Loa loa, the African eyeworm, is a major filarial pathogen of humans. Unlike most filariae, Loa loa does not contain the obligate intracellular Wolbachia endosymbiont. Over the past four years, we were able to generated a 20x draft genome sequence of Loa loa, and a 12x draft genome of the related filarial parasite Wuchereria bancrofti for comparative purposes. The L. loa genome is 91.4 Mb with 14,907 predicted genes. By comparing its genome to those of two other pathogenic human filarial parasites, Wuchereria bancrofti and Brugia malayi, and to several non-parasitic nematode genomes, we were able to demonstrate conservation of gene order (synteny) is highly conserved between L. loa and the related parasites W. bancrofti and Brugia malayi but to the other non-filarial nematodes (see illustration below). The L. loa genome is enriched for numerous gene classes relative to the other filarial parasites, including zinc-finger transcription factors and chemosensors. Using orthology to C. elegans, we profiled the metabolic biosynthesis and transport capabilities of the three filarial worms, in addition to five other free-living and parasitic nematodes. All nematodes showed remarkable conservation of metabolic pathways, with the exception of purine biosynthesis which has been lost independently in multiple lineages. Despite lacking intracellular Wolbachia, Loa loa shows no new metabolic synthesis or transport capabilities relative to the other filarial parasites, and no evidence of extensive lateral gene transfer from Wolbachia or from other bacteria. These results suggest the metabolic role of Wolbachia symbionts is likely more subtle than previously thought and reveal marked differences between parasitic and non-parasitic nematodes. By using whole genome sequencing of single adult male parasites of O. volvulus and W. bancrofti, we were able to accomplish 2 additional important goals. The first was to obtain complete sequences of the Wolbachia of O. volvulus and W. bancrofti, analyses of these genomes being in progress. Secondly, by using single adult parasites we were able to generate allelic differences (SNPs) across the entire genomes of these 2 parasites, information that has set the stage for studies in population biology of W. bancrofti and for studies of drug resistance in O. volvulus. We extended this initial proteomic study to a large-scale proteomic characterization of almost all the major mammalian stages of Brugia malayi, resulting in the identification of more than 62% of the products predicted from the Brugia malayi draft genome. The analysis also yielded much of the proteome of Wolbachia, the obligate endosymbiont of Bm. 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 demonstrated stage-specific expression by the Bm endosymbiont Wolbachia as well. To understand better the developmental programs that underscore the transition between the mosquito-derived infective stage larvae (L3) to mammalian adapted L3s and to L4s following a molt, and the initial week of adaptation to the human host, we adapted an in vitro system that allowed for L3 development and subsequent molting to the L4. Using microarray and proteomic assessments at multiple times through this 9 day process we have not only identified those genes/pathways that are critical for the L3/L4 transition but we have also demonstrated by both pharmacologic inhibition (cysteine protease inhibition) and RNAi (of the critical CPLs) the critical role played by cysteine proteases in the early development of mammalian adapted L3s to L4s In that S stercoralis is a major pathogen in both normal and immunocompromised hosts and very little is known, at the molecular level, about its makeup. Having previously provided cDNA libraries both the filariform and rhabditiform larvae that formed the core of the Nematode EST project (based at Washington University at St. Louis), we were able to manufacture microarrays comprising all 3571 clusters from first and infective third stage larvae (L1, L3i). We have since performed two important set of analyses, the first comparing L1 to L3i that identified the major transcriptional differences between infective and noninfective S. stercoralis larvae 29 that identified potential therapeutic and vaccine targets that were then tested experimentally 30. The second set of gene expression studies compared infective third stage larvae before (L3i) and 72 hours after (L3+) host invasion and showed that S. stercoralis larvae markedly downregulate expression of extracellular matrix and energy metabolism genes, and increase expression of genes encoding catalytic enzymes providing important clues toward an understanding of how Ss establishes itself following host invasion. From a pool of over 1,800 L. loa microfilaria (mf) expressed sequence tags, 18 candidate L. loa (Ll) mf-specific PCR targets were identified. Real-time PCR (qPCR) assays were developed for two targets (LLMF72 and LLMF269) and these have been used in the quantitative assessment of Loa loa microfilarial levels. A similar approach was used to assemble into contigs 2048 expressed sequence tags (ESTs) from the L3 infective larvae of W. bancrofti (Wb) that were these were next assessed for homology to known proteins and nucleotides and to similar assemblies of L3 larval ESTs of B. malayi (n = 5068), O. volvulus (n = 4166), and L. loa (n = 3315). Nineteen potential L3- and Wb- -specific antigens were identified and 1, termed Wb123 has been used both as a rapid, high throughput tool to diagnose individual Wb infections and as a sensitive method for early detection of recrudescent infections in areas of control and for mapping new areas of Wb transmission.

Project Start
Project End
Budget Start
Budget End
Support Year
26
Fiscal Year
2013
Total Cost
$533,574
Indirect Cost
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State
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