Extense variant gene family repertoire overlap in Western Amazon Plasmodium falciparum isolates

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Abstract

In order to find a molecular basis for observations of relatively fast developing immunity to malarial infections in the Western Amazon region, the partial var, stevor and rif gene repertoires of nine different Plasmodium falciparum isolates collected in 1985 and 2000–2004 were evaluated. In contrast to previous results from South East Asia, the variant gene repertoire in Brazilian isolates is rather small and redundant. While the individual var repertoire sizes of Brazilian strains did not differ from Southeast Asian/African isolates, we found an over three times higher overlap of var sequence repertoires in Amazonian strains which was also conserved over time, suggesting the ongoing circulation of a similar var gene repertoire. Coincidently, almost 40% of the sequences identified herein showed the highest degree of similarity to var genes from either Brazilian or Venezuelan isolates, indicating a limited var repertoire of P. falciparum in the Amazon Basin as a whole. The intrastrain similarities of var genes were slightly but significantly lower than in Southeast Asian/African samples suggesting a higher selective pressure for diversification in Amazonian isolates. Despite of higher copy numbers per genome, rif genes also showed a significant repertoire overlap. stevor genes, which share the same predominant subtelomeric localization as var and rif genes, showed a still higher repertoire overlap and were highly similar to 3D7 stevor genes, indicating stronger functional conservation than var and rif genes. This is the first study that reveals that P. falciparum variant gene repertoires of certain areas can be limited. This has important implications for the strain-specific immunity against variant antigens occurring in these areas.

Introduction

The infection of humans with the apicomplexan parasite Plasmodium falciparum continues to be one of the most serious health problems in the world and 1–2 million people, mainly sub-Saharan African infants, succumb each year to the disease [1]. In the Amazon basin, where most cases of the American continent occur, the epidemiologic situation seems less dramatic. Although in Brazil around 450,000 new infections per year are reported which are caused mainly by Plasmodium vivax and to a lesser extent by P. falciparum [2], very few persons die from falciparum malaria.

P. falciparum uses antigenic variation in order to escape the immune attack and establish long-term infections. A major pathogenic factor is families of variant proteins localized on the infected erythrocyte (IRBC) surface which interact with endothelial and other receptors, thus avoiding spleen passage of the IRBC, or with unknown functions. The antigens which are responsible for receptor mediated IRBC-adherence were termed P. falciparum erythrocyte membrane proteins 1 (PfEMP1 [3]) and are encoded by a highly polymorphic gene family known as var genes [4], [5], [6]. Each parasite genome contains between 35 and 60 var genes [5], [7] most of which are located in subtelomeric parts of each chromosome [8], [9] and expressed in a highly regulated manner [10], [11], [12], [13], [14]. Another, still larger family of IRBC surface variant antigens termed RIFINs, encoded by the rif genes [15], [16], [17], share the chromosomal localization with the var genes [9]. Although an association to rosetting was proposed [17], the biological function of RIFINs genes is not yet clear. Gene families similar in structure and number to rif genes are found in all human and rodent Plasmodium species studied so far (analysis in Ref. [18]). The family of stevor genes, also located in clusters with var and rif genes, codes for antigens of yet unknown function which are present in Maurer's clefts in the asexual blood stage parasites, but are found on the surface of gametocytes [19]. The majority of var, rif and stevor genes are located in subtelomeric regions of chromosomes, which seem to recombine not only by homologous but also by ectopic recombination [20]. Therefore, it seems reasonable to expect that the subtelomeric variant gene repertoire is unlimited and ever-developing. Accordingly, Fowler et al. found only two shared var sequences between any two isolates when analyzing var gene repertoires from nine different strains from South East Asia and Africa [7]. Similar data were recently found in a number of isolates from infected children in Kilifi, Kenya [21]. However, a significant number of identical transcripts in different parasite isolates was observed [22], [23] when measuring var gene transcription in Brazilian Western Amazon P. falciparum. Also, a significant number of transcribed var sequences, shared between different isolates from different locations and time points, was detected in another var transcript analysis in isolates from all over Brazil [24]. Taking in account (i) observations of rapidly developing immunity in isolated Amazonian riverine populations which were infected with genetically highly related parasites [25] and considering that (ii) PfEMP1 and RIFIN are important targets of the strain-specific host immune defense [26], [27], we asked if a smaller variant antigen repertoire of Amazonian isolates could be the reason for these findings. Also, rif and stevor gene sequence repertoires and their polymorphisms were never systematically studied in field isolates. In the present study, we simultaneously analyzed partial repertoires of var, rif and stevor gene families of nine isolates from the Western Amazonian Amazon.

Section snippets

Parasite isolates

P. falciparum isolates 123 and 154 were obtained during a short term survey study in 1985 in the surroundings of Ariquemes, Rondônia State, Western Brazil. Isolate 106 was obtained in July 2001 from a citizen of the Candelária suburb, Porto Velho, Rondônia, Brazil (200 km from Ariquemes). Isolates 42 and 112, 114, 115 and 134 were obtained in August 2003 and March 2004, respectively, from patients seeking medical advice in Porto Velho and who were infected within a radius of 50 km of the

The analyzed parasite populations were genetically homogenous

Following microsatellite analysis, all parasites showed only one main genotype (Table S2 A). Importantly, no minor peaks (>5% intensity of the main peak) were observed, indicating the absence of co-circulating genetically different parasite populations (data not shown), even in the uncloned parasite lines. None of the parasites shared identical combinations of microsatellite sizes, indicating that all genomes were different from each other. The average genetic distance based on microsatellite

Discussion

Var genes are a major virulence factor in the infection with P. falciparum and it was previously shown that the var-encoded P. falciparum erythrocyte membrane proteins are a target of a protective immune response [26]. Similarly, RIFINs, the proteins encoded by rif genes, are also recognized by immunoglobulins of frequently infected individuals [27]. The pathogenic properties of RIFINs are currently unknown. To our knowledge, no data are currently available regarding the immune response against

Acknowledgments

The authors would like to thank Dr. Juan Miguel Salcedo Villalobos, Dr. Dhelio Batista Pereira, Dr. Mauro Shugiro Tada, Prof. Luiz Hildebrando Pereira da Silva and the staff of the CEPEM for continuing support, Wolfgang Fischer and Daniele Barbosa for sample preparation and sequencing. We also thank Dr. Silvano Wendel, Director of the Banco de Sangue do Hospital Sírio Libanês, for the kind supply of blood and serum for the in vitro culture of P. falciparum, and Jeffrey J. Shaw for critical

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    Note: Sequences from this work were deposited under GenBank entries: DQ265158DQ265705.

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