The absence of molecular tools for manipulation of gene expression in the pathogenic free-living amoeba Naegleria fowleri has historically limited our understanding of gene function in the organism and has coincidently impacted the identification of potential druggable pathways and proteins. Here, we describe the development of approaches for the generation of transgenic amoebae using polyethyleneimine nanoparticles to deliver plasmids designed to confer antibiotic resistance and fluorescence to the cells. Through a series of optimization steps, we found that transfection of plasmids encoding the fluorescent protein mCherry fused by a T2A self-cleaving peptide to a codon-optimized puromycin acetyltransferase selectable marker yielded fluorescent cells that were resistant up to 100 µg/mL puromycin. Transfected trophozoites harbored between 45 and 65 copies of the transgene per cell and both fluorescence and resistance were persistent in the presence of selector through continued passages. The development of these approaches is anticipated to enable application of an array of genetic manipulation techniques including forward and reverse genetics to the study of this important pathogen.

The absence of molecular tools for manipulation of gene expression in the pathogenic free-living amoeba Naegleria fowleri has historically limited our understanding of gene function in the organism and has coincidently impacted the identification of potential druggable pathways and proteins. Here, we describe the development of approaches for the generation of transgenic amoebae using polyethyleneimine nanoparticles to deliver plasmids designed to confer antibiotic resistance and fluorescence to the cells. Through a series of optimization steps, we found that transfection of plasmids encoding the fluorescent protein mCherry fused by a T2A self-cleaving peptide to a codon-optimized puromycin acetyltransferase selectable marker yielded fluorescent cells that were resistant up to 100 µg/mL puromycin. Transfected trophozoites harbored between 45 and 65 copies of the transgene per cell and both fluorescence and resistance were persistent in the presence of selector through continued passages. The development of these approaches is anticipated to enable application of an array of genetic manipulation techniques including forward and reverse genetics to the study of this important pathogen.

Brain infection by the pathogenic free-living amoebae Naegleria fowleri is a life-threatening illness, with a mortality rate >95% in the US. One of the challenges faced in treating these infections is the lack of efficacious drugs. Here, we further characterize two leads. The first, a N. fowleri enolase inhibitor (HEX ((1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid)), emerged from a target-based approach while the other, BDGR-20191 (rac-(4aR,13bS)-10,11-dichloro-4-(oxetan-3-ylmethyl)-1,2,3,4,4a,5,6,13b-octahydro-8H-[1,6]naphthyridino[5,6-b]quinazolin-8-one), was identified in a phenotypic screen for anti-amoebic agents. Chronic low dose exposure to these agents led to cell lines that were tolerant to each. However, the tolerance phenotypes were reversed in the absence of selection with the agent. Differential gene expression analyses suggest that HEX tolerant cells upregulate cellular components that could impact levels of accumulated glycolytic intermediates, while BDGR-20191 tolerant cells could be targeting membrane biosynthesis. Unbiased metabolomics of the lines supported these possibilities. While HEX and BDGR-20191 are not synergistic with one another or with other standard-of-care drugs, their additive nature suggests they could be a useful addition to current therapeutic cocktails.