The Effects of Ivermectin on Brugia malayi Females In Vitro: A Transcriptomic Approach

BackgroundLymphatic filariasis and onchocerciasis are disabling and disfiguring neglected tropical diseases of major importance in developing countries. Ivermectin is the drug of choice for mass drug administration programs for the control of onchocerciasis and lymphatic filariasis in areas where the diseases are co-endemic. Although ivermectin paralyzes somatic and pharyngeal muscles in many nematodes, these actions are poorly characterized in adult filariae. We hypothesize that paralysis of pharyngeal pumping by ivermectin in filariae could result in deprivation of essential nutrients, especially iron, inducing a wide range of responses evidenced by altered gene expression, changes in metabolic pathways, and altered developmental states in embryos. Previous studies have shown that ivermectin treatment significantly reduces microfilariae release from females within four days of exposure in vivo, while not markedly affecting adult worms. However, the mechanisms responsible for reduced production of microfilariae are poorly understood.Conclusion/SignificanceThese changes provide insight into the mechanisms involved in ivermectin-induced reduction in microfilaria output and impaired fertility, embryogenesis, and larval development.     

In vitro effects of antibiotics on Brugia malayi worm survival and reproduction

Recent studies have suggested that intracellular Wolbachia spp. endobacteria are necessary for the reproduction and survival of filarial nematodes. The effects of antibiotics that are active against related bacteria on adult worms and microfilariae (Mf) of Brugia malayi in vitro were investigated. Antibiotics tested were doxycycline (Doxy), tetracycline (Tet), rifampicin (Rif), azithromycin (Azith), and chloramphenicol (Chlor). Doxy, Tet, Rif, and Azith reduced release of Mf by adult female worms. The minimum effective concentrations that reduced Mf release by 50% were 5 microg/ml for Doxy, 20 microg/ml for Tet, 40 microg/ml for Rif, and 100 microg/ml for Azith. The same drugs (at higher concentrations) killed adult worms and Mf. Embryograms showed that Tets blocked embryogenesis in female worms. Electron microscopy (EM) showed that the Tets, Rif, and Azith cleared Wolbachia spp. from adult worms and damaged developing embryos. These studies show that antibiotics active against Rickettsiaceae affect adult B. malayi worms and Mf in vitro. Additional studies will be needed to elucidate the mechanisms of action of these antibiotics on Wolbachia and filarial worms.     

In Vitro Silencing of Brugia malayi Trehalose-6-Phosphate Phosphatase Impairs Embryogenesis and In Vivo Development of Infective Larvae in Jirds

Background

The trehalose metabolic enzymes have been considered as potential targets for drug or vaccine in several organisms such as Mycobacterium, plant nematodes, insects and fungi due to crucial role of sugar trehalose in embryogenesis, glucose uptake and protection from stress. Trehalose-6-phosphate phosphatase (TPP) is one of the enzymes of trehalose biosynthesis that has not been reported in mammals. Silencing of tpp gene in Caenorhabditis elegans revealed an indispensable functional role of TPP in nematodes.

Methodology and Principal Findings

In the present study, functional role of B. malayi tpp gene was investigated by siRNA mediated silencing which further validated this enzyme to be a putative antifilarial drug target. The silencing of tpp gene in adult female B. malayi brought about severe phenotypic deformities in the intrauterine stages such as distortion and embryonic development arrest. The motility of the parasites was significantly reduced and the microfilarial production as well as their in vitro release from the female worms was also drastically abridged. A majority of the microfilariae released in to the culture medium were found dead. B. malayi infective larvae which underwent tpp gene silencing showed 84.9% reduced adult worm establishment after inoculation into the peritoneal cavity of naïve jirds.

Conclusions/Significance

The present findings suggest that B. malayi TPP plays an important role in the female worm embryogenesis, infectivity of the larvae and parasite viability. TPP enzyme of B. malayi therefore has the potential to be exploited as an antifilarial drug target.     

In vitro killing of microfilariae of Brugia pahangi and Brugia malayi by eosinophil granule proteins

Eosinophil infiltration and degranulation around the tissue-invasive stages of several species of helminths have been observed. Release of eosinophil granule contents upon the worms is supported by localization of two of the major granule proteins, major basic protein (MBP) and eosinophil peroxidase (EPO), on and around species of trematodes, nematodes, and cestodes. In the case of filarial worms, MBP is deposited on degenerating microfilariae (mf) of Onchocerca volvulus. Here, we performed in vitro assays of the toxicity of four purified eosinophil granule proteins, namely, MBP, EPO, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN), for the mf of Brugia pahangi and Brugia malayi. MBP, ECP, and EDN killed these worms in a dose-related manner although relatively high concentrations of EDN were necessary. EPO, in the presence of a H2O2-generating system and a halide, was the most potent toxin on a molar basis; here, the most potent halide was I- followed by Br- and Cl-. Surprisingly, EPO in the absence of H2O2 killed mf at concentrations comparable to those required for MBP and ECP. The toxicity of EPO + H2O2 + halide was inhibited by heparin, catalase, or 1% BSA, whereas the toxicity of EPO alone was inhibited only by heparin. Heparin also inhibited killing by both MBP and ECP. Despite the homology of ECP with certain RNases, placental RNasin, an RNase inhibitor, was unable to inhibit ECP-mediated toxicity. These results indicate that all of the eosinophil granule proteins are toxic to mf and they support the hypothesis that eosinophil degranulation causes death of mf in vivo.     

In Silico and In Vitro Studies on the Protein-Protein Interactions between Brugia malayi Immunomodulatory Protein Calreticulin and Human C1q

Filarial parasites modulate effective immune response of their host by releasing a variety of immunomodulatory molecules, which help in the long persistence of the parasite within the host. The present study was aimed to characterize an immunomodulatory protein of Brugia malayi and its interaction with the host immune component at the structural and functional level. Our findings showed that Brugia malayi Calreticulin (BmCRT) is responsible for the prevention of classical complement pathway activation via its interaction with the first component C1q of the human host. This was confirmed by inhibition of C1q dependent lysis of immunoglobulin-sensitized Red Blood Cells (S-RBCs). This is possibly the first report which predicts CRT-C1q interaction on the structural content of proteins to explain how BmCRT inhibits this pathway. The molecular docking of BmCRT-C1q complex indicated that C1qB chain (IgG/M and CRP binding sites on C1q) played a major role in the interaction with conserved and non-conserved regions of N and P domain of BmCRT. Out of 37 amino acids of BmCRT involved in the interaction, nine amino acids (Pro¹²⁶, Glu¹³², His¹⁴⁷, Arg¹⁵¹, His¹⁵³, Met¹⁵⁴, Lys¹⁵⁶, Ala¹⁹⁶ and Lys²¹²) are absent in human CRT. Both ELISA and in silico analysis showed the significant role of Ca⁺² in BmCRT-HuC1q complex formation and deactivation of C1r₂–C1s₂. Molecular dynamics studies of BmCRT-HuC1q complex showed a deviation from ∼0.4 nm to ∼1.0 nm. CD analyses indicated that BmCRT is composed of 49.6% α helix, 9.6% β sheet and 43.6% random coil. These findings provided valuable information on the architecture and chemistry of BmCRT-C1q interaction and supported the hypothesis that BmCRT binds with huC1q at their targets (IgG/M, CRP) binding sites. This interaction enables the parasite to interfere with the initial stage of host complement activation, which might be helpful in parasites establishment. These results might be utilized for help in blocking the C1q/CRT interaction and preventing parasite infection.     

Functional Analysis of the Cathepsin-Like Cysteine Protease Genes in Adult Brugia malayi Using RNA Interference

Background

Cathepsin-like enzymes have been identified as potential targets for drug or vaccine development in many parasites, as their functions appear to be essential in a variety of important biological processes within the host, such as molting, cuticle remodeling, embryogenesis, feeding and immune evasion. Functional analysis of Caenorhabditis elegans cathepsin L (Ce-cpl-1) and cathepsin Z (Ce-cpz-1) has established that both genes are required for early embryogenesis, with Ce-cpl-1 having a role in regulating in part the processing of yolk proteins. Ce-cpz-1 also has an important role during molting.

Methods and Findings

RNA interference assays have allowed us to verify whether the functions of the orthologous filarial genes in Brugia malayi adult female worms are similar. Treatment of B. malayi adult female worms with Bm-cpl-1, Bm-cpl-5, which belong to group Ia of the filarial cpl gene family, or Bm-cpz-1 dsRNA resulted in decreased numbers of secreted microfilariae in vitro. In addition, analysis of the intrauterine progeny of the Bm-cpl-5 or Bm-cpl Pro dsRNA- and siRNA-treated worms revealed a clear disruption in the process of embryogenesis resulting in structural abnormalities in embryos and a varied differential development of embryonic stages.

Conclusions

Our studies suggest that these filarial cathepsin-like cysteine proteases are likely to be functional orthologs of the C. elegans genes. This functional conservation may thus allow for a more thorough investigation of their distinct functions and their development as potential drug targets.     

Effect of CGP 20376 on Brugia malayi and parasite antigenemia in jirds

This study was designed to investigate the activity of CGP 20376, a benzothiazole derivative, against Brugia malayi in jirds and to illustrate the utility of parasite antigen detection as a means of monitoring drug efficacy in filariasis. Drug treatment was 100% effective in jirds treated 3 or 24 days after infection. Microfilaria and adult worm counts were reduced (relative to counts in sham-treated control animals) by 96% and 95%, respectively, in animals treated 153 days after infection. Four of 6 animals in this treatment group cleared their microfilaremias and were free of adult worms 5 mo after treatment. Thus, CGP 20376 was effective against all life cycle stages of B. malayi in jirds. Parasite antigen levels in jird sera were consistent with parasitological results in all treatment groups, but antigen clearance was incomplete in some cases after apparently successful treatment of mature and immature infections.     

Adult 175 kDa collagenase antigen of Setaria cervi in immunoprophylaxis against Brugia malayi in jirds

A 175 kDa antigen fraction with collagenase activity was isolated and purified from somatic extracts of adult Setaria cervi females using column chromatography involving consecutive steps of DEAE-Sepharose CL6B and Sephadex G-100. The optimum pH for 175 kDa collagenase was found to be pH 7.0. Sensitivities to a variety of inhibitors and activators indicated that the 175 kDa coIlagenolytic enzyme was metalloserine in nature. The enzyme hydrolysed a variety of protein substrates such as haemoglobin, casein, azocasein (general substrates) and collagen, FALGPA (furanoyl-acryloyl-leu-gly-pro-ala), the specific substrate of collagenase. The enzyme showed 57% inhibition by jird anti-somatic collagenase antibodies and reacted insignificantly with normal jird sera. Further analysis was undertaken on the immunoprophylactic potential of 175 kDa collagenase in inducing immunity against Brugia malayi (a human filarial parasite) in jirds (Meriones unguiculatus) in vitro and in situ. Immune sera of jirds raised against this antigen promoted partial adherence of peritoneal exudate cells to B. malayi microfilariae (mf) and infective larvae (L3) in vitro and induced partial cytotoxicity to the parasites within 48 h. The anti-S. cervi 175 kDa antigen serum was more effective in inducing cytotoxicity to B. malayi L3, than mf. In the microchambers implanted inside immune jirds, host cells could migrate and adhere to the mf and infective larvae thereby killing them partially within 48 h.     

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.     

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.     

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 survey of Brugia malayi infection on the Heugsan Islands,Korea

Lymphatic filariasis due to Brugia malayi infection was endemic in several areas of South Korea. The infection was controlled, or disappeared, in most areas, with the exception of the remote southwestern islands of Jeonranam-do, including the Heugsan Islands. To discover its current situation, a small-scale survey was performed on the Heugsan Islands in September 2000. A total of 378 people, 151 male and 227 female, living in 8 villages (6 on Daeheugsan-do, 1 on Daejang-do, and 1 on Yeongsan-do) were subjected to a night blood survey for microfilaremia, and physical examination for elephantiasis on the extremities. There were 6 (1.6%) microfilaria positive cases, all in females aged 57-72 years, and from only two villages of the Daeheugsan-do area. There were 4 patients with lower leg elephantiasis, but they showed no microfilaremia. The results show that a low-grade endemicity of filariasis remains on the Daeheugsan-do.     

Exome and Transcriptome Sequencing of Aedes aegypti Identifies a Locus That Confers Resistance to Brugia malayi and Alters the Immune Response

Many mosquito species are naturally polymorphic for their abilities to transmit parasites, a feature which is of great interest for controlling vector-borne disease. Aedes aegypti, the primary vector of dengue and yellow fever and a laboratory model for studying lymphatic filariasis, is genetically variable for its capacity to harbor the filarial nematode Brugia malayi. The genome of Ae. aegypti is large and repetitive, making genome resequencing difficult and expensive. We designed exome captures to target protein-coding regions of the genome, and used association mapping in a wild Kenyan population to identify a single, dominant, sex-linked locus underlying resistance. This falls in a region of the genome where a resistance locus was previously mapped in a line established in 1936, suggesting that this polymorphism has been maintained in the wild for the at least 80 years. We then crossed resistant and susceptible mosquitoes to place both alleles of the gene into a common genetic background, and used RNA-seq to measure the effect of this locus on gene expression. We found evidence for Toll, IMD, and JAK-STAT pathway activity in response to early stages of B. malayi infection when the parasites are beginning to die in the resistant genotype. We also found that resistant mosquitoes express anti-microbial peptides at the time of parasite-killing, and that this expression is suppressed in susceptible mosquitoes. Together, we have found that a single resistance locus leads to a higher immune response in resistant mosquitoes, and we identify genes in this region that may be responsible for this trait.     

The Secreted Triose Phosphate Isomerase of Brugia malayi Is Required to Sustain Microfilaria Production In Vivo

Human lymphatic filariasis is a major tropical disease transmitted through mosquito vectors which take up microfilarial larvae from the blood of infected subjects. Microfilariae are produced by long-lived adult parasites, which also release a suite of excretory-secretory products that have recently been subject to in-depth proteomic analysis. Surprisingly, the most abundant secreted protein of adult Brugia malayi is triose phosphate isomerase (TPI), a glycolytic enzyme usually associated with the cytosol. We now show that while TPI is a prominent target of the antibody response to infection, there is little antibody-mediated inhibition of catalytic activity by polyclonal sera. We generated a panel of twenty-three anti-TPI monoclonal antibodies and found only two were able to block TPI enzymatic activity. Immunisation of jirds with B. malayi TPI, or mice with the homologous protein from the rodent filaria Litomosoides sigmodontis, failed to induce neutralising antibodies or protective immunity. In contrast, passive transfer of neutralising monoclonal antibody to mice prior to implantation with adult B. malayi resulted in 60–70% reductions in microfilarial levels in vivo and both oocyte and microfilarial production by individual adult females. The loss of fecundity was accompanied by reduced IFNγ expression by CD4⁺ T cells and a higher proportion of macrophages at the site of infection. Thus, enzymatically active TPI plays an important role in the transmission cycle of B. malayi filarial parasites and is identified as a potential target for immunological and pharmacological intervention against filarial infections.     

Identification of Brugia malayi immunogens by an immunoproteomics approach

Filariasis remains a health problem in tropical countries. Identification of immunogens from its causative organism would lead to development of a better diagnostic test, as well as vaccine discovery to effectively prevent this disease. We applied immunoproteomics to define potential immunogens of adult Brugia malayi that were recognized by IgM, IgG1 and IgG4 in sera of patients with four distinct clinical spectra of filariasis, including endemic asymptomatic, lymphangitis, elephantiasis and microfilaremia (n=5/group). Sera of healthy individuals (n=5) from non-endemic area served as the negative control. Brugian proteins were resolved by 2-DE and subjected to 2-D Western blot analysis probed with these sera. A total of 30 immunoreactive proteins recognized by IgM, IgG1 and IgG4 in sera from all four filarial groups were identified by Q-TOF MS and MS/MS analyses. Interestingly, only three immunogens were recognized by IgM in lymphangitis, elephantiasis and microfilaremia, but not in endemic asymptomatic group. IgG1 recognized 20 immunogens in endemic asymptomatic, lymphangitis and microfilaremia (mostly in endemic asymptomatic group), but not in elephantiasis, whereas IgG4 recognized 28 immunogens in all four filarial groups (mostly in microfilaremia). This large data set is an important resource for further development of a new diagnostic test and/or vaccine for filariasis.     

The genome of Brugia malayi - all worms are not created equal

Filarial nematode parasites, the causative agents of elephantiasis and river blindness, undermine the livelihoods of over one hundred million people in the developing world. Recently, the Filarial Genome Project reported the draft sequence of the ~95 Mb genome of the human filarial parasite Brugia malayi - the first parasitic nematode genome to be sequenced. Comparative genome analysis with the prevailing model nematode Caenorhabditis elegans revealed similarities and differences in genome structure and organization that will prove useful as additional nematode genomes are completed. The Brugia genome provides the first opportunity to comprehensively compare the full gene repertoire of a free-living nematode species and one that has evolved as a human pathogen. The Brugia genome also provides an opportunity to gain insight into genetic basis for mutualism, as Brugia, like a majority of filarial species, harbors an endosybiotic bacterium (Wolbachia). The goal of this review is to provide an overview of the results of genomic analysis and how these observations provide new insights into the biology of filarial species.