Delayed parasite elimination in human infections treated with clindamycin parallels 'delayed death' of Plasmodium falciparum in vitro

Clindamycin is safe and effective for the treatment of Plasmodium falciparum malaria, but its use as monotherapy is limited by unacceptably slow initial clinical response rates. To investigate whether the protracted action is due to an accumulative, time of exposure-dependent or a delayed effect on parasite growth, we studied the in vivo and in vitro pharmacodynamic profiles of clindamycin against P. falciparum. In vivo, elimination of young, circulating asexual parasite stages during treatment with clindamycin displayed an unusual biphasic kinetic: a plateau phase was followed by a precipitated decline of asexual parasite densities to nearly undetectable levels after 72 and 60 h in adult patients and asymptomatic children, respectively, suggesting an uninhibited capacity to establish a second, but not third, infectious cycle. In vitro, continuous exposure of a laboratory-adapted P. falciparum strain to clindamycin with concentrations of up to 100 microM for two replication cycles (96 h) did not produce inhibitory effects of >50% compared with drug-free controls as measured by the production of P. falciparum histidine-rich protein II (PfHRP2). PfHRP2 production was completely arrested after the second cycle (96-144h) (>10,000-fold decrease of mean half-inhibitory concentrations measured at 96-144h compared to 48-96h). Furthermore, incubation with clindamycin during only the first (0-48h) versus three (0-144h) parasite replication cycles led to comparable inhibition of PfHRP2 production in the third infectious cycle (96-144h) (mean IC(99) of 27 and 22nM, respectively; P=0.2). When parasite cultures were exposed to different concentrations of clindamycin ranging from 50 to 1,000nM for 72h and followed up in an experiment designed to simulate a typical 3-day treatment regimen, parasitaemia was initially suppressed below the microscopic detection threshold. Nonetheless, parasites reappeared in a dose-dependent manner after removal of drug at 72h but not in continuously drug-exposed controls. The delayed, but potent, antimalarial effect of clindamycin appears to be of greatest potential benefit in new combinations of clindamycin with rapidly acting antimalarial combination partners.     

Within-host competition and drug resistance in the human malaria parasite Plasmodium falciparum

Infections with the malaria parasite Plasmodium falciparum typically comprise multiple strains, especially in high-transmission areas where infectious mosquito bites occur frequently. However, little is known about the dynamics of mixed-strain infections, particularly whether strains sharing a host compete or grow independently. Competition between drug-sensitive and drug-resistant strains, if it occurs, could be a crucial determinant of the spread of resistance. We analysed 1341 P. falciparum infections in children from Angola, Ghana and Tanzania and found compelling evidence for competition in mixed-strain infections: overall parasite density did not increase with additional strains, and densities of individual chloroquine-sensitive (CQS) and chloroquine-resistant (CQR) strains were reduced in the presence of competitors. We also found that CQR strains exhibited low densities compared with CQS strains (in the absence of chloroquine), which may underlie observed declines of chloroquine resistance in many countries following retirement of chloroquine as a first-line therapy. Our observations support a key role for within-host competition in the evolution of drug-resistant malaria. Malaria control and resistance-management efforts in high-transmission regions may be significantly aided or hindered by the effects of competition in mixed-strain infections. Consideration of within-host dynamics may spur development of novel strategies to minimize resistance while maximizing the benefits of control measures.     

Dihydroorotase of human malarial parasite Plasmodium falciparum differs from host enzyme

Plasmodium falciparum, the causative agent of human malaria, is totally dependent on de novo pyrimidine biosynthetic pathway. A gene encoding P. falciparum dihydroorotase (pfDHOase) was cloned and expressed in Escherichia coli as monofunctional enzyme. PfDHOase revealed a molecular mass of 42 kDa. In gel filtration chromatography, the major enzyme activity eluted at 40 kDa, indicating that it functions in a monomeric form. This was similarly observed using the native enzyme purified from P. falciparum. Interestingly, kinetic parameters of the enzyme and inhibitory effect by orotate and its 5-substituted derivatives parallel that found in mammalian type I DHOase. Thus, the malarial enzyme shares characteristics of both type I and type II DHOases. This study provides the monofunctional property of the parasite DHOase lending further insights into its differences from the human enzyme which forms part of a multifunctional protein.     

4-Thiophenoxy-2-trichloromethyquinazolines display in vitro selective antiplasmodial activity against the human malaria parasite Plasmodium falciparum

A series of original quinazolines bearing a 4-thiophenoxy and a 2-trichloromethyl group was synthesized in a convenient and efficient way and was evaluated toward its in vitro antiplasmodial potential. The series revealed global good activity against the K1-multi-resistant Plasmodium falciparum strain, especially with hit compound 5 (IC(50)=0.9 μM), in comparison with chloroquine and doxycycline chosen as reference-drugs. Both the in vitro cytotoxicity study which was conducted on the human HepG2 cell line and the in vitro antitoxoplasmic screening against Toxoplasma gondii indicate that this series presents an interesting selective antiplasmodial profile. Structure-activity- and toxicity relationships highlight that the trichloromethyl group plays a key role in the antiplasmodial activity and also show that the modulation of the thiophenol moiety influences the toxicity/activity ratio.     

Plasmodium falciparum: food vacuole localization of nitric oxide-derived species in intraerythrocytic stages of the malaria parasite

Nitric oxide (NO) has diverse biological functions. Numerous studies have documented NO’s biosynthetic pathway in a wide variety of organisms. Little is known, however, about NO production in intraerythrocytic Plasmodium falciparum. Using diaminorhodamine-4-methyl acetoxymethylester (DAR-4M AM), a fluorescent indicator, we obtained direct evidence of NO and NO-derived reactive nitrogen species (RNS) production in intraerythrocytic P. falciparum parasites, as well as in isolated food vacuoles from trophozoite stage parasites. We preliminarily identified two gene sequences that might be implicated in NO synthesis in intraerythrocytic P. falciparum. We showed localization of the protein product of one of these two genes, a molecule that is structurally similar to a plant nitrate reductase, in trophozoite food vacuole membranes. We confirmed previous reports on the antiproliferative effect of NOS (nitric oxide synthase) inhibitors in P. falciparum cultures; however, we did not obtain evidence that NOS inhibitors had the ability to inhibit RNS production or that there is an active NOS in mature forms of the parasite. We concluded that a nitrate reductase activity produce NO and NO-derived RNS in or around the food vacuole in P. falciparum parasites. The food vacuole is a critical parasitic compartment involved in hemoglobin degradation, heme detoxification and a target for antimalarial drug action. Characterization of this relatively unexplored synthetic activity could provide important clues into poorly understood metabolic processes of the malaria parasite.     

Expression and characterization of human malaria parasite Plasmodium falciparum origin recognition complex subunit 1

In eukaryotes, the origin recognition complex (ORC) is essential for the initiation of DNA replication. The largest subunit of this complex (ORC1) has a regulatory role in origin activation. Here we report the cloning and functional characterization of Plasmodium falciparum ORC1 homolog. Using immunofluorescence and immunoelectron microscopy, we show here that PfORC1 is expressed in the nucleus during the late trophozoite and schizont stages where maximum amount of DNA replication takes place. Homology modelling of the carboxy terminal region of PfORC1 (781-1033) using Saccharomyces pombe Cdc6/Cdc18 homolog as a template reveals the presence of a similar AAA+ type nucleotide-binding fold. This region shows ATPase activity in vitro that is important for the origin activity. To our knowledge, this is the first evidence of an individual ORC subunit that shows ATPase activity. These observations strongly suggest that PfORC1 might be involved in DNA replication initiation during the blood stage of the parasitic life cycle.     

Plasmodium falciparum: effect of radiation on levels of gene transcripts in sporozoites

Humans immunized by the bites of irradiated Plasmodium falciparum (Pf) sporozoite-infected mosquitoes are protected against malaria. Radiation attenuates the sporozoites preventing them from fully developing and replicating in hepatocytes, but the effects of radiation on gene expression in sporozoites are unknown. We used RT-PCR (35 cycles of PCR followed by densitometry) to assess the expression of ten genes in Pf sporozoites, and in sporozoites irradiated with 15,000cGy. Irradiation reduced expression substantially (>60%) of two DNA repair genes; moderately (30-60%) of PfUIS3, the Pf orthologue of PbUIS3, a gene up-regulated in Plasmodium berghei sporozoites and of a third DNA repair gene; and minimally (<30%) of the Pf18S ribosomal RNA, PfCSP, PfSSP2/TRAP, and PfCELTOS genes. Irradiation increased expression of PfSPATR minimally. PfLSA1 RNA was not detectable in sporozoites. These results establish that radiation of sporozoites affects gene expression levels and provide the foundation for studies to identify specific genes involved in attenuation and protective immunity.     

Polyamine synthesis and salvage pathways in the malaria parasite Plasmodium falciparum

We demonstrate, for the first time, a functional polyamine biosynthetic pathway in the malaria parasite Plasmodium falciparum that culminates in the synthesis of spermine. Additionally, we also report putrescine and spermidine salvage in the malaria parasite. Putrescine and spermidine transport in P. falciparum infected red blood cells is a highly specific, carrier mediated and active process, mediated by new transporters that differ from the transporters of uninfected red blood cells in their kinetic parameters, Vmax and km, as well as in their activation energy.     

Orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase exist as multienzyme complex in human malaria parasite Plasmodium falciparum

Plasmodium falciparum, the causative agent of the most lethal form of human malaria, totally depends on de novo pyrimidine biosynthetic pathway. Orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC), the fifth and sixth enzymes in the pathway catalyzing formation of uridine 5'-monophosphate (UMP), remain largely uncharacterized in the protozoan parasite. In this study, we achieved purification of OPRT and OMPDC to near homogeneity from P. falciparum cultivated in vitro. The OPRT and OMPDC activities were co-eluted in all chromatographic columns during purification, suggesting the purified proteins exist as a multienzyme complex with a molecular mass of 140+/-8 kDa and contain two subunits each of OPRT and OMPDC. Monomeric forms of OPRT and OMPDC had molecular masses of 32+/-3 and 38+/-3 kDa, respectively, in agreement with those of proteins predicted from P. falciparum genome database. Interestingly, kinetic parameters and inhibitory constants of both OPRT and OMPDC activities were found to be different to those of the bifunctional human red cell UMP synthase. Our evidence provides the first example of OPRT and OMPDC existing as a multienzyme complex.     

Glyoxalase I of the malarial parasite Plasmodium falciparum: evidence for subunit fusion

Recombinant Plasmodium falciparum glyoxalase I (PfGlx I) was characterized as monomeric Zn(2+)-containing enzyme of 44 kDa. The K(M) value of the methylglyoxal-glutathione adduct is 77+/-15 microM, the k(cat) value being 4000 min(-1) at 25 degrees C and pH 7.0. PfGlx I consists of two halves, each of which is homologous to the small 2-domain glyoxalase I of man. Both parts of the pfglx I gene were overexpressed; the C-terminal half of PfGlx I was found to be a stable protein and formed an enzymatically active dimer. These results support the hypothesis of domain-swapping and subunit fusion as mechanisms in glyoxalase I evolution.     

Conflicting immune responses can prolong the length of infection in Plasmodium falciparum malaria

We have recently proposed a new model for antigenic variation in Plasmodium falciparum that relies on a network of partially cross-protective immune responses to orchestrate this complex immune evasion process. In addition to exhibiting prolonged oscillations of single variants that resemble the sequential dominance of immunologically distinct antigenic types, the model implies that a higher efficacy of cross-reactive immunity actually increases the length of infection while reducing severity of disease. Here, we analyse the behaviour of a reduced system under conditions of perfect synchrony between variants to demonstrate that these features of this system can be attributed to the antagonism between cross-reactive and variant-specific responses.     

Density-dependent impact of the human malaria parasite Plasmodium falciparum gametocyte sex ratio on mosquito infection rates

Malaria parasites produce male and female life cycle stages (gametocytes) that must fertilize to achieve successful colonization of the mosquito. Gametocyte sex ratios have been shown to be under strong selection pressure both as an adaptive response to a worsening blood environment for transmission and according to the number of co-infecting clones in the vertebrate. Evidence for an impact of sex ratio on the transmission success of Plasmodium falciparum has, however, been more controversial. Theoretical models of fertilization predict that increasingly male sex ratios will be favoured at low gametocyte densities to ensure fertilization. Here, we analyse in vitro transmission studies of P. falciparum to Anopheles gambiae mosquitoes and test this prediction. We find that there is a discernible effect of sex ratio on transmission but which is dependent upon the gametocyte density. While increasingly male sex ratios do give higher transmission success at low gametocyte densities, they reduce success at higher densities. This therefore provides empirical confirmation that sex ratio has an immediate impact on transmission success and that it is density-dependent. Identifying the signals used by the parasite to alter its sex ratio is essential to determine the success of transmission-blocking vaccines that aim to impede the fertilization process.     

Cyclosporin-binding proteins of Plasmodium falciparum

A number of cyclosporins, including certain non-immunosuppressive ones, are potent inhibitors of the intraerythrocytic growth of the human malarial parasite Plasmodium falciparum. The major cyclosporin-binding proteins of P. falciparum were investigated by affinity chromatography on cyclosporin-Affigel followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting, and peptide mass fingerprinting. The two bands obtained on gels were shown to correspond to cyclophilins, PfCyP-19A (formerly PfCyP-19) and PfCyP-19B, whose genes had been characterised previously. PfCyP-19B was an abundant protein of intraerythrocytic P. falciparum (up to 0.5% of parasite protein) that was present in the highest amounts in schizont-stage parasites. Unexpectedly, given its apparent signal sequence, it was located primarily in the cytosol of the parasite. The peptidyl-prolyl cis-trans isomerase activity of recombinant PfCyP-19B had the same profile of susceptibility to cyclosporin derivatives as the bulk isomerase activity of crude P. falciparum extracts. The binding of cyclosporins to cyclophilins may be relevant to the mechanism of action of the drug in the parasite.     

Transformation of sporozoites of Plasmodium berghei into exoerythrocytic forms in the liver of its mammalian host

Summary Intrahepatocytic transformation in vivo of the rodent malaria sporozoite of Plasmodium berghei, into the young trophic exoerythrocytic tissue stage was studied by immunofluorescence, light- and electron microscopy. The first 20 h of intracellular life were involved entirely in dedifferentiation with limited proliferation of organelles. From about 20 h onwards nuclear division commenced, rough endoplasmic reticulum became markedly expanded, and mitochondria increased in numbers. However, remains of the sporozoite pellicle (i.e., inner membranes and subpellicular microtubules) persisted for at least 28 h, which correlates with the persisting reaction of young exoerythrocytic forms with antisporozoite antibodies. In general, the basic mechanism of transformation resembles that of the ookinete into oocyst and that of the merozoite into erythrocytic trophozoite.     

Thioredoxin and glutathione system of malaria parasitePlasmodium falciparum

Summary Plasmodium falciparum is the causative agent of malaria tropica. Due to the increasing resistance towards the commonly used plasmodicidal drugs there is an urgent need to identify and assess new targets for the chemotherapeutic intervention of parasite development in the human host. It is established thatP. falciparum-infected erythrocytes are vulnerable to oxidative stress, and therefore efficient antioxidative systems are required to ensure parasite development within the host cell. The thioredoxin and glutathione redox systems represent two powerful means to detoxify reactive oxygen species and this article summarizes some of the recent work which has led to a better understanding of these systems in the parasite and will help to assess them as potential targets for the development of new chemotherapeutics of malaria.Abbreviation BSO L-buthionine-(S,R)-sulphoximdne     

Dynamic changes in white blood cell counts in uncomplicated Plasmodium falciparum and P. vivax malaria

Total and differential white blood cell (WBC) counts are basic and essential indicators in any type of illness resulting from infection. In malaria, WBC counts are generally characterized as low to normal during treatment. WBC-counts data, before and during treatment with artemisinin derivatives, was gathered for patients with either Plasmodium falciparum or Plasmodium vivax infection (at 28-day follow-up), to investigate dynamic changes in WBC count. We analyzed and compared the WBC counts of 1310 inpatients presenting with uncomplicated P. falciparum and P. vivax malaria at the Hospital for Tropical Diseases, in Bangkok, Thailand. Before-treatment, a statistically significant negative correlation was found between initial WBC count and highest temperature on admission. Before and during treatment, WBC counts were significantly lower in P. falciparum than P. vivax infection on days 0 and 7, but the numerical difference was small. We also found clinically significantly low WBC counts during the acute stages of both types of malaria, which subsequently normalized by day 28 follow-up. This finding has important clinical implications for the conventional method of estimating parasitemia using an assumed WBC count of 8000 cells/μL. The most significant finding in our analysis is that WBC counts in acute P. falciparum and P. vivax malaria are significantly lower than previously assumed for estimating malaria-parasite density. However, these abnormalities returned to normal within several weeks after artemisinin-derivative-based treatment.     

Plasmodium falciparum TryThrA antigen synthetic peptides block in vitro merozoite invasion to erythrocytes

Tryptophan-threonine-rich antigen (TryThrA) is a Plasmodium falciparum homologue of Plasmodium yoelii-infected erythrocyte membrane pypAg-1 antigen. pypAg-1 binds to the surface of uninfected mouse erythrocytes and has been used successfully in vaccine studies. The two antigens are characterized by an unusual tryptophan-rich domain, suggesting similar biological properties. Using synthetic peptides spanning the TryThrA sequence and human erythrocyte we have done binding assays to identify possible TryThrA functional regions. We describe four peptides outside the tryptophan-rich domain having high activity binding to normal human erythrocytes. The peptides termed HABPs (high activity binding peptides) are 30884 ((61)LKEKKKKVLEFFENLVLNKKY(80)) located at the N-terminal and 30901 ((401)RKSLEQQFGDNMDKMNKLKKY(420)), 30902 ((421)KKILKFFPLFNYKSDLESIM(440)) and 30913 ((641)DLESTAEQKAEKKGGKAKAKY(660)) located at the C-terminal. Studies with polyclonal goat antiserum against synthetic peptides chosen to represent the whole length of the protein showed that TryThrA has fluorescence pattern similar to PypAg-1 of P. yoelii. All HABPs inhibited merozoite in vitro invasion, suggesting that TryThrA protein may be participating in merozoite-erythrocyte interaction during invasion.     

Cyclin-dependent kinase homologues of Plasmodium falciparum

The intraerythrocytic asexual cycle of the malarial parasite is complex and atypical: during schizogony the parasite undergoes multiple rounds of DNA replication and asynchronous nuclear division without cytokinesis. This cell cycle deviates from the classical eukaryotic cell cycle model where, 'DNA replicates only once per cell cycle'. A clear understanding of the molecular switches that control this unusual developmental cycle would be of great interest, both in terms of fundamental Plasmodium biology and in terms of novel potential drug target identification. In recent years considerable effort has been made to identify the malarial orthologues of the cyclin-dependent kinases, which are key regulators of the orderly progression of the eukaryotic cell cycle. This review focuses on the current state-of-knowledge of Plasmodium falciparum cyclin-dependent kinase-like kinases and their regulators.