Elsevier

Experimental Parasitology

Volume 104, Issues 3–4, July–August 2003, Pages 96-103
Experimental Parasitology

Haemonchus contortus: molecular characterisation of a small heat shock protein

This manuscript is dedicated to the loving memory of Dr. Susan Newton. A tremendous scientist and leader who instilled in many of us a respect and love for the wonderful world of parasitology. Sue, we will forever miss you.
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Abstract

A cDNA encoding a predicted small heat shock protein, HSP20, was isolated from the parasitic nematode Haemonchus contortus. This cDNA encoded a predicted protein of 156 amino acids, which had high sequence identity with other nematode small heat shock proteins. Southern blot analysis of genomic DNA suggested that in H. contortus HSP20 is encoded by a single copy gene. The HSP20 transcript and protein were expressed in the infective larvae (L3), early L4 and adult stages, but expression was not increased by heat shock treatment. In situ hybridisation analysis was used to localise expression of HSP20 mRNA in the adult parasite. Similar HSPs (heat shock protein) were detected by Western blotting in Ancylostoma caninum, Dictyocaulus viviparus, and Toxocara canis, but not in Trichostronglyus colubriformis. The conservation of HSP20 in several different nematode species may reflect its importance to parasites that require mammalian hosts as a part of their development.

Index Descriptors and Abbreviations: Haemonchus contortus; nematode; small heat shock protein; L3, infective larvae; xL3, exsheathed L3; eL4, early L4; EST, expressed sequence tag; HSP20, heat shock protein 20; sHSP, small heat shock protein

Introduction

Parasitism by worms is one of the most challenging problems to livestock industries, with an estimated cost of £1000 million per annum attributed to parasitism (Newton and Munn, 1999). The abomasal nematode, Haemonchus contortus, is predominantly a parasite of sheep, with the pathological effects of infection being largely due to the worm’s blood feeding behaviour. The transition from free living larval stages to parasitism of a mammalian host describes a period where parasites encounter stress due to dramatic environmental changes. Small heat shock proteins (sHSPs), with molecular weights ranging from 12 to 43 kDa, have been identified in virtually all eukaryotes and are believed to interact within cells as molecular chaperones, with the level of expression being inducible through exposure to particular environments including stress (reviewed in Arrigo and Landry, 1994). Some sHSP genes are developmentally regulated, including those of parasitic nematodes, where sHSPs may play a role in transition from free-living or insect vector stages to the mammalian host (Raghavan et al., 1999; Thompson et al., 1996; Tweedie et al., 1993). In this study, a small heat shock protein from the parasitic nematode H. contortus was identified and characterised at the molecular level. The transcript and protein were constitutively expressed in the parasitic stages, and expression levels were not elevated by heat shock treatment.

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Materials and methods

Infective larvae (L3), exsheathed L3 (xL3), in vitro cultured early L4 (eL4), and adult H. contortus were prepared by methods previously described (Nikolaou et al., 2002). Trichostrongylus colubriformis were provided by Novartis Animal Health; Oesophagostomum dentatum, Ancylostoma caninum, and Toxocara canis were provided by Peter Boag (Victorian Institute of Animal Science); and Dictyocaulus viviparus were obtained from an infected bovine calf at postmortem examination.

Total RNA for cDNA

Results

Clone G0056 was identified from an adult H. contortus intestinal cDNA library as part of an expressed sequence tag (EST) sequencing project. The full-length DNA sequence of the G0056 insert was determined in both directions (refer to Genbank Accession No. AY130968). The cDNA insert of 558 bp included an open reading frame of 468 nucleotides, encoding a predicted protein of 156 amino acids with a predicted molecular weight of 18.35 kDa. The sequence surrounding the predicted initiating methionine

Discussion

Small HSPs have been described previously for the rat intestinal nematode N. brasiliensis (Tweedie et al., 1993), canine heartworm, Dirofilaria immitis (Lillibridge et al., 1996), and the human filarioids Brugia malayi (Raghavan et al., 1999) and B. pahangi (Thompson et al., 1996), but this is the first report of a sHSP from a gastrointestinal nematode of ruminants.

With the advent of EST sequencing programs, sequences for putative sHSPs have now been deposited in the databases for a wide range

Acknowledgements

Project support was provided by Novartis Animal Health Australasia, the Department of Natural Resources and Environment, Victoria and Agriculture Victoria. We thank the Molecular Vaccines team for dissecting the nematode intestines, preparation of the Northern Blots, and parasite extracts. We also thank Tamarae Griffiths and Toby Williams (Novartis Animal Health Australia) for immunisation of sheep, preparation of antisera and provision of L3 H. contortus and adult T. colubriformis; and Peter

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The sequence data reported herein have been submitted to the Genbank, EMBL, and DDBJ databases and assigned Accession No. AY130968.

1

Present address: School of Engineering and Science, Swinburne University of Technology, Hawthorn, Vic. 3122, Australia.

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