Red blood cells (RBCs) parasitized byPlasmodium falciparum are rigid and poorly deformable and show abnormal circulatory behavior. During parasite development, knob-associated histidinerich protein (KAHRP) and P falciparum erythrocyte membrane protein 3 (PfEMP3) are exported from the parasite and interact with the RBC membrane skeleton. Using micropipette aspiration, the membrane shear elastic modulus of RBCs infected with transgenic parasites (with kahrp or pfemp3 genes deleted) was measured to determine the contribution of these proteins to the increased rigidity of parasitized RBCs (PRBCs). In the absence of either protein, the level of membrane rigidification was significantly less than that caused by the normal parental parasite clone. KAHRP had a significantly greater effect on rigidification than PfEMP3, contributing approximately 51% of the overall increase that occurs in PRBCs compared to 15% for PfEMP3. This study provides the first quantitative information on the contribution of specific parasite proteins to altered mechanical properties of PRBCs. IntroductionMalaria caused by Plasmodium falciparum remains the most serious and widespread parasitic disease of humans. Clinical symptoms of malaria occur during the asexual stage of the parasite's life cycle, when it multiplies within red blood cells (RBCs). The extreme virulence of P falciparum and the occurrence of severe, often fatal clinical complications is related to the ability of RBCs parasitized by mature forms of the parasite to accumulate in the microvasculature of a variety of organs. 1 This abnormal circulatory behavior for RBCs appears to be directly related to parasite-induced alteration of its mechanical and adhesive properties.During the last 2 decades, the altered adhesive properties of parasitized RBCs (PRBCs) have been studied intensively (see 2,3 for recent reviews). In contrast, alterations of their mechanical properties, and the molecular mechanisms underpinning these changes, have been relatively ignored. Previous studies have clearly demonstrated that the deformability of intact PRBCs is profoundly reduced. [4][5][6] The overall increase in red cell rigidity is due, in part, to the presence of the large, nondeformable intracellular parasite and to a number of stage-specific parasite-encoded proteins that associate with the RBC membrane skeleton. 3,5,6 Paulitschke and Nash 6 used micropipette aspiration to measure the rigidity of RBCs parasitized by a number of unrelated parasite lines of knobby and knobless phenotypes. In general, the membranes of knobby PRBCs were more rigid than those lacking knobs; however, there was considerable variation in rigidity, particularly between knobby lines, with some knobby PRBCs only slightly more rigid than others infected with knobless parasite lines. Unfortunately, in their study, there was no characterization of the parasite genotype or immunohistochemical analysis of the PRBCs to determine precisely which parasite proteins were or were not expressed in different parasite lines. As such, thoug...

The high mortality of Plasmodium falciparum malaria is the result of a parasite ligand, PfEMP1 (P. falciparum) erythrocyte membrane protein 1), on the surface of infected red blood cells (IRBCs), which adheres to the vascular endothelium and causes the sequestration of IRBCs in the microvasculature. PfEMP1 transport to the IRBC surface involves Maurer's clefts, which are parasite-derived membranous structures in the IRBC cytoplasm. Targeted gene disruption of a Maurer's cleft protein, SBP1 (skeleton-binding protein 1), prevented IRBC adhesion because of the loss of PfEMP1 expression on the IRBC surface. PfEMP1 was still present in Maurer's clefts, and the transport and localization of several other Maurer's cleft proteins were unchanged. Maurer's clefts were altered in appearance and were no longer found as close to the periphery of the IRBC. Complementation of mutant parasites with sbp1 led to the reappearance of PfEMP1 on the IRBC surface and the restoration of adhesion. Our results demonstrate that SBP1 is essential for the translocation of PfEMP1 onto the surface of IRBCs and is likely to play a pivotal role in the pathogenesis of P. falciparum malaria.

Resonance Raman spectra of beta-hematin and hemin are reported for a range of excitation wavelengths including 406, 488, 514, 568, 633, 780, 830, and 1064 nm. Dramatic enhancement of A(1g) modes (1570, 1371, 795, 677, and 344 cm(-1)), ring breathing modes (850-650 cm(-1)), and out-of-plane modes including iron-ligand modes (400-200 cm(-1)) were observed when irradiating with 780- and 830-nm laser excitation wavelengths for beta-hematin and to a lesser extent hemin. Absorbance spectra recorded during the transformation of hemin to beta-hematin showed a red-shift of the Soret and Q (0-1) bands, which has been interpreted as excitonic coupling resulting from porphyrin aggregation. A small broad electronic transition observed at 867 nm was assigned to a z-polarized charge-transfer transition d(xy) --> e(g)(pi). The extraordinary band enhancement observed when exciting with near-infrared excitation wavelengths in beta-hematin when compared to hemin is explained in terms of an aggregated enhanced Raman scattering hypothesis based on the intermolecular excitonic interactions between porphyrinic units. This study provides new insight into the electronic structure of beta-hematin and therefore hemozoin (malaria pigment). The results have important implications in the design and testing of new anti-malaria drugs that specifically interfere with hemozoin formation.

Micro-Raman spectra of hemozoin encapsulated within the food vacuole of a Plasmodium falciparum-infected erythrocyte are presented. The spectrum of hemozoin is identical to the spectrum of L L-hematin at all applied excitation wavelengths. The unexpected observation of dramatic band enhancement of A 1g modes including X X 4 (1374 cm 31 ) observed when applying 780 nm excitation enabled Raman imaging of hemozoin in the food vacuole. This unusual enhancement, resulting from excitonic coupling between linked porphyrin moieties in the extended porphyrin array, enables the investigation of hemozoin within its natural environment for the ¢rst time.