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Acyl-CoA oxidase ACOX-1 interacts with a peroxin PEX-5 to play roles in larval development of Haemonchus contortus
['Hengzhi Shi', 'Institute Of Preventive Veterinary Medicine', 'Zhejiang Provincial Key Laboratory Of Preventive Veterinary Medicine', 'College Of Animal Sciences', 'Zhejiang University', 'Hangzhou', 'Zhejiang', 'Xiaocui Huang', 'Shanghai Institute Of Materia Medica', 'Chinese Academy Of Science']
Date: None

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.

Haemonchus contortus is one of the most pathogenic nematodes causing substantial economic losses worldwide that undergo facultative arrest (diapause) to survive harsh environmental and host conditions. However, the molecular basis (e.g., fatty acid oxidation and dauer pheromone synthesis) underlying such survival mechanisms has not yet been elucidated in parasitic nematodes. Here, we show that an acyl-CoA oxidase ACOX-1 interacts with a peroxin PEX-5 and functions in fatty acid oxidation in peroxisomes. Further, knockdown of ACOX-1 and PEX-5 results in a lethal phenotype of the infective stage and likely developmental parasitic larval stage within host animals. This data demonstrates an essential role of ACOX-1-PEX-5 and potentially ascarosides signalling in the facultative arrested larval development of parasitic nematodes, and suggests possible intervention targets.

In our previous work [ 37 – 40 ], three orthologues of genes maoc-1, dhs-28 and daf-22 involved in the fatty acid β-oxidation pathway have been identified in H. contortus (Trichostrongylidae; the barber’s pole worm), one of the most pathogenic worms that enter developmental arrest in the infective juvenile stage and diapause of the early fourth larval stage (L4) within small ruminants [ 41 – 43 ]. Here, we identified and functionally characterised the previously unknown gene acox-1 in the fatty acid β-oxidation pathway of this parasitic nematode of global economic importance. Fatty acid oxidation activities of acox-1 encoded proteins tested in vitro and in vivo. Subcellular localisation, tissue expression, developmental transcription and RNA interference (RNAi)-mediated gene knockdown analyses were conducted to investigate the functions of ACOX-1 in the fatty acid β-oxidation and developmental arrest of the parasitic nematode H. contortus.

The peroxisomal fatty acid β-oxidation cycle functions in long-chain fatty acid degradation, and in biosynthesis of the short-chain ascarosides in C. elegans [ 10 , 11 , 33 , 34 ]. This β-oxidation cycle involves an acyl-CoA oxidase (ACOX-1), an enoyl-CoA hydratase (MAOC-1), a (3R)-hydroxyacyl-CoA dehydrogenase (DHS-28) and a 3-ketoacyl-CoA thiolase (DAF-22) that function in the oxidation, hydration, dehydration and thiolysis of fatty acids, respectively [ 35 , 36 ]. In particular, mutation of these molecules leads to compromised production of short-chain ascarosides in C. elegans [ 10 , 11 ], suggesting their important roles in dauer pheromone synthesis. Nonetheless, little is known about this fatty acid β-oxidation pathway at the molecular level in major parasitic nematodes of socioeconomic importance.

Dauer pheromones play crucial roles in the developmental arrest of C. elegans, representing a class of 3, 6-dideoxy-L-sugar ascarylose linked with a fatty acid-derived side chain (ascarosides) [ 14 , 15 ]. At least five ascarosides, such as asc-C6-MK (C6; ascr#2), asc-ΔC9 (C9; ascr#3), asc-ωC3 (C3; ascr#5), asc-ΔC7-PABA (ascr#8) and IC-asc-C5 (C5; icas#9), serve as dauer pheromones and play roles in developmental regulation of C. elegans [ 15 – 17 ]. These molecules are sensed by G protein-coupled receptors (GPCRs) of specific chemosensory neurons [ 16 , 18 – 20 ], inhibiting cyclic guanosine monophosphate (cGMP) [ 21 , 22 ], transforming growth factor β (TGF-β) [ 23 , 24 ], and insulin/insulin-like growth factor 1 (IGF-1) [ 25 – 27 ], which converge on steroid hormone receptor inactivation for a molecular decision of dauer formation [ 28 , 29 ]. Apart from C. elegans, ascarosides have also been detected in a range of free-living and parasitic nematodes, suggesting conserved ascarosides synthesis and signalling machinery among nematodes [ 30 – 32 ]. However, the genetic basis for such machinery has not yet been elucidated in parasitic nematodes, particularly species of veterinary and medical importance.

Temporary cessation of development among nematodes in response to certain circumstances or within certain host animals, known as facultative developmental arrest or hypobiosis, is an ability to interrupt the life cycle to survive harsh conditions [ 1 – 3 ]. For instance, in the free-living nematode Caenorhabditis elegans, larvae arrest their development at the third-larval stage (L3) and enter a stress-resistant dauer stage under adverse conditions such as food shortage, high population density, or high temperature [ 4 , 5 ]. Similarly, in parasitic nematodes (e.g., Ancylostomatidae, Ascaridae, Strongyloididae, Trichostrongylidae, Trichonematidae), infective larvae can also cease their development at an early parasitic stage in response to seasonal/host factors, and do not resume their development until conditions become favourable [ 1 , 6 , 7 ]. Mechanisms underlying such developmental arrest of nematodes and related phenomena have been proposed and investigated for decades [ 1 – 3 ], with advanced understanding achieved mostly in the model organism C. elegans [ 8 – 13 ].

(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.

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.

Different transcriptional patterns were observed for the three transcripts Hc-acox-1.1, -1.2 and -1.3 among key developmental stages of H. contortus. Specifically, Hc-acox-1.1 was highly transcribed in the egg and diapause stages, particularly in the latter ( Fig 6A ); Hc-acox-1.2 was predominantly transcribed in the egg, L1, L2 and L3 stages collected from the environment while transcribed at low level in the diapause stage collected from the host animals ( Fig 6B ); Hc-acox-1.3 was highly transcribed in the early developmental stages (i.e., egg, L1, L2 and L3), with a relatively higher transcriptional level detected in the diapause stage of H. contortus ( Fig 6C ). None of the three transcripts was highly expressed in the L4 and adult female and male of this parasitic nematode ( Fig 6A–6C ). The relative transcriptional level of Hc-acox-1.1 in egg and diapaused L4 stage was significantly higher than that in other stages ( Fig 6D ).

Polyclonal antibodies generated against each recombinant protein rHc-ACOX-1.1, -1.2 and -1.3 recognised the native Hc-ACOX-1 proteins of H. contortus, but could not distinguish the proteins ( S4 Fig ). Using the polyclonal antiserum (anti-rHc-ACOX-1.1), protein distribution of Hc-ACOX-1 was shown to predominate in the intestine and hypodermis of L4s ( Fig 5A ) and the adults of H. contortus (Figs 5B and S5A ).

(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.

A punctate distribution was observed for Hc-ACOX-1 protein in the cytoplasm of HEK293T cells ( Fig 3A ), which colocalised with peroxisomes, rather than mitochondria ( S2 Fig ). Without a PTS1 sequence, Hc-ACOX-1 could not be translocated to peroxisomes and was homogenously distributed in the cytoplasm ( Fig 3B ). The peroxisomal distribution of Hc-ACOX-1 indicated potential interactions between Hc-ACOX-1 and peroxins, which was verified by the growth of positive yeast clones expressing Hc-ACOX-1 and Hc-PEX-5 on QDO plates (quadruple dropout supplements, SD/-Leu/-Trp/-His/-Ade) in the yeast two hybrid (Y2H) assay ( S3 Fig ). By contrast, yeast expressing Hc-ACOX-1 without PTS1 could not grow on QDO plates, in accordance with the untargeted peroxisomal distribution of PTS1-lossing Hc-ACOX-1 ( S3 Fig ). In addition, an interaction between flag-tagged Hc-ACOX-1 and HA-tagged Hc-PEX-5 was confirmed in a co-immunoprecipitation (Co-IP) assay, and PTS1 also played an essential role in the recognition of Hc-ACOX-1 by Hc-PEX-5 ( Fig 4 ).

(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 OD 600 = 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.

The potential role of Hc-ACOX-1 in fatty acid β-oxidation was evaluated by testing the palmitoyl-CoA oxidase activity of the recombinant protein in vitro ( S1A Fig ). Specifically, optimum temperatures for the activity of rHc-ACOX-1.1, -1.2 and -1.3 were determined by measuring activity at 37°C, 42°C and 42°C, respectively, and a consistent optimum pH at 7.5 ( S1C and S1D Fig ). Under optimum conditions, rHc-ACOX-1.1, -1.2 and -1.3 showed different potencies of oxidase activity, with maximum reaction rate (Vmax) measured at 0.53 μM/min, 1.35 μM/min and 1.01 μM/min, respectively, and Michaelis constant (Km) at 78.79 μM, 6.83 μM and 12.64 μM, respectively ( Fig 2A and 2C ). The function of Hc-ACOX-1 in fatty acid oxidation was further confirmed in Saccharomyces cerevisiae. Specifically, supplementation of oleic acid in the culture medium promoted the growth of wild type yeast expressing Hc-ACOX-1, and the deficiency of Δpox1 (peroxisomal acyl-CoA oxidase loss-of-function strain) in utilising oleic acid and growing in YNBO medium (YNB medium supplemented oleic acid as the sole carbon source) was rescued by expressing Hc-ACOX-1, with different potencies detected for the three proteins ( Fig 2D and 2E ). Deleting the PTS1 of Hc-ACOX-1 led to compromised growth of wild-type yeast in YNBO medium ( Fig 2D ). Introducing site (i.e., binding and active sites) mutations into the amino acid sequences of Hc-ACOX-1 compromised the growth of Δpox1 on the YNBO plate. However, mutations at Thr (151) and Glu (433) in Hc-ACOX-1.1 showed weak effects on the growth of Δpox1 ( Fig 2F and 2G ). Although single mutation in Hc-ACOX-1.1 (151A or 190A) did not show obvious effect on the growth of Δpox1 on the YNBO plate, multiple mutations compromised the growth of Δpox1 ( Fig 2G ).

(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.

Three transcripts were identified by mapping molecularly cloned sequences to the reference genome of H. contortus, matching two gene loci Hc-acox-1.1 (chromosome IV: 9780094–9786809) and Hc-acox-1.2 (chromosome IV: 9789018–9796292) in chromosome IV of this parasitic nematode ( Fig 1A ). Specifically, Hc-acox-1.1 was a full-length transcript (2019 nt in length; GenBank accession number: MZ229680), whereas Hc-acox-1.2 (1338 nt in length; GenBank accession number: MZ229681) ( Fig 1A ). A similar transcript (Hc-acox-1.3) (1338 nt in length with 91.93% identity to Hc-acox-1.2; GenBank accession number: MZ229682) was partially matched with the Hc-acox-1.2 gene model, representing a possible locus not part of the current genome assembly ( Fig 1A ). Three amino acid sequences Hc-ACOX-1.1, Hc-ACOX-1.2 and Hc-ACOX-1.3 were deduced from the gene models of Hc-acox-1 in H. contortus. Specifically, two flavin adenine dinucleotide (FAD)-binding sites Thr (151) and Gly (190) and an active site Glu (433) were predicted in Hc-ACOX-1.1, with only active site Glu (206) indicated in the deduced partial Hc-ACOX-1.2 and Hc-ACOX-1.3 ( Fig 1B ). A peroxisomal targeting signal (PTS1) was predicted at the carboxyl terminal of each deduced Hc-ACOX-1 protein ( Fig 1B ).

Discussion

In this work, we identified a key gene, Hc-acox-1, that is likely involved in the peroxisomal fatty acid β-oxidation pathway of H. contortus, a parasitic nematode of economic significance worldwide. Hc-acox-1.1, -1.2 and -1.3 encode three proteins, which all show a fatty acid oxidation activity and an interaction with PEX-5 in peroxisomes. Hc-acox-1 is predominantly detected in the intestine and hypodermis of H. contortus, particularly in the early larval stages and the diapaused L4s. Gene knockdown analysis indicates that Hc-acox-1 and Hc-pex-5 are crucial for early larval development and likely in the facultative developmental arrest (hypobiosis) of H. contortus.

Identification of the previously unknown Hc-acox-1 fills a gap in the peroxisomal fatty acid β-oxidation pathway in parasitic nematodes, particularly H. contortus. Peroxisomal β-oxidation participates in lipid metabolism by oxidizing long-chain fatty acids and branched-chain fatty acids, which is crucial for the biosynthesis of dauer pheromones, short-chain ascarosides secreted by the free-living nematode C. elegans in response to environmental variants [4,16,44,45]. Although ascarosides have been identified in a broad range of free-living and parasitic nematodes and might play roles in mediating a variety of behaviours [30,46], little is known about these aspects in H. contortus and related species of the order Strongylida–one of the largest groups of pathogenic worms in animals. In addition, specific ascaroside profiles were also indicated among species, suggesting species specificity of ascaroside synthesis and signalling among nematodes [47]. However, little is known about mechanisms underlying the species specificity of ascaroside biosynthesis in parasitic nematodes. In our previous work [37–40], three (maoc-1, dhs-28 and daf-22) of four genes that are known to be involved in the fatty acid β-oxidation have been identified in H. contortus, which implies a relatively conserved fatty acid β-oxidation pathway between this parasitic nematode and the model organism C.elegans. In the current work, identification of the last gene homologue Hc-acox-1 confirmed the genetic basis for fatty acid β-oxidation cycles and short-chain ascaroside biosynthesis in this parasitic nematode. On this genetic basis, experiments for detecting specific ascarosides and exploring their biological roles in H. contortus and related parasitic nematodes can be performed with greater confidence.

Our heterologous expression studies support that Hc-ACOX-1 plays a role in fatty acid oxidation in peroxisomes by interacting with Hc-PEX-5. Although encoded by three transcripts of two genes, enzymatic activities of all the three proteins in fatty acid oxidation have been verified both in vitro and vivo. Interestingly, Hc-ACOX-1.2 and Hc-ACOX-1.3 proteins without FAD-binding site showed higher enzymatic activities, suggesting a likely regulatory role of this binding-site in the fatty acid oxidation, which should be further investigated. In particular, heterologous expression of Hc-ACOX-1 rescued the deficiency of Δpox1 strain of S. cerevisiae in utilising oleic acid and promoted the growth of wild-type S. cerevisiae. Importantly, we found that the peroxisomal targeting signal (PTS1) was essential for the acyl-CoA oxidase activity of Hc-ACOX-1. Without the PTS1 sequence, Hc-ACOX-1 was homogenously distributed in the cytoplasm and could not be translocated into the peroxisomes [48,49], which explains the failed rescue of Δpox1 strain of S. cerevisiae using a PTS1-lossing mutant [50,51]. The essential role of PTS1 was further confirmed by Y2H and Co-IP assays, in which Hc-ACOX-1 is recognised by Hc-PEX-5 via the PTS1 [51]. Peroxins (PEX) are required for peroxisome biogenesis [52], particularly in importing peroxisomal matrix proteins synthesised in the cytosol into the peroxisomes [53]. Proteins with a PTS1 can be recognised by PEX which serves as a receptor for matrix-targeted proteins [54–57]. From this, we conclude that function of Hc-ACOX-1 in peroxisome is Hc-PEX-5-dependant, suggesting a class of potential intervention targets of fatty acid oxidation in H. contortus.

ACOX-1 and PEX-5 might play important roles in the post-embryonic larval development and facultative developmental arrest of parasitic nematodes. This statement can be supported firstly by the developmental transcription analysis of Hc-acox-1, which showed high mRNA levels of specific spliced variants in the early larval stages (free-living stages in the environment) of H. contortus, as well as in the diapause stage (early parasitic stage in the host animals) of this nematode. It is likely a molecular response in the free-living larvae to the environmental variants, as there is no stable food availability, temperature and moisture before entering into a host. It is also a possible molecular alteration for facultative developmental arrest of L4s within host animals, particularly when seasonal and host immune or physiological conditions are unfavourable for a continuous development of this parasitic nematode [58]. In addition, the potential roles of ACOX-1 and PEX-5 in early larval development and developmental arrest of H. contortus are also supported by the gene knockdown analyses. For instance, knockdown of Hc-acox-1 and Hc-pex-5 resulted in significantly changed body length and width of the L2s and L3s of H. contortus.

There are also some questions pertaining to acox-1 and pex-5 that still need to be further investigated in H. contortus and related parasitic nematodes. In particular, the different transcriptional patterns of spliced variants of Hc-acox-1 and their specific functions (e.g., synthesis of ascarosides with specific side-chain lengths) are not clear. Knockdown of Hc-pex-5 resulted in a lethal phenotype in H. contortus at the second larval stage, which is of major interest to be understood in detail as it might play multiple roles in the essential biological processes of this parasitic nematode. Additionally, little is known about the functions (particularly the essentiality) of Hc-acox-1 and Hc-pex-5 gene homologues in related species of the order Strongylida, and other socioeconomically important parasitic nematodes. Moreover, functional interactions between ACOX-1 and other peroxins, as well as functional relationships among different ACOXs warrant further investigation [35,59]. A better understanding of these aspects should provide insights into the developmental biology of parasitic worms and the discovery of novel targets to control major parasitic diseases.

In conclusion, we identified a palmitoyl-CoA oxidase ACOX-1 in H. contortus, which appeared to be required for normal post-embryotic larval development in the environment and likely in hypobiosis within host animals. Perturbation of this molecule and its interacting protein PEX-5 results in shortened lifespan and even lethality in H. contortus, suggesting novel potential targets for the control of parasitic diseases of socioeconomical significance.

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