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Transposon-mediated Chromosomal Integration of Transgenes in the Parasitic Nematode Strongyloides ratti and Establishment of Stable Transgenic Lines  [PDF]
Hongguang Shao equal contributor,Xinshe Li equal contributor,Thomas J. Nolan,Holman C. Massey Jr.,Edward J. Pearce,James B. Lok
PLOS Pathogens , 2012, DOI: 10.1371/journal.ppat.1002871
Abstract: 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.
Identification of novel aspartic proteases from Strongyloides ratti and characterisation of their evolutionary relationships, stage-specific expression and molecular structure
Luciane V Mello, Helen O'Meara, Daniel J Rigden, Steve Paterson
BMC Genomics , 2009, DOI: 10.1186/1471-2164-10-611
Abstract: 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.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.Adaptation of nematode taxa to a parasitic life-style represents an evolutionary challenge that is likely to be met, in part, by gene duplication in a gene family and subsequent acquisition of novel gene function among its paralogous members. One group of genes that has received considerable interest in this respect are aspartic proteases. Aspartic proteases are defined by h
Identification of a Bacteria-Like Ferrochelatase in Strongyloides venezuelensis, an Animal Parasitic Nematode  [PDF]
Eiji Nagayasu, Sohta A. Ishikawa, Shigeru Taketani, Gunimala Chakraborty, Ayako Yoshida, Yuji Inagaki, Haruhiko Maruyama
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0058458
Abstract: Heme is an essential molecule for vast majority of organisms serving as a prosthetic group for various hemoproteins. Although most organisms synthesize heme from 5-aminolevulinic acid through a conserved heme biosynthetic pathway composed of seven consecutive enzymatic reactions, nematodes are known to be natural heme auxotrophs. The completely sequenced Caenorhabditis elegans genome, for example, lacks all seven genes for heme biosynthesis. However, genome/transcriptome sequencing of Strongyloides venezuelensis, an important model nematode species for studying human strongyloidiasis, indicated the presence of a gene for ferrochelatase (FeCH), which catalyzes the terminal step of heme biosynthesis, whereas the other six heme biosynthesis genes are apparently missing. Phylogenetic analyses indicated that nematode FeCH genes, including that of S. venezuelensis (SvFeCH) have a fundamentally different evolutionally origin from the FeCH genes of non-nematode metazoa. Although all non-nematode metazoan FeCH genes appear to be inherited vertically from an ancestral opisthokont, nematode FeCH may have been acquired from an alpha-proteobacterium, horizontally. The identified SvFeCH sequence was found to function as FeCH as expected based on both in vitro chelatase assays using recombinant SvFeCH and in vivo complementation experiments using an FeCH-deficient strain of Escherichia coli. Messenger RNA expression levels during the S. venezuelensis lifecycle were examined by real-time RT-PCR. SvFeCH mRNA was expressed at all the stages examined with a marked reduction at the infective third-stage larvae. Our study demonstrates the presence of a bacteria-like FeCH gene in the S. venezuelensis genome. It appeared that S. venezuelensis and some other animal parasitic nematodes reacquired the once-lost FeCH gene. Although the underlying evolutionary pressures that necessitated this reacquisition remain to be investigated, it is interesting that the presence of FeCH genes in the absence of other heme biosynthesis genes has been reported only for animal pathogens, and this finding may be related to nutritional availability in animal hosts.
RNAseq Analysis of the Parasitic Nematode Strongyloides stercoralis Reveals Divergent Regulation of Canonical Dauer Pathways  [PDF]
Jonathan D. Stoltzfus,Samuel Minot,Matthew Berriman,Thomas J. Nolan,James B. Lok
PLOS Neglected Tropical Diseases , 2012, DOI: 10.1371/journal.pntd.0001854
Abstract: The infectious form of many parasitic nematodes, which afflict over one billion people globally, is a developmentally arrested third-stage larva (L3i). The parasitic nematode Strongyloides stercoralis differs from other nematode species that infect humans, in that its life cycle includes both parasitic and free-living forms, which can be leveraged to investigate the mechanisms of L3i arrest and activation. The free-living nematode Caenorhabditis elegans has a similar developmentally arrested larval form, the dauer, whose formation is controlled by four pathways: cyclic GMP (cGMP) signaling, insulin/IGF-1-like signaling (IIS), transforming growth factor β (TGFβ) signaling, and biosynthesis of dafachronic acid (DA) ligands that regulate a nuclear hormone receptor. We hypothesized that homologous pathways are present in S. stercoralis, have similar developmental regulation, and are involved in L3i arrest and activation. To test this, we undertook a deep-sequencing study of the polyadenylated transcriptome, generating over 2.3 billion paired-end reads from seven developmental stages. We constructed developmental expression profiles for S. stercoralis homologs of C. elegans dauer genes identified by BLAST searches of the S. stercoralis genome as well as de novo assembled transcripts. Intriguingly, genes encoding cGMP pathway components were coordinately up-regulated in L3i. In comparison to C. elegans, S. stercoralis has a paucity of genes encoding IIS ligands, several of which have abundance profiles suggesting involvement in L3i development. We also identified seven S. stercoralis genes encoding homologs of the single C. elegans dauer regulatory TGFβ ligand, three of which are only expressed in L3i. Putative DA biosynthetic genes did not appear to be coordinately regulated in L3i development. Our data suggest that while dauer pathway genes are present in S. stercoralis and may play a role in L3i development, there are significant differences between the two species. Understanding the mechanisms governing L3i development may lead to novel treatment and control strategies.
Immunological Responses Elicited by Different Infection Regimes with Strongyloides ratti  [PDF]
Steve Paterson, Clare Wilkes, Colin Bleay, Mark E. Viney
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0002509
Abstract: Nematode infections are a ubiquitous feature of vertebrate life. In nature, such nematode infections are acquired by continued exposure to infective stages over a prolonged period of time. By contrast, experimental laboratory infections are typically induced by the administration of a single (and often large) dose of infective stages. Previous work has shown that the size of an infection dose can have significant effects on anti-nematode immune responses. Here we investigated the effect of different infection regimes of Strongyloides ratti, comparing single and repeated dose infections, on the host immune response that was elicited. We considered and compared infections of the same size, but administered in different ways. We considered infection size in two ways: the maximum dose of worms administered and the cumulative worm exposure time. We found that both infection regimes resulted in Th2-type immune response, characterised by IL4 and IL13 produced by S. ratti stimulated mesenteric lymph node cells, anti-S. ratti IgG1 and intestinal rat mast cell protease II (RMCPII) production. We observed some small quantitative immunological differences between different infection regimes, in which the concentration of IL4, IL13, anti-S. ratti IgG1 and IgG2a and RMCPII were affected. However, these differences were quantitatively relatively modest compared with the temporal dynamics of the anti-S. ratti immune response as a whole.
Strongyloides stercoralis age-1: A Potential Regulator of Infective Larval Development in a Parasitic Nematode  [PDF]
Jonathan D. Stoltzfus, Holman C. Massey, Thomas J. Nolan, Sandra D. Griffith, James B. Lok
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0038587
Abstract: Infective third-stage larvae (L3i) of the human parasite Strongyloides stercoralis share many morphological, developmental, and behavioral attributes with Caenorhabditis elegans dauer larvae. The ‘dauer hypothesis’ predicts that the same molecular genetic mechanisms control both dauer larval development in C. elegans and L3i morphogenesis in S. stercoralis. In C. elegans, the phosphatidylinositol-3 (PI3) kinase catalytic subunit AGE-1 functions in the insulin/IGF-1 signaling (IIS) pathway to regulate formation of dauer larvae. Here we identify and characterize Ss-age-1, the S. stercoralis homolog of the gene encoding C. elegans AGE-1. Our analysis of the Ss-age-1 genomic region revealed three exons encoding a predicted protein of 1,209 amino acids, which clustered with C. elegans AGE-1 in phylogenetic analysis. We examined temporal patterns of expression in the S. stercoralis life cycle by reverse transcription quantitative PCR and observed low levels of Ss-age-1 transcripts in all stages. To compare anatomical patterns of expression between the two species, we used Ss-age-1 or Ce-age-1 promoter::enhanced green fluorescent protein reporter constructs expressed in transgenic animals for each species. We observed conservation of expression in amphidial neurons, which play a critical role in developmental regulation of both dauer larvae and L3i. Application of the PI3 kinase inhibitor LY294002 suppressed L3i in vitro activation in a dose-dependent fashion, with 100 μM resulting in a 90% decrease (odds ratio: 0.10, 95% confidence interval: 0.08–0.13) in the odds of resumption of feeding for treated L3i in comparison to the control. Together, these data support the hypothesis that Ss-age-1 regulates the development of S. stercoralis L3i via an IIS pathway in a manner similar to that observed in C. elegans dauer larvae. Understanding the mechanisms by which infective larvae are formed and activated may lead to novel control measures and treatments for strongyloidiasis and other soil-transmitted helminthiases.
Strongyloides ratti: In Vitro and In Vivo Activity of Tribendimidine  [PDF]
Jennifer Keiser ,Kai Thiemann,Yvette Endriss,Jürg Utzinger
PLOS Neglected Tropical Diseases , 2008, DOI: 10.1371/journal.pntd.0000136
Abstract: Background Strongyloidiasis is a truly neglected tropical disease, but its public health significance is far from being negligible. At present, only a few drugs are available for the treatment and control of strongyloidiasis. Methodology/Principal Findings We investigated the activity of tribendimidine against third-stage larvae (L3) of Strongyloides ratti in vitro and against juvenile and adult stages of the parasite in vivo. S. ratti larvae incubated in PBS buffer containing 10–100 μg/ml tribendimidine died within 24 hours. A single 50 mg/kg oral dose of tribendimidine administered to rats infected with 1-day-old S. ratti showed no effect. The same dose administered to rats harboring a 2-day-old infection showed a moderate reduction of the intestinal parasite load. Three days post-exposure a significant reduction of the immature worm burden was found. Administration of tribendimidine at doses of 50 mg/kg and above to rats harboring mature S. ratti resulted in a complete elimination of the larval and adult worm burden. For comparison, we also administered ivermectin at a single 0.5 mg/kg oral dose to rats infected with adult S. ratti and found a 90% reduction of larvae and a 100% reduction of adult worms. Conclusion/Significance Tribendimidine exhibits activity against S. ratti in vitro and in vivo. The effect of tribendimidine in humans infected with S. stercoralis should be assessed.
Microarray-Based Analysis of Differential Gene Expression between Infective and Noninfective Larvae of Strongyloides stercoralis  [PDF]
Roshan Ramanathan ,Sudhir Varma,José M. C. Ribeiro,Timothy G. Myers,Thomas J. Nolan,David Abraham,James B. Lok,Thomas B. Nutman
PLOS Neglected Tropical Diseases , 2011, DOI: 10.1371/journal.pntd.0001039
Abstract: Background Differences between noninfective first-stage (L1) and infective third-stage (L3i) larvae of parasitic nematode Strongyloides stercoralis at the molecular level are relatively uncharacterized. DNA microarrays were developed and utilized for this purpose. Methods and Findings Oligonucleotide hybridization probes for the array were designed to bind 3,571 putative mRNA transcripts predicted by analysis of 11,335 expressed sequence tags (ESTs) obtained as part of the Nematode EST project. RNA obtained from S. stercoralis L3i and L1 was co-hybridized to each array after labeling the individual samples with different fluorescent tags. Bioinformatic predictions of gene function were developed using a novel cDNA Annotation System software. We identified 935 differentially expressed genes (469 L3i-biased; 466 L1-biased) having two-fold expression differences or greater and microarray signals with a p value<0.01. Based on a functional analysis, L1 larvae have a larger number of genes putatively involved in transcription (p = 0.004), and L3i larvae have biased expression of putative heat shock proteins (such as hsp-90). Genes with products known to be immunoreactive in S. stercoralis-infected humans (such as SsIR and NIE) had L3i biased expression. Abundantly expressed L3i contigs of interest included S. stercoralis orthologs of cytochrome oxidase ucr 2.1 and hsp-90, which may be potential chemotherapeutic targets. The S. stercoralis ortholog of fatty acid and retinol binding protein-1, successfully used in a vaccine against Ancylostoma ceylanicum, was identified among the 25 most highly expressed L3i genes. The sperm-containing glycoprotein domain, utilized in a vaccine against the nematode Cooperia punctata, was exclusively found in L3i biased genes and may be a valuable S. stercoralis target of interest. Conclusions A new DNA microarray tool for the examination of S. stercoralis biology has been developed and provides new and valuable insights regarding differences between infective and noninfective S. stercoralis larvae. Potential therapeutic and vaccine targets were identified for further study.
Cryo-Microtome sections of coproculture larvae of Strongyloides stercoralis and Strongyloides ratti as antigen sources for the immunodiagnosis of human strongyloidiasis
COSTA-CRUZ, Julia Maria;BULLAMAH, Carina Barbosa;GON?ALVES-PIRES, Maria do Rosário F.;CAMPOS, Dulcinéa Maria B.;VIEIRA, Miguel Alípio;
Revista do Instituto de Medicina Tropical de S?o Paulo , 1997, DOI: 10.1590/S0036-46651997000600001
Abstract: cryo-microtome sections of larvae of strongyloides stercoralis and s. ratti respectively obtained from human and rat feces cultures, were used as antigens. fluoresceinate conjugates against human igg were employed at the ideal titer of 10 for s. stercoralis and 100 for s. ratti. the sensitivity of the indirect immunofluorescence reaction (iif) was 94.4% and 92.5% and the specificity 94.2% and 97.1% for the two specific larval antigens, respectively. sera from 123 persons (54 from carriers of s. stercoralis infections and 69 from controls) were submitted to the reaction. the titers of different sera varied from 20 to 2560. there was a significant linear correlation (r = 0.85 p £ 0.001) between the antibodies from the two species of larval antigens. we conclude that both antigens may be used in the iif reaction for the diagnosis of human strongyloidiasis. due to the feasibility of safe and low-cost mass production of s. ratti larvae in the laboratory with a considerable economy of conjugate, their utilization in the serum diagnosis of human strongyloidiasis is recommended
Cryo-Microtome sections of coproculture larvae of Strongyloides stercoralis and Strongyloides ratti as antigen sources for the immunodiagnosis of human strongyloidiasis  [cached]
COSTA-CRUZ Julia Maria,BULLAMAH Carina Barbosa,GON?ALVES-PIRES Maria do Rosário F.,CAMPOS Dulcinéa Maria B.
Revista do Instituto de Medicina Tropical de S?o Paulo , 1997,
Abstract: Cryo-microtome sections of larvae of Strongyloides stercoralis and S. ratti respectively obtained from human and rat feces cultures, were used as antigens. Fluoresceinate conjugates against human IgG were employed at the ideal titer of 10 for S. stercoralis and 100 for S. ratti. The sensitivity of the indirect immunofluorescence reaction (IIF) was 94.4% and 92.5% and the specificity 94.2% and 97.1% for the two specific larval antigens, respectively. Sera from 123 persons (54 from carriers of S. stercoralis infections and 69 from controls) were submitted to the reaction. The titers of different sera varied from 20 to 2560. There was a significant linear correlation (r = 0.85 p £ 0.001) between the antibodies from the two species of larval antigens. We conclude that both antigens may be used in the IIF reaction for the diagnosis of human strongyloidiasis. Due to the feasibility of safe and low-cost mass production of S. ratti larvae in the laboratory with a considerable economy of conjugate, their utilization in the serum diagnosis of human strongyloidiasis is recommended
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