Amoebicidal effect of pyocyanin and its mechanism of action against Naegleria fowleri, the "brain-eating" amoeba.

Naegleria fowleri is a free-living amoeba that causes primary amoebic meningoencephalitis (PAM) in healthy humans. Infection occurs through deep inhalation of contaminated water during water-related activities. Treatment of PAM is complicated due to the low efficacy and high toxicity of available drugs. Therefore, novel therapeutics and control agents are urgently needed. Since this amoeba shares an aquatic habitat with other microbes, it is logical to search for bioactive molecules produced by aquatic bacteria to find novel therapeutic and control agents to manage N. fowleri infection. Previously, pyocyanin (PCN), a secondary metabolite produced by Pseudomonas aeruginosa, was proven to have anti-amoebic activity against N. fowleri. However, its mechanism of action needs clarification. This study aimed to investigate the molecular mechanism of PCN against N. fowleri. Trophozoites of N. fowleri were treated with 300 μg/ml of PCN, and their survival was assessed at different time points. Morphological changes, DNA fragmentation, mitochondrial dysfunction, reactive oxygen species (ROS) accumulation, autophagy induction, and cytotoxicity were investigated. PCN was found to have an amoebicidal effect against N. fowleri in a time-dependent manner. Trophozoite survival decreased significantly to 34% and 26% at 12 and 24 h, respectively. Treatment of N. fowleri with PCN caused a morphological alteration. Trophozoites exposed to a high dose of PCN were fragmented. Mechanistically, PCN induced mitochondrial dysfunction similar to amphotericin B, a standard drug. Moreover, it also induced ROS accumulation but did not cause DNA fragmentation. Autophagy was also induced in PCN-treated trophozoites, as evidenced by the increased expression of autophagy-related genes. These findings indicate that PCN showed an amoebicidal effect against N. fowleri by inducing mitochondrial dysfunction-driven cell death with autophagic features. Reduction of toxicity by structural modification is necessary in the case of use as a therapeutic or control agent for the management of N. fowleri infection. Overall, this study highlights the potential of PCN as a candidate anti-N. fowleri agent, either as a standalone treatment or in combination with other drugs used for PAM management.