The human malaria parasite, Plasmodium falciparum, possesses a broad repertoire of proteins that are proposed to be trafficked to the erythrocyte cytoplasm or surface, based upon the presence within these proteins of a Pexel/VTS erythrocyte-trafficking motif. This catalog includes large families of predicted 2 transmembrane (2TM) proteins, including the Rifin, Stevor and Pfmc-2TM superfamilies, of which each possesses a region of extensive sequence diversity across paralogs and between isolates that is confined to a proposed surface-exposed loop on the infected erythrocyte. Here we express epitope-tagged versions of the 2TM proteins in transgenic NF54 parasites and present evidence that the Stevor and Pfmc-2TM families are exported to the erythrocyte membrane, thus supporting the hypothesis that host immune pressure drives antigenic diversity within the loop. An examination of multiple P.falciparum isolates demonstrates that the hypervariable loop within Stevor and Pfmc-2TM proteins possesses sequence diversity across isolate boundaries. The Pfmc-2TM genes are encoded within large amplified loci that share profound nucleotide identity, which in turn highlight the divergences observed within the hypervariable loop. The majority of Pexel/VTS proteins are organized together within sub-telomeric genome neighborhoods, and a mechanism must therefore exist to differentially generate sequence diversity within select genes, as well as within highly defined regions within these genes.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) exerts its multiple functions by activating two receptor tyrosine kinases, Flt-1 (VEGFR-1) and KDR (VEGFR-2), both of which are selectively expressed on primary vascular endothelium. To dissect the respective signaling pathways and biological functions mediated by these receptors in primary endothelial cells with two receptors intact, we, recently developed chimeric receptors (EGDR and EGLT) in which the extracellular domain of the epidermal growth factor receptor was fused to the transmembrane domain and intracellular domain of KDR and Flt-1, respectively. With these fusion receptors, we have shown that KDR is solely responsible for VPF/VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and migration, whereas Flt-1 showed an inhibitory effect on KDR-mediated proliferation but not migration. To further characterize the VPF/VEGF-stimulated HUVEC proliferation and migration here, we have created several EGDR mutants by site-directed mutagenesis. We show that tyrosine residues 1059 and 951 of KDR are essential for VPF/VEGF-induced HUVEC proliferation and migration, respectively. Furthermore, the mutation of tyrosine 1059 to phenylanaline results in the complete loss of KDR/EGDR-mediated intracellular Ca 2؉ mobilization and MAPK phosphorylation, but the mutation of tyrosine 951 to phenylanaline did not affect these events. Our results suggest that KDR mediates different signaling pathways for HUVEC proliferation and migration and, moreover, intracellular Ca 2؉ mobilization and MAPK phosphorylation are not essential for VPF/VEGFinduced HUVEC migration.

The human malaria parasite Plasmodium falciparum utilizes a mechanism of antigenic variation to avoid the antibody response of its human host and thereby generates a long-term, persistent infection. This process predominantly results from systematic changes in expression of the primary erythrocyte surface antigen, a parasite-produced protein called PfEMP1 that is encoded by a repertoire of over 60 var genes in the P. falciparum genome. var genes exhibit extensive sequence diversity, both within a single parasite's genome as well as between different parasite isolates, and thus provide a large repertoire of antigenic determinants to be alternately displayed over the course of an infection. While significant work has recently been published documenting the extreme level of diversity displayed by var genes found in natural parasite populations, little work has been done regarding the mechanisms that lead to sequence diversification and heterogeneity within var genes. In the course of producing transgenic lines from the original NF54 parasite isolate, we cloned and characterized a parasite line, termed E5, which is closely related to but distinct from 3D7, the parasite used for the P. falciparum genome nucleotide sequencing project. Analysis of the E5 var gene repertoire, as well as that of the surrounding rif and stevor multi-copy gene families, identified examples of frequent recombination events within these gene families including an example of a duplicative transposition indicating recombination events play a significant role in the generation of diversity within the antigen encoding genes of P. falciparum.

Infection of erythrocytes with the human malaria parasite, Plasmodium falciparum, results in dramatic changes to the host cell structure and morphology. The predicted functional localization of the STEVOR proteins at the erythrocyte surface suggests that they may be involved in parasite-induced modifications of the erythrocyte membrane during parasite development. To address the biologic function of STEVOR proteins, we subjected a panel of stevor transgenic parasites and wild-type clonal lines exhibiting different expression levels for stevor genes to functional assays exploring parasite-induced modifications of the erythrocyte membrane. Using this approach, we show that stevor expression impacts deform-ability of the erythrocyte membrane. This process may facilitate parasite sequestra-tion in deep tissue vasculature. (Blood. 2012;119(2):e1-e8) Introduction Erythrocytes infected with the human malaria parasite, Plasmo-dium falciparum, undergo dramatic changes in structure and morphology, in part because of the export of a broad repertoire of parasite proteins as well as influences of the shape and volume of the developing parasite itself. Alterations of the erythrocyte membrane include the appearance of cytoadherent knobs, the acquisition of novel adhesive and serologic properties, an increase in membrane rigidity, and the activation of solute permeability pathways, termed the new permeability pathways, for nutrient uptake and waste removal. The increased rigidity and adhesive properties of infected erythrocytes are major factors in the survival and virulence of the parasite. 1 Uninfected erythrocytes are highly deformable because of their high surface area-to-volume ratio and the elasticity of the erythrocyte membrane and cytoskeleton. 2 In contrast, infection with P falciparum results in a loss of deformabil-ity, and this perhaps increases the pathogenesis of the parasite by facilitating sequestration of infected erythrocytes and blockage of microcapillaries. 3 The knob-associated parasite protein KAHRP has been shown to associate with the erythrocyte cytoskeletal proteins spectrin, actin, and ankyrin, and these interactions are correlated with increased membrane rigidity. 4 In cultured parasite lines, truncations of nonessential telomeric regions are well documented, and one such shortening of P falciparum chromosome 2 leads to loss of KAHRP and, as a consequence, a knobless phenotype. 5 KAHRP() parasites propagate in culture at normal rates, supporting a role for rigidity and adhesion solely during in vivo infections. Targeted gene deletion of KAHRP, as well as knockout of another parasite gene, PfEMP3, results in a significant decrease in erythrocyte rigidity; however, the observed deformability remains less than that of uninfected erythrocytes, indicating that other factors also contribute to parasite-infected erythrocyte rigidity. 4,6 The erythro-cyte-exported RESA protein has been shown to contribute to the increased rigidity of ring-stage infected erythrocytes, although not of late-stage infected erythr...