Antiamoebic Activity of Rutamycin B and Inhibition of Naegleria fowleri F₁F_O ATP Synthase Activity

Naegleria fowleri induces fatal human infection known as primary amoebic meningoencephalitis (PAM). Currently, the recommended treatment does not guarantee uniform survival. Thus, there is an urgent need to find an effective and fast-acting treatment for PAM. Our previous study identified anti-N. fowleri activity of marine microbial metabolite mixtures. We recently isolated a pure compound rutamycin B from an amoebicidal metabolite. We sought to determine the activity of rutamycin B and to identify its target in N. fowleri. We determined the dose–response of rutamycin B against N. fowleri and two mammalian cells and measured potency at different time points. We investigated the combined effect of rutamycin B and amphotericin B on both N. fowleri and mammalian cells. For target identification in N. fowleri, we (i) determined the mitochondrial ATP synthase activity of N. fowleri treated with rutamycin B, and (ii) quantified the production of intracellular reactive oxygen species (ROS) in the presence of the compound. Rutamycin B exhibited potency between 0.02 and 0.3 µM against five strains of N. fowleri and had a selectivity index of 200–300. It achieved nanomolar potency on all five strains within four hours, and the combination of rutamycin B with amphotericin B was synergistic without eliciting toxicity on mammalian cells. Significant inhibition of N. fowleri ATP synthase activity and subsequent ROS production suggest that rutamycin B may target F₁F_O ATP synthase of the pathogen. Our findings identified a potent marine-derived compound and its potential target in N. fowleri and support further pharmacological studies of rutamycin B for the treatment of PAM.