Molecular characterization, expression and localization of a peroxiredoxin from the sheep scab mite, Psoroptes ovis.

The sheep scab mite, Psoroptes ovis, induces an intensely pruritic exudative dermatitis which is responsible for restlessness, loss of appetite and weight loss. Within the first 24 h of infection, there is a rapid inflammatory influx of eosinophils and apoptosis of the keratinocytes at the site of infection. The former cell type is capable of a sustained respiratory burst, toxic products of which may directly damage the mite and also contribute to lesion formation. Analysis of a P. ovis expressed sequence tag (EST) database identified a number of antioxidant enzyme-encoding sequences, including peroxiredoxin (thioredoxin peroxidase EC 1.11.1.15), all of which may help the mite endure the potentially toxic skin environment. A full length sequence encoding Po-TPx, a protein of 206 amino acids which showed high homology to a peroxiredoxin from the salivary gland of the tick Ixodes scapularis, was amplified from P. ovis cDNA. Recombinant Po-TPx was expressed in bacteria and antiserum to this protein was used to localize native Po-TPx in mite sections. Peroxiredoxin was localized, amongst other sites, to a subpharyngeal region in mite sections. The recombinant protein was recognized by sera from sheep infested with the mite suggesting that it may be secreted or excreted by the mite and interact with the host immune response.

Immune recognition of the surface associated antigen, Tc-SAA-1, from infective larvae of Teladorsagia circumcincta.

A cDNA encoding a surface-associated antigen was amplified by reverse transcriptase polymerase chain reaction (PCR) from RNA extracted from Teladorsagia circumcincta exsheathed third stage larvae (xL3). The protein encoded by this cDNA, Tc-SAA-1, displays 77% identity over 162 amino acid residues to a surface associated antigen from Ancylostoma caninum (Ac-SAA-1). Antiserum raised against a bacterially-expressed recombinant form of Tc-SAA-1 reacted with a native protein in somatic and surface extracts of xL3 but not with L4 or adult parasites. Limited binding of anti-Tc-SAA-1 antibody was observed on the cuticular surface of xL3 s, however, regions of localization underlying the cuticle were observed. Incubation of xL3 T. circumcincta with anti-SAA rabbit serum failed to significantly inhibit penetration of the abomasal mucosa in vitro. IgA in abomasal mucus derived from sheep that had received a trickle infection of T. circumcincta bound recombinant Tc-SAA-1.

Identification and characterization of an asparaginyl proteinase (legumain) from the parasitic nematode, Haemonchus contortus.

Asparaginyl proteinases (or legumains) are a recently identified, novel class of cysteine proteinase which specifically hydrolyse peptide bonds after asparagine residues. Legumains have been implicated in the activation of cysteine proteases, particularly cathepsin B-like proteinases which are thought to help degrade the bloodmeal in blood-feeding helminths such as schistosomes, hookworms and other nematode species. An EST sequence representing a full-length legumain was identified from the Haemonchus contortus dataset. This encoded a protein with a predicted Mr of 49 kDa, the amino acid sequence of which showed good homology (34-40% identity) to legumains from Schistosoma mansoni, human and rat and contained a legumain-like active site. RT-PCR indicated that the legumain transcript was expressed from the L4 life-cycle stage onwards. The coding sequence was expressed in E. coli and antibodies to the resultant recombinant protein indicated that the enzyme was expressed in the microvillar surface of the intestinal cells. Legumain activity was detected in extracts of the adult parasite but not the host protective Thiol-Sepharose-binding fraction, although it was detectable in the latter by immunoblot. Activity was relatively insensitive to E64, an inhibitor of cysteine proteinases and completely inhibited by the alkylating agent, N-ethylmaleimide, consistent with inhibitor effects on previously characterized legumains.