The crystal structure of superoxide dismutase from Plasmodium falciparum

Background: Superoxide dismutases (SODs) are important enzymes in defence against oxidative stress. In Plasmodium falciparum, they may be expected to have special significance since part of the parasite life cycle is spent in red blood cells where the formation of reactive oxygen species is likely to be promoted by the products of haemoglobin breakdown. Thus, inhibitors of P. falciparum SODs have potential as anti-malarial compounds. As a step towards their development we have determined the crystal structure of the parasite's cytosolic iron superoxide dismutase. Results: The cytosolic iron superoxide dismutase from P. falciparum (PfFeSOD) has been overexpressed in E. coli in a catalytically active form. Its crystal structure has been solved by molecular replacement and refined against data extending to 2.5 angstrom resolution. The structure reveals a two-domain organisation and an iron centre in which the metal is coordinated by three histidines, an aspartate and a solvent molecule. Consistent with ultracentrifugation analysis the enzyme is a dimer in which a hydrogen bonding lattice links the two active centres. Conclusion: The tertiary structure of PfFeSOD is very similar to those of a number of other iron-and manganese-dependent superoxide dismutases, moreover the active site residues are conserved suggesting a common mechanism of action. Comparison of the dimer interfaces of PfFeSOD with the human manganese-dependent superoxide dismutase reveals a number of differences, which may underpin the design of parasite-selective superoxide dismutase inhibitors

Identification of Plasmodium falciparum var1CSA and var2CSA domains that bind IgM natural antibodies

Malaria in pregnancy is responsible for maternal anaemia, low-birth-weight babies and infant deaths. Plasmodium falciparum infected erythrocytes are thought to cause placental pathology by adhering to host receptors such as chondroitin sulphate A (CSA). CSA binding infected erythrocytes also bind IgM natural antibodies from normal human serum, a process that may facilitate placental adhesion or promote immune evasion. The parasite ligands that mediate placental adhesion are thought to be members of the variant erythrocyte surface antigen family P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var genes. Two var gene sub-families, var1CSA and var2CSA, have been identified as parasite CSA binding ligands and are leading candidates for a vaccine to prevent pregnancy-associated malaria. We investigated whether these two var gene subfamilies implicated in CSA binding are also the molecules responsible for IgM natural antibody binding. By heterologous expression of domains in COS-7 cells, we found that both var1CSA and var2CSA PfEMP1 variants bound IgM, and in both cases the binding region was a DBL epsilon domain occurring proximal to the membrane. None of the domains from a control non-IgM-binding parasite (R29) bound IgM when expressed in COS-7 cells. These results show that PfEMP1 is a parasite ligand for non-immune IgM and are the first demonstration of a specific adhesive function for PfEMP1 epsilon type domains

Genome-wide profiling of chromosome interactions in Plasmodium falciparum characterizes nuclear architecture and reconfigurations associated with antigenic variation.

Spatial relationships within the eukaryotic nucleus are essential for proper nuclear function. In Plasmodium falciparum, the repositioning of chromosomes has been implicated in the regulation of the expression of genes responsible for antigenic variation, and the formation of a single, peri-nuclear nucleolus results in the clustering of rDNA. Nevertheless, the precise spatial relationships between chromosomes remain poorly understood, because, until recently, techniques with sufficient resolution have been lacking. Here we have used chromosome conformation capture and second-generation sequencing to study changes in chromosome folding and spatial positioning that occur during switches in var gene expression. We have generated maps of chromosomal spatial affinities within the P. falciparum nucleus at 25 Kb resolution, revealing a structured nucleolus, an absence of chromosome territories, and confirming previously identified clustering of heterochromatin foci. We show that switches in var gene expression do not appear to involve interaction with a distant enhancer, but do result in local changes at the active locus. These maps reveal the folding properties of malaria chromosomes, validate known physical associations, and characterize the global landscape of spatial interactions. Collectively, our data provide critical information for a better understanding of gene expression regulation and antigenic variation in malaria parasites

A well-conserved Plasmodium falciparum var gene shows an unusual stage-specific transcript pattern

The var multicopy gene family encodes Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variant antigens, which, through their ability to adhere to a variety of host receptors, are thought to be important virulence factors. The predominant expression of a single cytoadherent PfEMP1 type on an infected red blood cell, and the switching between different PfEMP1 types to evade host protective antibody responses, are processes thought to be controlled at the transcriptional level. Contradictory data have been published on the timing of var gene transcription. Reverse transcription-polymerase chain reaction (RT-PCR) data suggested that transcription of the predominant var gene occurs in the later (pigmented trophozoite) stages, whereas Northern blot data indicated such transcripts only in early (ring) stages. We investigated this discrepancy by Northern blot, with probes covering a diverse var gene repertoire. We confirm that almost all var transcript types were detected only in ring stages. However, one type, the well-conserved varCSA transcript, was present constitutively in different laboratory parasites and does not appear to undergo antigenic variation. Although varCSA has been shown to encode a chondroitin sulphate A (CSA)-binding PfEMP1, we find that the presence of full-length varCSA transcripts does not correlate with the CSA-binding phenotype

Plasmodium falciparum Variant Surface Antigen Expression Patterns during Malaria

The variant surface antigens expressed on Plasmodium falciparum–infected erythrocytes are potentially important targets of immunity to malaria and are encoded, at least in part, by a family of var genes, about 60 of which are present within every parasite genome. Here we use semi-conserved regions within short var gene sequence “tags” to make direct comparisons of var gene expression in 12 clinical parasite isolates from Kenyan children. A total of 1,746 var clones were sequenced from genomic and cDNA and assigned to one of six sequence groups using specific sequence features. The results show the following. (1) The relative numbers of genomic clones falling in each of the sequence groups was similar between parasite isolates and corresponded well with the numbers of genes found in the genome of a single, fully sequenced parasite isolate. In contrast, the relative numbers of cDNA clones falling in each group varied considerably between isolates. (2) Expression of sequences belonging to a relatively conserved group was negatively associated with the repertoire of variant surface antigen antibodies carried by the infected child at the time of disease, whereas expression of sequences belonging to another group was associated with the parasite “rosetting” phenotype, a well established virulence determinant. Our results suggest that information on the state of the host–parasite relationship in vivo can be provided by measurements of the differential expression of different var groups, and need only be defined by short stretches of sequence data

Patterns of gene recombination shape var gene repertoires in Plasmodium falciparum: comparisons of geographically diverse isolates

BACKGROUND: Var genes encode a family of virulence factors known as PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) which are responsible for both antigenic variation and cytoadherence of infected erythrocytes. Although these molecules play a central role in malaria pathogenesis, the mechanisms generating variant antigen diversification are poorly understood. To investigate var gene evolution, we compared the variant antigen repertoires from three geographically diverse parasite isolates: the 3D7 genome reference isolate; the recently sequenced HB3 isolate; and the IT4/25/5 (IT4) parasite isolate which retains the capacity to cytoadhere in vitro and in vivo. RESULTS: These comparisons revealed that only two var genes (var1csa and var2csa) are conserved in all three isolates and one var gene (Type 3 var) has homologs in IT4 and 3D7. While the remaining 50 plus genes in each isolate are highly divergent most can be classified into the three previously defined major groups (A, B, and C) on the basis of 5' flanking sequence and chromosome location. Repertoire-wide sequence comparisons suggest that the conserved homologs are evolving separately from other var genes and that genes in group A have diverged from other groups. CONCLUSION: These findings support the existence of a var gene recombination hierarchy that restricts recombination possibilities and has a central role in the functional and immunological adaptation of var genes