Transcription profiling of Strongyloides ratti to host immunity
ABSTRACT: Strongyloides ratti is a parasitic nematode of rats and a laboratory model for nematode infection more generally. The response of two lines of S. ratti were compared in contrasting immunological environments: (i) day 5 post infection (p.i.) in naive rats; (ii) day 12 p.i. in naive rats; day 5 p.i. in rats previously immunised with 10 iL3s; and day 12 p.i. in rats previously immunised with 10 iL3s. The gene expression response of parasitic females were assayed using cDNA microarrays. Large numbers of responding genes were found (but with modest fold changes) and clusters of co-expressed genes identified with differences observed between worms taken from naive and previously exposed hosts and from the two time points.
Project description:Strongyloides ratti is a parasitic nematode of rats and a laboratory model for nematode infection more generally. The aim of this experiment was to determine the gene expression response of parasitic females to abiotic factors in its environment ex vivo that may be relevant to its natural environment in the gut in vivo. Thus, we used cDNA arrays to assay transcriptional responses to high and low salt, to RPMI versus PBS media and to 37C versus 40C. A moderate number of gene expression changes were observed.
Project description:In previous studies we have shown that the two adult females morphs of S. ratti have very different lifespans. This experiment was designed to try to identify differentially expressed genes in these two adult morphs that may account for these differing lifespans. The genes expressed by S. ratti parasitic females at day 6 p.i. were compared to the genes expressed by S. ratti free living females at 3 days 19 degrees C. This comparison was done using a microarray chip that is spotted with PCR fragments from the libraries that were generated from parasitic females extracted at day 6 and day 15 p.i., and a microarray chip that is spotted with PCR fragments from the libraries that were generated from free-living larval stages L1, L2 and infective L3s and from free-living males and females.
Project description:This experiment exploits the life-cycle of Strongyloides ratti, which is a parasitic nematode of brown rats that exhibits three adult stages within its life-cycle - parasitic females, freeliving females and free-living males. We use a cDNA microarray to examine patterns of (i) gender-biased gene expression by contrasting free-living females against free-living males, and (ii) parasitic-biased expression by contrasting parasitic females against free-living females. Of the 3688 distinct transcripts represented on our array, 20% exhibited male-biased expression 19% exhibit female-biased expression, 11% exhibit parasitic-biased expression and 8% exhibit free-living-biased expression. Among the top responding genes, an orthologue of major sperm protein is upregulated in males, distinct aspartic protease orthologues are upregulated in either parasitic or in free-living females, and orthologues of hsp-17 chaperone are upregulated in parasitic females. Upon a global analysis of gene expression, we find that female-biased expression is associated with genes involved in reproductive processes and larval development, that male-biased expression is associated with genes involved in metabolism, and that free-living biased expression is associated with genes involved in regulation of body fluids and response to external stimulus. The association of gene ontology with parasite-biased expression is less clear. Our results provide an initial gene expression analysis of gender- and parasite-biased expression in S. ratti, may be more generally applicable to other parasitic nematodes, and may help to refine the search for novel drug or vaccine targets against parasitic nematodes.
Project description:Strongyloides ratti is a parasitic nematode of rats and a laboratory model for nematode infection more generally. Selected lines were generated over the course of 20 - 30 generations such that eggs were harvested either at the beginning or towards the end of an infection, termed 'fast' and 'slow' lines, respectively. Phenotypic differences in these lines in their fecundity and response to host immunity were observed. The gene expression response of these lines in both permissive and restrictive immune environments were assayed using cDNA microarrays. Large numbers of responding genes were found (but with modest fold changes) and clusters of co-expressed genes identified. Genes exhibiting female-biased expression responded to host immunity, consistent with increased investment into transmission in restrictive immune environments.
Project description:We wished to determine the changes in gene expression that occurred in S. ratti parasitic females as an infection progressed and thus as these stages are exposed to an anti-S. ratti immune response. To do this we compared gene expression in parasitic females recovered 6 days p.i. (i.e. no or very low immune response) with those recovered at 15 days p.i. (i.e. high immune response); for convenience, we refer to these as parasitic females subject to "low immune pressure" and "high immune pressure", respectively. These days were chosen because previous analyses of S. ratti parasitic females have shown significant differences in the size, appearance etc. of worms at these time points. The experimental design used, was to have at least three biological replicates for each sample (i.e. three independent preparations of the relevant worm samples and their RNA) and to have at least three technical replicates (i.e. independent, separate cDNA synthesis, amplification and hybridization etc.) for each biological replicate. For each hybridisation (below) a dye-swap was used i.e. each sample to be used in a hybridisation was labelled, separately, with each of the two dyes (below).<br> <br> 21,085 ESTs were sequenced from various S. ratti stage-specific libraries, of which 14,761 resulted in sequence data above a quality threshold that were then submitted to public databases. 11,551 clones were derived from the S. ratti parasitic libraries of which 7,385 produced sequence data (above a quality threshold); all of these 7,385 clones were arrayed together with a random sample of 1,619 of clones for which no sequence data were available. These 7,385 ESTs are highly redundant since they represent 2,963 contigs and 2,125 clusters, both including 1,220 singletons (i.e. clusters or contigs containing only one EST). Notwithstanding this redundancy, they were used in the microarray construction for two reasons: (i) this approach was less error-prone than attempting to select a unique clone set and (ii) this in-built redundancy provides many replicates of individual contigs and clusters, which can be exploited in quality-control analyses. In addition to these 9,004 S. ratti clones, the following controls were included: 281 EST clones from the mixed iL3/free-living adult library (representing 173 contigs and 167 clusters, 67 of which are singletons) to ensure that gene expression in the parasitic and free-living stages could be differentiated; 230 commercially available controls (Amersham Biosciences UK, Ltd.). 12 poly-A and 457 spotting buffer-only controls. Thus, in total 9,984 spots were arrayed.
Project description:SUMMARY Strongyloides venezuelensis is a parasitic nematode that infects rodents. Although Strongyloides species described to date are known to exhibit parthenogenetic reproduction in the parasitic stage of their life cycle and sexual reproduction in the free-living stage, we did not observe any free-living males in S. venezuelensis in our strain, suggesting that the nematode is likely to depend on parthenogenetic reproduction. We confirmed by cytological analysis that S. venezuelensis produces eggs by parthenogenesis during the parasitic stage of its life cycle. Phylogenetic analysis using nearly the full length of 18S and D3 region of 28S ribosomal RNA gene suggested that S. venezuelensis is distantly related to another rodent parasite, namely Strongyloides ratti, but more closely related to a ruminant parasite, Strongyloides papillosus. Karyotype analysis revealed S. venezuelensis reproduces with mitotic parthenogenesis, and has the same number of chromosomes as S. papillosus (2n = 4), but differs from S. ratti (2n = 6) in this regard. These results, taken together, suggest that S. venezuelensis evolved its parasitism for rodents independently from S. ratti and, therefore, is likely to have a different reproductive strategy.
Project description:BACKGROUND: Aspartic proteases are known to play an important role in the biology of nematode parasitism. This role is best characterised in blood-feeding nematodes, where they digest haemoglobin, but they are also likely to play important roles in the biology of nematode parasites that do not feed on blood. In the present work, we investigate the evolution and expression of aspartic proteases in Strongyloides ratti, which permits a unique comparison between parasitic and free-living adult forms within its life-cycle. RESULTS: We identified eight transcribed aspartic protease sequences and a further two genomic sequences and compared these to homologues in Caenorhabditis elegans and other nematode species. Phylogenetic analysis demonstrated a complex pattern of gene evolution, such that some S. ratti sequences had a one-to-one correspondence with orthologues of C. elegans but that lineage-specific expansions have occurred for other aspartic proteases in these two nematodes. These gene duplication events may have contributed to the adaptation of the two species to their different lifestyles. Among the set of S. ratti aspartic proteases were two closely-related isoforms that showed differential expression during different life stages: ASP-2A is highly expressed in parasitic females while ASP-2B is predominantly found in free-living adults. Molecular modelling of the ASP-2 isoforms reveals that their substrate specificities are likely to be very similar, but that ASP-2B is more electrostatically negative over its entire molecular surface than ASP-2A. This characteristic may be related to different pH values of the environments in which these two isoforms operate. CONCLUSIONS: We have demonstrated that S. ratti provides a powerful model to explore the genetic adaptations associated with parasitic versus free-living life-styles. We have discovered gene duplication of aspartic protease genes in Strongyloides and identified a pair of paralogues differentially expressed in either the parasitic or the free-living phase of the nematode life-cycle, consistent with an adaptive role for aspartic proteases in the evolution of nematode parasitism.
Project description:A wide range of biomolecules, including proteins, are excreted and secreted from helminths and contribute to the parasite's successful establishment, survival, and reproduction in an adverse habitat. Excretory and secretory proteins (ESP) are active at the interface between parasite and host and comprise potential targets for intervention. The intestinal nematode Strongyloides spp. exhibits an exceptional developmental plasticity in its life cycle characterized by parasitic and free-living generations. We investigated ESP from infective larvae, parasitic females, and free-living stages of the rat parasite Strongyloides ratti, which is genetically very similar to the human pathogen, Strongyloides stercoralis. Proteomic analysis of ESP revealed 586 proteins, with the largest number of stage-specific ESP found in infective larvae (196), followed by parasitic females (79) and free-living stages (35). One hundred and forty proteins were identified in all studied stages, including anti-oxidative enzymes, heat shock proteins, and carbohydrate-binding proteins. The stage-selective ESP of (1) infective larvae included an astacin metalloproteinase, the L3 Nie antigen, and a fatty acid retinoid-binding protein; (2) parasitic females included a prolyl oligopeptidase (prolyl serine carboxypeptidase), small heat shock proteins, and a secreted acidic protein; (3) free-living stages included a lysozyme family member, a carbohydrate-hydrolyzing enzyme, and saponin-like protein. We verified the differential expression of selected genes encoding ESP by qRT-PCR. ELISA analysis revealed the recognition of ESP by antibodies of S. ratti-infected rats. A prolyl oligopeptidase was identified as abundant parasitic female-specific ESP, and the effect of pyrrolidine-based prolyl oligopeptidase inhibitors showed concentration- and time-dependent inhibitory effects on female motility. The characterization of stage-related ESP from Strongyloides will help to further understand the interaction of this unique intestinal nematode with its host.
Project description:Strongyloides and related genera are advantageous subjects for transgenesis in parasitic nematodes, primarily by gonadal microinjection as has been used with Caenorhabditis elegans. Transgenesis has been achieved in Strongyloides stercoralis and in Parastrongyloides trichosuri, but both of these lack well-adapted, conventional laboratory hosts in which to derive transgenic lines. By contrast, Strongyloides ratti develops in laboratory rats with high efficiency and offers the added advantages of robust genomic and transcriptomic databases and substantial volumes of genetic, developmental and immunological data. Therefore, we evaluated methodology for transgenesis in S. stercoralis as a means of transforming S. ratti. S. stercoralis-based GFP reporter constructs were expressed in a proportion of F1 transgenic S. ratti following gonadal microinjection into parental free-living females. Frequencies of transgene expression in S. ratti, ranged from 3.7% for pAJ09 to 6.8% for pAJ20; respective frequencies for these constructs in S. stercoralis were 5.6% and 33.5%. Anatomical patterns of transgene expression were virtually identical in S. ratti and S. stercoralis. This is the first report of transgenesis in S. ratti, an important model organism for biological investigations of parasitic nematodes. Availability of the rat as a well-adapted laboratory host will facilitate derivation of transgenic lines of this parasite.
Project description:The genus Strongyloides spp. include important human parasites. There is also a well studied rodent model, S. ratti. Uniquely among parasitic nematodes, the Strongyloides life-cycle includes both a parasitic female stage and a genetically identical free-living female stage. Differences between these two female forms must be epigenetic, presumably controlled by altered transcription and translation. This is a project to compare the proteome and transcriptome of the parasitic and free-living females of S. ratti. From this we will define the genes and gene products of the parasitic female stage. This approach exploits the currently advanced S. ratti genome sequencing project. This work will give an understanding of the molecular basis of nematode parasitism, and so define new potential drug targets. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/