Protist Oxygen Sensing in Human Disease Protist Oxygen Sensing in Human Disease

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Sensing and responding to changes in environmental oxygen is critical for aerobic organisms. Metazoans accomplish this by destabilizing a transcription factor called hypoxia inducible factor (HIF) in oxygen sufficiency. This is achieved by an oxygen-dependent prolyl 4-hydroxylase (PHD) that hydroxylates proline residues in the HIF-1α transcription factor that targets it for ubiquitin-dependent proteasomal degradation. Protozoans have PHDs but lack HIF and thus respond to changes in oxygen differently and we use the evolutionary distant protists Dictyostelium and Toxoplasma to address this question. Initial studies in Dictyostelium revealed that its PHD, DdPHYa, modifies a proline in Skp1, which is a component of the Skp1/Cullin1/F-box protein/Rbx1 polyubiquitin ligase complex. Skp1 prolyl hydroxylation does not affect its stability, but allows it to be modified by a pentasaccharide that acts to alter the repertoire of associated F- Box proteins. Genome analysis and biochemical assays demonstrated that this Skp1 modification pathway is conserved in the protozoan parasite Toxoplasma, but not in metazoans. Loss of Toxoplasma PHYa leads decreases virulence in vivo and decreased growth in vitro under limited O2 and amino acid conditions. Unlike Dictyostelium, Toxoplasma expresses a second PHD, PHYb, which is required for colonization of oxygen rich tissues as well as Toxoplasma growth at high oxygen. In contrast to PHYa, PHYb functions by regulating elongation during protein synthesis and specifically does so at elevated O2 levels. Because of its medical importance, we will focus on Toxoplasma and pursue three specific aims: i) Determine how PHYa mediates growth at low oxygen and amino acids; ii) Define how PHYb regulates elongation; and iii) Test whether PHYa and PHYb work in tandem as an oxygen-sensing rheostat to grow in whatever oxygen tension in encounters as it infects a host and causes disease.