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.

DNA vaccine encoding the moonlighting protein Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) leads to partial protection in a mouse model of human filariasis.

River blindness, caused by the filarial parasite Onchocerca volvulus, is a major socio-economic and public health problem in Sub-Saharan Africa. In January 2015, The Onchocerciasis Vaccine for Africa (TOVA) Initiative has been launched with the aim of providing new tools to complement mass drug administration (MDA) of ivermectin, thereby promoting elimination of onchocerciasis in Africa. In this context we here present Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH) as a possible DNA vaccine candidate. We report that in a laboratory model for filariasis, immunization with Ov-GAPDH led to a significant reduction of adult worm load and microfilaraemia in BALB/c mice after challenge infection with the filarial parasite Litomosoides sigmodontis. Mice were either vaccinated with Ov-GAPDH.DNA plasmid (Ov-pGAPDH.DNA) alone or in combination with recombinantly expressed Ov-GAPDH protein (Ov-rGAPDH). During the following challenge infection of immunized and control mice with L. sigmodontis, those formulations which included the DNA plasmid, led to a significant reduction of adult worm loads (up to 57% median reduction) and microfilaraemia (up to 94% reduction) in immunized animals. In a further experiment, immunization with a mixture of four overlapping, synthetic Ov-GAPDH peptides (Ov-GAPDHpept), with alum as adjuvant, did not significantly reduce worm loads. Our results indicate that DNA vaccination with Ov-GAPDH has protective potential against filarial challenge infection in the mouse model. This suggests a transfer of the approach into the cattle Onchocerca ochengi model, where it is possible to investigate the effects of this vaccination in the context of a natural host-parasite relationship.

Molecular cloning and analysis of Ancylostoma ceylanicum glutamate-cysteine ligase.

Glutamate-cysteine ligase (GCL) is a heterodimer enzyme composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM). This enzyme catalyses the synthesis of γ-glutamylcysteine, a precursor of glutathione. cDNAs of the putative glutamate-cysteine ligase catalytic (Ace-GCLC) and modifier subunits (Ace-GCLM) of Ancylostoma ceylanicum were cloned using the RACE-PCR amplification method. The Ace-gclc and Ace-gclm cDNAs encode proteins with 655 and 254 amino acids and calculated molecular masses of 74.76 and 28.51kDa, respectively. The Ace-GCLC amino acid sequence shares about 70% identity and 80% sequence similarity with orthologs in Loa loa, Onchocerca volvulus, Brugia malayi, and Ascaris suum, whereas the Ace-GCLM amino acid sequence has only about 30% sequence identity and 50% similarity to homologous proteins in those species. Real-time PCR analysis of mRNA expression in L3, serum stimulated L3 and adult stages of A. ceylanicum showed the highest level of Ace-GCLC and Ace-GCLM expression occurred in adult worms. No differences were detected among adult hookworms harvested 21 and 35dpi indicating expression of Ace-gclc and Ace-gclm in adult worms is constant during the course of infection. Positive interaction between two subunits of glutamate-cysteine ligase was detected using the yeast two-hybrid system, and by specific enzymatic reaction. Ace-GCL is an intracellular enzyme and is not exposed to the host immune system. Thus, as expected, we did not detect IgG antibodies against Ace-GCLC or Ace-GCLM on days 21, 60 and 120 of A. ceylanicum infection in hamsters. Furthermore, vaccination with one or both antigens did not reduce worm burdens, and resulted in no improvement of clinical parameters (hematocrit and hemoglobin) of infected hamsters. Therefore, due to the significant role of the enzyme in parasite metabolism, our analyses raises hope for the development of a successful new drug against ancylostomiasis based on the specific GCL inhibitor.

Localization of a filarial phosphate permease that is up-regulated in response to depletion of essential Wolbachia endobacteria.

Wolbachia of filarial nematodes are essential, obligate endobacteria. When depleted by doxycycline worm embryogenesis, larval development and worm survival are inhibited. The molecular basis governing the endosymbiosis between Wolbachia and their filarial host is still being deciphered. In rodent filarial nematode Litomosoides sigmodontis, a nematode encoded phosphate permease gene (Ls-ppe-1) was up-regulated at the mRNA level in response to Wolbachia depletion and this gene promises to have an important role in Wolbachia-nematode endosymbiosis. To further characterize this gene, the regulation of phosphate permease during Wolbachia depletion was studied at the protein level in L. sigmodontis and in the human filaria Onchocerca volvulus. And the localization of phosphate permease (PPE) and Wolbachia in L. sigmodontis and O. volvulus was investigated in untreated and antibiotic treated worms. Depletion of Wolbachia by tetracycline (Tet) resulted in up-regulation of Ls-ppe-1 in L. sigmodontis. On day 36 of Tet treatment, compared to controls (Con), >98% of Wolbachia were depleted with a 3-fold increase in mRNA levels of Ls-ppe-1. Anti-Ls-PPE serum used in Western blots showed up-regulation of Ls-PPE at the protein level in Tet worms on day 15 and 36 of treatment. Immunohistology revealed the localization of Wolbachia and Ls-PPE in the embryos, microfilariae and hypodermis of L. sigmodontis female worms and up-regulation of Ls-PPE in response to Wolbachia depletion. Expression of O. volvulus phosphate permease (Ov-PPE) studied using anti-Ov-PPE serum, showed up-regulation of Ov-PPE at the protein level in doxycycline treated Wolbachia depleted O. volvulus worms and immunohistology revealed localization of Ov-PPE and Wolbachia and up-regulation of Ov-PPE in the hypodermis and embryos of doxycycline treated worms. Ls-PPE and Ov-PPE are upregulated upon Wolbachia depletion in same tissues and regions where Wolbachia are located in untreated worms, reinforcing a link between Wolbachia and this nematode encoded protein. The function of nematode phosphate permease in the endosymbiosis is unknown but could involve transportation of phosphate to Wolbachia, which encode all the genes necessary for de novo nucleotide biosynthesis. Electron microscopic localization of PPE and Wolbachia and RNAi mediated knock-down of PPE in filarial nematodes will bring further insights to the functions of PPE in the Wolbachia-nematode symbiosis.

Filarial parasites possess an antizyme but lack a functional ornithine decarboxylase.

In eukaryotes, the key player in polyamine metabolism is the ornithine decarboxylase (ODC) that catalyses the first and rate limiting step in cellular polyamine synthesis. The half life of ODC is strictly regulated by the antizyme (AZ), which promotes its degradation. Older reports on the polyamine situation in filarial parasites indicate a lack of ornithine decarboxylation activity and an increased uptake of polyamines. Our in silico analysis of the Brugia malayi genome revealed only an ODC-like protein that lacks essential residues. Consequently, the recombinant protein had no enzymatic ODC activity. Furthermore, only ODC-like genes were found in the available draft genomes of other filarial parasites. In this ODC-free scenario, we set out to investigate the AZ of O. volvulus (OvAZ). The expression of the recombinant protein allowed us to analyse the localization of OvAZ in different O. volvulus stages as well as to identify it as target for the human humoral immune response. Strong immunostaining was observed in the outer zone of the uterine epithelium as well as in the uterus lumen around the periphery of the developing parasite, indicating a potential role of the OvAZ in the control of polyamine levels during embryonic development. By employing a novel in vivo method using Caenorhabditis elegans, we postulate that the OvAZ enters the secretory pathway. Even though the ODCs are absent in filarial parasites, OvAZ has the ability to bind to various ODCs, thereby demonstrating the functionality of the conserved AZ-binding domains. Finally, pull-down assays show an interaction between B. malayi AZ and the B. malayi ODC-like protein, indicating that the B. malayi ODC-like protein might function as an AZI. Taken together, our results suggest that filarial species do not possess the ODC while retaining the ODC-regulatory proteins AZ and AZI. It is tempting to speculate that both proteins are retained for the regulation of polyamine transport systems.

Effect of Onchocerca volvulus chitinase on MUC5B expression in human airway epithelial cells.

BACKGROUND: Chitin is an essential structural component of the wall of fungal cells and is present in the exoskeleton of arthropods. It has been generally assumed that mammals lack the ability to produce chitinase proteins, the enzymes responsible for chitin degradation. However, recent studies have indicated that mammals produce chitinases and chitinase-like proteins, and chitinase plays a potential role in human asthma and allergic inflammation. In this study, the effect and brief signaling pathway of chitinase on MUC5B expression were investigated in human airway epithelial cells. METHODS: In the mucin-producing human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells, the effect and signaling pathway of chitinase on MUC5B expression were investigated using the reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). RESULTS: In the mucin-producing human NCI-H292 airway epithelial cells, chitinase increased MUC5B expression. Chitinase significantly activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) but not the phosphorylation of extracellular signa-l-related kinase (ERK) 1/2. The SB203580 (p38 MAPK inhibitor) significantly attenuated chitinase-induced MUC5B mRNA expression, but U0126 (ERK1/2 inhibitor) did not. Knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked chitinase-induced MUC5B expression. In the primary cultures of normal nasal epithelial cells, chitinase significantly increased MUC5B gene expression and this was significantly attenuated after pretreatment with SB203580. CONCLUSION: These results suggest that chitinase induces MUC5B expression by activation of the p38 MAPK signaling pathway in human airway epithelial cells.

Functional characterization and immune recognition of the extracellular superoxide dismutase from the human pathogenic parasite Onchocerca volvulus (OvEC-SOD).

Onchocerca volvulus is a human pathogenic filarial nematode causing chronic onchocerciasis, a disease characterized by chronic skin and eye lesions. Despite attempts to control this infection from many perspectives, it still remains a threat to public health because of adverse effects of available drugs and recent reports of drug resistance. Under control of an intact immune system, O. volvulus survives for a long time in the host by employing a variety of strategies including the utility of antioxidant enzymes. In the present study, we focus on the extracellular superoxide dismutase from O. volvulus (OvEC-SOD) found in the excretory/secretory products of adult worms. Contrary to previous studies, the OvEC-SOD was found to have a 19 amino acid long signal peptide that is cleaved off during the process of maturation. To validate this result, we designed a novel method based on Caenorhabditis elegans cup5(ar465) mutants to specifically evaluate signal peptide-mediated secretion of nematodal proteins. Following purification, the recombinant OvEC-SOD was active as a dimer. Site-directed mutagenesis of the three cysteines present in the OvEC-SOD shows that enzyme activity is markedly reduced in the Cys-192 mutant. A homology model of the OvEC-SOD underlines the importance of Cys-192 for the stabilization of the adjacent active site channel. The generation of a humoral immune response to secretory OvEC-SOD was indicated by demonstrating IgG reactivity in sera from patients infected with O. volvulus while the cross-reactivity of IgG in plasma samples from cows, infected with the most closely related parasite Onchocerca ochengi, occurred only marginally. High IgG1 and IgM titres were recorded in sera from mice infected with the filaria Litomosoides sigmodontis, however, low or no cellular proliferative responses were observed. Thus, the present data suggest that secretory OvEC-SOD is a target of the humoral immune response in human onchocerciasis and induced strongest IgG responses in hyperreactive onchocerciasis. Furthermore, humoral response during murine infection induced SOD-specific IgG that cross-reacted with OvEC-SOD.

A computational analysis of the binding mode of closantel as inhibitor of the Onchocerca volvulus chitinase: insights on macrofilaricidal drug design.

Onchocerciasis is a leading cause of blindness with at least 37 million people infected and more than 120 million people at risk of contracting the disease; most (99%) of this population, threatened by infection, live in Africa. The drug of choice for mass treatment is the microfilaricidal Mectizan(®) (ivermectin); it does not kill the adult stages of the parasite at the standard dose which is a single annual dose aimed at disease control. However, multiple treatments a year with ivermectin have effects on adult worms. The discovery of new therapeutic targets and drugs directed towards the killing of the adult parasites are thus urgently needed. The chitinase of filarial nematodes is a new drug target due to its essential function in the metabolism and molting of the parasite. Closantel is a potent and specific inhibitor of chitinase of Onchocerca volvulus (OvCHT1) and other filarial chitinases. However, the binding mode and specificity of closantel towards OvCHT1 remain unknown. In the absence of a crystallographic structure of OvCHT1, we developed a homology model of OvCHT1 using the currently available X-ray structures of human chitinases as templates. Energy minimization and molecular dynamics (MD) simulation of the model led to a high quality of 3D structure of OvCHIT1. A flexible docking study using closantel as the ligand on the binding site of OvCHIT1 and human chitinases was performed and demonstrated the differences in the closantel binding mode between OvCHIT1 and human chitinase. Furthermore, molecular dynamics simulations and free-energy calculation were employed to determine and compare the detailed binding mode of closantel with OvCHT1 and the structure of human chitinase. This comparative study allowed identification of structural features and properties responsible for differences in the computationally predicted closantel binding modes. The homology model and the closantel binding mode reported herein might help guide the rational development of novel drugs against the adult parasite of O. volvulus and such findings could be extrapolated to other filarial neglected diseases.

Design, synthesis, and biological activities of closantel analogues: structural promiscuity and its impact on Onchocerca volvulus.

Onchocerciasis, or river blindness, is a neglected tropical disease that affects more than 37 million people worldwide, primarily in Africa and Central and South America. We have disclosed evidence that the larval-stage-specific chitinase, OvCHT1, may be a potential biological target for affecting nematode development. On the basis of screening efforts, closantel, a known anthelmintic drug, was discovered as a potent and highly specific OvCHT1 inhibitor. Originally, closantel's anthelmintic mode of action was believed to rely solely on its role as a proton ionophore; thus, the impact of each of its biological activities on O. volvulus L3 molting was investigated. Structure-activity relationship studies on an active closantel fragment are detailed, and remarkably, by use of a simple salicylanilide scaffold, compounds acting only as protonophores or chitinase inhibitors were identified. From these data, unexpected synergistic protonophore and chitinase inhibition activities have also been found to be critical for molting in O. volvulus L3 larvae.

The Onchocerca volvulus cysteine proteinase inhibitor, Ov-CPI-2, is a target of protective antibody response that increases with age.

BACKGROUND: Despite considerable efforts, a suitable vaccine against Onchocerca volvulus infection has remained elusive. Herein, we report on the use of molecular tools to identify and characterize O. volvulus antigens that are possibly associated with the development of concomitant immunity in onchocerciasis. METHODOLOGY/PRINCIPAL FINDINGS: Third-stage larvae (L3) and molting L3 (mL3) O. volvulus stage-specific cDNA libraries were screened with a pool of sera from chronically infected patients who had likely developed such immunity. The 87 immunoreactive clones isolated were grouped into 20 distinct proteins of which 12 had already been cloned and/or characterized before and 4 had been proven to be protective in a small O. volvulus animal model. One of these, onchocystatin (Ov-CPI-2), a previously characterized O. volvulus cysteine proteinase inhibitor was, overall, the most abundant clone recognized by the immune sera in both the L3 and mL3 cDNA libraries. To further characterize its association with protective immunity, we measured the IgG subclass and IgE class specific responses to the antigen in putatively immune (PI) and infected (INF) individuals living in a hyperendemic area in Cameroon. It appeared that both groups had similar IgG3 and IgE responses to the antigen, but the INF had significantly higher IgG1 and IgG4 responses than the PI individuals (p<0.05). In the INF group, the IgG3 levels increased significantly with the age of the infected individuals (r = 0.241; p<0.01). The IgG1 responses in the INF were high regardless of age. Notably, culturing L3 in vitro in the presence of anti-Ov-CPI-2 monospecific human antibodies and naïve neutrophils resulted in almost complete inhibition of molting of L3 to L4 and to cytotoxicity to the larvae. CONCLUSIONS/SIGNIFICANCE: These results add to the knowledge of protective immunity in onchocerciasis and support the possible involvement of anti-Ov-CPI-2 IgG1 and/or IgG3 cytophilic antibodies in the development of protective immunity in the PI and the INF. The results further support the consideration of Ov-CPI-2 as a leading target for an anti-L3 vaccine.

The secretory omega-class glutathione transferase OvGST3 from the human pathogenic parasite Onchocerca volvulus.

Onchocerciasis or river blindness, caused by the filarial nematode Onchocerca volvulus, is the second leading cause of blindness due to infectious diseases. The protective role of the omega-class glutathione transferase 3 from O. volvulus (OvGST3) against intracellular and environmental reactive oxygen species has been described previously. In the present study, we continue our investigation of the highly stress-responsive OvGST3. Alternative splicing of two exons and one intron retention generates five different transcript isoforms that possess a spliced leader at their 5'-end, indicating that the mechanism of mature mRNA production involves alternative-, cis- and trans-splicing processes. Interestingly, the first two exons of the ovgst3 gene encode a signal peptide before sequence identity to other omega-class glutathione transferases begins. Only the recombinant expression of the isoform that encodes the longest deduced amino acid sequence (OvGST3/5) was successful, with the purified enzyme displaying modest thiol oxidoreductase activity. Significant IgG1 and IgG4 responses against recombinantly expressed OvGST3/5 were detected in sera from patients with the generalized as well as the chronic hyperreactive form of onchocerciasis, indicating exposure of the secreted protein to the human host's immune system and its immunogenicity. Immunohistological localization studies performed at light and electron microscopy levels support the extracellular localization of the protein. Intensive labeling of the OvGST3 was observed in the egg shell at the morula stage of the embryo, indicating extremely defined, stage-specific expression for a short transient period only.

Structure of the extracellular glutathione S-transferase OvGST1 from the human pathogenic parasite Onchocerca volvulus.

Onchocerciasis or river blindness, caused by the filarial worm Onchocerca volvulus, is the world's second leading infectious cause of blindness. In order to chronically infect the host, O. volvulus has evolved molecular strategies that influence and direct immune responses away from the modes most damaging to it. The O. volvulus GST1 (OvGST1) is a unique glutathione S-transferase (GST) in that it is a glycoprotein and possesses a signal peptide that is cleaved off in the process of maturation. The mature protein starts with a 25-amino-acid extension not present in other GSTs. In all life stages of the filarial worm, it is located directly at the parasite-host interface. Here, the OvGST1 functions as a highly specific glutathione-dependent prostaglandin D synthase (PGDS). The enzyme therefore has the potential to participate in the modulation of immune responses by contributing to the production of parasite-derived prostanoids and restraining the host's effector responses, making it a tempting target for chemotherapy and vaccine development. Here, we report the crystal structure of the OvGST1 bound to its cofactor glutathione at 2.0 A resolution. The structure reveals an overall structural homology to the haematopoietic PGDS from vertebrates but, surprisingly, also a large conformational change in the prostaglandin binding pocket. The observed differences reveal a different vicinity of the prostaglandin H(2) binding pocket that demands another prostaglandin H(2) binding mode to that proposed for the vertebrate PGDS. Finally, a putative substrate binding mode for prostaglandin H(2) is postulated based on the observed structural insights.

Molecular and biochemical characterization of nematode cofactor independent phosphoglycerate mutases.

Phosphoglycerate mutase (PGM, EC 5.4.2.1) catalyzes the isomerization of 3-phosphoglycerate and 2-phosphoglycerate in glycolysis and gluconeogenesis. Two distinct types of PGM exist in nature, one that requires 2,3-bisphosphoglycerate as a cofactor (dPGM) and another that does not (iPGM). The two enzymes are structurally distinct and possess different mechanisms of action. In any particular organism, one form may exist or both. Nematodes possess the iPGM form whereas mammals have dPGM. In the present study, we have cloned and expressed iPGM from Onchocerca volvulus and described the catalytic properties of O. volvulus, Brugia malayi and Caenorhabditis elegans iPGM enzymes. Temperature and pH optima were determined for each enzyme. Like other iPGM enzymes, the activities of the nematode iPGM enzymes were dependent on the presence of divalent ions. Inactivation by EDTA could be restored most effectively by magnesium and manganese ions. Kinetic parameters and specific activities of the various recombinant enzymes were determined. The high similarity in catalytic properties among the enzymes indicates that a single enzyme inhibitor would likely be effective against all nematode enzymes. Inhibition of iPGM activity in vivo may lead to lethality as indicated by RNAi studies in C. elegans. Our results support the development of iPGM as a promising drug target in parasitic nematodes.

Identification and characterization of onchoastacin, an astacin-like metalloproteinase from the filaria Onchocerca volvulus.

The tissue-invasive nematode Onchocerca volvulus causes skin and eye pathology in human onchocerciasis. While the adult females reside sessile in subcutaneous nodules, the microfilariae are abundantly released from the nodules, males and juvenile worms migrate through the host tissue. Matrix-degrading metallo- and serine proteinases have been detected in excretory-secretory worm products that may be essential for migration of the mobile stages. In this study, a 1713bp long cDNA encoding for a putative proteinase of O. volvulus has been isolated. The predicted protein sequence includes a signal peptide indicating secretion to the extracellular space, a propeptide, an astacin-like protease domain, an EGF-like and a CUB-domain, thereby identifying the protein as a member of the astacin family of zinc endopeptidases. Onchoastacin, Ov-AST-1, is most closely related to a subfamily comprising nematode astacins including Caenorhabditis and Ancylostoma. Ov-AST-1 was expressed as a recombinant protein in baculovirus-infected insect cells and exhibited enzymatic activity. The exposure of onchoastacin to the host immune system is indicated by demonstration of IgG reacting with the recombinant Ov-AST-1 and with two peptides of the protein. Since a homologous metalloproteinase is part of a promising hookworm vaccine, Ov-AST-1 may be a candidate for intervention strategies in filarial infections.

Cloning, characterization and DNA immunization of an Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH).

In the search for Onchocerca volvulus antigens possibly involved in protection against human onchocerciasis, partial amino acid sequence analysis of one of the O. volvulus antigens of the serologically identified proteins showed a close relationship to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein family. Subsequent adult worm cDNA library screening and cloning produced a clone of 1650 bp. An open reading frame spans over 1020 bp encoding for a protein of 340 amino acids with an apparent molecular weight of 38000. Comparison of the complete amino acid sequence identified this protein as a member of the GAPDH protein family. The recombinantly expressed protein shows GAPDH enzymatic activity as well as plasminogen-binding capacity. DNA sequence analysis of the corresponding gene revealed the presence of two introns. Using immunohistology Ov-GAPDH was observed in microfilariae, infective larvae, and adult male and female worms. Most striking was the labelling of the musculature of the body wall. Labelling was also observed in the pseudocoeloma cavity and in a subset of cell nuclei, suggesting additional, non-glycolytic functions of the Ov-GAPDH. Gene gun immunization with the DNA-construct in cattle led to specific humoral immune responses. Thus, the protective potential of the DNA-construct of Ov-GAPDH can be evaluated in vaccination trials using animal models such as the cattle/Onchocerca ochengi model.

Structure of the major cytosolic glutathione S-transferase from the parasitic nematode Onchocerca volvulus.

Onchocerciasis is a debilitating parasitic disease caused by the filarial worm Onchocerca volvulus. Similar to other helminth parasites, O. volvulus is capable of evading the host's immune responses by a variety of defense mechanisms, including the detoxification activities of the glutathione S-transferases (GSTs). Additionally, in response to drug treatment, helminth GSTs are highly up-regulated, making them tempting targets both for chemotherapy and for vaccine development. We analyzed the three-dimensional x-ray structure of the major cytosolic GST from O. volvulus (Ov-GST2) in complex with its natural substrate glutathione and its competitive inhibitor S-hexylglutathione at 1.5 and 1.8 angstrom resolution, respectively. From the perspective of the biochemical classification, the Ov-GST2 seems to be related to pi-class GSTs. However, in comparison to other pi-class GSTs, in particular to the host's counterpart, the Ov-GST2 reveals significant and unusual differences in the sequence and overall structure. Major differences can be found in helix alpha-2, an important region for substrate recognition. Moreover, the binding site for the electrophilic co-substrate is spatially increased and more solvent-accessible. These structural alterations are responsible for different substrate specificities and will form the basis of parasite-specific structure-based drug design investigations.

RNA interference targeting cathepsin L and Z-like cysteine proteases of Onchocerca volvulus confirmed their essential function during L3 molting.

We describe the successful use of RNA interference (RNAi) to investigate gene function in the human filarial parasite Onchocerca volvulus third-stage larvae (L3). We targeted two specific gene products, the O. volvulus cathepsin L (Ov-CPL) and cathepsin Z-like (Ov-CPZ) cysteine proteases, which were proposed to function during O. volvulus L3 molting. We show that fluorescent-labeled Cy3-dsRNA corresponding to cpl or cpz regions encoding the mature enzymes can enter the larvae. The molting rate of larvae treated overnight with 0.5 mg ml(-1) cpl was reduced by 92% and 86% in comparison to normal control worms. It appeared that although the larvae started the molting process the last stage of molting, ecdysis was inhibited. The effect was gene specific, as larvae that did not molt in the presence of cpl or cpz dsRNA expressed the other cysteine protease, CPZ and CPL, respectively. This was confirmed by immunoelectron microscopy using antibodies directed against each enzyme. Our present study validate conclusively that both enzymes are essential for the molting of O. volvulus L3 to fourth-stage larvae. We also confirmed that the activity of the enzymes is specific to the changes that occur during the molting process on days 1-3, when the separation between the cuticles is in progress. The development of RNAi in O. volvulus L3 could further help study many of the abundant L3 and molting L3 genes identified through the filarial genome project, many of which, although have no attributed function, were identified as vaccine candidates or potential drug targets.

Onchocerca volvulus: expression and immunolocalization of a nematode cathepsin D-like lysosomal aspartic protease.

The N-terminal region of the cathepsin D-like aspartic protease from the human filarial parasite Onchocerca volvulus was expressed as His-tag fusion protein. Light and electron microscopic immunohistology using antibodies against the recombinant protein showed labeling of lysosomes in the hypodermis and epithelia of the intestine and the reproductive organs of Onchocerca. While developing oocytes were negative, mature oocytes and early morulae showed strong labeling. In older embryos and mature microfilariae, stained lysosomes were only found in a few cells. Cell death in degenerating microfilariae of patients untreated and treated with microfilaricidal drugs was associated with strong expression of aspartic protease. IgG1, IgG4, and IgE antibodies reactive with the recombinant protein were demonstrated in sera from onchocerciasis patients indicating exposure and recognition of the enzyme by the host's defence system. The aspartic protease of O. volvulus appears to function in intestinal digestion and tissue degradation of the filaria.

Crystallization of the major cytosolic glutathione S-transferase from Onchocerca volvulus.

Glutathione S-transferases (GSTs) are a family of detoxification enzymes that catalyse the conjugation of glutathione to xenobiotic and endogenous electrophilic compounds, thus facilitating their elimination from cells. The recombinant Onchocerca volvulus GST2 has been expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion technique. Two different crystal forms were grown under identical conditions. They belong to space groups P2(1)2(1)2 and P2(1), respectively. The unit-cell parameters obtained are a = 112.6, b = 84.3, c = 45.1 A for the P2(1)2(1)2 crystal form and a = 51.6, b = 82.3, c = 56.7 A, beta = 95.89 degrees for the P2(1) form. Complete data sets to 2.6 and 1.5 A, respectively, have been collected at 100 K with synchrotron radiation.