Plasmodium falciparum: analysis of transcribed var gene sequences in natural isolates from the Brazilian Amazon region☆
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).
Section snippets
Study area and population. Clinical and parasitological studies
Patients participating in this study were residents of the periurban areas of Porto Velho and nearby rural areas along the BR364 highway leaving Porto Velho in the direction of southern parts of Rondônia State. Patients attended the Ambulatory Unit of the Centro de Pesquisa em Medicina Tropical (CEPEM) for diagnosis and treatment of malaria and acute febrile syndromes in the period of August 1999–February 2000. Malaria in this area presents a hypoendemic profile with seasonal variation and
Cytoadhesion phenotype of trophozoite stage IRBC from natural infections
Cytoadherence assays were performed using IRBC suspensions prepared from isolates after maturation to trophozoite stage (see Section 2). Maturation of original ring stage parasites was controlled microscopically and the quantification of the different parasite forms after the maturation and concentration procedures showed that less than 5% of rings (usually 1–2% only) were still present in the final suspension (Table 2). Most assays were performed with IRBC suspensions with parasitemias varying
Discussion
In the present study, natural isolates of P. falciparum from patients suffering from falciparum malaria in the state of Rondônia (Western Amazon region, Brazil) were analysed for their cytoadherence properties and for the repertoire of the transcribed var gene repertoire. The clinical symptoms in all but one of the studied cases corresponded to symptomatic mild malaria and the symptoms of the only severe malaria case in this study reflected a multivisceral failure of organ function (data not
Acknowledgements
The authors thank W. Fischer for assistance during sequencing and Dr. E.P. Camargo for technical support. The authors thank Dr. L.A. Pirrit for critical reading of the manuscript. P.A.N. was supported by a CNPq doctoral fellowship, G.W. was supported by a FAPESP postdoctoral fellowship and J.D.C.N supported by a M.Sc. fellowship from CNPq. This work was supported by grants from FAPESP (Grant 1998/12107-2), CNPq, PRONEX and the EC (IC18CT98-0362).
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2008, Trends in ParasitologyCitation Excerpt :Another area of research where molecular techniques can be used in field studies is the understanding of variable antigens on the surface of infected erythrocytes. In an endeavor to identify those PfEMP1 molecules that are associated with a certain disease presentation, numerous studies have been conducted to analyze the expression of var genes from field samples [7,8,28–32]. Not only is this a major difficulty because RNA work has to be conducted under field conditions but also the value of the studies is brought into question by a recent publication by Montgomery et al. [33] from Malawi, who were able to show using post-mortem samples that tissue-specific var gene expression occurs.
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