The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over three months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6–48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.

Summary Whereas mammalian cells and most other organisms can synthesize polyamines from basic amino acids, the protozoan parasite Trypanosoma cruzi is incapable of polyamine biosynthesis de novo and therefore obligatorily relies upon putrescine acquisition from the host to meet its nutritional requirements. The cell surface proteins that mediate polyamine transport into T. cruzi, as well as most eukaryotes, however, have by-in-large eluded discovery at the molecular level. Here we report the identification and functional characterization of two polyamine transporters, TcPOT1.1 and TcPOT1.2, encoded by alleles from two T. cruzi haplotypes. Overexpression of the TcPOT1.1 and TcPOT1.2 genes in T. cruzi epimastigotes revealed that TcPOT1.1 and TcPOT1.2 were high-affinity transporters that recognized both putrescine and cadaverine but not spermidine or spermine. Furthermore, the activities and subcellular locations of both TcPOT1.1 and TcPOT1.2 in intact parasites were profoundly influenced by extracellular putrescine availability. These results establish TcPOT1.1 and TcPOT1.2 as key components of the T. cruzi polyamine transport pathway, an indispensable nutritional function for the parasite that may be amenable to therapeutic manipulation.

c Endochin-like quinolones (ELQs) are potent and specific inhibitors of cytochrome bc 1 from Plasmodium falciparum and Toxoplasma gondii and show promise for novel antiparasitic drug development. To determine whether the mitochondrial electron transport chain of Leishmania parasites could be targeted similarly for drug development, we investigated the activity of 134 structurally diverse ELQs. A cohort of ELQs was selectively toxic to amastigotes of Leishmania mexicana and L. donovani, with 50% inhibitory concentrations (IC 50 s) in the low micromolar range, but the structurally similar hydroxynaphthoquinone buparvaquone was by far the most potent inhibitor of electron transport, ATP production, and intracellular amastigote growth. Cytochrome bc 1 is thus a promising target for novel antileishmanial drugs, and further improvements on the buparvaquone scaffold are warranted for development of enhanced therapeutics.

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Gene expression in kinetoplastid parasites is regulated via post-transcriptional mechanisms that modulate mRNA turnover, translation rate, and/or post-translational protein stability. To facilitate the analysis of post-transcriptional regulation, a dual luciferase system was developed in which firefly and Renilla luciferase reporters genetically fused to compatible drug resistance genes are integrated in place of one allele of the gene of interest and of an internal control gene, respectively, in a manner that preserves the cognate pre-mRNA processing signals. The sensitivity and reproducibility of the assay coupled with the ability to rapidly assemble reporter integration constructs render the dual luciferase system suitable for analysis of multiple candidates derived from global expression analysis platforms. To demonstrate the utility of the system, regulation of three genes in response to purine starvation was examined in Leishmania donovani promastigotes. This dual luciferase system should be directly applicable to the analysis of post-transcriptional regulation in other kinetoplastids.