The infective schistosome cercaria develops within the intramolluscan daughter sporocyst from an undifferentiated germ ball, during which synthesis of proteins essential for infection occurs. When the aquatic cercaria locates the mammalian host it rapidly penetrates into the epidermis using glandular secretions. It then undergoes metamorphosis into the schistosomulum, including replacement of its tegument surface membranes, a process taking several days before it exits the skin. Patterns of gene expression underlying this transition have been characterised. All gene models from the S. mansoni genome (www. GeneDB. org) were incorporated into a high-density oligonucleotide array. Double-stranded cDNA from germ balls, cercariae, and day 3 schistosomula was hybridised to the array without amplification. Statistical analysis was performed using Bioconductor to reveal differentially transcribed loci. Genes were categorised on the basis of biological process, tissue association or molecular function to aid understanding of the complex processes occurring. Genes necessary for DNA replication were enriched only in the germ ball, while those involved in translation were up-regulated in the germ ball and/or day 3 schistosomulum. Different sets of developmental genes were up-regulated at each stage. A large number of genes encoding elastases and invadolysins, and some venom allergen-like proteins were up-regulated in the germ ball, those encoding cysteine and aspartic proteases in the cercaria and schistosomulum. Micro exon genes encoding variant secreted proteins were highly up-regulated in the schistosomulum along with tegument and gut-associated genes, coincident with remodelling of the parasite body. Genes encoding membrane proteins were prominently up-regulated in the cercaria and/or day 3 schistosomulum. Our study highlights an expanded number of transcripts encoding proteins potentially involved in skin invasion. It illuminates the process of metamorphosis into the schistosomulum and highlights the very early activation of gut-associated genes whilst revealing little change in the parasite' s energy metabolism or stress responses.
Schistosomiasis mansoni remains an important water-borne disease of humans in Sub-Saharan Africa and parts of South America. Transmission between humans and the aquatic intermediate molluscan host is effected by a miracidium. This free-swimming larva hatches from eggs excreted in the faeces and penetrates into the snail. There follows a phase of asexual multiplication within the snail, before the non-feeding infective cercariae emerge. These have a free life of only hours during which they must locate a human host or perish. Infection occurs when a cercaria penetrates through the skin and transforms into the schistosomulum stage. There is then a period of physiological and morphological adaptation to the new environment, lasting several days, before the parasite locates a blood or lymphatic vessel to exit the skin and begin its intravascular migration to the portal system. The biological processes associated with the transition from snail haemolymph via fresh water to mammalian tissues are the key to an understanding of the infection process. Indeed, it could be argued that preventing parasite establishment would provide the optimum control strategy for the disease. The secretions used by the cercaria to enter the skin have been a focus of interest for decades, latterly using biochemical [1] and proteomic techniques [2], [3]. These studies have revealed the importance of serine- and metallo-proteases plus potential immunomodulators released by the parasite to gain entry and establish in the skin. However, the relative paucity of parasite material coupled with the limited sensitivity of proteomic techniques means that we have only a partial picture of one small aspect of the transition from snail to human. For example, virtually nothing is known about processes associated with development of the germ balls that mature into cercariae within the daughter sporocyst, nor is the transformation into the schistosomulum well characterised. The cercarial tegument is known to be shed and replaced by the novel double bilayer structure [4] accompanied by the appearance of glucose transporters on the schistosomulum surface, doubtless to facilitate nutrient uptake [5]. The switch from aerobic to anaerobic metabolism has also been noted [6]. The highly sensitive methods now available to characterise gene expression present an opportunity to gain a deeper understanding of this important transition in the life cycle. Microarrays have become a widely used tool for comparing transcription levels between different biological samples; it is good practice to use at least three biological replicates (i. e. material from separate organisms) to get statistically significant results for each comparison made. A variety of schistosome arrays has previously been designed and used to answer distinct questions about the parasite' s biology. These have included differences in gene transcription between female and male adult worms [7], [8], with laser-capture micro-dissection added to facilitate comparison of gastrodermis and vitellaria [9], and changes in transcription between different life cycle stages [10]–[13]. Germane to our current study, a custom cDNA array, comprising 6000 features from the lung stage larva was used to identify transcripts enriched at the lung stage compared to six other life cycle stages [12]. The same array was also used to characterise differences in transcription pattern between schistosomula transformed from normal and radiation-attenuated cercariae, and cultured in vitro for four, seven and ten days [14]. Other arrays, comprising 12–38,000 synthetic oligonucleotide probes have been used to investigate a range of life cycle stages. These arrays were based on ESTs and contigs available pre-2006 at the DCFI S. mansoni Gene Index (formerly the TIGR Gene Index) [11], [15] or ESTs and contigs available at GenBank and the Wellcome Trust Sanger Institute ftp site in May 2005 [13]. Infected snail hepatopancreas, cercariae and adult worms, have been compared using such oligonucleotide arrays, with uninfected snail tissue as a control for the first of these samples [11]. It was reported that the intramolluscan parasite has high levels of transcripts encoding proteins involved in translation and quality control, cell death and ubiquitination. In the cercaria, highly expressed genes were mainly involved in mitochondrial function, enabling the energy production necessary for swimming. However, it was noted that the cercaria was less transcriptionally active than the other stages studied. Transcription levels at 15 distinct points throughout the parasite life cycle have also been compared [13]. Data analysis focussed on three gene families, fucosyl transferases, tetraspanins, and G protein-coupled receptors (GPCRs) proffered as potential intervention targets. Finally transcription in 3 hr and 5 day schistosomula, cultured in vitro +/− erythrocytes, was compared with cercariae as the baseline [15]. The most apparent changes were the up-regulation of genes involved in blood feeding, tegument and cytoskeleton development, cell adhesion and stress. Although the annotated S. mansoni genome with standardised nomenclature for predicted gene models was published in 2009 [16], the above microarray studies have used other nomenclatures and annotations for their constituent ESTs and contigs. This makes specific inter-study comparisons about changes in the transcription of named genes extremely cumbersome and when attempted, points up considerable discrepancies in annotation. We report here the design and use of the most comprehensive microarray platform for S. mansoni to gain insights into infection of the mammalian host. The array was used to probe transcripts from three life cycle stages, intramolluscan germ balls, free-living cercariae, and ‘skin’ schistosomula. We use the term ‘germ ball’ to encompass all stages of embryonic development up to, but excluding, the mature cercaria that comprises approximately 1000 cells [17], differentiated into tissues and organs. The germ balls were essential as it has been shown that many proteins used by the cercaria for host entry are transcribed and translated during its development in the snail [2], [18]. They were obtained by microdissection of snails 22–26 days post-infection. Day 3 schistsosomula were chosen because by that point, metamorphosis from the cercaria is nearing completion, they have adapted to life in mammalian tissues and are ready to begin intravascular migration. Such schistosomula, transformed and cultured by the methods we used, are able to mature if transferred into the murine host [19]. They are biologically comparable to ex vivo worms and can be produced in large quantities. Sufficient RNA was obtained from all three life cycle stages for hybridisation to the array without PCR amplification. A comprehensive analysis of greater than two-fold changes in transcription between the life cycle stages is presented.
The procedures involving animals were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986, as authorised on personal and project licences issued by the UK Home Office. The study protocol was approved by the Biology Department Ethical Review Committee at the University of York. All parasite material was from a Puerto Rican isolate of S. mansoni maintained at the University of York by passage through NMRI strain mice and albino Biomphalaria glabrata snails. Developing germ balls from daughter sporocysts were obtained from snails infected with 40 miracidia each and dissected carefully in filter-sterilised 50% PBS (pH 7. 4.) 22–26 days later, before cercarial maturity. Obvious snail material was removed and freed germ balls at all stages of development were accumulated on ice until use. Cercariae were collected from snails infected with 10 miracidia each. Five weeks after infection the snails were placed in the dark for two days and then illuminated in approximately 10 mls aerated tap water for two hours to induce shedding. The emerging cercariae were gravity-concentrated by cooling on ice for one hour, which prevented swimming. Skin stage schistosomula were obtained by mechanical transformation of cercariae and separation of their bodies which were cultured for three days in vitro as previously described [19]. They were then recovered and washed twice in RPMI before processing [19]. RNA was extracted from the three larval stages by homogenisation in TRIzol (Invitrogen, Paisley, UK) at approximately 1 ml per 100 µl tissue. The RNA was extracted as per the manufacturer' s instructions, with the addition of DEPC-treated high salt solution (0. 8 M sodium citrate and 1. 2 M NaCl) at the isopropanol step, to remove glycoprotein. RNA was isopropanol-precipitated overnight at −80°C with 1 µl Glycoblue (15 mg/ml; Ambion) to aid the process, and visualisation of the pellet. It was recovered by centrifugation at 12,000× g for 30 minutes at 4°C. The pellets were washed with 70% ethanol, and allowed to air-dry at room temperature before being resuspended in 300 µl DEPC-treated water. RNA was quantified using a Nanodrop ND-1000 Spectophotometer (Nanodrop Products Fisher, Wilmington, Delawere, USA) and quality assessed using a 2100 Bioanalyzer PicoChip (Agilent, Wokingham, UK). The predicted genes from version D of the S. mansoni genome assembly as of June 2008 (www. GeneDB. org) formed the input for the array design along with all S. mansoni ESTs available at GeneDB. org whose direction was known, compiled using phrap (http: //www. phrap. org/phredphrapconsed. html). The input data were broken up into sequential 50mers offset by one base each time, and redundant sequences were removed using FAlite. pm (Ian Korf; http: //homepage. mac. com/iankorf/) and associated Perl scripts. The unique sequences were mapped back to the genome assembly using ‘exonerate’ (http: //www. ebi. ac. uk/~guy/exonerate/). From a map-ordered list, every 13th 50mer was chosen as a probe. No selection was made for the number of probes per predicted transcript. The design was sent to Roche-NimbleGen, who made some minor refinements for ease of synthesis and constructed the arrays using digital micromirror technology [20]. There were 385K features on the array comprising 377,598 S. mansoni sequences and 11,613 random sequences for hybridisation controls. Double stranded cDNA for hybridisation was synthesised from total RNA using SuperScript Double-Stranded cDNA synthesis kits (Invitrogen) according to the protocol supplied by Roche-NimbleGen. The resulting cDNA was pooled such that separate biological replicates were obtained i. e. no parasite homogenates were split across replicates. Roche-NimbleGen were supplied with at least 2. 7 µg of double stranded cDNA for three biological replicates each from germ balls, cercariae, and day 3 schistosomula to perform the hybridisations. Each biological replicate of cDNA was labelled with Cy3 and hybridised to the array for 16 to 20 hours at 42°C. Slides were washed, and dried before fluorescence data were read using a Roche-NimbleGen MS 2000 Scanner with NimbleScan software. Roche-NimbleGen supplied background-corrected data. All subsequent statistical analysis was carried out using programmes from the Bioconductor suite [21]. The data were quality-assessed by visual inspection of graphical representations of the raw probe level data. Box plots were drawn using the boxplot function from the graphics package. Correlation data were calculated using the cor function from the stats package and heatmaps were made by calling the heatmap. 2 function in the gplots package. All arrays passed the quality assessment. Next, the data were quantile-normalised using the normalizeBetweenArrays function in the limma package. This ensures identical distributions of the data [22]. Following normalisation, the probe level data were summarised to yield ‘gene level’ data. The probes were re-mapped to the S. mansoni gene predictions at www. geneDB. org (version F) using ‘exonerate’. If a probe matched an S. mansoni predicted gene (Smp) locus with an e value <1−05 by both nucleotide and protein BLAST, the probe was annotated to that Smp locus. The intensity value for each locus was the mean intensity of all the probes by which it was represented. The resulting gene level data were the input for the differential expression analysis, which was carried out using the limma package. First a linear model fit was performed. This reduced the data for each gene to a mean value from each of the life cycle stages. Next, differential expression data were obtained by performing a contrast analysis. This compares the transcription level of each gene in the following contrasts: Multiple testing was corrected for using the eBayes function which employs the method of Benjamini and Hochberg [23]. This gives an adjusted P value (adjP). Genes which were differentially expressed above a two fold cut-off between any two stages with an B>3 were chosen for further analysis. The B value is the log (odds that a gene is differentially expressed). For example if B = 3, the odds that a gene is expressed is e3 = 20, or 1 in 20, corresponding to a probability of 95%. Log2 quantile-normalised probe level data from the array are deposited at the public database Gene Expression Omnibus (http: //www. ncbi. nlm. nih. gov/geo) under accession numbers GSE22037 and GPL10466. For ease of visualisation, statistically significant expression values were converted to relative fold change, with the stage having the lowest expression for each gene set to unity. This facilitates analysis of changes in gene expression associated with the infection process. The actual baseline expression values may be different for each gene, and comparisons of absolute expression levels between genes, based on the charts and tables shown here, are not valid. However, the patterns of gene expression may be compared between genes. Where a data bar is missing for a particular life cycle stage, this is because either no statistically significant gene expression was observed for that life cycle stage, or there was a less than two-fold difference. Finally a hypergeometric test was carried out using the Category package to discover whether particular GO terms were over-represented in the differentially expressed genes compared to the ‘gene universe’ of Smp gene models on the array. As an aid to understanding biological processes associated with the transition of the parasite from the snail, via fresh water to the mammalian host tissues, we grouped the S. mansoni genes into useful categories. Some of these (DNA replication, translation, energy metabolism, lipid metabolism, and membrane) were based purely on the classification of genes by Gene Ontology (http: //www. geneontology. org/). In creating a muscle tissue category, we used the GO term ‘actin binding’. Two of the tissue categories, ‘tegument’ and ‘alimentary tract’ were created using our proteomic analyses [24]–[28]. In the case of ‘alimentary tract’, as four proteins containing saposin domains had already been identified in worm vomitus, we searched the genome database for other genes encoding saposins and added them to this category. The custom category ‘defence against stress’ was compiled from the literature on schistosomes [29]–[31]. The VAL and MEG categories were based on recently published compilations [32], [33]. Genes encoding proteins involved in lipid synthesis were culled from supplementary table 11 of the genome paper [16]. The lists of proteases classified by catalytic type (http: //merops. sanger. ac. uk/) were abstracted from the S. mansoni genome supplementary table 18 [16]. The ‘development’ category comprised the neural development and TGFβ signalling genes highlighted in the genome paper (supplementary tables 9 and 15, respectively [16]) along with those genes annotated to the GO term ‘Wnt receptor signaling pathway’ while the protein glycosylation category was created by assiduous interrogation of the S. mansoni genome database. RNA from each of the biological replicates was reserved for qPCR to validate the microarray results. Total RNA (0. 413 µg) from each sample was reverse-transcribed using Superscript II (Invitrogen) and primed with oligo-dT according to the manufacturer' s instructions, in a reaction volume of 20 µl. After the reaction, the volume was made up to 100 µl with DEPC-treated water. Relative quantitation was carried out using SYBR green PCR Master Mix (Applied Biosystems) with 1 µl of cDNA per 25 µl reaction. The PCR was carried out using an ABI7300 (Applied Biosystems) according to the manufacturer' s instructions with 18S ribosomal RNA [14] as the endogenous control, run for each sample on each plate. Genes chosen and their primer pairs are detailed in Table S1, with primers designed using PrimerExpress (Applied Biosystems). There were three technical replicates per biological replicate for each gene. Relative quantification was calculated by the ΔΔCT method using Applied Biosystems Sequence Detection Software version 1. 2. 3 7000 system.
This term encompasses the basic functions that might be expected to occur in the ‘average’ schistosome cell. This category encompasses the mechanisms involved in the development and differentiation of tissues and the transcription of genes linked to three specific tissues, namely muscle, gut and tegument. This category encompasses groups of genes either with a similar organization (the MEGs) or those that encode proteins with a related molecular structure or function. In this paper we have described in detail the changes in gene transcription throughout the mammalian infection process. Evidence for DNA replication and cell division was only seen in the germ ball, while each stage up-regulated different genes involved in development and morphogenesis, including cell-cell adhesion. Thus, neural patterning genes were enriched in the germ ball, while there was considerable activation of genes involved in nerve function in the schistosomulum, coincident with body remodelling. Forward planning was seen throughout, with genes encoding skin penetration enzymes transcribed in the germ ball, and those involved in transformation, gut activity and tegument replacement beginning their up-regulation in the cercaria. Our data suggest there are vastly more proteases involved in skin penetration than hitherto envisaged. Likewise the number of possible gut proteases and transporters is expanded. The up-regulation of many receptors in the schistosomulum raises the thorny issue of whether they interact with host or endogenous ligands. Surprisingly, few stress-related genes were up-regulated in the schistosomulum and, apart from the cercarial enrichment of genes encoding proteins involved in aerobic respiration, there was little change in energy metabolism during the transitions studied. The inscrutable VALs are deployed early in infection, whilst the majority of the MEGS, also of unknown function, are transcribed later with extremely high fold changes. Many of the genes identified throughout our study warrant detailed investigation.
The schistosome cercaria develops from undifferentiated germ balls within the daughter sporocyst located in the hepatopancreas of its snail intermediate host. This is where the proteins it uses to infect humans are synthesised. After a brief free life in fresh water, if the cercaria locates a host, it infects by direct penetration through the skin. It then transforms into the schistosomulum stage, adapted for life in human tissues. We have designed a large scale array comprising probes representing all known schistosome genes and used it in hybridisation experiments to establish which genes are turned on or off in the parasite during these stages in its life cycle. Genes encoding proteins involved in cell division were prominent in the germ ball along with those for proteases and potential immunomodulators, deployed during skin penetration. The non-feeding cercaria was the least active at synthesising proteins. Conversion to the schistosomulum was accompanied by transcription of genes involved in body remodeling, including production of a new outer surface, and gut activation long before ingestion of red blood cells begins. Our data help us to understand better the proteins deployed to achieve infection, and subsequent adaptations necessary for establishment of the parasite in the human host.
medicine
infectious diseases
schistosomiasis
functional genomics
gene expression
genetics
biology
genomics
parasitic diseases
genetics and genomics
dna transcription
Gene Expression Patterns in Larval Schistosoma mansoni Associated with Infection of the Mammalian Host