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Molecular characterization of the Haemonchus contortus phosphoinositide-dependent protein kinase-1 gene (Hc-pdk-1)
Fa-Cai Li1, Robin B Gasser2,3, James B Lok4
1State Key Laboratory of Agricultural Microbiology, Key Laboratory of Development of Veterinary Products, Ministry of Agriculture, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China. li78561270@163.com.
Insights
This study characterizes the pdk-1 gene in Haemonchus contortus, revealing its expression across developmental stages, particularly in infective larvae. Findings offer insights into the nematode
Area of Science:
- Molecular Biology
- Parasitology
- Genetics
Background:
- Phosphoinositide-dependent protein kinase-1 (PDK-1) regulates vital cellular processes and is key in Caenorhabditis elegans insulin signaling.
- The role of PDK-1 in parasitic nematodes, like Haemonchus contortus, remains largely unknown.
- Haemonchus contortus is a significant gastric nematode parasite of ruminants.
Purpose of the Study:
- To isolate and characterize the pdk-1 gene (Hc-pdk-1) and its protein product (Hc-PDK-1) in Haemonchus contortus.
- To investigate the transcriptional profiles and gene expression patterns of Hc-pdk-1 and Hc-PDK-1.
- To compare the expression of Hc-pdk-1 with its Caenorhabditis elegans ortholog.
Main Methods:
- Gene isolation and characterization of Hc-pdk-1 and Hc-PDK-1 from H. contortus.
- In silico analysis of Hc-PDK-1 functional domains.
- In vitro and in vivo studies of Hc-pdk-1 transcription and Hc-PDK-1 expression patterns.
- Transgenic C. elegans expressing Hc-pdk-1 promoter-GFP constructs for localization studies.
Main Results:
- In silico analysis revealed conserved protein kinase and PH domains in Hc-PDK-1, crucial for signaling.
- Hc-pdk-1 transcription was detected in all H. contortus developmental stages, peaking in infective third-stage larvae (iL3).
- Hc-pdk-1 promoter-driven GFP expression in C. elegans localized to the intestine and neurons, differing from Ce-pdk-1 expression patterns.
Conclusions:
- This study presents the first characterization of pdk-1/PDK-1 in a trichostrongyloid nematode.
- The findings provide initial evidence for Hc-pdk-1 involvement in the insulin-like signaling pathway in H. contortus.
- Understanding Hc-pdk-1 function may offer targets for controlling H. contortus infections.
Background:
Phosphoinositide-dependent protein kinase-1 (PDK-1), which functions downstream of phosphoinositide 3-kinase (AGE-1) and activates protein kinases of the AGC family, plays critical roles in regulating biology processes, such as metabolism, growth, development and survival. In the free-living nematode Caenorhabditis elegans, PDK-1 is a key component of the insulin-like signalling pathway, regulating the entry into and exit from dauer (arrested development). Although it is proposed that similar molecular mechanisms control the transition from the free-living to the parasitic stages of nematodes, nothing is known about PDK-1 in Haemonchus contortus, a socioeconomically important gastric nematode of ruminants.
Methods:
Here, we isolated and characterized the pdk-1 gene (Hc-pdk-1) and its inferred product (Hc-PDK-1) from H. contortus. Using in vitro and in vivo methods, we then studied the transcriptional profiles of Hc-pdk-1 and anatomical gene expression patterns of Hc-PDK-1 in different developmental stages of C. elegans.
Results:
In silico analysis of Hc-PDK-1 displayed conserved functional domains, such as protein kinase and pleckstrin homology (PH) domains and two predicted phosphorylation sites (Thr226/Tyr229), which are crucial for the phosphorylation of downstream signalling. The Hc-pdk-1 gene is transcribed in all of the main developmental stages of H. contortus, with its highest transcription in the infective third-stage larvae (iL3) compared with other stages. Transgene constructs, in which respective promoters were fused to the coding sequence for green fluorescent protein (GFP), were used to transform C. elegans, and to localize and compare the expression of Hc-pdk-1 and Ce-pdk-1. The expression of GFP under the control of the Hc-pdk-1 promoter was localized to the intestine, and head and tail neurons, contrasting somewhat the profile for the C. elegans ortholog, which is expressed in pharynx, intestine and head and tail neurons.
Conclusions:
This is the first characterization of pdk-1/PDK-1 from a trichostrongyloid nematode. Taken together, the findings from this study provide a first glimpse of the involvement of Hc-pdk-1 in the insulin-like signalling pathway in H. contortus.
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