Microarray-based comparative genomic analyses of the human malaria parasite Plasmodium falciparum using Affymetrix arrays

Microarray-based comparative genomic hybridization (CGH) provides a powerful tool for whole genome analyses and the rapid detection of genomic variation that underlies virulence and disease. In the field of Plasmodium research, many of the parasite genomes that one might wish to study in a high throughput manner are not laboratory clones, but clinical isolates. One of the key limitations to the use of clinical samples in CGH, however, is the miniscule amounts of genomic DNA available. Here we describe the successful application of multiple displacement amplification (MDA), a non-PCR-based amplification method that exhibits clear advantages over all other currently available methods. Using MDA, CGH was performed on a panel of NF54 and IT/FCR3 clones, identifying previously published deletions on chromosomes 2 and 9 as well as polymorphism in genes associated with disease pathology.     

Invasion profiles of Brazilian field isolates of Plasmodium falciparum: phenotypic and genotypic analyses

The invasion of red blood cells (RBCs) by Plasmodium falciparum is dependent on multiple molecular interactions between erythrocyte receptors and parasite ligands. Invasion studies using culture-adapted parasite strains have indicated significant receptor heterogeneity. It is not known whether this heterogeneity reflects the parasite invasion arsenal in the field. We have studied the invasion phenotypes of 14 distinct field isolates from the Legal Amazon areas of Brazil by using erythrocyte invasion assays to investigate invasion into normal, enzyme-treated, and clinical-mutant RBCs. Analysis of these isolates revealed four distinct invasion profiles. Using En(a-) cells to get an unequivocal estimate of the use of glycophorin A (GPA) as a receptor, we found that the 175-kDa erythrocyte-binding antigen (EBA-175)/GPA pathway was used by a minority of the parasite isolates studied. Although polymorphism of region II domains at specific amino acid positions in both EBA-140 and EBA-181 was found in these field isolates, this did not correlate with invasion profiles and thus receptor selectivity. These studies have further confirmed the existence of a significant diversity of invasion pathways in nature and suggest that additional parasite ligands will have to be targeted to devise global vaccines that will work in the field.     

Mitochondrial DNA of the human malarial parasite Plasmodium falciparum

Covalently closed circular DNA molecules were isolated from Plasmodium falciparum total DNA by isopycnic centrifugation in CsCl gradients containing either ethidium bromide or 2',6-diamidino-2-phenylindole. The circular molecules had an average contour length of 11.1 +/- 0.5 micron, similar to the analogous molecules previously isolated from the simian malaria parasite P. knowlesi. Both circular molecules shared considerable sequence homology and conserved restriction sites. The nucleotide sequence of one 936 bp fragment of the P. falciparum molecule was determined and identified, by a data base homology search, as part of a mitochondrial small rRNA subunit, thus confirming the mitochondrial origin of the circular DNAs of both malarial species.     

Hydrophobic interactions in Plasmodium falciparum invasion into human erythrocytes

Human glycophorins block in vitro invasion of Plasmodium falciparum merozoites into human erythrocytes. A segment of glycophorin A which appears to be involved in the inhibition, is at, or adjacent to, the membrane-spanning domain of the molecule. To study the role of hydrophobic interactions in the inhibition, a series of proteins were derivatized with lipophilic side groups, and tested for inhibitory activity. Glycophorin A became five times more inhibitory after derivatization with nitrobenzylfurazan groups. Bovine serum albumin was derivatized to different degrees with nitrobenzylfurazan, dinitrobenzyl, trinitrobenzyl, dansyl, disulfonic stilbene, and fluorescein groups. The presence of hydrophobic side groups on the protein rendered it highly inhibitory to invasion, whereas the presence of hydrophilic substitutes such as disulfonic stilbenes did not. Other soluble proteins such as human serum albumin, transferrin, ovalbumin, fetuin and casein derivatized with dinitrobenzyl groups, were also found to block invasion. Inhibition was not a result of toxic effects of the protein derivatives on parasite metabolism or development. A minimum of ten hydrophobic side groups per bovine serum albumin was required in order to elicit appreciable inhibition. The invasion blocking activity was highly correlated with the rate and affinity of binding of the derivatized macromolecules to heptyl-Sepharose. The latter provided a quantitative measure for the capacity of amphiphiles to undergo hydrophobic interactions with insoluble matrices. The results of the present study indicate that hydrophobic interactions may be an essential component in the invasion of P. falciparum merozoites into human erythrocytes.     

Plasmodium falciparum induces apoptosis in human mononuclear cells.

The level of spontaneous apoptosis in short-term lymphocyte cultures was evaluated in different human immunodeficiency virus-negative groups of either healthy control individuals or patients with clinical malaria. The mean percentage of spontaneous apoptosis found in patients during a malaria attack was significantly higher than in sex- and age-matched healthy controls. The healthy asymptomatic controls were individuals with different degrees of exposure to Plasmodium falciparum as reflected by their various mean levels of specific anti-P. falciparum (immunoglobulin G and M) antibodies. The percentages of apoptotic nuclei were found to be significantly higher in lymphocytes from subjects living in an area where malaria is holoendemic than in lymphocytes from subjects less exposed. Concentrations of soluble plasma interleukin-2 receptor were also higher in subjects from areas where malaria is endemic than in other groups, revealing different levels of lymphocyte activation. Of particular relevance to the in vivo situation, a P. falciparum schizont-rich extract induced a systematic and significant elevation of apoptotic nuclei at day 6 in 87.5% (35 of 40) of the subjects tested. In additional studies with different concentrations of extract, [3H]thymidine incorporation was concomitant with a low or limited level of apoptosis. Taken together, our results strongly suggest that acute as well as chronic asymptomatic P. falciparum infections were consistently associated with a marked increase in the level of mononuclear cell apoptosis. This process could be implicated in some of the alterations reported for the proliferative T-cell responses in areas where malaria is endemic.     

Plasmodium falciparum biosynthesizes sulfoglycosphingolipids

Sulfated glycosphingolipids are present on the surface of a variety of cells. They are active participants in adhesion processes in many systems and appear to be involved in the regulation of cell proliferation, differentiation and other developmental cellular events. However, the body of knowledge about synthesis, structure, and function of glycolipids in parasitic protozoa is very limited so far. In this work, we show by metabolic incorporation of [(14)C]palmitic acid, [(14)C]glucose and Na(2)(35)SO(4) that sulfoglycosphingolipids are biosynthesized in the three intraerythrocytic stages of Plasmodium falciparum. After saponification, purification of the labelled acidic components was achieved and two components named SPf1 and SPf2 were characterized. Chemical degradations and TLC analysis pointed out to sulfolipidic structures. Analysis by UV-MALDI-TOF mass spectrometry in the negative ion mode using nor-harmane as matrix showed for SPf2 a structure consisting in a disulfated hexose linked to a 20:1 sphingosine acylated with C18:0 fatty acid. Interestingly, parasite treatment with low concentrations of d,l-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) caused an arrest on parasite development associated to the inhibition of sulfoglycolipid biosynthesis. Taking into account that sulfoglycolipidic structures are currently involved in adhesion processes, our findings open the possibility to study the participation of this type of structures in the described aggregation phenomena in severe malaria and may contribute to clarify the pathogenesis of the disease. This report shows for the first time the synthesis of sulfoglycolipids in Apicomplexa.     

Mutational prevalence of chloroquine resistance transporter gene among Plasmodium falciparum field isolates in Assam and Arunachal Pradesh,India

Objective: The present study aims to find out the mutational prevalence of Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene in Assam and Arunachal Pradesh, India. Methods: To fulfil the objective of the study, a total of 54 P. falciparum malaria positive samples were attempted for genotyping of Pfcrt gene using polymerase chain reaction (PCR) and direct DNA sequencing method. Results: The K76T mutation was observed in 77.78% cases followed by M74I (61.11%), N75E (61.11%) and C72S (16.67%). Triple mutant allele M74I+N75E+K76T was found in 61.11% P. falciparum field isolates. Double mutant allele C72S+K76T was seen among 16.67% samples. Mutation studies have shown existence of three different haplotypes of which two were associated with chloroquine resistance. The haplotype CVIET was found preponderance and circulated in all four districts. The other haplotype SVMNT was observed only in Karbi Anglong district of Assam. Maximum haplotype diversity was also recorded from Karbi Anglong district of Assam. Conclusion: Our study has confirmed high prevalence of mutant Pfcrt genotypes associated with chloroquine resistance in Assam and Arunachal Pradesh, India.     

Evolution and selection of human leukocyte antigen alleles by Plasmodium falciparum infection

Infection by Plasmodium falciparum malaria was one of the major driving forces for the selection of various genes, some of which might be involved in protection against this infection. The human leukocyte antigen (HLA) system is a highly polymorphic supergene complex with extensive diversity across different populations. In areas traditionally endemic for malaria for centuries, there seems to be some selection of certain HLA alleles that may somehow be involved in protection against the infection. One of the major conundrums is the lack of homogeneity in the HLA alleles selected by P. falciparum across different populations. Various factors like microheterogeneity in parasite species, genetic drift in parasitic antigens, varying intensities of transmission, different polymorphisms of red cell antigens, and diversity in the HLA system have exerted selection pressure, which probably determined the emergence of different dominant HLA antigens in different endemic populations. The complex life cycle of P. falciparum, with different antigens becoming important at different phases of the cycle and invasion of different tissues causing different clinical manifestations of the same disease, is also another significant factor contributing to a selection pattern. Evolutionary selection pressure probably selected different HLA antigens for modulations of different components of the disease as well as the severity of the disease. A coevolution, where the parasite polymorphisms meet the host heterogeneity, is likely to have occurred, resulting in the selection of a few HLA antigens associated with P. falciparum infection. Data might have been overwhelmed by the noise of additional selection pressure exerted by other infectious agents prevalent in endemic areas of P. falciparum malaria.     

Plasmodium falciparum products enhance human lymphocyte transformation by Epstein-Barr virus

Supernatants obtained from the in vitro culture of Plasmodium falciparum infected erythrocytes induced prolonged lymphocyte survival in culture for more than 8 weeks in six cultures and permanent cell lines were established in four of these. The cells in the latter showed lymphoblastoid features similar to those seen in parallel cultures to which transforming Epstein-Barr (EB) virus instead of P. falciparum derived substances had been added. Cells from the same donors stimulated with other mitogens (pokeweed mitogen, Salmonella paratyphi culture supernatants) ceased to proliferate and died after 3-4 weeks. A 195 Kd polypeptide obtained from P. falciparum parasites also exhibited the potential to transform normal lymphocytes. Characterization of the cell lines indicated a B lymphocyte origin and the presence of EB virus in these lines suggests the possibility that P. falciparum products may activate latent EB virus genomes. These observations appear relevant to both the choice of P. falciparum derived antigens as vaccines, and to the interaction of EB virus and malaria in the aetiology of African Burkitt's lymphoma (BL).     

Biosynthesis of glycosphingolipids de-novo by the human malaria parasite Plasmodium falciparum

Glycolipids are important components of cellular membranes involved in various biological functions. In this report we describe the identification of the de-novo synthesis of glycosphingolipids by intraerythrocytic, asexual stages of the malaria parasite, Plasmodium falciparum. Parasite-specific glycolipids were identified in organic solvent extracts of parasites metabolically labeled with tritiated serine and glucosamine and characterised as sphingolipids based on their insensitivity towards alkaline treatment. While the de-novo synthesis of parasite glycosphingolipids was affected by fumonisin B1, threo-PPMP, cyclo-serine and myriocin, these well established inhibitors of de-novo ceramide biosynthesis were unable to arrest the intraerythrocytic development of the parasites in culture.     

Altered expression of human monocyte Fc receptors in Plasmodium falciparum malaria

The state of activation of human peripheral blood monocytes was examined by using a rosette assay that detects changes in Fc receptor expression. Monocytes from patients with uncomplicated Plasmodium falciparum malaria showed a significant increase in the number of rosettes relative to healthy controls. In addition, the monocytes from these patients were tested for their ability to phagocytose Candida albicans, but this ability did not differ from that of normal individuals. Finally, the monocytes from patients with cerebral malaria were also tested for Fc receptor expression. In contrast to the results from uncomplicated cases, the activity of the monocytes from these patients was no different from that of controls. We concluded that uncomplicated P. falciparum malaria caused an increase in monocyte Fc receptor expression which did not occur in cerebral malaria and that this difference in activation may be important in the pathogenesis of cerebral malaria.     

Molecular markers for detection and differentiation of Plasmodium falciparum and Plasmodium vivax in human blood samples by pyrosequencing

PCR amplification coupled with pyrosequencing was used to measure molecular markers that could be used to detect and differentiate Plasmodium falciparum and Plasmodium vivax in human blood samples. The detection rates were in agreement with the results of Giemsa-stained film microscopy, which is the current gold standard for detection. This method provides an exciting alternative for malaria diagnosis.     

Identification in Plasmodium falciparum of a protein specifically binding human fibronectins

A fibronectin binding protein (FnBp) was identified in 3H isoleucine labeled P. falciparum schizonts using affinity chromatography on human fibronectin (Fn) coupled to Sepharose 4B. After incubation of Nonidet-P 40 parasite lysate with Fn-Sepharose, elution was performed with SDS-PAGE buffer. Analysis of FnBp by SDS-PAGE demonstrated a major band which migrated with an apparent Mr of 70,000 under reducing conditions. This band was not found when human or rabbit IgG coupled Sepharose 4B were used instead of Fn as control.     

Response of sensitized and unsensitized human lymphocyte subpopulations to Plasmodium falciparum antigens.

Antigen preparations derived from Plasmodium falciparum-infected erythrocytes (but not from uninfected erythrocytes) can stimulate the in vitro proliferation of peripheral blood lymphocytes from malaria-sensitized as well as nonsensitized donors. The possibility that the nonspecific responses might be due to a parasite-derived B-cell mitogen has been previously suggested since polyclonal hypergammaglobulinemia is a frequent accompaniment of malaria infection. To test this hypothesis, we investigated the in vitro proliferative responses of purified T- and B-cell populations to malaria antigens. T but not B cells responded to the antigens. The addition of small numbers of T cells restored the ability of purified B cells to respond to lectin mitogens but not to malaria antigens. Falciparum malaria infection was associated with an increase in T-cell but not in B-cell proliferation in vivo, as assessed by the spontaneous tritiated thymidine incorporation of lymphocytes during a brief incubation in vitro. Our observations suggest that extracts of malaria parasites do not contain a B-cell mitogen but are antigenic as well as mitogenic for T cells.