A Cell-Based Screen Reveals that the Albendazole Metabolite, Albendazole Sulfone, Targets Wolbachia

Targets Wolbachia. PLoS Pathog 8(9): e1002922. doi:10.1371/journal.ppat.1002922 A Cell-Based Screen Reveals that the Albendazole Metabolite, Albendazole Sulfone, Targets Wolbachia Laura R. Serbus 0 Frederic Landmann 0 Walter M. Bray 0 Pamela M. White 0 Jordan Ruybal 0 R. Scott Lokey 0 Alain Debec 0 William Sullivan 0 David S. Schneider, Stanford University, United States of America 0 1 Molecular, Cell and Developmental Biology, University of California, Santa Cruz, California, United States of America, 2 Department of Chemistry and Biochemistry, University of California, Santa Cruz, California, United States of America, 3 Polarity and Morphogenesis Group, Jacques Monod Institute, CNRS, University Paris Diderot, UPMC Batiment Buffon , Paris , France Wolbachia endosymbionts carried by filarial nematodes give rise to the neglected diseases African river blindness and lymphatic filariasis afflicting millions worldwide. Here we identify new Wolbachia-disrupting compounds by conducting high-throughput cell-based chemical screens using a Wolbachia-infected, fluorescently labeled Drosophila cell line. This screen yielded several Wolbachia-disrupting compounds including three that resembled Albendazole, a widely used anthelmintic drug that targets nematode microtubules. Follow-up studies demonstrate that a common Albendazole metabolite, Albendazole sulfone, reduces intracellular Wolbachia titer both in Drosophila melanogaster and Brugia malayi, the nematode responsible for lymphatic filariasis. Significantly, Albendazole sulfone does not disrupt Drosophila microtubule organization, suggesting that this compound reduces titer through direct targeting of Wolbachia. Accordingly, both DNA staining and FtsZ immunofluorescence demonstrates that Albendazole sulfone treatment induces Wolbachia elongation, a phenotype indicative of binary fission defects. This suggests that the efficacy of Albendazole in treating filarial nematode-based diseases is attributable to dual targeting of nematode microtubules and their Wolbachia endosymbionts. - Funding: This work was supported by an NSF Frontiers in Integrative Biological Research grant (EF-0328363), the Ruth L. Kirschstein National Research Service Award (F32 GM080192), the NIH Initiative to Maximize Student Diversity (R25 GM058903), the Anti-Wolbachia Consortium based at the Liverpool School of Tropical Medicine, and an NSF BIO Innovation Activities grant (MCB-1122252). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Wolbachia are intracellular maternally transmitted bacteria present in the majority of all insect species as well as some mites, crustaceans and filarial nematodes [1,2]. Wolbachia were initially studied in insects because they induce unconventional reproductive phenotypes including sperm-egg cytoplasmic incompatibility, feminization of males, male-killing, and parthenogenesis [3,4]. Wolbachia are essential endosymbionts of some filarial nematodes and recent studies demonstrated that they are the causative agent of African river blindness and also contribute to lymphatic filariasis [5,6]. One sixth of the world population is at risk of infection by Wuchereria bancrofti, Brugia timori and Brugia malayi, the filarial nematode species that cause lymphatic filariasis [7]. Wolbachia released from filarial nematodes into the human body trigger an inflammatory reaction that underlies the lymphedema and corneal occlusion associated with these neglected diseases [8,9,10,11, 12,13,14,15,16,17,18]. Lymphatic filariasis and African river blindness have traditionally been treated through the administration of three drugs, singly or in combination: diethylcarbamazine (DEC), ivermectin (IVM) and albendazole (ALB). These drugs target the filarial nematodes associated with these diseases, namely Onchocerca volvulus, B. timori, B. malayi, and W. bancrofti [5,6,19]. DEC disrupts the nematodes by targeting the arachidonic acid metabolic pathway in the host [20]. IVM disrupts glutamate-gated chloride channels in the nematode that control release of excretory/secretory vesicles that would normally suppress the immune response [21,22]. ALB is a benzimidazole used to disrupt the nematode microtubule cytoskeleton [23]. Orally administered ALB is rapidly metabolized by in the intestinal mucosa and liver into albendazole sulfoxide (ALB-SO) and albendazole sulfone (ALB-SO2) [24,25]. ALB-SO is normally considered to be the active, form of Albendazole against systemic parasites, while ALB-SO2 is considered to be an inactive form of the drug [26]. All three drugs exhibit microfilaricidal effects [19]. The macrofilaricidal effects of ALB are not clear, though specific formulations induce worm sterility in animal models [27]. In addition, a number of clinical trials demonstrate that ALB when used in combination with DEC or IVM is macrofilaricidal [28,29,30,31,32]. Wolbachia are obligate symbionts of filarial nematodes required for normal embryogenesis, larval development and perhaps most significantly adult survival [33,34,35,36,37,38,39,40,41]. A recent study demonstrated that loss of Wolbachia in the adult results in high levels of apoptosis throughout the nematode [37]. Studies have also found that much of the pathology associated with filarial Wolbachia-based neglected diseases currently threaten one sixth of the world population. Millions of people are infected with filarial nematodes that rely upon endosymbiotic Wolbachia bacteria for their survival. These Wolbachia ultimately induce an immune response that gives rise to African river blindness or lymphatic filariasis. Thus, targeting Wolbachia will prevent induction of disease symptoms while also eliminating the filarial nematode infection. To identify new, fast-acting anti-Wolbachia drugs, we tested candidate compounds in Wolbachiainfected insect cells using automated robotics. Some of the anti-Wolbachia compounds we discovered closely resembled Albendazole, an FDA-approved drug already used worldwide to combat filarial nematode infections. We found that a common Albendazole metabolite strongly suppresses Wolbachia density in fruit flies and filarial nematodes by disrupting Wolbachia growth. These findings suggest that Albendazole is effective in treating filarial nematode-based diseases because it independently targets both the nematode and its essential Wolbachia endosymbionts. This has immediate implications for treating lymphatic filariasis and African river blindness. nematode diseases is due to induction of innate and adaptive host immune responses upon release of Wolbachia from their nematode hosts [8,9,10,11,12,13,14,15,16,17,18]. These discoveries suggest that compounds directly targeting Wolbachia may be a powerful alternative to the more traditional approaches for trea (...truncated)