Heavy metals are required by all organisms for normal function, but high levels of heavy metals are toxic. Therefore, homeostasis of these metals is crucial. In the human malaria-causing agent Plasmodium falciparum, the mechanisms of heavy metal transport have yet to be characterized. We have developed a P. falciparum line resistant to heavy metals from a wild-type line sensitive to heavy metals. A molecular and biochemical analysis of the involvement of the P. falciparum multidrug resistance 2 ( pfmdr2) gene, an ABC-type transporter, in heavy metal homeostasis was studied. Using a novel uptake assay applied on these two strains, it was demonstrated that, when exposed to heavy metals, the sensitive line accumulates metal, whereas no accumulation was observed in the resistant line. The accumulation occurs within the parasite itself and not in the cytoplasm of the red blood cell. This difference in the accumulation pattern is not a result of amplification of the pfmdr2 gene or of a change in the expression pattern of the gene in the two lines. Sequencing of the gene from both lines revealed a major difference; a stop codon is found in the sensitive line upstream of the normal termination, resulting in a truncated protein that lacks 188 amino acids that contain a portion of the essential cytoplasmatic transporter domain, thereby rendering it inactive. In contrast, the resistant line harbors a full-length, active protein. These findings strongly suggest that the PFMDR2 protein acts as an efflux pump of heavy metals.Malaria, caused in the majority of cases by the protozoan parasite Plasmodium falciparum, is a major global disease (1). Annual morbidity and mortality are estimated to reach overwhelming numbers of 300 -500 million and 1-3 million, respectively. In addition, malaria tolls a severe economic and social price, adding more afflictions to many third world countries already stricken by poverty and social unrest (2). Some of the reasons for this situation are the lack of an adequate vaccine and suitable antimalarial drugs, the latter due to the appearance of drug-resistant parasites. Our insufficient understanding of the biology of the malaria-causing agents limits our ability to vigorously combat this disease (1-3).A clear example of a poorly defined metabolic pathway of P. falciparum is its mechanism of heavy metal homeostasis. Heavy metals are defined as metals with a density higher than 5 g/cm 3 . Of 90 naturally occurring elements, 53 are heavy metals. Based on their solubility under physiological conditions, some 20 of them are available to living cells. Of these, only some are essential (iron, molybdenum, manganese, zinc, nickel, copper, vanadium, cobalt, and selenium), whereas the remaining ones appear in cells as a result of their wide distribution in various ecosystems. Regardless of their essentiality to life, most heavy metals are toxic to cells when their concentrations increase above a certain level. As such, mechanisms have evolved in different organisms to allow homeostasis of heavy metals. To d...

Protein kinase C (PKC) functions as a core component of the immunological synapse and serves as a key protein in the integrated T-cell antigen receptor (TCR)/CD28-induced signaling cascade leading to T-cell activation. However, the involvement of PKC in host-mediated immune responses to pathogens has not been thoroughly investigated. We tested the consequences of PKC ablation on the host response to infection by Plasmodium berghei ANKA (PbA). We found that both PKC ؉/؉ and PKC ؊/؊ C57BL/6J mice are susceptible to infection with PbA. However, despite a similar parasite burden, PKC ؉/؉ mice had an earlier onset of neurological signs, characteristics of experimental cerebral malaria (ECM), resulting in an earlier death. These mice suffered from an early and pronounced splenomegaly with a concomitant increase in the total number of CD4 ؉ splenic T cells. In contrast, a large proportion of PbA-infected PKC ؊/؊ mice overcame the acute phase characterized by neurological symptoms and survived longer than PKC ؉/؉ mice. The partial resistance of PKC ؊/؊ mice to ECM was associated with an impaired production of Th1-type cytokines, including gamma interferon and tumor necrosis factor alpha/lymphotoxin-␣, which are known to exacerbate symptoms leading to ECM. In addition, PbA infection-induced LFA-1 expression in CD8 ؉ T cells was suppressed in PKC-deficient T cells, suggesting a diminished ability to adhere to endothelial cells and sequester in brain microvasculature, which may explain the decrease in neurological symptoms. These data implicate PKC in CD4 ؉ Th1 ؉ and CD8 ؉ T-cell-mediated immune responses during PbA infection that contribute to the development of ECM.

1% of the genes of the human malaria causing agent Plasmodium falciparum belong to the heterogeneous var gene family which encodes P. falciparum erythrocyte membrane protein 1 (PFEMP1). This protein mediates part of the pathogenesis of the disease by causing adherence of infected erythrocytes (IE) to the host endothelium. At any given time, only one copy of the family is expressed on the IE surface. The cues which regulate the allelic exclusion of these genes are not known. We show the existence of a differential expression pattern of these genes upon exposure to biological stress in relation to their positional placement on the chromosome – expression of centrally located var genes is induced while sub-telomeric copies of the family are repressed - this phenomenon orchestrated by the histone deacetylase pfsir2. Moreover, stress was found to cause a switch in the pattern of the expressed var genes thus acting as a regulatory cue. By using pharmacological compounds which putatively affect pfsir2 activity, distinct changes of var gene expression patterns were achieved which may have therapeutic ramifications. As disease severity is partly associated with expression of particular var gene subtypes, manipulation of the IE environment may serve as a mechanism to direct transcription towards less virulent genes.

BackgroundThe study of the Plasmodium falciparum heavy metal transporter gene pfmdr2 employed radioactive labelled heavy metal. As the use of radioactive isotopes shrank considerably during the last few years, resulting in the cessation of the production of some isotopes, amongst them Cadmium109 which was used for that purpose, a different approach had to be developed. Herein, a dual fluorescent labelling of heavy metals accumulation in the P. falciparum parasite is proposed as an alternative to the use of radioactive labelled heavy metals.MethodsPlasmodium falciparum Cd resistant and sensitive strains at the trophozoite stage were used in this study. The cells were cultured at different CdCl2 concentrations and for different time periods followed by staining of the infected red blood cells with Fluo-3/AM for Cd detection and Hoechst 33342 for parasite DNA labelling. The fluorescent analysis was done by flow cytometry and confocal microscopy.ResultsThe results show that the sensitive strain has a higher Fluo-3/AM fluorescence in a Cd concentration and time dependent manner, whereas in the resistant strain Fluo-3/AM fluorescence levels were negligible and increased only at high concentrations of Cd and at long incubation periods, but to a much lesser extent than the sensitive strain. No Cd uptake is observed in uninfected red blood cells populations originating from cultures infected with either sensitive or resistant strain. In addition, confocal microscopy overlay of Fluo-3/AM and Hoechst staining shows that the Cd metal accumulates in the parasite itself.ConclusionsThe dual fluorescent labelling is a valid method for detecting heavy metal accumulation in P. falciparum. Furthermore, in contrast to the use of radioactive labelled heavy metal, the fluorescent labelling enables us to differentiate between the different populations existing in a P. falciparum infected red blood cells cultures and thus actually study a phenomenon at the level of a single cell.