Defining the filarial N-glycoproteome by glycosite mapping in the human parasitic nematode Brugia malayi.

N-linked glycosylation is a critical post translational modification of eukaryotic proteins. N-linked glycans are present on surface and secreted filarial proteins that play a role in host parasite interactions. Examples of glycosylated Brugia malayi proteins have been previously identified but there has not been a systematic study of the N-linked glycoproteome of this or any other filarial parasite. In this study, we applied an enhanced N-glyco FASP protocol using an engineered carbohydrate-binding protein, Fbs1, to enrich N-glycosylated peptides for analysis by LC-MS/MS. We then mapped the N-glycosites on proteins from three host stages of the parasite: adult female, adult male and microfilariae. Fbs1 enrichment of N-glycosylated peptides enhanced the identification of N-glycosites. Our data identified 582 N-linked glycoproteins with 1273 N-glycosites. Gene ontology and cell localization prediction of the identified N-glycoproteins indicated that they were mostly membrane and extracellular proteins. Comparing results from adult female worms, adult male worms, and microfilariae, we find variability in N-glycosylation at the protein level as well as at the individual N-glycosite level. These variations are highlighted in cuticle N-glycoproteins and adult worm restricted N-glycoproteins as examples of proteins at the host parasite interface that are well positioned as potential therapeutic targets or biomarkers.

Infrared light photobiostimulation mediates periodicity in Dirofilaria immitis microfilariae.

Microfilariae (Mfs) of filarial nematode parasites exhibit nocturnal periodicity, with their numbers in peripheral blood peaking at night and decreasing during the day. However, the reason for their appearance at night remains unknown. In this study, in vitro photobiostimulation experiments showed that Mfs exhibited positive phototaxis toward infrared light with lower photon flux densities of infrared light at wavelengths of 890 and 700 nm, in particular, mediating paradoxically higher velocity than intense ones. Microarray analysis revealed that infrared light stimulation influenced gene expression in Mfs and induced significant upregulation of genes, with phosphorylation- and neurogenesis-related genes being highly enriched. Weaker natural infrared beams from the atmosphere only at midnight may induce microfilaria periodicity, and the nature of the periodic pattern is innate and plastic, as demonstrated by artificially changing the light-dark cycle. This is the first report of positive phototaxis toward infrared light in Dirofilaria immitis Mfs. The notable finding is that they moved in union despite the lack of a fluid current inside the container, indicating that infrared light appears to control nocturnal periodicity in D. immitis Mfs. The newly developed culture medium and the adoption of charge-coupled device (CCD) camera and time-lapse VHS videocassette recorder used in this study made possible to be a long observation.

Loa loa Microfilariae in Skin Snips: Consequences for Onchocerciasis Monitoring and Evaluation in L. loa-Endemic Areas.

The specificity of skin snips for onchocerciasis diagnoses is considered to be almost 100%. Our molecular methods revealed that microfilariae emerging from skin snips collected from highly microfilaremic Loa loa-infected individuals were largely misidentified as Onchocerca volvulus. This has important implications for onchocerciasis diagnostic testing in Loa-endemic areas.

Isolation and characterization of endochitinase and exochitinase of Setaria cervi.

Chitin metabolism has been shown to have a role in the development of parasitic nematodes including filarial parasites and the enzymes associated with chitin metabolism have been considered as potential vaccine and drug target. Chitinases are members of the enzyme superfamily of glycoside hydrolases, which are characterized by the ability to hydrolyze glycosidic bonds in chitin chain by either an endolytic or an exolytic mechanism. In the present study, we have demonstrated the chitinase (exochitinase and endochitinase) activity in different stages of Setaria cervi (bovine filarial parasite) and have also purified and characterized the endochitinase from microfilarial stage of the parasite. The chitinase activity has been detected in adult and microfilarial stages of S. cervi using the fluorescent substrates. The S. cervi adult stage was found to have high activity of exochitinase (28.72±0.25 nmol/min/mg) while microfilarial stage showed high activity of endochitinase (24.40±0.25 nmol/min/mg). Native polyacrylamide gel electrophoresis, followed by staining of enzyme activity with fluorescent substrates, revealed single isoenzymic form of exochitinase in adults and endochitinase in microfilariae of S. cervi. The endochitinase from S. cervi microfilariae was purified employing chitin affinity matrix and DEAE-Sephacel ion-exchange chromatography. The enzyme was purified about 55 fold with an enzyme recovery of 22.33%. The purified enzyme exhibited a doublet of protein bands on SDS-PAGE at 65-70 kDa. The closantel (chitinase inhibitor) strongly inhibited the enzyme activity of S. cervi microfilariae endochitinase with a Ki value of 4.3±0.18 µM.

Ethanolic extract of Azadirachta indica (A. Juss.) causing apoptosis by ROS upregulation in Dirofilaria immitis microfilaria.

Dirofilaria immitis is the causative agent of cardiopulmonary dirofilariasis in the Canine family. The aim of the study was to evaluate the efficacy of the ethanolic extract of Azadirachta indica leaves (EEA) against the microfilaria (mf) of D. immitis in vitro. EEA was evaluated for different compound classes through HPTLC. Relative motility, mortality and morphological alterations were observed in the mf after exposure to EEA. The effect of EEA on redox status in the treated mf was evaluated by some key enzymatic and non-enzymatic parameters. An enhanced reactive oxygen species (ROS) level in the treated mf along with altered redox status was evident. DNA fragmentation and terminal-deoxynucleotidyl-transferase dUTP nick end labeling (TUNEL) confirmed apoptosis. In addition, western blotting revealed down-regulation of anti-apoptotic protein and up-regulation of pro-apoptotic proteins. Taken together, the microfilaricidal activity of EEA can be attributed to its capacity to inflict oxidative stress culminating in apoptosis.

Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro.

The macrocyclic lactones are the only anthelmintics used to prevent heartworm disease, but it is very difficult to reproduce their in vivo efficacy against Dirofilaria immitis larvae in experiments in vitro. These assays typically measure motility, suggesting that paralysis is not the mode of action of the macrocyclic lactones against D. immitis. We isolated peripheral blood mononuclear cells (PBMC) and neutrophils from uninfected dogs and measured their adherence to D. immitis microfilariae in the presence of varying concentrations of ivermectin. We found that adherence of PBMC to the microfilariae was increased in the presence of ivermectin concentrations ≥100 nM and adherence of neutrophils was increased in drug concentrations ≥10 nM. Up to 50% of microfilariae had adherent PBMC in the presence of the drug, and binding was maximal after 40 h incubation. Neutrophil adherence was maximal after 16 h, with approximately 20% of the microfilariae having at least one cell adhered to them. Adherent neutrophils showed morphological evidence of activation. These results are consistent with a model in which the macrocyclic lactones interfere with the parasites ability to evade the host's innate immune system.

A deep sequencing approach to comparatively analyze the transcriptome of lifecycle stages of the filarial worm, Brugia malayi.

BACKGROUND: Developing intervention strategies for the control of parasitic nematodes continues to be a significant challenge. Genomic and post-genomic approaches play an increasingly important role for providing fundamental molecular information about these parasites, thus enhancing basic as well as translational research. Here we report a comprehensive genome-wide survey of the developmental transcriptome of the human filarial parasite Brugia malayi. METHODOLOGY/PRINCIPAL FINDINGS: Using deep sequencing, we profiled the transcriptome of eggs and embryos, immature (≤3 days of age) and mature microfilariae (MF), third- and fourth-stage larvae (L3 and L4), and adult male and female worms. Comparative analysis across these stages provided a detailed overview of the molecular repertoires that define and differentiate distinct lifecycle stages of the parasite. Genome-wide assessment of the overall transcriptional variability indicated that the cuticle collagen family and those implicated in molting exhibit noticeably dynamic stage-dependent patterns. Of particular interest was the identification of genes displaying sex-biased or germline-enriched profiles due to their potential involvement in reproductive processes. The study also revealed discrete transcriptional changes during larval development, namely those accompanying the maturation of MF and the L3 to L4 transition that are vital in establishing successful infection in mosquito vectors and vertebrate hosts, respectively. CONCLUSIONS/SIGNIFICANCE: Characterization of the transcriptional program of the parasite's lifecycle is an important step toward understanding the developmental processes required for the infectious cycle. We find that the transcriptional program has a number of stage-specific pathways activated during worm development. In addition to advancing our understanding of transcriptome dynamics, these data will aid in the study of genome structure and organization by facilitating the identification of novel transcribed elements and splice variants.

Transforming growth factor-ß1 variant Leu10Pro is associated with both lack of microfilariae and differential microfilarial loads in the blood of persons infected with lymphatic filariasis.

Antigen testing and ultrasound detection have shown that many persons are infected with Wuchereria bancrofti even though they do not have microfilariae (Mf) in the blood. To ascertain the role of human host immunogenetics on the lack of circulating Mf in the blood, 152 lymphatic filariasis (LF)-infected patients comprising 118 patients with microfilaremic (Mf+, patent) infection and 34 patients with latent (Mf-, antigen-positive) infection were recruited and genotyped for association of single nucleotide polymorphisms of TGF-ß1 and differential Mf load and/or lack of Mf in the blood from infected persons in Ghana. An association was found between the TGF-ß1 Leu10Pro variant and lack of Mf in the blood. Patients with latent infection had a higher frequency of the Leu/Leu genotype than patients with patent infection (p = 0.03). Secondary analysis revealed an association among the three possible Leu10Pro genotypes and different Mf loads in the blood. In conclusion, the differential Mf loads and the lack of Mf in the blood of patients is likely to have a genetic basis. Because the adult worms are responsible for pathology, these results underscore the need for a review of using only Mf detection in blood smears for diagnosis of LF infection in endemic areas. This information is also important for the mapping and surveillance activities of national and global programs for elimination of LF.

ABC transporters influence sensitivity of Brugia malayi to moxidectin and have potential roles in drug resistance.

Some ABC transporters play a significant role in human health and illness because they confer multidrug resistance (MDR) through their overexpression. Compounds that inhibit the drug efflux mechanism can improve efficacy or reverse resistance. Of the eight described ABC transporter subfamilies, those proteins conferring MDR in humans are in subfamilies A, B, C, and G. In nematodes, transporters in subfamilies B and C are suggested to confer resistance to ivermectin. The Brugia malayi ABC transporter superfamily was examined to assess their potential to influence sensitivity to moxidectin. There was an increase in expression of ABC transporters in subfamilies A, B, C, and G following treatment. Co-administration of moxidectin with inhibitors of ABC transporter function did not enhance sensitivity to moxidectin in males; however, sensitivity was significantly enhanced in females and microfilariae. The work suggests that ABC transporters influence sensitivity to moxidectin and have a potential role in drug resistance.

Functional and phenotypic characteristics of alternative activation induced in human monocytes by interleukin-4 or the parasitic nematode Brugia malayi.

Human monocytes from patients with patent filarial infections are studded with filarial antigen and express markers associated with alternative activation of macrophages (MΦ). To explore the role of filaria-derived parasite antigen in differentiation of human monocytes, cells were exposed to microfilariae (mf) of Brugia malayi, and their phenotypic and functional characteristics were compared with those of monocytes exposed to factors known to generate either alternatively (interleukin-4 [IL-4]) or classically (macrophage colony-stimulating factor [MCSF]) activated MΦ. IL-4 upregulated mRNA expression of CCL13, CCL15, CCL17, CCL18, CCL22, CLEC10A, MRC1, CADH1, CD274, and CD273 associated with alternative activation of MΦ but not arginase 1. IL-4-cultured monocytes had a diminished ability to promote proliferation of both CD4(+) and CD8(+) T cells compared to that of unexposed monocytes. Similar to results with IL-4, exposure of monocytes to live mf induced upregulation of CCL15, CCL17, CCL18, CCL22, CD274, and CD273 and downregulation of Toll-like receptor 3 (TLR3), TLR5, and TLR7. In contrast to results with MCSF-cultured monocytes, exposure of monocytes to mf resulted in significant inhibition of the phagocytic ability of these cells to the same degree as that seen with IL-4. Our data suggest that short exposure of human monocytes to IL-4 induces a phenotypic characteristic of alternative activation and that secreted filarial products skew monocytes similarly.

Characterization of the DMAE-modified juvenile excretory-secretory protein Juv-p120 of Litomosoides sigmodontis.

Juv-p120 is an excretory-secretory 160 kDa glycoprotein of juvenile female Litomosoides sigmodontis and exhibits features typical for mucins. 50% of its molecular mass is attributed to posttranslational modifications with the unusual substituent dimethylaminoethanol (DMAE). By that Juv-p120 corresponds to the surface proteins of the microfilarial sheath, Shp3 and Shp3a. The secreted protein consists of 697 amino acids, organized in two different domains of repeat elements separated by a stretch of polar residues. The N-terminal domain shows fourteen P/S/T/F-rich repeat elements highly modified with phospho-DMAE substituted O-glycans confering a negative charge to the protein. The C-terminal domain is extremely rich in glutamine (35%) and leucine (25%) in less organized repeats and may play a role in oligomerization of Juv-p120 monomers. A protein family with a similar Q/L-rich region and conserved core promoter region was identified in Brugia malayi by homology screening and in Wuchereria bancrofti and Loa loa by database similarity search. One of the Q/L-rich proteins in each genus has an extended S/T-rich region and due to this feature is supposed to be a putative Juv-p120 ortholog. The corresponding modification of Juv-p120 and the microfilarial sheath surface antigens Shp3/3a explains the appearance of anti-sheath antibodies before the release of microfilariae. The function of Juv-p120 is unknown.

In vivo transfection of developmentally competent Brugia malayi infective larvae.

Transient transfection of isolated Brugia malayi embryos by biolistics has proven to be useful in defining promoter structure and function in this parasite. However, isolated transfected embryos are developmentally incompetent. A method of producing developmentally competent transfected parasites is therefore needed. We report that L3 parasites can be chemically transfected in situ in the peritoneal cavity of a gerbil with a construct consisting of a secreted luciferase reporter gene containing a promoter, the 3' untranslated region and first intron derived from the B. malayi 70 kDa heat shock protein gene. The in situ chemically transfected parasites are developmentally competent, producing adult parasites with an efficiency similar to that obtained from implanted untreated L3s. Cultured adult parasites and progeny microfilariae (mf) derived from L3s transfected with this construct secreted luciferase into the culture medium. When the transfected mf were fed to mosquitoes and the resulting L3s collected, the L3s also secreted luciferase into the culture medium. Progeny mf from transgenic adult parasites contained transgenic DNA, and the transgenic mRNA produced in these parasites was found to be correctly cis- and trans-spliced. In situ chemical transformation thus results in developmentally competent transfected B. malayi in which the transgenic sequences remain transcriptionally active in all life cycle stages and are present in the subsequent generation.

In vitro effect of four herbal plants on the motility of Brugia malayi microfilariae.

BACKGROUND & OBJECTIVE: Disease burden due to lymphatic filariasis is disproportionately high despite mass drug administration with conventional drugs. Usage of herbal drugs in traditional medicine is quite well known but largely empirical. Hence the present study was designed to screen the in vitro antifilarial effect of four herbal plants on Brugia malayi. METHODS: Motility of microfilariae of B. malayi after incubation for 48 h with aqueous/methanol extracts of Vitex negundo L. (roots), Butea monosperma L. (roots and leaves), Ricinus communis L. (leaves), and Aegle marmelos Corr. (leaves) was explored in the concentration range of 20 to 100 ng/ml for possible antifilarial effect by comparing with suitable solvent control. RESULTS: Butea monosperma leaves and roots, Vitex negundo root and Aegle marmelo leaves showed significant inhibition of motility of microfilariae as compared to controls whereas inhibitory activity demonstrated by Ricinus communis L. leaves was not significant. Antifilarial effects imparted by all these extracts were found to be a function of their relative concentrations. Inhibitory concentrations (IC(50)) for the plant extracts with significant antifilarial activity against Brugia malayi microfilariae in in vitro system have been derived to be 82, 83 and 70 ng/ml for Vitex negundo L., Butea monosperma L. and Aegle marmelos Corr. respectively. INTERPRETATION & CONCLUSION: The present study recorded significant antifilarial effect of all plant extracts studied except for Ricinus communis L. leaves and contributes to the development of database for novel drug candidates for human lymphatic filariasis.

Identification of 38kDa Brugia malayi microfilarial protease as a vaccine candidate for lymphatic filariasis.

A FPLC purified 38kDa protease (Bm mf S-7) isolated from B. malayi microfilarial soluble antigen was identified. It showed pronounced reactivity with sera collected from 'putatively immune' asymptomatic and amicrofilaraemic individuals residing in an endemic area for bancroftian filariasis. Further the immune protective activity of Bm mf S-7 antigen was evaluated in susceptible hosts, jirds (Meriones unguiculatus) against B. malayi filarial infection. The antigen showed 89% cytotoxicity against mf and 87-89% against infective (L3) larvae in in vitro antibody dependent cellular cytotoxicity Assay (ADCC) and in situ micropore chamber methods. Bm mf S-7 immunized jirds after challenge infection showed 81.5% reduction in the adult worm burden. The present study has shown that, the 38kDa microfilarial proteases (Bm mf S-7) could stimulate a strong protective immune response against microfilariae and infective larvae in jird model to block the transmission of filariasis. Analysis of IgG subclasses against Bm mf S-7 revealed a significant increase in IgG2 and IgG3 antibodies in endemic normals. Lymphocyte proliferation to Bm mf S-7 was significantly high in endemic normal group as compared to that in clinical and microfilarial carriers. Significantly enhanced levels of IFN-gamma in the culture supernatant of PBMC of endemic normals followed by stimulation with Bm mf S-7 suggest that the cellular response in this group is skewed towards Th 1 type.

Hsp90 is essential in the filarial nematode Brugia pahangi.

The development of a compound with activity against filarial nematodes (a 'macrofilaricide') has been a long-standing goal of the World Health Organization. However, adult filariae have proved remarkably difficult to kill. To some extent this reflects a lack of understanding of key pathways and processes in filarial nematodes that may be suitable targets for chemotherapy. In this paper we show that geldanamycin (GA), a specific inhibitor of the activity of the heat shock protein 90 (Hsp90) family, kills adult worms and microfilariae (Mf) of Brugia pahangi at nanomolar concentrations. In addition, release of Mf from adult worms is inhibited within 24 h of exposure to GA and is not recoverable, demonstrating that GA effectively sterilises the worm. Similar results were obtained with a second filarial worm Acanthocheilonema viteae. In contrast GA has no effect on the free-living nematode Caenorhabditis elegans despite a high degree of conservation between the nematode Hsp90 sequences. In keeping with these findings, Brugia Hsp90 binds GA in a solid phase pull-down assay while the binding of C. elegans Hsp90 to immobilised GA is undetectable. In other eukaryotes, GA is known to bind in the N-terminal ATP pocket of Hsp90, disrupting its interactions with client proteins which are then targeted for degradation via the proteasome pathway. Thus, Hsp90 or some of its client proteins may provide novel targets for the chemotherapy of filarial infection.

Poorer NF-kappa B signaling by microfilariae in macrophages from BALB/c mice affects their ability to produce cytotoxic levels of nitric oxide to kill microfilariae.

Upon activation with microfilariae (mf), macrophages from C57Bl/6 mice showed higher nuclear factor-kappa B (NF-kappa B) but lower activating protein 1 DNA-binding activity as compared to BALB/c macrophages. The C57Bl/6 macrophages produced cytotoxic levels of nitric oxide (NO) to kill Setaria cervi mf as compared to BALB/c macrophages. Inhibition of the NF-kappa B signal by pyrrolidine dithiocarbamate (PDTC) blocked NO production and microfilaricidal activity of C57Bl/6 macrophages and inclusion of the exogenous NO generator (SNP) in the PDTC treated C57Bl/6 macrophage cultures induced mf cytotoxicity. These results underscore that the NF-kappa B signal (induced in response to mf) is important for the NO-mediated microfilaricidal activity of macrophages.

Assessing density dependence in the transmission of lymphatic filariasis: uptake and development of Wuchereria bancrofti microfilariae in the vector mosquitoes.

Understanding density dependence in the transmission of lymphatic filariasis is essential for assessing the prospects of elimination. This study seeks to quantify the relationship between microfilaria (Mf) density in human blood and the number of third stage (L3) larvae developing in the mosquito vectors Aedes polynesiensis Marks and Culex quinquefasciatus Say (Diptera: Culicidae) after blood-feeding. Two types of curves are fitted to previously published data. Fitting a linearized power curve through the data allows for correction for measurement error in human Mf counts. Ignoring measurement error leads to overestimation of the strength of density dependence; the degree of overestimation depends on the accuracy of measurement of Mf density. For use in mathematical models of transmission of lymphatic filariasis, a hyperbolic saturating function is preferable. This curve explicitly estimates the Mf uptake and development at lowest Mf densities and the average maximum number of L3 that can develop in mosquitoes. This maximum was estimated at 23 and 4 for Ae. polynesiensis and Cx. quinquefasciatus, respectively.

Synthesis and antifilarial evaluation of N1,Nn- xylofuranosylated diaminoalkanes.

A series of N(1),N(n)-xylofuranosylated diaminoalkanes (3-9 and 11-18) has been synthesized either by reductive amination of deoxy xylouloses (2a, 2b) with amines followed by one pot reduction with NaBH(4) or NaCNBH(3); or by 1,4-conjugate addition of amines to glycosyl olefinic esters (10a, 10b). The compounds were screened for their interference with filarial worms' glutathione metabolism, a potential target for chemotherapeutic attack. Interestingly, these compounds affected intracellular glutathione, gamma-glutamyl cysteine synthetase, glutathione reductase and glutathione-S-transferase(s) of bovine filarial worms to varying degrees. Some of the compounds though effected the motility and MTT reduction potential of filarial worms Brugia malayi, however, little microfilaricidal and macrofilaricidal were noted with compounds at 50mg/kg oral dose. Compounds 6, 16 and 17 were evaluated also for in vivo activity.

Brugia malayi: resistance of cuticular lipids to oxidant-induced damage and detection of alpha-tocopherol in the neutral lipid fraction.

We have examined the susceptibility of cuticular membrane lipids of Brugia malayi to oxidants generated in vitro. Live parasites as well as extracted cuticular lipids were treated with hydrogen peroxide and hypochlorous acid and the extent of lipid peroxidation was quantified. The cuticular membranes of B. malayi were found to be resistant to lipid peroxidation at hydrogen peroxide concentrations which were lethal to the organism. This resistance was partly due to the inherently low unsaturation indices of the fatty acyl residues, but complete protection was afforded by lipid-soluble antioxidants present in the neutral lipid fraction of the parasites. We have identified alpha-tocopherol as a major antioxidant present in both adult and microfilarial B. malayi. In addition, we report that although hypochlorous acid chemically modifies isolated parasite lipids, the latter do not appear to be the primary substrate for the oxidant in live worms. The data are discussed in terms of the susceptibility of B. malayi to products of the respiratory burst from activated myeloid cells.

Acanthocheilonema viteae: stage-specific expression of G-protein alpha-subunits.

We have previously demonstrated by Western blot analysis that the adult stage of the filarial nematode Acanthocheilonema viteae expresses the alpha-subunits of heterotrimeric G-proteins corresponding to GS and Gq. We now show, using the same technique, that these two alpha-subunits are not detectable in the microfilaria stage of the parasite. Conversely, microfilariae contain Go, an alpha-subunit not expressed by the adult worm. No other G-protein alpha-subunits were found in microfilariae by Western blotting. However, reverse transcriptase-polymerase chain reaction (RT-PCR) with degenerate G-protein oligonucleotide primers, followed by hybridisation analysis, using oligonucleotides specific for individual G-protein alpha-subunits, not only confirmed expression of Go, but also detected Gi1 and G11 alpha-subunits. G-protein expression in infective larvae was also investigated by RT-PCR analysis: this stage of the organism was found to resemble the adult more than the microfilaria but differed from the adult in that GS was absent and Gi3 was present. The significance of these stage-specific differences in G-protein expression is discussed with respect to their possible role in parasite development and survival.