Stage-specific proteomic expression patterns of the human filarial parasiteBrugia malayiand its endosymbiontWolbachia

Abstract

Global proteomic analyses of pathogens have thus far been limited to unicellular organisms (e.g., protozoa and bacteria). Proteomic analyses of most eukaryotic pathogens (e.g., helminths) have been restricted to specific organs, specific stages, or secretomes. We report here a large-scale proteomic characterization of almost all the major mammalian stages of Brugia malayi, a causative agent of lymphatic filariasis, resulting in the identification of more than 62% of the products predicted from the Bm draft genome. The analysis also yielded much of the proteome of Wolbachia, the obligate endosymbiont of Bm that also expressed proteins in a stage-specific manner. Of the 11,610 predicted Bm gene products, 7,103 were definitively identified from adult male, adult female, blood-borne and uterine microfilariae, and infective L3 larvae. Among the 4,956 gene products (42.5%) inferred from the genome as "hypothetical," the present study was able to confirm 2,336 (47.1%) as bona fide proteins. Analysis of protein families and domains coupled with stage-specific expression highlight the important pathways that benefit the parasite during its development in the host. Gene set enrichment analysis identified extracellular matrix proteins and those with immunologic effects as enriched in the microfilarial and L3 stages. Parasite sex-and stage-specific protein expression identified those pathways related to parasite differentiation and demonstrates stage-specific expression by the Bm endosymbiont Wolbachia as well.filaria | nematode D isease associated with infection by Brugia malayi (Bm) and Wuchereria bancrofti, the two major causative organisms of human lymphatic filariasis, is the second leading cause of morbidity/disability worldwide, in large part because of the parasites' ability to alter the structural and functional integrity of the lymphatics, leading to lymphedema and elephantiasis. Invasion, establishment of infection within the host and development are essential processes within the complex parasite life cycle (SI Appendix, Fig. S1), with many of the parasitic stages being targets for therapeutic intervention or vaccines. Each of the filarial life cycle stages has characteristics that are shared and others that are stage-specific.Filarial infections are often characterized by a series of discrete host responses directed at the parasite and its endosymbiont Wolbachia that evolve during the course of infection. Because proteins are usually the effectors of most biological functions, proteomic data enable a more direct understanding of these important processes compared with those inferred from genomic studies. Absolute quantification of genome-wide expressed proteins is not yet within our reach for most eukaryotes. However, spectral counts of massive MS-based data (e.g., observed frequencies of each peptide) allow for relative quantification. Proteomic data also allow for clearer genomic curation by improving annotation and the identification of translational sites, stop codon read-throughs, frame shifts, and predict...

Quiescent Innate Response to Infective Filariae by Human Langerhans Cells Suggests a Strategy of Immune Evasion

“…Both TLR3 and TLR4 have been shown previously to be functionally repressed by the mf stage of B. malayi (12), a stage that shares some (but not all) proteins with the L3 (30), while TLR2 was activated by mf (12). The finding that L3 exposure failed to alter TLR expression or responses to TLR ligands in LC points to the concept of parasite stage specificity (30) and the anatomical location and/or specificity of LC (12).…”

“…Using proteomics to identify the L3 secretome, only relatively few secreted proteins have been identified: several thioredoxin peroxidases known to neutralize host reactive oxygen (33). Among the nonsecreted proteins that may modulate the host immune response is a TGF-␤ homologue (TGH-2) that was found to be expressed primarily in the L3 stage of the parasite and that may mimic human TGF-␤ (30). Studies are ongoing in our laboratory to identify additional immunomodulatory proteins that are parasite derived.…”

“…To date, there are no data on the receptors required for L3 antigen internalization or recognition. A homologue of Acanthocheilonema viteae-derived ES-62, is expressed in molting L3 (30) of B. malayi and is recognized by TLR4 (32), a TLR known to be expressed on LC that may represent a target for antigen internalization by LC. Using proteomics to identify the L3 secretome, only relatively few secreted proteins have been identified: several thioredoxin peroxidases known to neutralize host reactive oxygen (33).…”

Effects of soluble extracts from Leishmania infantum promastigotes, Toxoplasma gondii tachyzoites on TGF-β mediated pathways in activated CD4+ T lymphocytes

“…In malaria infection accessory cells engulfed with parasites as well as activated T lymphocytes produce high amounts of TGF-b and IL-10 which might easily induce the conversion of bystander effector T cells to the regulatory phenotype [11]. Furthermore several parasites directly interfere with TGF-b mediated pathway to subvert protective immune responses: a homolog of human TGF-b is produced by most filarial parasites [12,13] while enzymatic molecules activating latent TGF-b are produced by Plasmodium falciparum, Leishmania chagasi, and by the intestinal helminth parasite Heligmosomoides polygyrus [14e16]. Activation of latent TGF-b on the surface of activated T cells is a requirement for triggering signals from TGF-b receptor which regulate the expression of Foxp3 gene, the master regulator of T regulatory cell function [17,18].…”

Analysis of gene expression from theWolbachiagenome of a filarial nematode supports both metabolic and defensive roles within the symbiosis

“…To perform a comparison of the predominant protein composition in wOo and wBm, we ranked Wolbachia proteins by the number of unique peptides detected in adult female worms of B. malayi (Bennuru et al 2011) and O. ochengi (Supplemental Table S11). Remarkably, this rank ordering showed substantial differences, even on the simple measure of presence in the top 20; although GroEL, DnaK, a single ortholog of Wolbachia surface protein (WSP; wOo07430/wBm0432), and a putative outer membrane protein (wOo08110/wBm0010) predicted to be a porin (Supplemental Table S14) clearly dominated the proteome in both strains.…”