The authors have declared that no competing interests exist.
Schistosomes are parasitic blood flukes that survive for many years within the mammalian host vasculature. How the parasites establish a chronic infection in the hostile bloodstream environment, whilst evading the host immune response is poorly understood. The parasite develops morphologically and grows as it migrates to its preferred vascular niche, avoiding or repairing damage from the host immune system. In this study, we investigated temporal changes in gene expression during the intra-mammalian development of
The life cycle of the parasitic flatworm
The blood fluke
Susceptible aquatic snails transmit schistosomes between mammalian hosts by releasing infectious larvae called cercariae that seek out a mammalian host, penetrate through its skin and transform into a juvenile form known as schistosomula. The schistosomula enter the bloodstream and by day 6 post-infection are mainly present in the lung capillaries [
Large-scale transcriptional changes have been extensively described across the life-cycle of
In contrast to adult stages, gene expression in early stages of intra-mammalian development has been poorly studied. Studying several specific developmental stages has been compromised by difficulties in obtaining parasite material. This has resulted in a complex picture being pieced together using material obtained
In order to fill these knowledge gaps, we have analysed the
All procedures involving mice were performed by authorised personnel according to the UK Animals (Scientific Procedures) Act 1986 with project license held by MJD (number PPL 40/3595). The work was approved by the Ethical Review Committee of the University of Nottingham and was carried out in strict accordance with UK Home Office regulations for animal welfare and amelioration of suffering.
Mice were infected with cercariae of
Mice were infected with indicated numbers of cercariae for parasite collection at six time points post-infection. The number of mice used for each time point is shown on the left. The method for parasite collection at day 6 involves mincing and incubation of the lung. Collections at other time points were done by portal perfusion.
On the indicated day post-infection, mice were culled using an overdose of pentobarbitone containing 10 U/ml heparin. Day-6 lung-stage parasites were collected as described [
For all other time points, parasites were collected by portal perfusion with approximately 30 ml of perfusion media (Dulbecco's Modified Eagle's Medium (DMEM) high glucose, with 10 U/ml heparin). Parasites were left to settle for 30 minutes at room temperature and washed twice with DMEM before being recovered with a Pasteur pipette from the bottom of the tube.
Parasites were transferred to a Petri dish for imaging using an Olympus SZ61dissecting microscope with a Euromex Cmex10 camera and Image Focus 4.0 software. A subset of the parasites from each mouse was imaged for morphology scoring. After imaging, parasites were transferred into a 2 ml Eppendorf tube and centrifuged at 150 x g for 3 minutes before replacing the supernatant with 1 ml TRIzol (Thermo Fisher), left at room temperature for up to 1 hour, transferred to dry ice for transport, and later stored at -80 ºC until RNA extraction. An average of 31 worms were imaged per mouse (range 18–75 worms); this did not represent a total number of parasites collected.
Parasite morphology was classified using numerical scores based on published categories [
RNA was extracted from parasite material using a modified phenol-chloroform method and column purification. Briefly, frozen samples in TRIzol reagent were thawed on ice, resuspended by gently pipetting and transferred to 2 ml tubes containing ceramic beads (MagNA Lyser Green Beads, Roche). The parasites were homogenised in a MagNA Lyser Instrument (FastPrep-24) at maximum speed twice for 20 seconds, with a 1-minute rest on ice in between. Next, 200 μl of chloroform-isoamyl alcohol 24:1 was added to each tube, followed by vigorous shaking for 5 seconds. The tubes were centrifuged at 13,000 x g for 15 minutes at 4°C to separate the aqueous and organic solvent layers. The aqueous layer was transferred into a RNase-free 1.5 ml tube and one volume of 100% ethanol added and mixed by pipetting. The mixture was transferred to Zymo RNA Clean & Concentrator-5 column (Zymo Research) and processed according to the manufacturer's protocol. To elute the RNA, 15 μl of RNase-free water was added to the column and centrifuged for 30 seconds at 13,000 x g. The RNA concentration and integrity were measured by Agilent RNA 6000 Nano kit (Agilent Technologies), and its purity assessed using a NanoDrop spectrophotometer.
One to 2.8 μg of RNA was used to prepare each sequencing library. The libraries were produced using TruSeq Stranded RNA Sample Preparation v2 Kits (Illumina). Libraries were amplified using 10–14 cycles of PCR and cleaned using Agencourt AMPure XP Beads (Beckman Coulter). The libraries were quantified by qPCR before sequencing using the Illumina HiSeq 2500 platform. All sequencing data were produced as 75 bp paired-end reads and are available through ENA study accession number ERP113121.
Sequencing reads (
Analyses were performed using RStudio version 0.99.489 [
Genes were clustered using self-organising maps constructed in the R package Kohonen (version 2.0.19) [
Gene Ontology (GO) annotations for the
Amino acid sequences for proteins of interest were obtained from
Protein domains were identified from amino acid sequences using InterProScan online server (v60, v61 and v78) [
Changes in the morphology and transcriptome during intra-mammalian development of
Lung schistosomula were morphologically homogeneous, whereas circulating larvae that had left the lungs (days 13 to 28) were heterogeneous in size and developmental progression (
A) Morphological scoring of
Parasites from individual mice were pooled and each of these pools was considered a biological replicate. At least 3 biological replicates were obtained for each time point and changes in their transcriptomes were measured using RNA-seq. A principle components analysis showed tight clustering of biological replicates and a large variation among the time points (
PCA plot of all transcriptomic data based on rlog-transformed normalised read counts. Each dot represents the transcriptome from a pool of parasites collected from an individual mouse, i.e. one biological replicate.
Comparing the transcriptomes of parasites collected 6- and 13-days post-infection provided information on the transition between lung schistosomula and circulating juveniles, i.e. parasites that have already left the lungs and have entered the systemic circulation
As the parasites develop from circulating juveniles to adult forms, up-regulated genes identified in the liver stage (day 21) compared to pre-egg-laying adult stage (day 28) were involved in cell division, differentiation, and developmental regulation (
Between days 28 and 35, the parasites become fully established in the portal system within the mesentery veins and lay large numbers of eggs. Expectedly, amongst the 72 genes that were up-regulated during the progression from day 28 to day 35, many were related to egg production (
To further explore transcriptomic changes across all developmental stages analysed, genes were clustered into 96 groups based on their expression profile over the whole time-course (
Expression profiles of genes showing differential expression in at least one time point, clustered into 96 groups. The clustering was based on mean-normalised rlog-transformed raw read counts over six time points. The y-axes are scaled independently to emphasise the differences between clusters. Plots with a single y-axis scale are shown in
Given the scarcity of data on lung schistosomula, we investigated gene expression at this stage by performing pairwise comparisons between the lung stage and day-13 parasites, and by focusing on data that was clustered over multiple time points but showed striking differences in the lung. High expression in the lung stage, compared with other intra-mammalian stages, was seen mainly in three clusters (8, 24 and 32), where high expression on day 6 precipitously dropped to a low baseline for the rest of the time-course (
Genes related to developmental control were also over-represented in 11 clusters that showed high expression in lung stage followed by a steady decline towards adult (cluster 5, 6, 7, 13, 14, 15, 16, 21, 22, 23, 31;
Compared to day-13 parasites, the relative expression of multiple genes related to signalling processes was high in the lung stage; amongst 864 highly expressed genes, the top-four enriched GO terms were
Gene identifier | Adjusted p-value | Product name | |
---|---|---|---|
Smp_138080 | 12.62 | 1.48E-19 | MEG-3 (Grail) family |
Smp_138070 | 11.99 | 3.58E-30 | MEG-3 (Grail) family |
Smp_159810 | 11.22 | 1.02E-48 | MEG-2 (ESP15) family |
Smp_159800 | 9.66 | 5.10E-28 | MEG-2 (ESP15) family |
Smp_181510 | 9.58 | 1.15E-13 | hypothetical protein |
Smp_032990 | 8.98 | 7.14E-13 | Calmodulin 4 (Calcium binding protein Dd112) |
Smp_159830 | 8.69 | 1.00E-05 | MEG-2 (ESP15) family |
Smp_138060 | 8.06 | 3.76E-09 | MEG-3 (Grail) family |
Smp_203400 | 7.34 | 1.88E-07 | rhodopsin orphan GPCR |
Smp_005470 | 7.31 | 3.34E-08 | dynein light chain |
Smp_077610 | 6.61 | 6.19E-07 | hypothetical protein |
Smp_166350 | 6.59 | 1.31E-06 | hypothetical protein |
Smp_180330 | 5.97 | 2.91E-32 | MEG 2 (ESP15) family |
Smp_205660 | 5.84 | 4.52E-15 | hypothetical protein |
Smp_033250 | 5.80 | 4.43E-05 | hypothetical protein |
Smp_132500 | 5.73 | 4.50E-04 | ras and EF hand domain containing protein |
Smp_152730 | 5.68 | 3.41E-11 | histone lysine N methyltransferase MLL3 |
Smp_241430 | 5.61 | 3.89E-23 | Aquaporin 12A |
Smp_125060 | 5.55 | 6.70E-04 | kinase suppressor of Ras (KSR) |
Smp_198060 | 5.51 | 4.73E-13 | hypothetical protein |
*Log2FC (lung/D13), logarithm of the fold change in expression level between lung stage and day-13 schistosomula
The GO term
Micro-exon genes (MEGs), whose structures mainly comprise short exons with lengths that are multiples of three bases, are an abundant feature in parasitic helminths [
Genes involved in defence against oxidative stress were highly expressed in lung schistosomula, presumably to neutralise reactive oxygen species (ROS) produced during inflammation. For instance, expression of
These three genes with particularly striking up-regulation in the lung stage belong to the same cluster (cluster 72). Given the possible roles of cluster 72 genes in counteracting oxidative stress and in host immune system evasion, other genes from this cluster were explored in more detail. Cluster 72 contained seven additional genes (
Gene identifier | Product name |
---|---|
Smp_059480 | thioredoxin peroxidase |
Smp_059980 | arginase |
Smp_074570 | hypothetical protein |
Smp_114660 | hypothetical protein |
Smp_134870 | early growth response protein |
Smp_147730 | single Kunitz protease inhibitor; serine type protease inhibitor |
Smp_156510 | PDZ and LIM domain protein 7 |
Smp_166920 | PDZ and LIM domain protein Zasp |
Smp_174810 | Extracellular superoxide dismutase (Cu Zn) |
Smp_182770 | hypothetical protein |
Cluster 64 contained genes with similar expression profiles to cluster 72 and like the latter cluster, was annotated with GO terms enriched for redox processes (
With four genes out of ten in cluster 72 potentially involved in host immune interactions, the three that encoded hypothetical proteins (Smp_074570, Smp_114660, Smp_182770) were investigated further. Peptides encoded by Smp_074570 are abundant (top 10) in secreted extracellular vesicles produced by
(A) Three-dimensional structure of Smp_182770 predicted using the I-TASSER server. (B) Alignment between the predicted structure (blue) and 3D structure of human CFH (red) in 250 mM NaCl buffer, obtained from PDB (entry 3GAW). (C) Domain components of human CFH identified from multiple databases using the InterProScan web server [
Homologues of the Smp_182770 are present in other
A) Homologues of Smp_182770. Information on homologues of genes was obtained from WormBase ParaSite, release 8 [
We have described a complete transcriptome time-course of
In early stages of
Our data revealed that multiple genes with potential immune evasion or protective roles are up-regulated. One example was the
Multiple genes with tentative roles in immune evasion displayed particularly high expression in the lung stage, moderately high expression in adult stages and lower expression in the liver stages. In particular, a previously uncharacterised gene, annotated as a hypothetical protein, with this expression pattern was predicted to be structurally similar to the human complement factor H (CFH), a regulator of the complement cascade. In mammals, CFH cleaves C3b, a central protein in the complement cascade [
Certain limitations need to be borne in mind when interpreting the data from this study. First, it has been shown that gene expression can change with the types of hosts [
The data produced from this study will serve as a unique resource for the research community to explore changes across intra-mammalian stages of schistosome development. Our particular focus on the lung stage demonstrated consistency with previous observations and introduced potential new players in host-parasite interactions and parasite development. Further investigation and functional validation of genes identified here will help to decipher mechanisms for parasite long-term survival within the mammalian host, exposing vulnerabilities that can be exploited to develop new control strategies for this neglected tropical pathogen.
Expression profile of genes differentially expressed in at least one time point clustered into 96 groups. The clustering was done on mean-normalised regularized log transformation (rlog- transformed) of raw read counts. X-axes represent six time points from this dataset; y-axes represent the mean-normalised rlog-transformed. Unlike
(TIF)
Each dot represents one replicate from each of the time points. Y-axis represents normalised counts from DESeq2.
(TIF)
Each dot represents one replicate from each of the time points. Y-axis represents normalised counts from DESeq2.
(TIF)
Expression of Smp_174810 and Smp_095980 with y axes on log scale. Each dot represents one replicate from each of the time points. Y-axis represents normalised counts from DESeq2. Smp_095980 was identified as differentially expressed (
(TIF)
Each dot represents one replicate from each of the time points. Y-axis represents normalised counts from DESeq2. Log2FC between D13/D06 is -0.52, adjusted p-value for differential expression between D13/D06 is 4.10e-21.
(TIF)
Each dot represents one replicate from each of the time points. Y-axis represents normalised counts from DESeq2.
(TIF)
A) Predicted 3D structure of Smp_334090 (resulting from a merge of Smp_182770 and Smp_038730 in the most recent version of the
(TIF)
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Total number of reads and % mapped to
(DOCX)
GFF file containing annotation for
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A zipped folder containing final counts from HTSeq-count. The folder contains the following files: D06_SM_1_17675_4_1.htseq-count.txt, D06_SM_2_17675_4_2.htseq-count.txt, D06_SM_3_17675_4_3.htseq-count.txt, D06_SM_4_17675_4_4.htseq-count.txt, D06_SM_5_17675_4_5.htseq-count.txt, D06_SM_6_17675_4_6.htseq-count.txt, D06_SM_7_17675_4_7.htseq-count.txt, D13_SM_1_17675_4_8.htseq-count.txt, D13_SM_2_17675_4_9.htseq-count.txt, D13_SM_3_17675_4_10.htseq-count.txt, D17_SM_1_17675_4_11.htseq-count.txt, D17_SM_2_17675_4_12.htseq-count.txt, D17_SM_3_17675_4_13.htseq-count.txt, D21_SM_1_17675_4_14.htseq-count.txt, D21_SM_2_17675_4_15.htseq-count.txt, D21_SM_3_17675_4_16.htseq-count.txt, D28_SM_1_17675_4_17.htseq-count.txt, D28_SM_2_17675_4_18.htseq-count.txt, D28_SM_3_17675_4_19.htseq-count.txt, D35_SM_1_17675_4_20.htseq-count.txt, D35_SM_2_17675_4_21.htseq-count.txt, D35_SM_3_17675_4_22.htseq-count.txt.
(ZIP)
We thank Prof Karl Hoffmann and Dr Cinzia Cantacessi for their comments on the study and the first version of this manuscript. We thank multiple members of the Parasite Genomics team at the Wellcome Sanger Institute for their comments and input for the experimental design and analysis; in particular, we thank Hayley Bennett, Lia Chappell, James Cotton, Stephen Doyle, Magda Lotkowska, Thomas Otto, Kate Rawlinson, Adam Reid, Alan Tracey and Gavin Rutledge. The infrastructure used for the analysis is maintained by the core IT Service and the Pathogen Informatics teams at the Wellcome Sanger Institute.
Dear Dr Berriman:
Thank you very much for submitting your manuscript "Transcriptome of the parasitic flatworm Schistosoma mansoni during intra-mammalian development" (PNTD-D-19-01474) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified a few aspects of the manuscript that should be improved.
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Reviewer's Responses to Questions
As you describe the new analyses required for acceptance, please consider the following:
-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?
-Is the study design appropriate to address the stated objectives?
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-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?
-Were correct statistical analysis used to support conclusions?
-Are there concerns about ethical or regulatory requirements being met?
Reviewer #1: See below
Reviewer #2: The objectives of the study are clear and study design is appropriate as presented, but I am concerned that previous knowledge from studies of additional life cycle stages are not discussed.
The sample size is adequate and there are no ethical concerns.
Reviewer #3: The authors’ submitted manuscript “Transcriptome of the parasitic flatworm Schistosoma mansoni during intra-mammalian development” investigated the temporal changes in the gene expression of S. mansoni in mice. This work has highlighted gaps in current understanding of S. mansoni infection and has provided valuable data adding to the current knowledge of the possible molecular mediators during host-parasite infection by studying gene expression of six developmental stages of the parasite. The authors used RNA-seq to analyze the transcriptomic profiles of the parasites and used bioinformatics tools to further analyze the biological significance of the gene expression. The authors have also done a thorough analysis and speculating signaling pathways that might be involved during the infection based on the expression profiles of genes showing differential expression at different developmental stages by performing cluster and GO term analysis. The authors further investigated the three hypothetical proteins that are involved in host immune interactions by protein structure prediction. Based on differential expression analysis the authors found micro-exon-genes (MEGs) to be highly up-regulated in day 6 as compared to day-16 schistosomula recapitulated some of the data shown in previous studies. In addition, data from the manuscript showed that the major signaling pathways regulated during these six developmental stages are those related to developmental control, cell differentiation and host interaction (including oxidative stress, iron homeostasis and inflammation) and found that one of the proteins aligned with the structure of human CFH, a regulator of the complement cascade. Overall this manuscript is clearly written and easy to follow. Methods used to generate the transcriptome data were also well presented.
Minor comments:
1. (Line 77) Previous studies on intra-mammalian development of S. mansoni were mentioned and it would have been helpful for the general audience to include statements on the major findings/summaries from those in vitro/in vivo studies.
2. Authors should include details of the raw RNA-seq data including number of reads obtained and % of mapping.
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-Does the analysis presented match the analysis plan?
-Are the results clearly and completely presented?
-Are the figures (Tables, Images) of sufficient quality for clarity?
Reviewer #1: See below
Reviewer #2: The analyses presented match the study plan and the results are clearly and completely described and the figures are clear.
Reviewer #3: see above
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-Are the conclusions supported by the data presented?
-Are the limitations of analysis clearly described?
-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?
-Is public health relevance addressed?
Reviewer #1: See below
Reviewer #2: The conclusions are supported but the limitations could be better addressed. The authors clearly describe how the results can advance the field.
Reviewer #3: see above
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Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.
Reviewer #1: See below
Reviewer #2: None
Reviewer #3: see above
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Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.
Reviewer #1: Although the paper and approach are not entirely novel, the work presented herein presents a new and authoritative understanding of gene expression during development of Schistosome mansoni in its experimental host. The work uncovers the profiles of molecules involved in a range of activities at the host-parasite interface. The separation of lung-phase worms appears to be quite rapid compared with those of previous studies and there is some confidence that the transcriptome(s) of this stages approaches the native state.
The manuscript is clear and well presented and will make a fine contribution. Some minor comments are made below to assist improve an already strong manuscript.
1. Line 262. Upregulation of molecules associated with neuronal function. One very interesting analysis of Fasciola development in its mammal host by MV Sukhdeo of Rutgers University suggested that trematodes followed fixed actions patterns of behaviour, that is, the parasites displayed certain behaviours that facilitated their development to a certain point. Having reached that point, a whole new set of behaviours were initiated. The lung stage of Schistosoma seems critical in development and host-parasite interaction and some major morphological changes occur in the parasites in the time they are present in the lungs. So, just as a thought exercise, I wonder if it is worthwhile adding that the neuronal activity is also associated with a transition to a whole new phase of development?
2. I was happy to see reference to iron metabolism and the putative iron transporters and related genes. The findings of ferritin are interesting. Work on S mansoni (Schüssler et al Mol Reprod Dev. 1995 Jul;41(3):325-30) and S japonicum (Jones 2007;39(9):1646-58) demonstrate that one form of ferritin (called yolk ferritin) is associated with vitelline cells. Iron was proposed to be either stored in vitelline cells as part of the requirements for development in the snail (Schüssler) or to stabilise the egg shells (Jones). One would expect or hope to see one ferritin shoot up in its transcriptomic profiles in older worms. Is there any evidence of this occurring. Given the statement above, does the drop in ferritin levels at day 13 seem somewhat strange?
3. I may have missed it, but do the authors have a way of determining the numbers of males and females in each host at different stage? Is the gender balance the same in all mice? Implicit in this question is the concept that males and females will, or be expected to, have distinct transcriptional profiles at all stages. Would a difference in gender balance between mice or between stages skew the transcriptomes? Thus, is there a way of, using either male-only or female-only genes as markers of the numbers of respective gender balance?
Reviewer #2: The research presented here in the manuscript of Wangwiwatsin et al, is experimentally sound and examines difficult to study in vivo lifecycle stages with the power of and quantitative advantages of RNA-seq. Of the work that is presented I have almost no concerns and several interesting findings, especially the CHF and other immune-related findings. My concerns and request for major revision are focused on what is not presented. In particular:
1) The source of the S. mansoni parasites used is not presented.
2) There is no discussion of potential parasite gene expression differences in a mouse vs human host.
3) The references to previous RNA-seq analysis of Schistosoma gene expression including S. mansoni is quite lacking, only much older literature is cited. In fact, this manuscript fails to cite a previous Berriman bioRxiv analysis of existing RNA-seq data doi:
4) Despite the fact that the S. mansoni genome is now on version 8, with version 7 publicly released by the Wellcome Trust for more than a year in WormBase Parasite, the much older assembly version 5 was sued. This seems odd. In fact, the MS has to go to this newer version to highlight a gene of interest that is mis-assembled in version 5.
4) Most significantly, this manuscript only focuses on the data they have generated and fail to include gene expression insights from the rest the lifecycle. It is impossible to declare, as in line 321 "Lung-stage specific" when all stages are not considered. Given that the RNA-seq data for other stages exists and findings from these studies and microarrays exist these new data should be analyzed in that context.
5) Minor comment line 379 "appeared to upregulated" what is the uncertainty?
Reviewer #3: (No Response)
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Dear Dr. Berriman,
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