Plasmodium falciparum: analysis of transcribed var gene sequences in natural isolates from the Brazilian Amazon region

Abstract

Parasite isolates from Brazilian Western Amazonian patients suffering from uncomplicated falciparum malaria were matured in vitro and their var gene transcripts were analysed by RT-PCR and sequencing. Additionally, the cytoadherence patterns of these isolates were determined by panning techniques using transfected CHO cell lines expressing different surface receptors. All of the isolates tested showed between 4 and 13 different var gene transcripts per isolate. Several of these transcripts were present in more than one isolate and three sequences appeared to be preferentially expressed in natural infections. In most of the isolates, cytoadherence occurred to the receptors ICAM-1 and CD36. Several isolates showed a multiadherent profile. Analysis of MSP1 and MSP2 allelic polymorphism indicated polyclonal infections, that could be responsible for the multiadherent phenotype.

Introduction

Cytoadhesion of mature parasitized red blood cells to vascular endothelial cells (sequestration) and binding of unparasitized red cells to parasitized ones (rosetting) are considered important virulence factors in Plasmodium falciparum infections. Both phenomena depend on the expression of variant parasite proteins that are exposed on the surface of infected red blood cells. Sequestration and rosetting have been associated with severe malaria and in particular with cerebral malaria which is responsible for high infant mortality in Africa (reviewed in Newbold et al., 1999; Wahlgren et al., 1999). The molecular basis of cytoadhesion and rosetting has been extensively investigated in recent years and various molecules, from both the host cells and the parasitized cell membrane have been identified in these host–parasite interactions (Craig and Scherf, 2001).

Several endothelial cell membrane proteins belonging to the integrin and adhesin families were shown to be receptors involved in sequestration. These include CD36, ICAM-1 (inter cellular adhesion molecule 1), VCAM (vascular cell adhesion molecule), trombospondin and E-selectin. More recently, the role of Chondroitin sulfate A (CSA), a glycosaminoglycan present in thrombomodulin, and binding to human IgG was shown in the sequestration in placenta syncytiotrophoblasts (Buffet et al., 1999; Flick et al., 2001; Fried and Duffy, 1996; Reeder et al., 1999).

The PfEMP-1 antigens of P. falciparum (erythrocyte membrane protein-1), large molecules of 200–350 kDa were identified as parasite ligands responsible for receptor binding. They are a family of highly polymorphic and antigenically variant proteins (Leech et al., 1984) encoded by a multigenic family—the var genes—composed of 40–50 members per haploid genome. The var genes are distributed in all of the parasite’s chromosomes (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995) with a preferential subtelomeric localisation (Fischer et al., 1997; Hernandez Rivas et al., 1997).

A considerable amount of information has been accumulated regarding the structure, chromosome location, mechanisms of antigenic shifting, expression of var genes and binding specificity of their variant PfEMP-1 products (see reviews by Newbold et al., 1999; Wahlgren et al., 1999). The high interest in this field is related to the high immunogenicity of these antigens and the possibility that they could represent important targets for asexual blood stage vaccines. Indeed, it was shown that PfEMP-1 variants are targets of antibodies agglutinating infected red blood cells (IRBCs) (Bull et al., 1998; Smith et al., 1995), which is consistent with the idea that such antibodies are related to variant specific immunity.

To date, little data exists on the transcription of var genes in field isolates. Although previous studies have shown that specific var gene sequences appear in different isolates from infections occurring in the same geographical location or even in distant areas (Kirchgatter et al., 2000), nothing so far is known about the redundancy of transcripts in different infections. Nor is it known if there are a number of different transcripts present (indicating a number of antigenically different circulating IRBC) at a given time point during a single infection. To address these questions and to evaluate the cytoadherence patterns in Brazilian P. falciparum isolates, we analysed the var gene repertoire transcribed by parasites from falciparum malaria patients from the Porto Velho area of Rondônia (Brazilian Western Amazon region).