| Haemonchus contortus is an abomasum-infected parasitic nematode that can lead to important disease of grazing sheep worldwide, causing weight reduction, death and enormous economic loss. As there is no effective vaccine available, treatment of parasitic nematode infections relies heavily on anthelmintics. However, the widespread use of these anthelmintics has resulted in serious resistance and drug residue problems worldwide. Therefore, it is imperative to develop new intervention strategies. One of the possibilities is the rational design of anti-parasite drugs and/or vaccines, built on the deep understanding of the biological and developmental processes in these parasites. For instance, for parasitic nematodes, clear insights into the developmental transition from free-living to parasitic stages might identify key switches as new drug targets.The characterization of the nuclear genomes and transcriptomes of some key parasitic nematodes, including Ascaris suum, Brugia malayi, Dirofilaria immitis, Haemonchus contortus, Loa loa and Necator americanus, provides a solid foundation for investigating developmental processes using complementary molecular (i.e. genetic, genomic, proteomic and metabolomic) tools. However, a lack of effective genetic and genomic tools for some parasitic nematodes and an inability to maintain their entire life cycle in the laboratory hampers detailed functional studies. In contrast, Caenorhabditis elegans can be readily maintained in the laboratory and used to explore fundamental processes and mechanisms, such as dauer formation. This free-living nematode belongs to clade V and is relatively closely related to H. contortus. Published information also indicates similarity in dauer induction and recovery between C. elegans and strongylids. The dauer state occurs in C. elegans when the nematode encounters unfavorable environmental conditions, including high temperature, starvation and/or crowding. The dauer form can survive for several months and resume development to reproductive adults when the environmental conditions improve. Consistent with C. elegans, strongylid nematodes have a similar infective L3(iL3), which is resistant to unfavorable conditions and does not feed because it is enveloped by a sheath. The "dauer hypothesis" or "daf-c paradigm" holds that the resumption of iL3development in parasitic nematodes is developmentally and functionally analogous to the recovery from dauer in C. elegans and is governed by similar molecular mechanisms. Dauer development in C. elegans is regulated by several signalling pathways, including an insulin/IGF-1-like signaling pathway, which contains a number of components, such as the insulin-like receptor kinase DAF-2, the PI3kinase AGE-1,3-phosphoinositide dependent protein kinase PDK-1and the FOXO-class transcription factor DAF-16. Mutations in the daf-2, age-1and pdk-1genes result in dauer constitutive (daf-c) phenotypes, whereas mutations in daf-16give dauer-defective (daf-d) phenotypes. Signalling through DAF-2activates AKT-1/2by phosphorylation, which, in turn, negatively regulates DAF-16, which functions as a central mediator of multiple biological processes, including longevity, development and stress resistance.(1) The structural and functional characterizations of the insulin-like receptor(Hc-daf-2)Using a PCR-based approach, we identified and characterized a gene (Hc-daf-2) and its inferred product (Hc-DAF-2) in Haemonchus contortus (a socioeconomically important parasitic nematode of ruminants). The sequence of Hc-DAF-2displays significant sequence homology to insulin receptors in both vertebrates and invertebrates, and contains conserved structural domains. A sequence encoding an important proteolytic motif (RKRR) identified in the predicted peptide sequence of Hc-DAF-2is consistent with that of the human insulin receptor (HIR), suggesting that it is involved in the formation of the insulin-receptor complex. The Hc-daf-2gene was transcribed in all life stages of H. contortus, with a significant up-regulation in the infective third-stage (iL3) compared with other stages. To compare patterns of expression between Hc-daf-2and Ce-daf-2, reporter constructs fusing the Ce-daf-2or Hc-daf-2promoter to sequence encoding the green fluorescent protein (GFP) were micro injected into the N2strain of C. elegans, and transgenic lines were established and examined. Both genes showed similar patterns of expression in amphidial (head) neurons, which relate to sensation and signal transduction. Further study by heterologous genetic complementation in a daf-2-deficient strain of C. elegans (CB1370) showed partial rescue of function by Hc-daf-2.(2) The structural and functional characterizations of the phosphatidykinositol3-kinasePI3K (Hc-AGE-1and Hc-AAP-1)We identified and characterized the PI3K catalytic subunit (Hc-AGE-1) and regulatory subunit (Hc-AAP-1) from Haemonchus contortus. The sequence of Hc-AGE-1and Hc-AAP-1show high homology to PI3K in both vertebrates and invertebrates, and contain conserved structural domains. The Hc-age-1and Hc-aap-1genes were transcribed in all life stages of H. contortus, with up-regulation in the eggs, infective third-stage (iL3) and female adults compared with other stages. To compare patterns of expression between Hc-age-1, Ce-age-1and Hc-aap-1, Ce-aap-1, reporter constructs fusing the promoter to sequence encoding the green fluorescent protein (GFP) were microinjected into the N2strain of C. elegans, respectively, and transgenic lines were established and examined. Hc-age-1and Hc-aap-1show the expression pattern in intestine, and Ce-age-1and Ce-aap-1display in intestine and neurons. Yeast two-hybrid system showed the p85binding domain of Hc-age-1and Hc-aap-1can interact with each other strongly. Further study by heterologous genetic complementation in an oge-1-deficient strain of C. elegans (CY246) showed no rescue of function by Hc-age-1.(3) Characterizations of the3-phosphoinositide dependent protein kinase (Hc-PDK-1)We identified and characterized the3-phosphoinositide dependent protein kinase from Haemonchus contortus. The Hc-PDK-1shows high homology with Ce-PDK-1(about46%), and contains catalytic kinase domain and PH domain. PH domain is a functional regulator of a group of protein kinases and plays a center role in cell signalling transduction and cytoskeletal function. The Hc-pdk-1genes were transcribed in all life stages of H. contortus. To compare patterns of expression between Hc-pdk-1and Ce-pdk-1, reporter constructs fusing the promoter to sequence encoding the green fluorescent protein (GFP) were microinjected into the N2strain of C. elegans, respectively. The expression location of Hc-pdk-1is in intestine, and Ce-pdk-1is in head neurons, pharynx and intestine.Taken together, these findings provide a first insight into the roles of Hc-daf-2/Hc-DAF-2, Hc-age-1/Hc-AGE-1, Hc-aap-1/Hc-AAP-1and Hc-pdk-1/Hc-PDK-1in the biology and development of H. contortus, particularly in the transition to parasitism. |
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