Plasmodium falciparum malaria parasites were transformed with plasmids containing P. falciparum or Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (dhfr-ts) coding sequences that confer resistance to pyrimethamine. Under pyrimethamine pressure, transformed parasites were obtained that maintained the transfected plasmids as unrearranged episomes for several weeks. These parasite populations were replaced after 2 to 3 months by parasites that had incorporated the transfected DNA into nuclear chromosomes. Depending upon the particular construct used for transformation, homologous integration was detected in the P. falciparum dhfr-ts locus (chromosome 4) or in hrp3 and hrp2 sequences that were used in the plasmid constructs as gene control regions (chromosomes 13 and 8, respectively). Transformation by homologous integration sets the stage for targeted gene alterations and knock-outs that will advance understanding of P. falciparum.

Chloroquine resistance in a P. falciparum cross maps as a Mendelian trait to a 36 kb segment of chromosome 7. This segment harbors cg2, a gene encoding a unique approximately 330 kDa protein with complex polymorphisms. A specific set of polymorphisms in 20 chloroquine-resistant parasites from Asia and Africa, in contrast with numerous differences in 21 sensitive parasites, suggests selection of a cg2 allele originating in Indochina over 40 years ago. One chloroquine-sensitive clone exhibited this allele, suggesting another resistance component. South American parasites have cg2 polymorphisms consistent with a separate origin of resistance. CG2 protein is found at the parasite periphery, a site of chloroquine transport, and in association with hemozoin of the digestive vacuole, where chloroquine inhibits heme polymerization.

SignificanceProtozoal proteasome is a validated target for antimalarial drug development, but species selectivity of reported inhibitors is suboptimal. Here we identify inhibitors with improved selectivity for malaria proteasome β5 subunit over each active subunit of human proteasomes. These compounds kill the parasite in each stage of its life cycle. They interact synergistically with a β2 inhibitor and with artemisinin. Resistance to the β5 inhibitor arose through a point mutation in the nonproteolytic β6 subunit. The same mutation made the mutant strain more sensitive to a β2 inhibitor and less fit to withstand irradiation. These findings reveal complex interplay among proteasome subunits and introduce the prospect that combined inhibition of β2 and β5 subunits can afford synergy and thwart resistance.

Malaria parasites replicate asexually within their mammalian hosts as haploid cells and are subject to DNA damage from the immune response and chemotherapeutic agents that can significantly disrupt genomic integrity. Examination of the annotated genome of the parasite Plasmodium falciparum identified genes encoding core proteins required for the homologous recombination (HR) pathway for repairing DNA double-strand breaks (DSBs), but surprisingly none of the components of the canonical non-homologous end joining (C-NHEJ) pathway were identified. To better understand how malaria parasites repair DSBs and maintain genome integrity, we modified the yeast I-SceI endonuclease system to generate inducible, site-specific DSBs within the parasite’s genome. Analysis of repaired genomic DNA showed that parasites possess both a typical HR pathway resulting in gene conversion events as well as an end joining (EJ) pathway for repair of DSBs when no homologous sequence is available. The products of EJ were limited in number and identical products were observed in multiple independent experiments. The repair junctions frequently contained short insertions also found in the surrounding sequences, suggesting the possibility of a templated repair process. We propose that an alternative end-joining pathway rather than C-NHEJ, serves as a primary method for repairing DSBs in malaria parasites.

Summary Resistance to antimalarials targeting the folate pathway is widespread. GTP-cyclohydrolase (gch1), the first enzyme in this pathway, exhibits extensive copy number variation (CNV) in parasite isolates from areas with a history of longstanding antifolate use. Increased CN of gch1 is associated with a greater number of point mutations in enzymes targeted by the antifolates, pyrimethamine and sulfadoxine. While these observations suggest that increases in gch1 CN are an adaptation to drug pressure, changes in CN have not been experimentally demonstrated to directly alter drug susceptibility. To determine if changes in gch1 expression alone modify pyrimethamine sensitivity, we manipulated gch1 CN in several parasite lines to test the effect on drug sensitivity. We report that increases in gch1 CN alter pyrimethamine resistance in most parasites lines. However we find evidence of a detrimental effect of very high levels of gch1 overexpression in parasite lines with high endogenous levels of gch1 expression, revealing the importance of maintaining balance in the folate pathway and implicating changes in gch1 expression in preserving proper metabolic flux. This work expands our understanding of parasite adaptation to drug pressure and provides a possible mechanism for how specific mutations become fixed within parasite populations.

The ability of Toxoplasma gondii tachyzoites to differentiate into latent bradyzoite forms is essential for pathogenesis of clinical disease. We examined the effects of cyclic nucleotides on T. gondii bradyzoite differentiation in vitro. Differentiation of tachyzoites to bradyzoites was measured in an immunofluorescence assay using ME49 or its clonal derivative PLK, two well-characterized T. gondii strains. Treatment of human fibroblast cultures infected with T. gondii with 8-(4-chlorophenylthio)-cyclic GMP (CPT-cGMP), a membranepermeable, nonhydrolyzable analogue of cGMP, resulted in an increased percentage of bradyzoite-positive vacuoles. Cyclic AMP (cAMP) also induced in vitro conversion of PLK, but the method of cAMP elevation was critical. Forskolin raises cAMP levels transiently and induced bradyzoites, whereas agents predicted to cause sustained elevation of cAMP were inhibitory to parasite conversion. Levels of cAMP were measured in host cells and extracellular tachyzoites. Forskolin, CPT-cGMP, and agents known to induce bradyzoite formation elevated cAMP in host cells and PLK parasites. These data suggest cyclic nucleotide signaling pathways are important in the stress-induced conversion of T. gondii tachyzoites to bradyzoites. Furthermore, because cAMP elevation was seen in PLK but not RH, a T. gondii strain that did not differentiate well in our assay, cAMP signaling within the parasite is likely to be critical.Toxoplasma gondii is an obligate intracellular apicomplexan parasite responsible for encephalitis in immunocompromised individuals and birth defects in children infected in utero. Although some individuals present with toxoplasmosis during acute infection, most clinically apparent disease results from reactivation of dormant bradyzoites and their conversion to tachyzoites. Unchecked multiplication of the rapidly growing tachyzoite is thought to be responsible for disease, and control of tachyzoites by the immune system results in their conversion to latent bradyzoite forms. Thus, elucidation of the signaling pathways responsible for tachyzoite-bradyzoite interconversion is critical for understanding pathogenesis of toxoplasmosis.Recent studies by several investigators have established that a variety of stress conditions including pH shock, heat shock, mitochondrial inhibitors, chemical stress, and nitric oxide induce bradyzoite formation (2,3,21,23). Induction of a variety of heat shock proteins (HSPs) including HSP70 is associated with bradyzoite transition (19,25), and knockout of a bradyzoite-specific small HSP gene, BAG1, results in reduced numbers of bradyzoites in mouse brains (29). These data collectively suggest that the transition from tachyzoite to bradyzoite is a stress-induced differentiation response. Because of the remarkable conservation of cyclic nucleotide signaling pathways in the stress response in a wide variety of organisms including other eukaryotic pathogens, we examined the role of cyclic nucleotide signaling in bradyzoite differentiation in T. gondii.Our data suggest th...

The human malaria parasite Plasmodium falciparum utilizes a mechanism of antigenic variation to avoid the antibody response of its human host and thereby generates a long-term, persistent infection. This process predominantly results from systematic changes in expression of the primary erythrocyte surface antigen, a parasite-produced protein called PfEMP1 that is encoded by a repertoire of over 60 var genes in the P. falciparum genome. var genes exhibit extensive sequence diversity, both within a single parasite's genome as well as between different parasite isolates, and thus provide a large repertoire of antigenic determinants to be alternately displayed over the course of an infection. While significant work has recently been published documenting the extreme level of diversity displayed by var genes found in natural parasite populations, little work has been done regarding the mechanisms that lead to sequence diversification and heterogeneity within var genes. In the course of producing transgenic lines from the original NF54 parasite isolate, we cloned and characterized a parasite line, termed E5, which is closely related to but distinct from 3D7, the parasite used for the P. falciparum genome nucleotide sequencing project. Analysis of the E5 var gene repertoire, as well as that of the surrounding rif and stevor multi-copy gene families, identified examples of frequent recombination events within these gene families including an example of a duplicative transposition indicating recombination events play a significant role in the generation of diversity within the antigen encoding genes of P. falciparum.

, a zoonotic intraerythrocytic parasite, is the primary etiological agent of human babesiosis in the United States. Human infections range from subclinical illness to severe disease resulting in death, with symptoms being related to host immune status. Despite advances in our understanding and management of , the incidence of infection in the United States has increased. Therefore, research focused on eradicating disease and optimizing clinical management is essential. Here we review this remarkable organism, with emphasis on the clinical, diagnostic, and therapeutic aspects of human disease.