Functional alteration of red blood cells by a megadalton protein of Plasmodium falciparum.

Proteins exported from Plasmodium falciparum parasites into red blood cells (RBCs) interact with the membrane skeleton and contribute to the pathogenesis of malaria. Specifically, exported proteins increase RBC membrane rigidity, decrease deformability, and increase adhesiveness, culminating in intravascular sequestration of infected RBCs (iRBCs). Pf332 is the largest (>1 MDa) known malaria protein exported to the RBC membrane, but its function has not previously been determined. To determine the role of Pf332 in iRBCs, we have engineered and analyzed transgenic parasites with Pf332 either deleted or truncated. Compared with RBCs infected with wild-type parasites, mutants lacking Pf332 were more rigid, were significantly less adhesive to CD36, and showed decreased expression of the major cytoadherence ligand, PfEMP1, on the iRBC surface. These abnormalities were associated with dramatic morphologic changes in Maurer clefts (MCs), which are membrane structures that transport malaria proteins to the RBC membrane. In contrast, RBCs infected with parasites expressing truncated forms of Pf332, although still hyperrigid, showed a normal adhesion profile and morphologically normal MCs. Our results suggest that Pf332 both modulates the level of increased RBC rigidity induced by P falciparum and plays a significant role in adhesion by assisting transport of PfEMP1 to the iRBC surface.

A Maurer's cleft-associated protein is essential for expression of the major malaria virulence antigen on the surface of infected red blood cells.

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.

Raman imaging of hemozoin within the food vacuole of Plasmodium falciparum trophozoites.

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 beta-hematin at all applied excitation wavelengths. The unexpected observation of dramatic band enhancement of A(1g) modes including nu(4) (1374 cm(-1)) 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 first time.

An exported kinase (FIKK4.2) that mediates virulence-associated changes in Plasmodium falciparum-infected red blood cells.

Alteration of the adhesive and mechanical properties of red blood cells caused by infection with the malaria parasite Plasmodium falciparum underpin both its survival and extreme pathogenicity. A unique family of parasite putative exported kinases, collectively called FIKK (Phenylalanine (F) - Isoleucine (I) - Lysine (K) - Lysine (K)), has recently been implicated in these pathophysiological processes, however, their precise function in P. falciparum-infected red blood cells or their likely role in malaria pathogenesis remain unknown. Here, for the first time, we demonstrate that one member of the FIKK family, FIKK4.2, can function as an active kinase and is localised in a novel and distinct compartment of the parasite-infected red blood cell which we have called K-dots. Notably, targeted disruption of the gene encoding FIKK4.2 (fikk4.2) dramatically alters the parasite's ability to modify and remodel the red blood cells in which it multiplies. Specifically, red blood cells infected with fikk4.2 knockout parasites were significantly less rigid and less adhesive when compared with red blood cells infected with normal parasites from which the transgenic clones had been derived, despite expressing similar levels of the major cytoadhesion ligand, PfEMP1, on the red blood cell surface. Notably, these changes were accompanied by dramatically altered knob-structures on infected red blood cells that play a key role in cytoadhesion which is responsible for much of the pathogenesis associated with falciparum malaria. Taken together, our data identifies FIKK4.2 as an important kinase in the pathogenesis of P. falciparum malaria and strengthens the attractiveness of FIKK kinases as targets for the development of novel next-generation anti-malaria drugs.

Contribution of parasite proteins to altered mechanical properties of malaria-infected red blood cells.

Red blood cells (RBCs) parasitized by Plasmodium falciparum are rigid and poorly deformable and show abnormal circulatory behavior. During parasite development, knob-associated histidine-rich 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.

Assignment of functional roles to parasite proteins in malaria-infected red blood cells by competitive flow-based adhesion assay.

Adhesion of parasitized red blood cells (PRBCs) to endothelial cells and subsequent accumulation in the microvasculature are pivotal events in the pathogenesis of falciparum malaria. During intraerythrocytic development, numerous proteins exported from the parasite associate with the RBC membrane skeleton but the precise function of many of these proteins remain unknown. Their cellular location, however, suggests that some may play a role in adhesion. The adhesive properties of PRBCs are best studied under flow conditions in vitro; however, experimental variation in levels of cytoadherence in currently available assays make subtle alterations in adhesion difficult to quantify. Here, we describe a flow-based assay that can quantify small differences in adhesion and document the extent to which a number of parasite proteins influence adhesion using parasite lines that no longer express specific proteins. Loss of parasite proteins ring-infected erythrocyte surface antigen (RESA), knob-associated histidine-rich protein (KAHRP) or Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) had a significant effect on the ability of PRBCs to adhere, whereas loss of mature parasite-infected erythrocyte surface antigen (MESA) had no effect. Our studies indicate that a number of membrane skeleton-associated parasite proteins, although not exposed on the RBC surface, can collectively affect the adhesive properties of PRBCs and further our understanding of pathophysiologically relevant structure/function relationships in malaria-infected RBCs.

Resonance Raman spectroscopy reveals new insight into the electronic structure of beta-hematin and malaria pigment.

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.