Infected erythrocyte choline carrier inhibitors: exploring some potentialities inside Plasmodium phospholipid metabolism for eventual resistance acquisition

We have developed a model for designing antimalarial drugs based on interference with an essential metabolism developed by Plasmodium during its intraerythrocytic cycle, phospholipid (PL) metabolism. The most promising drug interference is choline transporter blockage, which provides Plasmodium with a supply of precursor for synthesis of phosphatidylcholine (PC), the major PL of infected erythrocytes. Choline entry is a limiting step in this metabolic pathway and occurs by a facilitated-diffusion system involving an asymmetric carrier operating according to a cyclic model. Choline transport in the erythrocytes is not sodium dependent nor stereospecific as demonstrated using stereoisomers of alpha and beta methylcholine. These last two characteristics along with distinct effects of nitrogen substitution on transport rate demonstrate that choline transport in the infected erythrocyte possesses characteristics quite distinct from that of the nervous system. This indicates a possible discrimination between the antimalarial activity (inhibition of choline transport in the infected erythrocyte) and a possible toxic effect through inhibition of choline entry in synaptosomes. Apart from the de novo pathway of choline, PC can be synthesized by N-methylation from phosphatidylethanolamine (PE). There is a de novo pathway for PE biosynthesis from ethanolamine in infected cells but phosphatidylserine (PS) decarboxylation also occurs. In addition, PE can be directly and abundantly synthesized from serine decarboxylation into ethanolamine, a pathway which is absent from the host. The variety of the pathways that exist for the biosynthesis of one given PL led us to investigate whether an equilibrium can occur between all PL metabolic pathways. Indeed, if alternative (compensative) pathway(s) can operate after blockage of the de novo PC biosynthesis pathway this would indicate a potential mechanism for resistance acquisition. Up until now, there is no evidence of such a compensative process occurring in Plasmodium-infected erythrocytes under physiological conditions. Besides, the discovery of a highly parasite-specific pathway (serine decarboxylation and the presence of PS synthase) constitutes a very attractive and promising target, which could be attacked if resistances are built up against choline analogs. Indeed, potential inhibitions of the serine decarboxylase pathway could be very useful in acting instead of, or in surgery with, choline analogs.

Simian malaria at two sites in the Brazilian Amazon: I-The infection rates of Plasmodium brasilianum in non-human primates

The parasite that causes simian malaria in the Brazilian Amazon, Plasmodium brasilianum, is infective to man. In this region, where humans live within and in close proximity to the forest, it was suspected that this parasite could be the cause of a zoonosis. A study was performed in the areas surrounding two hydroelectric plants in the Amazon, Balbina and Samuel, aiming at determining the zoonotic potential of this parasite. P. brasilianum was detected in, respectively, 15.8% and 9.9% of 126 and 252 primates belonging to seven and eight species examined from Balbina and Samuel. The highest malaria infection rates were found among the red-howler monkey Alouatta seniculus straminea (32.3%), the bearded-saki Chiropotes satanas chiropotes (50%) and the spider-monkey Ateles paniscus paniscus (2[1+]) from Balbina and in the squirrel-monkey Saimiri ustus (21%) and the black-faced-spider-monkey Ateles paniscus chamek (28.6%) from Samuel.

Compositional Constraints in the Extremely GC-poor Genome of Plasmodium falciparum

We have analyzed the compositional properties of coding (protein encoding) and non-coding sequences of Plasmodium falciparum, a unicellular parasite characterized by an extremely AT-rich genome. GC% levels, base and dinucleotide frequencies were studied. We found that among the various factors that contribute to the properties of the sequences analyzed, the most relevant are the compositional constraints which operate on the whole genome

Identification of Novel Membrane Structures in Plasmodium falciparum Infected Erythrocytes

Little is known about the molecular mechanisms underlying the release of merozoites from malaria infected erythrocytes. In this study membranous structures present in the culture medium at the time of merozoite release have been characterized. Biochemical and ultrastructural evidence indicate that membranous structures consist of the infected erythrocyte membrane, the parasitophorous vacuolar membrane and a residual body containing electron dense material. These are subcellular compartments expected in a structure that arises as a consequence of merozoite release from the infected cell. Ultrastructural studies show that a novel structure extends from the former parasite compartment to the surface membrane. Since these membrane modifications are detected only after merozoites have been released from the infected erythrocyte, it is proposed that they might play a role in the release of merozoites from the host cell

Allelic Diversity at the Merozoite Surface Protein-1 (MSP-1) Locus in Natural Plasmodium falciparum Populations: a Brief Overview

The merozoite surface protein-1 (MSP-1) locus of Plasmodium falciparum codes for a major asexual blood-stage antigen currently proposed as a major malaria vaccine candidate. The protein, however, shows extensive polymorphism, which may compromise its use in sub-unit vaccines. Here we compare the patterns of allelic diversity at the MSP-1 locus in wild isolates from three epidemiologically distinct malaria-endemic areas: the hypoendemic southwestern Brazilian Amazon (n = 54), the mesoendemic southern Vietnam (n = 238) and the holoendemic northern Tanzania (n = 79). Fragments of the variable blocks 2, 4a, 4b and 6 or 10 of this single-copy gene were amplified by the polymerase chain reaction, and 24 MSP-1 gene types were defined as unique combinations of allelic types in each variable block. Ten different MSP-1 types were identified in Brazil, 23 in Vietnam and 13 in Tanzania. The proportion of genetically mixed infections (isolates with parasites carrying more than one MSP-1 version) ranged from 39% in Brazil to 44% in Vietnam and 60% in Tanzania. The vast majority (90%) of the typed parasite populations from Brazil and Tanzania belonged to the same seven most frequent MSP-1 gene types. In contrast, these seven gene types corresponded to only 61% of the typed parasite populations from Vietnam. Non-random associations were found between allelic types in blocks 4a and 6 among Vietnamese isolates, the same pattern being observed in independent studies performed in 1994, 1995 and 1996. These results suggest that MSP-1 is under selective pressure in the local parasite population. Nevertheless, the finding that similar MSP-1 type frequencies were found in 1994 and 1996 argues against the prominence of short-term frequency-dependent immune selection of MSP-1 polymorphisms. Non-random associations between MSP-1 allelic types, however, were not detected among isolates from Brazil and Tanzania. A preliminary analysis of the distribution of MSP-1 gene types per host among isolates from Tanzania, but not among those from Brazil and Vietnam, shows significant deviation from that expected under the null hypothesis of independent distribution of parasites carrying different gene types in the human hosts. Some epidemiological consequences of these findings are discussed

Evaluation of DNA Recombinant Methodologies for the Diagnosis of Plasmodium falciparum and their Comparison with the Microscopy Assay

Since 1984, DNA tests based on the highly repeated subtelomeric sequences of Plasmodium falciparum (rep 20) have been frequently used in malaria diagnosis. Rep 20 is very specific for this parasite, and is made of 21 bp units, organized in repeated blocks with direct and inverted orientation. Based in this particular organization, we selected a unique consensus oligonucleotide (pf-21) to drive a PCR reaction coupled to hybridization to non-radioactive labeled probes. The pf-21 unique oligo PCR (pf-21-I) assay produced DNA amplification fingerprints when was applied on purified P. falciparum DNA samples (Brazil and Colombia), as well as in patient's blood samples from a large area of Venezuela. The performance of the Pf-21-I assay was compared against Giemsa stained thick blood smears from samples collected at a malaria endemic area of the Bolívar State, Venezuela, at the field station of Malariología in Tumeremo. Coupled to non-radioactive hybridization the pf-21-I performed better than the traditional microscopic method with a r=1.7:1. In the case of mixed infections the r value of P. falciparum detection increased to 2.5:1. The increased diagnostic sensitivity of the test produced with this homologous oligonucleotide could provide an alternative to the epidemiological diagnosis of P. falciparum being currently used in Venezuela endemic areas, where low parasitemia levels and asymptomatic malaria are frequent. In addition, the DNA fingerprint could be tested in molecular population studies

Increase of a Calcium Independent Transglutaminase Activity in the Erythrocyte during the Infection with Plasmodium falciparum

We have studied the activity of a calcium dependent transglutaminase (EC 2.3.2.13) during the growth of the parasite Plasmodium falciparum inside the infected human erythrocyte. There is only one detectable transglutaminase in the two-cell-system, and its origin is erythrocytic. No activity was detected in preparations of the parasite devoid of erythrocyte cytoplasm. The Michaelis Menten constants (Km) of the enzyme for the substrates N'N'dimethylcaseine and putrescine were undistinguishable whether the cell extracts used in their determination were obtained from normal or from infected red cells. The total activity of transglutaminase in stringently synchronized cultures, measured at 0.5mM Ca2+, decreased with the maturation of the parasite. However, a fraction which became irreversibly activated and independent of calcium concentration was detected. The proportion of this fraction grew with maturation; it represented only 20% of the activity in 20 hr-old-trophozoites while in 48-hr-schizonts it was more than 85% of the total activity. The activation of this fraction of transglutaminase did not depend on an increase in the erythrocyte cytoplasmic calcium, since most of the calcium was shown to be located in the parasite.

Estimated Financial Impact of Trypanosoma vivax on the Brazilian Pantanal and Bolivian Lowlands

The financial impact of the first outbreak of Trypanosoma vivax in the Brazilian Pantanal wetland is estimated. Results are extended to include outbreaks in the Bolivian lowlands providing a notion of the potential influence of the disease and an analytical basis. More than 11 million head of cattle, valued at more than US$3 billion are found in the Brazilian Pantanal and Bolivian lowlands. The total estimated cost of the 1995 outbreak of T. vivax is the sum of the present values of mortality, abortion, and productivity losses and treatment costs, or about 4% of total brood cow value on affected ranches. Had the outbreak gone untreated, the estimated losses would have exceeded 17% of total brood cow value.

Plasmodium falciparum malaria: rosettes are disrupted by quinine, artemisinin, mefloquine, primaquine, pyrimethamine, chloroquine and proguanil

An assay was developed measuring the disruption of rosettes between Plasmodium falciparuminfected (trophozoites) and uninfected erythrocytes by the antimalarial drugs quinine, artemisinin mefloquine, primaquine, pyrimethamine, chloroquine and proguanil. At 4 hr incubation rosettes were disrupted by all the drugs in a dose dependent manner. Artemisinin and quinine were the most effective anti-malarials at disrupting rosettes at their therapeutic concentrations with South African RSA 14, 15, 17 and The Gambian FCR-3 P. falciparum strains. The least effective drugs were proguanil and chloroquine. A combination of artemisinin and mefloquine was more effective than each drug alone. The combinations of pyrimethamine or primaquine, with quinine disrupted more rosettes than quinine alone. Quinine may be an effective drug in the treatment of severe malaria because the drug efficiently reduces the number of rosettes.

Antimalarial drug susceptibility testing of Plasmodium falciparum in Brazil using a radioisotope method

From March 1996 to August 1997, a study was carried out in a malaria endemic area of the Brazilian Amazon region. In vivo sensitivity evaluation to antimalarial drugs was performed in 129 patients. Blood samples (0.5 ml) were drawn from each patient and cryopreserved to proceed to in vitro studies. In vitro sensitivity evaluation performed using a radioisotope method was carried out with the cryopreserved samples from September to December 1997. Thirty-one samples were tested for chloroquine, mefloquine, halofantrine, quinine, arteether and atovaquone. Resistance was evidenced in 96.6% (29/30) of the samples tested for chloroquine, 3.3% (1/30) for quinine, none (0/30) for mefloquine and none for halofantrine (0/30). Overall low sensitivity was evidenced in 10% of the samples tested for quinine, 22.5% tested for halofantrine and in 20% tested for mefloquine. Means of IC 50 values were 132.2 (SD: 46.5) ng/ml for chloroquine, 130.6 (SD: 49.6) ng/ml for quinine, 3.4 (SD: 1.3) ng/ml for mefloquine, 0.7 (SD: 0.3) ng/ml for halofantrine, 1 (SD: 0.6) ng/ml for arteether and 0.4 (SD: 0.2) ng/ml for atovaquone. Means of chloroquine IC 50 of the tested samples were comparable to that of the chloroquine-resistant strain W2 (137.57 ng/ml) and nearly nine times higher than that of the chloroquine-sensitive strain D6 (15.09 ng/ml). Means of quinine IC 50 of the tested samples were 1.7 times higher than that of the low sensitivity strain W2 (74.84 ng/ml) and nearly five times higher than that of the quinine-sensitive strain D6 (27.53 ng/ml). These results disclose in vitro high resistance levels to chloroquine, low sensitivity to quinine and evidence of decreasing sensitivity to mefloquine and halofantrine in the area under evaluation.