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.     

Effects of Doxycycline on gene expression in Wolbachia and Brugia malayi adult female worms in vivo

Background

Most filarial nematodes contain Wolbachia symbionts. The purpose of this study was to examine the effects of doxycycline on gene expression in Wolbachia and adult female Brugia malayi.

Methods

Brugia malayi infected gerbils were treated with doxycycline for 6-weeks. This treatment largely cleared Wolbachia and arrested worm reproduction. RNA recovered from treated and control female worms was labeled by random priming and hybridized to the Version 2- filarial microarray to obtain expression profiles.

Results and discussion

Results showed significant changes in expression for 200 Wolbachia (29% of Wolbachia genes with expression signals in untreated worms) and 546 B. malayi array elements after treatment. These elements correspond to known genes and also to novel genes with unknown biological functions. Most differentially expressed Wolbachia genes were down-regulated after treatment (98.5%). In contrast, doxycycline had a mixed effect on B. malayi gene expression with many more genes being significantly up-regulated after treatment (85% of differentially expressed genes). Genes and processes involved in reproduction (gender-regulated genes, collagen, amino acid metabolism, ribosomal processes, and cytoskeleton) were down-regulated after doxycycline while up-regulated genes and pathways suggest adaptations for survival in response to stress (energy metabolism, electron transport, anti-oxidants, nutrient transport, bacterial signaling pathways, and immune evasion).

Conclusions

Doxycycline reduced Wolbachia and significantly decreased bacterial gene expression. Wolbachia ribosomes are believed to be the primary biological target for doxycycline in filarial worms. B. malayi genes essential for reproduction, growth and development were also down-regulated; these changes are consistent with doxycycline effects on embryo development and reproduction. On the other hand, many B. malayi genes involved in energy production, electron-transport, metabolism, anti-oxidants, and others with unknown functions had increased expression signals after doxycycline treatment. These results suggest that female worms are able to compensate in part for the loss of Wolbachia so that they can survive, albeit without reproductive capacity. This study of doxycycline induced changes in gene expression has provided new clues regarding the symbiotic relationship between Wolbachia and B. malayi.     

Tetracycline treatment targeting Wolbachia affects expression of an array of proteins in Brugia malayi parasite

Wolbachia is an intracellular endosymbiont of Brugia malayi parasite whose presence is essential for the survival of the parasite. Treatment of B. malayi‐infected jirds with tetracycline eliminates Wolbachia, which affects parasite survival and fitness. In the present study we have tried to identify parasite proteins that are affected when Wolbachia is targeted by tetracycline. For this Wolbachia depleted parasites (B. malayi) were obtained by tetracycline treatment of infected Mongolian jirds (Meriones unguiculatus) and their protein profile after 2‐DE separation was compared with that of untreated parasites harboring Wolbachia. Approximately 100 protein spots could be visualized followed by CBB staining of 2‐D gel and included for comparative analysis. Of these, 54 showed differential expressions, while two new protein spots emerged (of 90.3 and 64.4 kDa). These proteins were subjected to further analysis by MALDI‐TOF for their identification using Brugia coding sequence database composed of both genomic and EST sequences. Our study unravels two crucial findings: (i) the parasite or Wolbachia proteins, which disappeared/down‐regulated appear be essential for parasite survival and may be used as drug targets and (ii) tetracycline treatment interferes with the regulatory machinery vital for parasites cellular integrity and defense and thus could possibly be a molecular mechanism for the killing of filarial parasite. This is the first proteomic study substantiating the wolbachial genome integrity with its nematode host and providing functional genomic data of human lymphatic filarial parasite B. malayi.     

Computational prediction of essential genes in an unculturable endosymbiotic bacterium, Wolbachia of Brugia malayi

Background  

Wolbachia (wBm) is an obligate endosymbiotic bacterium of Brugia malayi, a parasitic filarial nematode of humans and one of the causative agents of lymphatic filariasis. There is a pressing need for new drugs against filarial parasites, such as B. malayi. As wBm is required for B. malayi development and fertility, targeting wBm is a promising approach. However, the lifecycle of neither B. malayi nor wBm can be maintained in vitro. To facilitate selection of potential drug targets we computationally ranked the wBm genome based on confidence that a particular gene is essential for the survival of the bacterium.     

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.     

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: 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.     

Biochemical characterization of Recombinase A from Wolbachia endosymbiont of filarial nematode Brugia malayi (wBmRecA)

Lymphatic filariasis is a debilitating disease that affects over 890 million people in 49 countries. A lack of vaccines, non-availability of adulticidal drugs, the threat of emerging drug resistance against available chemotherapeutics and an incomplete understanding of the immunobiology of the disease have sustained the problem. Characterization of Wolbachia proteins, the bacterial endosymbiont which helps in the growth and development of filarial worms, regulates fecundity in female worms and mediates immunopathogenesis of Lymphatic Filariasis, is an important approach to gain insights into the immunopathogenesis of the disease. In this study, we carried out extensive biochemical characterization of Recombinase A from Wolbachia of the filarial nematode Brugia malayi (wBmRecA) using an Electrophoretic Mobility Shift Assay, an ATP binding and hydrolysis assay, DNA strand exchange reactions, DAPI displacement assay and confocal microscopy, and evaluated anti-filarial activity of RecA inhibitors. Confocal studies showed that wBmRecA was expressed and localised within B. malayi microfilariae (Mf) and uteri and lateral chord of adult females. Recombinant wBmRecA was biochemically active and showed intrinsic binding capacity towards both single-stranded DNA and double-stranded DNA that were enhanced by ATP, suggesting ATP-induced cooperativity. wBmRecA promoted ATP hydrolysis and DNA strand exchange reactions in a concentration-dependent manner, and its binding to DNA was sensitive to temperature, pH and salt concentration. Importantly, the anti-parasitic drug Suramin, and Phthalocyanine tetrasulfonate (PcTs)-based inhibitors Fe-PcTs and 3,4-Cu-PcTs, inhibited wBmRecA activity and affected the motility and viability of Mf. The addition of Doxycycline further enhanced microfilaricidal activity of wBmRecA, suggesting potential synergism. Taken together, the omnipresence of wBmRecA in B. malayi life stages and the potent microfilaricidal activity of RecA inhibitors suggest an important role of wBmRecA in filarial pathogenesis.     

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.     

Asymmetric Wolbachia Segregation during Early Brugia malayi Embryogenesis Determines Its Distribution in Adult Host Tissues

Wolbachia are required for filarial nematode survival andfertility and contribute to the immune responses associated with human filarialdiseases. Here we developed whole-mount immunofluorescence techniques tocharacterize Wolbachia somatic and germline transmissionpatterns and tissue distribution in Brugia malayi, a nematoderesponsible for lymphatic filariasis. In the initial embryonic divisions,Wolbachia segregate asymmetrically such that they occupyonly a small subset of cells in the developing embryo, facilitating theirconcentration in the adult hypodermal chords and female germline.Wolbachia are not found in male reproductive tissues andthe absence of Wolbachia from embryonic germline precursors inhalf of the embryos indicates Wolbachia loss from the malegermline may occur in early embryogenesis. Wolbachia rely onfusion of hypodermal cells to populate adult chords. Finally, we detectWolbachia in the secretory canal lumen suggesting livingworms may release bacteria and/or their products into their host.