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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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S. stercoralis L3i activation is attenuated by the PI3 kinase inhibitor LY294002.In vitro activation of S. stercoralis L3i under host-like culture conditions by incubation in DMEM, 10% canine serum, and 12.5 mM reduced glutathione for 24 hours at 37°C and 5% CO2. The percentage of L3i that resumed feeding, a hallmark of activation, was scored by ingestion of FITC into the pharynx. Conditions included DMSO (carrier) positive control and M9 buffer negative control. The PI3 kinase inhibitor LY294002 was evaluated at 100 µM, 50 µM, and 10 µM, with each condition compared to the DMSO control using a logistic regression analysis. Error bars represent +1 SEM and parenthetical integers show the total number of L3i evaluated for each condition.
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pone-0038587-g005: S. stercoralis L3i activation is attenuated by the PI3 kinase inhibitor LY294002.In vitro activation of S. stercoralis L3i under host-like culture conditions by incubation in DMEM, 10% canine serum, and 12.5 mM reduced glutathione for 24 hours at 37°C and 5% CO2. The percentage of L3i that resumed feeding, a hallmark of activation, was scored by ingestion of FITC into the pharynx. Conditions included DMSO (carrier) positive control and M9 buffer negative control. The PI3 kinase inhibitor LY294002 was evaluated at 100 µM, 50 µM, and 10 µM, with each condition compared to the DMSO control using a logistic regression analysis. Error bars represent +1 SEM and parenthetical integers show the total number of L3i evaluated for each condition.

Mentions: We assessed the percentage of L3i feeding in three concentrations of LY294002, 10–100 µM, and compared this to a DMSO (carrier) positive control. We observed a significant, dose-dependent reduction in L3i activation in response to LY294002 (Figure 5). Logistic regression analysis revealed a 90% decrease (odds ratio: 0.10, 95% confidence interval: 0.08–0.13) in the odds of resumption of feeding for L3i at 100 µM, a 67% decrease (odds ratio: 0.33, 95% confidence interval: 0.28–0.40) in the odds of resumption of feeding for L3i at 50 µM, and a 25% decrease (odds ratio: 0.75, 95% confidence interval: 0.63–0.89) in the odds of resumption of feeding for L3i at 10 µM, all in comparison to the DMSO positive control. All odds ratios were significant at conventional statistical probabilities (p<0.001). At 100 µM of the PI3 kinase inhibitor, resumption of feeding by L3i was nearly inhibited to the level observed in the M9 buffer negative control. These data support the hypothesis that IIS signaling through the PI3 kinase, composed of Ss-age-1 and Ss-aap-1, is necessary for resumption of L3i development in S. stercoralis upon encountering a host.


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)

S. stercoralis L3i activation is attenuated by the PI3 kinase inhibitor LY294002.In vitro activation of S. stercoralis L3i under host-like culture conditions by incubation in DMEM, 10% canine serum, and 12.5 mM reduced glutathione for 24 hours at 37°C and 5% CO2. The percentage of L3i that resumed feeding, a hallmark of activation, was scored by ingestion of FITC into the pharynx. Conditions included DMSO (carrier) positive control and M9 buffer negative control. The PI3 kinase inhibitor LY294002 was evaluated at 100 µM, 50 µM, and 10 µM, with each condition compared to the DMSO control using a logistic regression analysis. Error bars represent +1 SEM and parenthetical integers show the total number of L3i evaluated for each condition.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038587-g005: S. stercoralis L3i activation is attenuated by the PI3 kinase inhibitor LY294002.In vitro activation of S. stercoralis L3i under host-like culture conditions by incubation in DMEM, 10% canine serum, and 12.5 mM reduced glutathione for 24 hours at 37°C and 5% CO2. The percentage of L3i that resumed feeding, a hallmark of activation, was scored by ingestion of FITC into the pharynx. Conditions included DMSO (carrier) positive control and M9 buffer negative control. The PI3 kinase inhibitor LY294002 was evaluated at 100 µM, 50 µM, and 10 µM, with each condition compared to the DMSO control using a logistic regression analysis. Error bars represent +1 SEM and parenthetical integers show the total number of L3i evaluated for each condition.
Mentions: We assessed the percentage of L3i feeding in three concentrations of LY294002, 10–100 µM, and compared this to a DMSO (carrier) positive control. We observed a significant, dose-dependent reduction in L3i activation in response to LY294002 (Figure 5). Logistic regression analysis revealed a 90% decrease (odds ratio: 0.10, 95% confidence interval: 0.08–0.13) in the odds of resumption of feeding for L3i at 100 µM, a 67% decrease (odds ratio: 0.33, 95% confidence interval: 0.28–0.40) in the odds of resumption of feeding for L3i at 50 µM, and a 25% decrease (odds ratio: 0.75, 95% confidence interval: 0.63–0.89) in the odds of resumption of feeding for L3i at 10 µM, all in comparison to the DMSO positive control. All odds ratios were significant at conventional statistical probabilities (p<0.001). At 100 µM of the PI3 kinase inhibitor, resumption of feeding by L3i was nearly inhibited to the level observed in the M9 buffer negative control. These data support the hypothesis that IIS signaling through the PI3 kinase, composed of Ss-age-1 and Ss-aap-1, is necessary for resumption of L3i development in S. stercoralis upon encountering a host.

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