The Annotated Genome of Wolbachia from the Filarial Nematode Brugia malayi: What It Means for Progress in Antifilarial Medicine

Filarial nematodes contain endosymbiotic bacteria of the genus Wolbachia. As described in the April 2005 issue of PLoS Biology, Foster et al. have sequenced the genome of the Wolbachia that lives in the nematode Brugia malayi. What are the clinical implications?


Filariasis
The fi larial nematodes Wuchereria bancrofti, Brugia spp., and Onchocerca volvulus are insect-borne parasites that cause lymphatic or cutaneous fi lariasis. Lymphangitis, hydrocele, and elephantiasis are pathologies that result from W. bancrofti and Brugia spp. infections. O. volvulus infections can present with severe skin pathologies (acute and chronic dermatitis, atrophy) and blindness (onchocerciasis, or river blindness). The nematodes infect more than 140 million people in 90 mostly tropical countries. An additional one billion people are at risk of contracting the diseases caused by these nematodes [1,2].
Current control efforts, both vector control and mass antifi larial chemotherapies, have shown initial success, but sustainability is uncertain. For example, vector control, used for onchocerciasis in west African savannah areas, has become too expensive and therefore been stopped. Current and planned elimination programs will rely on mass administration of antifi larial drugs that require annual administration for 5-10 years for lymphatic fi lariasis and more than 20 years for onchocerciasis.
Control programs based on drug administration require long treatment durations because the adult female worms, which produce thousands of larvae daily, survive many years (over 14 years for onchocerciasis) and are not killed by current drugs [1]. The success of such control programs, as shown by mathematical modeling, depends on a minimum of 60% of the people in an endemic area participating every year [3]. This is probably too optimistic an estimate, as a recent review of onchocerciasis therapy in regions that have had 10-12 years of ivermectin treatment still show infection levels of 2%-3%. These levels are enough to establish the infection within a few years after the end of annual drug administration [4]. Additionally, there is evidence that some geographic areas have worms with apparent resistance to ivermectin [5]. Therefore, it is essential that we fi nd new drugs that kill or sterilize adult worms.

Enter Wolbachia-Endosymbionts of Filarial Nematodes
For almost 30 years, it has been known that fi larial nematodes contain endosymbiotic bacteria. These endobacteria are found in the hypodermis of male and female worms, and in the oocytes, embryos, and larval stages ( Figure 1). As in many animal fi larial species, endobacteria are present in the human fi lariae W. bancrofti, Brugia spp., and O. volvulus [1,6,7], but not in Loa loa [8,9]. Recently, these endosymbionts were classifi ed at the molecular level to be of the genus Wolbachia, a genus of bacteria that are common endosymbionts of arthropods. The next nearest relatives of Wolbachia are Rickettsia, Ehrlichia, Cowdria, and Anaplasma [10].
Studies of the effect of antirickettsials such as tetracycline and rifampicin in animals infected with fi larial nematodes have shown, using immunohistochemistry, that these drugs deplete the Wolbachia from the worms. After the Wolbachia are depleted, the worms develop a distinct phenotype. Monitoring the microfi laria (Mf) levels in the blood showed that the number of Mf in the treated animals was lower than the number in the control groups, and that the number of Mf in the treated group neared zero with time. Examination

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The Perspectives section is for experts to discuss the clinical practice or public health implications of a published article that is freely available online. of the adult worms showed that embryogenesis was blocked and the uteri contained degenerated embryos [1]. A study done in cattle infected with O. ochengi even showed killing of adult worms [11].
The success of antibiotic treatment against animal fi lariae has been extended to human fi larial infections. Trials of doxycycline have been completed for populations infected with O. volvulus and W. bancrofti. For both species, larval levels were zero or near zero after treatment [6,12]. Examination of worms from extirpated nodules of patients with onchocerciasis showed that, as in the animal studies, depletion of Wolbachia led to a block in embryogenesis that appears to be permanent [2]. Most recently, there has been evidence for the killing of adult W. bancrofti by doxycycline therapy [13].
Wolbachia stimulate infl ammatory responses via Toll-like receptor (TLR) 2 and TLR4 [14], pattern recognition receptors that recognize a variety of bacterial molecules [15]. Working with a mouse model of O. volvulus-induced corneal infl ammation (keratitis), it has been shown that Wolbachia antigens presented via TLR4 are required for the development of pathology [16]. Wolbachia have also been associated with adverse reactions seen in infected patients after antifi larial therapy.
Recent studies supporting a role for Wolbachia in adverse reactions after antifi larial treatment have shown that doxycycline given before ivermectin reduced Wolbachia loads as well as the number and severity of adverse reactions in patients with lymphatic fi lariasis ( [17]; J. Turner, S. Mand, A. Y. Debrah, J. Muehlfeld J, K. M. Pfarr, et al., unpublished data). Thus, doxycycline fulfi lls the role for a new antifi larial therapy in that it produces sterility in lymphatic fi lariasis and onchocerciasis, kills adult worms in lymphatic fi lariasis, and prevents or lessens adverse reactions due to the rapid killing of Mf by microfi laricidal drugs. However, the treatment time of four weeks is still longer than that desired for new antifi larial therapies. Wolbachia are ideal targets for antifi larial drugs that have the same effect as doxycycline, but that work in a shorter interval. Potential drug targets may be found by analyzing the genome of Wolbachia.

Sequencing the Wolbachia Genome
As part of the effort to fi nd antiwolbachial drugs that act in less time than the current four-week regime for doxycycline, a consortium was established to sequence the genomes of the Wolbachia species that inhabit human fi larial nematodes. In the April 2005 issue of PLoS Biology, Foster and colleagues report on the completion of the sequencing and annotation of the genome from the Wolbachia of Brugia malayi (wBm) (Figure 2) [18]. This is the fi rst complete Wolbachia genome from a fi larial nematode. The authors compare the wBm genome to the fi rst sequenced genome of the Wolbachia of Drosophila melanogaster (wMel), which is strictly parasitic, and the genomes of other endosymbiotic bacteria, pointing out potential metabolites that wBm may supply to the nematode.

Features of the Genome and Metabolites that wBm May Provide to Its Host
The genome of wBm is 1.1 million nucleotides, smaller than wMel and Rickettsia prowazekii, but larger than that of Mycobacterium. The reduced genome size is indicative of long-term symbiosis, and refl ects the loss of all genes necessary to make all but one amino acid, genes needed to infect new DOI: 10.1371/journal.pmed.0020110.g002 hosts, and genes needed to evade the host immune system. Also lacking from the wBm genome are several genes needed for DNA repair and genes required for RNA modifi cation. Many of these genes have also been lost from other endosymbiotic bacteria. wBm cannot synthesize lipopolysaccharide, a component of the cell membrane in most bacteria. This is astounding because, as noted above, Wolbachia stimulate an infl ammatory reaction via TLR4. Endobacteria antigens presented via TLR4 are also responsible for adverse reactions after antifi larial treatment [17], and for pathology in onchocerciasis [16]. wBm lack the genes necessary to cross-link and degrade the carbohydrate backbone of peptidoglycan. The Wolbachia peptidoglycan structure resembles the peptidoglycan-derived cytotoxins produced by Neisseria gonorrhoeae and Bordetella pertussis, which are made up of muramyl peptides [19]. These muramyl peptides are known to stimulate an infl ammatory response and pathology via TLR2.
However, to understand the Wolbachia-fi laria endosymbiosis, it is not only important what wBm has lost from the genome during its long symbiosis with B. malayi, but also what has been kept. The endobacteria have retained all of the genes necessary for the synthesis of the co-factors ribofl avin, fl avin adenine dinucleotide, and heme. The genome also contains the genes necessary to make glutathione, although it lacks the genes needed for glutathione-mediated methylglyoxal detoxifi cation [20], which is the most common reason intracellular bacteria need glutathione. Finally, in contrast to most endosymbiotic bacteria, wBm have retained the genes necessary to make all nucleotides.
As the wBm genome has the genes for a type IV secretion system, used by intracellular bacteria for exporting molecules to nonbacterial (e.g., host) cells, the above described metabolites could be supplied to the nematode host by Wolbachia. To date, there is no evidence of genes for ribofl avin and heme synthesis in the B. malayi genome [21]. Heme from Wolbachia could be vital to worm embryogenesis, as there is evidence that molting and reproduction are controlled by ecdysteroid-like homones [22], whose synthesis requires heme. Depletion of Wolbachia would therefore halt production of these hormones and block embryogenesis. Wolbachia could be a source of glutathione which, aside from its role in the detoxifi cation of methylglyoxal, is required for protection against oxidative stress from oxygen-reactive species secreted by mammalian immune cells [23]. Finally, Wolbachia may be an essential source of nucleotides during embryogenesis. Wolbachia as a source of the above metabolites would explain the block in embryogenesis and the sterility seen in worms after depletion of the endobacteria.

Conclusion
The completion of the wBm genome offers a wealth of information for both basic and applied science. With the completion of this genome, one can now compare close relatives that infect different hosts and have different symbiotic lifestyles, i.e., parasitic versus mutualistic. Such a comparison of the differences could help to defi ne genes necessary for invading host cells. Examining the genome of wBm will help us understand the molecular basis for the endosymbiosis between fi larial nematodes and Wolbachia. Researchers now know which metabolites the endobacteria require from the nematode host and which might be provided by Wolbachia to the nematode. This is exciting because it opens up the possibility to fi nd and test drugs already registered for use in humans that might inhibit key biochemical pathways in the Wolbachia-and lead to sterility or killing of the adult worms in shorter treatment times-and that could be given to all infected persons. Given the huge challenges that still lie ahead for the programs to eliminate fi lariasis, such a need has been and will be repeatedly stated. The sequencing and annotation of the wBm genome will aid researchers in fulfi lling this goal by focusing research on those biochemical events that are essential to the mutualistic symbiosis between fi larial nematodes and their Wolbachia.