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PubMed1992

The actin genes of Onchocerca volvulus.

The genome of Onchocerca volvulus was found to contain 2 actin gene classes (called 1 and 2) of 2 genes each. The 4 genes are located in 2 clusters (called A and B), each containing a gene class member. Five short introns of 122-207 bp occur within each gene. The sequences of the fourth intron and the 5' and 3' untranslated regions of all the 2 gene classes are completely different even though their coding regions share 95% identity. Mature transcripts from the actin genes have the nematode spliced leader (SL) at their 5' ends. One actin cDNA was found to be derived from an actin pre-mRNA which locks both the 5 introns and the 5' SL, suggesting that in at least some transcripts cis-splicing is completed before trans-splicing occurs.

Actins↗
PubMed1992

Comparison of the body wall myosin heavy chain sequences from Onchocerca volvulus and Brugia malayi.

The complete coding sequence of Onchocerca volvulus myosin heavy chain has been determined from a series of overlapping cDNAs. The protein sequences from the 2 filarids, one responsible for subcutaneous filariasis, the other for lymphatic filariasis, show 92% identity, and are 1957 amino acids long. Each protein sequence is also equally related, with 75% identity, to MHC-B, the protein encoded by the unc-54 gene of the free-living nematode C.elegans. Such analysis is useful in phylogenetic studies among nematodes, as well as in structure-function relationships among myosin isolates.

Amino Acid Sequence↗
PubMed1992

Identification and characterization of an Onchocerca volvulus cDNA clone encoding a microfilarial surface-associated antigen.

The identification and characterization of a recombinant cDNA clone (OV103) expressing a microfilarial surface-associated antigen of Onchocerca volvulus is described. OV103 was identified and isolated from a lambda gt11 cDNA expression library derived from adult O. volvulus mRNA using a chimpanzee antiserum, taken 2 years after infection with third-stage larvae of O. volvulus. The cDNA clone encodes a 12.5-kDa protein that corresponds to a 15-kDa parasite protein present in microfilariae and adult female worms. The antigen encoded by this clone is located in the basal layer of the cuticle and the hypodermis of the female adult worm, and on the surface of microfilariae. OV103 fusion polypeptide is recognized only by some sera from onchocerciasis infected subjects (57%), but more significantly (89%) by sera from individuals that have low levels of patent infection. In addition, the antibody response to this protein developed before appearance of microfilariae in the skin of chimpanzees that had developed non-patent or low level patent infections, while the antibody response in chimpanzees with high levels of microfilariae appeared later at the time of appearance of microfilariae. Preliminary experiments indicated that affinity purified antibodies directed against OV103 fusion polypeptide mediated killing of nodular microfilariae in vitro in the presence of normal peripheral blood granulocytes.

Amino Acid Sequence↗
PubMed1994

The Onchocerca volvulus homologue of the multifunctional polypeptide protein disulfide isomerase.

Protein disulfide isomerase (PDI) functions to catalyze the formation of correct disulfide bonds in nascent proteins, and also acts as one of the subunits of prolyl-4 hydroxylase, the enzyme responsible for the oxidative maturation of procollagen. Since the cuticle of parasitic nematodes consists primarily of a network of collagen molecules which are connected through intermolecular disulfide bonds, PDI might be expected to be involved in the process of cuticle biosynthesis. The isolation and characterization of a cDNA encoding the PDI homologue of Onchocerca volvulus is described. This cDNA contains a single, long open reading frame that encodes sequence motifs identical to the two known active sites of PDI for isomerase activity. The O. volvulus PDI appears to be encoded by a single copy gene. Both in situ hybridization and immunolocalization data suggest that PDI is both spatially and temporally regulated in O. volvulus. The pattern of spatial and temporal regulation is consistent with the involvement of PDI in the biosynthesis of the parasite cuticle. The parasite protein appears to be an antigen recognized by a minority of individuals exposed to O. volvulus.

Amino Acid Sequence↗
PubMed1995

Molecular cloning of a gene expressed during early embryonic development in Onchocerca volvulus.

Little attention has been paid to the reproductive biology of filarial nematode parasites as a possible target for immunological or chemotherapeutic intervention. An interruption of the reproductive process would, in addition to breaking the cycle of transmission, reduce the morbidity associated with certain filarial infections. As part of our efforts to define molecules that have important functions during filarial embryogenesis, antibodies against embryo-associated proteins were used to identify a 6308-bp cDNA sequence (ovt1) from an Onchocerca volvulus cDNA expression library. The ovt1 cDNA contained an open reading frame that coded for 2022 amino acids. The deduced amino acid sequence was highly hydrophilic, alpha-helical in nature and included two leucine zipper domains. OVT1 also contained a single Arg-Gly-Asp (RGD) site. The results of Southern blot analyses demonstrated that an ovt1-like gene occurs in a number of different species of filarial nematodes. In situ hybridization experiments to identify tissues that contain ovt1 transcripts showed that ovt1 was transcribed at high levels in the late morula/early blastocyst stage of embryonic development. Transcripts for ovt1 were also detected in O. volvulus larvae and in the hypodermal cells of adult parasites. Two fragments of ovt1 were expressed as fusion proteins and the fusion proteins were used to produce antibodies in rabbits. Both antibodies recognized a native protein with an apparent molecular mass of 230 kDa in extracts from gravid female O. volvulus. In addition, the antibodies reacted with a restricted number of lower-molecular mass bands which may represent the products of post-transcriptional or post-translational processing. The predicted coiled-coil structure and the sites of transcription suggest that OVT1 may be a component of the extracellular matrix.

Amino Acid Sequence↗
PubMed1994

Molecular characterisation and localisation of an Onchocerca volvulus pi-class glutathione S-transferase.

Glutathione S-transferases (GSTs) constitute a major detoxification mechanism in helminth organisms and are regarded vaccine candidates against helminth infections. Onchocerca volvulus glutathione-binding proteins were purified from the aqueous soluble fraction of homogenised adult females by affinity chromatography on glutathione-agarose. The eluted proteins had a specific GST activity of 1.6 mumol min-1 mg-1. Immunohistochemical studies localised these antigens in the hypodermis, the wall of the seminal receptacle and spermatozoa of adult worms. A lambda gt11 clone was isolated from an expression library of O. volvulus by immunoscreening. Sequence analysis revealed that it encoded a pi-class GST with 60% identity with Caenorhabditis elegans and up to 45% identity with mammalian pi-class GSTs. Antibodies affinity selected with recombinant GST demonstrated cross-reactivity between Litomosoides sigmodontis and O. volvulus GSTs.

Amino Acid Sequence↗
PubMed1994

Characterization of enzymatically active Onchocerca volvulus Cu/Zn superoxide dismutase expressed in Escherichia coli.

The Onchocerca volvulus superoxide dismutase was expressed in Escherichia coli, using a protocol designed to produce the native enzyme rather than a fusion protein. The recombinant O. volvulus superoxide dismutase (rOVSOD) was found in the cytosol of the disrupted bacteria and represented > 10% of the total bacterial protein. The enzyme was purified to homogeneity using DEAE-Sepharose chromatography, followed by phenyl-Sepharose chromatography. The rOVSOD was enzymatically active which was demonstrated by its reactivity with O2.- produced either by the xanthine-xanthine oxidase system or by stimulated eosinophils. The specific activity was determined to be 4668 U mg-1. This activity could be blocked by rabbit antiserum raised against the rOVSOD. The maximal activity was obtained upon supplementation of the bacterial growth media and enzyme buffer with copper and zinc ions. Activity characteristics in the presence of inhibitors was also characteristic of a Cu/Zn superoxide dismutase. The rOVSOD has an apparent subunit molecular mass of 16,000 in SDS-PAGE. The active enzyme behaves as a dimer of 32 kDa as determined by gel filtration.

Amino Acid Sequence↗
PubMed1994

Identification and characterization of an Onchocerca volvulus cDNA clone encoding a highly immunogenic calponin-like protein.

The identification and characterization of a recombinant cDNA clone, designated OV9M, expressing an antigen present in Onchocerca volvulus infective larvae and adult stages is described. Clone OV9M was identified by screening a lambda gt11 cDNA expression library derived from adult O. volvulus mRNA using pooled rabbit antisera raised against the third (L3) and fourth (L4) stage larvae of the parasite. The cDNA clone encodes an open reading frame of 238 amino acids corresponding to a 27-kDa polypeptide. This polypeptide contains a series of five highly conserved repeats of 25 amino acids that are similar to repeats found in calponin, a protein previously only identified in vertebrate smooth muscle. Extension of the 5' end of the cDNA clone revealed two additional repeats extending the sequence to 378 amino acids, encoding a 41.8-kDa protein. Affinity purified antibodies, which bound specifically to the glutathione S-transferase-OV9M fusion polypeptide, recognize a series of antigens in extracts of O.volvulus microfilariae, L3, L4 and adult stages. The apparent molecular weight of the native OV9M protein in the adult is 45 kDa. Similar proteins are present in extracts of other nematodes including Caenorhabditis elegans, and antibodies from other filarial infections are cross reactive with glutathione S-transferase-OV9M fusion polypeptide. Immunoelectron microscopy revealed that the antigen encoded by this clone is present in the longitudinal muscles of the various larval stages and adult worms. Antibodies to the OV9M protein are present in 40-60% of both patently infected and non-patent individuals residing in onchocerciasis endemic areas.

Amino Acid Sequence↗
PubMed1995

Characterization of genes encoding members of the nuclear hormone receptor superfamily from Onchocerca volvulus.

Several lines of evidence suggest that molting in parasitic nematodes is controlled through the action of steroid molting hormones, or ecdysones. In other organisms, the central mediator of steroid hormone action is the hormone receptor. These receptor molecules are members of a superfamily of proteins called the nuclear hormone receptor family. Using an oligonucleotide derived from the amino-acid sequence of the Drosophila melanogaster ecdysone receptor, genes encoding homologues of the nuclear hormone receptor family were identified in the genome of the human filarial parasite Onchocerca volvulus. The O. volvulus genome contains at least three genes that encode putative members of the nuclear hormone receptor superfamily. A complete cDNA for one of these genes, designated OvNHR-1, has been isolated and characterized. The OvNHR-1 cDNA was 2378 bp in length, and contained a single open reading frame of 1104 bp. The open reading frame encoded a peptide with all of the features characteristic of a member of the nuclear hormone receptor superfamily of proteins. OVNHR-1 appeared to be encoded by a single-copy gene. Expression of the mRNA corresponding to OvNHR-1 was developmentally regulated, with maximal expression occurring during early embryogenesis. The polypeptide encoded by the OvNHR-1 open reading frame is antigenic in a minority of individuals exposed to O. volvulus.

Amino Acid Sequence↗
PubMed1996

Chitinase genes expressed by infective larvae of the filarial nematodes, Acanthocheilonema viteae and Onchocerca volvulus.

State-specific products of 220 and 75 kDa were identified by metabolic labelling of infective larvae of the filarial nematode Acanthocheilonema viteae in ticks. Synthesis was temperature sensitive, occurring at 27 degrees C but not at 37 degrees C. These products were secreted 3-6 days after leaving the vector during post-infective development, but subsequent expression was not detected. The smaller protein with a pI of 6.2, was purified by two-dimensional electrophoresis and the N-terminal amino acid sequence was derived. This provisionally identified the protein as a chitinase, which was confirmed biochemically by glycol-chitin substrate gel electrophoresis. The polymerase chain reaction was used to amplify a product from a cDNA library of A. viteae infective larvae. The nucleotide sequence codes for a putative signal peptide of 20 amino acids and a mature protein of 504 residues (Mr 56 kDa), exhibiting 69% identity (81% similarity allowing for conservative substitutions) with the MF1 chitinase described from microfilariae of Brugia malayi. N-linked glycosylation may account for some, or all, of the discrepancy in Mr between the predicted polypeptide and the native parasite product (75 kDa). Primers based on the A. viteae sequence were used to amplify a related sequence from a cDNA library of Onchocerca volvulus infective larvae. The O. volvulus cDNA codes for a 20-amino acid signal peptide followed by 477 residues with an Mr of 54 kDa, and shares 67% identity with the A. viteae chitinase (80% similarity allowing for conservative substitutions) and 69% identity with the B. malayi MF1 molecule. It is proposed that chitinases expressed by infective stages of these filarial nematodes may play a role in ecdysis during post-infective development.

Amino Acid Sequence↗
PubMed1996

Biochemical analysis, gene structure and localization of the 24 kDa glutathione S-transferase from Onchocerca volvulus.

Survival of Onchocerca volvulus, a pathogenic human filarial parasite, is likely to depend upon the detoxification activities of the glutathione S-transferases (GSTs). The 24 kDa O. volvulus GST, OvGST2, was expressed in a bacterial system and the recombinant protein was purified to homogeneity by affinity chromatography. Specific activities of the recombinant OvGST2 (rOvGST2) with a variety of substrates, and in the presence of inhibitors, were determined. With the universal substrate 1-chloro-2,4-dinitrobenzene, the specific activity of rOvGST2 was 2130 nmol min-1 mg-1. The rOvGST2 showed relatively limited selenium-independent glutathione peroxidase activity, but secondary products of lipid peroxidation, namely members of the trans,trans-alka-2,4-dienal,trans-alk-2-enal and 4-hydroxyalk-2-enal series, were conjugated to glutathione via OvGST2 dependent activity. The gene encoding the OvGST2 was isolated and the nucleotide sequence determined. The ovgst2 gene was found to possess seven exons with six intervening sequences, with all except one having consensus splice-site junctions. This intron/exon organisation of the ovgst2 gene is almost identical with those described for the mammalian Pi class GST genes, consistent with the protein structural evidence that the OvGST2 is related to the Pi class GSTs. Southern blot analysis with total parasite genomic DNA indicated a single copy gene, with a restriction pattern consistent with that of the isolated gene. The tissue distribution of the OvGST2 was examined in O. volvulus by immunohistochemistry and was shown to be distinct from that of the OvGST1. The OvGST2 was located throughout the syncytial hypodermis of male and female adult worms, as well as in the uterine epithelium. Microfilariae, and infective third stage larvae of O. volvulus, isolated from Simulium neavei, were immunopositive for OvGST2.

Amino Acid Sequence↗
PubMed1996

Cloning and characterization of cDNAs encoding putative glutamate transporters from Caenorhabditis elegans and Onchocerca volvulus.

We report the identification and partial characterization of cDNAs encoding for putative glutamate transporters from the free-living nematode Caenorhabditis elegans and the filarial parasite Onchocerca volvulus. Glutamate transporters can be used as reliable markers for identifying cells and neurons that synaptically release glutamate and aspartate. An amplified PCR fragment containing a highly conserved amino acid heptamer found in all vertebrate glutamate transporters was used to screen a C. elegans cDNA library. Two full-length cDNA sequences from C. elegans were deduced from the isolated cDNA clones and RT-PCR products with the splice leader. The two C. elegans cDNA sequences differ by only 97 nucleotides at the 5' end. The C. elegans glutamate transporter gene glt-1 spans at least 2.9 kb of chromosomal DNA and possesses nine exons and eight introns. Primers directed to the CeGlt cDNA were used with O. volvulus first-strand cDNA to amplify and isolate the O. volvulus cDNA homolog. The C. elegans and O. volvulus glutamate transporters are 98% identical over 492 amino acids to each other and 52 to 58% identical to the mammalian glutamate transporters. Antibodies generated against partial coding regions of the C. elegans glutamate transporter recognized a protein of approximately 66 kDa in C. elegans and O. volvulus protein extracts.

ATP-Binding Cassette Transporters↗
PubMed1991

Onchocerca fasciata Railliet and Henry, 1910 and its nodule development in camels in Saudi Arabia.

A total of 192 male camels of three age groups (young, adult and old) from Saudi Arabia were examined for Onchocerca fasciata infection by detection of microfilariae in skin snips and nodules in the nuchal ligaments and subcutaneous tissues of the neck and shoulder. The overall prevalence rates were 10.9 and 33.3%, respectively. The prevalence rate by the skin snip technique and the number of microfilariae per gram of skin were higher in young and adult camels than in old camels. However, the prevalence rate by the detection of nodules and the number of nodules per infected camel, increased with increase in age of the camels. An increase in size and weight of nodules was reported with an increase in age of the camels. Nodules varied in diameter from 2 to 36 mm and in weight from 0.5 to 5.0 g. The overall percentage of soft viable and calcified nodules was 42.5 and 57.5%, respectively. The viability of worms decreased, but calcification increased with increased age of the camels. Four levels of degeneration and calcification of worms were described following scanning electron microscopy.

Animals↗
PubMed1994

Cloning of a cDNA encoding a putative nicotinic acetylcholine receptor subunit of the human filarial parasite Onchocerca volvulus.

A partial cDNA clone encoding a putative nicotinic acetylcholine receptor (ACHR) subunit of the human filarial parasite, Onchocerca volvulus, has been isolated and sequenced. A truncated open reading frame of 1308 bp capable of encoding 436 amino acids with a calculated M(r) of 51,209 was identified. The absence of the double cysteine residues necessary for neurotransmitter binding indicates that the cloned ACHR is a non-alpha subunit.

Amino Acid Sequence↗
PubMed1996

Onchocerca volvulus: identification of cDNAs encoding a putative phosphatidyl-ethanolamine-binding protein and a putative partially processed mRNA precursor.

We have identified and sequenced cDNAs of the parasitic nematode Onchocerca volvulus which encode a homologue of phosphatidylethanolamine-binding proteins from mammals. These clones are also closely related to the O. volvulus Ov16 cDNA (Lobos et al., 1990). One identified cDNA clone appears to represent a partially processed precursor. These results suggest that these cDNAs are derived from a complex genomic locus, raising the possibility of polycistronic transcription in this nematode.

Amino Acid Sequence↗
PubMed2003

Sensitive and specific serodiagnosis of river blindness using Onchocerca ochengi antigens.

Despite the large-scale application of ivermectin for the treatment of human onchocerciasis, this filarial disease has remained a public health hazard, requiring rapid and sensitive diagnostic methods for more efficient management. Several serological methods based on homologous native or recombinant diagnostic antigens have been developed. However, these antigens have remained scarce in the endemic areas. We report herein the preparation and evaluation of an Onchocerca ochengi total worm extract together with its low molecular weight (< 31 kDa) and 14.4 kDa antigens for the diagnosis of human onchocerciasis. The 14.4 kDa antigen and the low molecular weight antigen fraction were identified by Western blot experiments. The antigens were employed for the detection of IgG4 in sera of 43 onchocerciasis patients, 35 normal Africans, 15 schistosomiasis patients, 11 ascariasis patients, 9 loaisis patients and 3 healthy Europeans. The diagnostic specificity values were 94.5, 87.5 and 100%, while the sensitivities were 90.7, 80 and 76.6% using the worm extract, low molecular weight antigen fraction and the 14.4 kDa antigen, respectively. We conclude that total worm extracts of O. ochengi can conveniently replace recombinant antigen cocktails in poor settings that do not have access to the latter.

Adolescent↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2004

Wolbachia endosymbionts of Onchocerca volvulus express a putative periplasmic HtrA-type serine protease.

Wolbachia are intracellular bacteria of many filarial nematodes. A mutualistic interaction between the endobacteria and the filarial host is likely, because the clearance of Wolbachia by tetracycline leads to the obstruction of embryogenesis and larval development. Databases were searched for exported molecules to identify candidates involved in this mutualism. Fragments of a Wolbachia serine protease from the human filarial parasite Onchocerca volvulus were obtained (Wol-Ov-HtrA) by the use of a PCR technique and primers based on the Rickettsia prowazekii genome. The deduced amino acid sequence exhibited 87% and 81% identity to the homologous Wolbachia proteases identified from Brugia malayi and Drosophila melanogaster, respectively. The full-length cDNA encodes 494 amino acids with a calculated mass of 54 kDa. Three characteristic features, (i) a catalytic triad of serine proteases, (ii) two PDZ domains and (iii) a putative signal peptide, classify the endobacterial protein as a member of the periplasmic HtrA family of proteases known to express chaperone and regulator activity of apoptosis. Using a rabbit antiserum raised against a recombinantly expressed 33-kDa fragment of Wol-Ov-HtrA, strong labelling of the antigen was found associated with endobacteria in hypodermis, oocytes, zygotes, all embryonic stages and microfilariae of O. volvulus. Staining of hypodermal cytoplasm surrounding the endobacteria indicated a possible release of the protein from the Wolbachia. The demonstration of Wol-Ov-HtrA-reactive IgG1 antibodies in sera of O. volvulus-infected persons indicated the exposure to the protein and its recognition by the human immune system. Wol-Ov-HtrA is a candidate for an exported Wolbachia protein that may interact with the filarial host metabolism.

Amino Acid Sequence↗