Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Plasmodium vivax is completely dependent on interaction with the Duffy blood group antigen to invade human erythrocytes. The P. vivax Duffy-binding protein, which binds the Duffy antigen during invasion, belongs to a family of erythrocyte-binding proteins that also includes Plasmodium falciparum sialic acid binding protein and Plasmodium knowlesi Duffy binding protein. The receptor binding domains of these proteins lie in a conserved, N-terminal, cysteine-rich region, region II, found in each of these proteins. Here, we have expressed P. vivax region II (PvRII), the P. vivax Duffy binding domain, in Escherichia coli. Recombinant PvRII is incorrectly folded and accumulates in inclusion bodies. We have developed methods to refold and purify recombinant PvRII in its functional conformation. Biochemical, biophysical, and functional characterization confirms that recombinant PvRII is pure, homogeneous, and functionally active in that it binds Duffy-positive human erythrocytes with specificity. Refolded PvRII is highly immunogenic and elicits high titer antibodies that can inhibit binding of P. vivax Duffy-binding protein to erythrocytes, providing support for its development as a vaccine candidate for P. vivax malaria. Development of methods to produce functionally active recombinant PvRII is an important step for structural studies as well as vaccine development.The invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions between host receptors and parasite ligands (1). Plasmodium vivax and the related simian malaria parasite Plasmodium knowlesi require interaction with the Duffy blood group antigen to invade human erythrocytes (2, 3). P. knowlesi can also invade rhesus erythrocytes using alternative Duffy-independent receptors (4). P. falciparum commonly uses sialic acid residues of glycophorin A as invasion receptors (5-9). Like P. knowlesi, P. falciparum also invades erythrocytes by multiple pathways and is not completely dependent on sialic acid residues of glycophorin A (8,10,12,13).Parasite ligands that bind host receptors to mediate erythrocyte invasion include P. vivax and P. knowlesi Duffy-binding proteins, P. knowlesi ␤ and ␥ proteins, which bind Duffy-independent receptors on rhesus erythrocytes, and P. falciparum sialic acid-binding protein (also known as EBA-175), which binds sialic acid residues on glycophorin A (4, 14 -18). These parasite ligands share similar features and belong to a family of erythrocyte-binding proteins (19). The extracellular domain of each erythrocyte-binding protein contains two conserved cysteine-rich regions, regions II and VI, at the amino and carboxyl ends, respectively. P. falciparum EBA-175 contains a tandem duplication (F1 and F2) of the N-terminal, conserved, cysteine-rich region. The functional receptor binding domain of each erythrocyte-binding protein lies in region II (20, 21). In the case of EBA-175, region F2 was found to have receptor b...

Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Whereas Plasmodium vivax and Plasmodium knowlesi use the Duffy blood group antigen, Plasmodium falciparum uses sialic acid residues of glycophorin A as receptors to invade human erythrocytes. P. knowlesi uses the Duffy antigen as well as other receptors to invade rhesus erythrocytes by multiple pathways. Parasite ligands that bind these receptors belong to a family of erythrocyte-binding proteins (EBP). The EBP family includes the P. vivax and P. knowlesi Duffy-binding proteins, P. knowlesi ␤ and ␥ proteins, which bind alternate receptors on rhesus erythrocytes, and P. falciparum erythrocyte-binding antigen (EBA-175), which binds sialic acid residues of human glycophorin A. Binding domains of each EBP lie in a conserved N-terminal cysteinerich region, region II, which contains around 330 amino acids with 12 to 14 conserved cysteines. Regions containing binding residues have now been mapped within P. vivax and P. knowlesi ␤ region II. Chimeric domains containing P. vivax region II sequences fused to P. knowlesi ␤ region II sequences were expressed on the surface of COS cells and tested for binding to erythrocytes. Binding residues of P. vivax region II lie in a 170-aa stretch between cysteines 4 and 7, and binding residues of P. knowlesi ␤ region II lie in a 53-aa stretch between cysteines 4 and 5. Mapping regions responsible for receptor recognition is an important step toward understanding the structural basis for the interaction of these parasite ligands with host receptors. Invasion of erythrocytes by Plasmodium merozoites is mediated by specific molecular interactions between erythrocyte receptors and parasite ligands (1). Plasmodium vivax and Plasmodium knowlesi bind the Duffy blood group antigen to invade human erythrocytes (2, 3). Duffy-negative human erythrocytes are completely resistant to invasion by these parasites. In contrast, P. knowlesi can use the Duffy antigen as well as alternate receptors to invade rhesus erythrocytes by multiple pathways (4). Plasmodium falciparum, the most important parasite for human malaria, commonly uses sialic acid residues on glycophorin A as receptors to invade human erythrocytes (5, 6).The parasite ligands that bind these receptors belong to the erythrocyte-binding protein (EBP) family (7). The EBP family includes the Duffy-binding proteins of P. vivax and P. knowlesi, P. knowlesi ␤ and ␥ proteins, which bind alternate receptors on rhesus erythrocytes, and P. falciparum sialic acid-binding protein, also known as erythrocyte-binding antigen (EBA-175), which binds sialic acid residues of glycophorin A (7). Each EBP contains two cysteine-rich domains, region II and region VI, which contain conserved cysteines and hydrophobic amino acid residues. The functional binding domains of EBPs lie in region II, the conserved N-terminal cysteine-rich region (8-10). These functional domains are referred to as Duffy-binding-like (DBL) domains after region II of the P. vivax Duffy-binding pro...

dMycobacterium tuberculosis expresses the 28-kDa protein HupB (Rv2986c) and the Fe 3؉ -specific high-affinity siderophores mycobactin and carboxymycobactin upon iron limitation. The objective of this study was to understand the functional role of HupB in iron acquisition. A hupB mutant strain of M. tuberculosis, subjected to growth in low-iron medium (0.02 g Fe ml ؊1 ), showed a marked reduction of both siderophores with low transcript levels of the mbt genes encoding the MB biosynthetic machinery. Complementation of the mutant strain with hupB restored siderophore production to levels comparable to that of the wild type. We demonstrated the binding of HupB to the mbtB promoter by both electrophoretic mobility shift assays and DNA footprinting. The latter revealed the HupB binding site to be a 10-bp AT-rich region. While negative regulation of the mbt machinery by IdeR is known, this is the first report of positive regulation of the mbt operon by HupB. Interestingly, the mutant strain failed to survive inside macrophages, suggesting that HupB plays an important role in vivo.

The genomic era has seen a remarkable increase in the number of genomes being sequenced and annotated. Nonetheless, annotation remains a serious challenge for compositionally biased genomes. For the preliminary annotation, popular nucleotide and protein comparison methods such as BLAST are widely employed. These methods make use of matrices to score alignments such as the amino acid substitution matrices. Since a nucleotide bias leads to an overall bias in the amino acid composition of proteins, it is possible that a genome with nucleotide bias may have introduced atypical amino acid substitutions in its proteome. Consequently, standard matrices fail to perform well in sequence analysis of these genomes. To address this issue, we examined the amino acid substitution in the AT-rich genome of Plasmodium falciparum, chosen as a reference and reconstituted a substitution matrix in the genome's context. The matrix was used to generate protein sequence alignments for the parasite proteins that improved across the functional regions. We attribute this to the consistency that may have been achieved amid the target and background frequencies calculated exclusively in our study. This study has important implications on annotation of proteins that are of experimental interest but give poor sequence alignments with standard conventional matrices.

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