Acyl-CoA oxidase ACOX-1 interacts with a peroxin PEX-5 to play roles in larval development of Haemonchus contortus

Abstract

Hypobiosis (facultative developmental arrest) is the most important life-cycle adaptation ensuring survival of parasitic nematodes under adverse conditions. Little is known about such survival mechanisms, although ascarosides (ascarylose with fatty acid-derived side chains) have been reported to mediate the formation of dauer larvae in the free-living nematode Caenorhabditis elegans. Here, we investigated the role of a key gene acox-1, in the larval development of Haemonchus contortus, one of the most important parasitic nematodes that employ hypobiosis as a routine survival mechanism. In this parasite, acox-1 encodes three proteins (ACOXs) that all show a fatty acid oxidation activity in vitro and in vivo, and interact with a peroxin PEX-5 in peroxisomes. In particular, a peroxisomal targeting signal type1 (PTS1) sequence is required for ACOX-1 to be recognised by PEX-5. Analyses on developmental transcription and tissue expression show that acox-1 is predominantly expressed in the intestine and hypodermis of H. contortus, particularly in the early larval stages in the environment and the arrested fourth larval stage within host animals. Knockdown of acox-1 and pex-5 in parasitic H. contortus shows that these genes play essential roles in the post-embryonic larval development and likely in the facultative arrest of this species. A comprehensive understanding of these genes and the associated β-oxidation cycle of fatty acids should provide novel insights into the developmental regulation of parasitic nematodes, and into the discovery of novel interventions for species of socioeconomic importance.

Fig 1. Complementary DNA-confirmed gene models of acox-1 in Haemonchus contortus.

(A) Gene structures of Hc-acox-1.1 and Hc-acox-1.2 are defined in H. contortus based on complementary DNA sequences. Arrow boxes indicate two adjacent gene loci of Hc-acox-1, with a 91.93% identity indicated between Hc-acox-1.2 and Hc-acox-1.3, a possible locus not part of the current genome, based on NCBI blastn searching. Black blocks represent exons that were matched with cloned sequences, and horizontal lines represent introns indicated by transcripts. The numbers above and below the boxes indicate the start and end of gene loci. The lines above exons represent the sequences targeted in RNAi (red) and in qPCR (blue), respectively. (B) Predicted key sites in the deduced amino acid sequences of Hc-ACOX-1.1, -1.2 and -1.3, with a 97.53% similarity indicated between the latter two proteins based on NCBI blastp searching. Predicted flavin adenine dinucleotide binding sites 151 (T, threonine) and 190 (G, glycine), and active site 206/433 (E, glutamic acid) of deduced Hc-ACOX-1 are indicated. The amino acid sequences without SKL are used for polyclonal antibodies preparation. SKL represents peroxisomal targeting signal type 1 (PTS1). S: serine, K: lysine, L: leucine.

Fig 2. Enzyme activity and spotting assay of Hc-ACOX-1 in vivo and in vitro.

(A-C) Linear double-reciprocal plots of Hc-ACOX-1.1 (A), Hc-ACOX-1.2 (B) and Hc-ACOX-1.3 (C) constructed based on reciprocal reaction velocity (1/V) and reciprocal value of palmitoyl-CoA concentration (1/[S]). Panels D-G: The letters (1.1, 1.2 and 1.3) in square brackets represent the corresponding protein of Hc-ACOX-1 and the characters in parentheses indicate the key sites that are replaced with Alanine. 151A/190A represents the 151st and 190th amino acids are replaced with Alanine. Yeast concentrations are marked at top with a starting concentration of OD₆₀₀ = 1. (D) Growth of wild-type Saccharomyces cerevisiae on YNBO plates (YNB supplemented with oleic acid as sole carbon source). (-) represents mutation without peroxisomal targeting signal type 1. (E) Growth of S. cerevisiae Δpox1 strain rescued with Hc-acox-1 on YNBO plates. (F) Effect of key site of Hc-ACOX-1.2 and -1.3 on the growth of Δpox1. (G) Effect of key sites of Hc-ACOX-1.1 on the growth of Δpox1. Multiple mutations in Hc-ACOX-1.1 affect the growth of Δpox1 on the YNBO plate, whereas single mutation at 151A or 190A did not. WT and Δpox1 represent wild-type and POX (ACOX homologue) mutant strain of S. cerevisiae, respectively.

Fig 3. Co-localisation of Hc-ACOX-1 and peroxisomes in HEK293T cells.

(A) Subcellular localisation of Hc-ACOX-1 and peroxisomes in HEK293T cells. (B) Subcellular localisation of Hc-ACOX-1 without peroxisomal targeting signal type 1 (PTS1) and peroxisomes in HEK293T cells. Hc-ACOX-1 without PTS1 is designated as Hc-ACOX-1 (-). Green fluorescent protein (GFP)-fused ACOX-1 is expressed in HEK293T cells and the nuclei are stained with 4’,6-diamidino-2-phenylindole (DAPI). Red fluorescence indicates RFP/peroxisome protein expressed in peroxisome. Scale bar: 10 μm.

Fig 4. Interaction of Hc-ACOX-1 and Hc-PEX-5 via peroxisomal targeting signal type 1 (PTS1).

(A-B) Co-expression of Hc-ACOX-1-FLAG with (A) or without (B) PTS1 and HA-tagged Hc-PEX-5 in HEK293T cells. Anti-FLAG antibody is used in immunoprecipitation (IP, top two panels) of the total cellular lysates (input, bottom tow panels). The immunoprecipitated portion and input are individually subjected to Western blot using anti-DYKDDDDK (FLAG) tag and anti-HA tag antibodies as labelled. The letters a, b and c represent Hc-ACOX-1.1, -1.2, and -1.3, respectively. Hc-ACOX-1.2 is used as positive control in (B). ΔSKL represents the deletion of PTS1.

Fig 5. Tissue immunolocalisation of Hc-ACOX-1 in the fourth-stage larvae (L4s) and adult of Haemonchus contortus.

Tissues of L4s (A) and female adults (B) of H. contortus are probed with rabbit anti-rHc-ACOX-1.1 polyclonal antibodies followed by fluorescein conjugated-goat anti-rabbit IgG (H+L) as secondary antibody. Nuclei are counterstained with 4’,6-diamidino-2-phenylindole (DAPI). DIC: differential interference contrast, ct: cuticle, in: intestine, hd: hypodermis, nu: nucleus. Sections incubated with pre-immune serum (negative control) are provided in supplementary S5B Fig. Scale bar: 50 μm.

Fig 6. Transcriptional dynamics of Hc-acox-1 in different life cycle stages of Haemonchus contortus.

Transcripts of Hc-acox-1.1 (A), Hc-acox-1.2 (B) and Hc-acox-1.3 (C) in the egg, first- (L1), second- (L2), third- (L3), fourth-larval (L4) stages, diapaused L4 (dL4) and adult female (Af) and male (Am) of H. contortus were detected by quantitative real-time PCR using Hc-β-tubulin as an internal control. Expressional level of Hc-acox-1 at the egg stage is set as one unit, and those of the other life cycle stage are relative to eggs. (D) Relative expression levels of three transcripts compared with each other in different stages of H. contortus. The statistical analysis was performed using 2^−ΔΔCt method in Excel 2016 and one-way ANOVA with Dunnett post-hoc test in GraphPad Prism 5. All Data are resented by mean ± SEM. Three technical replicates are included for three independent experiments. Statistical analysis is performed using one-way ANOVA with Dunnett post-hoc test. *P<0.05, **P<0.01, ***P<0.001, ns: no significance.

Fig 7. Effects of gene knockdown of acox-1 and pex-5 on Haemonchus contortus larval development and survival.

(A) Relative transcriptional levels of Hc-acox-1.1, -acox-1.2, -acox-1.3 and -pex-5 in RNA interference (RNAi)-treated worms. Hc-β-tubulin is used as an internal control. Arabidopsis thaliana light harvesting complex gene (Lhcb4.3) is used as a negative control. H. contortus tropomyosin (Hc-tmy-1) is used as a positive control. The relative abundance of each transcripts in treated worms is compared with that of the negative control. (B-D) Death rates of the first- (L1, B), second- (L2, C) and third- (L3, D) stage larvae of RNAi-treated worms. (E-F) Changes in body length (E) and body width (F) of RNAi-treated L2s of H. contortus. (G-H) Changes in body length (G) and body width (H) of RNAi-treated L3s of H. contortus. Data in all panels are presented in mean ± SEM (n = 20). Statistical analysis is performed using one-way ANOVA with Dunnett post-hoc test. *P<0.05, **P<0.01, ***P<0.001, ns: no significance.