Identification and characterization of a conserved, stage-specific gene product of Plasmodium falciparum recognized by parasite growth inhibitory antibodies

Abstract

We have identified a novel conserved protein of Plasmodium falciparum, designated D13, that is stage-specifically expressed in asexual blood stages of the parasite. The predicted open reading frame (ORF) D13 contains 863 amino acids with a calculated molecular mass of 99.7 kDa and displays a repeat region composed of pentapeptide motives. Northern blot analysis with lysates of synchronized blood stage parasites showed that D13 is highly expressed at the mRNA level during schizogony. The first N'-terminal 138 amino acids of D13 were expressed in Escherichia coli and the purified protein was used to generate anti-D13 monoclonal antibodies (MAbs). Using total lysates of blood stage parasites and Western blot analysis, these MAbs stained one single band of approximately 100 kDa, corresponding to the predicted molecular mass of ORF D13. Western blot analysis demonstrated further that D13 is expressed during schizogony, declines rapidly in early ring stages and is undetectable in trophozoites. D13 protein is localized in individual merozoites in a distinct area, as demonstrated by indirect immunofluorescence analysis. After subcellular fractionation, D13 was confined to the pelleted fraction of the parasite lysate and its extraction by alkaline carbonate buffer treatment indicated that D13 is not a membrane-integral protein. Inclusion of certain anti-D13 MAbs into in vitro cultures of blood stage parasites resulted in considerable reduction in parasite growth. The N'-terminal domain encompassing 158 amino acids is 94 and 95%, respectively, identical at the amino acid level between Plasmodium knowlesi, Plasmodium yoelii, and P. falciparum. In summary, we describe a novel stage-specifically expressed, highly conserved gene product of P. falciparum that is recognized by parasite growth inhibitory antibodies.

FIG. 1.

Alignment of deduced amino acid sequences of D13 orthologues of P. falciparum, P. knowlesi, and P. yoelii. The alignment was done with ClustalW and prepared for display using BOXSHADE (http://bioweb.pasteur.fr). Gaps were inserted to give the best fit. (Sequences for Plasmodium spp. are available from the PlasmoDB database [http://www.plasmoDB.org].)

FIG. 2.

(A) Northern blot analysis of synchronized asexual blood stage of P. falciparum. Panel I shows total RNA (25 μg) from in vitro-grown synchronized P. falciparum blood stage parasites that was separated on agarose gels, blotted onto nylon membrane, and hybridized to an [α-³²P]dCTP-labeled probe corresponding to the 5′terminal 422-bp fragment of D13. Time points analyzed were 0 to 6 h, 6 to 12 h, 12 to 18 h, 18 to 24 h, 24 to 30 h, 30 to 36 h, 36 to 42 h and 42 to 48 h (lanes 1 to 8) after synchronization. Panel II shows the blot rehybridized with an [α-³²P]dCTP-labeled probe of cDNA of pfGAPDH (5). (B) Western blot analysis of total lysates of blood stage parasites. Total lysates of infected RBC were separated by SDS-10% PAGE under reducing conditions and blotted onto a nitrocellulose membrane. Samples were taken at 6, 12, 18, 24, 30, 36, 42, and 48 h (lanes 1 to 8) after synchronization. Lysates of unsynchronized infected RBC (lane 9) and uninfected RBC (lane 10) were also loaded. The blot was incubated with anti-D13 MAb DD1.1 and developed using the ECL system. MAb DD1.1 recognized a single protein band of about 100 kDa in most lanes containing lysates of infected, but not in uninfected RBC.

FIG. 3.

Immunofluorescence analysis of schizonts, segmenter, and released merozoites of P. falciparum using anti-D13 MAb DD1.1 and confocal microscopy. In column I, staining with MAb DD1.1 is shown, while in column II the phase contrast of the corresponding parasites is presented.

FIG. 4.

Association of D13 with the sediment fraction of infected erythrocytes. Schizonts were enriched by Percoll-gradient centrifugation and hypotonically lysed by repeated cycles of freeze-thaw in water. The sediment (lane 1) and supernatant (lane 2) fractions were obtained by centrifugation. Aliquots of membrane fractions were further processed by incubation with alkaline carbonate (lanes 3 and 4) or water (lanes 5 and 6), respectively, and separated by ultracentrifugation in sediment lanes (lanes 3 and 5) and supernatant lanes (lanes 4 and 6) fractions, respectively. The samples were electrophoresed and probed with anti-D13 MAb DD1.1 (Fig. 4A) or anti-MSP-1 MAb 7/27 (Fig. 4B).

FIG. 5.

Parasite growth inhibitory activity of anti-D13 MAbs. The vertical bars indicate the 95% confidence intervals. The filled symbols represent results of three separate experiments conducted with MAb DD1.1, while results with the parasite nonbinding MAb DD1.3 are represented by the open symbol (⋄).