Philip J. Rosenthal

Tetracyclines are effective but slow-acting antimalarial drugs whose mechanism of action remains uncertain. To characterize the antimalarial mechanism of tetracyclines, we evaluated their stage-specific activities, impacts on parasite transcription, and effects on two predicted organelle targets, the apicoplast and the mitochondrion, in cultured Plasmodium falciparum . Antimalarial effects were much greater after two 48-h life cycles than after one cycle, even if the drugs were removed at the end of the first cycle. Doxycycline-treated parasites appeared morphologically normal until late in the second cycle of treatment but failed to develop into merozoites. Doxycycline specifically impaired the expression of apicoplast genes. Apicoplast morphology initially appeared normal in the presence of doxycycline. However, apicoplasts were abnormal in the progeny of doxycycline-treated parasites, as evidenced by a block in apicoplast genome replication, a lack of processing of an apicoplast-targeted protein, and failure to elongate and segregate during schizogeny. Replication of the nuclear and mitochondrial genomes and mitochondrial morphology appeared normal. Our results demonstrate that tetracyclines specifically block expression of the apicoplast genome, resulting in the distribution of nonfunctional apicoplasts into daughter merozoites. The loss of apicoplast function in the progeny of treated parasites leads to a slow but potent antimalarial effect.

Falcipain-2 (FP2) is a papain family cysteine protease and important hemoglobinase of erythrocytic Plasmodium falciparum parasites. Inhibitors of FP2 block hemoglobin hydrolysis and parasite development, suggesting that this enzyme is a promising target for antimalarial chemotherapy. FP2 and related plasmodial cysteine proteases have an unusual 14-aa motif near the C terminus of the catalytic domain. Recent solution of the structure of FP2 showed this motif to form a β-hairpin that is distant from the enzyme active site and protrudes out from the protein. To evaluate the function of this motif, we compared the activity of the wild-type enzyme with that of a mutant lacking 10 aa of the motif ( ^Δ10 FP2). ^Δ10 FP2 had nearly identical activity to that of the wild-type enzyme against peptide substrates and the protein substrates casein and gelatin. However, ^Δ10 FP2 demonstrated negligible activity against hemoglobin or globin. FP2 that was inhibited with trans -epoxysuccinyl- l -leucylamido-(4-guanidino)butane (FP2 ^E-64 ) formed a complex with hemoglobin, but ^Δ10 FP2 ^E-64 did not, indicating that the motif mediates binding to hemoglobin independent of the active site. A peptide encoding the motif blocked hemoglobin hydrolysis, but not the hydrolysis of casein. Kinetics for the inhibition of ^Δ10 FP2 were very similar to those for FP2 with peptidyl and protein inhibitors, but ^Δ10 FP2 was poorly inhibited by the inhibitory prodomain of FP2. Our results indicate that FP2 utilizes an unusual motif for two specific functions, interaction with hemoglobin, its natural substrate, and interaction with the prodomain, its natural inhibitor.

Among potential new targets for antimalarial chemotherapy are Plasmodium falciparum cysteine proteases, known as falcipains. Falcipain-2 and falcipain-3 are food vacuole hemoglobinases that may have additional functions. The function of falcipain-1 remains uncertain. To better characterize the role of falcipain-1 in erythrocytic parasites, we disrupted the falcipain-1 gene and characterized recombinant parasites. Disruption of the falcipain-1 gene was confirmed with Southern blots, and loss of expression of falcipain-1 was confirmed with immunoblots and by loss of labeling with a specific protease inhibitor. Compared with wild-type parasites, falcipain-1 knockout parasites developed normally, with the same morphology, multiplication rate, and invasion efficiency, and without significant differences in sensitivity to cysteine protease inhibitors. In wild-type and knockout parasites, cysteine protease inhibitors blocked hemoglobin hydrolysis in trophozoites, with a subsequent block in rupture of erythrocytes by mature schizonts, but they did not inhibit erythrocyte invasion by merozoites. Our results indicate that although falcipain-1 is expressed by erythrocytic parasites, it is not essential for normal development during this stage or for erythrocyte invasion.

Summary: Sulphadoxine/pyrimethamine (SP) has become the first‐line treatment of uncomplicated malaria in a number of African countries. Molecular surveillance of resistance‐mediating mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) has been proposed as a means of predicting SP treatment outcomes, but optimal methods of surveillance in different populations have not been well established. To investigate the relationship between molecular markers of SP resistance, host immunity, and response to therapy, we evaluated the association between the presence of five key dhfr and dhps mutations at enrollment and clinical outcome in children and adults treated with SP for uncomplicated malaria in Kampala, Uganda. Clinical treatment failure was 11% at 14 days, increasing to 30% at 28 days, after excluding new infections. Outcomes varied markedly based on the number of dhfr and dhps mutations and on the age of treated subjects. All infections with less than two dhfr/dhps mutations were successfully treated. Treatment failure associated with any two, three, or four dhfr/dhps mutations occurred in nine of 24 (38%) children up to 5 years, but not in older patients (0/20). In the presence of all five mutations, treatment failure occurred equally in children aged 5 years or younger [7/16 (44%)] and in older patients [8/16 (50%)]. Our results showed that age, a surrogate marker of antimalarial immunity, had a major impact on the relationship between polymorphisms in SP target enzymes and treatment outcomes. The use of molecular markers of SP resistance to predict treatment failure rates should take age into account.