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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 expressed at a low level in all examined S. stercoralis life stages.Ss-age-1 (white bars) transcript levels in comparison to transcript levels of two reference genes, Ss-act-2 (dark blue bars) and Ss-gapdh (light green bars), in six S. stercoralis developmental stages: free-living females (FL Female), post-free-living first-stage larvae (PFL L1), infectious third-stage larvae (L3i), parasitic females (P Female), predominantly (>95%) heterogonically developing post-parasitic first-stage larvae (PP L1), and post-parasitic approximately third-stage larvae heterogonically developing to free-living adults (PP L3). Transcript levels were normalized to Ss-age-1 in free-living females and log transformed. Error bars represent +1 SEM.
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pone-0038587-g002: Ss-age-1 is expressed at a low level in all examined S. stercoralis life stages.Ss-age-1 (white bars) transcript levels in comparison to transcript levels of two reference genes, Ss-act-2 (dark blue bars) and Ss-gapdh (light green bars), in six S. stercoralis developmental stages: free-living females (FL Female), post-free-living first-stage larvae (PFL L1), infectious third-stage larvae (L3i), parasitic females (P Female), predominantly (>95%) heterogonically developing post-parasitic first-stage larvae (PP L1), and post-parasitic approximately third-stage larvae heterogonically developing to free-living adults (PP L3). Transcript levels were normalized to Ss-age-1 in free-living females and log transformed. Error bars represent +1 SEM.

Mentions: To determine whether Ss-age-1 is transcriptionally regulated or constitutively expressed over the course of the S. stercoralis life cycle, we performed reverse transcription quantitative PCR (RT-qPCR) for six developmental stages. Transcript abundance was calculated for Ss-age-1, as well as two reference genes, actin-2 (Ss-act-2) and glyceraldehyde 3-phosphate dehydrogenase (Ss-gapdh), using 50 ng of total RNA isolated from three biological replicates of each developmental stage. We calculated transcript abundances, which were normalized to an arbitrarily determined mean of 10 copies of Ss-age-1 in free-living females and log transformed. We observed Ss-age-1 expression at low, but consistent, levels in comparison to both Ss-act-2 and Ss-gapdh for all life stages examined (Figure 2). Although expression of Ss-age-1 was at its nadir in free-living females, the biological relevance of this difference is questionable, since similar levels of variability between developmental stages were also observed for Ss-act-2 and Ss-gapdh. Overall, these data suggest that, like Ce-age-1, Ss-age-1 is expressed at a low level throughout the course of the S. stercoralis life cycle. This is consistent with the hypothesis that IIS signaling through AGE-1 is regulated post-translationally and not at the transcriptional level.


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 expressed at a low level in all examined S. stercoralis life stages.Ss-age-1 (white bars) transcript levels in comparison to transcript levels of two reference genes, Ss-act-2 (dark blue bars) and Ss-gapdh (light green bars), in six S. stercoralis developmental stages: free-living females (FL Female), post-free-living first-stage larvae (PFL L1), infectious third-stage larvae (L3i), parasitic females (P Female), predominantly (>95%) heterogonically developing post-parasitic first-stage larvae (PP L1), and post-parasitic approximately third-stage larvae heterogonically developing to free-living adults (PP L3). Transcript levels were normalized to Ss-age-1 in free-living females and log transformed. Error bars represent +1 SEM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368883&req=5

pone-0038587-g002: Ss-age-1 is expressed at a low level in all examined S. stercoralis life stages.Ss-age-1 (white bars) transcript levels in comparison to transcript levels of two reference genes, Ss-act-2 (dark blue bars) and Ss-gapdh (light green bars), in six S. stercoralis developmental stages: free-living females (FL Female), post-free-living first-stage larvae (PFL L1), infectious third-stage larvae (L3i), parasitic females (P Female), predominantly (>95%) heterogonically developing post-parasitic first-stage larvae (PP L1), and post-parasitic approximately third-stage larvae heterogonically developing to free-living adults (PP L3). Transcript levels were normalized to Ss-age-1 in free-living females and log transformed. Error bars represent +1 SEM.
Mentions: To determine whether Ss-age-1 is transcriptionally regulated or constitutively expressed over the course of the S. stercoralis life cycle, we performed reverse transcription quantitative PCR (RT-qPCR) for six developmental stages. Transcript abundance was calculated for Ss-age-1, as well as two reference genes, actin-2 (Ss-act-2) and glyceraldehyde 3-phosphate dehydrogenase (Ss-gapdh), using 50 ng of total RNA isolated from three biological replicates of each developmental stage. We calculated transcript abundances, which were normalized to an arbitrarily determined mean of 10 copies of Ss-age-1 in free-living females and log transformed. We observed Ss-age-1 expression at low, but consistent, levels in comparison to both Ss-act-2 and Ss-gapdh for all life stages examined (Figure 2). Although expression of Ss-age-1 was at its nadir in free-living females, the biological relevance of this difference is questionable, since similar levels of variability between developmental stages were also observed for Ss-act-2 and Ss-gapdh. Overall, these data suggest that, like Ce-age-1, Ss-age-1 is expressed at a low level throughout the course of the S. stercoralis life cycle. This is consistent with the hypothesis that IIS signaling through AGE-1 is regulated post-translationally and not at the transcriptional level.

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