Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachia's principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease.

Parasitic nematodes that cause elephantiasis and river blindness threaten hundreds of millions of people in the developing world. We have sequenced the approximately 90 megabase (Mb) genome of the human filarial parasite Brugia malayi and predict approximately 11,500 protein coding genes in 71 Mb of robustly assembled sequence. Comparative analysis with the free-living, model nematode Caenorhabditis elegans revealed that, despite these genes having maintained little conservation of local synteny during approximately 350 million years of evolution, they largely remain in linkage on chromosomal units. More than 100 conserved operons were identified. Analysis of the predicted proteome provides evidence for adaptations of B. malayi to niches in its human and vector hosts and insights into the molecular basis of a mutualistic relationship with its Wolbachia endosymbiont. These findings offer a foundation for rational drug design.

Although common among bacteria, lateral gene transfer-the movement of genes between distantly related organisms-is thought to occur only rarely between bacteria and multicellular eukaryotes. However, the presence of endosymbionts, such as Wolbachia pipientis, within some eukaryotic germlines may facilitate bacterial gene transfers to eukaryotic host genomes. We therefore examined host genomes for evidence of gene transfer events from Wolbachia bacteria to their hosts. We found and confirmed transfers into the genomes of 4 insect and 4 nematode species that range from nearly the entire Wolbachia genome (>1 megabase) to short (<500 base pairs) insertions. Potential Wolbachia to host transfers were also detected computationally in three additional sequenced insect genomes. We also show that some of these inserted Wolbachia genes are transcribed within eukaryotic cells lacking endosymbionts. Therefore, heritable lateral gene transfer occurs into eukaryotic hosts from their prokaryote symbionts, potentially providing a mechanism for acquisition of new genes and functions.

High throughput DNA sequencing methodology (next generation sequencing; NGS) has rapidly evolved over the past 15 years and new methods are continually being commercialized. As the technology develops, so do increases in the number of corresponding applications for basic and applied science. The purpose of this review is to provide a compendium of NGS methodologies and associated applications. Each brief discussion is followed by web links to the manufacturer and/or web-based visualizations. Keyword searches, such as with Google, may also provide helpful internet links and information. © 2018 by John Wiley & Sons, Inc.

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Here we report the complete genome sequence of Teredinibacter turnerae T7901. T. turnerae is a marine gamma proteobacterium that occurs as an intracellular endosymbiont in the gills of wood-boring marine bivalves of the family Teredinidae (shipworms). This species is the sole cultivated member of an endosymbiotic consortium thought to provide the host with enzymes, including cellulases and nitrogenase, critical for digestion of wood and supplementation of the host's nitrogen-deficient diet. T. turnerae is closely related to the free-living marine polysaccharide degrading bacterium Saccharophagus degradans str. 2–40 and to as yet uncultivated endosymbionts with which it coexists in shipworm cells. Like S. degradans, the T. turnerae genome encodes a large number of enzymes predicted to be involved in complex polysaccharide degradation (>100). However, unlike S. degradans, which degrades a broad spectrum (>10 classes) of complex plant, fungal and algal polysaccharides, T. turnerae primarily encodes enzymes associated with deconstruction of terrestrial woody plant material. Also unlike S. degradans and many other eubacteria, T. turnerae dedicates a large proportion of its genome to genes predicted to function in secondary metabolism. Despite its intracellular niche, the T. turnerae genome lacks many features associated with obligate intracellular existence (e.g. reduced genome size, reduced %G+C, loss of genes of core metabolism) and displays evidence of adaptations common to free-living bacteria (e.g. defense against bacteriophage infection). These results suggest that T. turnerae is likely a facultative intracellular ensosymbiont whose niche presently includes, or recently included, free-living existence. As such, the T. turnerae genome provides insights into the range of genomic adaptations associated with intracellular endosymbiosis as well as enzymatic mechanisms relevant to the recycling of plant materials in marine environments and the production of cellulose-derived biofuels.

Wolbachia endosymbiotic bacteria have been implicated in the inflammatory pathogenesis of filariasis. Inflammation induced by Brugia malayi female worm extract (BMFE) is dependent on Toll-like receptors 2 and 6 (TLR2/6) with only a partial requirement for TLR1. Removal of Wolbachia, lipids, or proteins eliminates all inflammatory activity. Wolbachia bacteria contain the lipoprotein biosynthesis genes Ltg and LspA but not Lnt, suggesting Wolbachia proteins cannot be triacylated, accounting for recognition by TLR2/6. Lipoprotein databases revealed 3–11 potential lipoproteins from Wolbachia. Peptidoglycan-associated lipoprotein (PAL) and Type IV secretion system-VirB6 were consistently predicted, and B. malayi Wolbachia PAL (wBmPAL) was selected for functional characterization. Diacylated 20-mer peptides of wBmPAL (Diacyl Wolbachia lipopeptide (Diacyl WoLP)) showed a near identical TLR2/6 and TLR2/1 usage compared with BMFE and bound directly to TLR2. Diacyl WoLP induced systemic tumor necrosis factor-α and neutrophil-mediated keratitis in mice. Diacyl WoLP activated monocytes induce up-regulation of gp38 on human lymphatic endothelial cells and induced dendritic cell maturation and activation. Dendritic cells primed with BMFE generated a non-polarized Th1/Th2 CD4+ T cell profile, whereas priming with Wolbachia depleted extracts (following tetracycline treatment; BMFEtet) polarized to a Th2 profile that could be reversed by reconstitution with Diacyl WoLP. BMFE generated IgG1 and IgG2c antibody responses, whereas BMFEtet or inoculation of TLR2 or MyD88−/− mice produced defective IgG2c responses. Thus, in addition to innate inflammatory activation, Wolbachia lipoproteins drive interferon-γ-dependent CD4+ T cell polarization and antibody switching.

Human disease caused by parasitic filarial nematodes is a major cause of global morbidity. The parasites are transmitted by arthropod intermediate hosts and are responsible for lymphatic filariasis (elephantiasis) or onchocerciasis (river blindness). Within these filarial parasites are intracellular alpha-proteobacteria, Wolbachia, that were first observed almost 30 years ago. The obligate endosymbiont has been recognized as a target for anti-filarial nematode chemotherapy as evidenced by the loss of worm fertility and viability upon antibiotic treatment in an extensive series of human trials. While current treatments with doxycycline and rifampicin are not practical for widespread use due to the length of required treatments and contraindications, anti-Wolbachia targeting nevertheless appears a promising alternative for filariasis control in situations where current programmatic strategies fail or are unable to be delivered and it provides a superior efficacy for individual therapy. The mechanisms that underlie the symbiotic relationship between Wolbachia and its nematode hosts remain elusive. Comparative genomics, bioinfomatic and experimental analyses have identified a number of potential interactions, which may be drug targets. One candidate is de novo heme biosynthesis, due to its absence in the genome sequence of the host nematode, Brugia malayi, but presence in Wolbachia and its potential roles in worm biology. We describe this and several additional candidate targets, as well as our approaches for understanding the nature of the host-symbiont relationship.