Hc-daf-2 encodes an insulin-like receptor kinase in the barber’s pole worm, Haemonchus contortus, and restores partial dauer regulation

Abstract

Infective L3s (iL3s) of parasitic nematodes share common behavioural, morphological and developmental characteristics with the developmentally arrested (dauer) larvae of the free-living nematode Caenorhabditis elegans. It is proposed that similar molecular mechanisms regulate entry into or exit from the dauer stage in C. elegans, and the transition from free-living to parasitic forms of parasitic nematodes. In C elegans, one of the key factors regulating the dauer transition is the insulin-like receptor (designated Ce-DAF-2) encoded by the gene Ce-daf-2. However, nothing is known about DAF-2 homologues in most parasitic nematodes. Here, using a PCR-based approach, we identified and characterised 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-2 displays significant sequence homology to insulin receptors (IR) 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-2 is consistent with that of the human IR, suggesting that it is involved in the formation of the IR complex. The Hc-daf-2 gene was transcribed in all life stages of H. contortus, with a significant up-regulation in the iL3 compared with other stages. To compare patterns of expression between Hc-daf-2 and Ce-daf-2, reporter constructs fusing the Ce-daf-2 or Hc-daf-2 promoter to sequence encoding GFP were microinjected into the N2 strain 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. Taken together, these findings provide a first insight into the roles of Hc-daf-2/Hc-DAF-2 in the biology and development of H. contortus, particularly in the transition to parasitism.

Exploring the role of two interacting phosphoinositide 3-kinases of Haemonchus contortus

“…PI3Ks, which are relatively conserved enzymes from unicellular eukaryotes to multicellular organisms, play important roles in intracellular trafficking, cell metabolism, differentiation and/or survival [53][54][55][56]. Recently, components of insulinlike signalling have been identified in parasitic nematodes, including A. caninum [37,38], H. contortus [39,40] and S. stercoralis [30,41,42,57], suggesting that similar signalling is utilized by both free-living and parasitic nematodes. In the present study, two PI3K genes were identified in H. contortus using available data [33], including a Hc-age-1 encoding a p110 catalytic subunit and a Hc-aap-1 encoding a p50/p55-like regulatory subunit, consistent with those in C. elegans and S. stercoralis [12,13,30].…”

“…Heterologous complementation showed that the main components of insulin-like signalling, including daf-16, daf-2, age-1 and daf-12 in parasitic nematodes [37,[39][40][41]67], have similar functions to C. elegans orthologues. However, it is, nonetheless, interesting to note that the roles of PI3K from H. contortus appear to be divergent.…”

“…uk/Tools/msa/clustalo/). Genomic sequences upstream of the genes Hc-age-1 and Hc-aap-1 were sequenced as described previously [40].…”

“…However, a lack of effective in vitro-culture and genetic methods for parasitic nematodes has hampered the study of the developmental biology of these nematodes [28,[34][35][36]. In contrast, C. elegans provides a powerful surrogate system to shed light on gene function in parasitic nematodes, such as Ancylostoma caninum [37,38], H. contortus [39,40] and S. stercoralis [41,42].…”

Expression of nicotinic acetylcholine receptor subunits from parasitic nematodes in Caenorhabditis elegans

“…In recent years, a number of groups, including us, have reported the successful expression of proteins from parasitic nematodes in C. elegans [1–8]. However, most, if not all, of these proteins are active as either monomers or homomeric multimers.…”