Characterization of a novel glycosylated glutathione transferase of Onchocerca ochengi, closest relative of the human river blindness parasite.

Filarial nematodes possess glutathione transferases (GSTs), ubiquitous enzymes with the potential to detoxify xenobiotic and endogenous substrates, and modulate the host immune system, which may aid worm infection establishment, maintenance and survival in the host. Here we have identified and characterized a σ class glycosylated GST (OoGST1), from the cattle-infective filarial nematode Onchocerca ochengi, which is homologous (99% amino acid identity) with an immunodominant GST and potential vaccine candidate from the human parasite, O. volvulus, (OvGST1b). Onchocerca ochengi native GSTs were purified using a two-step affinity chromatography approach, resolved by 2D and 1D SDS-PAGE and subjected to enzymic deglycosylation revealing the existence of at least four glycoforms. A combination of lectin-blotting and mass spectrometry (MS) analyses of the released N-glycans indicated that OoGST1 contained mainly oligomannose Man5GlcNAc2 structure, but also hybrid- and larger oligommanose-type glycans in a lower proportion. Furthermore, purified OoGST1 showed prostaglandin synthase activity as confirmed by Liquid Chromatography (LC)/MS following a coupled-enzyme assay. This is only the second reported and characterized glycosylated GST and our study highlights its potential role in host-parasite interactions and use in the study of human onchocerciasis.

Repositioning of an existing drug for the neglected tropical disease Onchocerciasis.

Onchocerciasis, or river blindness, is a neglected tropical disease caused by the filarial nematode Onchocerca volvulus that affects more than 37 million people, mainly in third world countries. Currently, the only approved drug available for mass treatment is ivermectin, however, drug resistance is beginning to emerge, thus, new therapeutic targets and agents are desperately needed to treat and cure this devastating disease. Chitin metabolism plays a central role in invertebrate biology due to the critical structural function of chitin for the organism. Taken together with its absence in mammals, targeting chitin is an appealing therapeutic avenue. Importantly, the chitinase OvCHT1 from O. volvulus was recently discovered, however, its exact role in the worm's metabolism remains unknown. A screening effort against OvCHT1 was conducted using the Johns Hopkins Clinical Compound Library that contains over 1,500 existing drugs. Closantel, a veterinary anthelmintic with known proton ionophore activities, was identified as a potent and specific inhibitor of filarial chitinases, an activity not previously reported for this compound. Notably, closantel was found also to completely inhibit molting of O. volvulus infective L3 stage larvae. Closantel appears to target two important biochemical processes essential to filarial parasites. To begin to unravel closantel's effects, a retro-fragment-based study was used to define structural elements critical for closantel's chitinase inhibitor function. As resources towards the development of new agents that target neglected tropical diseases are scant, the finding of an existing drug with impact against O. volvulus provides promise in the hunt for new therapies against river blindness.

An assessment of genetic variability in the mitochondrial cytochrome c oxidase subunit 1 gene of Cercopithifilaria sp. (Spirurida, Onchocercidae) from dog and Rhipicephalus sanguineus populations.

This study investigates sequence variation in mitochondrial cytochrome c oxidase subunit 1 gene within Cercopithifilaria sp. recorded recently in Italy. Fourteen sequence types (haplotypes) were characterized for 163 (7.7%) amplicons from 2111 Genomic DNA samples prepared from skin samples from dogs and from Rhipicephalus sanguineus (ticks) from different geographical areas of the Mediterranean basin (i.e., Italy, Spain and Greece). The most prevalent sequence types represented haplotypes I (70.5%) and X (16.0%), followed by haplotype VIII (4.9%) and other 11 haplotypes (8.6%). Three haplotypes (II, V and VI) were found exclusively in ticks. The overall intraspecific nucleotide variation among pcox1 haplotypes ranged from 0.4 to 3.5% (mean = 1.6%), whereas a mean interspecific difference of 9.5% was detected as compared with other onchocercids. Phylogenetic analysis of the nucleotide sequence data showed a clustering of Cercopithifilaria sp. with the other Cercopithifilaria species (with strong statistical support) to the exclusion of other onchocercids. The number of haplotypes identified here might be explained by complex ecology and transmission patterns as well as the high mutation rate of mitochondrial DNA and/or inbreeding associated with hosts and their vectors.

Detection, purification and characterisation of a secretory alkaline phosphatase from Onchocerca species.

An Onchocerca secretory alkaline phosphatase (E.C. 3.1.3.1) of molecular weight 90 kDa when in crude extract, but which dimerises to about 180 kDa upon purification, was detected, purified and characterised. The enzyme was found to be secreted by both O. ochengi and O. volvulus worms. It was shown to be of Onchocerca origin by Western blotting with bovine onchocerciasis sera and by its time-dependent release in cultures. The O. ochengi enzyme was purified to near homogeneity by a combination of polyethylene glycol precipitation, DEAE-cellulose chromatography and preparative electrophoresis. About 0.96 mg of the active enzyme was purified from 48.4 mg of the crude parasite-released products, giving a purification fold of 71.45 and a yield of 8.7%. The purified enzyme exhibited a typical Michaelis-Menten kinetics with optimum activity on p-nitrophenylphosphate (p-NPP) at pH 10.2. Its apparent K(m) for p-NPP was 0.56+/-0.03 mM and it required Mg(2+) and dithiothreitol (DTT) for stability throughout its purification. Sodium dodecyl sulphate at 2% (w/v) did not inhibit the enzyme activity, but apparently stabilised it during freezing. Inorganic phosphate inhibited the enzyme competitively with an apparent inhibition constant (K(i)) of 3.33+/-0.04 mM, whereas l-phenylalanine inhibited it in a mixed way with a K(i) of 3.18+/-0.03 mM. While contributing to the understanding of metabolism in Onchocerca, the present apparently unique enzyme which is likely to serve in the nutrition of the parasite could be further characterised as a macrofilaricide target or diagnostic marker in onchocerciasis.

Molecular genetic comparison of Onchocerca sp. infecting dogs in Europe with other spirurid nematodes including Onchocerca lienalis.

In the past 15 years, subconjunctival onchocercosis has been reported from 63 dogs in south-western United States (Arizona, California, Utah) and Southern and Central Europe (Germany, Greece, Hungary, Portugal, Switzerland). To reveal the taxonomic status of the parasite responsible for these infections, fragments of the mitochondrial cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) genes of three European strains of canine Onchocerca sp. and the 16S ribosomal RNA (16S rRNA) gene of their Wolbachia endosymbionts were sequenced and compared to the homologous sequences of other spirurid nematodes. The evolutionary divergence between COI and ND5 gene sequences of Greek, Hungarian and Portuguese strains of canine Onchocerca sp. were similar in magnitude to that seen within Thelazia callipaeda or Onchocerca lienalis. The evolutionary divergence between the sequences of canine Onchocerca sp. and other Onchocerca spp. including O. lienalis were similar or higher in magnitude to that seen between other Onchocerca spp. The results of the current and earlier phylogenetic analyses indicate that canine Onchocerca sp. separated from other Onchocerca spp. early in the evolution. Based on the similar clinical pictures, the identical morphology of nematodes and the sequence analyses of COI and ND5 genes of the worms and 16S rRNA gene of their wolbachiae, the Onchocerca worms isolated from European dogs appear to belong to the same species. The results support the earlier biological and morphological arguments that a distinct species, most likely O. lupi originally described from the subconjunctival tissues of a Caucasian wolf is responsible for canine ocular onchocercosis in Europe.

Detection of adenosine diphosphate-ribosyl transferase activity in the filarial worm, Onchocerca volvulus.

The existence of the nuclear enzyme ADP-ribosyl transferase in the filarial worm Onchocerca volvulus was demonstrated. The enzyme activity was observed in the nuclear preparation from the parasitic organism. Poly(ADP-ribose) was identified as the reaction product by the isolation of phosphoribosyl-AMP and 5'AMP as the major products of snake-venom phosphodiesterase digestion. The temperature and pH optima for the enzyme were 25 degrees C and 8.5, respectively. The apparent Km value exhibited by the substrate NAD+, is 750 microM and the activity of the enzyme is inhibited by four chemical classes of inhibitors, nicotinamides, methylxanthines, thymidine and aromatic amides.

Chitinase in female Onchocerca gibsoni and its inhibition by allosamidin.

Chitinase activity has been detected in female worms of Onchocerca gibsoni. With 3,4-dinitrophenyl-tetra-N-acetylchitotetraoside as substrate 50% of maximum activity was achieved at about 25 microM substrate, with inhibition above 50 microM substrate. The antibiotic allosamidin very strongly inhibited the chitinase activity, 50% inhibition being achieved by 200 pM allosamidin in the presence of 45 microM substrate.

Differential susceptibility of filarial and human erythrocyte glutathione reductase to inhibition by the trivalent organic arsenical melarsen oxide.

The glutathione reductases (GR) from two cattle filariae (Setaria digitata and Onchocerca gutturosa) have been isolated and their properties have been compared to those of human erythrocyte GR. In general, the enzymes appear to be very similar with respect to substrate-specificity for glutathione disulfide and NADPH, molecular mass (97 kDa vs. 98 kDa) and oligomeric organisation (subunit size of 51 kDa vs. 50 kDa). However, studies on the inhibition of the enzymes by the trivalent melaminophenyl arsenical melarsen oxide revealed that the human GR is less susceptible to inhibition by the arsenical than the filarial enzymes. Further, it was found that the mechanism of inactivation differs for the host and filarial enzymes. The human enzyme is inhibited by melarsen oxide in a competitive manner with a Ki of 23.7 microM, whereas the filarial GRs are inhibited in two stages: an immediate partial inactivation followed by a time-dependent stage with saturable pseudo-first-order kinetics. Ki values for the S. digitata and O. gutturosa GRs are 38.3 microM and 4.5 microM, respectively, with maximum second-stage inactivation rates of 1.0 x 10(-4) s-1 and 24.3 x 10(-4) s-1, respectively. These differences between host and parasite enzyme might reflect differences in the primary and secondary structure of the proteins which might be exploitable for the design of new specific macrofilaricidal drugs.

Inhibition of NADP-linked malic enzyme from Onchocerca volvulus and Dirofilaria immitis by suramin.

NADP-linked malic enzyme (malate dehydrogenase (oxaloacetate-decarboxylating) NADP+, EC 1.1.1.40) has been partially purified from adult Onchocerca volvulus and Dirofilaria immitis. Suramin was found to inhibit the activity of malic enzyme from both filarial worms. The inhibition constants for suramin were calculated to be 0.011 microM and 0.015 microM for the enzymes from O. volvulus and D. immitis, respectively. In the case of NADP-linked malic enzyme from Trypanosoma brucei and chicken liver the inhibition by suramin was less pronounced. The inhibition constants were found to be 0.8 microM and 2.5 microM for the protozoan and vertebrate enzymes, respectively. The type of inhibition was competitive with respect to malate. The Michaelis constants for malate and pyruvate were determined to be 0.9 and 4.5 mM for O. volvulus and 0.85 and 5.0 mM for D. immitis, respectively. The low Km values for malate compared to those for pyruvate and the about 15-fold greater turnover in the direction of decarboxylation compared to carboxylation indicated that malic enzyme from both filarial sources might be involved in an alternative pathway leading from phosphoenolpyruvate via oxaleacetate, malate and pyruvate to lactate. It is suggested, that the inhibition of malic enzyme activity from O. volvulus by suramin might interfere with the generation of NADPH for biosynthetic reactions.

Dolichol kinase in Ascaris suum and Onchocerca volvulus.

Dolichol kinase was demonstrated in the microsomal fraction of Ascaris suum and Onchocerca volvulus. The enzyme from nematodes exhibited specificity for CTP as phosphoryl donor and was found to be inhibited by the reaction product CDP. Enzyme activity was optimal at pH 7.4, in the temperature range between 30 degrees and 37 degrees C, and in the presence of 0.5% Triton X-100. In addition, the enzyme was found to depend on divalent cations for activity; Mg2+ being more effective than Mn2+ and Ca2+. The dolichol kinase from both nematodes was shown to be independent of Ca2+-calmodulin for activity. The apparent Km values for dolichol were determined to be 7.5 and 9.0 micrograms ml-1 for the enzyme from A. suum and O. volvulus, respectively. Those for CTP were estimated to be 0.85 and 0.75 mM, respectively.

Putrescine N-acetyltransferase in Onchocerca volvulus and Ascaris suum, an enzyme which is involved in polyamine degradation and release of N-acetylputrescine.

A novel type of N-acetyltransferase, clearly different from the nuclear and cytosolic polyamine N-acetyltransferases of mammals, was recently found in the intestinal nematode Ascaris suum. The occurrence of this putrescine N-acetylating enzyme has also been noted in the filarial parasite Onchocerca volvulus. The enzyme was partially purified from adults of O. volvulus and A. suum by chromatography on DEAE-cellulose and cadaverine-Sepharose. Substrate specificities of the filarial enzyme resemble those of the N-acetyltransferase from A. suum, with respect to its preference for putrescine and other diamines above polyamines and histones. Additionally, both nematode enzymes acetylated histamine, whereas dopamine and serotonin were not accepted as substrates. The activities of the N-acetyltransferase from O. volvulus and A. suum were potently inhibited by the drug berenil; the type of inhibition was competitive with respect to putrescine. The inhibition constants for berenil were determined as 4.2 and 1.2 microM for the enzymes of O. volvulus and A. suum, the Km values for putrescine were found to be 330 microM and 250 microM, respectively. Putrescine N-acetyltransferase is discussed as a regulatory step in the degradation of excessive polyamines via polyamine oxidase to putrescine. At this branching point, putrescine is retained in the cell for de novo synthesis of spermidine and spermine, catabolized via diamine oxidase or acetylated to a suitable transport product for excretion.

Studies on a filarial antigen with collagenase activity.

We examined the ability of two filarial species, Onchocerca volvulus and Brugia malayi, to solubilize collagen molecules from native collagen fibrils. Collagenolytic activity was detected in extracts of adult worms, in living microfilariae of O. volvulus and in live infective larvae and adult female worms of B. malayi. Excretion-secretion factors produced in vitro by infective larvae of B. malayi also contained large amounts of collagenase. Studies with enzyme inhibitors suggest that the latter may be a metallo-protease. Antibodies to filarial collagenase were present in sera from patients with onchocerciasis and brugian filariasis and from mice immunized with B. malayi. These antibodies and a monoclonal antibody raised against O. volvulus antigens immunoprecipitate filarial collagenase but appear not to be directed against the active site of the enzyme.

Parasite-specific inhibition of 5'-nucleotidase from Onchocerca volvulus and Dirofilaria immitis by the amoscanate-derivative CGP 8065.

The presence of 5'-nucleotidase was demonstrated in Onchocerca volvulus and Dirofilaria immitis; the bulk of activity was found in the particulate fraction. The enzyme of filarial worms exhibited a broad pH-optimum between 6.4 and 8.0 and substrate specificity for nucleotides compared to glucose-6-phosphate and p-nitrophenyl phosphate. The apparent Km-values for AMP were found to be 0.15 mM and 0.22 mM for the enzyme from O. volvulus and D. immitis, respectively. The activity of 5'-nucleotidase from both filarial worms was effectively inhibited by the filaricidal compound CGP 8065, a dithiocarbamate-derivative of amoscanate, whereas the 5'-nucleotidase from rat liver was not affected. The parasite-specific inhibition by CGP 8065 was found to be reversible and to be competitive with respect to the substrate AMP. The inhibition constants were calculated to be 24 microM and 8 microM for the enzyme from O. volvulus and D. immitis, respectively.

The detoxification of xenobiotic compounds by Onchocerca gutturosa (Nematoda: Filarioidea).

In common with other helminths O. gutturosa appears to lack cytochrome P450 linked phase 1 enzymes and so its ability to metabolize aromatic nuclei may be severely restricted. The parasite could reduce azo- but not nitro-compounds and low levels of epoxide hydrolase activity were also detected. Glutathione S-transferase was the only phase 2 enzyme which could be demonstrated in O. gutturosa. High levels of glyoxalase I and in particular glyoxalase II were found in the parasite, suggesting an important role for these enzymes in detoxification.

Onchocerca volvulus: enzyme polymorphism in relation to the differentiation of forest and savannah strains of this parasite.

Isozyme analysis was carried out on Onchocerca volvulus worms collected from Liberia, Ivory Coast, Burkina Faso and Sudan to see whether this technique could detect differences between forest and savannah populations of this parasite. A total of 243 forest and 189 savannah individual female worms were electrophoresed and stained for seven enzymes. Four showed some polymorphism, LDH, MDH, PGM and MPI and the other three, GAPDH, PEP and GPI were invariant. Statistical analysis of the results showed that the relative proportions of genotypes from within the different countries conformed to Hardy-Weinberg expectations. Pairwise comparisons of allele frequencies between countries showed that populations from Liberia and Ivory Coast had a very similar composition; there was some divergence between all the other pairs of populations and the genetic distance was calculated to summarize the degree of divergence. The number of loci examined was small and the genetic distances were within the range expected for separate geographical populations of the same species. The usefulness of this technique in worm identification is discussed.

Detection of protein kinase substrates in extracts of Onchocerca volvulus.

Extracts of Onchocerca volvulus were phosphorylated in the presence of (gamma 32P)ATP and Mg2+ by endogenous protein kinase activity and exogenous rabbit muscle catalytic sub-unit of the adenosine 3'5' monophosphate dependent protein kinase (E.C. 2.7.1.37). Sodium dodecylsulfate polyacrylamide gel electrophoretic analysis of the 32P-labelled extracts revealed at least seven (32P)-phosphoproteins with apparent Mr of 92,000; 86,000; 40,000; 27,000; 26,000; 23,000 and 17,000. The phosphorylation of the components with apparent Mr of 23,000 and 17,000 was catalysed by both endogenous and exogenous protein kinases, whereas the other components required exogenous protein kinase for their phosphorylation. The endogenous protein kinase activity was inhibited by suramin and the heat-stable protein inhibitor of the adenosine 3'5' monophosphate dependent protein kinase. The (32P)phosphoproteins identified in this investigation are probably candidate regulatory molecules in O. volvulus; though their physiological functions remain to be determined.

Preliminary data on the genetic differentiation of Onchocerca volvulus in Africa (Nematoda: Filarioidea).

Data are reported on the genetic structure of three Onchocerca volvulus populations, respectively from Mali (savanna), Ivory Coast (forest), and Zaire (forest gallery in savanna). Electrophoretic analysis, carried out on 25 gene-enzyme systems, has shown a remarkable genetic heterogeneity existing within O.volvulus. Zaire and West Africa populations appear chiefly differentiated at Mdh-1 and 6Pgdh loci, their average Nei's genetic distance being 0.11. In West Africa Nei's D found between the savanna and forest samples is 0.04. The savanna population from Zaire is more similar to the savanna one from Mali (D = 0.09) than to the forest one from Ivory Coast (D = 0.13). This appears mainly due to the loci Ldh and Hbdh (possibly linked), some alleles of which seem to be selected for in forest populations (Ldh110, Hbdh108), while others in the savanna ones (Ldh100, Hbdh100). The hypothesis that the discrepant epidemiological patterns of human onchocerciasis are related to intrinsic differences in the parasite seems supported by the obtained data. The differences in allele frequencies found at the reported loci appear strong enough to allow biochemical identification of O. volvulus populations from different geographic regions and different habitats.