Brian M. Cooke

Targeted Gene Disruption Shows That Knobs Enable Malaria-Infected Red Cells to Cytoadhere under Physiological Shear Stress

Knobs at the surface of erythrocytes infected with Plasmodium falciparum have been proposed to be important in adherence of these cells to the vascular endothelium. This structure contains the knob-associated histidine-rich protein (KAHRP) and the adhesion receptor P. falciparum erythrocyte membrane protein 1. We have disrupted the gene encoding KAHRP and show that it is essential for knob formation. Knob-transfectants adhere to CD36 in static assays; when tested under flow conditions that mimic those of postcapillary venules, however, the binding to CD36 was dramatically reduced. These data suggest that knobs on P. falciparum-infected erythrocytes exert an important influence on adherence of parasitized-erythrocytes to microvascular endothelium, an important process in the pathogenesis of P. falciparum infections.

Adhesion of Plasmodium falciparum-infected erythrocytes to hyaluronic acid in placental malaria

Infection with Plasmodium falciparum during pregnancy leads to the accumulation of parasite-infected erythrocytes in the placenta, and is associated with excess perinatal mortality, premature delivery and intrauterine growth retardation in the infant, as well as increased maternal mortality and morbidity. P. falciparum can adhere to specific receptors on host cells, an important virulence factor enabling parasites to accumulate in various organs. We report here that most P. falciparum isolates from infected placentae can bind to hyaluronic acid, a newly discovered receptor for parasite adhesion that is present on the placental lining. In laboratory isolates selected for specific high-level adhesion, binding to hyaluronic acid could be inhibited by dodecamer or larger oligosaccharide fragments or polysaccharides, treatment of immobilized receptor with hyaluronidase, or treatment of infected erythrocytes with trypsin. In vitro flow-based assays demonstrated that high levels of adhesion occurred at low wall shear stress, conditions thought to prevail in the placenta. Our findings indicate that adhesion to hyaluronic acid is involved in mediating placental parasite accumulation, thus changing the present understanding of the mechanisms of placental infection, with implications for the development of therapeutic and preventative interventions.

Malaria parasite proteins that remodel the host erythrocyte

Exported proteins of the malaria parasite Plasmodium falciparum interact with proteins of the erythrocyte membrane and induce substantial changes in the morphology, physiology and function of the host cell. These changes underlie the pathology that is responsible for the deaths of 1–2 million children every year due to malaria infections. The advent of molecular transfection technology, including the ability to generate deletion mutants and to introduce fluorescent reporter proteins that track the locations and dynamics of parasite proteins, has increased our understanding of the processes and machinery for export of proteins in P. falciparum-infected erythrocytes and has provided us with insights into the functions of the parasite protein exportome. We review these developments, focusing on parasite proteins that interact with the erythrocyte membrane skeleton or that promote delivery of the major virulence protein, PfEMP1, to the erythrocyte membrane.

Novel roles for erythroid Ankyrin-1 revealed through an ENU-induced null mouse mutant

Insights into the role of ankyrin-1 (ANK-1) in the formation and stabilization of the red cell cytoskeleton have come from studies on the nb/nb mice, which carry hypomorphic alleles of Ank-1. Here, we revise several paradigms established in the nb/nb mice through analysis of an N-ethyl-N-nitrosourea (ENU)-induced Ank-1-null mouse. Mice homozygous for the Ank-1 mutation are profoundly anemic in utero and most die perinatally, indicating that Ank-1 plays a nonredundant role in erythroid development. The surviving pups exhibit features of severe hereditary spherocytosis (HS), with marked hemolysis, jaundice, compensatory extramedullary erythropoiesis, and tissue iron overload. Red cell membrane analysis reveals a complete loss of ANK-1 protein and a marked reduction in beta-spectrin. As a consequence, the red cells exhibit total disruption of cytoskeletal architecture and severely altered hemorheologic properties. Heterozygous mutant mice, which have wild-type levels of ANK-1 and spectrin in their RBC membranes and normal red cell survival and ultrastructure, exhibit profound resistance to malaria, which is not due to impaired parasite entry into RBC. These findings provide novel insights into the role of Ank-1, and define an ideal model for the study of HS and malarial resistance.

New guidelines for hemorheological laboratory techniques

This document, supported by both the International Society for Clinical Hemorheology and the European Society for Clinical Hemorheology and Microcirculation, proposes new guidelines for hemorheolog ...

The malaria-infected red blood cell: structural and functional changes.

Abstract The asexual stage of malaria parasites of the genus Plasmodium invade red blood cells of various species including humans. After parasite invasion, red blood cells progressively acquire a new set of properties and are converted into more typical, although still simpler, eukaryotic cells by the appearance of new structures in the red blood cell cytoplasm, and new proteins at the red blood cell membrane skeleton. The red blood cell undergoes striking morphological alterations and its rheological properties are considerably altered, manifesting as red blood cells with increased membrane rigidity, reduced deformability and increased adhesiveness for a number of other cells including the vascular endothelium. Elucidation of the structural changes in the red blood cell induced by parasite invasion and maturation and an understanding of the accompanying functional alterations have the ability to considerably extend our knowledge of structure-function relationships in the normal red blood cell. Furthermore, interference with these interactions may lead to previously unsuspected means of reducing parasite virulence and may lead to the development of novel antimalarial therapeutics.

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 Maurers clefts, which are parasite-derived membranous structures in the IRBC cytoplasm. Targeted gene disruption of a Maurers 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 Maurers clefts, and the transport and localization of several other Maurers cleft proteins were unchanged. Maurers 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.

Targeted mutagenesis of Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) disrupts cytoadherence of malaria‐infected red blood cells

Adhesion of parasite‐infected red blood cells to the vascular endothelium is a critical event in the pathogenesis of malaria caused by Plasmodium falciparum. Adherence is mediated by the variant erythrocyte membrane protein 1 (PfEMP1). Another protein, erythrocyte membrane protein‐3 (PfEMP3), is deposited under the membrane of the parasite‐infected erythrocyte but its function is unknown. Here we show that mutation of PfEMP3 disrupts transfer of PfEMP1 to the outside of the P.falciparum‐infected cell. Truncation of the C‐terminal end of PfEMP3 by transfection prevents distribution of this large (>300 kDa) protein around the membrane but does not disrupt trafficking of the protein from the parasite to the cytoplasmic face of the erythrocyte membrane. The truncated PfEMP3 accumulates in structures that appear to be associated with the erythrocyte membrane. We show that accumulation of mutated PfEMP3 blocks the transfer of PfEMP1 onto the outside of the parasitized cell surface and suggest that these proteins traffic through an erythrocyte membrane‐associated compartment that is involved in the transfer of PfEMP1 to the surface of the parasite‐infected red blood cell.

The Deformability of Red Blood Cells Parasitized by Plasmodium falciparum and P. vivax

Red blood cells (RBCs) must deform considerably during their multiple passages through the microvasculature and the sinusoids of the spleen. RBCs infected with Plasmodium falciparum (Pf-IRBCs) become increasingly rigid as they mature but avoid splenic clearance by sequestering in venules and capillaries. In contrast, RBCs infected with P. vivax (Pv-IRBCs) do not sequester. We compared the effects of P. vivax and P. falciparum infection on RBC deformability in a laminar shear flow system. Pf-IRBCs became more rigid as the parasite matured, but equivalent maturation of Pv-IRBCs resulted in a doubling of flexibility. Coincidentally, the IRBC surface area increased from 56.7+/-1.3 microm2 to 74.7+/-0.6 microm2 to 90.9+/-1.1 microm2 in ring-, trophozoite-, and schizont-stage Pv-IRBCs, respectively, whereas Pf-IRBCs did not increase in size. P. vivax increases the deformability of IRBCs and thereby avoids splenic entrapment.

Plasmodium falciparum–Mediated Induction of Human CD25hiFoxp3hi CD4 T Cells Is Independent of Direct TCR Stimulation and Requires IL-2, IL-10 and TGFβ

CD4+CD25+Foxp3+ regulatory T cells (Tregs) regulate disease-associated immunity and excessive inflammatory responses, and numbers of CD4+CD25+Foxp3+ Tregs are increased during malaria infection. The mechanisms governing their generation, however, remain to be elucidated. In this study we investigated the role of commonly accepted factors for Foxp3 induction, TCR stimulation and cytokines such as IL-2, TGFβ and IL-10, in the generation of human CD4+CD25+Foxp3+ T cells by the malaria parasite Plasmodium falciparum. Using a co-culture system of malaria-infected red blood cells (iRBCs) and peripheral blood mononuclear cells from healthy individuals, we found that two populations of Foxp3hi and Foxp3int CD4+CD25hi T cells with a typical Treg phenotype (CTLA-4+, CD127low, CD39+, ICOS+, TNFRII+) were induced. Pro-inflammatory cytokine production was confined to the Foxp3int subset (IFNγ, IL-4 and IL-17) and inversely correlated with high relative levels of Foxp3hi cells, consistent with Foxp3hi CD4 T cell–mediated inhibition of parasite-induced effector cytokine T cell responses. Both Foxp3hi and Foxp3int cells were derived primarily from proliferating CD4+CD25− T cells with a further significant contribution from CD25+Foxp3+ natural Treg cells to the generation of the Foxp3hi subset. Generation of Foxp3hi, but not Foxp3int, cells specifically required TGFβ1 and IL-10. Add-back experiments showed that monocytes expressing increased levels of co-stimulatory molecules were sufficient for iRBC-mediated induction of Foxp3 in CD4 T cells. Foxp3 induction was driven by IL-2 from CD4 T cells stimulated in an MHC class II–dependent manner. However, transwell separation experiments showed that direct contact of monocytes with the cells that acquire Foxp3 expression was not required. This novel TCR-independent and therefore antigen-non specific mechanism for by-stander CD4+CD25hiFoxp3+ cell induction is likely to reflect a process also occurring in vivo as a consequence of immune activation during malaria infection, and potentially a range of other infectious diseases.