Vaccination is a feasible strategy for controlling the haematophagous poultry red mite Dermanyssus gallinae. A cDNA library enriched for genes upregulated after feeding was created to identify potential vaccine antigens. From this library, a gene (Dg-CatD-1) encoding a 383 amino acid protein (Dg-CatD-1) with homology to cathepsin D lysosomal aspartyl proteinases was identified as a potential vaccine candidate. A second gene (Dg-CatL-1) encoding a 341 amino acid protein (Dg-CatL-1) with homology to cathepsin L cysteine proteinases was also selected for further study. IgY obtained from naturally infested hens failed to detect Dg-CatD-1 suggesting that it is a concealed antigen. Conversely, Dg-CatL-1 was detected by IgY derived from natural-infestation, indicating that infested hens are exposed to Dg-CatL-1. Mortality rates 120 h after mites had been fed anti-Dg-CatD-1 were significantly higher than those fed control IgY (PF<0·01). In a survival analysis, fitting a proportional hazards model to the time of death of mites, anti-Dg-CatD-1 and anti-Dg-CatL-1 IgY had 4·42 and 2·13 times higher risks of dying compared with controls (PF<0·05). Dg-CatD-1 and L-1 both have potential as vaccine antigens as part of a multi-component vaccine and have the potential to be improved as vaccine antigens using alternative expression systems.

Introduction. Leishmania donovani, the causative agent of visceral leishmaniasis in the Indian subcontinent, has been reported to be genetically homogeneous. In order to support ongoing initiatives to eliminate the disease, highly discriminative tools are required for documenting the parasite population and dynamics. Methods. Thirty-four clinical isolates of L. donovani from Nepal were analysed on the basis of size and restriction endonuclease polymorphisms of PCR amplicons from kinetoplast minicircle DNA, 5 nuclear microsatellites, and nuclear loci encoding glycoprotein 63, cysteine proteinase B, and hydrophilic acylated surface protein B. We present and validate a procedure allowing standardized analysis of kDNA fingerprint patterns. Results. Our results show that parasites are best discriminated on the basis of kinetoplast minicircle DNA (14 genotypes) and 1 microsatellite defining 7 genotypes, while the remaining markers discriminated 2 groups or were monomorphic. Combination of all nuclear markers revealed 8 genotypes, while extension with kDNA data yielded 18 genotypes. Conclusion. We present tools that allow discrimination of closely related L. donovani strains circulating in the Terai region of Nepal. These can be used to study the micro-epidemiology of parasite populations, determine the geographical origin of infections, distinguish relapses from re-infection, and monitor the spread of particular variants.

Since Fasciola sp. contained proteolytic enzyme(s), it was confirmed that degradation took place in protein components in extracts of the liver flukes, which resulted in lack of clarity of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Degradation was shown to occur mostly during a heating process of the extract samples. The proteolytic activity in the extracts was completely blocked and electrophoretic patterns were improved only by the use of cysteine proteinase inhibitor N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). Great improvement was also noted in electrophoretic patterns of the extracts of other trematodes, such as Paragonimus westermani, P. miyazakii and Clonorchis sinensis, when their extracts were treated with E-64.

Sensitive assays capable of detecting proteinases in single females of the phytoparasite Globodera pallida have been developed and used to define the proteinase activity of young adult females. Digestion of the large subunit of the plant protein Rubisco established a pH optimum for the proteinase activity at pH 5·7. The activity was inhibited by the cysteine proteinase inhibitors p-chloromercuribenzoic acid (PMBA) and p-chloromercurisulphonic acid (PMSA) and stimulated by both cysteine and dithiothreitol (DTT). It was moderately reduced by L-trans-epoxysuccinyl-leucylamido-(4- guanidino) butane (E64) but not by specific inhibitors of serine, aspartate or metallo-proteinases. The activity separated into 3 bands on a non-denaturing gel but only I proteinase of 62 kDa was recovered following a combination of anion-exchange chromatography and affinity chromatography using PMBA. The effect of inhibitors was similar to that reported previously for some of the cysteine proteinase activity recovered from Caenorhabditis elegans but is apparently not that for which the corresponding gene has been cloned in this nematode and Haemonchus contortus. The proteinase may have a major role in digestion of dietary protein and so offers an exciting target for future control of this important plant-parasitic nematode.

A new method is described which has made possible the long-term axenic cultivation of Leishmania mexicana amastigotelike forms in Schneider's Drosophila medium supplemented with 2% (v/v) foetal calf serum. Unlike previous methods, it utilizes direct culture of parasites obtained from the lesions of infected animals rather than adaptation of promastigotes in vitro. Ultrastructural (possession of megasomes), biochemical (cysteine proteinase activity and gelatin SDS-PAGE banding pattern) and infectivity (in vivo) data are presented which show the close similarity of the cultured forms to lesion amastigotes. The axenically cultured forms grew optimally at a temperature of 32–33 °C, providing further evidence for their amastigote nature. It was found that adjustment of the pH of the growth medium to 5·4 was required in order to retain the amastigote morphology of the cultured parasites. This supports the notion that leishmanial amastigotes are acidophiles.

The excreted/secreted proteinases of adult and juvenile Fasciola hepatica maintained in vitro were found to hydrolyse the fluorogenic substrates Cbz-Phe-Arg- and Cbz-Arg-Arg-NHMec. This activity was demonstrated to have a classical cysteine proteinase inhibitor profile, with turn-over of both substrates being blocked by pre-incubation with E64 and peptidyl diazomethanes. The Cbz-Arg-Arg-NHMec hydrolysing activity of the mature fluke exhibited an alkaline stability not characteristic of its mammalian lysosomal counterparts. Further, the biotinylated affinity reagents biotin-Phe-Ala- CHN2 and biotin-Phe-Cys(SBzyl)-CHN2 were used to label and characterize these cysteine proteinases in terms of apparent molecular weight and subsite specificity. Adult fluke media were found to contain four species of molecular weights 66, 58, 50 and 25–26 kDa; juvenile media contained three species of molecular weights 66, 54 and 25–26 kDa. The major 25–26 kDa cysteine proteinase common to both stages was shown to have a subsite specificity similar to that of mammalian cathepsin B.

A cDNA encoding the immunogen Pso o 1 from Psoroptes ovis was obtained by polymerase chain reaction (PCR) amplification. The amplicon contained the entire coding sequence for the prepro-enzyme in an open reading frame (ORF) of 966 bp. This gene encoded a predicted protein of 322 amino acids (aa) with 64% aa identity (80% similarity) to the major house dust mite faecal allergen Der f 1. The pro-enzyme form of Pso o 1 was expressed as a recombinant protein in the Pichia pastoris-eukaryotic expression system. Maturation of the recombinant pro-enzyme by autocatalytic activation was not observed, and such maturation could not be achieved using a number of techniques known to activate recombinant Der p 1 and Der f 1 expressed in the same system. Serum raised against recombinant Pso o 1 cross-reacted with mature Der p 1 and allowed Pso o 1 to be immunolocalized to the gut of P. ovis.

Infection of the central nervous system by Taenia solium cysticerci is the cause of human neurocysticercosis, a major neurological infection in the Third World and an emerging infectious disease in the United States. We previously isolated a cysteine proteinase from cysticerci of Taenia crassiceps and demonstrated that it degrades human IgG in vitro. We have now isolated a 48 kDa thiol-dependent proteinase from T. solium. The T. solium enzyme also degrades human IgG, but does not significantly degrade albumin. IgG degradation was inhibited by cysteine proteinase inhibitors, but not significantly by inhibitors of aspartic, serine, or metalloproteinases. The peptide substrate specificity and pH optimum resemble cathepsin L. The Km for the peptide substrate Z-Phe-Arg-AFC was calculated to be 7·0×10−6M, the Kcat was 1·98×105 s−1, and the Kcat/Km 2·84×109M−1 s−1, a value which is within the diffusion control limit for highly catalytic enzymes. We propose that immunoglobulin degradation by the T. solium cysteine proteinase may play a key role in the host-parasite interface and could be employed as a target for chemotherapy.

Promastigotes of Leishmania mexicana mutants lacking the multicopy CPB cysteine proteinase genes (ΔCPB) are markedly less able than wild-type parasites to infect macrophages in vitro. ΔCPB promastigotes invade macrophages in large numbers but are unable to survive in the majority of the cells. In contrast, ΔCPB amastigotes invade and survive within macrophages in vitro. This extreme in vitro stage-specific difference was not mimicked in vivo; both promastigotes and amastigotes of ΔCPB produced lesions in BALB/c mice, but in each case the lesions grew considerably more slowly than those caused by wild-type parasites and only small lesions resulted. Inhibition of CPB in situ using cell-permeant peptidyldiazomethylketones had no measurable effect on parasite growth or differentiation axenically in vitro. In contrast, N-benzoyloxycarbonyl-phe-ala-diazomethylketone reduced the infectivity of wild-type parasites to macrophages by 80%. Time-course experiments demonstrated that application of the inhibitor caused effects not seen with ΔCPB, suggesting that CPB may not be the prime target of this inhibitor. The data show that the CPB genes of L. mexicana encode enzymes that have important roles in intracellular survival of the parasite and more generally in its interaction with its mammalian host.

CrmA is an unusual viral serpin that inhibits both cysteine and serine proteinases involved in the regulation of host inflammatory and apoptosis processes. It differs from other members of the serpin superfamily by having a reactive center loop that is one residue shorter, and by its apparent inability to form SDS-stable covalent complexes with cysteine proteinases. To obtain insight into the inhibitory mechanism of crmA, we determined the crystal structure of reactive center loop-cleaved crmA to 2.9 Å resolution. The structure, which is the first of a viral serpin, suggests that crmA can inhibit cysteine proteinases by a mechanism analogous to that used by other serpins against serine proteinases. However, one striking difference from other serpins, which may be significant for in vivo function, is an additional highly charged antiparallel strand for β sheet A, whose sequence and length are unique to crmA.

Cysteine proteinases have been implicated in the protection conferred by vaccination with detergent-soluble extracts of Haemonchus contortus. In the present study, antisera from sheep refractory to Haemonchus challenge following vaccination with a ‘proteinase-enriched’ Haemonchus gut membrane extract, were employed to screen a cDNA expression library of the adult parasite. This resulted in the isolation of 3 cDNAs (designated hmcp1, 4 and 6) encoding cathepsin B-like cysteine proteinases. Immunocytochemical studies specifically localized the products of these genes to the microvillar surface of the parasite's gut and RT–PCR experiments revealed that these were developmentally regulated, being expressed exclusively during the blood-feeding parasitic stages. In addition, a generic PCR approach was adopted in order to identify the predominant cysteine proteinases in a UK strain of Haemonchus. A panel of 5 cDNAs, including hmcp1 and 4, was amplified in this way. Genomic Southern blot analysis indicated that some of these enzymes were encoded by single-copy genes, whereas others were encoded by multi-copy genes. Subsequent sequence analysis revealed that the proteases identified in this study were distinct from those previously reported in USA strains of the parasite.

We compared a Trypanosoma cruzi clone unable to infect or induce pathology in mice (CL-14), with virulent T. cruzi (Y and CL strains) in terms of cruzipain expression, subcellular distribution and functional activity. Our results showed that (1) intracellular Y amastigotes expressed R1 (carboxy-terminal) and R2 (catalytic) domains concentrated in cytoplasmic vesicles, while CL-14 presented R1 labelling on membrane clusters and R2 in intracellular compartments, (2) CL-14-trypomastigotes presented R1 and R2 staining preferentially on flagellar and cellular membranes, similar to CL, but different from Y strain intracellular labelling pattern, (3) flow-cytometry revealed higher expression of R1 by CL-14-trypomastigotes than virulent strains, but R2 staining similar to CL-trypomastigotes, (4) CL-14-trypomastigotes presented normal cruzipain activity in gelatin gels, but different banding patterns were found in CL-14 versus CL and Y strains. Our data rule out failure in cruzipain expression, activity or subcellular distribution as an explanation for CL-14 biological behaviour, but suggest the expression of a different isoform. These results also cast doubt on the putative role of cruzipain as a target of immunopathological responses, since high levels of functional cruzipain are expressed by a non-pathogenic T. cruzi.

Two cDNAs encoding cysteine proteinases were isolated from a cDNA library constructed from feeding females of Heterodera glycines. The library was screened with a cysteine proteinase gene fragment originally amplified from cDNA of H. glycines. Database searches predict that 1 cDNA (hgcp-I) encodes a cathepsin L-like proteinase, while the second (hgcp-II) encodes a cathepsin S-like enzyme. Both predicted proteins contain a short secretion signal sequence, a long pro-peptide and a mature protein of 219 amino acids. Southern blot analysis suggests that the cathepsin S-like enzyme, HGCP-II, is encoded by a single-copy gene in contrast to the cathepsin L-like proteinase, HGCP-I which may have 2 homologues. The regions encoding the mature proteinases were cloned into an expression vector and recombinant protein produced in E. coli. HGCP-I was shown, after refolding, to cleave the synthetic peptide Z-Phe-Arg-AMC, and this activity could be inhibited by the engineered rice cystatin Oc-IΔD86. HGCP-II showed no activity against the synthetic substrates tested. The knowledge gained from these studies will improve our understanding of plant nematode proteinases and aid the development of a rational proteinase inhibitor-based approach to plant nematode resistance.

Based on substrate preferences, cercariae of Schistosoma mansoni were seen to express both cathepsin L and cathepsin B cysteine proteinases, although the former activity was many-fold greater. Two cathepsin L activities identified in cercarial extracts by zymography co-migrated with activities in extracts of 3 h and 24 h schisotosomula and in extracts of adult worms. Since these enzymes have been implicated in haemoglobin digestion by adult worms, they may perform a similar function in schistosomula. Immunolocalization using scanning electron micrographs showed that cathepsin L and cathepsin B proteinases were present in the cercarial post-acetabular glands. In addition, cercarial serine proteinase activities considered to facilitate skin penetration efficiently cleaved the substrates Z-Gly-Pro-Arg-NHMec and Z-Gly-Pro-Lys-NHMec. Cercariae release most of this serine proteinase activity when induced to secrete the contents of their acetabular glands. In contrast, newly transformed 3 h and 24 h schistosomula did not express this activity.