International Journal for Parasitology 44 (2014) 775–786
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Hc-fau, a novel gene regulating diapause in the nematode parasite Haemonchus contortus Baolong Yan a, Xiaolu Guo a, Qianjin Zhou b, Yi Yang a, Xueqiu Chen a, Weiwei Sun c, Aifang Du a,⇑ a
Institute of Preventive Veterinary Medicine & Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310058, China School of Marine Science, Ningbo University, Ningbo 315211, China c Laboratory for Evolution & Development, Institute of Evolution & Marine Biodiversity and Department of Marine Biology, Ocean University of China, Qingdao 266003, China b
a r t i c l e
i n f o
Article history: Received 6 March 2014 Received in revised form 27 May 2014 Accepted 28 May 2014 Available online 21 July 2014 Keywords: Nematode Haemonchus contortus Hc-fau Diapause Caenorhabditis elegans
a b s t r a c t Diapause induced in the early fourth stage of Haemonchus contortus is a strategy to adapt this nematode to hostile environmental conditions. In this study, we identified a new gene, Hc-fau, a homologue of human fau and Caenorhabditis elegans Ce-rps30. Hc-fau encodes two proteins through alternative RNA splicing, Hc-FAUA and Hc-FAUB, consisting of 130 and 107 amino acids, respectively. Hc-FAU possesses a diverged ubiquitin-like (UBiL) protein domain and a conserved ribosome protein S30 domain. The protein is ubiquitously expressed, except in the gonad. However Hc-fau transcripts decrease significantly in diapausing L4s of H. contortus. In C. elegans, knockdown of Ce-rps30 confers an extended lifespan, increased lipid storage in the intestine and shortened body length. These morphological characteristics are comparable with dauer larvae of C. elegans, in which the gonad is condensed considerably. In contrast, a shortened lifespan is observed in C. elegans over-expressing Hc-faua, and especially Hc-faub, with hatching failure detected. The genes of insulin/IGF-1 signalling (IIS), TGF-b, cGMP, dafachronic acid (DA), apoptosis (AP) and fatty acids (FA) metabolism are all down-regulated in Ce-rps30RNAi (RNA interference) worms, except for akt-1 and daf-16. However, daf-16 up-regulation is inconsistent with its target gene down-regulation and the result from a heat stress assay in these worms. Daf-16 RNAi conducted in Cerps30 (tm6034/nt1) mutants failed to rescue the worms. The S30 domain stays in the nucleus, while UBiL accumulates in the cytoplasm. Compared with Hc-FAUA, results of UBiL domain and S30 domain overexpression indicate synergism between UBiL and S30 in regulating lifespan and reproduction. These results suggest the potential functions of Hc-fau in regulating larval diapause in H. contortus. Ó 2014 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.
1. Introduction Nematodes are capable of periods of arrested development (Sommerville and Davey, 2002). Dauer, a specialised stage in the free-living nematode Caenorhabditis elegans, is one form of arrested development, which occurs in L3s (Golden and Riddle, 1984). Dauer larvae of C. elegans survive hostile environmental conditions such as scarcity of food, high temperature and high population density (Riddle and Albert, 1997). Dormancy terminates when conditions become favourable for normal activity (Sommerville and Davey, 2002). In the parasitic nematode Haemonchus contortus, diapause, another form of arrested development, occurs in the early fourth stage in abomasums of ruminants (Blitz and Gibbs, 1971). ⇑ Corresponding author. Postal address: Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China. Tel.: +86 571 8898 2583. E-mail address: [email protected] (A. Du).
Diapause is a strategy to adapt this nematode to hostile environmental conditions such as low oxygen tension, short photoperiod, low temperature and host immune response (Sommerville and Davey, 2002). The dauer stage, regulated by four molecular pathways, cGMP (Ren et al., 1996), insulin (Kimura et al., 1997), TGF-b (Birnby et al., 2000) and dafachronic acid (DA) signalling via the nuclear hormone receptor DAF-12 (Motola et al., 2006; Crook, 2014), has been extensively studied in the model organism C. elegans (Angelo and Van Gilst, 2009). It has been suggested that the infective L3s (iL3s) of parasitic nematodes are analogous to dauer larvae of C. elegans (Rogers and Sommerville, 1963; Crook, 2014; Zhang et al., 2013), and some conserved pathways have also been determined in parasitic nematodes (Crook, 2014). However, the diapausing parasitic nematodes (L4s) do not, according to the definition in this paper, equate with the dauer larvae of C. elegans (Sommerville and Davey, 2002). In this study, we use C. elegans as a tool to explore the in vivo functions of a gene we cloned from H. contortus (Hc-fau).
http://dx.doi.org/10.1016/j.ijpara.2014.05.011 0020-7519/Ó 2014 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.
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We identified Hc-fau from a mRNA differential display assay between normal larvae and diapausing larvae of H. contortus. It is a homologue of human fau (H.s-fau) (Kas et al., 1992) and Ce-rps30 (C26F1.4). The fox (FBR osteosarcoma X) sequence from the FinkelBiskis-Reilly murine sarcoma virus (FBR-MuSV) is an antisense sequence to fau (FBR-MuSV associated ubiquitously expressed gene) (Van Beveren et al., 1984; Michiels et al., 1993). Expression of fox increases the transforming capability and tumorigenicity of the virus, presumably by inactivating fau expression in the mouse (Michiels et al., 1993). Expression of fau is down-regulated in both breast cancer (Pickard et al., 2009) and ovarian cancer (Moss et al., 2010). A sequence antisense to fau is able to inhibit apoptosis (AP) induced by dexamethasone, UV or cisplatin in W7.2c cells (Mourtada-Maarabouni et al., 2004). FAU was also found to regulate apoptosis in human T-cell lines and HEK293/17 cells (Pickard et al., 2009). fau encodes a ubiquitin-like protein (UBiL), fused to ribosomal protein S30 (S30) as a carboxy-terminal extension (Kas et al., 1992). These two products are thought to result from post-translational cleavage (Pickard et al., 2011). Human UBiL has 37% amino acid sequence identity (57% sequence similarity) to ubiquitin and retains the C-terminal G-G dipeptide motif that participates in isopeptide bond formation between ubiquitin and lysine of target proteins. A lack of internal lysine residues, sites of poly ubiquitin chain formation, indicates that the biological function of UBiL is distinct from that of ubiquitin (Kas et al., 1992; Pickard et al., 2011). The identification of UBiL covalently bound to Bcl-G, a member of the Bcl-2 family of apoptosis control proteins (Nakamura and Tanigawa, 2003), suggests a pro-apoptotic regulatory role for fau, mediated via Bcl-G (Mourtada-Maarabouni et al., 2004; Pickard et al., 2011). In this report, we describe the cloning, initial characterisation and functional study of the novel gene Hc-fau from H. contortus, with the aim of investigating its role in regulating nematode parasite diapause.
Caenorhabditis elegans strains (N2) were maintained on Nematode Growth Media (NGM) agar plates at 20 °C as described previously (Brenner, 1974). Worms were fed Escherichia coli strain OP50 unless otherwise stated. Sheep used as experimental animals were treated according to the recommendation in the Guide for the Regulation for the Administration of Affairs concerning Experimental Animals of the People’s Republic of China. Animal experiments were approved by Zhejiang University Experimental Animal Ethics Committee (Permit Number: 2009/01). The care and maintenance of sheep followed the guidelines of this institution.
2. Materials and methods
2.4. Isolation of full-length genomic DNA of Hc-fau
2.1. Propagation of H. contortus and C. elegans
Full-length genomic DNA of Hc-fau from the ZJ strain of H. contortus was obtained by a genome walking kit (Takara Biotechnology Co., Ltd.), using primers designed based on the acquired cDNA sequence (Supplementary Table S1), following the manufacturer’s instructions. The third-round PCR products were cloned into a pMD18-T vector and sequenced.
Sheep, maintained under helminth-free conditions, were infected intraruminally with 8,000 iL3s of H. contortus ZJ strain, which is maintained and propagated in Zhejiang University, China. The presence of parasites was confirmed at day 24 by detecting strongylid eggs in the faeces using the flotation method (Cox and Todd, 1962). All sheep were held in metabolism cages in concrete-floored pens, where the faeces could be collected in the metal trays in order to preclude the possibility of re-infection occurring during this period. L1s, L2s and iL3s were collected after 1, 3 and 7 days of incubation of faeces at 28 °C, respectively. For infection, larvae were counted by a dilution technique. Exsheathment of L3 worms (xL3s) was carried out with NaOCl using the methods described by Rothwell and Sangster (1993). Haemonchus contortus diapause ZJ strains were obtained according to Blitz and Gibbs (1971). Experimentally infected ewes (5 months old) were orally administered 8,000 to 10,000 H. contortus iL3s. Dexamethasone sodium phosphate (1 ml:5 mg) was injected i.m. in doses of 0.5 mg/kg liveweight 1 day before and 3 days p.i. Before autopsy, all the lambs were held for 60 days in metabolism cages where re-infection did not occur during this period. After euthanizing the animals and opening the abomasums, the ingesta and washings were fixed in 7% formol saline and abomasal mucosa was digested in peptic-HCl. Then the worms were detected under an anatomical lens (Motic, China). Adults of H. contortus ZJ strain were collected from sheep abomasums at necropsy using fine forceps and washed extensively in chilled (4 °C) PBS. All worm samples were stored in liquid nitrogen until use.
2.2. Isolation, purification, treatment and storage of nucleic acids Total genomic DNA was extracted from adult worms using a small-scale genomic DNA extraction kit (Takara Biotechnology Co., Ltd., Japan). Total RNA was extracted from worms at different developmental stages employing Trizol reagent (Invitrogen, USA), followed by treatment with 2 U of DNase I (Takara Biotechnology Co., Ltd.). First strand cDNA was obtained using the M-MLV RTase cDNA Synthesis Kit (Takara Biotechnology Co., Ltd.). Both DNA and RNA samples were stored at 70 °C. 2.3. Isolation of full-length cDNA encoding Hc-fau from H. contortus Using gene-specific primer pairs (Supplementary Table S1), designed based on the available expressed sequence tag (EST) sequence, two partially overlapping cDNA fragments were obtained from total RNA isolated from adult H. contortus using 50 - and 30 - rapid amplification of cDNA ends (RACE) (Takara Biotechnology Co., Ltd.). Products were cloned into a pMD18-T vector and sequenced. Based on these sequences, additional primers (Supplementary Table S1) were designed to amplify full-length Hc-faua and Hc-faub.
2.5. Whole-mount in situ hybridisation (WISH) Haemonchus contortus xL3s, diapausing L4s and C. elegans L3s were used for WISH. Hc-fau and Ce-rps30 cloned into the vector pGEM-T Easy were linearised using endonuclease NcoI, antisense probes were synthesised using Sp6 RNA polymerase and primers were as listed in Supplementary Table S1. WISH was performed as described by Thisse and Thisse (2008), and images were taken under a microscope (Nikon, Japan). 2.6. Caenorhabditis elegans dauer formation in liquid Caenorhabditis elegans dauer larvae were produced according to the method described (Gottlieb and Ruvkun, 1994). To induce synchronous dauer formation in liquid, eggs were isolated by bleach treatment from gravid hermaphrodites and incubated in S Basal (Sulston and Hodgkin, 1988) in the absence of food for 12–16 h at 25 °C to synchronise all animals at the L1 stage. Then a large number of synchronised L1s were placed into a flask (50 ml) containing 10 ml of S Medium (Sulston and Hodgkin, 1988), 5 ll of a crude pheromone preparation (prepared as described in Golden and Riddle, 1984) and 600 ll of a 4% solution of streptomycin-treated
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bacteria E. coli OP50. The flask was then placed in a shaking water bath for 72 h at 25 °C. 2.7. Quantitative real-time PCR (qRT-PCR) analysis qRT-PCR was performed to determine the abundance of Hc-fau transcripts in different developmental stages (L1, L2, L3, diapause, adult) of H. contortus, Ce-rps30 transcripts in adults and dauer larvae of C. elegans, and some candidate gene transcripts in Cerps30RNAi (RNA interference) C. elegans. Gene expression levels were determined by RT-PCR using SYBRÒ Green PCR Master Mix and a 7500 Real-Time PCR System (Applied Biosystems, USA). Relative gene expression was compared with actin (act-1) or b-tubulin as an internal loading control. The target genes and the primers used are listed in Supplementary Table S1. Statistical analysis was conducted using a one-way ANOVA; P 6 0.05 was set as the criterion for significance. 2.8. RNAi feeding experiments To generate Ce-rps30- and daf-16-specific RNAi vectors, Ce-rps30 and daf-16 cDNAs were cloned into the L4440 vector. Plasmids were transformed into E. coli strain HT115. Primers used for PCR analysis are listed in Supplementary Table S1. RNAi plates and media were prepared according to Kwon et al. (2010). Gravid adults of C. elegans were allowed to lay eggs overnight on the RNAi plates and adult worms were picked off. Empty vector-containing E. coli were used on separate plates as negative controls. 2.9. Transgenic worms To analyse promoter activity of Hc-fau, the promoter regions were amplified and cloned into plasmid pPD95.77 to construct pHc-fau::gfp and pCe-rps30::gfp. To perform over-expression of Hc-FAU, Ce-RPS30 and their functional domains, fragments were amplified accordingly and cloned into pPD95.77 to construct plasmids with the promoter of Ce-rps30. Hc-FAU/Ce-RPS30 were expressed in separate transgenic lines as fusion proteins with red fluorescent protein (RFP) upstream and GFP downstream, and Hc-FAUA-UBiL/Ce-RPS30-UBiL with RFP upstream, Hc-FAU-S30/ Ce-RPS30-S30 with GFP downstream, Hc-FAUB-UBiL with RFP upstream and GFP downstream (Fig. 1). All primers used are listed in Supplementary Table S1.
Fig. 1. The constructs of over-expression plasmids. Plasmids encoding for Haemonchus contortus FAU/Caenorhabditis elegans RPS30 (Hc-FAU/Ce-RPS30) were constructed with red fluorescent protein (RFP) upstream and GFP downstream, and Hc-FAUA-ubiquitin-like (UBiL)/Ce-RPS30-UBiL with RFP upstream, Hc-FAU-S30/ Ce-RPS30-S30 with GFP downstream, and Hc-FAUB-UBiL with RFP upstream and GFP downstream. Fragments were cloned into pPD95.77 plasmids with the promoter of Ce-rps30.
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Recombinant plasmids were each microinjected into the gonad of young, adult wild-type (N2) C. elegans hermaphrodites as described (Mello et al., 1991), together with plasmid pRF4 containing a dominant mutant allele of rol-6 gene, each at a final concentration of 50 lg/mL in the same mixture, using pPD95.77 (pCe-rps30::gfp) and pRF4 plasmid mixture as a control. The F2 and subsequent generations with a roller phenotype were analysed and selected to examine the expression patterns of GFP or RFP, using a fluorescent microscope (Olympus IX71). A minimum of three independent lines expressing each transgene were evaluated.
2.10. Lipid staining Oil Red O staining was performed as described by Soukas et al. (2009). Worms were washed off the NGM or RNAi plates and incubated in PBS buffer for 30 min on a shaker at room temperature. The worms were then fixed in Modified Ruvkun’s witches brew (MRWB) buffer containing 1% paraformaldehyde (Soukas et al., 2009). After three rounds of freezing/thawing, the worms were dehydrated in 60% isopropanol followed by addition of saturated Oil-Red-O (Sigma, USA) solution. Fixed worms were incubated overnight on a shaker at room temperature, mounted on slides and viewed using a microscope with differential interference contrast optics (Nikon, Japan).
2.11. Heat stress assays Ce-rps30RNAi (10 days old) and N2 (L4) C. elegans were picked onto fresh NGM plates (a total of 21 plates) seeded with E. coli and incubated at 20 °C for 18 h, followed by an upshift to 37 °C. At hourly intervals, three plates of each strain were removed directly from 37 °C and worms were allowed to recover at 20 °C for approximately 12 h after which individual C. elegans were assessed as alive or dead. Heat stress-induced mortality was determined by tapping the worms with a platinum wire to check for motility; unresponsive individuals were scored as dead. Statistical analyses for survival were conducted using the chi-square-based log rank test (Kwon et al., 2010).
2.12. Lifespan measurement, brood size and body size Measurement of body length was performed as described (Mörck and Pilon, 2006). Different stages of worms were picked and photographed. Body length measurements were performed with the free Java image processing program ImageJ. Larvae and adults were measured from the nose to the tail tip. The brood size was determined as described (Wong et al., 1995). Larvae were placed singly onto fresh plates and incubated at 20 °C until they had matured and laid the first few eggs. The hermaphrodites were then transferred onto fresh plates daily to prevent overcrowding until egg laying ceased. The progeny were counted 3 days after removal of the parents. A lifespan assay was carried out according to the method described (Wong et al., 1995). The L4s were placed at 20 °C until they laid the first few eggs, then the adult worms were picked out and eggs allowed to hatch to larvae. Larvae were placed singly onto fresh plates, and monitored once daily until death. The animals were transferred once daily while producing eggs to keep them separate from their progeny. Animals were scored as dead when they no longer responded with movement to light prodding of the head. All C. elegans were kept at 20 °C. Animals that crawled off the plates during the assay were excluded from calculations. P values were derived from Student’s t-tests.
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2.13. Whole animal DAPI staining
3.2. Identification and sequence analysis of the Hc-fau gene and cDNAs
Haemonchus contortus diapause L4 worms and iL3s were fixed in ice cold, 100% methanol (5 min, 20 °C) and washed once with PBS. Fixed animals were incubated (30 min, room temperature, protected from light) in DAPI solution at a final concentration of 500 ng/ml. Stained animals were washed once in PBS and transferred to a glass slide. Images were captured with FV1000 spectral confocal and fluorescent (Olympus IX71) microscopes.
An EST was identified from a mRNA differential display experiment comparing L4s with arrested L4s of H. contortus (data not shown), that had a predicted polypeptide sequence with similarity to the RPS30 superfamily domain, and with most similarity to the human H.s-FAU and C. elegans Ce-RPS30 proteins. The full-length cDNAs of two transcripts, Hc-faua and Hc-faub, were isolated by RACE from H. contortus. The full-length cDNA of Hc-faua was 521 bp in length, including an open reading frame (ORF) of 393 bp (including stop codon), a 50 -untranslated region (UTR) of 55 bp, and a 30 -UTR of 73 bp. The full-length cDNA of Hc-faub was 602 bp in length, containing an ORF of 324 bp (including stop codon), a 50 -UTR of 205 bp and a 30 -UTR of 73 bp (Fig. 3A, B). The cDNAs of Hc-faua and Hc-faub encoded predicted proteins of 130 and 107 amino acids, respectively (Fig. 3A). An alignment of the two protein sequences revealed that the 103 amino acid C-terminal domains of both (positions 28–130 for Hc-FAUA and positions 5–107 for Hc-FAUB) were identical (Fig. 3A). The predicted amino acid sequences of Hc-FAUA and Hc-FAUB were aligned with H.s-FAU and Ce-RPS30 (Fig. 3A). The alignment showed that the C-terminal S30 domains were conserved (Hc-FAU-S30 versus
3. Results 3.1. Identification of H. contortus diapause In sheep killed 60 days p.i., only two distinct populations of H. contortus were found: mature parasites and early L4s (Fig. 2A). The larvae were characterised by similar length, measuring 1067 ± 97 lm (n = 18) and with rod-like crystalline inclusions in the intestinal cells (Fig. 2B, C), corresponding to the morphological characteristics described previously (Blitz and Gibbs, 1971). Genital primordia were present in diapausing H. contortus L4s (Fig. 2D–F), but these were not present in iL3s (Fig. 2G–I).
Fig. 2. Morphological characterisation of Haemonchus contortus diapause. (A) Anterior end of the H. contortus diapause larva. (B, C) The crystalline inclusion in the intestinal cells (black arrows), and intestinal lumen (white arrow). (D–F) Whole animal DAPI staining revealed a genital primordium in an H. contortus diapausing L4, and a vulval ovejector primordium (white arrow). (G–I) Whole animal DAPI staining revealed no genital primordium in an H. contortus infective L3 (iL3).
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Fig. 3. Sequence analysis of Haemonchus contortus Hc-fau cDNAs. (A) Alignments of the Hc-FAUA, Hc-FAUB, human FAU (H.s-FAU) and Caenorhabditis elegans Ce-RPS30. Identical and similar residues are shown in black and grey blocks, respectively. The potential cleavage sites (Gly-Gly) of the fusion protein (ubiquitin-like; UBiL-ribosome protein S30; S30) are indicated with black arrows (upstream and downstream sequences are UBiL and S30 domains, respectively). The predicted nuclear location signals in the S30 domains are indicated with a blue block. (B) The exon–intron organisation of the Hc-fau gene. The Hc-fau gene spans 2590 bp, with alternative splicing at the 50 -end of exon 2 accounting for the production of the Hc-FAUA and Hc-FAUB proteins, where the splice-acceptor sites at nucleotide position 307 versus 1279 in the gene are utilised for Hc-FAUA and Hc-FAUB, respectively. The positions of the start and termination codons are indicated, with coding regions in grey blocks and non-coding 50 - and 30 untranslated region (UTR) sequences in empty blocks. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Ce-RPS30-S30 and H.s-FAU-S30, 88.1% and 79.7% similarity, respectively), whereas the N-terminal UBiL domains were divergent (HcFAUA-UBiL versus Ce-RPS30-UBiL and H.s-FAU-UBiL, 33.8% and 31.4% similarity, respectively). The complete Hc-fau gene, including its 50 flanking sequence, was isolated by Genome Walking (see Section 2.4) and sequenced. Comparison with the cDNA sequences suggests an alternative mRNA splicing mechanism in which a different splice acceptor site associated with exon 2 is utilised, resulting in a change in the proximal reading frame (Fig. 3B). The 50 UTR contains 11 consecutive pyrimidines (TCTTTCTTTCC), which are found at the 50 end of eukaryotic ribosomal protein mRNAs (Olvera and Wool, 1993), and are hypothesised to play a role in the regulation of translation (Levy et al., 1991). The 30 UTR harbours the hexamer AATAAA (positions, 56 downstream of the TAA). Another 387 bp sequence was amplified from the genomic DNA of H. contortus. Sequence analysis revealed that it was 91.7% identical to the Hc-faua cDNA sequence (Supplementary Fig. S1). However, it lacks both introns and the complete 50 UTR of Hc-faua. Several point and deletion mutations in the sequence lead to a premature stop codon and ORFs for Hc-FAUA/B no longer exist. Thus, we defined the sequence as a processed pseudogene: Hc-fau-ps.
Transgenic lines showing the roller phenotype were selected. Expression of pHc-fau::gfp was restricted to the distal intestine of C. elegans in all stages from L1 to adult (Fig. 4A, B; only adult shown), whereas pCe-rps30::gfp was expressed in almost all cells except for the gonad (Fig. 4C). In situ localisation revealed that Hc-fau transcripts are present in all cells in H. contortus xL3s (Fig. 4E), but absent in diapausing L4s of the parasite (Fig. 4F). However, the probe used detected both mRNAs encoding Hc-FAUA and Hc-FAUB. Ce-rps30 mRNA was detected widely in C. elegans L3s (Fig. 4D), consistent with H. contortus xL3s. qRT-PCR was performed to determine the relative abundance of Hc-fau transcripts in different developmental stages throughout the life cycle of H. contortus (Fig. 4H) and of Ce-rps30 in C. elegans dauer (Fig. 4I). We observed expression of Hc-fau at approximately the same level as the control genes in all stages, except in diapausing L4s. Hc-fau expression was significantly down-regulated in H. contortus diapausing L4s (Fig. 4H), consistent with Ce-rps30 expression in C. elegans dauers (Fig. 4I) which was significantly down-regulated in dauer L3s of C. elegans.
3.3. The expression patterns of Hc-FAU
To investigate Hc-FAU in vivo functions, Hc-FAU, Ce-RPS30 and their functional domains were over-expressed in transgenic C. elegans, using the Ce-rps30 50 -flanking region as the promoter. Hc-FAU and Ce-RPS30 were expressed in separate transgenic lines as fusion proteins with RFP upstream and GFP downstream, and the other
The 2,000 bp sequence upstream of the Hc-fau 50 -UTR was used as a putative promoter and cloned to construct a promoter-gfp fusion in order to analyse transcriptional activity in C. elegans.
3.4. Consequences of post-embryonic development and lifespan deficiency in C. elegans overexpressing Ce-rps30/Hc-fau
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Fig. 4. Expression patterns of Haemonchus contortus Hc-FAU. (A–C) The promoter activity of Hc-fau/Ce-rps30 in Caenorhabditis elegans. Expression of GFP is restricted in intestinal cells in pHc-fau::gfp worms, but is ubiquitous in pCe-rps30::gfp worms, except in the gonad. Arrows indicate the following tissues: i, intestinal; m, muscle; n, neuron; p, pharynx; g, gonad. (D–H) In situ hybridisations indicating that Hc-fau/Ce-rps30 mRNA is localised ubiquitously in both H. contortus exsheathed L3 (xL3) and C. elegans L3, but cannot be detected in H. contortus diapausing L4. (F) Worms were viewed by fluorescence and differential interference contrast (DIC) microscopy. (H) The abundance of Hc-fau transcripts was quantified by quantitative real-time PCR (qRT-PCR) in all H. contortus life stages. (I) The abundance of Ce-rps30 transcript was quantified in C. elegans dauer larvae.
domains with RFP upstream or GFP downstream, respectively (Fig. 1). Caenorhabditis elegans expressing pCe-rps30::gfp were used as a control transgenic line for comparison (Supplementary Fig. S2). The proteins, Hc-FAU, Ce-RPS30 and their functional domains, were ubiquitously expressed, except in the gonad (Fig. 5A–W), consistent with pCe-rps30::gfp (Fig. 4C; Supplementary Fig. S2). In worms over-expressing Hc-faua, Hc-FAUA was cleaved into two segments (Fig. 5G), UBiL and S30, as described previously (Rossman et al., 2003). The two segments were both able to enter the nucleus and UBiL assembles in the cytoplasm (Fig. 5G). The Hcfaua transgene shortened the lifespan of C. elegans (N2) significantly (Fig. 6E and Table 1). The Hc-faua transgenic line could not produce eggs (Fig. 6A). The body length (258 ± 12 lm), comparable with a L1, was significantly shorter than that of N2 adults, L4, L3 or L2 (Fig. 6B). Worms over-expressing Hc-faua-ubil could grow to adults (Fig. 5I–K). Hc-faua-ubil transgene had less effect on lifespan (Fig. 6E and Table 1), while brood size was substantially reduced compared with N2 (pCe-rps30::gfp) (Fig. 6A). There were no
significant differences between Hc-faua-ubil transgenic worms and N2 adults in length (Fig. 6B). Eggs over-expressing Hc-faub in the F2 generation did not hatch (Fig. 6E and Table 1). Worms over-expressing Hc-faub-ubil could also grow to adults, and Hc-FAUB-UBiL assembled significantly in the cytoplasm of Hcfaub-ubil transgenic worms (Fig. 5L–Q and Supplementary Fig. S3). The Hc-faub-ubil transgene shortened the lifespan of C. elegans (N2) significantly (Fig. 6E and Table 1). Brood size was substantially reduced compared with N2 (pCe-rps30::gfp) (Fig. 6A). There were also no significant differences between Hc-faua-ubil or Hc-faub-ubil transgenic worms and N2 adults in length (Fig. 6B). Worms over-expressing Hc-fau-s30 could grow to sexual maturity. However, the adults were deficient in egg laying (Figs. 5R–W and 6A). The Hc-fau-s30 transgene shortened the lifespan of wild type (N2) C. elegans significantly (Fig. 6E and Table 1). The body length of Hc-fau-s30 transgenic adults (372 ± 24 lm) was comparable with wild type L2s in length, and significantly shorter than that of N2 adults (Fig. 6B).
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Fig. 5. Post-embryonic development of Caenorhabditis elegans worms over-expressing Ce-rps30/Hc-fau. (A–G) Effects of Hc-fau/Ce-rps30 over-expression. (G) Fusion protein is cleaved into an ubiquitin-like (UBiL) domain (arrow) and a S30 domain (arrowhead). An intestinal cell is outlined with a white dotted line. Red colour (arrow), red fluorescent protein (RFP) and UBiL domain fusion protein; green colour, GFP and S30 domain fusion protein; yellow colour (arrowhead), merging red into green. The red one (UBiL) assembles dominantly in the cytoplasm, and the green one (S30) remains in the nucleus, combined with the red one. (I–K) Hc-faua-ubil/Ce-rps30-ubil over-expression. RFP is expressed ubiquitously, except in the gonad (white dotted line). The vulva is marked with an arrowhead. (L–Q) Haemonchus contortus Hc-faua-ubil over-expression. GFP accumulates in body cells (arrow). (R–W) Effects of Hc-fau-s30/Ce-rps30-s30 over-expression. GFP is not expressed in the gonad; uterine eggs are marked with arrows. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. Post-embryonic development and lifespan in Ce-rps30/Hc-fau over-expression and knockdown Caenorhabditis elegans worms. (A) Brood size of worms over-expressing Hc-faua, Hc-faua-ubil, Hc-faub-ubil, Hc-fau-s30, using pPD95.77 (pCe-rps30::gfp) and pRF4 plasmid mixture as a control. Values represent mean ± S.E.M. (B) Body lengths of 20 adult worms of each transgenic line or RNA interference (RNAi) knockdown cohort. (C) Lifespan of Ce-rps30RNAi worms grown on RNAi plates, using L4440 empty vectorcontaining Escherichia coli on separate plates as a negative control. (D) Brood size of Ce-rps30RNAi worms, using L4440 empty vector containing E. coli on separate plates as a negative control. Values represent means ± S.E.M. (E) Lifespan of each transgenic line grown in Nematode Growth Media (NGM).
The transcriptional expression patterns and other consequences of Ce-RPS30, Ce-RPS30-UBiL and Ce-RPS30-S30 expression in C. elegans were consistent with those of Hc-FAUA, Hc-FAUA-UBiL and HcFAUA-S30 expression in C. elegans (Figs. 5A–K, R–W and 6A, B, E). 3.5. Post-embryonic development and lifespan of Ce-rps30RNAi worms Hc-fau expression was significantly down-regulated in diapausing L4s (Fig. 4H). In order to predict the effect of Hc-fau knockdown in H. contortus, RNAi was conducted in C. elegans, targeted to the homologous gene Ce-rps30. Ce-rps30RNAi prolonged the lifespan of wild type (N2) C. elegans significantly (Fig. 6C and Table 1). There were no worms grown to adults in Ce-rps30RNAi C. elegans. Ce-
rps30RNAi worms produced no eggs during their life cycles (Fig. 6A). The degree of gonad development and body length of 3-day-old Ce-rps30RNAi worms were both comparable with L1L2 stages of C. elegans (Figs. 6B and 7A, D), indicating a substantial reduction in the development and growth rate (from egg to L1–L2): 3 days to the L1–L2 stage, compared with 20–32 h in N2 (L4440 vector) (data not shown). The body lengths of 10- and 20-dayold Ce-rps30RNAi worms were 368 ± 9 lm and 403 ± 17 lm, respectively (Fig. 6B), and the latter is similar to the length of dauer larvae (Fig. 6B). However, the gonads of 20-day-old Ce-rps30RNAi worms were considerably condensed (Fig. 7C, F) in contrast to that of dauer larvae, in which the gonad is similar to an early L2 stage (Russell and Cassada, 1975; Riddle, 1988).
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B. Yan et al. / International Journal for Parasitology 44 (2014) 775–786 Table 1 Effects of Haemonchus contortus Hc-fau and Caenorhabditis elegans Ce-rps30 genes on lifespan. Experiment
Genotype a
Mean LS ± S.E.M. (days)
Number of worms
P value versus control
RNAi
Control Ce-rpa30RNAi
15.4 ± 0.2 26.6 ± 0.3
54 50
Control
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International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara
Hc-fau, a novel gene regulating diapause in the nematode parasite Haemonchus contortus Baolong Yan a, Xiaolu Guo a, Qianjin Zhou b, Yi Yang a, Xueqiu Chen a, Weiwei Sun c, Aifang Du a,⇑ a
Institute of Preventive Veterinary Medicine & Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310058, China School of Marine Science, Ningbo University, Ningbo 315211, China c Laboratory for Evolution & Development, Institute of Evolution & Marine Biodiversity and Department of Marine Biology, Ocean University of China, Qingdao 266003, China b
a r t i c l e
i n f o
Article history: Received 6 March 2014 Received in revised form 27 May 2014 Accepted 28 May 2014 Available online 21 July 2014 Keywords: Nematode Haemonchus contortus Hc-fau Diapause Caenorhabditis elegans
a b s t r a c t Diapause induced in the early fourth stage of Haemonchus contortus is a strategy to adapt this nematode to hostile environmental conditions. In this study, we identified a new gene, Hc-fau, a homologue of human fau and Caenorhabditis elegans Ce-rps30. Hc-fau encodes two proteins through alternative RNA splicing, Hc-FAUA and Hc-FAUB, consisting of 130 and 107 amino acids, respectively. Hc-FAU possesses a diverged ubiquitin-like (UBiL) protein domain and a conserved ribosome protein S30 domain. The protein is ubiquitously expressed, except in the gonad. However Hc-fau transcripts decrease significantly in diapausing L4s of H. contortus. In C. elegans, knockdown of Ce-rps30 confers an extended lifespan, increased lipid storage in the intestine and shortened body length. These morphological characteristics are comparable with dauer larvae of C. elegans, in which the gonad is condensed considerably. In contrast, a shortened lifespan is observed in C. elegans over-expressing Hc-faua, and especially Hc-faub, with hatching failure detected. The genes of insulin/IGF-1 signalling (IIS), TGF-b, cGMP, dafachronic acid (DA), apoptosis (AP) and fatty acids (FA) metabolism are all down-regulated in Ce-rps30RNAi (RNA interference) worms, except for akt-1 and daf-16. However, daf-16 up-regulation is inconsistent with its target gene down-regulation and the result from a heat stress assay in these worms. Daf-16 RNAi conducted in Cerps30 (tm6034/nt1) mutants failed to rescue the worms. The S30 domain stays in the nucleus, while UBiL accumulates in the cytoplasm. Compared with Hc-FAUA, results of UBiL domain and S30 domain overexpression indicate synergism between UBiL and S30 in regulating lifespan and reproduction. These results suggest the potential functions of Hc-fau in regulating larval diapause in H. contortus. Ó 2014 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.
1. Introduction Nematodes are capable of periods of arrested development (Sommerville and Davey, 2002). Dauer, a specialised stage in the free-living nematode Caenorhabditis elegans, is one form of arrested development, which occurs in L3s (Golden and Riddle, 1984). Dauer larvae of C. elegans survive hostile environmental conditions such as scarcity of food, high temperature and high population density (Riddle and Albert, 1997). Dormancy terminates when conditions become favourable for normal activity (Sommerville and Davey, 2002). In the parasitic nematode Haemonchus contortus, diapause, another form of arrested development, occurs in the early fourth stage in abomasums of ruminants (Blitz and Gibbs, 1971). ⇑ Corresponding author. Postal address: Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China. Tel.: +86 571 8898 2583. E-mail address: [email protected] (A. Du).
Diapause is a strategy to adapt this nematode to hostile environmental conditions such as low oxygen tension, short photoperiod, low temperature and host immune response (Sommerville and Davey, 2002). The dauer stage, regulated by four molecular pathways, cGMP (Ren et al., 1996), insulin (Kimura et al., 1997), TGF-b (Birnby et al., 2000) and dafachronic acid (DA) signalling via the nuclear hormone receptor DAF-12 (Motola et al., 2006; Crook, 2014), has been extensively studied in the model organism C. elegans (Angelo and Van Gilst, 2009). It has been suggested that the infective L3s (iL3s) of parasitic nematodes are analogous to dauer larvae of C. elegans (Rogers and Sommerville, 1963; Crook, 2014; Zhang et al., 2013), and some conserved pathways have also been determined in parasitic nematodes (Crook, 2014). However, the diapausing parasitic nematodes (L4s) do not, according to the definition in this paper, equate with the dauer larvae of C. elegans (Sommerville and Davey, 2002). In this study, we use C. elegans as a tool to explore the in vivo functions of a gene we cloned from H. contortus (Hc-fau).
http://dx.doi.org/10.1016/j.ijpara.2014.05.011 0020-7519/Ó 2014 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.
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We identified Hc-fau from a mRNA differential display assay between normal larvae and diapausing larvae of H. contortus. It is a homologue of human fau (H.s-fau) (Kas et al., 1992) and Ce-rps30 (C26F1.4). The fox (FBR osteosarcoma X) sequence from the FinkelBiskis-Reilly murine sarcoma virus (FBR-MuSV) is an antisense sequence to fau (FBR-MuSV associated ubiquitously expressed gene) (Van Beveren et al., 1984; Michiels et al., 1993). Expression of fox increases the transforming capability and tumorigenicity of the virus, presumably by inactivating fau expression in the mouse (Michiels et al., 1993). Expression of fau is down-regulated in both breast cancer (Pickard et al., 2009) and ovarian cancer (Moss et al., 2010). A sequence antisense to fau is able to inhibit apoptosis (AP) induced by dexamethasone, UV or cisplatin in W7.2c cells (Mourtada-Maarabouni et al., 2004). FAU was also found to regulate apoptosis in human T-cell lines and HEK293/17 cells (Pickard et al., 2009). fau encodes a ubiquitin-like protein (UBiL), fused to ribosomal protein S30 (S30) as a carboxy-terminal extension (Kas et al., 1992). These two products are thought to result from post-translational cleavage (Pickard et al., 2011). Human UBiL has 37% amino acid sequence identity (57% sequence similarity) to ubiquitin and retains the C-terminal G-G dipeptide motif that participates in isopeptide bond formation between ubiquitin and lysine of target proteins. A lack of internal lysine residues, sites of poly ubiquitin chain formation, indicates that the biological function of UBiL is distinct from that of ubiquitin (Kas et al., 1992; Pickard et al., 2011). The identification of UBiL covalently bound to Bcl-G, a member of the Bcl-2 family of apoptosis control proteins (Nakamura and Tanigawa, 2003), suggests a pro-apoptotic regulatory role for fau, mediated via Bcl-G (Mourtada-Maarabouni et al., 2004; Pickard et al., 2011). In this report, we describe the cloning, initial characterisation and functional study of the novel gene Hc-fau from H. contortus, with the aim of investigating its role in regulating nematode parasite diapause.
Caenorhabditis elegans strains (N2) were maintained on Nematode Growth Media (NGM) agar plates at 20 °C as described previously (Brenner, 1974). Worms were fed Escherichia coli strain OP50 unless otherwise stated. Sheep used as experimental animals were treated according to the recommendation in the Guide for the Regulation for the Administration of Affairs concerning Experimental Animals of the People’s Republic of China. Animal experiments were approved by Zhejiang University Experimental Animal Ethics Committee (Permit Number: 2009/01). The care and maintenance of sheep followed the guidelines of this institution.
2. Materials and methods
2.4. Isolation of full-length genomic DNA of Hc-fau
2.1. Propagation of H. contortus and C. elegans
Full-length genomic DNA of Hc-fau from the ZJ strain of H. contortus was obtained by a genome walking kit (Takara Biotechnology Co., Ltd.), using primers designed based on the acquired cDNA sequence (Supplementary Table S1), following the manufacturer’s instructions. The third-round PCR products were cloned into a pMD18-T vector and sequenced.
Sheep, maintained under helminth-free conditions, were infected intraruminally with 8,000 iL3s of H. contortus ZJ strain, which is maintained and propagated in Zhejiang University, China. The presence of parasites was confirmed at day 24 by detecting strongylid eggs in the faeces using the flotation method (Cox and Todd, 1962). All sheep were held in metabolism cages in concrete-floored pens, where the faeces could be collected in the metal trays in order to preclude the possibility of re-infection occurring during this period. L1s, L2s and iL3s were collected after 1, 3 and 7 days of incubation of faeces at 28 °C, respectively. For infection, larvae were counted by a dilution technique. Exsheathment of L3 worms (xL3s) was carried out with NaOCl using the methods described by Rothwell and Sangster (1993). Haemonchus contortus diapause ZJ strains were obtained according to Blitz and Gibbs (1971). Experimentally infected ewes (5 months old) were orally administered 8,000 to 10,000 H. contortus iL3s. Dexamethasone sodium phosphate (1 ml:5 mg) was injected i.m. in doses of 0.5 mg/kg liveweight 1 day before and 3 days p.i. Before autopsy, all the lambs were held for 60 days in metabolism cages where re-infection did not occur during this period. After euthanizing the animals and opening the abomasums, the ingesta and washings were fixed in 7% formol saline and abomasal mucosa was digested in peptic-HCl. Then the worms were detected under an anatomical lens (Motic, China). Adults of H. contortus ZJ strain were collected from sheep abomasums at necropsy using fine forceps and washed extensively in chilled (4 °C) PBS. All worm samples were stored in liquid nitrogen until use.
2.2. Isolation, purification, treatment and storage of nucleic acids Total genomic DNA was extracted from adult worms using a small-scale genomic DNA extraction kit (Takara Biotechnology Co., Ltd., Japan). Total RNA was extracted from worms at different developmental stages employing Trizol reagent (Invitrogen, USA), followed by treatment with 2 U of DNase I (Takara Biotechnology Co., Ltd.). First strand cDNA was obtained using the M-MLV RTase cDNA Synthesis Kit (Takara Biotechnology Co., Ltd.). Both DNA and RNA samples were stored at 70 °C. 2.3. Isolation of full-length cDNA encoding Hc-fau from H. contortus Using gene-specific primer pairs (Supplementary Table S1), designed based on the available expressed sequence tag (EST) sequence, two partially overlapping cDNA fragments were obtained from total RNA isolated from adult H. contortus using 50 - and 30 - rapid amplification of cDNA ends (RACE) (Takara Biotechnology Co., Ltd.). Products were cloned into a pMD18-T vector and sequenced. Based on these sequences, additional primers (Supplementary Table S1) were designed to amplify full-length Hc-faua and Hc-faub.
2.5. Whole-mount in situ hybridisation (WISH) Haemonchus contortus xL3s, diapausing L4s and C. elegans L3s were used for WISH. Hc-fau and Ce-rps30 cloned into the vector pGEM-T Easy were linearised using endonuclease NcoI, antisense probes were synthesised using Sp6 RNA polymerase and primers were as listed in Supplementary Table S1. WISH was performed as described by Thisse and Thisse (2008), and images were taken under a microscope (Nikon, Japan). 2.6. Caenorhabditis elegans dauer formation in liquid Caenorhabditis elegans dauer larvae were produced according to the method described (Gottlieb and Ruvkun, 1994). To induce synchronous dauer formation in liquid, eggs were isolated by bleach treatment from gravid hermaphrodites and incubated in S Basal (Sulston and Hodgkin, 1988) in the absence of food for 12–16 h at 25 °C to synchronise all animals at the L1 stage. Then a large number of synchronised L1s were placed into a flask (50 ml) containing 10 ml of S Medium (Sulston and Hodgkin, 1988), 5 ll of a crude pheromone preparation (prepared as described in Golden and Riddle, 1984) and 600 ll of a 4% solution of streptomycin-treated
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bacteria E. coli OP50. The flask was then placed in a shaking water bath for 72 h at 25 °C. 2.7. Quantitative real-time PCR (qRT-PCR) analysis qRT-PCR was performed to determine the abundance of Hc-fau transcripts in different developmental stages (L1, L2, L3, diapause, adult) of H. contortus, Ce-rps30 transcripts in adults and dauer larvae of C. elegans, and some candidate gene transcripts in Cerps30RNAi (RNA interference) C. elegans. Gene expression levels were determined by RT-PCR using SYBRÒ Green PCR Master Mix and a 7500 Real-Time PCR System (Applied Biosystems, USA). Relative gene expression was compared with actin (act-1) or b-tubulin as an internal loading control. The target genes and the primers used are listed in Supplementary Table S1. Statistical analysis was conducted using a one-way ANOVA; P 6 0.05 was set as the criterion for significance. 2.8. RNAi feeding experiments To generate Ce-rps30- and daf-16-specific RNAi vectors, Ce-rps30 and daf-16 cDNAs were cloned into the L4440 vector. Plasmids were transformed into E. coli strain HT115. Primers used for PCR analysis are listed in Supplementary Table S1. RNAi plates and media were prepared according to Kwon et al. (2010). Gravid adults of C. elegans were allowed to lay eggs overnight on the RNAi plates and adult worms were picked off. Empty vector-containing E. coli were used on separate plates as negative controls. 2.9. Transgenic worms To analyse promoter activity of Hc-fau, the promoter regions were amplified and cloned into plasmid pPD95.77 to construct pHc-fau::gfp and pCe-rps30::gfp. To perform over-expression of Hc-FAU, Ce-RPS30 and their functional domains, fragments were amplified accordingly and cloned into pPD95.77 to construct plasmids with the promoter of Ce-rps30. Hc-FAU/Ce-RPS30 were expressed in separate transgenic lines as fusion proteins with red fluorescent protein (RFP) upstream and GFP downstream, and Hc-FAUA-UBiL/Ce-RPS30-UBiL with RFP upstream, Hc-FAU-S30/ Ce-RPS30-S30 with GFP downstream, Hc-FAUB-UBiL with RFP upstream and GFP downstream (Fig. 1). All primers used are listed in Supplementary Table S1.
Fig. 1. The constructs of over-expression plasmids. Plasmids encoding for Haemonchus contortus FAU/Caenorhabditis elegans RPS30 (Hc-FAU/Ce-RPS30) were constructed with red fluorescent protein (RFP) upstream and GFP downstream, and Hc-FAUA-ubiquitin-like (UBiL)/Ce-RPS30-UBiL with RFP upstream, Hc-FAU-S30/ Ce-RPS30-S30 with GFP downstream, and Hc-FAUB-UBiL with RFP upstream and GFP downstream. Fragments were cloned into pPD95.77 plasmids with the promoter of Ce-rps30.
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Recombinant plasmids were each microinjected into the gonad of young, adult wild-type (N2) C. elegans hermaphrodites as described (Mello et al., 1991), together with plasmid pRF4 containing a dominant mutant allele of rol-6 gene, each at a final concentration of 50 lg/mL in the same mixture, using pPD95.77 (pCe-rps30::gfp) and pRF4 plasmid mixture as a control. The F2 and subsequent generations with a roller phenotype were analysed and selected to examine the expression patterns of GFP or RFP, using a fluorescent microscope (Olympus IX71). A minimum of three independent lines expressing each transgene were evaluated.
2.10. Lipid staining Oil Red O staining was performed as described by Soukas et al. (2009). Worms were washed off the NGM or RNAi plates and incubated in PBS buffer for 30 min on a shaker at room temperature. The worms were then fixed in Modified Ruvkun’s witches brew (MRWB) buffer containing 1% paraformaldehyde (Soukas et al., 2009). After three rounds of freezing/thawing, the worms were dehydrated in 60% isopropanol followed by addition of saturated Oil-Red-O (Sigma, USA) solution. Fixed worms were incubated overnight on a shaker at room temperature, mounted on slides and viewed using a microscope with differential interference contrast optics (Nikon, Japan).
2.11. Heat stress assays Ce-rps30RNAi (10 days old) and N2 (L4) C. elegans were picked onto fresh NGM plates (a total of 21 plates) seeded with E. coli and incubated at 20 °C for 18 h, followed by an upshift to 37 °C. At hourly intervals, three plates of each strain were removed directly from 37 °C and worms were allowed to recover at 20 °C for approximately 12 h after which individual C. elegans were assessed as alive or dead. Heat stress-induced mortality was determined by tapping the worms with a platinum wire to check for motility; unresponsive individuals were scored as dead. Statistical analyses for survival were conducted using the chi-square-based log rank test (Kwon et al., 2010).
2.12. Lifespan measurement, brood size and body size Measurement of body length was performed as described (Mörck and Pilon, 2006). Different stages of worms were picked and photographed. Body length measurements were performed with the free Java image processing program ImageJ. Larvae and adults were measured from the nose to the tail tip. The brood size was determined as described (Wong et al., 1995). Larvae were placed singly onto fresh plates and incubated at 20 °C until they had matured and laid the first few eggs. The hermaphrodites were then transferred onto fresh plates daily to prevent overcrowding until egg laying ceased. The progeny were counted 3 days after removal of the parents. A lifespan assay was carried out according to the method described (Wong et al., 1995). The L4s were placed at 20 °C until they laid the first few eggs, then the adult worms were picked out and eggs allowed to hatch to larvae. Larvae were placed singly onto fresh plates, and monitored once daily until death. The animals were transferred once daily while producing eggs to keep them separate from their progeny. Animals were scored as dead when they no longer responded with movement to light prodding of the head. All C. elegans were kept at 20 °C. Animals that crawled off the plates during the assay were excluded from calculations. P values were derived from Student’s t-tests.
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2.13. Whole animal DAPI staining
3.2. Identification and sequence analysis of the Hc-fau gene and cDNAs
Haemonchus contortus diapause L4 worms and iL3s were fixed in ice cold, 100% methanol (5 min, 20 °C) and washed once with PBS. Fixed animals were incubated (30 min, room temperature, protected from light) in DAPI solution at a final concentration of 500 ng/ml. Stained animals were washed once in PBS and transferred to a glass slide. Images were captured with FV1000 spectral confocal and fluorescent (Olympus IX71) microscopes.
An EST was identified from a mRNA differential display experiment comparing L4s with arrested L4s of H. contortus (data not shown), that had a predicted polypeptide sequence with similarity to the RPS30 superfamily domain, and with most similarity to the human H.s-FAU and C. elegans Ce-RPS30 proteins. The full-length cDNAs of two transcripts, Hc-faua and Hc-faub, were isolated by RACE from H. contortus. The full-length cDNA of Hc-faua was 521 bp in length, including an open reading frame (ORF) of 393 bp (including stop codon), a 50 -untranslated region (UTR) of 55 bp, and a 30 -UTR of 73 bp. The full-length cDNA of Hc-faub was 602 bp in length, containing an ORF of 324 bp (including stop codon), a 50 -UTR of 205 bp and a 30 -UTR of 73 bp (Fig. 3A, B). The cDNAs of Hc-faua and Hc-faub encoded predicted proteins of 130 and 107 amino acids, respectively (Fig. 3A). An alignment of the two protein sequences revealed that the 103 amino acid C-terminal domains of both (positions 28–130 for Hc-FAUA and positions 5–107 for Hc-FAUB) were identical (Fig. 3A). The predicted amino acid sequences of Hc-FAUA and Hc-FAUB were aligned with H.s-FAU and Ce-RPS30 (Fig. 3A). The alignment showed that the C-terminal S30 domains were conserved (Hc-FAU-S30 versus
3. Results 3.1. Identification of H. contortus diapause In sheep killed 60 days p.i., only two distinct populations of H. contortus were found: mature parasites and early L4s (Fig. 2A). The larvae were characterised by similar length, measuring 1067 ± 97 lm (n = 18) and with rod-like crystalline inclusions in the intestinal cells (Fig. 2B, C), corresponding to the morphological characteristics described previously (Blitz and Gibbs, 1971). Genital primordia were present in diapausing H. contortus L4s (Fig. 2D–F), but these were not present in iL3s (Fig. 2G–I).
Fig. 2. Morphological characterisation of Haemonchus contortus diapause. (A) Anterior end of the H. contortus diapause larva. (B, C) The crystalline inclusion in the intestinal cells (black arrows), and intestinal lumen (white arrow). (D–F) Whole animal DAPI staining revealed a genital primordium in an H. contortus diapausing L4, and a vulval ovejector primordium (white arrow). (G–I) Whole animal DAPI staining revealed no genital primordium in an H. contortus infective L3 (iL3).
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Fig. 3. Sequence analysis of Haemonchus contortus Hc-fau cDNAs. (A) Alignments of the Hc-FAUA, Hc-FAUB, human FAU (H.s-FAU) and Caenorhabditis elegans Ce-RPS30. Identical and similar residues are shown in black and grey blocks, respectively. The potential cleavage sites (Gly-Gly) of the fusion protein (ubiquitin-like; UBiL-ribosome protein S30; S30) are indicated with black arrows (upstream and downstream sequences are UBiL and S30 domains, respectively). The predicted nuclear location signals in the S30 domains are indicated with a blue block. (B) The exon–intron organisation of the Hc-fau gene. The Hc-fau gene spans 2590 bp, with alternative splicing at the 50 -end of exon 2 accounting for the production of the Hc-FAUA and Hc-FAUB proteins, where the splice-acceptor sites at nucleotide position 307 versus 1279 in the gene are utilised for Hc-FAUA and Hc-FAUB, respectively. The positions of the start and termination codons are indicated, with coding regions in grey blocks and non-coding 50 - and 30 untranslated region (UTR) sequences in empty blocks. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Ce-RPS30-S30 and H.s-FAU-S30, 88.1% and 79.7% similarity, respectively), whereas the N-terminal UBiL domains were divergent (HcFAUA-UBiL versus Ce-RPS30-UBiL and H.s-FAU-UBiL, 33.8% and 31.4% similarity, respectively). The complete Hc-fau gene, including its 50 flanking sequence, was isolated by Genome Walking (see Section 2.4) and sequenced. Comparison with the cDNA sequences suggests an alternative mRNA splicing mechanism in which a different splice acceptor site associated with exon 2 is utilised, resulting in a change in the proximal reading frame (Fig. 3B). The 50 UTR contains 11 consecutive pyrimidines (TCTTTCTTTCC), which are found at the 50 end of eukaryotic ribosomal protein mRNAs (Olvera and Wool, 1993), and are hypothesised to play a role in the regulation of translation (Levy et al., 1991). The 30 UTR harbours the hexamer AATAAA (positions, 56 downstream of the TAA). Another 387 bp sequence was amplified from the genomic DNA of H. contortus. Sequence analysis revealed that it was 91.7% identical to the Hc-faua cDNA sequence (Supplementary Fig. S1). However, it lacks both introns and the complete 50 UTR of Hc-faua. Several point and deletion mutations in the sequence lead to a premature stop codon and ORFs for Hc-FAUA/B no longer exist. Thus, we defined the sequence as a processed pseudogene: Hc-fau-ps.
Transgenic lines showing the roller phenotype were selected. Expression of pHc-fau::gfp was restricted to the distal intestine of C. elegans in all stages from L1 to adult (Fig. 4A, B; only adult shown), whereas pCe-rps30::gfp was expressed in almost all cells except for the gonad (Fig. 4C). In situ localisation revealed that Hc-fau transcripts are present in all cells in H. contortus xL3s (Fig. 4E), but absent in diapausing L4s of the parasite (Fig. 4F). However, the probe used detected both mRNAs encoding Hc-FAUA and Hc-FAUB. Ce-rps30 mRNA was detected widely in C. elegans L3s (Fig. 4D), consistent with H. contortus xL3s. qRT-PCR was performed to determine the relative abundance of Hc-fau transcripts in different developmental stages throughout the life cycle of H. contortus (Fig. 4H) and of Ce-rps30 in C. elegans dauer (Fig. 4I). We observed expression of Hc-fau at approximately the same level as the control genes in all stages, except in diapausing L4s. Hc-fau expression was significantly down-regulated in H. contortus diapausing L4s (Fig. 4H), consistent with Ce-rps30 expression in C. elegans dauers (Fig. 4I) which was significantly down-regulated in dauer L3s of C. elegans.
3.3. The expression patterns of Hc-FAU
To investigate Hc-FAU in vivo functions, Hc-FAU, Ce-RPS30 and their functional domains were over-expressed in transgenic C. elegans, using the Ce-rps30 50 -flanking region as the promoter. Hc-FAU and Ce-RPS30 were expressed in separate transgenic lines as fusion proteins with RFP upstream and GFP downstream, and the other
The 2,000 bp sequence upstream of the Hc-fau 50 -UTR was used as a putative promoter and cloned to construct a promoter-gfp fusion in order to analyse transcriptional activity in C. elegans.
3.4. Consequences of post-embryonic development and lifespan deficiency in C. elegans overexpressing Ce-rps30/Hc-fau
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Fig. 4. Expression patterns of Haemonchus contortus Hc-FAU. (A–C) The promoter activity of Hc-fau/Ce-rps30 in Caenorhabditis elegans. Expression of GFP is restricted in intestinal cells in pHc-fau::gfp worms, but is ubiquitous in pCe-rps30::gfp worms, except in the gonad. Arrows indicate the following tissues: i, intestinal; m, muscle; n, neuron; p, pharynx; g, gonad. (D–H) In situ hybridisations indicating that Hc-fau/Ce-rps30 mRNA is localised ubiquitously in both H. contortus exsheathed L3 (xL3) and C. elegans L3, but cannot be detected in H. contortus diapausing L4. (F) Worms were viewed by fluorescence and differential interference contrast (DIC) microscopy. (H) The abundance of Hc-fau transcripts was quantified by quantitative real-time PCR (qRT-PCR) in all H. contortus life stages. (I) The abundance of Ce-rps30 transcript was quantified in C. elegans dauer larvae.
domains with RFP upstream or GFP downstream, respectively (Fig. 1). Caenorhabditis elegans expressing pCe-rps30::gfp were used as a control transgenic line for comparison (Supplementary Fig. S2). The proteins, Hc-FAU, Ce-RPS30 and their functional domains, were ubiquitously expressed, except in the gonad (Fig. 5A–W), consistent with pCe-rps30::gfp (Fig. 4C; Supplementary Fig. S2). In worms over-expressing Hc-faua, Hc-FAUA was cleaved into two segments (Fig. 5G), UBiL and S30, as described previously (Rossman et al., 2003). The two segments were both able to enter the nucleus and UBiL assembles in the cytoplasm (Fig. 5G). The Hcfaua transgene shortened the lifespan of C. elegans (N2) significantly (Fig. 6E and Table 1). The Hc-faua transgenic line could not produce eggs (Fig. 6A). The body length (258 ± 12 lm), comparable with a L1, was significantly shorter than that of N2 adults, L4, L3 or L2 (Fig. 6B). Worms over-expressing Hc-faua-ubil could grow to adults (Fig. 5I–K). Hc-faua-ubil transgene had less effect on lifespan (Fig. 6E and Table 1), while brood size was substantially reduced compared with N2 (pCe-rps30::gfp) (Fig. 6A). There were no
significant differences between Hc-faua-ubil transgenic worms and N2 adults in length (Fig. 6B). Eggs over-expressing Hc-faub in the F2 generation did not hatch (Fig. 6E and Table 1). Worms over-expressing Hc-faub-ubil could also grow to adults, and Hc-FAUB-UBiL assembled significantly in the cytoplasm of Hcfaub-ubil transgenic worms (Fig. 5L–Q and Supplementary Fig. S3). The Hc-faub-ubil transgene shortened the lifespan of C. elegans (N2) significantly (Fig. 6E and Table 1). Brood size was substantially reduced compared with N2 (pCe-rps30::gfp) (Fig. 6A). There were also no significant differences between Hc-faua-ubil or Hc-faub-ubil transgenic worms and N2 adults in length (Fig. 6B). Worms over-expressing Hc-fau-s30 could grow to sexual maturity. However, the adults were deficient in egg laying (Figs. 5R–W and 6A). The Hc-fau-s30 transgene shortened the lifespan of wild type (N2) C. elegans significantly (Fig. 6E and Table 1). The body length of Hc-fau-s30 transgenic adults (372 ± 24 lm) was comparable with wild type L2s in length, and significantly shorter than that of N2 adults (Fig. 6B).
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Fig. 5. Post-embryonic development of Caenorhabditis elegans worms over-expressing Ce-rps30/Hc-fau. (A–G) Effects of Hc-fau/Ce-rps30 over-expression. (G) Fusion protein is cleaved into an ubiquitin-like (UBiL) domain (arrow) and a S30 domain (arrowhead). An intestinal cell is outlined with a white dotted line. Red colour (arrow), red fluorescent protein (RFP) and UBiL domain fusion protein; green colour, GFP and S30 domain fusion protein; yellow colour (arrowhead), merging red into green. The red one (UBiL) assembles dominantly in the cytoplasm, and the green one (S30) remains in the nucleus, combined with the red one. (I–K) Hc-faua-ubil/Ce-rps30-ubil over-expression. RFP is expressed ubiquitously, except in the gonad (white dotted line). The vulva is marked with an arrowhead. (L–Q) Haemonchus contortus Hc-faua-ubil over-expression. GFP accumulates in body cells (arrow). (R–W) Effects of Hc-fau-s30/Ce-rps30-s30 over-expression. GFP is not expressed in the gonad; uterine eggs are marked with arrows. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. Post-embryonic development and lifespan in Ce-rps30/Hc-fau over-expression and knockdown Caenorhabditis elegans worms. (A) Brood size of worms over-expressing Hc-faua, Hc-faua-ubil, Hc-faub-ubil, Hc-fau-s30, using pPD95.77 (pCe-rps30::gfp) and pRF4 plasmid mixture as a control. Values represent mean ± S.E.M. (B) Body lengths of 20 adult worms of each transgenic line or RNA interference (RNAi) knockdown cohort. (C) Lifespan of Ce-rps30RNAi worms grown on RNAi plates, using L4440 empty vectorcontaining Escherichia coli on separate plates as a negative control. (D) Brood size of Ce-rps30RNAi worms, using L4440 empty vector containing E. coli on separate plates as a negative control. Values represent means ± S.E.M. (E) Lifespan of each transgenic line grown in Nematode Growth Media (NGM).
The transcriptional expression patterns and other consequences of Ce-RPS30, Ce-RPS30-UBiL and Ce-RPS30-S30 expression in C. elegans were consistent with those of Hc-FAUA, Hc-FAUA-UBiL and HcFAUA-S30 expression in C. elegans (Figs. 5A–K, R–W and 6A, B, E). 3.5. Post-embryonic development and lifespan of Ce-rps30RNAi worms Hc-fau expression was significantly down-regulated in diapausing L4s (Fig. 4H). In order to predict the effect of Hc-fau knockdown in H. contortus, RNAi was conducted in C. elegans, targeted to the homologous gene Ce-rps30. Ce-rps30RNAi prolonged the lifespan of wild type (N2) C. elegans significantly (Fig. 6C and Table 1). There were no worms grown to adults in Ce-rps30RNAi C. elegans. Ce-
rps30RNAi worms produced no eggs during their life cycles (Fig. 6A). The degree of gonad development and body length of 3-day-old Ce-rps30RNAi worms were both comparable with L1L2 stages of C. elegans (Figs. 6B and 7A, D), indicating a substantial reduction in the development and growth rate (from egg to L1–L2): 3 days to the L1–L2 stage, compared with 20–32 h in N2 (L4440 vector) (data not shown). The body lengths of 10- and 20-dayold Ce-rps30RNAi worms were 368 ± 9 lm and 403 ± 17 lm, respectively (Fig. 6B), and the latter is similar to the length of dauer larvae (Fig. 6B). However, the gonads of 20-day-old Ce-rps30RNAi worms were considerably condensed (Fig. 7C, F) in contrast to that of dauer larvae, in which the gonad is similar to an early L2 stage (Russell and Cassada, 1975; Riddle, 1988).
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B. Yan et al. / International Journal for Parasitology 44 (2014) 775–786 Table 1 Effects of Haemonchus contortus Hc-fau and Caenorhabditis elegans Ce-rps30 genes on lifespan. Experiment
Genotype a
Mean LS ± S.E.M. (days)
Number of worms
P value versus control
RNAi
Control Ce-rpa30RNAi
15.4 ± 0.2 26.6 ± 0.3
54 50
Control
