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New effective antimalarial agents are urgently needed due to increasing drug resistance of Plasmodium falciparum. Phosphorothioate antisense oligodeoxynucleotides (ODNs) silencing of malarial topoisomerase II gene have shown to possess promising features as anti malarial agents. In order to improve stability and to increase intracellular penetration, ODNs were complexed with the biodegradable polymer chitosan to form solid nanoparticles with an initial diameter of approximately 55 nm. The particle zetapotential depended on the chitosan/ODN mass ratio. Nanoparticles with mass ratio of 2:1 displayed a positive surface charge (+15 mV) whereas particles with 1:1 mass ratio were negatively charged (-20 mV). Additionally nanoparticles were found to protect ODNs from nuclease degradation. P. falciparum K1 strain was exposed to the chitosan/ODN-nanoparticles for 48 h in order to examine the effects of chitosan/antisense (AS) and chitosan/sense (S) oligodeoxynucleotide nanoparticles on malaria parasite growth. Both negatively and positively charged antisense nanoparticles as well as free antisense ODNs (in a final concentration of 0.5 microM) showed sequence specific inhibition compared with sense sequence controls. However, nanoparticles were much more sequence specific in their antisense effect than free ODNs. Nanoparticles with negative surface charge exhibited a significantly stronger inhibitory effect ( approximately 87% inhibition) on the parasite growth in comparison to the positive ones ( approximately 74% inhibition) or free ODNs ( approximately 68% inhibition). This is the first study demonstrating the susceptibility of P. falciparum to antisense nanoparticles.

Threonine-peptidases of the T1-family are multi-subunit complexes with broad substrate specificity. In eukaryotes, at least 14 genes encode subunits of the prototypic T1 threonine-peptidase, the proteasome. The proteasome determines the turnover of most proteins and thereby plays a fundamental role in diverse processes such as protein quality control, signal transduction, and cell cycle regulation. While eukaryotes and archaea possess a proteasome, bacteria generally express a second member of the T1-family, the proteasomal predecessor ClpQ/hslV that has a similar structure but is encoded by only one gene. The plasmodial genome is an exception because it encodes proteasomal subunits as well as a ClpQ/hslV-orthologe (Plasmodium falciparum-hslV; PfhslV). Structure, expression, and function of both types of peptidase-complex in P. falciparum are presently unknown. Our aim was to analyze both the coding sequences and derived proteins of both peptidase-complexes because highly specific and potent inhibitors can be designed against this class of enzymes. The proteasome was found expressed throughout the cell cycle, whereas PfhslV was detectable in schizonts and merozoites only. Treatment of P. falciparum with the threonine-peptidase inhibitor epoxomicin blocked two of three catalytically active proteasome subunits. This led to the accumulation of ubiquitinated proteins and, finally, to parasite death. In conclusion, we provide the first functional analysis of plasmodial threonine-peptidase-complexes and identify a lead compound for the development of a novel class of antimalarial drugs.

A Plasmodium falciparum gene closely linked to the chloroquine resistance locus encodes PfCG2, a predicted 320-330kDa protein. In the parasitized erythrocyte, PfCG2 expression rises sharply in the trophozoite stage and is detected in electron-dense patches along the parasitophorous vacuolar membrane (PVM), in the cytoplasm and in the digestive vacuole (DV). Results of extraction and partitioning experiments show that PfCG2 is a peripheral membrane protein. Exposure of trophozoite-infected erythrocytes to trypsin-containing buffer after streptolysin O permeabilization indicates that PfCG2 is exposed to the erythrocyte cytosol at the outer face of the PVM. PfCG2 is highly susceptible to hydrolysis by aspartic and cysteine proteases and shows dose-dependent accumulation in the presence of protease inhibitors. These results suggest that PfCG2 is delivered from the outside face of the PVM to the DV, where it is broken down by parasite proteases. PfCG2 interacts with erythrocyte cytoplasm and may be associated with processes of hemoglobin uptake and digestion by erythrocytic-stage parasites.

When bloodstream forms of Trypanosoma brucei differentiate into procyclic forms they rapidly synthesise a new surface coat composed of procyclins. Procyclin genes are transcribed in bloodstream forms at approximately one-tenth of the rate in procyclic forms, but little, if any, mRNA can be detected, indicating that further down-regulation must occur post-transcriptionally. We have examined the role of the 297 bp procyclin 3' untranslated region (UTR) in regulating expression in bloodstream forms and have identified three discrete elements: a dominant, negative element between positions 101 and 173, and two positive elements. When chloramphenicol acetyl transferase (CAT) was used as the reporter gene, deletion of the negative element caused a approximately 6-fold increase in the level of steady state mRNA and > 30-fold increase in CAT activity, suggesting that both RNA stability and translation were affected. Similar results were obtained with glutamic acid/alanine-rich protein (GARP), the T. congolense analogue of procyclin, indicating that the 3' UTR acts independently of the coding region. In contrast, when trypanosomes were stably transformed with a construct in which the procyclin coding region was linked to a truncated form of the 3' UTR which lacked the negative element, they expressed high levels of mRNA, but no protein could be detected in cell lysates or culture supernatants. These results imply that the procyclin coding region exerts yet another layer of control which prevents inappropriate expression of the protein in the mammalian host.

Nucleoside transport may play a critical role in successful intracellular parasitism by Toxoplasma gondii. This protozoan is incapable of de novo purine synthesis, and must salvage purines from the host cell. We characterized purine transport by extracellular T. gondii tachyzoites, focusing on adenosine, the preferred salvage substrate. Although wild-type RH tachyzoites concentrated [3H]adenosine 1.8-fold within 30 s, approx. half of the [3H]adenosine was converted to nucleotide, consistent with the known high parasite adenosine kinase activity. Studies using an adenosine kinase deficient mutant confirmed that adenosine transport was non-concentrative. [14C]Inosine, [14C]hypoxanthine and [3H]adenine transport was also rapid and non-concentrative. Adenosine transport was inhibited by dipyridamole (IC50 approx. 0.7 microM), but not nitrobenzylthioinosine (15 microM). Transport of inosine, hypoxanthine and adenine was minimally inhibited by 10 microM dipyridamole, however. Competition experiments using unlabeled nucleosides and bases demonstrated distinct inhibitor profiles for [3H]adenosine and [14C]inosine transport. These results are most consistent with a single, dipyridamole-sensitive, adenosine transporter located in the T. gondii plasma membrane. Additional permeation pathways for inosine, hypoxanthine, adenine and other purines may also be present.

Appropriate functioning of mitochondria is critical for survival and growth of erythrocytic stages of malarial parasites, making it an attractive target for antimalarial drugs which may take advantage of unique features of parasite mitochondrial metabolism. We have sequenced the presumptive mitochondrial DNA, the 6-kb element, of Plasmodium falciparum, permitting an analysis of the predicted structure of parasite electron transport proteins. Although the overall structures of the 3 polypeptides, cytochrome c oxidase subunit 1, cytochrome c oxidase subunit 3, and cytochrome b (cyt b), were similar to those from other species, some striking differences were observed, especially for the cyt b. Analysis of the cyt b structure showed that the critical quinone binding sites of the protein are quite divergent from those of other species. Comparative analysis suggests that these changes are the likely cause for the resistance of parasite cytochrome bc1 complex to antimycin and related inhibitors. We suggest that the same features are responsible for increased affinity of the parasite cyt b for antimalarial compounds of class 8-aminoquinolines and hydroxynaphthoquinones, explaining the therapeutic value of these drugs.