The role of Plasmodium falciparum var genes in malaria in pregnancy.

Sequestration of Plasmodium falciparum-infected erythrocytes in the placenta is responsible for many of the harmful effects of malaria during pregnancy. Sequestration occurs as a result of parasite adhesion molecules expressed on the surface of infected erythrocytes binding to host receptors in the placenta such as chondroitin sulphate A (CSA). Identification of the parasite ligand(s) responsible for placental adhesion could lead to the development of a vaccine to induce antibodies to prevent placental sequestration. Such a vaccine would reduce the maternal anaemia and infant deaths that are associated with malaria in pregnancy. Current research indicates that the parasite ligands mediating placental adhesion may be members of the P. falciparum variant surface antigen family PfEMP1, encoded by var genes. Two relatively well-conserved subfamilies of var genes have been implicated in placental adhesion, however, their role remains controversial. This review examines the evidence for and against the involvement of var genes in placental adhesion, and considers whether the most appropriate vaccine candidates have yet been identified.

Sequence of Plasmodium falciparum chromosomes 1, 3-9 and 13.

Since the sequencing of the first two chromosomes of the malaria parasite, Plasmodium falciparum, there has been a concerted effort to sequence and assemble the entire genome of this organism. Here we report the sequence of chromosomes 1, 3-9 and 13 of P. falciparum clone 3D7--these chromosomes account for approximately 55% of the total genome. We describe the methods used to map, sequence and annotate these chromosomes. By comparing our assemblies with the optical map, we indicate the completeness of the resulting sequence. During annotation, we assign Gene Ontology terms to the predicted gene products, and observe clustering of some malaria-specific terms to specific chromosomes. We identify a highly conserved sequence element found in the intergenic region of internal var genes that is not associated with their telomeric counterparts.

Antigenic variation at the infected red cell surface in malaria.

Many pathogens that either rely on an insect vector to complete their life cycle (e.g., Trypanosoma spp. and Borrelia spp.) or exist in a unique ecological niche where transmission from host to host is sporadic (e.g., Neisseria spp.) have evolved strategies to maintain infection of their mammalian hosts for long periods of time in order to ensure their survival. Because they have to survive in the face of a fully functional immune system, a common feature of many of these organisms is their development of sophisticated strategies for immune evasion. For the above organisms and for malaria parasites of the genus Plasmodium, a common theme is the ability to undergo clonal antigenic variation. In all cases, surface molecules that are important targets of the humoral immune response are encoded in the genome as multicopy, nonallelic gene families. Antigenic variation is accomplished by the successive expression of members of these gene families that show little or no immunological cross-reactivity. In the case of malaria parasites, however, some of the molecules that undergo antigenic variation are also major virulence factors, adding an additional level of complication to the host-parasite interaction. In this review, we cover the history of antigenic variation in malaria and then summarize the more recent data with particular emphasis on Plasmodium falciparum, the etiological agent of the most severe form of human malaria.

Decoding the language of var genes and Plasmodium falciparum sequestration.

Sequestration and rosetting are key determinants of Plasmodium falciparum pathogenesis. They are mediated by a large family of variant proteins called P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 proteins are multispecific binding receptors that are transported to parasite-induced, 'knob-like' binding structures at the erythrocyte surface. To evade immunity and extend infections, parasites clonally vary their expressed PfEMP1. Thus, PfEMP1 are functionally selected for binding while immune selection acts to diversify the family. Here, we describe a new way to analyse PfEMP1 sequence that provides insight into domain function and protein architecture with potential implications for malaria disease.

Genetic restriction of Plasmodium falciparum in an area of stable transmission: an example of island evolution?

To date, a high degree of polymorphism has been demonstrated at both the MSP1 and MSP2 loci in parasites from areas of stable malaria transmission. As a consequence, in such areas it is rare to find parasites of the same 2-locus genotype in more than 1 subject. We have studied MSP1 and MSP2 diversity in parasites collected from subjects with both symptomatic (n = 86) and asymptomatic (34) malaria living on the island of Santo, Vanuatu, an area of stable malaria transmission. Polymorphism at the MSP1 and MSP2 loci was considerably less than previously reported: only 5 MSP1 and 5 MSP2 alleles were detected and these showed no size variation within alleles. Santo is unique amongst the areas studied so far in that it is a small island at the limit of the malaria belt in the South Pacific. Thus, the evolution of the parasite population may have been affected by the small size and isolation of this island population. Moreover, limited parasite diversity may explain the unusually mild nature of Plasmodium falciparum disease on Santo. Islands have fascinated biologists for centuries and fuelled the advancement of evolutionary theory, since they are natural laboratories for the study of evolution. The simplicity of the Vanuatu P. falciparum population may facilitate the use and interpretation of sequence level analyses to address the mechanisms by which genetic diversity is generated and maintained in natural populations.

Identification of a Plasmodium falciparum intercellular adhesion molecule-1 binding domain: a parasite adhesion trait implicated in cerebral malaria.

Binding of infected erythrocytes to brain venules is a central pathogenic event in the lethal malaria disease complication, cerebral malaria. The only parasite adhesion trait linked to cerebral sequestration is binding to intercellular adhesion molecule-1 (ICAM-1). In this report, we show that Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) binds ICAM-1. We have cloned and expressed PfEMP1 recombinant proteins from the A4tres parasite. Using heterologous expression in mammalian cells, the minimal ICAM-1 binding domain was a complex domain consisting of the second Duffy binding-like (DBL) domain and the C2 domain. Constructs that contained either domain alone did not bind ICAM-1. Based on phylogenetic criteria, there are five distinct PfEMP1 DBL types designated alpha, beta, gamma, delta, and epsilon. The DBL domain from the A4tres that binds ICAM-1 is DBLbeta type. A PfEMP1 cloned from a distinct ICAM-1 binding variant, the A4 parasite, contains a DBLbeta domain and a C2 domain in tandem arrangement similar to the A4tres PfEMP1. Anti-PfEMP1 antisera implicate the DBLbeta domain from A4var PfEMP1 in ICAM-1 adhesion. The identification of a P. falciparum ICAM-1 binding domain may clarify mechanisms responsible for the pathogenesis of cerebral malaria and lead to interventions or vaccines that reduce malarial disease.

Entering the post-genomic era of malaria research.

The sequencing of the genome of Plasmodium falciparum promises to revolutionize the way in which malaria research will be carried out. Beyond simple gene discovery, the genome sequence will facilitate the comprehensive determination of the parasite's gene expression during its developmental phases, pathology, and in response to environmental variables, such as drug treatment and host genetic background. This article reviews the current status of the P. falciparum genome sequencing project and the unique insights it has generated. We also summarize the application of bioinformatics and analytical tools that have been developed for functional genomics. The aim of these activities is the rational, information-based identification of new therapeutic strategies and targets, based on a thorough insight into the biology of Plasmodium spp.

A study of var gene transcription in vitro using universal var gene primers.

The polymorphic multigene family, var, encodes the variant antigen, Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), present on the surface of erythrocytes infected with the human malaria parasite, P. falciparum. PfEMP1 has been implicated in the pathology of malaria through its ability to bind to host endothelial receptors and uninfected erythrocytes. Understanding the relationship between host pathology, immune response and parasite variation is crucial, but requires a method of reliably detecting and differentiating all possible var genes. Several primer pairs used to date are biased and limited in their detection capacity. Here we describe a set of PCR primers that amplify the majority of var genes in the laboratory isolates 3D7 and A4, and appear to work equally well on all isolates tested. We use these universal primers to examine the relationship between var gene transcription as assessed by reverse transcriptase-PCR (RT-PCR) with that measured by Northern analysis of parasite RNA. Phenotypically selected young parasites have multiple transcripts detected by RT-PCR, but the full-length transcript appears to be homogeneous. In addition, we demonstrate that the choice of primers used for RT-PCR is crucial in data interpretation.

Cytoadherence, pathogenesis and the infected red cell surface in Plasmodium falciparum.

The particular virulence of Plasmodium falciparum compared with the other malaria species which naturally infect humans is thought to be due to the way in which the parasite modifies the surface of the infected red cell. Approximately 16 hours into the asexual cycle, parasite encoded proteins appear on the red cell surface which mediate adherence to a variety of host tissues. Binding of infected red cells to vascular endothelium, a process which occurs in all infections, is thought to be an important factor in the pathogenesis of severe disease where concentration of organisms in particular organs such as the brain occurs. Binding to uninfected red cells to form erythrocyte rosettes, a property of some isolates, is linked to disease severity. Here we summarise the data on the molecular basis of these interactions on both the host and parasite surfaces and review the evidence for the involvement of particular receptors in specific disease syndromes. Finally we discuss the relevance of these data to the development of new treatments for malaria.

The complete nucleotide sequence of chromosome 3 of Plasmodium falciparum.

Analysis of Plasmodium falciparum chromosome 3, and comparison with chromosome 2, highlights novel features of chromosome organization and gene structure. The sub-telomeric regions of chromosome 3 show a conserved order of features, including repetitive DNA sequences, members of multigene families involved in pathogenesis and antigenic variation, a number of conserved pseudogenes, and several genes of unknown function. A putative centromere has been identified that has a core region of about 2 kilobases with an extremely high (adenine + thymidine) composition and arrays of tandem repeats. We have predicted 215 protein-coding genes and two transfer RNA genes in the 1,060,106-base-pair chromosome sequence. The predicted protein-coding genes can be divided into three main classes: 52.6% are not spliced, 45.1% have a large exon with short additional 5' or 3' exons, and 2.3% have a multiple exon structure more typical of higher eukaryotes.

Rifins: a second family of clonally variant proteins expressed on the surface of red cells infected with Plasmodium falciparum.

Many pathogens evade the host immune response or adapt to their environment by expressing surface proteins that undergo rapid switching. In the case of Plasmodium falciparum, products of a multigene family known as var are expressed on the surface of infected red cells, where they undergo clonal antigenic variation and contribute to malaria pathogenesis by mediating adhesion to a variety of host endothelial receptors and to uninfected red blood cells by forming rosettes. Herein we show that a second gene family, rif, which is associated with var at subtelomeric sites in the genome, encodes clonally variant proteins (rifins) that are expressed on the infected red cell surface. Their high copy number, sequence variability, and red cell surface location indicate an important role for rifins in malaria host-parasite interaction.

Analysis of adhesive domains from the A4VAR Plasmodium falciparum erythrocyte membrane protein-1 identifies a CD36 binding domain.

The A4VAR is a variant antigen expressed by a clonal line that binds CD36 and intercellular adhesion molecule-1, ICAM-1. We have cloned and sequenced the extracellular domain coded by the A4var gene. To probe the relationship between A4var expression and parasite adhesion to ICAM-1, var mRNA and protein expression were analyzed in an enriched population of A4 parasites that displayed higher ICAM-1 binding. By Northern analyses, A4var was the predominant var message and antisera raised against a recombinant A4VAR protein reacted with the majority of infected erythrocytes, reinforcing previous conclusions that A4VAR binds ICAM-1. A4VAR contains five Duffy-binding like (DBL) domains, and two cysteine-rich interdomain regions (CIDR) domains. DBL and CIDR domains from A4VAR were expressed in mammalian cells to determine which regions mediate binding to CD36 and ICAM-1. Using several different binding assays, the A4VAR CIDR1 was the only domain found to bind CD36. In contrast, the same assays were unable to identify the ICAM-1 binding domain in A4VAR. This is the first time that each of the DBL and CIDR domains from a Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) have been systematically expressed and tested for binding. These results confirm that CIDR1 is sufficient to bind CD36 without any apparent contribution from other domains.

Limited spatial clustering of individual Plasmodium falciparum alleles in field isolates from coastal Kenya.

We describe Plasmodium falciparum genetic diversity in coastal Kenya, typing S-antigen and the merozoite surface proteins 1 and 2 (MSP-1 and MSP-2) in field isolates by the polymerase chain reaction (PCR). Malaria in coastal Kenya is characterized by low seasonal transmission, and a relatively high incidence of severe disease, which tends to occur in time-space clusters. We chose the highly polymorphic S-antigen as a marker for localized parasite diversity because it has been shown to vary in serotype prevalence in time and space. A total of 261 children (up to nine years of age) in two neighboring locations with different transmission rates were sampled for blood-stage parasites in cross-sectional surveys before and after the main transmission period in 1991, and also in a concomitant one-year longitudinal survey tracing clinical infections. Six major sequence types of S-antigen were identified, which were subdivided into 70 alleles; however, only 50% of isolates were typeable. The S-antigen sequence types varied qualitatively between locations, over time, and between asymptomatic and clinical disease infections, but not between different age groups. The MSP-1 and MSP-2 sequence type prevalences, in contrast, did not differ in any of these comparisons. We describe the use of the Mantel test for assessing clustering of individual parasite alleles at the household level, and demonstrate low-level clustering of MSP-1 and MSP-2 alleles and S-antigen sequence types, at the end of a long period of low transmission.

A high frequency African coding polymorphism in the N-terminal domain of ICAM-1 predisposing to cerebral malaria in Kenya.

The malarial parasite Plasmodium falciparum has acted as a potent selective force on the human genome. The particular virulence of this organism is thought to be due to the adherence of parasitised red blood cells to small vessel endothelium through several receptors, including CD36, thrombospondin and intercellular adhesion molecule 1 (ICAM-1, CD54), and parasite isolates differ in their ability to bind to each. Immunohistochemical studies have implicated ICAM-1 as of potential importance in the pathogenesis of cerebral malaria, leading us to reason that if any single receptor were involved in the development of cerebral malaria, then in view of the high mortality of that complication, natural selection should have produced variants with reduced binding capacity. We therefore sequenced the N-terminal domain of ICAM-1 from a number of Africans and discovered a single mutation present at high frequency. Genotypes at this locus from samples from a case-control study indicated an association of the polymorphism with the severity of clinical malaria such that individuals homozygous for the mutation have increased susceptibility to cerebral malaria with a relative risk of two. These counterintuitive results have implications for the mechanism of malaria pathogenesis, resistance to other infectious agents and transplantation immunology.

PfEMP1, polymorphism and pathogenesis.

The virulence of Plasmodium falciparum relative to the other species of malarial parasite which infect humans is thought to be due to this parasite's ability to adhere to endothelial cells lining small blood vessels and, in some cases, to its ability to form rosettes with uninfected erythrocytes. The latter phenotype has been found more frequently in cases of severe disease. The former property means that only the younger, asexual, intra-erythrocytic forms circulate whereas the more mature developmental stages are sequestered in the vasculature of a variety of organs. When large numbers of parasites accumulate in a vulnerable target organ such as the brain, the the life-threatening condition of cerebral malaria may result. While the factors that control whether or not cerebral malaria develops are not clearly defined, one crucial determinant my be the endothelial receptors utilised by the infecting isolate. Many such receptors have been identified, including CD36, thrombospondin, ICAM-1, VCAM, E-selectin and chondroitin-4-sulphate. The results of laboratory, field, post-mortem and direct receptor-binding studies indicate that, of the receptors currently identified, ICAM-1 binding is more likely to be associated with the development of cerebral malaria. The molecule expressed on the surface of the infected erythrocyte which mediates adherence to endothelium belongs to a large family of clonally variable antigens encoded by the var genes. The evidence for this conclusion and progress in defining the regions of var-gene products responsible to receptor-specific binding are discussed. Finally, the organization of the var genes within and between parasites is discussed in relation to the evolution of the var-gene family and its functions of antigenic variation and endothelial adhesion.

Plasmodium falciparum: a method for the amplification of S antigens and its application to laboratory and field samples.

Polymerase chain reaction amplification of several polymorphic genes has been used to study the population biology of Plasmodium falciparum. S antigen is particularly suitable for such studies, but difficulties in the amplification of this gene have precluded its use. Here we describe a simple method for the amplification of S antigen and show why previous attempts may have been unsuccessful. Data are presented from both laboratory and field isolates.