Glucose and Glycogen Metabolism in Brugia malayi Is Associated with Wolbachia Symbiont Fitness

Wolbachia are endosymbiotic bacteria found in the majority of arthropods and filarial nematodes of medical and veterinary importance. They have evolved a wide range of symbiotic associations. In filarial nematodes that cause human lymphatic filariasis (Wuchereria bancrofti, Brugia malayi) or onchocerciasis (Onchocerca volvulus), Wolbachia are important for parasite development, reproduction and survival. The symbiotic bacteria rely in part on nutrients and energy sources provided by the host. Genomic analyses suggest that the strain of Wolbachia found in B. malayi (wBm) lacks the genes for two glycolytic enzymes—6-phosphofructokinase and pyruvate kinase—and is thus potentially unable to convert glucose into pyruvate, an important substrate for energy generation. The Wolbachia surface protein, wBm00432, is complexed to six B. malayi glycolytic enzymes, including aldolase. In this study we characterized two B. malayi aldolase isozymes and found that their expression is dependent on Wolbachia fitness and number. We confirmed by immuno-transmission electron microscopy that aldolase is associated with the Wolbachia surface. RNAi experiments suggested that aldolase-2 plays a significant role in both Wolbachia survival and embryogenesis in B. malayi. Treatment with doxycycline reduced Wolbachia fitness and increased the amount of both glucose and glycogen detected in the filarial parasite, indicating that glucose metabolism and glycogen storage in B. malayi are associated with Wolbachia fitness. This metabolic co-dependency between Wolbachia and its filarial nematode indicates that glycolysis could be a shared metabolic pathway between the bacteria and B. malayi, and thus a potential new target for anti-filarial therapy.     

Interaction of a Wolbachia WSP-like protein with a nuclear-encoded protein of Brugia malayi

The Brugia malayi endosymbiont Wolbachia has recently been shown to be essential for its host’s survival and development. However, relatively little is known about Wolbachia proteins that interact with the filarial host and which might be important in maintaining the obligate symbiotic relationship. The Wolbachia surface proteins (WSPs) are members of the outer membrane protein family and we hypothesise that they might be involved in the Wolbachia-Brugia symbiotic relationship. Notably, immunolocalisation studies of two WSP members, WSP-0432 and WSP-0284 in B. malayi female adult worms showed that the corresponding proteins are not only present on the surface of Wolbachia but also in the host tissues, with WSP-0284 more abundant in the cuticle, hypodermis and the nuclei within the embryos. These results confirmed that WSPs might be secreted by Wolbachia into the worm’s tissue. Our present studies focus on the potential involvement of WSP-0284 in the symbiotic relationship of Wolbachia with its filarial host. We show that WSP-0284 binds specifically to B. malayi crude protein extracts. Furthermore, a fragment of the hypothetical B. malayi protein (Bm1_46455) was found to bind WSP-0284 by panning of a B. malayi cDNA library. The interaction of WSP-0284 and this protein was further confirmed by ELISA and pull-down assays. Localisation by immunoelectron microscopy within Wolbachia cells as well as in the worm’s tissues, cuticle and nuclei within embryos established that both proteins are present in similar locations within the parasite and the bacteria. Identifying such specific interactions between B. malayi and Wolbachia proteins should lead to a better understanding of the molecular basis of the filarial nematode and Wolbachia symbiosis.     

Brugia malayi: effects of gamma radiation on adult worms and their intracellular Wolbachia bacteria

Prior studies have shown that intracellular Wolbachia endobacteria are necessary for the normal development, reproduction, and survival of filarial nematodes. The purpose of this study was to examine effects of gamma radiation on Wolbachia and reproduction in Brugia malayi adult worms. Worms were exposed to 0, 10, 25, 45, 75, and 105 krad of gamma radiation from a 137cesium source and cultured in vitro for 10 days. Irradiation reduced production of microfilariae in a dose-dependent manner. Embryograms of irradiated female worms showed dose-related abnormalities with arrested development at the early embryo stage. Irradiation reduced the viability of adult worms in a dose-dependent manner, but no lethal effect was observed. Electron microscopy studies showed that irradiation cleared Wolbachia from worm tissues. Real-time polymerase chain reaction studies demonstrated greatly reduced Wolbachia DNA in irradiated worms. These effects are essentially the same as those observed in adult worms treated with doxycycline. These studies suggest that effects of irradiation on reproduction in Brugia malayi may be caused by effects of irradiation on Wolbachia.     

The Wolbachia genome of Brugia malayi: endosymbiont evolution within a human pathogenic nematode

Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachia's principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease.     

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.     

Molecular characterization of NAD+-dependent DNA ligase from Wolbachia endosymbiont of lymphatic filarial parasite Brugia malayi

The lymphatic filarial parasite, Brugia malayi contains Wolbachia endobacteria that are essential for development, viability and fertility of the parasite. Therefore, wolbachial proteins have been currently seen as the potential antifilarial drug targets. NAD(+)-dependent DNA ligase is characterized as a promising drug target in several organisms due to its crucial, indispensable role in DNA replication, recombination and DNA repair. We report here the cloning, expression and purification of NAD(+)-dependent DNA ligase of Wolbachia endosymbiont of B. malayi (wBm-LigA) for its molecular characterization. wBm-LigA has all the domains that are present in nearly all the eubacterial NAD(+)-dependent DNA ligases such as N-terminal adenylation domain, OB fold, helix-hairpin-helix (HhH) and BRCT domain except zinc-binding tetracysteine domain. The purified recombinant protein (683-amino acid) was found to be biochemically active and was present in its native form as revealed by the circular dichroism and fluorescence spectra. The purified recombinant enzyme was able to catalyze intramolecular strand joining on a nicked DNA as well as intermolecular joining of the cohesive ends of BstEII restricted lamda DNA in an in vitro assay. The enzyme was localized in the various life-stages of B. malayi parasites by immunoblotting and high enzyme expression was observed in Wolbachia within B. malayi microfilariae and female adult parasites along the hypodermal chords and in the gravid portion as evident by the confocal microscopy. Ours is the first report on this enzyme of Wolbachia and these findings would assist in validating the antifilarial drug target potential of wBm-LigA in future studies.     

Single-channel recording from adult Brugia malayi

Lymphatic filariasis is a significant cause of morbidity in humans. One of the causative agents is Brugia malayi a clade III nematode. Current therapeutic agents are effective against the microfilaria but less so against the adults residing in the host lymphatics. A large number of anthelmintics act on nematode ion channels including the nicotinic receptors found on nematode somatic muscle. The purpose of this study was to develop a preparation from adult B. malayi that was amenable to patch-clamp recording to facilitate the study of the ion channels present in this organism. We also present a preliminary characterization of the single-channel properties of nicotinic receptors from the adult musculature.     

Molecular cloning and characterization of Brugia malayi hexokinase

5' EST from filarial gene database has been subjected to 3' rapid amplification of cDNA ends (RACE), semi-nested PCR and PCR to obtain full-length cDNA of Brugia malayi. Full-length hexokinase gene was obtained from cDNA using gene specific primers. The elicited PCR product was cloned, sequenced and expressed as an active enzyme in Escherichia coli. Sequence analysis of B. malayi hexokinase (BmHk) revealed 59% identity with nematode Caenorhabditis elegans but low similarity with all other available hexokinases including human. BmHk, an apparent tetramer with subunit molecular mass of 72 kDa, was able to phosphorylate glucose, fructose, mannose, maltose and galactose. The Km values for glucose, fructose and ATP were found to be 0.035+/-0.005, 75+/-0.3 and 1.09+/-0.5 mM respectively. BmHk was strongly inhibited by ADP, glucosamine, N-acetyl glucosamine and mannoheptulose. The recombinant enzyme was found to be activated by glucose-6-phosphate. ADP exhibited noncompetitive inhibition with the substrate glucose (Ki=0.55 mM) while, mixed type of inhibition was observed with inorganic pyrophosphate (PPi) when ATP was used as substrate (Ki=9.92 microM). The enzyme activity is highly dependent on maintenance of free sulfhydryl groups. CD analysis indicated that BmHk is composed of 37% alpha-helices and 26% beta-sheets. The observed differences in kinetic properties of BmHk as compared to host enzyme may facilitate designing of specific inhibitors against BmHk.     

Paramyosin-enhanced clearance of Brugia malayi microfilaremia in mice

Progress in development of a vaccine against human filariasis has been hampered by lack of knowledge of the biochemical structure of specific Ag that induce protective immunity in experimental hosts. In the current study, antiserum to infective third-stage larvae of Brugia malayi was used to select potentially protective Ag shared by microfilariae (mf) and adult worms. A major Ag of 97 kDa (Bm 97) was identified by immunoblotting and isolated by electroelution. Immunization of mice with 2 micrograms electroeluted Bm 97 induced partial resistance to subsequent i.v. challenge with live B. malayi mf (40 to 60% reduction in parasitemia compared to controls, p less than 0.05). Immunoblot studies of B. malayi mf and adult worm lysates showed reactivity of a 97-kDa molecule with monospecific antiserum to Schistosoma mansoni paramyosin. In addition, mouse antibody to Bm 97 reacted with a 97-kDa molecule contained in wild-type Caenorhabditis elegans but not in two mutant strains deficient for paramyosin. Subcutaneous injection of mice with paramyosin (5 micrograms twice at a 2-wk interval) purified from C. elegans or B. malayi by salt precipitation induced resistance to microfilaremia (21 to 60% lower intensities than controls, p less than 0.01). These data indicate that the invertebrate muscle protein paramyosin enhances clearance of blood-borne stages of lymphatic filariae. Examination of the ability of paramyosin to induce resistance in third-stage larvae-challenged hosts is warranted.     

A Potential Role for the Interaction of Wolbachia Surface Proteins with the Brugia malayi Glycolytic Enzymes and Cytoskeleton in Maintenance of Endosymbiosis

The human filarial parasite Brugia malayi harbors an endosymbiotic bacterium of the genus Wolbachia. The Wolbachia represent an attractive target for the control of filarial induced disease as elimination of the bacteria affects molting, reproduction and survival of the worms. The molecular basis for the symbiotic relationship between Wolbachia and their filarial hosts has yet to be elucidated. To identify proteins involved in this process, we focused on the Wolbachia surface proteins (WSPs), which are known to be involved in bacteria-host interactions in other bacterial systems. Two WSP-like proteins (wBm0152 and wBm0432) were localized to various host tissues of the B. malayi female adult worms and are present in the excretory/secretory products of the worms. We provide evidence that both of these proteins bind specifically to B. malayi crude protein extracts and to individual filarial proteins to create functional complexes. The wBm0432 interacts with several key enzymes involved in the host glycolytic pathway, including aldolase and enolase. The wBm0152 interacts with the host cytoskeletal proteins actin and tubulin. We also show these interactions in vitro and have verified that wBm0432 and B. malayi aldolase, as well as wBm0152 and B. malayi actin, co-localize to the vacuole surrounding Wolbachia. We propose that both WSP protein complexes interact with each other via the aldolase-actin link and/or via the possible interaction between the host''s enolase and the cytoskeleton, and play a role in Wolbachia distribution during worm growth and embryogenesis.     

Identification of anti-filarial leads against aspartate semialdehyde dehydrogenase of Wolbachia endosymbiont of Brugia malayi: combined molecular docking and molecular dynamics approaches

Lymphatic filariasis is a debilitating vector borne parasitic disease that infects human lymphatic system by nematode Brugia malayi. Currently available anti-filarial drugs are effective only on the larval stages of parasite. So far, no effective drugs are available for humans to treat filarial infections. In this regard, aspartate semialdehyde dehydrogenase (ASDase) in lysine biosynthetic pathway from Wolbachia endosymbiont Brugia malayi represents an attractive therapeutic target for the development of novel anti-filarial agents. In this present study, molecular modeling combined with molecular dynamics simulations and structure-based virtual screening were performed to identify potent lead molecules against ASDase. Based on Glide score, toxicity profile, binding affinity and mode of interactions with the ASDase, five potent lead molecules were selected. The molecular docking and dynamics results revealed that the amino acid residues Arg103, Asn133, Cys134, Gln161, Ser164, Lys218, Arg239, His246, and Asn321 plays a crucial role in effective binding of Top leads into the active site of ASDase. The stability of the ASDase-lead complexes was confirmed by running the 30 ns molecular dynamics simulations. The pharmacokinetic properties of the identified lead molecules are in the acceptable range. Furthermore, density functional theory and binding free energy calculations were performed to rank the lead molecules. Thus, the identified lead molecules can be used for the development of anti-filarial agents to combat the pathogenecity of Brugia malayi.     

Guanidine hydrochloride and urea-induced unfolding of Brugia malayi hexokinase

Guanidine hydrochloride and urea-induced unfolding of B. malayi hexokinase (BmHk), a tetrameric protein, was examined in detail by using various optical spectroscopic techniques, enzymatic activity measurements, and size-exclusion chromatography. The equilibrium unfolding of BmHk by guanidine hydrochloride (GdmCl) and urea proceeded through stabilization of several unique oligomeric intermediates. In the presence of low concentrations of GdmCl, stabilization of an enzymatically active folded dimer of BmHk was observed. However an enzymatically inactive dimer of BmHk was observed for urea-treated BmHk. This is the first report of an enzymatically active dimer of hexokinase from any human filarial parasite. Furthermore, although complete recovery of the native enzyme was observed on refolding of BmHk samples denatured by use of low concentrations of GdmCl or urea, no recovery of the native enzyme was observed for BmHk samples denatured by use of high concentrations of GdmCl or urea.     

Variable infectivity of third-stage larvae of Brugia malayi

Infectivity of third-stage larvae of Brugia malayi was assessed following intraperitoneal inoculation into jirds, Meriones unguiculatus. Larvae were of two ages and were derived from two sites in Aedes aegypti mosquitoes, i.e., specimens collected from the thorax 11 days after infection and from the head on day 14. Larvae from the thorax had just completed the second moult and measured 990 to 1100 micron in length. Only 6% of these specimens developed to adult worms in jirds. Larvae that migrated to the head were 1400 to 1700 micron long on day 14 and, in contrast, 23% of the inocula developed to adult worms. This study establishes that all third-stage larvae, regardless of their age or location in the arthropod host, are potentially infective. However, pronounced physical maturation does seem to be accompanied by a marked increase in infectivity.