RNAi-mediated knockdown of daf-12 in the model parasitic nematode Strongyloides ratti.

The gene daf-12 has long shown to be involved in the dauer pathway in Caenorhabditis elegans (C. elegans). Due to the similarities of the dauer larvae of C. elegans and infective larvae of certain parasitic nematodes such as Strongyloides spp., this gene has also been suspected to be involved in the development of infective larvae. Previous research has shown that the application of dafachronic acid, the steroid hormone ligand of DAF-12 in C. elegans, affects the development of infective larvae and metabolism in Strongyloides. However, a lack of tools for either forward or reverse genetics within Strongyloides has limited studies of gene function within these important parasites. After determining whether Strongyloides had the requisite proteins for RNAi, we developed and report here the first successful RNAi by soaking protocol for Strongyloides ratti (S. ratti) and use this protocol to study the functions of daf-12 within S. ratti. Suppression of daf-12 in S. ratti severely impairs the formation of infective larvae of the direct cycle and redirects development towards the non-infective (non-dauer) free-living life cycle. Further, daf-12(RNAi) S. ratti produce slightly but significantly fewer offspring and these offspring are developmentally delayed or incapable of completing their development to infective larvae (L3i). Whilst the successful daf-12(RNAi) L3i are still able to infect a new host, the resulting infection is less productive and shorter lived. Further, daf-12 knockdown affects metabolism in S. ratti resulting in a shift from aerobic towards anaerobic fat metabolism. Finally, daf-12(RNAi) S. ratti have reduced tolerance of temperature stress.

Comparative characterization of two galectins excreted-secreted from intestine-dwelling parasitic versus free-living females of the soil-transmitted nematode Strongyloides.

Helminths are complex pathogens that ensure their long-term survival by influencing the immune responses of their host. Excretory/secretory products (ESP) can exert immunoregulatory effects which foster parasite survival. Galectins represent a widespread group of ß-galactoside-binding proteins which are involved in a multitude of biological processes operative in parasite-host interaction. We had earlier identified seven galectins in Strongyloides ratti, four of them detected in the ESP of distinct developmental stages of the parasite. In the present report, we focused on the characterization of two of them, Sr-galectin-1 (Sr-Gal-1) and Sr-galectin-3 (Sr-Gal-3). While Sr-Gal-3 expression was strongest in parasitic females, Sr-Gal-1 was predominantly expressed in free-living females. Both proteins were cloned and recombinantly expressed in an E. coli expression system. Their glycan-binding activity was verified by haemagglutination and glycan array analysis. Furthermore, primary immunological activities of the Sr-galectins were initially investigated by the application of an in vitro mucosal 3D-culture model, comprising of mucosa-associated epithelial and dendritic cells. The Sr-galectins stimulated preferentially the release of the type 2 cytokines thymic stromal lymphopoietin and IL-22, a first indication for immunoregulatory activity. In addition, the Sr-galectins dose-dependently fostered cell migration. Our results confirm the importance of these carbohydrate-binding proteins in host-parasite-interaction by indicating possible interaction with the host mucosa-associated cells.

Transposon-mediated chromosomal integration of transgenes in the parasitic nematode Strongyloides ratti and establishment of stable transgenic lines.

Genetic transformation is a potential tool for analyzing gene function and thereby identifying new drug and vaccine targets in parasitic nematodes, which adversely affect more than one billion people. We have previously developed a robust system for transgenesis in Strongyloides spp. using gonadal microinjection for gene transfer. In this system, transgenes are expressed in promoter-regulated fashion in the F1 but are silenced in subsequent generations, presumably because of their location in repetitive episomal arrays. To counteract this silencing, we explored transposon-mediated chromosomal integration of transgenes in S. ratti. To this end, we constructed a donor vector encoding green fluorescent protein (GFP) under the control of the Ss-act-2 promoter with flanking inverted tandem repeats specific for the piggyBac transposon. In three experiments, free-living Strongyloides ratti females were transformed with this donor vector and a helper plasmid encoding the piggyBac transposase. A mean of 7.9% of F1 larvae were GFP-positive. We inoculated rats with GFP-positive F1 infective larvae, and 0.5% of 6014 F2 individuals resulting from this host passage were GFP-positive. We cultured GFP-positive F2 individuals to produce GFP-positive F3 L3i for additional rounds of host and culture passage. Mean GFP expression frequencies in subsequent generations were 15.6% in the F3, 99.0% in the F4, 82.4% in the F5 and 98.7% in the F6. The resulting transgenic lines now have virtually uniform GFP expression among all progeny after at least 10 generations of passage. Chromosomal integration of the reporter transgenes was confirmed by Southern blotting and splinkerette PCR, which revealed the transgene flanked by S. ratti genomic sequences corresponding to five discrete integration sites. BLAST searches of flanking sequences against the S. ratti genome revealed integrations in five contigs. This result provides the basis for two powerful functional genomic tools in S. ratti: heritable transgenesis and insertional mutagenesis.

A pharmacological study of NLP-12 neuropeptide signaling in free-living and parasitic nematodes.

NLP-12a and b have been identified as cholecystokinin/sulfakinin-like neuropeptides in the free-living nematode Caenorhabditis elegans. They are suggested to play an important role in the regulation of digestive enzyme secretion and fat storage. This study reports on the identification and characterization of an NLP-12-like peptide precursor gene in the rat parasitic nematode Strongyloides ratti. The S. ratti NLP-12 peptides are able to activate both C. elegans CKR-2 receptor isoforms in a dose-dependent way with affinities in the same nanomolar range as the native C. elegans NLP-12 peptides. The C-terminal RPLQFamide sequence motif of the NLP-12 peptides is perfectly conserved between free-living and parasitic nematodes. Based on systemic amino acid replacements the Arg-, Leu- and Phe- residues appear to be critical for high-affinity receptor binding. Finally, a SAR analysis revealed the essential pharmacophore in C. elegans NLP-12b to be the pentapeptide RPLQFamide.

Transgenesis in the parasitic nematode Strongyloides ratti.

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.

In silico secretome analysis approach for next generation sequencing transcriptomic data.

BACKGROUND: Excretory/secretory proteins (ESPs) play a major role in parasitic infection as they are present at the host-parasite interface and regulate host immune system. In case of parasitic helminths, transcriptomics has been used extensively to understand the molecular basis of parasitism and for developing novel therapeutic strategies against parasitic infections. However, none of transcriptomic studies have extensively covered ES protein prediction for identifying novel therapeutic targets, especially as parasites adopt non-classical secretion pathways. RESULTS: We developed a semi-automated computational approach for prediction and annotation of ES proteins using transcriptomic data from next generation sequencing platforms. For the prediction of non-classically secreted proteins, we have used an improved computational strategy, together with homology matching to a dataset of experimentally determined parasitic helminth ES proteins. We applied this protocol to analyse 454 short reads of parasitic nematode, Strongyloides ratti. From 296231 reads, we derived 28901 contigs, which were translated into 20877 proteins. Based on our improved ES protein prediction pipeline, we identified 2572 ES proteins, of which 407 (1.9%) proteins have classical N-terminal signal peptides, 923 (4.4%) were computationally identified as non-classically secreted while 1516 (7.26%) were identified by homology to experimentally identified parasitic helminth ES proteins. Out of 2572 ES proteins, 2310 (89.8%) ES proteins had homologues in the free-living nematode Caenorhabditis elegans and 2220 (86.3%) in parasitic nematodes. We could functionally annotate 1591 (61.8%) ES proteins with protein families and domains and establish pathway associations for 691 (26.8%) proteins. In addition, we have identified 19 representative ES proteins, which have no homologues in the host organism but homologous to lethal RNAi phenotypes in C. elegans, as potential therapeutic targets. CONCLUSION: We report a comprehensive approach using freely available computational tools for the secretome analysis of NGS data. This approach has been applied to S. ratti 454 transcriptomic data for in silico excretory/secretory proteins prediction and analysis, providing a foundation for developing new therapeutic solutions for parasitic infections.

Genes important in the parasitic life of the nematode Strongyloides ratti.

Parasitic nematodes are important pathogens of humans and other animals. The genus Strongyloides has both a parasitic and a free-living adult generation. S. ratti infections of its rat host are negatively affected by the host immune response, such that a month after infection, worms are lost from the hosts. Here we have investigated the changes in parasite gene expression that occur as the anti-S. ratti immune pressure increases. Existing S. ratti expressed sequence tags were used to construct a microarray consisting of 2227 putative genes. This was probed with cDNA prepared from parasites subject to low or high immune pressures. There are significant changes in the gene expression of S. ratti when subject to different immune pressures. Most of the genes whose expression changes have no significant alignment to known genes. These data together with previous S. ratti EST data were then used to identify genes that we hypothesise are central to the parasitic life of S. ratti and, perhaps, other parasitic nematodes. These analyses have identified genes likely to play a key role in the parasitic life of S. ratti; these genes should be the priority for further investigation.

daf-7 and the development of Strongyloides ratti and Parastrongyloides trichosuri.

daf-7 is a key ligand in one of the three pathways that control dauer larva development in Caenorhabditis elegans. Given the similarities between dauer larvae of free-living nematodes and third stage infective larvae of animal parasitic nematodes, we hypothesised that daf-7 may be involved in the development of these infective larvae. To investigate this, we cloned daf-7 orthologues from Strongyloides ratti and Parastrongyloides trichosuri and analysed their RNA level by semi-quantitative RT-PCR during the S. ratti and P. trichosuri life cycles and in a range of in vitro and in vivo conditions. We found that, in both species, the RNA level of daf-7 was low in free-living stages but peaked in the infective L3 (iL3) stage with little or no expression in the parasitic stages. This contrasts with the daf-7 RNA level in C. elegans, which peaks in L1, decreases thereafter, and is absent in dauer larvae. The RNA level of daf-7 in infective larvae was reduced by larval penetration of host skin or development in the host, but not by a shift to the body temperature of the host.

Steady-state content of glycolytic/tricarboxylic acid-cycle intermediates, adenine nucleotide pools and the cellular redox-status in the infective (L3) larvae of (homogonic) Strongyloides ratti.

Infective (L3) larvae of Strongyloides ratti (homogonic strain) were freeze-clamped (-196 degrees C) and the steady-state content of the glycolytic, Krebs tricarboxylic acid (KTA)-cycle intermediates and adenine nucleotides analysed. Comparison of the mass-action ratios (MARs) of the glycolytic enzymes with their apparent equilibrium constants (K9eq) indicate that phosphoglucomutase, glucosephosphate isomerase, triosephosphate isomerase, phosphoglyceromutase and phosphopyruvate hydratase reactions were all at or near equilibrium, whilst hexokinase, phosphofructokinase and pyruvate kinase were displaced from equilibrium. The S. ratti aldolase and myokinase appear to be somewhat displaced from equilibrium and thus may have pseudoregulatory roles. The adenylate energy charge (AEC), ATP/ADP ratio and the available adenylate energy (AAE) indices were 0.9 +/- 0.04, 8.76 +/- 1.5 and 397 +/- 43, respectively. The free [NAD+]/[NADH+H+] ratio of the cytoplasmic compartment of S. ratti L3 larvae calculated employing the steady-state content of the oxidised and reduced substrates of lactate dehydrogenase (E.C. 1.1.1.27) and the combined glyceraldehyde 3-phosphate dehydrogenase (E.C. 1.2.1.12)/3-phosphoglycerate kinase (E.C. 2.7.2.3) system were ca. 523 and 1200, respectively. The free[NAD+]/[NADH+H+] ratio in the mitochondrial compartment of S. ratti L3 larvae calculated using the malate dehydrogenase (E.C. 1.1.1.37) equilibrium was found to be 1962:1. The data is discussed with respect to the predominantly aerobic nature of the energy metabolism of the L3 larvae.