Nitroxoline treatment of Balamuthia mandrillaris triggers genomic crisis, transcriptional collapse, and incomplete encystment via copper and iron chelation

Balamuthia mandrillaris is a free-living amoeba that can infect the central nervous system to cause granulomatous amebic encephalitis (GAE), a highly fatal disease with mortality rates up to 90%. GAE is typically treated with a six-drug regimen with limited efficacy and significant toxicity. Our lab previously identified nitroxoline, a quinolone antibiotic, as a promising therapeutic complement to the existing regimen, and in 2021 nitroxoline was first successfully used to treat a human patient. In this study, we elucidate the mechanism of action for nitroxoline using a combination of genomics, transcriptomics, chemical complementation, growth assays, and electron microscopy. In service of this goal, an annotated draft genome of B. mandrillaris strain CDC:V039 was generated using long read sequencing, yielding 88 telomere-to-telomere contigs and over 25,000 protein coding genes. Leveraging this resource, comparative transcriptomics were used to characterize encystment responses to three different cellular stressors: nitroxoline, galactose, and hypoxia. Our findings reveal that nitroxoline-induced cysts undergo transcriptional collapse and genomic and structural destabilization via copper and iron chelation. These data provide insight into the multifaceted impact of nitroxoline on B. mandrillaris growth and cellular processes and advance our understanding of the transcriptional landscape associated with stress induced encystment. Our findings support the continued clinical study of nitroxoline.