Glucose metabolism is the sole source of ATP for the infectious lifecycle stage of the African trypanosome, Trypanosoma brucei. Mis-regulation of the first enzyme in the pathway, hexokinase, is toxic to the parasite. However, little is known concerning the regulatory mechanisms that modulate expression of the two genes that encode this essential enzyme activity. The goal of this application is to identify the mechanisms the parasite employs to regulate hexokinases at the gene expression and enzyme activity levels in response to distinct environmental conditions. Preliminary data indicates the trypanosome hexokinases are dynamically regulated in response to environmental glucose levels through mechanisms that include both modulation of transcript steady-state abundance and expression, as well as changes in enzyme oligomer composition. Regulation of transcript abundance in the African trypanosome occurs primarily via post-transcriptional mechanisms as a result of information encoded in gene 3'UTRs. Elements that influence hexokinase steady-state transcript abundance will be identified by monitoring transcript levels of a reporter gene construct harboring a series of mutated hexokinase 3'UTRs. The impact of these constructs on gene expression will also be considered by scoring enzyme activity of the reporter gene. At the protein level, hexokinase hexamer composition, which the parasite can alter based on growth conditions, influences enzyme activity, including sensitivity to regulatory molecules. To understand the differences in sensitivity to regulatory molecules, site-directed mutagenesis will be used to identify domains and residues required for inhibitor binding. The impact of variants that are engineered to no longer be susceptible to regulation in vitro will be assessed in vivo by expression in T. brucei. Through the characterization of regulatory mechanisms required for hexokinase expression, new means of targeting glucose metabolism, a required parasite pathway, will be identified.
The proposed research is important to public health as mechanisms identified here will yield new targets for desperately needed therapeutic development for the African trypanosome while expanding our understanding of the fundamental cellular process of glucose sensing, topics that are supported by the mission of the NIH.
|Flaherty, Daniel P; Harris, Michael T; Schroeder, Chad E et al. (2017) Optimization and Evaluation of Antiparasitic Benzamidobenzoic Acids as Inhibitors of Kinetoplastid Hexokinase?1. ChemMedChem 12:1994-2005|
|Hackler, Amber; Patrick, Stephen L; Kahney, Elizabeth W et al. (2017) Antiparasitic lethality of sulfonamidebenzamides in kinetoplastids. Bioorg Med Chem Lett 27:755-758|
|Hackler, Amber L; Qiu, Yijian; Patrick, Stephen L et al. (2015) Characterization of an African trypanosome mutant refractory to lectin-induced death. Biochem Biophys Rep 4:33-38|
|Harris, M T; Mitchell, W G; Morris, J C (2014) Targeting protozoan parasite metabolism: glycolytic enzymes in the therapeutic crosshairs. Curr Med Chem 21:1668-78|
|Baker, Carol J; Carey, Vincent J; Rench, Marcia A et al. (2014) Maternal antibody at delivery protects neonates from early onset group B streptococcal disease. J Infect Dis 209:781-8|
|Lin, Sheng; Morris, Meredith T; Ackroyd, P Christine et al. (2013) Peptide-targeted delivery of a pH sensor for quantitative measurements of intraglycosomal pH in live Trypanosoma brucei. Biochemistry 52:3629-37|
|Harris, Michael T; Walker, Dawn M; Drew, Mark E et al. (2013) Interrogating a hexokinase-selected small-molecule library for inhibitors of Plasmodium falciparum hexokinase. Antimicrob Agents Chemother 57:3731-7|
|Joice, April C; Lyda, Todd L; Sayce, Andrew C et al. (2012) Extra-glycosomal localisation of Trypanosoma brucei hexokinase 2. Int J Parasitol 42:401-9|
|Stoner, Kevin A; Rabe, Lorna K; Austin, Michele N et al. (2011) Incidence and epidemiology of Streptococcus pseudoporcinus in the genital tract. J Clin Microbiol 49:883-6|
|Dodson, Heidi C; Morris, Meredith T; Morris, James C (2011) Glycerol 3-phosphate alters Trypanosoma brucei hexokinase activity in response to environmental change. J Biol Chem 286:33150-7|
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