Human malaria infections resulting from Plasmodium falciparum have become increasingly difficult to treat due to the emergence of drug-resistant parasites. The P. falciparum purine salvage enzyme purine nucleoside phosphorylase (PfPNP) is a potential drug target. Previous studies, in which PfPNP was targeted by transition state analogue inhibitors, found that those inhibiting human PNP and PfPNPs killed P. falciparum in vitro. However, many drugs have off-target interactions, and genetic evidence is required to demonstrate single target action for this class of potential drugs. We used targeted gene disruption in P. falciparum strain 3D7 to ablate PNP expression, yielding transgenic 3D7 parasites (⌬pfpnp). Lysates of the ⌬pfpnp parasites showed no PNP activity, but activity of another purine salvage enzyme, adenosine deaminase (PfADA), was normal. When compared with wild-type 3D7, the ⌬pfpnp parasites showed a greater requirement for exogenous purines and a severe growth defect at physiological concentrations of hypoxanthine. Drug assays using immucillins, specific transition state inhibitors of PNP, were performed on wild-type and ⌬pfpnp parasites. The ⌬pfpnp parasites were more sensitive to PNP inhibitors that bound hPNP tighter and less sensitive to MT-ImmH, an inhibitor with 100-fold preference for PfPNP over hPNP. The results demonstrate the importance of purine salvage in P. falciparum and validate PfPNP as the target of immucillins.Each year, Plasmodium species infect 300 to 500 million people and cause nearly two million deaths, mostly in children under the age of five in sub-Saharan Africa (1). Most deaths are due to infection with Plasmodium falciparum. Only AIDS and tuberculosis are more lethal human infectious diseases. No vaccine is available, despite intensive international efforts. At present, control of malaria is dependent on prevention with bed nets, insecticides or chemoprophylaxis, and chemotherapeutic treatment of clinical cases. However, chemotherapy for malaria has been complicated by recent increases in mortality and morbidity due to the emergence of drug-resistant strains (2).Because parasitic protozoa are unable to synthesize purines de novo, purine salvage has been proposed as a potential target for chemotherapy of protozoan parasite infections, including those caused by Plasmodium spp. Malaria parasites are obligate intracellular parasites that carry out their asexual cycle in erythrocytes. Unlike most mammalian cells, erythrocytes have no biochemical machinery for de novo purine synthesis, but act as a rich source of purine salvage enzymes, particularly purine nucleoside phosphorylase (PNP) 4 and adenosine deaminase (ADA). The purine salvage pathway of Plasmodium begins with the deamination of adenosine to inosine by ADA, followed by conversion of inosine to hypoxanthine by PNP. The final enzyme in the pathway is hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT). Hypoxanthine is a precursor for all purines and is a central metabolite for nucleic acid synthesis in P. ...

In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

Apicomplexa are obligate intracellular parasites which cause various animal and human diseases including malaria, toxoplasmosis, and cryptosporidiosis. They proliferate by a unique mechanism that combines physically separated semi-closed mitosis of the nucleus and assembly of daughter cells by internal budding. Mitosis occurs in the presence of a nuclear envelope and with little appreciable chromatin condensation. A long standing question in the field has been how parasites keep track of their uncondensed chromatin chromosomes throughout their development, and hence secure proper chromosome segregation during division. Past work demonstrated that the centromeres, the region of kinetochore assembly at chromosomes, of Toxoplasma gondii remain clustered at a defined region of the nuclear periphery proximal to the main microtubule organizing center of the cell, the centrosome. We have proposed that this mechanism is likely involved in the process. Here we set out to identify underlying molecular players involved in centromere clustering. Through pharmacological treatment and structural analysis we show that centromere clustering is not mediated by persistent microtubules of the mitotic spindle. We identify the chromatin binding factor a homolog of structural maintenance of chromosomes 1 (SMC1). Additionally, we show that both TgSMC1, and a centromeric histone, interact with TgExportin1, a predicted soluble component of the nuclear pore complex. Our results suggest that the nuclear envelope, and in particular the nuclear pore complex may play a role in positioning centromeres in T. gondii.

Our knowledge of cell cycle regulatory mechanisms in apicomplexan parasites is very limited. In this study, we describe a novel Toxoplasma gondii factor, essential for chromosome replication 1 (ECR1), that has a vital role in chromosome replication and the regulation of cytoplasmic and nuclear mitotic structures. ECR1 was discovered by complementation of a temperature sensitive (ts) mutant that suffers lethal, uncontrolled chromosome replication at 40 o C similar to a ts-mutant carrying a defect in topoisomerase. ECR1 is a 52kDa protein containing divergent RING and TRAF-Sina like zinc-binding domains that is dynamically expressed in the tachyzoite cell cycle.ECR1 first appears in the centrocone compartment of the nuclear envelope in early S phase and then in the nucleus in late S phase where it reaches maximum expression. Following nuclear division, but before daughters resolve from the mother, ECR1 is down regulated and is absent in new daughter parasites. The proteomics of ECR1 identified interactions with the ubiquitinmediated protein degradation machinery and the minichromosome maintenance complex and the loss of ECR1 led to increased stability of a key member of this complex, MCM2. ECR1 also forms a stable complex with the CDK-related kinase, TgCrk5, which shares a similar cell cycle expression and localization during tachyzoite replication. Altogether, the results of this study suggest ECR1 may be a unique E3 ligase that regulates DNA licensing and other mitotic processes. Importantly, the localization of ECR1/TgCrk5 in the centrocone indicates this Apicomplexa-specific spindle compartment houses important regulatory factors that control the parasite cell cycle. IMPORTANCEParasites of the apicomplexan family are important causes of human disease including malaria, toxoplasmosis, and cryptosporidiosis. Parasite growth is the underlying cause of pathogenesis, yet despite this importance the molecular basis for parasite replication is poorly understood. Filling this knowledge gap cannot be accomplished by mining recent whole genome sequencing because apicomplexan cell cycles differ substantially and lack many of the key regulatory factors of wellstudied yeast and mammalian cell division models. We have utilized forward genetics to discover essential factors that regulate cell division in these parasites using the Toxoplasma gondii model.

Role of funding:The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.