predictions were accomplished using on-line tools at http://www.cbs.dtu.dk/services/SignalP and http://www.cbs.dtu.dk/services/ TMHMM. The subcellular localization of CDSs was predicted using PSORT II at http://www.psort.nibb.ac.jp/form2.html. Genetic polymorphisms. Candidate SNPs or indels were identified based on high-quality assembled contigs with multiple ESTs using revised stringent criteria [44,45] as detailed in Protocol S1. The ratio of dN/dS. Orthologous gene pairs between S. japonicum and S. mansoni were identified as reciprocal best BLAST (BLASTP version 2.2.12) hits using translated protein sequences. Alignments with greater than 50% similarity in length and with E < 10−10 were considered significant. The 1,514 orthologous gene pairs from S. japonicum and S. mansoni were globally aligned with ClustalW version 1.83 (default parameters). The dN and dS between pair-wise alignments were calculated with SNAP (http://www.hiv.lanl.gov/content/hiv-db/SNAP/WEBSNAP/SNAP.html) [60] based on the method of Nei and Gojobori [61]. 2D-nano-LC-MS. The protein mixtures from solubilized schistosome samples were digested and then fractionated into 20–30 subgroups by strong cation exchange (SCX) chromatography. The peptide mixture of each SCX fraction was sequentially loaded onto a reverse phase (RP) trap column, connected in-line to a C18 column (LC Packings Incorporated, San Francisco, California, United States), as published [10], and the peptide mixture was eluted into a QSTAR pulser i mass spectrometer coupled to a Protana NanoES electrospray ionization source. The remaining supernatant and the insoluble pellet were denatured in the loading buffer for SDS-PAGE and size-fractionated by one-dimensional electrophoresis. Each lane of the gel was cut equally into eight or 12 slices that were subsequently in-gel digested with trypsin. Silver staining and the in-gel tryptic digests were performed according to standard procedures [62]. Hydrolysates from gels were likewise analyzed by in-line RP-LC-MS as described above (further details in Protocol S1). MS data interpretation. The MS/MS spectra were searched against the rabbit or mouse and the S. japonicum protein databases, the latter deduced from CDSs obtained from ESTs in the present study, using MASCOT software (http://www.matrixscience.com, Matrix Science). The initial results were combined and further filtered using revised criteria [10]. Protein quantifications were carried out as previously described [11] (more detailed in Protocol S1). The proteomic data were further employed to identify the translated variants due to the nonsynonymous SNPs, indels, or microsatellites, where a special peptide database containing the protein variants through/beyond the genetic polymorphisms was established. The MS/MS spectra were searched against both the specific and common databases by MASCOT software. The matched peptide variants with MASCOT score higher than 30 were considered as significant, as the possibility that the MS/MS spectra were matched to other common peptides was excluded. Immunofluorescence assay. Immunofluorescence assays were carried out on 5-μm-thick frozen sections of adult worms embedded in OCT fixative. Slides were incubated in a humid atmosphere at 37 °C for 60 min with an anti-immunophilin (SjFKBP50) monoclonal mouse antibody, generated by immunizing mice with recombinant SjFKBP50 protein. The slides were washed and incubated for 60 min with a FITC-conjugated, rabbit anti-mouse immunoglobulin antibody (Nordic, Tilburg, Netherlands), diluted 1/40 in PBS containing 0.5 mg/ml Evans blue in a humid atmosphere at 37 °C. Antibody staining was visualized and recorded using a Leica DM-RB fluorescence microscope (Leica, Wetzlar, Germany) with the appropriate filter combination for FITC fluorescence. Supporting Information Figure S1 Schematic Representation of the Strategy for the Integrated S. japonicum Transcriptome and Proteome Analyses The boxes represent the main results or steps of the transcriptomic or proteomic analyses that were described in the main text. The arrows illustrate the direction of process procedures. (8 KB PDF) Click here for additional data file. Figure S2 Characteristics of the S. japonicum Clusters (A) The length distribution of all clusters, clusters with protein-coding genes, and with complete CDSs of S. japonicum. nt, nucleotide genes. (B) The length distribution of proteins deduced from all CDSs and complete CDSs of S. japonicum. aa, amino acid. (5 KB PDF) Click here for additional data file. Figure S3 Predicted Functions of S. japonicum CDSs and Proteins Based on GO (A) The distribution of GO categories based on molecular functions (left) and biological processes (right). The filled and open boxes represent the GO categories according to the transcriptomic and proteomic data, respectively. The classification integrated the numbers of known genes and InterPro domains assigned into different GO categories. (B) The distribution of GO categories throughout the life cycle, including C, cercariae; S, hepatic schistosomula; A, adults; E, eggs; and Mi, miracidia based on the proteomic data, according to biological processes (left) and molecular functions (right). The detailed list of GO assignments can be found in Tables S2 and S4. (12 KB PDF) Click here for additional data file. Figure S4 Correlation between Transcriptomic and Proteomic Data of Representative Proteins among Life-Cycle Stages and Sexes (A) Some developmental stage-enriched proteins were generally consistent with the transcriptomic data. C, cercariae; S, hepatic schistosomula; A, adults; E, eggs; and Mi, miracidia. (B) Gender-enriched proteins are also shown as colored boxes: F, females; M, males. The protein abundances were calculated as detailed in the Protocol S1. Proteins not detected in life-cycle stages are depicted as black blocks. The abundances of mRNAs are represented by the ratio of EST copy numbers to the total EST numbers in the libraries. (602 KB PDF) Click here for additional data file. Figure S5 Verification of SNPs by DNA Sequencing (A) Fructose 1,6 bisphosphate aldolase. (B) Putative preprocathepsin L. (C) SJCHGC00098, containing similarity to preprocathepsin cathepsin L, was checked by re-sequencing PCR-amplified products of field S. japonicum genomic DNA samples from five Chinese provinces, indicated on the right. The SNPs indicated by the red arrows were identical to the EST findings presented in this study. The single letter amino acid codes separated by a forward slash represent homozygous or heterogeneous SNPs in the genome, and the numbers indicate the position of the SNP sites on the cluster sequences. Replacements of amino acid residues due to missense mutations are illustrated based on the DNA sequences. (621 KB PDF) Click here for additional data file. Protocol S1 The Detailed Approaches for Transcriptomic and Proteomic Analyses (46 KB PDF) Click here for additional data file. Table S1 Summary of S. japonicum Transcriptomic Data (17 KB XLS) Click here for additional data file. Table S2 Integrated Information of the Transcriptome and Proteome of S. japonicum (7.4 MB XLS) Click here for additional data file. Table S3 Domain Analyses of S. japonicum Proteins (463 KB XLS) Click here for additional data file. Table S4 GO Classification of S. japonicum Transcripts Based on Similarity with Known Genes and Domains (30 KB XLS) Click here for additional data file. Table S5 Statistics of Polymorphisms of S. japonicum Clusters (18 KB XLS) Click here for additional data file. Table S6 Polymorphisms of S. japonicum Clusters (1.3 MB XLS) Click here for additional data file. Accession Numbers The nucleotide sequence described here has been deposited in public databases with accession numbers: EST sequences (CV671092–CV674724, CV581651–CV582043, CV693277–CV699272, CV681278–CV693276, CV736204–CV758494, and CX856533–CX863389); the full-length, partial cDNAs and hypothetical genes (AY812752–AY816180, AY808309–AY812729, and AY914876–AY915917). The GenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for schistosoma japonicum sequences described in this paper are acetylcholine receptor (alpha-3 chain) (AY815304), actins (AY813805), amidase (AY809279), 21.7 kDa antigen (AAD13338), budding uninhibited by benzimidazoles 1 (AY812514), calcineurin A (AY810505), calcium/calmodulin-dependent protein kinase II (delta isoform 3) (AY813551), calcium-binding protein Sj66 (AAC62193), calpain (AY808568), calreticulin (AAC00515), collagen (type I alpha 3) (AY810097), craniofacial development protein 1 (AY814915), cyclophilin B (AY816130), cytochrome c oxidase (subunit 1) (AAG13143), dioxin receptor (AY813606), dynein light chain 1 (AAD41626), eggshell protein (AAP05897 ), egumental antigen Sm20 (AY813791), endoplasmin (gp96) (AY813390), 14–3–3 epsilon (AY815015), extracellular superoxide dismutase (AY812195), F-box only protein 9 (AY815855), feline leukemia virus (subtype-B) receptor (AY813727), female-specific 800 protein (AY815492), fimbrin (AY809033), flavoprotein (Fp) (AY814217), forebrain embryonic zinc-finger like (Fezl) (AY808322), G protein alpha subunit (AY815795), GABA receptor (AY815726), glutathione-S-transferase (AY816103), gynecophoral canal protein (AY810721), high voltage-activated calcium channel (beta subunit 2) (AY812476), histone H2A (gonadal) (AY812081), HLA-B associated transcript 1 (AAP06453), Hsp60 (AY813151), immunophilin (FK506-binding protein 50) (AY815389), insulin receptor protein kinase RTK-2 (AY813034), karyopherin (AY810727), leucine aminopeptidase (AY814468), manganese superoxide dismutase (AY814748), MF3 (AY809998), microtubule-associated protein (AY812854), myosin (AY810340), nitric oxide synthase 1 (NOS1) (AY815837), nocturnin (AY812870), Notch receptor (AY810632), osteonectin (SPARC) (AY814549), p40 major egg antigen (AY813596), p48 eggshell protein (AY812971), paramyosin (AAD29285), peptidylprolyl isomerase (AY814078), phosphomannose isomerase (type I) (AY812397), platelet glycoprotein IIIa (GPIIIa) (AY810920), presenilin (AY809924), prion protein interacting protein (AY815835), prosaposin (AY815893), protein disulfide isomerase (AAC78302), purinergic receptor P2X (AY812469), putative C1-tetrahydrofolate synthase (AAP06003), Putative deoxyribodipyrimidine photo-lyase (DNA photolyase) (AY812553), Putative ribophorin II (AY809963), putative ribosome-associated protein P40 (AAP05908), putative sex-determining region Y protein (SRY) (AY813503), receptor tyrosine phosphatase (gamma and delta) (AY812724), regulator of G-protein signaling 2 (AY814274), SJCHGC01743 protein (AY813753), Sj-Ts4 (AY812897), SM22.6 antigens (A12) (AY813797), SnaK (AY808337), stage-specific protein SPO-1 (AY812887), Su(var)3–9 (AY815180), 22.6 kDa tegument membrane-associated antigen (AY815413), thioredoxin peroxidases (AY813893), transducin-like enhancer of split 3 (TLE3) (AY810007), trispanning orphan receptor (AY814912), tropomyosin (AY809967), tubulins (AY815746), twister (AY809513), ubiquitin-protein ligase NEDD4-like (AY812719), and vasopressin-activated calcium-mobilizing receptor (cullin 5) (AY812566). The Swissprot (http://www.ebi.ac.uk/swissprot) accession number for the schistosoma japonicum sequence described in this paper is antigen Sj32 (P42665). The PIR (http://pir.georgetown.edu) accession number for the schistosoma japonicum sequence described in this paper is cathepsin B-like cysteine protease precursor (pir||S31909). All transcriptomic and protemic data described here are freely available and can be downloaded from our Web site: http://www.function.chgc.sh.cn/sj-proteome/index.htm.
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- Abstract: found
- Article: not found
Hemoglobin digestion in blood-feeding ticks: mapping a multipeptidase pathway by functional proteomics.
Martin Horn, Martina Nussbaumerová, Miloslav Šanda … (2009)
Hemoglobin digestion is an essential process for blood-feeding parasites. Using chemical tools, we deconvoluted the intracellular hemoglobinolytic cascade in the tick Ixodes ricinus, a vector of Lyme disease and tick-borne encephalitis. In tick gut tissue, a network of peptidases was demonstrated through imaging with specific activity-based probes and activity profiling with peptidic substrates and inhibitors. This peptidase network is induced upon blood feeding and degrades hemoglobin at acidic pH. Selective inhibitors were applied to dissect the roles of the individual peptidases and to determine the peptidase-specific cleavage map of the hemoglobin molecule. The degradation pathway is initiated by endopeptidases of aspartic and cysteine class (cathepsin D supported by cathepsin L and legumain) and is continued by cysteine amino- and carboxy-dipeptidases (cathepsins C and B). The identified enzymes are potential targets to developing novel anti-tick vaccines.
Author and article information
Contributors
Pavel Roudnický:
ORCID: http://orcid.org/0000-0002-6904-7646
Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole:
MethodologyRole: VisualizationRole: Writing – original draft
David Potěšil: Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Supervision
Zbyněk Zdráhal: Role: Project administrationRole: ResourcesRole: Writing – review & editing
Milan Gelnar: Role: Funding acquisitionRole: Resources
Martin Kašný: Role: ConceptualizationRole: MethodologyRole: Project administrationRole: ResourcesRole:
SupervisionRole: Writing – review & editing
Matthew Bogyo: Role: Editor
Journal
Journal ID (nlm-ta): PLoS One
Journal ID (iso-abbrev): PLoS ONE
Journal ID (publisher-id): plos
Journal ID (pmc): plosone
Title:
PLoS ONE
Publisher:
Public Library of Science
(San Francisco, CA USA
)
ISSN
(Electronic):
1932-6203
Publication date
(Electronic):
17
June
2020
Publication date Collection: 2020
Volume: 15
Issue: 6
Electronic Location Identifier: e0231681
Affiliations
Author notes
* E-mail:
p.roudnicky@
123456mail.muni.cz
Author information
Pavel Roudnický
http://orcid.org/0000-0002-6904-7646
Article
Publisher ID: PONE-D-20-08758DOI: 10.1371/journal.pone.0231681
PMC ID: 7299319
PubMed ID: 32555742
SO-VID: 01df2419-7594-4daf-87cf-4b267f8427e8
Copyright © © 2020 Roudnický et al
License:
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
History
Date
received
: 27
March
2020
Date
accepted
: 25
May
2020
Page count
Figures: 5,
Tables: 3,
Pages: 15
Funding
Funded by: funder-id http://dx.doi.org/10.13039/501100001824, Grantová Agentura České Republiky;
Award ID: GBP505/12/G112
Funded by: funder-id http://dx.doi.org/10.13039/501100001824, Grantová Agentura České Republiky;
Award ID: GAP506/12/1258
Funded by: funder-id http://dx.doi.org/10.13039/501100001823, Ministerstvo Školství, Mládeže a Tělovýchovy;
Award ID: LM2018132
Funded by: funder-id http://dx.doi.org/10.13039/501100001823, Ministerstvo Školství, Mládeže a Tělovýchovy;
Award ID: LM2018127
Funded by: funder-id http://dx.doi.org/10.13039/501100001823, Ministerstvo Školství, Mládeže a Tělovýchovy;
Award ID: LQ1601
Funded by: funder-id http://dx.doi.org/10.13039/501100001823, Ministerstvo Školství, Mládeže a Tělovýchovy;
Award ID: LM2018140
This work was supported by the Czech Science Foundation (GBP505/12/G112, GAP506/12/1258)
and Grant Agency of Masaryk University (MUNI/A/0918/2018). We acknowledge the CF Genomics
CEITEC MU supported by the NCMG research infrastructure (LM2018132 funded by MEYS
CR) for their support with obtaining scientific data presented in this paper. CIISB
research infrastructure project LM2018127 funded by MEYS CR is gratefully acknowledged
for their financial support of LC-MS/MS measurements at the Proteomics Core Facility.
The work was supported by the project CEITEC 2020 (LQ1601), MEYS CR. Computational
resources were supplied by the project “e-Infrastruktura CZ” (e-INFRA LM2018140) provided
within the program Projects of Large Research, Development, and Innovations Infrastructures.
The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Categories
Subject:
Research Article
Subject:
Biology and Life Sciences
Subject:
Biochemistry
Subject:
Enzymology
Subject:
Enzymes
Subject:
Proteases
Subject:
Biology and Life Sciences
Subject:
Biochemistry
Subject:
Proteins
Subject:
Enzymes
Subject:
Proteases
Subject:
Biology and Life Sciences
Subject:
Anatomy
Subject:
Digestive System
Subject:
Gastrointestinal Tract
Subject:
Medicine and Health Sciences
Subject:
Anatomy
Subject:
Digestive System
Subject:
Gastrointestinal Tract
Subject:
Biology and Life Sciences
Subject:
Microbiology
Subject:
Virology
Subject:
Viral Structure
Subject:
Tegument Proteins
Subject:
Biology and Life Sciences
Subject:
Biochemistry
Subject:
Proteins
Subject:
Tissue Proteins
Subject:
Engineering and Technology
Subject:
Equipment
Subject:
Optical Equipment
Subject:
Lasers
Subject:
Research and Analysis Methods
Subject:
Database and Informatics Methods
Subject:
Biological Databases
Subject:
Proteomic Databases
Subject:
Biology and Life Sciences
Subject:
Biochemistry
Subject:
Proteomics
Subject:
Proteomic Databases
Subject:
Biology and Life Sciences
Subject:
Computational Biology
Subject:
Genome Analysis
Subject:
Transcriptome Analysis
Subject:
Biology and Life Sciences
Subject:
Genetics
Subject:
Genomics
Subject:
Genome Analysis
Subject:
Transcriptome Analysis
Subject:
Physical Sciences
Subject:
Chemistry
Subject:
Chemical Compounds
Subject:
Organic Compounds
Subject:
Amino Acids
Subject:
Hydroxyl Amino Acids
Subject:
Serine
Subject:
Physical Sciences
Subject:
Chemistry
Subject:
Organic Chemistry
Subject:
Organic Compounds
Subject:
Amino Acids
Subject:
Hydroxyl Amino Acids
Subject:
Serine
Subject:
Biology and Life Sciences
Subject:
Biochemistry
Subject:
Proteins
Subject:
Amino Acids
Subject:
Hydroxyl Amino Acids
Subject:
Serine
Custom metadata
Data Availability Proteomic data are now publicly available from PRIDE under accession number: PXD017275.
(
https://www.ebi.ac.uk/pride/archive/projects/PXD017275).
ScienceOpen disciplines: Uncategorized
Data availability:
ScienceOpen disciplines: Uncategorized
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