Plasmodium falciparum malaria is increasingly difficult to treat and control due to the emergence of parasite resistance to the major antimalarials, notably chloroquine. Recent work has shown that the chloroquine resistance phenotype can be conferred by multiple amino acid mutations in the parasite digestive vacuole transmembrane protein PfCRT. Here, we have addressed whether chloroquine resistance can also be affected by changes in expression levels of this protein.Transient transfection reporter assays revealed that truncation of the pfcrt 3-untranslated region just prior to putative polyadenylation sites resulted in a 10-fold decrease in luciferase expression levels. Using allelic exchange on a chloroquine-resistant line (7G8 from Brazil), this truncated 3-untranslated region was inserted downstream of the pfcrt coding sequence, in the place of the endogenous 3-untranslated region. The resulting pfcrt-modified "knockdown" clones displayed a marked decrease in pfcrt transcription and an estimated 30 -40% decrease in PfCRT protein expression levels. [ 3 H]hypoxanthine incorporation assays demonstrated up to a 40% decrease in chloroquine with or without verapamil IC 50 levels of pfcrt knockdown clones, relative to the 7G8 parent. Single-cell photometric analyses were consistent with an altered intracellular pH in the knockdown clones, providing further evidence for a relationship between PfCRT, pH regulation, and chloroquine resistance. Genetic truncation of 3-untranslated regions provides a useful approach for assessing the impact of candidate genes on drug resistance or other quantifiable phenotypes in P. falciparum.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) clusters at electron-dense knob-like structures on the surface of malaria-infected red blood cells and mediates their adhesion to the vascular endothelium. In parasites lacking knobs, vascular adhesion is less efficient, and infected red cells are not able to immobilize successfully under hemodynamic flow conditions even though PfEMP1 is still present on the exterior of the infected red cell. We examined the interaction between the knob-associated histidine-rich protein (KAHRP), the parasite protein upon which knob formation is dependent, and PfEMP1, and we show evidence of a direct interaction between KAHRP and the cytoplas- Plasmodium falciparum causes the most severe form of human malaria and is responsible for at least two million deaths worldwide each year. The excessive pathogenicity of P. falciparum appears to be related to an ability to cause infected red blood cells to adhere to vascular endothelium and sequester in

The Plasmodium falciparum mature parasite-infected erythrocyte surface antigen (MESA) is exported from the parasite to the infected red blood cell (

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. ...

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38-97 of PfEMP3 bound to purified spectrin with moderately high affinity (K(D(kin))=8.5 x 10(-8) M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K(D(kin))=3.8 x 10(-7) M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.

No abstract

Infection of erythrocytes by the malaria parasite Plasmodium falciparum results in the export of several parasite proteins into the erythrocyte cytoplasm. Changes occur in the infected erythrocyte due to altered phosphorylation of proteins and to novel interactions between host and parasite proteins, particularly at the membrane skeleton. In erythrocytes, the spectrin based red cell membrane skeleton is linked to the erythrocyte plasma membrane through interactions of ankyrin with spectrin and band 3. Here we report an association between the P. falciparum histidine-rich protein (PfHRP1) and phosphorylated proteolytic fragments of red cell ankyrin. Immunochemical, biochemical and biophysical studies indicate that the 89 kDa band 3 binding domain and the 62 kDa spectrin-binding domain of ankyrin are co-precipitated by mAb 89 against PfHRP1, and that native and recombinant ankyrin fragments bind to the 5' repeat region of PfHRP1. PfHRP1 is responsible for anchoring the parasite cytoadherence ligand to the erythrocyte membrane skeleton, and this additional interaction with ankyrin would strengthen the ability of PfEMP1 to resist shear stress.

No abstract