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

Li FC, Gasser RB, Lok JB, Korhonen PK, Wang YF, Yin FY, He L, Zhou R, Zhao JL, Hu M - Parasit Vectors (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, College of Veterinary Medicine, Huazhong Agricultural University, 1 Shizishan Street, Wuhan, 430070, Hubei, China. li78561270@163.com.

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.

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Related in: MedlinePlus

Developmental profile of transcriptional abundance forHc-age-1(white bar) andHc-aap-1(striped bar) in different developmental stages ofHaemonchus 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.
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Fig4: Developmental profile of transcriptional abundance forHc-age-1(white bar) andHc-aap-1(striped bar) in different developmental stages ofHaemonchus 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.

Mentions: Hc-age-1 and Hc-aap-1 were transcribed in all developmental stages of H. contortus. Highest transcription was measured in eggs, L3s and female adults (Hc-age-1), or eggs, L3s, female L4s and female adults (Hc-aap-1) (Figure 4). Expression was localized in C. elegans (N2) transformed with plasmid pL-Hagep containing the 1,900 bp-predicted promoter region of Hc-age-1 or plasmid pL-Haapp containing the 2,524 bp-predicted promoter region of Hc-aap-1, using pRF4 as a marker. In addition, C. elegans (N2) transformed with gfp fused to each Ce-age-1 and Ce-aap-1 constructs were used as controls. The transgenic lines were screened for the roller phenotype and GFP expression. GFP expression driven by the Ce-age-1 promoter was detected in amphidial neurons and the intestine, consistent with previous reports for S. stercoralis [30]. GFP expression driven by the Hc-age-1 promoter was detected in the entire intestine at L1 and L2 stages, but concentrated in the anterior intestine of the L3 stage (Figure 5c-f). Similarly, GFP expression driven by the Ce-aap-1 promoter was observed in intestine and amphidial/phasmidial neurons (Figure 5g-h) in accordance with previous report [13]. The expression of Hc-aap-1 was also predominantly present in anterior intestine, which is highly analogous to that of Hc-age-1 (Figure 5i-j). Despite some variation in expression among individual larvae, C. elegans containing Ce-age-1 p::gfp and Ce-aap-1 p::gfp showed a similar expression profile (amphidial/head neurons and intestine), as did C. elegans containing Hc-age-1 p::gfp and Hc-aap-1 p::gfp (anterior intestine). Based on their apparent co-location, Hc-AGE-1 and Hc-AAP-1 might interact as a heterodimer [52].Figure 4


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

Li FC, Gasser RB, Lok JB, Korhonen PK, Wang YF, Yin FY, He L, Zhou R, Zhao JL, Hu M - Parasit Vectors (2014)

Developmental profile of transcriptional abundance forHc-age-1(white bar) andHc-aap-1(striped bar) in different developmental stages ofHaemonchus 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4233088&req=5

Fig4: Developmental profile of transcriptional abundance forHc-age-1(white bar) andHc-aap-1(striped bar) in different developmental stages ofHaemonchus 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.
Mentions: Hc-age-1 and Hc-aap-1 were transcribed in all developmental stages of H. contortus. Highest transcription was measured in eggs, L3s and female adults (Hc-age-1), or eggs, L3s, female L4s and female adults (Hc-aap-1) (Figure 4). Expression was localized in C. elegans (N2) transformed with plasmid pL-Hagep containing the 1,900 bp-predicted promoter region of Hc-age-1 or plasmid pL-Haapp containing the 2,524 bp-predicted promoter region of Hc-aap-1, using pRF4 as a marker. In addition, C. elegans (N2) transformed with gfp fused to each Ce-age-1 and Ce-aap-1 constructs were used as controls. The transgenic lines were screened for the roller phenotype and GFP expression. GFP expression driven by the Ce-age-1 promoter was detected in amphidial neurons and the intestine, consistent with previous reports for S. stercoralis [30]. GFP expression driven by the Hc-age-1 promoter was detected in the entire intestine at L1 and L2 stages, but concentrated in the anterior intestine of the L3 stage (Figure 5c-f). Similarly, GFP expression driven by the Ce-aap-1 promoter was observed in intestine and amphidial/phasmidial neurons (Figure 5g-h) in accordance with previous report [13]. The expression of Hc-aap-1 was also predominantly present in anterior intestine, which is highly analogous to that of Hc-age-1 (Figure 5i-j). Despite some variation in expression among individual larvae, C. elegans containing Ce-age-1 p::gfp and Ce-aap-1 p::gfp showed a similar expression profile (amphidial/head neurons and intestine), as did C. elegans containing Hc-age-1 p::gfp and Hc-aap-1 p::gfp (anterior intestine). Based on their apparent co-location, Hc-AGE-1 and Hc-AAP-1 might interact as a heterodimer [52].Figure 4

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, College of Veterinary Medicine, Huazhong Agricultural University, 1 Shizishan Street, Wuhan, 430070, Hubei, China. li78561270@163.com.

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.

Show MeSH
Related in: MedlinePlus