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

Abstract

Background: Phosphoinositide 3-kinases (PI3Ks) are relatively conserved and important intracellular lipid kinases involved in signalling and other biological pathways. In the free-living nematode Caenorhabditis elegans, the heterodimeric form of PI3K consists of catalytic (AGE-1) and regulatory (AAP-1) subunits. These subunits are key components of the insulin-like signalling pathway and play roles in the regulation of the entry into and exit from dauer. Although, in parasitic nematodes, similar components are proposed to regulate the transition from free-living or arrested stages to parasitic larvae, nothing is known about PI3Ks in relation to the transition of third-stage larvae (L3s) to parasitism in Haemonchus contortus.

Methods: An integrated molecular approach was used to investigate age-1 and aap-1 of H. contortus (Hc-age-1 and Hc-aap-1) in C. elegans.

Results: The two genes Hc-age-1 and Hc-aap-1 were transcribed in all life stages, with the highest levels in the egg, infective L3 and adult female of H. contortus. The expression of these genes was localized to the intestine, contrasting the pattern of their orthologues in C. elegans (where they are expressed in both head neurons and the intestine). The yeast two-hybrid analysis demonstrated that the adaptor-binding domain of Hc-AGE-1 interacted strongly with the Hc-AAP-1; however, this complex did not rescue the function of its orthologue in age-1-deficient C. elegans.

Conclusions: This is the first time that the PI3K-encoding genes have been characterized from a strongylid parasitic nematode. The findings provide insights into the role of the PI3K heterodimer represented by Hc-age-1 and Hc-aap-1 in the developmental biology of H. contortus.

Figure 1

Main functional domains of phosphoinositide 3-kinase (PI3K) catalytic subunit Hc -AGE-1 and the regulatory subunit Hc -AAP-1 of Haemonchus contortus . (a) Colours represent different functional domains: adaptor-binding domain (yellow), Ras-binding domain (light blue), C2 domain (green), PIK domain (purple) and the catalytic domain (grey). (b) N-terminal and C-terminal SH2 domains (yellow). Numbers represent amino acid positions. Percentage under the amino and carboxylic terminal SH2 domain shows the similarity to the SH2 domains of homologues from various eukaryotes, including Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens.

Figure 2

Neighbor joining trees showing the relationship of Haemonchus contortus phosphoinositide 3-kinase (PI3K) catalytic subunit Hc -AGE-1 and regulatory subunit Hc -AAP-1. The trees were calculated using the Jones-Taylor-Thornton (JTT) model in the MEGA program version 5.0. Bootstrap values above or below the branches (1,000 iterations) are shown for robust clades (>50 %). (a) The species used in the analysis include six nematodes (Strongyloides stercoralis, Ss-AGE-1; Parastrongyloides trichosuri, Pt-AGE-1; Brugia malayi, Bm-AGE-1; Loa loa, Ll-AGE-1; Caenorhabditis elegans, Ce-AGE-1, Ce-PIKII and Ce-VPS34; C. briggsae, Cb-AGE-1) and three non-nematodes (Drosophila melanogaster, Dm-PI3K92E and Dm-PI3K59F; Homo sapiens, Hs-PI3KCA, Hs-PI3KC2 and Hs-PI3KC3; Saccharomyces cerevisiae, Sc-VPS34). Mus musculus EGFR AAA17899 was used as an outgroup. (b) AAP-1 used in the analysis included Caenorhabditis elegans, Ce-AAP-1; C. briggsae, Cb-AAP-1; C. remani, Cr-AAP-1; Strongyloides stercoralis, Ss-AAP-1; Ascaris sum, As-AAP-1; Brugia malayi, Bm-AAP-1; Loa loa, Ll-AAP-1; Trichinella spiralis, Ts-AAP-1; Drosophila melanogaster, Dm-P60; Homo sapiens, Hs-PI3K85). C. elegans Ce-LET-23 CAA93882 was used as an outgroup. GenBank accession numbers are listed on the right of each species.

Figure 3

Schematic diagram displaying the genomic organization of age-1 and aap-1 from Haemonchus contortus ( Hc-age-1 and Hc-aap-1 ), Caenorhabditis elegans ( Ce-age-1 and Ce-aap-1 ) and Strongyloides stercoralis ( Ss-age-1 and Ss-aap-1 ). Black boxes represent exons. Lines between the exons represent introns. Start (ATG) or stop (TAG/TGA/TAA) codons are indicated.

Figure 4

Developmental profile of transcriptional abundance for Hc-age-1 (white bar) and Hc-aap-1 (striped bar) in different developmental stages of Haemonchus contortus . Transcript abundance were compared among eight developmental stages, each in biological triplicate (n =3); eggs (E), the first-stage larvae (L1), the second-stage larvae (L2), the infective L3 (iL3), the fourth-stage males (L4m), the fourth-stage females (L4f), adult males (Am) and adult females (Af). Transcript abundances were counted as fragments per kilobase of coding exon per million mapped reads (FPKM). Error bars represent 95 % confidence intervals.

Figure 5

Expression patterns of Hc-age-1 and Hc-aap-1 . Representative expression patterns of Ce-age-1, Hc-age-1, Ce-aap-1 and Hc-aap-1 displayed in Caenorhabditis elegans transformed by either of the four green fluorescent protein (GFP) constructs pL-Cagep, pL-Hagep, pL-Caapp and pL-Haapp (Additional file 2). Three transgenic lines were produced for each of the four plasmids. (a and b) Differential interference contrast (DIC) (a) and fluorescence (b) images of transgenic third-stage larva (L3) of C. elegans expressing Ce-age-1p::gfp (pL-Cagep). (c and d) DIC (c) and fluorescence (d) images showing the expression pattern of Hc-age-1 p::gfp (pL-Hagep) in first-stage larva (L1). (e and f) DIC (e) and fluorescence (f) images, showing the expression pattern of Hc-age-1p::gfp (pL-Hagep) in L3. (g and h) DIC (g) and fluorescence (h) images showing expression pattern of Ce-aap-1 p::gfp (pL-Caapp) in L3. (I and J) DIC (i) and fluorescence (j) images showing expression pattern of Hc-aap-1 p::gfp (pL-Haapp) in L3. Scale bars =50 μm.