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

Microarray-Based Analysis of Differential Gene Expression between Infective and Noninfective Larvae of Strongyloides stercoralis

Strongyloides stercoralis is a soil-transmitted helminth that affects an estimated 30–100 million people worldwide. Chronically infected persons who are exposed to corticosteroids can develop disseminated disease, which carries a high mortality (87–100%) if untreated. Despite this, little is known about the fundamental biology of this parasite, including the features that enable infection. We developed the first DNA microarray for this parasite and used it to compare infective third-stage larvae (L3i) with non-infective first stage larvae (L1). Using this method, we identified 935 differentially expressed genes. Functional characterization of these genes revealed L3i biased expression of heat shock proteins and genes with products that have previously been shown to be immunoreactive in infected humans. Genes putatively involved in transcription were found to have L1 biased expression. Potential chemotherapeutic and vaccine targets such as far-1, ucr 2.1 and hsp-90 were identified for further study

Stage- and Gender-Specific Proteomic Analysis of Brugia malayi Excretory-Secretory Products

To succeed in infection, parasites must have ways to reach the host, penetrate its tissues and escape its defense systems. As they are not necessarily fatal, most helminth parasites remain viable within their host for many years, exerting a strong influence over the host immune function. Many of these functions are performed by products that are released from the parasite. We exploited the remarkable sensitivity of modern proteomics tools together with the availability of a sequenced genome to identify and compare the proteins released in vitro by adult males, adult females and the microfilariae of the filarial nematode Brugia malayi. This parasite is one of the etiological agents of lymphatic filariasis, a disease that poses continuing and significant threats to human health. The different forms of the parasite inhabit different compartments in the mammalian host. We found that the set of proteins released by each form is unique; they must reflect particular developmental processes and different strategies for evasion of host responses. The identification of these proteins will allow us to illuminate the biology of secretory processes in this organism and to establish a path for developing an understanding of how these parasite proteins function in immune evasion events

Helminth immunoregulation: The role of parasite secreted proteins in modulating host immunity

Helminths are masterful immunoregulators. A characteristic feature of helminth infection is a Th2-dominated immune response, but stimulation of immunoregulatory cell populations, such as regulatory T cells and alternatively activated macrophages, is equally common. Typically, Th1/17 immunity is blocked and productive effector responses are muted, allowing survival of the parasite in a “modified Th2” environment. Drug treatment to clear the worms reverses the immunoregulatory effects, indicating that a state of active suppression is maintained by the parasite. Hence, research has focussed on “excretory–secretory” products released by live parasites, which can interfere with every aspect of host immunity from initial recognition to end-stage effector mechanisms. In this review, we survey our knowledge of helminth secreted molecules, and summarise current understanding of the growing number of individual helminth mediators that have been shown to target key receptors or pathways in the mammalian immune system

Erythrocyte Invasion by Babesia bovis Merozoites Is Inhibited by Polyclonal Antisera Directed against Peptides Derived from a Homologue of Plasmodium falciparum Apical Membrane Antigen 1

Apical membrane antigen 1 (AMA-1) is a micronemal protein secreted to the surface of merozoites of Plasmodium species and Toxoplasma gondii tachyzoites in order to fulfill an essential but noncharacterized function in host cell invasion. Here we describe cloning and characterization of a Babesia bovis AMA-1 homologue designated BbAMA-1. The overall level of similarity of BbAMA-1 to P. falciparum AMA-1 was low (18%), but characteristic features like a transmembrane domain near the C terminus, a predicted short cytoplasmic C-terminal sequence with conserved sequence properties, and an extracellular domain containing 14 conserved cysteine residues putatively involved in disulfide bridge formation are typical of AMA-1. Rabbit polyclonal antisera were raised against three synthetic peptides derived from the N-terminal region and domains II and III of the putative extracellular domain and were shown to recognize specifically recombinant BbAMA-1 expressed in Escherichia coli. Immunofluorescence microscopy showed that there was labeling of the apical half of merozoites with these antisera. Preincubation of free merozoites with all three antisera reduced the efficiency of invasion of erythrocytes by a maximum of 65%. Antisera raised against the N-terminal peptide detected a 82-kDa protein on Western blots and a 69-kDa protein in the supernatant that was harvested after in vitro invasion, suggesting that proteolytic processing and secretion take place during or shortly after invasion. A combination of two-dimensional Western blotting and metabolic labeling allowing direct identification of spots reacting with the BbAMA-1 peptide antisera together with the very low silver staining intensity of these spots indicated that very low levels of BbAMA-1 are present in Babesia merozoites