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Strongyloides stercoralis age-1: a potential regulator of infective larval development in a parasitic nematode.

Stoltzfus JD, Massey HC, Nolan TJ, Griffith SD, Lok JB - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, United States of America.

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.

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Ss-AGE-1 is a homolog of the Ce-AGE-1 PI3 kinase catalytic subunit.(A) Intron-exon structure of the Ss-age-1 unspliced mRNA sequence. Grey boxes indicate the three exons, with the numbers above indicating the first and last base pairs of the exon. Introns are indicated by slanted lines between the exons, with the numbers indicating the total intron length. The 5′ and 3′ untranslated regions (UTR) are indicated with horizontal lines, with the italicized numbers indicating the total length of the UTR in base-pairs (bp). (B) Domain structure of the Ss-AGE-1 predicted protein. Shaded boxes represent the five protein family domains, with the numbers indicating the first and last amino acids of the domain. (C) Phylogenetic analysis of Ss-AGE-1. The predicted Ss-AGE-1 protein groups with other class I PI3 kinase catalytic subunits, including Ce-AGE-1. Abbreviations: Strongyloides stercoralis (Ss), Strongyloides ratti (Sr), Parastrongyloides trichosuri (Pt), Brugia malayi (Bm), Caenorhabditis briggsae (Cb), Caenorhabditis elegans (Ce), Homo sapiens (Hs), Drosophila melanogaster (Dm), and Saccharomyces cerevisiae (Sc). Accession numbers listed in Methods.
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pone-0038587-g001: Ss-AGE-1 is a homolog of the Ce-AGE-1 PI3 kinase catalytic subunit.(A) Intron-exon structure of the Ss-age-1 unspliced mRNA sequence. Grey boxes indicate the three exons, with the numbers above indicating the first and last base pairs of the exon. Introns are indicated by slanted lines between the exons, with the numbers indicating the total intron length. The 5′ and 3′ untranslated regions (UTR) are indicated with horizontal lines, with the italicized numbers indicating the total length of the UTR in base-pairs (bp). (B) Domain structure of the Ss-AGE-1 predicted protein. Shaded boxes represent the five protein family domains, with the numbers indicating the first and last amino acids of the domain. (C) Phylogenetic analysis of Ss-AGE-1. The predicted Ss-AGE-1 protein groups with other class I PI3 kinase catalytic subunits, including Ce-AGE-1. Abbreviations: Strongyloides stercoralis (Ss), Strongyloides ratti (Sr), Parastrongyloides trichosuri (Pt), Brugia malayi (Bm), Caenorhabditis briggsae (Cb), Caenorhabditis elegans (Ce), Homo sapiens (Hs), Drosophila melanogaster (Dm), and Saccharomyces cerevisiae (Sc). Accession numbers listed in Methods.

Mentions: To identify the S. stercoralis homolog of Ce-age-1, we performed BLAST searches of the nematode expressed sequence tag (EST) database and found an EST with homology to Ce-age-1 in Parastrongyloides trichosuri, a closely related parasitic nematode. We designed degenerate primers and amplified an S. stercoralis sequence from genomic DNA. Using successive rounds of inverse PCR, we elucidated the 3,999 base-pair (bp) genomic coding region for Ss-age-1 along with 1.3 kilobase (kb) of the upstream sequence and 1.8 kb of the downstream sequence (GenBank: JQ772018). To determine the 5′ and 3′ ends of the Ss-age-1 coding sequence, we performed rapid amplification of cDNA ends (RACE) using adapter-ligated cDNA. Subsequently, we cloned the 3,630 bp Ss-age-1 coding sequence from cDNA derived from free-living adults (Figure 1A) and inferred a predicted peptide of 1,209 amino acids (Figure 1B). The locations of the three introns in Ss-age-1 were not conserved in Ce-age-1.


Strongyloides stercoralis age-1: a potential regulator of infective larval development in a parasitic nematode.

Stoltzfus JD, Massey HC, Nolan TJ, Griffith SD, Lok JB - PLoS ONE (2012)

Ss-AGE-1 is a homolog of the Ce-AGE-1 PI3 kinase catalytic subunit.(A) Intron-exon structure of the Ss-age-1 unspliced mRNA sequence. Grey boxes indicate the three exons, with the numbers above indicating the first and last base pairs of the exon. Introns are indicated by slanted lines between the exons, with the numbers indicating the total intron length. The 5′ and 3′ untranslated regions (UTR) are indicated with horizontal lines, with the italicized numbers indicating the total length of the UTR in base-pairs (bp). (B) Domain structure of the Ss-AGE-1 predicted protein. Shaded boxes represent the five protein family domains, with the numbers indicating the first and last amino acids of the domain. (C) Phylogenetic analysis of Ss-AGE-1. The predicted Ss-AGE-1 protein groups with other class I PI3 kinase catalytic subunits, including Ce-AGE-1. Abbreviations: Strongyloides stercoralis (Ss), Strongyloides ratti (Sr), Parastrongyloides trichosuri (Pt), Brugia malayi (Bm), Caenorhabditis briggsae (Cb), Caenorhabditis elegans (Ce), Homo sapiens (Hs), Drosophila melanogaster (Dm), and Saccharomyces cerevisiae (Sc). Accession numbers listed in Methods.
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Related In: Results  -  Collection

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pone-0038587-g001: Ss-AGE-1 is a homolog of the Ce-AGE-1 PI3 kinase catalytic subunit.(A) Intron-exon structure of the Ss-age-1 unspliced mRNA sequence. Grey boxes indicate the three exons, with the numbers above indicating the first and last base pairs of the exon. Introns are indicated by slanted lines between the exons, with the numbers indicating the total intron length. The 5′ and 3′ untranslated regions (UTR) are indicated with horizontal lines, with the italicized numbers indicating the total length of the UTR in base-pairs (bp). (B) Domain structure of the Ss-AGE-1 predicted protein. Shaded boxes represent the five protein family domains, with the numbers indicating the first and last amino acids of the domain. (C) Phylogenetic analysis of Ss-AGE-1. The predicted Ss-AGE-1 protein groups with other class I PI3 kinase catalytic subunits, including Ce-AGE-1. Abbreviations: Strongyloides stercoralis (Ss), Strongyloides ratti (Sr), Parastrongyloides trichosuri (Pt), Brugia malayi (Bm), Caenorhabditis briggsae (Cb), Caenorhabditis elegans (Ce), Homo sapiens (Hs), Drosophila melanogaster (Dm), and Saccharomyces cerevisiae (Sc). Accession numbers listed in Methods.
Mentions: To identify the S. stercoralis homolog of Ce-age-1, we performed BLAST searches of the nematode expressed sequence tag (EST) database and found an EST with homology to Ce-age-1 in Parastrongyloides trichosuri, a closely related parasitic nematode. We designed degenerate primers and amplified an S. stercoralis sequence from genomic DNA. Using successive rounds of inverse PCR, we elucidated the 3,999 base-pair (bp) genomic coding region for Ss-age-1 along with 1.3 kilobase (kb) of the upstream sequence and 1.8 kb of the downstream sequence (GenBank: JQ772018). To determine the 5′ and 3′ ends of the Ss-age-1 coding sequence, we performed rapid amplification of cDNA ends (RACE) using adapter-ligated cDNA. Subsequently, we cloned the 3,630 bp Ss-age-1 coding sequence from cDNA derived from free-living adults (Figure 1A) and inferred a predicted peptide of 1,209 amino acids (Figure 1B). The locations of the three introns in Ss-age-1 were not conserved in Ce-age-1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, United States of America.

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.

Show MeSH
Related in: MedlinePlus