CD36-dependent adhesion and knob expression of the transmission stages of Plasmodium falciparum is stage-specific

Abstract

Plasmodium falciparum trophozoites sequester from the peripheral circulation by adherence to host endothelium. Gametocytes, also sequester during maturation. Analysis of the adhesion phenotype of stage I to V gametocytes of several isolates/clones was assessed by binding of infected cells to C32 melanoma cells (C32MC) and the purified adhesion proteins, antigen leucocyte differentiation (CD36) and intercellular adhesion molecule-1 (ICAM-1). These cells and proteins, have previously been shown to be receptors for adherence of trophozoites. Early gametocytes (stages I–IIA) were found to bind to C32MC as well as the purified receptor CD36 but not to ICAM-1. Early gametocytes bound to C32MC via CD36 and the parasite ligand involved in this binding was trypsin sensitive. Stage IIB to V gametocytes did not adhere to C32MC, CD36 nor ICAM-1. Electron-dense protruberances known as knobs and histidine rich protein 1 (HRP 1) expression have been associated with trophozite adhesion to CD36. Knobs were present at the surface of early but not late gametocyte infected cells. Stage-specific patterns of HRP 1 expression, consistent with a role for this molecule in CD36 adhesion of early gametocytes, were also observed. The adhesion phenotype of these young gametocytes was indistinguishable from that of the trophozoites by all criteria examined. These data support the hypothesis that other host receptors mediate the binding of late gametocytes.

Introduction

Erythrocytes infected with mature asexual stages of Plasmodium falciparum, known as trophozoites, sequester from the peripheral circulation by binding to the microvascular endothelium. Adhesion of these stages is thought to be a major virulence factor of P. falciparum for two reasons. Adherent parasites are found blocking the capillaries of the brains of subjects who have died of cerebral malaria 1, 2and parasites that have lost the ability to adhere produce low density, avirulent infections in certain primate hosts [3]. Gametocytes of P. falciparum, which are responsible for transmission to the mosquito vector, also sequester. Hawking et al. [4]have classified five stages of gametocyte development within the erythrocite. Stages I to IV sequester during maturation in the spleen and bone marrow 5, 6. Stage V gametocytes are found in the peripheral circulation and these stages become infectious for anopheline mosquitos after a futher 2 to 3 days in circulation.

It has been argued that adhesion of mature asexual stages, for the latter half of the 48 h erythrocytic cycle, evolved to prevent these stages from circulating through the spleen, thereby avoiding splenic mechanisms of clearance of infected erythrocytes with reduced deformability 3, 7. A similar argument can be made for the sexual stages, known as gametocytes, which require 8 to 10 days maturation before becoming infectious to anopheline vectors. During maturation, P. falciparum gametocytes become crescent shaped. This change of shape is associated with the development of an extensive subpellicular microtubule system under the parasite membrane [8]. These microtubules are lost upon maturation of stage V gametocytes with associated release of infectious gametocytes into the peripheral circulation ready for transmission.

The molecular mechanisms responsible for trophozoite adhesion have been characterised in terms of both host cell receptors and parasite ligands 7, 9. The leucocyte differentiation antigen CD36 10, 11, thrombospondin [12], the intercellular adhesion molecule 1 (ICAM-1) [13]vascular cell adhesion molecule 1, endothelial leucocyte adhesion molecule 1, [14]and chondroitin sulphate A [15]have been identified as potential sequestration receptors on human endothelium. All wild type P. falciparum isolates adhere to CD36 in vitro, whereas only a minority of isolates bind to the other host adhesion receptors in the absence of selection.

Erythrocytes infected with trophozoites develop surface membrane deformations known as knobs 16, 17, which appear to be necessary for adhesion in vivo [3]but not in vitro 18, 19. Immunoelectron microscopy studies have localised a protein of 80–110 kDa, designated histidine-rich protein 1 (also known as KAHRP), in the erythrocyte skeleton in the knobs [20]. Knobless lines of P. falciparum lack histidine rich protein (HRP) 1 expression [21]but can adhere via CD36 dependant mechanisms in vitro 18, 19. Disruption of the gene encoding HRP 1 shows that this molecule is necessary for knob formation and that knobless parasites are unable to adhere to CD36 under physiological shear stress conditions [22].

Adherent cells infected with trophozoites also express an agglutinogen on the surface of the infected erythrocyte that is highly polymorphic 23, 24. Surface radioiodination of these cells has led to identification of a trypsin sensitive, high molecular weight antigen designated P. falciparum erythrocyte membrane protein 1(PfEMP 1) [25]which has recently been shown to be the parasite ligand for trophozoite adhesion to CD36 [26]. PfEMP 1 has also been shown to undergo clonal antigenic variation in vitro 27, 28and is encoded by the recently described var gene family 29, 30, 31.

To date, there has been little work on the molecular mechanisms involved in gametocyte adhesion. Differences in site specificity of sequestration of sexual and asexual stages have led to the speculation that the mechanisms of adhesion are likely to be different in the two developmental pathways 5, 6, although a recent report suggests that this may not be the case. Rogers et al. [32]report low levels of adhesion of stages I to IV gametocytes (3D7 line) to C32 melanoma cells (C32MC), an in vitro correlate of adhesion in vivo [33], at high parasitaemia. This binding was inhibited by anti-CD36 and anti-ICAM-1 monoclonal antibodies (mAbs).

In the present study we have characterised stage-specific gametocyte adhesion, knob and HRP 1 expression of a number of isolates and clones of P. falciparum. These data, which differ significantly from those reported by Rogers et al. [32]for a single parasite line, are interpreted in the context of what is known about the adhesion mechanisms of trophozoites of P. falciparum.