CD36-dependent adhesion and knob expression of the transmission stages of Plasmodium falciparum is stage-specific
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.
Section snippets
Parasites
Parasite isolates 1776, 1933, 1916 and 1935 were collected from donors in Papua New Guinea as described previously [24]. Isolate MUZ12 was established in culture more recently after collection during a longitudinal cohort study in Papua New Guinea [34]. Cloned lines 3D7, HB3 and XP9 were obtained from Professor D. Walliker, Edinburgh, UK. The cloned line A4 selected by Roberts et al. [28]was used as an ICAM-1 binding positive control for receptor adhesion experiments. Gametocytes were produced
In vitro adhesion of gametocytes
Analysis of stage-specific parasitaemia before and (in the supernatant) after binding to C32MC showed depletion of early but not late gametocytes (Table 1A). Stage I gametocytes (small, round cell) and early stage II gametocytes (defined as IIA) which have lost their rounded shape and become elongated within the erythrocyte also bound to C32MC. Stage IIB gametocytes identified by a characteristic D shape, did not bind, nor did stage III, IV and V gametocytes. The same stage specificity of
Discussion
The data presented show that CD36 mediates adhesion of sexual stage parasites to C32MC. Interestingly, only early gametocytes (i.e. stage I and IIA) were capable of binding to these cells whereas, in vivo, these stages as well as stage IIB, III and IV gametocytes adhere 5, 6. The stage specificity of gametocytes binding to purified CD36 was identical to that observed for C32MC. Clearly other host adhesion receptors must be involved in the sequestration of late stage gametocytes (IIB-IV).
Knobs
Acknowledgements
K.P. Day would like to acknowledge Dr Patricia Graves for advice in establishing gametocyte culture methods in her laboratory. Support for preliminary experiments at The Walter and Eliza Hall Institute for Medical Research was provided by the Australian National Health and Medical Research Council and the John D. and Catherine T. MacArthur Foundation for K.P. Day and J. Culvenor. K.P. Day supported completion of the project by a programme grant from The Wellcome Trust and a grant from INCO-DC.
References (45)
- et al.
Evidence for cyclic development and short-lived maturity in the gametocytes of Plasmodium falciparum
Trans R Soc Trop Med Hyg
(1971) - et al.
The structure and development of Plasmodium falciparum gametocytes in the internal organs and peripheral circulation
Trans R Soc Trop Med Hyg
(1935) - et al.
Sequestration in Plasmodium falciparum malaria: sticky cells and sticky problems
Parasitol Today
(1990) - et al.
Localisation of Plasmodium falciparum histidine-rich protein 1 in the erythrocyte skeleton under knobs
Mol Biochem Parasitol
(1987) - et al.
Cloning the Plasmodium falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes
Cell
(1995) - et al.
Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes
Cell
(1995) - et al.
The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum infected erythrocytes
Cell
(1995) - et al.
Dynamics of malaria parasitaemia associated with febrile illness in children from a rural area of Madang, Papua New Guinea
Trans R Soc Trop Med Hyg
(1994) - et al.
Refractoriness of erythrocytes infected with Plasmodium falciparum gametocytes to lysis by sorbitol
Int J Parasitol
(1990) - et al.
Cellular location and temporal expression of the Plasmodium falciparum sexual stage antigen, Pfs16
Mol Biochem Parasitol
(1994)
Plasmodium falciparum: Adhesion of a knobless clone
Exp Parasitol
Human cerebral malaria. A quantitative ultrastructural analysis of parasitised erythrocyte sequestration
Am J Pathol
Human cerebral malaria
Am J Trop Med Hyg
Pathogenicity, stability and immunogenicity of a knobless clone of Plasmodium falciparum in Colombian owl monkeys
Infect Immun
The distribution of Plasmodium falciparum in the peripheral blood and bone marrow of Gambian children
Trans R Soc Trop Med Hyg
Malarial proteins at the membrane of Plasmodium falciparum infected erythrocytes and their involvement in adhesion to endothelial cells
Prog Allergy
Gametocyte and gamete development in Plasmodium falciparum
Proc R Soc London B
A human 88-kD membrane glycoprotein (CD36) functions in vitro as a receptor for a cytoadherence ligand on Plasmodium falciparum-infected erythrocytes
J Clin Invest
Identification of a platelet membrane glycoprotein as a falciparum malaria sequestrian receptor
Science
Thrombospondin binds falciparum malaria parasitised erythrocytes and may mediate adhesion
Nat Lond
Intercellular adhesion molecule-1 is an endothelial cell adhesion receptor for Plasmodium falciparum
Nat Lond
Human vascular endothelial cell adhesion receptors for Plasmodium falciparum-infected erythrocytes: roles for ELAM-1 and VCAM-1
J Exp Med
Cited by (62)
Repurposing of Plasmodium falciparum var genes beyond the blood stage
2022, Current Opinion in MicrobiologyErythrocyte remodeling by Plasmodium falciparum gametocytes in the human host interplay
2015, Trends in ParasitologyCitation Excerpt :Unlike asexual parasites, the mechanism responsible for gametocyte sequestration is poorly understood. Older studies, generally using stage II–IV gametocytes, produced conflicting data on the adhesion efficiency and possible involvement of PfEMP1 and showed low binding efficiency of immature gametocytes to host endothelial or non-endothelial cells such as C32 melanoma or bone marrow stromal cell lines and to purified host ligands such as ICAM-1 and CD36 [35–38]. One study reported that stage I gametocytes produce knobs on the surface of infected red blood cells (RBCs) and bind to CD36+ C32 melanoma cells with the same avidity as asexual trophozoites [36].
PfEMP1 - a parasite protein family of key importance in plasmodium falciparum malaria immunity and pathogenesis
2015, Advances in ParasitologyShape-shifting gametocytes: How and why does P. falciparum go banana-shaped?
2012, Trends in ParasitologyCitation Excerpt :There is debate about what host receptors are involved and whether PfEMP1 plays a role in the sequestration process. One var gene subset (type C) is transcribed in gametocytes [46], and an early report suggested that early-stage gametocytes adhere to CD36 via PfEMP1 but that the PfEMP1 type is switched in later-stage gametocytes, allowing adhesion to ICAM-1 [47]. Others report that sequestration does not involve a CD36 interaction, and instead involves ICAM-1 binding at all stages of development [48].