Parasitic organisms, including the intracellular parasite Toxoplasma gondii, are specialised at stealing nutrients from their hosts in order to survive and proliferate.
T. gondii, the causative agent of significant disease in humans and animals, is a successful intracellular pathogen capable of infecting a wide range of hosts, and within those hosts, a variety of tissue types. T. gondii parasites encounter diverse nutrient environments as they infect different tissues and must adapt to these differences. The scavenging of the essential amino acid arginine, is tightly regulated and its uptake is mediated by two plasma-membrane localised transporters, including the high-affinity transporter, TgApiAT1. We have previously shown that TgApiAT1 expression is highly regulated, with its protein abundance inversely correlated with arginine levels encountered by the parasite. In host organs where arginine levels are low (such as the liver), the parasite expresses higher levels of TgApiAT1 and in organs with high levels of arginine (like the kidneys), TgApiAT1 expression is repressed. Regulation of TgApiAT1 expression depends on a conserved upstream open reading frame (uORF) in the 5’ untranslated region (5’UTR) of the TgApiAT1 transcript. To identify factors involved in the arginine-dependent regulation of TgApiAT1, we developed a parasite strain expressing an arginine-sensitive fluorescent reporter and using this strain, we undertook a flow cytometry-based genome-wide CRISPR/Cas9 screen. This screen identified a nucleolus-localised SPOUT-family RNA methyltransferase (TgRMT) as an important regulator of TgApiAT1 expression. Homologs of TgRMT have recently been shown to mediate the methylation of a universally conserved uridine within the Peptidyl Transferase Center of ribosomal RNA in animals and plants. We provide evidence that the equivalent nucleotide is also modified by TgRMT, and the catalytic activity of TgRMT is required for the repression of TgApiAT1 expression in arginine-replete conditions. We have now begun developing approaches including ribosome profiling, to determine the mechanisms with which this rRNA modification mediates the uORF-mediated translational control of a key nutrient transporter. Overall, our findings highlight ways in which a model intracellular pathogen senses and responds to nutrient availability in its environment.