James C. Morris

The pathogenic free-living amoeba Naegleria fowleri is the cause of primary amebic meningoencephalitis (PAM), a central nervous system infection that is almost always lethal. One of the unusual features of the amoebae is the presence of ~4000 copies of a nucleolar-localized closed circular extrachromosomal ribosomal DNA element (CERE) that encodes the cell’s ribosomal RNA repertoire. It has historically been challenging to purify large quantities of CERE, limiting our understanding of the nucleic acid. Here, we describe a methodology for CERE purification that improves yield, reduces processing times, and maintains the integrity of the plasmid. This approach will enable the study of this unique DNA architecture, advancing our understanding of the pathobiology of the organism.

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.

The SAR of the N. fowleri enolase inhibitor HEX revealed that the activity of the HEX scaffold toward NfENO is rather sensitive to structural perturbations, confirming the necessity of both the hydroxamate and phosphonate groups to maintain potency.

Primary amoebic meningoencephalitis (PAM) is a human brain infection caused by Naegleria fowleri with a 97% mortality rate. Quinazolinones resulting from a Mannich-coupled domino rearrangement were recently identified as inhibitors of the amoeba. Herein, we resolved the effective concentrations for 25 pilot compounds and then, using the Mannich protocol and a key late-stage, N-demethylation/functionalization, we synthesized 53 additional analogs to improve potency, solubility and microsomal stability. We established an antiamoebic quinazolinone pharmacophore, culminating in (±)-trans-57b which featured the best combination of potency, selectivity index, solubility, and microsomal stability. Enantiomeric separation afforded (4aS,13bR)-57b (BDGR-20237) with a 41-fold potency advantage over its enantiomer. ADME and mouse pharmacokinetic profiling for BDGR-20237 revealed high brain penetrance but a limited half-life which did not statistically enhance the mouse survival in a pilot efficacy study. The pharmacophoric model, supported by 88 quinazolinones, several of which exhibit subnanomolar potency, will guide further scaffold optimization.

Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amoebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate these agents are potent inhibitors of N. fowleri ENO (NfENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC₅₀ = 0.14 ± 0.04 μM) that was toxic to trophozoites (EC₅₀ = 0.21 ± 0.02 μM) while the reported CC₅₀ was >300 μM. Molecular docking simulation revealed that HEX binds strongly to the active site of NfENO with a binding affinity of –8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of NfENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the end of the one-week observation, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. However, intranasal HEX delivery was not curative as brains of six of the eight survivors were positive for amoebae. These findings suggest that HEX requires further evaluation to develop as a lead for treatment of PAM.

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. Little is known about the cellular responses generated when environmental glucose levels change. In both bloodstream and procyclic form (insect stage) parasites, glycosomes house most of glycolysis. These organelles are rapidly acidified in response to glucose deprivation, which likely results in the allosteric regulation of glycolytic enzymes such as hexokinase. In previous work, localizing the chemical probe used to make pH measurements was challenging, limiting its utility in other applications. This paper describes the development and use of parasites that express glycosomally localized pHluorin2, a heritable protein pH biosensor. pHluorin2 is a ratiometric pHluorin variant that displays a pH (acid)-dependent decrease in excitation at 395 nm while simultaneously yielding an increase in excitation at 475 nm. Transgenic parasites were generated by cloning the pHluorin2 open reading frame into the trypanosome expression vector pLEW100v5, enabling inducible protein expression in either lifecycle stage. Immunofluorescence was used to confirm the glycosomal localization of the pHluorin2 biosensor, comparing the localization of the biosensor to the glycosomal resident protein aldolase. The sensor responsiveness was calibrated at differing pH levels by incubating cells in a series of buffers that ranged in pH from 4 to 8, an approach we have previously used to calibrate a fluorescein-based pH sensor. We then measured pHluorin2 fluorescence at 405 nm and 488 nm using flow cytometry to determine glycosomal pH. We validated the performance of the live transgenic pHluorin2-expressing parasites, monitoring pH over time in response to glucose deprivation, a known trigger of glycosomal acidification in PF parasites. This tool has a range of potential applications, including potentially being used in high-throughput drug screening. Beyond glycosomal pH, the sensor could be adapted to other organelles or used in other trypanosomatids to understand pH dynamics in the live cell setting.

Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate that these agents are potent inhibitors of N. fowleri ENO ( Nf ENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC ₅₀ value of 0.14 ± 0.04 µM) that was toxic to trophozoites (EC ₅₀ value of 0.21 ± 0.02 µM) while the reported CC ₅₀ was >300 µM. Molecular docking simulation revealed that HEX binds strongly to the active site of Nf ENO with a binding affinity of –8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of Nf ENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the conclusion of the experiment, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. Brains of six of the eight survivors were positive for amoebae, suggesting the agent at the tested dose suppressed, but did not eliminate, infection. These findings suggest that HEX is a promising lead for the treatment of PAM.