Melanisation of Teladorsagia circumcincta larvae exposed to sunlight: A role for GTP-cyclohydrolase in nematode survival

International Journal for Parasitology 42 (2012) 887–891

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Melanisation of Teladorsagia circumcincta larvae exposed to sunlight: A role for GTP-cyclohydrolase in nematode survival Rachael H. Baker a,b, Collette Britton c, Brett Roberts c, Curtis M. Loer d, Jacqueline B. Matthews a, Alasdair J. Nisbet a,⇑ a

Moredun Research Institute, Pentlands Science Park, Bush Loan, Edinburgh EH26 0PZ, UK Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian EH25 9RG, UK Institute of Infection, Immunity and Inflammation, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK d Department of Biology, University of San Diego, 5998 Alcalá Park, San Diego, CA 92110, USA b c

a r t i c l e

i n f o

Article history: Received 21 May 2012 Received in revised form 27 June 2012 Accepted 28 June 2012 Available online 1 August 2012 Keywords: GTP-cyclohydrolase Melanisation UV radiation Nematode Teladorsagia circumcincta

a b s t r a c t Trichostrongylid nematode parasites of livestock inhabit two very different niches during their life-cycle; within the host and free-living in the environment. UV radiation plays a significant role in the survival of free-living, pre-parasitic nematode larvae, with different species exhibiting differing levels of sensitivity. In many eukaryotes, melanisation is a key protective mechanism against UV damage, however there is little information about this process in parasitic nematodes. Caenorhabditis elegans cat-4 mutants, which are deficient in the enzyme guanosine triphosphate-cyclohydrolase I (GTP-CH), have both depleted levels of melanin in their cuticles and an increased sensitivity to anthelmintic drugs. Some parasitic nematodes have very high levels of GTP-CH transcript in their pre-parasitic stages, suggesting an important role for this biopterin synthetic enzyme. Here, we show that the Tci-cat-4 gene, which encodes GTP-CH in Teladorsagia circumcincta, has a role in melanisation and is also capable of rescuing C. elegans cat-4 mutants. In addition, following exposure of T. circumcincta L3s to sunlight, there is a 32% increase in GTP-CH enzyme activity (P = 0.019), and a 21% increase in levels of melanin (P = 0.031) compared with unexposed larvae. These data suggest that one explanation for the high level of GTP-CH present in pre-parasitic stages of trichostrongylid nematodes is to facilitate melanisation in response to UV exposure. Ó 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Infection of grazing livestock with parasitic nematodes has significant economic and animal welfare implications (Nieuwhof and Bishop, 2007). Teladorsagia circumcincta is the most prevalent gastrointestinal parasitic nematode of sheep and goats in temperate regions and infections can result in severe weight loss and diarrhoea (Bartley et al., 2003). These effects arise from damage caused by larvae developing in, and emerging from, the gastric glands and by adult activity on the mucosal surface of the abomasum (Taylor et al., 2007). Subclinical infections suppress the appetite (Greer et al., 2008) and this, combined with a loss of plasma protein into the gastrointestinal tract, results in reduced nutrient acquisition (McKellar, 1993). Even with relatively low levels of infection, lambs take longer to reach their target weight than uninfected animals and have poor body condition (Taylor et al., 2007). Teladorsagia circumcincta pre-parasitic stages must survive under a variety of environmental conditions. Eggs voided in the faeces can hatch within 24 h of excretion into L1s which feed on faecal ⇑ Corresponding author. Tel.: +44 0 131 445 5111; fax: +44 0 131 445 6235. E-mail address: [email protected] (A.J. Nisbet).

bacteria. After moulting, L2s continue feeding on bacteria. L3s retain the moulted L2 cuticle as a protective outer sheath, although this prevents further feeding (Keith et al., 1990). L3s are more resistant to extremes of temperature and humidity compared with eggs, L1s and L2s (O’Connor et al., 2006). Teladorsagia circumcincta L3s can survive up to 13 weeks at 10 °C (Pandey et al., 1993) and brief exposure to high temperature such as 90 min at 45 °C (Walker et al., 2007). Humidity is crucial for survival (Pandey et al., 1993), and is an important factor that determines L3 migration out of the faecal pellet (Young, 1983). In addition to being exposed to large fluctuations in environmental temperature and humidity, infective larvae must also be capable of surviving changes in levels of UV radiation, potentially over long periods. UV radiation causes damage to tissues through DNA modification (Ikehata and Ono, 2011) and death due to UV exposure has been proposed as a major contributor to the observed reduction in T. circumcincta L3 levels on pasture during the spring in the UK, when UV radiation levels increase (van Dijk et al., 2009; http://ozone-uv.defra.gov.uk). There is also evidence that L3 strongylid nematodes can survive for longer periods in faeces, or in shaded rough grazing, than when encountering higher UV radiation levels while on exposed grass (Gibson and Everett, 1967; Sargison et al., 2012).

0020-7519/$36.00 Ó 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijpara.2012.06.005

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Mechanisms through which parasitic nematode larvae may be protected against UV radiation have not been studied but, in other animals, melanisation plays a key role (Kollias et al., 1991). L3s of several strongylid nematode species contain high levels of transcript encoding guanosine triphosphate-cyclohydrolase I (GTPCH, EC 3.5.4.16), (Hoekstra et al., 2000; Moore et al., 2000; Nisbet et al., 2008; Baker et al., 2011). GTP-CH catalyses the first and rate-limiting step in synthesis of tetrahydrobiopterin (BH4), an essential cofactor for several enzymes including nitric oxide synthases, alkylglycerol monooxygenase and aromatic amino acid hydroxylases (Watschinger et al., 2010; Werner et al., 2011). As a cofactor for phenylalanine hydroxylase (PAH), biopterin is needed for conversion of phenylalanine to tyrosine, a first step in melanin production. Mutations in the cat-4 gene of Caenorhabditis elegans, which encodes GTP-CH, result in worms with lower levels of melanin (Calvo et al., 2008) and a loss of cuticular integrity, with an increase in sensitivity to external agents such as anthelmintics or sodium hypochlorite (Loer and Kenyon, 1993). To explore the function of GTP-CH in T. circumcincta in protecting pre-parasitic stages of the parasite against environmental impact, we examined the effects of sunlight on melanisation and GTP-CH levels in infective L3s and tested functional complementation and restoration of cuticular integrity of C. elegans cat-4 mutant worms with Tci-cat-4. 2. Materials and methods 2.1. Exposure of nematode larvae to sunlight The effect of natural sunlight exposure on melanin levels in T. circumcincta L3s was assessed by placing 20,000 L3s in 100 ml of tap water in a 150 mm Petri dish in direct sunshine in Edinburgh, UK, on each of 3 days in Spring, 2011. A second Petri dish of L3s was placed alongside, but was protected from direct sunshine by a loose covering of aluminium foil. After 5 h, L3s in each dish were reduced to 30 ll volume by centrifugation and the total melanin present was assessed using a methodology adapted from Haywood et al. (2006), Scoville and Pfrender (2010) and Herbert and Emery (1990). L3s were digested in 1 ml of 5 M NaOH for 24 h at 40 °C. As a standard, Sepia melanin (Sigma, UK) was mixed with 5 M NaOH in separate tubes and incubated for 24 h at 40 °C. After incubation, samples were centrifuged at 3,000g for 3 min to pellet any non-solubilised material. The Sepia melanin was diluted in a series of doubling dilutions to produce a standard series. The O.D. of standards and L3-derived material was measured at 350 nm using a spectrophotometer. Melanin concentrations of the L3-derived samples were obtained from the standard curve using Graphpad Prism 4 software. Measurements of UV intensity during the exposure were provided by the Health Protection Agency from a recording station in Glasgow, UK. 2.2. GTP-CH activity levels in T. circumcincta GTP-CH-catalysed conversion of GTP to 7–8-dihydroneopterin triphosphate was measured by deriving neopterin from the reaction product (Fig. 1) and measuring neopterin levels using a commercially-available kit. To measure GTP-CH activity in each

Fig. 1. The pathway used to derive neopterin in the current study as a measure of guanosine triphosphate-cyclohydrolase I (GTP-CH) activity.

developmental stage, PBS-soluble extracts (S1) were made from two biological sets (i.e. harvested from sheep on separate occasions) of eggs, L1, L3, L4, L5 and adult T. circumcincta as described previously (Smith et al., 1994). To measure the effect of UV exposure on GTP-CH activity, 1.2 million T. circumcincta L3s in 600 ml of tap water were divided equally between 6, 150 mm, Petri dishes. Three of the Petri dishes were placed outside in direct sunlight in Edinburgh, UK, for 2.5 h, while the remaining three dishes were protected from sunlight by aluminium foil. After exposure, L3s were concentrated by settling under gravity and excess water removed by aspiration. Samples were frozen at – 80 °C until S1 extracts were prepared. The protein concentrations in each of the S1 extracts were calculated using a bicinchoninic acid (BCA) assay (Pierce, UK) with BSA standards prepared in PBS. GTP-CH activity was measured, as described below, in triplicate for each S1 extract of each replicate. GTP (Sigma) was dissolved in Buffer A (0.05 M Tris, 0.05 M KCl, 2.5 mM EDTA, 10% glycerine (v/ v), pH 7.8) to a final concentration of 500 lM. Next, 90 ll of GTP/ Buffer A were combined with 10 ll of S1 extract and incubated for 1 h in darkness at 37 °C. Reactions were terminated by the addition of 590 ll of Buffer A and 100 ll of acidic iodine solution (1% I2 and 2% KI in 1 M HCl) and kept at room temperature for 15 min. After this, 100 ll of 1 M NaOH were added before a final incubation with 5 units of alkaline phosphatase (Promega, UK) at 37 °C for 45 min to produce neopterin. A commercially-available neopterin assay was performed on each sample as per the manufacturer’s protocol (ELItest; Brahms, Germany). Neopterin concentrations were calculated from the standard curve generated using neopterin in human serum samples provided with the kit. Neopterin concentrations were expressed as nmol per mg of S1 protein. 2.3. Functional complementation of the C. elegans cat-4 mutant Genomic DNA was extracted from the N2 (Bristol) strain of C. elegans using the DNeasy kit (Qiagen, UK). This was used as a template for PCR amplification of 1,500 bp of the 5’ untranslated region (UTR) of the Ce-cat-4 gene using primers that also introduced restriction sites for NcoI (forward primer), HindIII and SacII (reverse primer; all primer sequences are available on request from the authors). The PCR product was cloned into pGEMÒ-T plasmid using the restriction enzymes, NcoI and SacII, to form the pCe5’UTR plasmid. Full-length amplicons representing cat-4 genes from C. elegans and T. circumcincta were amplified by PCR from genomic DNA of each species using primers that added the restriction sites HindIII (forward) and SpeI (reverse) and these were then subcloned into the pCe5’UTR plasmid using the restriction enzymes, HindIII and SpeI, to form the pCe-cat-4 (control) and pTci-cat-4 plasmids. Finally, the 3’ UTR for the cat-4 gene from C. elegans was amplified by PCR using primers that added restriction sites for SpeI (forward) and NdeI (reverse), and subcloned into the pCe-cat-4 and pTci-cat4 plasmids using the restriction enzymes, SpeI and NdeI, to form the pRescue plasmids. After confirmation of the correct assembly of the construct by sequence analysis (Eurofins MWG), rescue plasmid DNA was purified using a plasmid mini kit (Qiagen). Rescue plasmid DNA (final concentration 20 lg/ml) was microinjected into the distal arm of the gonad of adult stage C. elegans cat4(tm773) deletion mutant strain LC81 (five-times out crossed) to generate transgenic lines. Caenorhabditis elegans rol-6 plasmid DNA (final concentration 100 lg/ml) was used as a co-injection marker to identify transformants by their ‘‘right-roller’’ phenotype (Kramer et al., 1990). Heritable extrachromosomal roller lines for both the C. elegans control rescue plasmid and the T. circumcincta rescue plasmid (referred to as Ce-rescue and Tc-rescue) were maintained on Nematode Growth Media (NGM) plates (Brenner, 1974). Individual, transgenic worms were picked and lysed and

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their DNA used as template for PCRs to confirm the absence of the C. elegans cat-4 gene in the C. elegans cat-4(tm773) deletion mutant strain LC81. Restoration of cuticular integrity in the transgenic worms by functional complementation with the orthologous Cecat-4 and Tci-cat-4 genes was measured by analysis of the sensitivity of live worms to sodium hypochlorite (Loer and Kenyon, 1993). Adult worms from each strain (C. elegans N2, LC81, Ce-rescue and Tc-rescue) were picked individually into a 10% NaClO solution (10–15% available chlorine; Sigma) and the time elapsed prior to paralysis was measured. This assay was performed with 24 individual worms from each strain. Total melanin concentrations in a sample of worms from each C. elegans strain (N2, LC81, Ce-rescue and Tc-rescue) were also measured. Briefly, synchronised cultures of each strain were grown on peptone-rich medium (1.2 g of NaCl, 20 g of peptone, 25 g of agarose, 1 ml of 5 mg/ml cholesterol, 1 ml of 1 M CaCl2, 1 ml of 1 M MgSO4, 25 ml of 1 M KPO4 per litre of water) seeded with the BL22 strain of Escherichia coli. When the individuals reached adulthood, 500 young adult worms were collected in M9 buffer (Brenner, 1974) and the volume reduced to 30 ll by aspiration before the addition of 100 ll of 6.5 M NaOH and digestion for 24 h at 40 °C. Following centrifugation at 12,000g for 10 min, 120 ll of the supernatant were passed through a WizardÒSV Minicolumn (Promega) by centrifugation at 12,000g for 1 min and the melanin concentration of 90 ll of the eluate was measured using a microtitre plate and a standard series of melanin solutions as described in Section 2.1. The experiment was repeated three times for each strain of C. elegans. 2.4. Statistical analyses For the comparison of melanin concentrations in UV-exposed and non-exposed T. circumcincta larvae, and in the transgenic C. elegans, data were analysed using Student’s t-tests. For the analysis of GTP-CH activity, mean neopterin concentrations were compared using ANOVA. 3. Results 3.1. Exposure of T. circumcincta to sunlight: melanisation

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Fig. 2. Guanosine triphosphate-cyclohydrolase I (GTP-CH) activity in different lifecycle stages of Teladorsagia circumcincta. GTP-CH activity is measured indirectly following conversion of its reaction product (7,8-dihydroneopterin triphosphate) to neopterin. Shaded bars and open bars represent each of two repetitions of the experiment using independent extracts of nematode material. Each bar represents the mean of three technical replicates from one protein extraction with error bars representing S.E.M.

3.3. Functional complementation of C. elegans cat-4 mutant Caenorhabditis elegans cat-4(tm773) mutant worms (LC81) died more rapidly (14 ± 1 s) when exposed to 10% sodium hypochlorite solution than did wild-type (WT) N2 worms (59 ± 2 s, P < 0.001 Fig. 4A). Transgenic cat-4(tm773) mutant worms carrying either WT C. elegans or T. circumcincta cat-4 gene plasmid constructs were rescued for the rapid death phenotype. These worms survived significantly longer than the Ce-cat-4 mutant (Ce-cat-4 rescue: 63 ± 2 s; Tc-cat-4 rescue: 59 ± 2 s; P < 0.001 for each). Rescued worms showed no statistically significant difference in time to death compared with WT N2 worms, indicating effective functional complementation. Gene-specific PCR analysis of C. elegans individual worm lysates confirmed that the Ce-cat-4 gene was only detectable in WT N2 and Ce-rescue strains, and not in the Tc-rescue strain, in which only the Tci-cat-4 gene was amplifiable (data not shown). Mean melanin concentrations were 21% higher in the N2 strain than in the Ce-cat-4 mutants (Fig. 4B, P = 0.036) and melanin levels

The effect of exposure to natural sunlight on melanin levels in T. circumcincta L3s was assessed by placing L3s in direct sunshine on each of 3 days in Spring, 2011. Melanin levels detected in T. circumcincta L3s exposed to UV radiation in direct sunlight were significantly higher (range from 2.2 to 4.4-fold increase) than in L3s that had been kept in darkness. Mean melanin concentrations in the extracts over the sampling periods were: sunlight exposed 202.2 lg/ml ± 46.3; non-exposed 64.3 lg/ml ± 4.7 (n = 3, P = 0.031). 3.2. GTP-CH activity assay Differences in GTP-CH activity, as detected via a neopterin assay, were observed across the life-cycle stages of T. circumcincta, with the highest levels observed in L1s and L3s (Fig. 2). Levels of enzyme activity in L5s and adult stages were significantly lower than those measured in the other stages (P < 0.05). In both biological replicates tested, there were significant differences in GTP-CH activity between pre-parasitic and L4 stages (Replicate 1, L3 levels were significantly higher than in L4 (P = 0.009); Replicate 2, L1 levels were significantly higher than in L4 (P = 0.03)). Exposure to sunlight resulted in increased GTP-CH activity in T. circumcincta L3s, with sunlight-exposed L3s possessing 32% higher activity on average (P = 0.019) than those which had been maintained in darkness (Fig. 3).

Fig. 3. Guanosine triphosphate-cyclohydrolase I (GTP-CH) activity in Teladorsagia circumcincta larvae exposed to UV radiation in natural sunlight. GTP-CH activity is measured indirectly following conversion of its reaction product (7,8-dihydroneopterin triphosphate) to neopterin. The mean neopterin concentrations per mg of protein in nematode extracts were measured in T. circumcincta L3s that had been exposed to UV radiation in direct sunlight or kept in darkness. Error bars represent the S.E.M.s of three technical replicates of three biological replicates for each sample.

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Fig. 4. Functional complementation of Caenorhabditis elegans cat-4 mutant worms with the homologous Ce-cat-4 gene and heterologous Tci-cat-4 gene from Teladorsagia circumcincta. (A) The duration of survival of the different strains of C. elegans when incubated in a 10% sodium hypochlorite solution. Error bars represent the S.E.M., n = 24 individual measurements. N2, wild-type strain; LC81, Ce-cat-4 deletion mutant strain; Ce-rescue and Tc-rescue, LC81 mutant strain rescued with the wild-type Ce-cat-4 or Tci-cat-4 gene, respectively. Bars annotated with the same letter are not significantly different (P < 0.05). (B) The melanin concentrations within extracts of the different strains of C. elegans. Error bars represent the S.E.s of three replicate measurements from extracts of 500 individuals. Bars annotated with the same letter are not significantly different (P < 0.05).

in both rescue strains were also statistically significantly higher than in the Ce-cat-4 mutant strain (Fig. 4B).

4. Discussion Here, we demonstrated the inducible nature of melanisation in free-living L3s of T. circumcincta. The proposed role of GTP-CH in this process was explored and it was demonstrated that T. circumcincta L3s exposed to natural sunlight had increased levels of GTPCH activity and increased melanin. Caenorhabditis elegans cat-4 mutants have lower levels of melanin in their cuticles than WT worms (Calvo et al., 2008 and Fig. 4B herein) and, through functional complementation, it was demonstrated that the Tci-cat-4 gene was capable of performing the same role as Ce-cat-4 in restoring melanisation. Together, these data demonstrate a role for GTP-CH in synthesising melanin in the pre-parasitic stages of T. circumcincta and in the maintenance of cuticular integrity. Previous studies, to determine the impact of UV exposure on larval death in T. circumcincta, exposed larvae to UV over a 24 h period (van Dijk et al., 2009). To avoid larval death, L3s were only exposed to sunlight for 5 h here. The change in colour of the supernatant derived from L3s after exposure to sunlight was visible by eye, and suggests a relatively rapid increase in melanin within the L3s; indeed this change occurred in less than 5 h. As has been shown previously (Baker et al., 2011), cat-4 transcript is present at high levels in T. circumcincta L1-L3. As excessive UV exposure can be lethal to

nematodes, prioritising resources to produce and/or store the transcript encoding the rate-limiting factor in a pathway that could prove critical for survival may be an adaptive strategy. As levels of GTP-CH enzyme activity have not been reported in nematodes previously, we sought to determine whether the high levels of transcript observed in the pre-parasitic stages of T. circumcincta (Nisbet et al., 2008; Baker et al., 2011) were reflected in the levels of the protein they encode. The data presented here demonstrate that this is the case, with higher GTP-CH activity detected in these pre-parasitic stages compared with L5s and adults. The differing activity levels in L4s, where transcript levels are lower than in pre-parasitic stages (Nisbet et al., 2008; Baker et al., 2011) may reflect variable persistence of active enzyme from earlier stages. The role and biochemical function of GTP-CH in melanisation in T. circumcincta was investigated successfully in this study using functional complementation in C. elegans. The use of this technique was central to understanding gene function in the study described herein as attempts to silence T. circumcincta cat-4 expression by RNA interference (RNAi) were unsuccessful (data not shown). Knockdown of another gene, which encodes b-tubulin isoform 1, was successful in these preliminary studies, suggesting that RNAi in larval T. circumcincta may be possible for some genes. Initial studies, using in vivo inhibition of the enzyme by a specific biochemical inhibitor (DAHP) in larval T. circumcincta were partially successful but confounded by high levels of mortality induced by the inhibitor (data not shown), underlining the utility of the functional complementation approach used here. In the present study we induced melanisation in T. circumcincta by exposure to sunlight and propose that this is a mechanism for protection against UV-induced DNA damage. The damaging effects of UV radiation on nematode larvae can be profound: survival of Ostertagia ostertagi L3s is significantly reduced if pasture is cut to 5–7 cm high on a regular basis, allowing deeper penetration of UV radiation into the sward (Fernandez et al., 2001). UV radiation has been shown to have a detrimental effect on the survival of the infective stages of other parasite species such as the intertidal trematode Maritrema novaezealandensis, with survival linked in a negative dose–response relationship to levels of UV radiation (Studer et al., 2012a). In M. novaezealandensis however, no mechanisms could be found within the cercariae that provided protection against the UV damage, despite the strong evolutionary pressures (Studer et al., 2012b). In addition to UV-protection, melanin is known to play a number of alternative or additive roles within eukaryotes (Riley, 1992). One function is to provide structural stability of invertebrate exoskeletons by cross-linking proteins. For insect cuticles this provides the main outer structural support (Riley, 1997) and, in C. elegans (and other nematodes), the structure of the outer cuticle also plays a key role in survival, providing an impervious barrier to prevent desiccation and protect the worm from harmful elements of the environment (Page and Johnstone, 2007). In nematodes, the main respiratory surface is the cuticle and as such, is exposed to the highest levels of oxygen (Fujii et al., 2011). Melanin has been hypothesised previously to protect C. elegans against the resulting oxidative stress (Calvo et al., 2008). As shown here Tci-cat-4 is capable of restoring melanisation of the Ce-cat-4 mutant cuticle, so it may be proposed that multiple roles of melanin and melanisation are shared between the two nematode species. In conclusion, we have demonstrated that T. circumcincta L3s respond to exposure to sunlight through melanisation, which we hypothesise provides protection against UV radiation. In order to produce the melanin, high levels of GTP-CH are required by the pre-parasitic larval stages. Acknowledgements We would like to thank Alison Morrison, Moredun Research Institute, UK, for the provision of parasite material. UV data were

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kindly provided by Andy Pearson at the Health Protection Agency, UK. RB is funded by The Perry Foundation, UK; JBM and AJN by Scottish Government RESAS. CML is supported by an endowment from the Fletcher Jones Foundation, USA. Some C. elegans strains were obtained from the Caenorhabditis Genetics Center, a National Institutes of Health, USA, Research Resource. The cat-4(tm773) deletion mutant was originally obtained from the National Bioresource Project for the Experimental Animal ‘Nematode C. elegans’ (Prof. Shohei Mitani, Tokyo Women’s Medical University, Japan).

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