Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Biochemistry and Molecular Biology

Committee Chair/Advisor

James Morris

Committee Member

Robert Anholt

Committee Member

Lesly Temesvari

Committee Member

Cheryl Ingram-Smith


The free-living amoeba, Naegleria fowleri, can cause a rare yet usually lethal infection of the brain called primary amebic meningoencephalitis. Because of poor diagnostics and limited treatment options, the mortality rate associated with the disease is >97%. Due to our finding that glucose is critical for trophozoite growth in culture, we have been interested in exploiting amoebae glucose metabolism to identify new potential drug targets. We have characterized the first enzyme of the glycolytic pathway, glucokinase (Glck), from N. fowleri and two other pathogenic free-living amoeba, Acanthamoeba castellanii and Balamuthia mandrillaris. We have assessed their biochemical properties and tested potential inhibitors on the recombinant Glcks, which revealed that these enzymes are sufficiently different from one another that developing pan-amoeba inhibitors may be challenging. However, their individual differences from the human host enzyme suggests that species-specific Glck inhibitors could be identified. We have also explored targeting the glucose metabolizing enzyme enolase in N. fowleri using a series of phosphonate human enolase 2 (ENO-2) specific inhibitors that were developed to treat human cancer. These compounds are curative for ENO-1 deleted glioblastoma in a rodent model, can cross the blood-brain barrier, and are of limited toxicity to non-human primates. The phosphonate inhibitors were toxic to N. fowleri in vitro with (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) being the most potent, with an EC50 value of 0.21 ± 0.02 µM, almost 1500-fold lower than the concentration required to impact human cells. Unbiased metabolomics indicates that glycolytic intermediates upstream of NfENO accumulate in HEX treated amoebae. In an effort to genetically validate new targets for therapeutic intervention, we have initiated efforts to develop molecular tools for use in N. fowleri. We have designed a vector for transient transfection of the amoebae that harbors portions of the 5’UTR of actin 1 (NF0111190) upstream of both eYFP and a hygromycin resistance gene, termed pJMJM1. We have tested a variety of approaches used in other parasite systems for plasmid delivery including the transfection reagent SuperFect, Amaxa Nucleofector technologies, and various electroporation settings. Transfection of N. fowleri flagellates with 5 µg pJMJM1 by electroporation (100 V, 500 µF, 400 Ω) yielded a population of fluorescent cells seven days after being treated with 300 µg/mL hygromycin, but this expression of eYFP was lost over time. More recently, we have used CRISPR/Cas9- mediated gene editing to successfully introduce an eYFP repair template into a predicted protein locus. While fluorescent cells were not noted in the culture, editing was confirmed by PCR analysis. Development of these molecular techniques will provide an important tool for uncovering potential target genes and allow for a better understanding of amoeba biology.

Author ORCID Identifier


Included in

Biochemistry Commons



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