Functional Characterization of Pneumocystis carinii Inositol Transporter 1

Abstract

ABSTRACT Fungi in the genus Pneumocystis live in the lungs of mammals, where they can cause a fatal pneumonia (PCP [ P neumo c ystis p neumonia]) in hosts with compromised immune systems. The absence of a continuous in vitro culture system for any species of Pneumocystis has led to limited understanding of these fungi, especially for the discovery of new therapies. We recently reported that Pneumocystis carinii , Pneumocystis murina , and most significantly, Pneumocystis jirovecii lack both enzymes necessary for myo- inositol biosynthesis but contain genes with homologies to fungal myo -inositol transporters. Since myo -inositol is essential for eukaryotic viability, the primary transporter, ITR1, was functionally and structurally characterized in P. carinii . The predicted structure of P. carinii ITR1 (PcITR1) contained 12 transmembrane alpha-helices with intracellular C and N termini, consistent with other inositol transporters. The apparent K m was 0.94 ± 0.08 (mean ± standard deviation), suggesting that myo- inositol transport in P. carinii is likely through a low-affinity, highly selective transport system, as no other sugars or inositol stereoisomers were significant competitive inhibitors. Glucose transport was shown to use a different transport system. The myo -inositol transport was distinct from mammalian transporters, as it was not sodium dependent and was cytochalasin B resistant. Inositol transport in these fungi offers an attractive new drug target because of the reliance of the fungi on its transport, clear differences between the mammalian and fungal transporters, and the ability of the host to both synthesize and transport this critical nutrient, predicting low toxicity of potential inhibitors to the fungal transporter. IMPORTANCE myo -Inositol is a sugarlike nutrient that is essential for life in most organisms. Humans and microbes alike can obtain it by making it, which involves only 2 enzymes, by taking it from the environment by a transport process, or by recycling it from other cellular constituents. Inspection of the genomes of the pathogenic fungi of the genus Pneumocystis showed that these pneumonia-causing parasites could not make myo -inositol, as they lacked the 2 enzymes. Instead, we found evidence of inositol transporters, which would import the sugar from the lungs where the fungi reside. In the present report, we characterized the transport of myo -inositol in the fungus and found that the transporter was highly selective for myo -inositol and did not transport any other molecules. The transport was distinct from that in mammalian cells, and since mammals can both make and transport myo -inositol, while Pneumocystis fungi must transport it, this process offers a potential new drug target.