Virulence and transmission success of the malarial parasite Plasmodium falciparum

Abstract

Virulence of Plasmodium falciparum is associated with the expression of variant surface antigens designated PfEMP1 (P. falciparum erythrocyte membrane protein 1) that are encoded by a family of var genes. Data presented show that the transmission stages of P. falciparum also express PfEMP1 variants. Virulence in this host-parasite system can be considered a variable outcome of optimizing the production of sexual transmission stages from the population of disease-inducing asexual stages. Immunity to PfEMP1 will contribute to the regulation of this trade-off by controlling the parasite population with potential to produce mature transmission stages.

Figure 1

Gametocytes express var genes and produce PfEMP1 protein. (A) RT-PCR of DBL1-encoding fragments from var genes run on agarose gels. Products were amplified with isolate 1776 mRNA isolated from trophozoites (T), early gametocytes (EG), and late gametocytes (LG) grown in parallel. Lanes designated − contain control reactions testing for genomic DNA contamination of the samples. (B) Sorbitol-purified gametocyte-infected RBCs of the isolate 1776 were permeabilized with methanol and reacted with a rat antiserum specific for the cytoplasmic region of the PfEMP1and then with FITC-conjugated goat anti-rat IgG. T, Mature trophozoite; IB, IIA, IIB, III, and IV, gametocyte stages. The antiserum was not reactive with uninfected RBCs. Infected RBCs were not detected by FITC-conjugated IgG alone. Gametocytes of cloned lines ITO4 and 3D7 and the isolate MUZ12 showed the same reactivity (data not shown). (Bar = 10 μm.)

Figure 2

Naturally acquired antibodies react with the surface of RBCs infected with early gametocytes but not late gametocytes. Samples of isolate 1776. Trophozoite-infected (A), early gametocyte-infected (stages I–IIA; B), and late gametocyte-infected (stages III–V; C) RBCs were analyzed by flow cytometry for reactivity with pooled HIS as detected by FITC-conjugated anti-human secondary antibodies. Trophozoites were then stained with ethidium bromide to distinguish infected from uninfected RBCs, and the samples were sorted so that reactivity with HIS was scored only in the population of cells positive for ethidium bromide staining. Samples containing early gametocytes were permeabilized and reacted with gametocyte-specific mAb 2G7 and phycoerythrin-conjugated goat anti-mouse IgG to distinguish gametocytes from asexual parasites. Similarly, the samples were sorted so that reactivity with HIS was scored only in the population of cells positive for phycoerythrin staining. Cells were scored for intensity of surface labeling with the HIS–FITC conjugate. The percentage of infected cells in each sample positive for reactivity with pooled serum (green) was calculated as described (44) to account for any nonspecific fluorescence from controls and is reported in each figure. Uninfected RBC-HIS–FITC (red) and infected RBC-normal human serum–FITC (blue) control reactions are shown for each sample.

Figure 3

Gametocytes and trophozoites of the isolate 1776 express the same var genes. (A) Sorbitol-purified early gametocytes (stained blue with 4,6-diamidino-2-phenylindole) and a separate culture of trophozoites (stained yellow with ethidium bromide) were both enriched to 10% parasitemias before combining and reacting with HIS in coagglutination assays. Agglutinates containing both parasite stages were also obtained with gametocytes and trophozoites from isolates MUZ12 and MUZ37 (data not shown). (Bar = 10 μm.) (B) Mixed agglutinates were observed in a direct fluorescence assay with cultures containing both parasite stages. Trophozoites and gametocytes could be distinguished by light microscopy; the distinctive granular pigment of the early gametocytes and the single spot of pigment of trophozoites were clearly visible. (Bar = 10 μm.)

Figure 4

(A) A schematic diagram of dot blots containing 37 unique DBL1 fragments obtained by genomic PCR. Each letter name represents a unique sequence type in the genome of the parasite isolate 1776. Trophozoite (B) and gametocyte (C) amplification products of expressed var gene fragments obtained by RT-PCR were radiolabeled and hybridized with the dot blots. Dots that were positive contained sequence types being expressed in the parasite population.