title: siRNA Screening Identifies the Host Hexokinase 2 (HK2) Gene as an Important Hypoxia-Inducible Transcription Factor 1 (HIF-1) Target Gene in Toxoplasma gondii-Infected Cells.
abstract: Although it is established that oxygen availability regulates cellular metabolism and growth, little is known regarding how intracellular pathogens use host factors to grow at physiological oxygen levels. Therefore, large-scale human small interfering RNA screening was performed to identify host genes important for growth of the intracellular protozoan parasite Toxoplasma gondii at tissue oxygen tensions. Among the genes identified by this screen, we focused on the hexokinase 2 (HK2) gene because its expression is regulated by hypoxia-inducible transcription factor 1 (HIF-1), which is important for Toxoplasma growth. Toxoplasma increases host HK2 transcript and protein levels in a HIF-1-dependent manner. In addition, parasite growth at 3% oxygen is restored in HIF-1-deficient cells transfected with HK2 expression plasmids. Both HIF-1 activation and HK2 expression were accompanied by increases in host glycolytic flux, suggesting that enhanced HK2 expression in parasite-infected cells is functionally significant. Parasite dependence on host HK2 and HIF-1 expression is not restricted to transformed cell lines, as both are required for parasite growth in nontransformed C2C12 myoblasts and HK2 is upregulated in vivo following infection. While HK2 is normally associated with the cytoplasmic face of the outer mitochondrial membrane at physiological O2 levels, HK2 relocalizes to the host cytoplasm following infection, a process that is required for parasite growth at 3% oxygen. Taken together, our findings show that HIF-1-dependent expression and relocalization of HK2 represent a novel mechanism by which Toxoplasma establishes its replicative niche at tissue oxygen tensions.
  
  
  OBJECTIVE
  Little is known regarding how the host cell contributes to the survival of the intracellular parasite Toxoplasma gondii at oxygen levels that mimic those found in tissues. Our previous work showed that Toxoplasma activates the expression of an oxygen-regulated transcription factor that is required for growth. Here, we report that Toxoplasma regulates the abundance and activity of a key host metabolic enzyme, hexokinase 2, by activating HIF-1 and by promoting dissociation of hexokinase 2 from the mitochondrial membrane. Collectively, our data reveal HIF-1/hexokinase 2 as a novel target for an intracellular pathogen that acts by reprograming the host cell's metabolism to create an environment conducive for parasite replication at physiological oxygen levels.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Menendez
authors.affiliation: Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Crystal
authors.initials: C
authors.lastname: Teygong
authors.affiliation: Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Kristin
authors.initials: K
authors.lastname: Wade
authors.affiliation: Department of Microbiology, SUNY at Buffalo School of Medicine, Buffalo, New York, USA.
authors.forename: Celia
authors.initials: C
authors.lastname: Florimond
authors.affiliation: iblader@buffalo.edu.
authors.forename: Ira J
authors.initials: IJ
authors.lastname: Blader
mesh_terms: D000818:Animals; D005786:Gene Expression Regulation / Q000187:drug effects*; D005820:Genetic Testing; D006019:Glycolysis; D006367:HeLa Cells; D006593:Hexokinase / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D064688:Metabolic Flux Analysis; D008810:Mice, Inbred C57BL; D009000:Monocytes / Q000201:enzymology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D014122:Toxoplasma / Q000502:physiology*; D014124:Toxoplasmosis, Animal / Q000473:pathology
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D034741:RNA, Small Interfering; D006593:Hexokinase