Developmental arrest of the human malaria parasite Plasmodium falciparum within the mosquito midgut via CTRP gene disruption.

Apicomplexan protozoa possess a family of micronemal and cell surface-associated proteins, each comprised a combination of cell-adhesive vertebrate von Willebrand factor (vWF)-like A domains and thrombospondin (TSP) type 1-like domains. The human malaria parasite Plasmodium falciparum has in the extracellular portion of the CS protein TRAP-related protein (CTRP) six tandemly arrayed A domains followed by seven TSP type 1-like domains, whereas a second member of this family, thrombospondin-related anonymous protein (TRAP), contains a single vWF-like A domain and a single TSP type 1-like domain. Here we show that CTRP transcripts are present within the infected mosquito midgut and that CTRP protein is expressed with a punctate distribution and a predominance at the apical end of mosquito midgut-stage ookinetes. This expression pattern is analogous to micronemal expression of TRAP in Plasmodium sporozoites. Disruption of the CTRP gene by homologous recombination in cultures of the human malaria parasite P. falciparum demonstrates that CTRP is essential for mosquito midgut development. Oocyst formation was never observed following membrane feeds of CTRP disruptant lines to Anopheline mosquitoes, despite the development of mature ookinetes. We propose that CTRP is involved in essential recognition or motility processes at the ookinete cell surface within the mosquito midgut.

Adherence of erythrocytes during exflagellation of Plasmodium falciparum microgametes is dependent on erythrocyte surface sialic acid and glycophorins.

Malaria male gametocytes within a newly ingested infected blood meal in the mosquito midgut emerge from erythrocytes and extrude approximately eight flagellar microgametes in a process termed exflagellation. In culture, and in blood removed from infected patients, emerging microgametes avidly adhere to neighboring uninfected and infected erythrocytes, as well as to emerged female macrogametes, creating "exflagellation centers". The mechanism of erythrocyte adherence is not known nor has it been determined for what purpose microgametes may bind to erythrocytes. The proposition of a function underlying erythrocyte adherence is supported by the observation of species-specificity in adhesion: microgametes of the human malaria Plasmodium falciparum can bind human erythrocytes but not chicken erythrocytes, whereas avian host Plasmodium gallinaceum microgametes bind chicken but not human erythrocytes. In this study we developed a binding assay in which normal, enzyme-treated, variant or null erythrocytes are identified by a cell surface fluorescent label and assayed for adherence to exflagellating microgametes. Neuraminidase, trypsin or ficin treatment of human erythrocytes eliminated their ability to adhere to Plasmodium falciparum microgametes, suggesting a role of sialic acid and one or more glycophorins in the binding to a putative gamete receptor. Using nulls lacking glycophorin A [En(a-)], glycophorin B (S-s-U-) or a combination of glycophorin A and B (Mk/Mk) we showed that erythrocytes lacking glycophorin B retain the ability to bind but a lack of glycophorin A reduced adherence by exflagellating microgametes. We propose that either the sialic acid moiety of glycophorins, predominantly glycophorin A, or a more complex interaction involving the glycophorin peptide backbone, is the erythrocyte receptor for adhesion to microgametes.

Cloning and cross-species comparison of the thrombospondin-related anonymous protein (TRAP) gene from Plasmodium knowlesi, Plasmodium vivax and Plasmodium gallinaceum.

To examine the structure of the Plasmodium sporozoite micronemal protein, thrombospondin-related anonymous protein (TRAP) we have isolated TRAP genes from three species of Plasmodium: P. gallinaceum (PgTRAP), P. knowlesi (PkTRAP) and P. vivax (PvTRAP). Thus it is now possible to compare the TRAP gene from a total of six species of Plasmodium. The overall structure of TRAP is conserved in all species; specifically, an amino-terminal A-domain similar to magnesium-binding domains of mammalian integrins; a thrombospondin-like sulfatide-binding domain similar to region II in Plasmodium circumsporozoite protein; an acidic asparagine/proline-rich repeat region; a trans-membrane domain and a short acidic cytoplasmic region with a highly conserved carboxy terminus. The overall structure of TRAP from P. gallinaceum and P. falciparum (PfTRAP) is conserved and phylogenetic analysis suggests a monophyletic relationship of avian P. gallinaceum and human P. falciparum. Comparison of the amino acid sequences of the A-domain of PgTRAP and PfTRAP indicates a more rapid divergence of this domain with respect to the rest of the protein in these two species. The structural differences of PgTRAP and PfTRAP may relate to the distinct invasion pathways, macrophage and endothelial cell invasion of P. gallinaceum sporozoites versus hepatocyte invasion of P. falciparum.

FGF-mediated aspects of skeletal muscle growth and differentiation are controlled by a high affinity receptor, FGFR1.

Fibroblast growth factors (FGFs) and FGF receptors (FGFRs) play major roles in vertebrate embryogenesis, including control of skeletal muscle growth and differentiation. Understanding their roles requires delineating the specific FGF and FGFR isoforms involved. This study analyzes the FGFR transcripts found in a model mouse skeletal myoblast cell line (MM14) during growth and terminal differentiation. MM14 cells express transcripts for FGFR1 (flg) but not FGFR2 (bek). The predominate FGFR1 transcript contains three immunoglobulin (Ig)-like domains in the extracellular ligand binding region. Approximately one-fourth of the three Ig-like domain transcripts possess a 6-nt deletion between the first and second Ig-like domains which after translation would result in deletion of an Arg-Arg pair. Cloning of mouse genomic DNA surrounding the region of the FGFR1 6-nt deletion indicates that the deletion is derived by alternative splicing of FGFR1 transcripts. Transcripts containing two Ig-like domains account for less than 5% of total FGFR1 mRNA in MM14 cells. A survey of RNA from mouse tissues indicated that two Ig-like domain FGFR1 transcripts are rare in all tissues except in lung, in which the two Ig-like domain form accounts for roughly 70% of the lung FGFR1 mRNA. PCR RACE cloning studies disclosed 162 nt of additional FGFR1 5'-flanking RNA which was highly GC-rich. FGFR1 transcripts decline 8- to 10-fold during low serum, (-)FGF-mediated differentiation of MM14 cultures. The kinetics of the FGFR1 mRNA decline is similar to the previously described differentiation-dependent decrease in cell surface FGF receptors.