Aberrant transcription and the failure of Plasmodium falciparum to differentiate into gametocytes.

In the human malaria parasite, Plasmodium falciparum, a DNA rearrangement is associated with erroneous transcription of the early gametocyte antigen gene, Pfg27/25, and the failure of the blood stages to differentiate into gametocytes. Altered blood stage-specific patterns of mRNA accumulation were observed for the Pfg27/25 gene, in mutant P. falciparum parasites that have lost the capacity to differentiate sexually. Each mutant demonstrates a DNA rearrangement upstream of the coding sequence. The location of the gene proximal breakpoint correlates with the complete absence of stable Pfg27/25 transcripts or with the accumulation of aberrant Pfg27/25 mRNAs in the asexual stages. The rearrangement identifies a labile region of the genome which contributes to accurate stage-specific Pfg27/25 gene transcription and may be necessary for a critical early step in sexual differentiation.

Structural features of Plasmodium cytochrome b that may underlie susceptibility to 8-aminoquinolines and hydroxynaphthoquinones.

Appropriate functioning of mitochondria is critical for survival and growth of erythrocytic stages of malarial parasites, making it an attractive target for antimalarial drugs which may take advantage of unique features of parasite mitochondrial metabolism. We have sequenced the presumptive mitochondrial DNA, the 6-kb element, of Plasmodium falciparum, permitting an analysis of the predicted structure of parasite electron transport proteins. Although the overall structures of the 3 polypeptides, cytochrome c oxidase subunit 1, cytochrome c oxidase subunit 3, and cytochrome b (cyt b), were similar to those from other species, some striking differences were observed, especially for the cyt b. Analysis of the cyt b structure showed that the critical quinone binding sites of the protein are quite divergent from those of other species. Comparative analysis suggests that these changes are the likely cause for the resistance of parasite cytochrome bc1 complex to antimycin and related inhibitors. We suggest that the same features are responsible for increased affinity of the parasite cyt b for antimalarial compounds of class 8-aminoquinolines and hydroxynaphthoquinones, explaining the therapeutic value of these drugs.

Pfs2400 can mediate antibody-dependent malaria transmission inhibition and may be the Plasmodium falciparum 11.1 gene product.

Monoclonal antibodies (mAb) have been raised against Plasmodium falciparum gametocyte stage protein extracts, in an effort to identify novel parasite antigens that might mediate malaria transmission-blocking immunity. mAb 1A1 identified Pfs2400, a sexual stage-specific antigen of greater than 2 megadaltons, that is associated with the outer leaflet of the parasitophorous vacuole membrane in mature circulating gametocyte-infected red blood cells. Upon induction of gametogenesis, Pfs2400 partitions between the gamete plasmalemma and the degenerating erythrocyte membrane. The antigen is no longer detectable in the fully emerged gamete. mAb 1A1 dramatically reduces the number of oocysts formed in P. falciparum gametocyte-fed mosquitoes. The cognate antigen is probably the product of the Pf11.1 gene (Scherf et al. 1988. EMBO [Eur. Mol. Biol. Organ.]J. 7:1129) on the basis that a peptide composed of two copies of the degenerate nine amino acid repeat sequence in the Pf11.1 protein, can inhibit binding of mAb1A1 to the native antigen. The mechanism of transmission inhibition mediated by the Pfs2400 is discussed.

A and T homopolymeric stretches mediate a DNA inversion in Plasmodium falciparum which results in loss of gene expression.

Ring-infected erythrocyte surface antigen-negative isolates of Plasmodium falciparum demonstrate a complex DNA rearrangement with inversion of 5' coding sequences, deletion of upstream and flanking sequences, and healing of the truncated chromosome by telomere addition. An inversion intermediate that results in the telomeric gene structure for RESA has been identified in the pathway. This inversion creates a mitotically stable substrate for the sequence-specific addition of telomere repeats at the deletion breakpoint.

Large deletions result from breakage and healing of P. falciparum chromosomes.

The human malaria parasite P. falciparum exhibits extensive strain-dependent chromosomal polymorphisms that have been implicated in the generation of antigenic variability in this organism. These polymorphisms can result in large deletions in chromosomes as determined by pulsed-field gradient gel electrophoresis. We have investigated the molecular basis for extensive deletions in chromosomes 2 and 8 in multiple geographic isolates of this parasite that result in the loss of expression of well-characterized parasite antigens. The structure of these polymorphic chromosomes reveal that a mechanism of chromosome breakage and healing by the addition of telomeric repeats most plausibly accounts for these karyotypes. Furthermore, the orientation of these gene fragments on their truncated chromosomes reveal that the healed chromosome originally associated with centromeric elements is mitotically stable and maintained. A model for the possible role of this mechanism in the complex parasite life-cycle is discussed.

Primary structure and subcellular localization of the knob-associated histidine-rich protein of Plasmodium falciparum.

Plasmodium falciparum-infected erythrocytes bind to venular endothelial cells by means of electron-dense deformations (knobs) on the parasitized erythrocyte surface. The primary structure of a parasite-derived histidine-rich protein associated with the knob structure was deduced from cDNA sequence analysis. The 634 amino acid sequence is rich in lysine and histidine and contains three distinct, tandemly repeated domains. Indirect immunofluorescence, using affinity-purified monospecific antibodies directed against recombinant protein synthesized in Escherichia coli, localized the knob-associated histidine-rich protein to the membrane of knobby infected erythrocytes. Immunoelectron microscopy established that the protein is clustered on the cytoplasmic side of the erythrocyte membrane and is associated with the electron-dense knobs. A role for this histidine-rich protein in knob structure and cytoadherence is suggested based upon these data.

A chromosomal rearrangement in a P. falciparum histidine-rich protein gene is associated with the knobless phenotype.

The significant morbidity and mortality associated with Plasmodium falciparum malaria results, in part, from the sequestration of parasitized erythrocytes in postcapillary venules, which may protect the parasite from splenic clearance and contribute to the pathogenesis of cerebral malaria. This sequestration has been linked to the expression of parasite-induced knob structures on the surface of the infected erythrocyte which mediate the cytoadherence phenomenon. While knobs are necessary for cytoadherence, they are not sufficient, requiring both parasite- and host-encoded proteins. Spontaneous mutants of P. falciparum have been isolated from in vitro cultures which lack the ability to express knobs and fail to cytoadhere. A histidine-rich protein has been described which is associated with the knobby phenotype and may be a constituent of the knob. We now report the isolation of complementary DNA clones for a knob-associated histidine-rich protein (KAHRP) and demonstrate that in knobless mutants the gene for this protein has undergone a rearrangement, resulting in a deletion in the 3' coding sequence. Moreover, the chromosome to which the KAHRP gene maps is rearranged in these mutants, producing a telomeric location of the truncated gene. These observations explain the loss of expression of the messenger RNA and protein in such mutants and may explain the loss of the knob itself. The implications for the generation of spontaneous mutations in the parasite by this novel mechanism are discussed.

Stimulation of human endothelial cell prostacyclin synthesis by select leukotrienes.

Cultured endothelial cells from human umbilical cord labeled with [3H]20:4 release radiolabel when exposed to leukotrienes C or D (LTC or LTD). The major radiolabeled 20:4 metabolite recovered in the culture medium was prostacyclin. Both leukotrienes produced a dose-dependent synthesis of prostacyclin, with a maximal response at 10(-7) M leukotriene. LTC promoted a twofold greater response than did LTD at all concentrations tested (10(-9) to 10(-7) M). In contrast, no release of radiolabel above basal levels was evident with a challenge of LTE or LTB at the same concentrations. Endothelial cells metabolize approximately 40-50% of exogenously supplied LTC to LTD and LTE in 60 min. Levels of alpha-glutamyltranspeptidase (gamma-GTPase), the ectoenzyme reported to convert LTC or LTD, were detected in intact endothelial cells with the chromogenic substrate L-gamma-glutamyl-p-nitroanilide at levels sufficient to account for the observed rate of LTC metabolism. High concentrations of the gamma-GTPase inhibitors, glutathione and AT-125, blocked the metabolism of LTC by endothelium. These results suggest that degradation of leukotrienes by endothelium may be one mechanism for inactivation of these lipid mediators.

Nature of the riboflavin interaction with the immunoglobulin IgGGAR: analogue binding studies.

The human immunoglobulin IgGGAR has been shown to bind riboflavin both in vivo and in vitro with a high binding constant [Farhangi & Osserman (1976), New Engl. J. Med. 294, 177-183; Chang et al. (1981b), Biochemistry 20, 2916-2921; Chang et al. (1981a), Biochemistry 20, 2922-2926] and, therefore, represents a natural human antibody hapten complex. In this study, the parts of the riboflavin molecule which are in intimate contact with the binding pocket have been identified, using a variety of riboflavin analogues. The binding constants of IgGGAR for these flavins were measured by fluorescence spectroscopy. The relative affinities indicate that the pyrimidine edge of the isoalloxazine interacts intimately with the combining site, particularly around N-3. The ribityl side chain and the dimethylbenzene edge of the flavin ring are not critical to the binding interaction and are probably exposed to solvent. Analogues of riboflavin with altered electronic structures have somewhat lowered affinities for IgGGAR, suggesting that the isoalloxazine ring forms stacking interactions. Comparison with other known flavin-binding proteins shows that IgGGAR binds riboflavin in a new way that is different from the sites in other proteins. The isoalloxazine ring binds similarly to the FMN-binding proteins; however, unlike virtually all the other proteins, the ribityl side chain is not essential to binding.