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

Abstract

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 mode-of-action and efficacy of the portfolio of anti-Wolbachia drugs. This work has demonstrated a consistent increase in autophagic flux with four broad-spectrum anti-Wolbachia antibiotics from different classes (doxycycline, rifampicin, moxifloxacin, and sparfloxacin) exposed to two insect cell lines (C6/36 and SF9) and in B. malayi. This autophagy activation was also observed for selected-candidates from the A-WOL consortium (flubentylosin [TylAMac], AWZ1066S and fusidic acid). Drugs ineffective against Wolbachia (levofloxacin, ciprofloxacin, amoxicillin, and streptomycin) failed to induce autophagic flux. Antibiotic-induced autophagy was observed in the absence of Wolbachia, indicating its effect occurs independent of the bacteria. The activation of autophagy flux by anti-Wolbachia drugs is not observed in mammalian cells, is restricted to insect cells and nematodes and is independent of ROS activation. Through concentration-dependency testing of anti-Wolbachia antibiotics, we show that only concentrations that induced autophagy resulted in effective Wolbachia depletion (of >90%), the empirical threshold of delivering the desired macrofilaricidal activity. The contribution of autophagy in the efficacy of anti-Wolbachia drugs and their ability to reduce bacterial viability was demonstrated in B. malayi microfilariaef and adult worms when autophagy was inhibited during anti-Wolbachia drug exposure. Moreover, a role for autophagy was observed in the continued decline in Wolbachia post-drug exposure. Wolbachia purified from C6/36 cells and exposed to anti-Wolbachia drugs showed no impact on their viability re-enforcing the essential requirement for autophagy flux activation in the efficacy of anti-Wolbachia drugs.