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

Lack of Evidence for the Direct Activation of Endothelial Cells by Adult Female and Microfilarial Excretory-Secretory Products

Lymphangiectasia (dilation of the lymphatic vessel (LV)) is pathognomonic for lymphatic filariasis. In both infected humans and animal models of infection, lymphangiectasia is not restricted to the site of the worm nest, but is found along the infected vessel. These observations argue that soluble products secreted by the worm could be mediating this effect by activating the lymphatic endothelial cells (LEC) lining the vessel. We tested the ability of filarial Excretory-Secretory products to activate LECs, but were unable to detect a direct effect of the Excretory-Secretory products on the activation of LEC as assessed by a variety of approaches including cellular proliferation, cell surface molecule expression and cytokine and growth factor production (although other mediators used as positive controls did induce these effects). Collectively, these results do not support the hypothesis that Excretory-Secretory products directly activate LECs

Unraveling cross-reactivity of anti-glycan IgG responses in filarial nematode infections

Parasitic nematodes responsible for filarial diseases cause chronic disablement in humans worldwide. Elimination programs have substantially reduced the rate of infection in certain areas, but limitations of current diagnostics for population surveillance have been pointed out and improved assays are needed to reach the elimination targets. While serological tests detecting antibodies to parasite antigens are convenient tools, those currently available are compromised by the occurrence of antibodies cross-reactive between nematodes, as well as by the presence of residual antibodies in sera years after treatment and clearance of the infection. We recently characterized the N-linked and glycosphingolipid derived glycans of the parasitic nematode Brugia malayi and revealed the presence of various antigenic structures that triggered immunoglobulin G (IgG) responses in infected individuals. To address the specificity of IgG binding to these glycan antigens, we screened microarrays containing Brugia malayi glycans with plasma from uninfected individuals and from individuals infected with Loa loa, Onchocerca volvulus, Mansonella perstans and Wuchereria bancrofti, four closely related filarial nematodes. IgG to a restricted subset of cross-reactive glycans was observed in infection plasmas from all four species. In plasma from Onchocerca volvulus and Mansonella perstans infected individuals, IgG binding to many more glycans was additionally detected, resulting in total IgG responses similar to the ones of Brugia malayi infected individuals. For these infection groups, Brugia malayi, Onchocerca volvulus and Mansonella perstans, we further studied the different IgG subclasses to Brugia malayi glycans. In all three infections, IgG1 and IgG2 appeared to be the major subclasses involved in response to glycan antigens. Interestingly, in Brugia malayi infected individuals, we observed a marked reduction in particular in IgG2 to parasite glycans post-treatment with anthelminthic, suggesting a promising potential for diagnostic applications. Thus, we compared the IgG response to a broad repertoire of Brugia malayi glycans in individuals infected with various filarial nematodes. We identified broadly cross-reactive and more specific glycan targets, extending the currently scarce knowledge of filarial nematode glycosylation and host anti-glycan antibody response. We believe that our initial findings could be further exploited to develop disease-specific diagnostics as part of an integrated approach for filarial disease control.Host-parasite interactio

Solution structure of a repeated unit of the ABA-1 nematode polyprotein allergen of ascaris reveals a novel fold and two discrete lipid-binding sites

Parasitic nematode worms cause serious health problems in humans and other animals. They can induce allergic-type immune responses, which can be harmful but may at the same time protect against the infections. Allergens are proteins that trigger allergic reactions and these parasites produce a type that is confined to nematodes, the nematode polyprotein allergens (NPAs). These are synthesized as large precursor proteins comprising repeating units of similar amino acid sequence that are subsequently cleaved into multiple copies of the allergen protein. NPAs bind small lipids such as fatty acids and retinol (Vitamin A) and probably transport these sensitive and insoluble compounds between the tissues of the worms. Nematodes cannot synthesize these lipids, so NPAs may also be crucial for extracting nutrients from their hosts. They may also be involved in altering immune responses by controlling the lipids by which the immune and inflammatory cells communicate. We describe the molecular structure of one unit of an NPA, the well-known ABA-1 allergen of Ascaris and find its structure to be of a type not previously found for lipid-binding proteins, and we describe the unusual sites where lipids bind within this structur

Lymphangiogenesis and Lymphatic Remodeling Induced by Filarial Parasites: Implications for Pathogenesis

Even in the absence of an adaptive immune system in murine models, lymphatic dilatation and dysfunction occur in filarial infections, although severe irreversible lymphedema and elephantiasis appears to require an intact adaptive immune response in human infections. To address how filarial parasites and their antigens influence the lymphatics directly, human lymphatic endothelial cells were exposed to filarial antigens, live parasites, or infected patient serum. Live filarial parasites or filarial antigens induced both significant LEC proliferation and differentiation into tube-like structures in vitro. Moreover, serum from patently infected (microfilaria positive) patients and those with longstanding chronic lymphatic obstruction induced significantly increased LEC proliferation compared to sera from uninfected individuals. Differentiation of LEC into tube-like networks was found to be associated with significantly increased levels of matrix metalloproteases and inhibition of their TIMP inhibitors (Tissue inhibitors of matrix metalloproteases). Comparison of global gene expression induced by live parasites in LEC to parasite-unexposed LEC demonstrated that filarial parasites altered the expression of those genes involved in cellular organization and development as well as those associated with junction adherence pathways that in turn decreased trans-endothelial transport as assessed by FITC-Dextran. The data suggest that filarial parasites directly induce lymphangiogenesis and lymphatic differentiation and provide insight into the mechanisms underlying the pathology seen in lymphatic filariasis

Genome-wide analysis of ivermectin response by Onchocerca volvulus reveals that genetic drift and soft selective sweeps contribute to loss of drug sensitivity

Treatment of onchocerciasis using mass ivermectin administration has reduced morbidity and transmission throughout Africa and Central/South America. Mass drug administration is likely to exert selection pressure on parasites, and phenotypic and genetic changes in several Onchocerca volvulus populations from Cameroon and Ghana-exposed to more than a decade of regular ivermectin treatment-have raised concern that sub-optimal responses to ivermectin's anti-fecundity effect are becoming more frequent and may spread.Pooled next generation sequencing (Pool-seq) was used to characterise genetic diversity within and between 108 adult female worms differing in ivermectin treatment history and response. Genome-wide analyses revealed genetic variation that significantly differentiated good responder (GR) and sub-optimal responder (SOR) parasites. These variants were not randomly distributed but clustered in ~31 quantitative trait loci (QTLs), with little overlap in putative QTL position and gene content between the two countries. Published candidate ivermectin SOR genes were largely absent in these regions; QTLs differentiating GR and SOR worms were enriched for genes in molecular pathways associated with neurotransmission, development, and stress responses. Finally, single worm genotyping demonstrated that geographic isolation and genetic change over time (in the presence of drug exposure) had a significantly greater role in shaping genetic diversity than the evolution of SOR.This study is one of the first genome-wide association analyses in a parasitic nematode, and provides insight into the genomics of ivermectin response and population structure of O. volvulus. We argue that ivermectin response is a polygenically-determined quantitative trait (QT) whereby identical or related molecular pathways but not necessarily individual genes are likely to determine the extent of ivermectin response in different parasite populations. Furthermore, we propose that genetic drift rather than genetic selection of SOR is the underlying driver of population differentiation, which has significant implications for the emergence and potential spread of SOR within and between these parasite populations

Attempts to Image the Early Inflammatory Response during Infection with the Lymphatic Filarial Nematode Brugia pahangi in a Mouse Model

Helminth parasites remain a major constraint upon human health and well-being in many parts of the world. Treatment of these infections relies upon a very small number of therapeutics, most of which were originally developed for use in animal health. A lack of high throughput screening systems, together with limitations of available animal models, has restricted the development of novel chemotherapeutics. This is particularly so for filarial nematodes, which are long-lived parasites with a complex cycle of development. In this paper, we describe attempts to visualise the immune response elicited by filarial parasites in infected mice using a non-invasive bioluminescence imaging reagent, luminol, our aim being to determine whether such a model could be developed to discriminate between live and dead worms for in vivo compound screening. We show that while imaging can detect the immune response elicited by early stages of infection with L3, it was unable to detect the presence of adult worms or, indeed, later stages of infection with L3, despite the presence of worms within the lymphatic system of infected animals. In the future, more specific reagents that detect secreted products of adult worms may be required for developing screens based upon live imaging of infected animals

Circulating Microbial Products and Acute Phase Proteins as Markers of Pathogenesis in Lymphatic Filarial Disease

Lymphatic filariasis can be associated with development of serious pathology in the form of lymphedema, hydrocele, and elephantiasis in a subset of infected patients. Dysregulated host inflammatory responses leading to systemic immune activation are thought to play a central role in filarial disease pathogenesis. We measured the plasma levels of microbial translocation markers, acute phase proteins, and inflammatory cytokines in individuals with chronic filarial pathology with (CP Ag+) or without (CP Ag−) active infection; with clinically asymptomatic infections (INF); and in those without infection (endemic normal [EN]). Comparisons between the two actively infected groups (CP Ag+ compared to INF) and those without active infection (CP Ag− compared to EN) were used preliminarily to identify markers of pathogenesis. Thereafter, we tested for group effects among all the four groups using linear models on the log transformed responses of the markers. Our data suggest that circulating levels of microbial translocation products (lipopolysaccharide and LPS-binding protein), acute phase proteins (haptoglobin and serum amyloid protein-A), and inflammatory cytokines (IL-1β, IL-12, and TNF-α) are associated with pathogenesis of disease in lymphatic filarial infection and implicate an important role for circulating microbial products and acute phase proteins

Integrated transcriptomic and proteomic analysis of the global response of Wolbachia to doxycycline-induced stress

The bacterium Wolbachia (order Rickettsiales), representing perhaps the most abundant vertically transmitted microbe worldwide, infects arthropods and filarial nematodes. In arthropods, Wolbachia can induce reproductive alterations and interfere with the transmission of several arthropod-borne pathogens. In addition, Wolbachia is an obligate mutualist of the filarial parasites that cause lymphatic filariasis and onchocerciasis in the tropics. Targeting Wolbachia with tetracycline antibiotics leads to sterilisation and ultimately death of adult filariae. However, several weeks of treatment are required, restricting the implementation of this control strategy. To date, the response of Wolbachia to stress has not been investigated, and almost nothing is known about global regulation of gene expression in this organism. We exposed an arthropod Wolbachia strain to doxycycline in vitro, and analysed differential expression by directional RNA-seq and label-free, quantitative proteomics. We found that Wolbachia responded not only by modulating expression of the translation machinery, but also by upregulating nucleotide synthesis and energy metabolism, while downregulating outer membrane proteins. Moreover, Wolbachia increased the expression of a key component of the twin-arginine translocase (tatA) and a phosphate ABC transporter ATPase (PstB); the latter is associated with decreased susceptibility to antimicrobials in free-living bacteria. Finally, the downregulation of 6S RNA during translational inhibition suggests that this small RNA is involved in growth rate control. Despite its highly reduced genome, Wolbachia shows a surprising ability to regulate gene expression during exposure to a potent stressor. Our findings have general relevance for the chemotherapy of obligate intracellular bacteria and the mechanistic basis of persistence in the Rickettsiales

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

Wolbachia are required for filarial nematode survival and fertility and contribute to the immune responses associated with human filarial diseases. Here we developed whole-mount immunofluorescence techniques to characterize Wolbachia somatic and germline transmission patterns and tissue distribution in Brugia malayi, a nematode responsible for lymphatic filariasis. In the initial embryonic divisions, Wolbachia segregate asymmetrically such that they occupy only a small subset of cells in the developing embryo, facilitating their concentration in the adult hypodermal chords and female germline. Wolbachia are not found in male reproductive tissues and the absence of Wolbachia from embryonic germline precursors in half of the embryos indicates Wolbachia loss from the male germline may occur in early embryogenesis. Wolbachia rely on fusion of hypodermal cells to populate adult chords. Finally, we detect Wolbachia in the secretory canal lumen suggesting living worms may release bacteria and/or their products into their host