An embryo-associated fatty acid-binding protein in the filarial nematode Brugia malayi

Brugia malayi is a filarial nematode parasite that causes lymphatic filariasis, a disease that affects millions of people in the tropics. Sexual reproduction of filarial worms occurs within the lymphatic vessels of the human host and is crucial for transmission of the parasite to the mosquito vector. We have previously identified several B. malayi genes that exhibit apparent gender-specific expression. One of these had significant sequence similarity to the Ascaris suum embryo-associated fatty acid binding protein, As-p18. The full length cDNA for the B. malayi female-associated fatty acid binding protein (Bm-FAB-1) encodes a 17.8 kDa protein (excluding a signal peptide) with 70% sequence identity with mature As-p18 and significant similarity to Caenorhabditis elegans and mammalian fatty acid-binding proteins (FABPs). Antibodies raised to Bm-FAB-1 bound to developing embryos within female worms, especially around early embryo cells and the surfaces of immature worms within eggs. Functional studies showed that recombinant Bm-FAB-1 binds to several long chain fatty acids including oleate, but not retinol. Taken together, these results demonstrate that Bm-FAB-1 is a member of an unusual nematode-specific, secreted lipid binding protein family. The existence of a novel class of lipid binding proteins in nematode embryos raises the possibility that drugs targeting these proteins could be developed with broad activity against nematode parasites of medical and veterinary importance.     

Isolation and partial sequence of a collagen gene from the human filarial parasite Brugia malayi

We report the isolation and sequence of a part of a gene encoding a collagen from the genome of the human filarial parasite Brugia malayi. The deduced amino acid sequence of the sequenced portion of this gene, which we have designated BmCol1, differs from the most catalogued nematode collagens in that it is composed predominantly of the glycine-X-Y motif, where either X or Y (or both) may be proline. The gene appears to be similar to two recently described Caenorhabditis elegans collagen genes whose deduced amino acid sequences resemble mammalian basement membrane collagens. BmCol1 is a single copy gene and appears to be present in several other parasitic nematodes examined.     

Induction of murine T-helper-cell responses to the filarial nematode Brugia malayi.

The purpose of this study was to examine the murine T-helper-cell (Th) cytokine response to the human filarial parasite Brugia malayi. In the first 14 days following intraperitoneal inoculation of live microfilariae into BALB/c mice, filarial antigen-driven splenic lymphoid cells produced gamma interferon (IFN-gamma) and little or no interleukin-5 (IL-5). After this time, IL-5 production increased (to 10 to 12 ng per 5 x 10(6) cells) coincident with a marked diminution in IFN-gamma generation. A single subcutaneous immunization with soluble microfilarial antigens also induced an IFN-gamma but no IL-5 response, whereas immunization three times elicited a predominant Th2-like reaction characterized by IL-4 and IL-5 production by CD4+ lymph node lymphocytes and a 10-fold increase in serum immunoglobulin E. The importance of IL-10 in establishing the balance between parasite-specific Th1 and Th2 responses was demonstrated by the ability of neutralizing monoclonal antibody to this cytokine to increase IFN-gamma production by splenic and lymph node cells from mice chronically exposed to live microfilariae or immunized multiple times with soluble filarial antigens.     

Profiling of gender-regulated gene transcripts in the filarial nematode Brugia malayi by cDNA oligonucleotide array analysis

Microarray technology permits high-throughput comparisons of gene expression in different parasite stages or sexes and has been used widely. We report the first use of this technology for analysis of gene expression in filarial male and female worms. The slide array (comprised of 65-mer oligos representing 3569 EST clusters) was spotted with sequences selected from the extensive Brugia malayi EST database (). Arrays were hybridized with Cy dye labeled male and female cDNA. The experimental design included both biological and technical (dye-flip) replicates. The data were normalized for background and probe intensity, and the relative abundance of hybridized cDNA for each spot was determined. Genes showing two-fold or greater differences with P<0.05 were considered gender-regulated candidates. One thousand one hundred and seventy of 2443 clusters (48%) with signals above threshold in at least one sex were considered as gender-regulated gene candidates. This included 520 and 650 clusters up-regulated in male and female worms, respectively. Fifty of 53 (94%) gender-regulated candidate genes identified by microarray analysis were confirmed by real-time RT-PCR. Approximately 61% of gender-regulated genes had significant similarity to known genes in other organisms such as Caenorhabditis elegans. Many C. elegans homologues of these genes have been reported to have reproductive phenotypes (sterility or abnormal embryo development) by RNA interference. This study has provided the first broad view of gender-regulated gene expression in B. malayi; this should lead to improved understanding of reproduction in filarial nematodes. More generally, this approach holds great promise as a means of studying stage-specific or tissue-specific gene expression in parasitic nematodes.     

APC from mice harbouring the filarial nematode, Brugia malayi, prevent cellular proliferation but not cytokine production

Specific T cell hyporesponsiveness and depressed antibody productionis a key feature of human infection with the filarial nematodes,Brugia malayi and Wuchereria bancrofti Despite this immune suppression,responses indicative of T_h2 subset activation are present, includingunusually high levels of specific lgG4. We tested the possibilitythat infection with filarial nematodes causes a reduction inthe co-stimulatory or antigen-presenting capacity of macrophagesresulting in a failure to activate specific T cells. Adherentperitoneal exudate cells (PEC) from mice implanted with adultB. malayi were used to present antigen to the conalbumin-specificT cell clone, D10.G4. Proliferation of the D10 cells at evenbackground levels was completely blocked by the presence ofimplant-derived adherent PEC. However, cytokine production bythese cells in response to antigen was intact, and thus PECfrom implanted mice are capable of functionally processing andpresenting antigen. The elicitation of a suppressive cell populationwas specific for live adults as cells from mice implanted withdead adult parasites effectively stimulated D10 proliferation.The block in cellular proliferation is not due to the productionof factors typically associated with macrophage suppressionsuch as nitric oxide, prostaglandins or catalase. These observationsare consistent with the T cell hyporesponsiveness seen in humancases of patent Brugia infection and may provide a murine modelfor the immune suppression seen in lymphatic filariasis.     

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.     

Isolation and characterization of a repetitive DNA element from the genome of the human filarial parasite, Brugia malayi

The genome of the human filarial parasite Brugia malayi contains at least two major repetitive DNA elements. One, referred to as the HhaI family, consists of 10(4)-10(5) tandemly arrayed copies per haploid genome of a monomer of 322 base pairs and does not contain a cleavage site for the restriction endonuclease MboI. We constructed a library of MboI-digested genomic B. malayi DNA in BamHI-cut M13mp18 resulting in the exclusion of the HhaI repeat family from the library. Hybridization of this genomic library with nick-translated genomic DNA yielded several copies of a repeat family which we have named the BmMboI family. From sequence analysis of more than 50 monomers, which differ from each other in sequence and length, we have been able to divide the monomers into several regions based on the level of sequence conservation. Southern blot analyses of B. malayi genomic DNA digested with a variety of restriction endonucleases and probed with the isolated repeat demonstrate multiple bands of varying sizes except with HindIII-cut DNA, where the repeat is found only in very high-molecular-weight DNA.     

Characterization of an hsp70 gene from the human filarial parasite, Brugia malayi (Nematoda).

We have previously shown that an Onchocerca volvulus cDNA clone in lambda gt-11 designated OvG15, potentially encoding a peptide homologous to the 70-kDa heat shock protein (Hsp70), was recognized by sera of many individuals living in a zone endemic for lymphatic filariasis and most strikingly by sera from amicrofilaremic individuals including endemic normals, those with chronic symptoms and TPE patients. Few asymptomatic microfilaremics recognized the Hsp70. We have now used the insert from the OvG15 clone to isolate the homologous gene from Brugia malayi and analyze its primary structure and expression. The data presented in this communication describe a heat-inducible member of the hsp70 gene family of B. malayi which demonstrates intriguing features of tissue specific basal level expression, developmental regulation and heat inducibility.     

The secretome of the filarial parasite, Brugia malayi: proteomic profile of adult excretory-secretory products

The secretome of a parasite in its definitive host can be considered to be its genome in trans, to the extent that secreted products encoded by the parasite fulfill their function in the host milieu. The 'extended phenotype' of the filarial parasite, Brugia malayi, is of particular interest because of the evidence that infection results in potent down-modulation of the host immune response. We collected B. malayi 'excretory-secretory' (BES) proteins from adult parasites and using a combination of shotgun LC-MS/MS and 2D gel electrophoresis, identified 80 B. malayi and two host proteins in BES, of which 31 (38%) were detectable in whole worm extract (BmA). Products which were enriched in BES relative to BmA included phosphatidylethanolamine-binding protein (PEB), leucyl aminopeptidase (LAP, homologue of ES-62 from the related filaria Acanthocheilonema viteae), N-acetylglucosaminyltransferase (GlcNAcT) and galectin-1, in addition to the previously described major surface glycoprotein, glutathione peroxidase (gp29, GPX-1) and the cytokine homologue macrophage migration inhibitory factor (MIF-1). One of the most abundant released proteins was triose phosphate isomerase (TPI), yet many other glycolytic enzymes (such as aldolase and GAPDH) were found only in the somatic extract. Among the more prominent novel products identified in BES were a set of 11 small transthyretin-like proteins, and three glutamine-rich-repeat mucin-like proteins. Notably, no evidence was found of any secreted protein corresponding to the genome of the Wolbachia endosymbiont present in B. malayi. Western blotting with anti-phosphorylcholine (PC) monoclonal antibody identified that GlcNAcT, and not the ES-62 homologue, is the major PC-bearing protein in BES, while probing with human filariasis sera showed preferential reactivity to galectin-1 and to processed forms of myotactin. Overall, this analysis demonstrates selective release of a suite of newly identified proteins not previously suspected to be involved at the host-parasite interface, and provides important new perspectives on the biology of the filarial parasite.     

Molecular cloning, purification and characterisation of myosin of human lymphatic filarial parasite Brugia malayi

Global efforts have been made towards development of vaccine for prevention of lymphatic filariasis. However, lack of thorough knowledge about developmental biology and pathogenesis of filarial parasite restricts us from developing an effective vaccine. A limited number of immunodominant antigens of human lymphatic filariid Brugia malayi have been characterised; however, none of these recombinant antigens so far induced significant degree of protective immunity to challenge infection. In the present study, we identified a ~2.0 Kb cDNA clone by immunoscreening of cDNA library of adult female Brugia malayi. The nucleotide sequence of the identified clone showed 94.3% homology with C-terminal part of myosin heavy chain gene of Brugia malayi. This cDNA insert was sub-cloned into pET28b vector and expressed in BL21(DE3). The recombinant protein was purified to near homogeneity by immobilised metal affinity chromatography (IMAC) with yield of ~25 mg/l. The purified protein was recognised in western blot with anti-His tag antibody as also with the antibodies present in the sera of human W. bancrofti patients of all categories and infected/immunized rodent serum demonstrating its functional role. Recombinant myosin induced marked cellular immune response as observed by lymphoproliferation assay. The present findings demonstrate the usefulness of B. malayi recombinant myosin as vaccine candidate against human lymphatic filariasis.     

Orthologues of the Drosophila melanogaster E75 molting control gene in the filarial parasites Brugia malayi and Dirofilaria immitis

Filarial parasites cause debilitating diseases in humans and domesticated animals. Brugia malayi and Dirofilaria immitis are transmitted by mosquitoes and infect humans and dogs, respectively. Their life cycle is punctuated by a series of cuticular molts as they move between different hosts and tissues. An understanding of the genetic basis for these developmental transitions may suggest potential targets for vaccines or chemotherapeutics. Nuclear receptor (NR) proteins have been implicated in molting in the free-living nematode Caenorhabditis elegans and have well characterized roles in molting during larval development of Drosophila melanogaster. For example, the D. melanogaster E75 (NR1D3) NR gene is required for molting and metamorphosis, as well as egg chamber development in adult females. We have identified Bm-nhr-11and Di-nhr-6, B. malayi and D. immitis orthologues of E75. Both genes encode canonical nuclear receptor proteins, are developmentally regulated, and are expressed in a sex-specific manner in adults.     

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.     

Mice genetically deficient in immunoglobulin E are more permissive hosts than wild-type mice to a primary,but not secondary,infection with the filarial nematode Brugia malayi

Primary and secondary murine and human infections with Brugia malayi are characterized by substantial increases in levels of immunoglobulin E (IgE). To investigate whether this is necessary for worm clearance, IgE(-/-) mice were subjected to primary- and secondary-infection protocols. Following a primary infection, IgE(-/-) mice displayed a profound deficit in their ability to clear an intraperitoneal injection of L3 infective-stage larvae in comparison to wild-type counterparts and maintained substantial worm burdens as late as 10 weeks postinfection. Although viable adult parasites were recovered at this late time point from IgE(-/-) mice, the majority of the mice remained free of microfilariae. IgE(-/-) cohorts subjected to a secondary-infection protocol were able to clear the challenge inoculation in an accelerated manner, with kinetics similar to that observed in the wild-type animals. Analysis of the humoral response in IgE(-/-) mice following infection demonstrates a defect in IgG1 and IgG2a production, in addition to the expected lack of IgE. The IgG1 deficiency is no longer evident following a secondary infection. These data imply that deficiencies other than IgE production (i.e., IgG1 production) deficiency may be responsible for the increased permissiveness of IgE(-/-) mice as hosts following infection with B. malayi.     

Role of gamma interferon and interleukin-4 in host defense against the human filarial parasite Brugia malayi

We have investigated the roles of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) in host defense against Brugia malayi. Our data suggest that the lack of either IFN-gamma or IL-4 prolongs the time required to achieve sterile immunity, suggesting that both canonical type 1 and type 2 responses are involved in the clearance of infection.