Autophagy is essential for anti-Wolbachia drug efficacy in Brugia malayi and insect cells

Introduction: Onchocerciasis and lymphatic filariasis (LF) cause a significant global public health burden with more than 900 million individuals at risk and over 60 million people living with symptomatic manifestations caused by filarial diseases. Due to the importance of Wolbachia for the survival of adult filarial nematodes, anti-Wolbachia therapy has been validated as a safe macrofilaricidal treatment for LF and onchocerciasis. The A-WOL consortium was established with the goals of defining registered anti-Wolbachia antibiotics, as well as developing new drugs effective in a regimen of 7 days or less. We have previously shown autophagy has a core role in the regulation of Wolbachia populations across a diverse range of associations. In this study, we investigated the role of autophagy in the efficacy of anti-Wolbachia drugs. Methods: Autophagic flux was assessed in response to broad-spectrum anti-Wolbachia antibiotics (doxycycline, rifampicin, moxifloxacin, and sparfloxacin) and A-WOL candidate compounds (flubentylosin [TylAMac], AWZ1066S, and fusidic acid), compared with antibiotics with no activity against Wolbachia (levofloxacin, ciprofloxacin, amoxicillin, and streptomycin). Autophagy was quantified through LC3B-I to LC3B-II conversion and p62 degradation in insect cell lines (C6/36 and Sf9) and in Brugia malayi. The role of autophagy was evaluated using early- and late-stage inhibitors. The generation of ROS was measured to assess its contribution to autophagy activation. Finally, the viability of purified extracellular Wolbachia following drug exposure was determined using live/dead staining and reinfection assays. Results: All effective anti-Wolbachia compounds induced autophagic flux in insect cells and B. malayi, whereas ineffective antibiotics did not. Autophagy activation occurred in the absence of Wolbachia, was restricted to insect and nematode systems, and was not preceded by the generation of ROS. Only concentrations that induced autophagy resulted in effective Wolbachia depletion (of >90%), the empirical threshold of delivering the desired macrofilaricidal activity, and autophagy inhibition reduced the efficacy of the drugs. Exposure of purified extracellular Wolbachia to anti-Wolbachia drugs showed no impact on their viability, indicating that host processes are required for depletion. Discussion: These results demonstrate the critical role of host autophagy in anti-wolbachia drug activity and a previously unrecognised host-directed mechanism in insect cells and B. malayi.