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The pigment hemozoin is a natural by-product of the metabolism of hemoglobin by the parasites which cause malaria. Previously, hemozoin was demonstrated to have a very high nonlinear optical response enabling third harmonic generation (THG) imaging. In this study, we present a complete characterization of the nonlinear THG response of natural hemozoin in malaria-infected red blood cells, as well as in pure isostructural synthesized hematin anhydride, in order to determine optimal imaging parameters for detection. Our study demonstrates the wavelength range for optimal pulsed femtosecond laser excitation of THG from hemozoin crystals. In addition, we show the hemozoin crystal detection as a function of crystal size, incident laser power, and the emission response of the hemozoin crystals to different incident laser polarization states. Our systematic measurements of the nonlinear optical response from hemozoin establish detection limits, which are essential for the optimal design of malaria detection technologies that exploit the THG response of hemozoin.

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Polymorphisms in Plasmodium falciparum chloroquine resistance transporter (or PfCRT) were shown to be causative of decreased sensitivity to diverse quinoline-based antimalarials. In this report we describe the identification of a post-translational variant of PfCRT using highly characterized antibodies raised against its N- and C-terminal cytoplasmic domains (e.g., 58 and 26 amino acids, respectively). Western blot analyses of P. falciparum protein extracts with anti N-PfCRT antiserum revealed two polypeptides with apparent molecular masses of 52 kDa and 42 kDa, relative to the calculated molecular mass of PfCRT of 48.7 kDa. The 52 kDa polypeptide was detectable with anti C-PfCRT antiserum, only after alkaline phosphatase treatment of P. falciparum extracts. Detailed epitope mapping of anti N- and C-PfCRT antisera revealed epitopes covering two previously identified phosphorylation sites, Ser411 and Thr416, whereby substitution of these residues with Asp amino acid, to mimic phosphorylated residues, dramatically inhibited anti C-PfCRT binding. Consistently, alkaline phosphatase treatment of P. falciparum extract unmasked the binding of anti C-PfCRT to the 52 kDa polypeptide, suggesting that the 52 kDa but not 42 kDa polypeptide is phosphorylated at its C-terminal Ser411 and Thr416. Interestingly, Pfcrt expressed in HEK-293F human kidney cells showed the same reactive polypeptides with anti N- and C-PfCRT antisera, consistent with PfCRT origin of the two polypeptides (e.g., 42 kDa and 52 kDa), but lacking PfCRT phosphorylation at its C-terminal. Immunohistochemical staining of late trophozoite-infected erythrocytes with anti N-or C-PfCRT antisera showed both polypeptides are localized to the parasite's digestive vacuole. Moreover, both polypeptides are detected in chloroquine-susceptible and -resistant strains of P. falciparum. This is the first report describing a post-translationally modified variant of PfCRT. The physiologic role of the 52 kDa phosphorylated PfCRT in P. falciparum remains to be determined. • Native PfCRT migrates as 42 kDa polypeptide is non-phosphorylated at its C-terminal. • Post-translationally modified PfCRT variant is phosphorylated and migrates as 52 kDa polypeptide. • Antibody to PfCRT N-terminal detects both native and post-translationally modified variant polypeptides. • Antibody to PfCRT C-terminal binds only non-phosphorylated native or dephosphorylated variant PfCRT. [ABSTRACT FROM AUTHOR]

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a phospho-protein with three identified phosphorylation sites (Ser33, Ser411 and Thr416) at its cytosolic N- and C-termini. In this study, we report on the characterization of PfCRT anti-serum and show the presence of three epitope-specific immunoglobulin (IgG) pools (i.e. , IgG-P1, P2, and P3), each recognizing a different epitope in PfCRT cytoplasmic C-terminal. IgG-P2 bound the heptapeptide sequence (408NEDSEGE414), including Ser411. The effect of Ser411 phosphorylation on the binding specificity of IgG-P2 was confirmed using heptapeptides and full-length PfCRT with substitutions of Ser411 with aspartic acid (phospho-serine mimic) and alanine residues. Moreover, using purified IgG-P2, we show the presence of PfCRT homodimer that has un-phosphorylated Ser411 and migrates with an apparent molecular mass of 90 kDa on SDS-PAGE. In addition, parasite lysates showed PfCRT to be more phosphorylated at Ser411 in CQ-sensitive (3D7) than CQ–resistant (Dd2-H) strains of P. falciparum. Taken together, the findings of this study suggest a role for Ser411 phosphorylation in PfCRT structure-function. • High resolution mapping of anti-PfCRT antisera to its C-terminal sequence. • Isolation of three reactive IgG pools in PfCRT antisera against different epitopes, with IgG pool 2 specific to Ser411 phosphorylation status. • Demonstrating the presence of PfCRT homodimer, ∼90 kDa, encoding un-phosphorylated Ser411. • PfCRT phosphorylation status at Ser411 may predict P. falciparum resistance to chloroquin. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum infection of mature erythrocytes leads to heightened oxidative stress that is tolerated in normal erythrocytes but not in erythrocytes from sickle cell, β-thalassaemia and glucose-6-phosphate dehydrogenase (G6PD) deficiency hosts. In this report, it was of interest to perturb the redox homeostasis of normal erythrocytes through drug-induced active efflux of glutathione via erythrocyte ABCC1, a member of the C-subfamily of the human ATP-binding cassette (ABC) transporters. To achieve this objective, we made use of apigenin (API), shown previously to activate ABCC1 glutathione efflux in mature erythrocytes. Our results show that API increased ABCC1-mediated efflux of calcein-AM from uninfected erythrocytes, which was reversed with MK571, an inhibitor of ABCC1 drug efflux. Similarly, addition of API to uninfected normal erythrocytes led to a dramatic increase in reactive oxygen species coupled with a significant decrease in intracellular glutathione. Moreover, using P. falciparum -infected normal erythrocytes, we demonstrate that increasing concentrations of API inhibited the proliferation of chloroquine-susceptible (3D7) and -resistant (Dd2) parasites in culture with similar 50% inhibitory concentration (IC 50 ) values (36.02 ± 2.4 µM and 34.45 ± 2.4 µM, respectively). Interestingly, the presence of API (25 µM) led to a three-fold decrease in the IC 50 of artemisinin compared with artemisinin alone (2.8 ± 0.7 nM vs. 7.09 ± 1.5 nM, respectively). Taken together, the findings of this study demonstrate the feasibility of our proposed approach for the development of novel antimalarials by modulating host protein functions that leads to heightened oxidative stress in P. falciparum -infected erythrocytes and inhibits parasite proliferation. [ABSTRACT FROM AUTHOR]

The Plasmodium falciparum genome encodes 16 members of ABC proteins, with one member of the ABCG subfamily (PfABCG). Analysis of PfABCG amino acid sequence shows equal sequence identity to hsABCG1 and G2. Using N-terminal directed antibody against a recombinant fragment of PfABCG, we show that PfABCG migrates with an apparent molecular mass of 65KDa polypeptide on SDS-PAGE. PfABCG is expressed in all four stages of the parasite erythrocytic life cycle, with lower and higher expression in ring and late trophozoite stages, respectively. The protein localizes to the plasma membrane and a novel spherical structure beneath the cell membrane. Similar localization is also observed in gametocytes where PfABCG is highly expressed. Analysis of PfABCG genomic sequences for polymorphisms and changes in protein expression between different strains of P. falciparum revealed identical nucleotide sequence among the different strains, but variable protein expression. PfABCG expression is least in HB3 chloroquine sensitive strain, while higher expression levels are seen in other chloroquine-sensitive and -resistant strains, with highest levels of expression in 7G8. The differential expression of PfABCG in three chloroquine-sensitive strains (e.g., 3D7, HB3 and D10) predicts the sensitivity of the different strains to ketotifen, an anti-histaminic drug, whereby low expression is associated with decreased sensitivity to ketotifen. Taken together, the results in this report provide the first description of native PfABCG expression and subcellular localization in asexual stages of the parasite and its localization in gametocytes. It remains to be determined if PfABCG is functionally equivalent to mammalian ABCG1, ABCG2 or both. [ABSTRACT FROM AUTHOR]

Chloroquine (CQ), the first cost-effective synthetic antimalarial, is rendered ineffective in malaria-endemic regions owing to the rise and spread of CQ-resistant Plasmodium falciparum . In this report, we show that a halogen derivative of CQ, namely 3-iodo-CQ, inhibits the proliferation of CQ-sensitive and -resistant P. falciparum in a verapamil-insensitive manner. Similar to CQ, the antimalarial activity of 3-iodo-CQ is likely due to its inhibition of β-haematin formation . Interestingly, the presence of non-inhibitory concentrations of 3-iodo-CQ potentiated the antiproliferative activity of CQ against CQ-resistant strains or P. falciparum transfectants expressing wild-type or mutant P. falciparum CQ resistance transporter (PfCRT) (C2 GC03 or C4 Dd2 , respectively). These findings demonstrate that halogenation of the third position of 4-aminoquinoline, with a simple one-step reaction from CQ, generates a novel derivative that is active against CQ-sensitive and -resistant P. falciparum , possibly by inhibiting the activity of mutant PfCRT. [ABSTRACT FROM AUTHOR]

Malaria is a major disease in the tropics where chemotherapy remains the main mode of treatment and as such the rise and spread of drug-resistant malaria can lead to human tragedy. Two membrane transport proteins, PfMDR1 (Plasmodium falciparum multidrug resistance protein 1) and PfCRT (P. falciparum chloroquine resistance transporter), have been shown to cause resistance to several antimalarials. Both PfMDR1 and PfCRT are localized to the digestive vacuolar membrane and appear to regulate the transport of drugs and physiological metabolites. In this study we have used MK571, a 2-amino quinoline, to explore its interaction with PfMDR1 and PfCRT in chloroquine-sensitive and -resistant strains of P. falciparum. Our results show that chloroquine-resistant strains (e.g., K1, Dd2, and 7G8) are consistently more sensitive to MK571 than chloroquine-sensitive strains (e.g., 3D7, 106/1 and D10). This association, however, was not maintained with the chloroquine-resistant strain FCB which IC50 value was similar to chloroquine-sensitive strains. Moreover, the susceptibility of chloroquine-sensitive and -resistant strains to MK571 does not correlate with mutated PfCRT, nor is it reversible with verapamil; but correlates with mutations in PfMDR1. Furthermore, MK571 appears to target the parasite's digestive vacuole (DV), as demonstrated by the ability of MK571 to: (1) block the accumulation of the fluorescent dye Fluo-4 AM, a PfMDR1 substrate, into the digestive vacuole; (2) reduce the transvacuolar pH gradient; and (3) inhibit the formation of β-hematin in vitro. Moreover, the presence of non-toxic concentrations of MK571 sensitized both chloroquine-sensitive and -resistant parasites to mefloquine and halofantrine, likely by competing against PfMDR1-mediated sequestering of the drugs into the DV compartment and away from the drugs' cytosolic targets. Our data, nevertheless, found only a minimal decrease in MK571 IC50 value in FCB parasite which second pfmdr1 copy was inactivated via gene disruption. Taken together, the findings of this study suggest that MK571 interacts with native and mutant PfMDR1 and modulates the import of drugs or solutes into the parasite's DV and, as such, MK571 may be a useful tool in the characterization of PfMDR1 drug interactions and substrate specificity. [ABSTRACT FROM AUTHOR]

In malaria, Plasmodium parasites produce hemozoin (Hz) as a route to detoxify free heme released from the catabolism of hemoglobin. Hz isolated from the parasites is encapsulated in an organic layer constituted by parasite and host components. This organic coating may play a role in Hz formation and in the immunomodulatory properties attributed to Hz, and they may influence the mode of action of antimalarials that block Hz formation. In this work, we analyze the organic layer adhered to Hz, and find Na, Cl, Si, Ca and P present, in addition to organic material. Our results suggest that Na, Cl, and P adsorb during Hz release from the red blood cells, while Si and Ca derive from components present during Hz biomineralization within the digestive vacuole of the parasite. Overall, we show that inorganic elements associated with Hz surface provide insights into the biological functions of Plasmodium parasites. Hemozoin crystals collected from malaria parasites present inorganic elements of silicon, calcium, phosphorus, sodium, and chlorine, in addition to organic material. The speciation of the inorganic elements is discussed and points out to unexplored functions in Plasmodium 's biology and possibly other Apicomplexan parasites. Unlabelled Image • Inorganic ions are associated with the organic coating adsorbed on hemozoin surface. • Sodium, chlorine and phosphorus adsorb during hemozoin release from the red blood cells. • Silicon and calcium derive from components present within the parasite. • Silicon is present as silica and, likely, silicon bound to cellular components. • These ions may be related to yet unexplored biological functions of the parasite. [ABSTRACT FROM AUTHOR]