An Oxygen Molecular Sensor, the HIF Prolyl 4-Hydroxylase, in the Marine Protist Perkinsus olseni

Abstract

Adaptations to changes in oxygen availability are crucial for survival and have been shown to be implicated in several disease states. Such adaptations are known to rely upon gene expression and regulation of hypoxia-inducible factors (HIF) and are mainly promoted by HIF Prolyl Hydroxylases (HPHs). These enzymes are involved in intracellular signaling and function as oxygen sensors. In the presence of molecular oxygen, HIF1α becomes hydroxylated at two specific prolyl residues and is targeted for destruction, whereas in the absence of oxygen (hypoxia conditions), HIF is not hydroxylated and thus remains free to activate HIF target genes. We have cloned the cDNAs corresponding to four different HPH transcripts from the bivalve parasite Perkinsus olseni, a unicellular eukaryotic organism, and determined the corresponding gene structure. Furthermore, we have analyzed the relationship between expression of this gene and parasite virulence. HPH-like genes were previously identified in other microorganisms, but only through comparative genomic analysis, and exclusively in pathogenic bacteria, thus suggesting that they may be involved in pathogen activity. To investigate a possible correlation between Perkinsus pathogenicity and HPH gene expression, pure cultures of this parasite were exposed to hemolymph from different bivalve species, either susceptible or resistant to Perkinsus, and HPH gene expression analyzed by real-time PCR. Our data show a massive increase in HPH expression in the presence of the susceptible host's hemolymph, indicative of the importance of HPH in infection mechanisms. In addition, the dependence of HIF-like enzymes on other factors or substrates known to be associated with their biological function, such as iron, 2-oxoglutarate (2OG), glycolytic enzymes and oxygen, was also investigated through the use of iron chelators, 2OG antagonists, glycolytic inhibitors and hypoxia conditions. A clear correlation was observed between the production of reactive oxygen species and HPH activity, in agreement with studies that indicate a stabilization of HIF by inhibition of HPH. Interestingly, it was also possible to conclude, through phylogenetic analysis, that Perkinsus HPH is closer to the actual HPH2 from mammals, thus providing additional evidence supporting previous data pointing to HPH2 as the most ancestral of the three HPH isoforms known in metazoans.