Onchocerca volvulus, O. gutturosa, Brugia malayi, and Dirofilaria immitis: a comparative study of the immunochemical properties of cuticular proteins from filarial parasites.

We compared the chemical and immunological properties of cuticular collagens from four species of filarial nematodes, Onchocerca volvulus, O. gutturosa, Brugia malayi, and Dirofilaria immitis. The electrophoretic mobility of the major polypeptides extracted from adult worms is characteristic for each species studied. Cuticular collagens from adult worms and infective larvae differ in their susceptibility to proteases that cleave vertebrate collagens and to collagenases prepared from different developmental stages of filarial parasites. The overall amino acid composition of filarial collagens resembles that of vertebrate interstitial collagens and differs from that reported for collagens from free-living or intestinal nematodes. However, cuticular proteins of the four filarial species studied significantly differed in amino acid composition and in their reactivity with antisera to interstitial and basement membrane collagens of vertebrates.

The sheaths of Brugia microfilariae: isolation and composition.

Burgia malayi and B. pahangi microfilariae were isolated from the blood of infected Mastomys natalensis, and were exsheathed by freezing, thawing and agitation. Pure sheaths were obtained by a filtration procedure. The sheaths were found to contain about 95 mol% of amino acids, with proline, glutamic acid/glutamine, alanine, cysteine/cystine and glycine being the major components, and 5 mol% of carbohydrates, notably (N-acetyl)galactosamine, but no (N-acetyl)glucosamine.

Localization and immunogenicity of tubulin in the filarial nematodes Brugia malayi and B. pahangi.

Tubulin was identified in the filarial nematodes Brugia malayi and B. pahangi by several approaches. Initially, a monoclonal antibody (6D8) was selected for its unusual binding to B. malayi microfilariae in indirect immunofluorescence assays: 6D8 showed granular, heterogeneously dispersed fluorescence on fixed parasites but did not bind to unfixed microfilariae. The microfilarial sheath did not bind 6D8, although it did bind fluoresceinated wheatgerm agglutinin. By Western blotting against microfilarial sonicate, 6D8 reacted with a 50,000-55,000 mol. wt protein, and also bound to purified chicken brain beta-tubulin. Additionally, this monoclonal antibody reacted with a recombinant fusion protein expressed by a clone (Bpa-7) originally isolated from an adult B. pahangi cDNA expression library by its reaction with chronic human filariasis serum. This clone encodes a small 40 amino acid C-terminal segment corresponding to residues 409-449 of beta-tubulin, and shows complete amino acid sequence homology with vertebrate beta-tubulin from 409 to 430 but 55% divergence (six amino acid substitutions, four insertions and one deletion) from human and chicken beta-tubulin over positions 431-449 at the C terminus. Antibody to both parasite and vertebrate (chicken) tubulin was found in filarial infection sera, with higher levels of autoreactive antibody apparent in amicrofilaraemic individuals. Immunogold electron microscopy was then used to localize beta-tubulin in B. malayi microfilariae and adult worms. Tubulin was shown not to be exposed on the microfilarial sheath or in the cuticle of either stage, but was found to be abundant in the somatic tissues. In microfilariae, 6D8 bound myofibril structures under the hypodermal layer, and also bound within cell nuclei. In the adult stage, tubulin was associated with muscle blocks, as well as the intestinal brush border and the embryonic uterine microfilariae.

Brugia malayi: microfilarial polyunsaturated fatty acid composition and synthesis.

The polyunsaturated fatty acid composition of Brugia malayi microfilariae was analyzed by gas chromatography and compared to that of sera from B. malayi-infected jirds. The essential fatty acid, linoleic acid (18:2 omega 6), was the most abundant fatty acid present in both microfilarial total lipids and phospholipids as well as in jird sera. In contrast, arachidonic acid (20:4 omega 6), as well as the 18:3 omega 6, 20:2 omega 6, and 20:3 omega 6 intermediates that are formed in the enzymatic conversion of linoleic acid to arachidonic acid, were proportionally more abundant in microfilariae than in jird sera. To assess the capacity of microfilariae to transform linoleic acid into arachidonic acid, B. malayi microfilariae were incubated with [14C]linoleic acid. Microfilarial lipids were extracted and resolved by high-pressure liquid chromatography and thin-layer chromatography. A portion of [14C]linoleic acid incorporated by microfilariae was converted to [14C]arachidonic acid. Thus, microfilariae can not only incorporate exogenous arachidonic acid, as previously demonstrated, but can also synthesize arachidonic acid from exogenous linoleic acid. The capacity of microfilariae to utilize specific host polyunsaturated fatty acids raises the possibility that intravascular filarial parasites may synthesize eicosanoid metabolites of arachidonic acid that could mediate filarial-host cell interactions.

A stage-specific calcium-binding protein from microfilariae of Brugia malayi (Filariidae).

The monoclonal antibody MF1 has been shown to cause transient clearance of blood-borne microfilariae in gerbils infected with Brugia malayi. The present study demonstrates that the MF1 antibody recognizes a proteinaceous determinant on two polypeptides of 70 and 75 kDa. Both the monoclonal antibody and antisera raised to the gel-purified antigen show binding restricted to mature microfilariae. Intrauterine and recently shed microfilariae apparently lack the MF1 molecules. Prominent 45Ca-binding molecules comigrate with the MF1 antigens in one-dimensional electrophoresis (under both reducing and non-reducing conditions), and co-purify through ion exchange chromatography. The MF1 antigen thus appears to be a developmentally regulated calcium-binding protein(s).

Use of Iodogen and sulfosuccinimidobiotin to identify and isolate cuticular proteins of the filarial parasite Brugia malayi.

The cuticle of filarial nematodes is a dynamic structure which may be an important target for protective host immune responses. Prior studies have employed radioiodination of intact parasites to demonstrate that the collagenous cuticle of filariids contains relatively few exposed proteins, some of which are stage and/or species-specific. In the present study, we have used sulfo-NHS-biotin to label and affinity purify cuticular components of living adult Brugia malayi. Results obtained by this method were compared with the widely used Iodogen method of surface radioiodination by SDS-PAGE analysis of detergent-solubilized worms and by ultrastructural analysis. Both labeling methods produced very similar electrophoretic patterns with major doublets at 70 and 100 kDa, a major band at 25 kDa, and minor bands between 60-200 kDa. Ultrastructural analysis showed that both methods labeled components throughout all levels of the parasite cuticle; underlying somatic tissues were not labeled. The biotinylated components were isolated from the total parasite extract by affinity chromatography on an avidin matrix. Further characterization of these surface-associated proteins may lead to improved methods for the control of filariasis.

Comparative analysis of surface components of adult, micro-filariae and infective larvae of Brugia malayi.

A comparative analysis of surface proteins of adult, microfilariae and infective larvae of Brugia malayi, the human filarial parasite, has been carried out using IODOGEN (1,3,4,6-tetrachloro-3,alpha 6 alpha-diphenyl-glycoluril) and lactoperoxidase methods. SDS-polyacrylamide gel electrophoretic and autoradiographic analyses revealed the presence of 9 proteins (15-200 kDa) in adults, while microfilariae and infective larvae showed 8 and 6 proteins (15-120 kDa), respectively. The pattern of proteins radiolabelled by IODOGEN method was very similar to that of proteins labelled by the lactoperoxidase method. Since these proteins are released by the protease treatment of whole parasites, they are likely to be present on the surface of the parasite.

Characterization of tubulin from Brugia malayi and Brugia pahangi.

The tubulins of Brugia malayi and B. pahangi were similar with respect to concentration (mg tubulin per mg soluble protein), electrophoretic and isoelectric mobility, reaction in Western blots with anti-tubulin monoclonal antibodies, and isoform patterns. Tubulin was estimated to account for 2.8% and 2.9% of soluble protein in B. malayi and B. pahangi extracts, respectively. Tubulins from Brugia nematodes have been partially purified by polylysine agarose chromatography and with taxol. Western blots with alpha- and beta-tubulin monoclonal antibodies confirmed the presence of tubulin. The mobility of Brugia tubulins on sodium dodecyl sulfate polyacrylamide gel electrophoresis was very similar to that of N. brasiliensis and rat brain tubulins. The isoelectric range for Brugia alpha- and beta-tubulin isoforms was pH 5.4-4.7. Western blots with anti-tubulin monoclonal antibodies revealed 4-5 isoforms of alpha-tubulin and 4-5 isoforms of beta-tubulin for Brugia nematodes.

Identification, synthesis and immunogenicity of cuticular collagens from the filarial nematodes Brugia malayi and Brugia pahangi.

The major structural proteins of the cuticle of the filarial nematode parasites Brugia malayi and Brugia pahangi were identified by extrinsic iodination and sensitivity to clostridial collagenase. At least 16 acidic components were identified in adult worms by 2-dimensional electrophoresis, with molecular weights ranging from 35,000 to 160,000. These proteins appear to be cross-linked by disulphide bonds, and localised in the basal and inner cortical layers of the cuticle. The outer cortex, containing the epicuticle, is insoluble in 1% sodium dodecyl sulphate and 5% 2-mercaptoethanol, and can be isolated free of cellular material. Despite their inaccessibility to the immune system in intact worms, antibodies to the cuticular collagens are provoked in humans infected with a variety of filarial parasites. Immunological cross-reactivity was demonstrated between a 35 kDa component and human type IV (basement membrane) collagen. Autoantibodies to type IV collagen were detected in a number of individuals with lymphatic filariasis, although no correlation could be drawn with observed pathology. Synthesis of cuticular collagens is discontinuous, occurs at negligible levels in mature adult male worms, and does not appear to involve the production of small molecular weight precursors, in contrast to Caenorhabditis elegans. Hybridisation with a heterologous cDNA probe coding for the alpha 2 chain of chicken type 1 collagen suggests that they are encoded by a multigene family.

Microfilarial surface carbohydrates as a function of developmental stage and ensheathment status in six species of filariids.

The lectin-binding properties of microfilariae of Onchocerca volvulus, O. lienalis, Brugia pahangi, Wuchereria bancrofti, Dirofilaria immitis, and Monanema (= Ackertia) marmotae share a number of characteristics. Carbohydrates specific for lectins are associated with the egg shell or sheath. N-acetyl-D-glucosamine is the predominant carbohydrate associated with the ensheathed forms with lesser quantities of D-galactose and/or alpha-lactose and D-galactosamine. The density of these carbohydrates on the sheath surface diminishes as the larvae undergo normal growth and development. Similar carbohydrates are not found on the cuticle as exsheathed microfilariae show virtually no ability to bind lectins.

DNA base composition of filarial nematodes.

We have determined the molar content of guanine + cytosine (GC content) of DNA of the filarial nematode (Brugia malayi, Brugia pahangi and Dirofilaria imitis) and of the free-living soil nematodes Caenorhabditis elegans and have analysed the DNA for the presence of methylcytosine. Two independent methods, thermal denaturation and direct analysis of base content by HPLC following enzymatic hydrolysis, reveal that the GC content of filarial nematodes is 26-28%. We have been unable to find methylcytosine in the DNA of B. malayi.

Monoclonal antibody to a unique surface epitope of the human filaria Brugia malayi identifies infective larvae in mosquito vectors.

We describe properties of an IgM monoclonal antibody (NEB-D1E5) raised against the human filarial parasite Brugia malayi. The antibody reacts with a stage- and species-specific determinant located on the surface of the infective-stage larva, as determined by indirect immunofluorescence. To use this reagent in epidemiological field studies, we developed an enzyme-linked immunoassay with which B. malayi larvae can be differentiated from other filarial parasites in mosquito vectors, including the morphologically indistinguishable parasite of animals Brugia pahangi. The immunoenzyme assay was 91-94% specific and 90-97% sensitive when performed on infected mosquitoes. In the absence of mosquito tissue, the levels of specificity and sensitivity increased to 100% and 97.5-100%, respectively. Binding of antibody to the surface of living larvae was abrogated by treatment of the worms with the enzymes pronase and proteinase K and with the detergents Triton X-100, octyl beta-D-glucopyranoside, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS). In contrast, treatment with trypsin, endoglycosidase-F, O-Glycanase, N-Glycanase, lipase, various phospholipases, boiling, 2-mercaptoethanol at 37 degrees C, or periodate did not reduce the antigenicity of the larval surface to antibody NEB-D1E5. These results suggest that the species-specific epitope is a peptide domain attached to a hydrophobic anchoring residue.

Brugia malayi antigens associated with lymphocyte activation in filariasis.

Filarial parasites induce immune response in humans which are still poorly characterized. To define the antigens responsible for inducing lymphocyte responses, fast protein liquid chromatography was used to fractionate the antigens of Brugia malayi adult worms which were then tested on lymphocytes from patients with filariasis and normal controls. From an anion exchange column (Mono Q), three peaks of lymphocyte-stimulating activity were eluted which were further fractionated by gel filtration (Superose-12). Peak I induced both a proliferative response as well as the production of filaria-specific antibody in patient lymphocytes. Peak II, capable of inducing only a proliferative response (without antibody production) in patient lymphocytes, was a glycoprotein with phosphocholine as one of the antigenic determinants. Peak III induced proliferative responses in both patient and normal lymphocytes and thus appears to be mitogenic. Two-dimensional gel electrophoresis was then used to identify changes in the major cellular proteins associated with the activation of patient lymphocytes by these partially purified antigens. Stimulation of patient lymphocytes with peak I resulted in increased synthesis of immunoglobulin heavy, light, and J chains. Further, these were the only major secreted proteins found in the culture supernatants. Peak II resulted in quantitative changes in proteins associated with T and not B lymphocyte stimulation. Further analysis of these antigens should help to elucidate the mechanism of host-parasite interaction at both the cellular and molecular levels.

Polyamine metabolism in filarial worms.

The human and animal filarial parasites Onchocerca volvulus, Dirofilaria immitis, Brugia patei and Litomosoides carinii contained low levels of putrescine but much higher levels of spermidine and spermine as estimated by ion-pair high pressure liquid chromatography; N-acetylated polyamines were present only in minute amounts. Enzyme activities of ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (EC 4.1.1.19), respectively, were not detectable. Experiments carried out with O. volvulus and D. immitis demonstrated the uptake and bioconversion of labeled polyamines. There is evidence for the existence of a complete reverse pathway generating putrescine from spermidine and spermine, respectively, in both worms. N-Acetylating enzyme activities were detected in 100,000 X g preparations of homogenates from D. immitis which were capable to acetylate putrescine, spermidine and spermine. Long term incubation of the worms in the presence of labeled polyamines resulted in the excretion of putrescine and N-acetylputrescine.

Lectin binding studies on adult filariae, intrauterine developing stages and microfilariae of Brugia malayi and Litomosoides carinii.

Sections of macrofilariae of Brugia malayi and Litomosoides carinii revealed binding of the gold-labelled lectins WGA, DBA and PNA. Specificity of binding was controlled by competitive inhibition with the respective sugars. N-acetyl-glucosamine, N-acetylgalactosamine and galactose residues seem to be present in the respective tissues. The lectins were bound preferentially to parts of the reproductive organs and to the fluid contents of their lumina. The results of the chitosan test and binding experiments with WGA-gold conjugate suggest the presence of chitin in the sheath of oocytes or zygotes. Binding of WGA could not be inhibited with 0.5 M N-acetylglucosamine, but only with 10 mM triacetyl chitotriose. In older stages, binding of WGA to the sheath could be inhibited by 0.5 M N-acetylglucosamine. In mature microfilariae, the outer surface of the sheath did not show affinity for WGA, but small amounts were bound to the inner surface. Therefore, the sheath of later developmental stages and microfilariae does not contain chitin but only N-acetylglucosamine residues. The degradation of the chitin content might enable the elongation and flexibility of the sheath of microfilariae.

An evaluation of different methods for labelling the surface of the filarial nematode Brugia pahangi with 125iodine.

The specificity of a range of 125I labelling techniques (Chloramine T, Iodogen, Bolton and Hunter reagent, lactoperoxidase and iodosulfanilic acid) to the surface of the filarial nematode Brugia pahangi was evaluated by autoradiography of sections of labelled worms and of dried SDS-polyacrylamide gels following electrophoresis of homogenised worm extracts. It was concluded that Bolton and Hunter reagent was not surface specific but labelled proteins throughout the body of the worm. At the light microscope level autoradiography of worms labelled using Chloramine T, Iodogen, lactoperoxidase and iodosulfanilic acid demonstrated that the 125I labelling was restricted to the worm surface. Electrophoresis and autoradiography showed that each method produced a different pattern of labelled polypeptide. A polypeptide of molecular weight 30 kDa was labelled using each method except Bolton and Hunter reagent, and appears to be a major surface component.

Biochemical surface components of Brugia pahangi microfilariae.

The sheath and cuticle of microfilariae of Brugia pahangi were examined by electron microscopy and the presence of various proteins, carbohydrate and enzymes sought. The epicuticle of microfilariae consists of a pentalaminate structure (24.0 +/- 1.4 nm), a cortex (13.7 +/- 3.6 nm) and a basal zone (27.8 +/- 4.8 nm) which is often banded in appearance. The pentalaminate layers are not continuous at the base of the interannular grooves. The sheath and the epicuticle of B. pahangi stained positively with concanavalin A and saccharated iron oxide. The sheath of approximately 50% of microfilariae showed activity for acid phosphatase, 5' nucleotidase and peroxidase, but not for ATPases, alkaline phosphatase or esterase. No enzymes were detected in the epicuticle although the cortex and basal layers of the cuticle did show enzymic activity. Structures beneath the cuticle in the main body of the worms contained considerable enzymic activity. Microfilariae directly isolated from the blood of infected cats were found by immunochemical means to carry serum proteins on their sheaths but not on their cuticles. These studies extend the definition of the outer structures of microfilariae and confirm that they significantly differ in morphology and enzyme content from typical mammalian cell membranes.

Brugia malayi: stage-specific expression of carbohydrates containing N-acetyl-D-glucosamine on the sheathed surfaces of microfilariae.

Microfilariae, infective larvae, and adult worms of Brugia malayi were incubated with a panel of seven lectins in order to study the expression of surface carbohydrates. Infective larvae and adult worms did not bind any of the lectins utilized. Microfilariae, on the other hand, bound wheat germ agglutinin. The binding of this lectin was saturable and specific, and attributed to the presence of N-acetyl-D-glucosamine. In addition, microfilariae derived in vitro bound concanavalin A, indicating the presence of glucose and/or mannose on this stage of the parasite. The fact that similar concanavalin A binding was not seen on microfilariae recovered directly from the infected host implies that there is masking or loss of parasite surface antigens as microfilariae mature in vivo.

Characterization of the exposed carbohydrates on the sheath surface of in vitro-derived Brugia pahangi microfilariae by analysis of lectin binding.

Adult female Brugia pahangi were maintained metabolically active in vitro for up to 35 days in Click's medium supplemented with 10% horse serum. For the first 14 to 18 days microfilariae were released into culture. Although these in vitro-derived microfilariae were morphologically identical to in vivo-derived microfilariae, they could be differentiated by their characteristic of binding to a panel of fluorescein-conjugated lectins. The results suggest that maturation and release of microfilariae are correlated with glycosidic alterations on the sheath surface.

Analysis of Brugia pahangi microfilariae surface carbohydrates: comparison of the binding of a panel of fluoresceinated lectins to mature in vivo-derived and immature in utero-derived microfilariae.

A fluoresceinated lectin binding assay was employed to detect carbohydrates on the sheath and cuticle of mature in vivo-derived, and immature in utero-derived Brugia pahangi microfilariae. The sheath of mature microfilariae bound concanavalin A and wheat germ agglutinin, indicating the presence of N-acetylglucosamine and glucose or mannose. In addition to binding concanavalin A and wheat germ agglutinin, the sheath of in utero-derived microfilariae also bound Limulus polyphemus agglutinin, peanut agglutinin, Ricinus communis agglutinin-I, and soybean agglutinin, indicating the presence of the additional sugars galactose, sialic acid, and N-acetylgalactosamine. There was no evidence of cuticle carbohydrates, as none of the tested fluoresceinated lectins bound to either mature or immature exsheathed microfilariae. The significance of these results in terms of the survival of microfilariae in the mammalian host, and development to third-stage larvae in the mosquito vector, is discussed.