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. 2008 Dec;7(12):2123-32.
doi: 10.1128/EC.00274-08. Epub 2008 Sep 26.

Deletion of the Plasmodium falciparum merozoite surface protein 7 gene impairs parasite invasion of erythrocytes

Madhusudan Kadekoppala  1 , Rebecca A O'Donnell, Munira Grainger, Brendan S Crabb, Anthony A Holder
Affiliations

Deletion of the Plasmodium falciparum merozoite surface protein 7 gene impairs parasite invasion of erythrocytes

Madhusudan Kadekoppala et al. Eukaryot Cell. 2008 Dec.

Abstract

Merozoite surface proteins have been implicated in the initial attachment to the host red blood cell membrane that begins the process of invasion, an important step in the life cycle of the malaria parasite. In Plasmodium falciparum, merozoite surface proteins include several glycosylphosphatidyl inositol-anchored proteins and peripheral proteins attached to the membrane through protein-protein interactions. The most abundant of these proteins is the merozoite surface protein 1 (MSP1) complex, encoded by at least three genes: msp1, msp6, and msp7. The msp7 gene is part of a six-member multigene family in Plasmodium falciparum. We have disrupted msp7 in the Plasmodium falciparum D10 parasite, as confirmed by Southern hybridization. Immunoblot and indirect immunofluorescence analyses confirmed the MSP7 null phenotype of D10DeltaMSP7 parasites. The synthesis, distribution, and processing of MSP1 were not affected in this parasite line. The level of expression and cellular distribution of the proteins MSP1, MSP3, MSP6, MSP9, and SERA5 remained comparable to those for the parental line. Furthermore, no significant change in the expression of MSP7-related proteins, except for that of MSRP5, was detected at the transcriptional level. The lack of MSP7 was not lethal at the asexual blood stage, but it did impair invasion of erythrocytes by merozoites to a significant degree. Despite this reduction in efficiency, D10DeltaMSP7 parasites did not show any obvious preference for alternate pathways of invasion.

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Figures

FIG. 1.
FIG. 1.
Disruption of Plasmodium falciparum D10 msp7 gene. (A) Schematic representation of the knockout plasmid construct (pHTKΔMSP7), the msp7 locus of the parental parasite line (D10), and the resultant msp7 knockout parasite (D10ΔMSP7) lines. The plasmid construct carries a viral thymidine kinase gene cassette (tk cassette) for negative selection with ganciclovir and an hDHFR cassette to insert into the D10 msp7 locus. Insertion of the hDHFR cassette by double homologous recombination within the 5′ and 3′ target regions of msp7 changes the hybridizing KpnI-HindIII restriction fragment sizes from 4.5 kb to 1.8 (with the 5′ target probe) or 3.4 kb (with the 3′ target probe). Relevant restriction sites—AvrII (A), BamHI (B), HindIII (H), KpnI (K), NcoI (N), and SacII (S)—are shown. (B) Southern hybridization of genomic DNA from D10 and transfected parasites digested with KpnI and HindIII and probed with msp7 3′ target probe. Lanes show transfected parasites (0) and ganciclovir-selected parasites after second (2g) or third (3g) WR22910 cycling. The signal at 7.7 kb in lane 0 corresponds to episomal DNA. (C and D) Hybridization analysis of genomic DNA from D10 (lane 1) or the D10ΔMSP7 clone (lane 2) with 5′ or 3′ target DNA probes, respectively. Molecular sizes of hybridizing DNA fragments are indicated.
FIG. 2.
FIG. 2.
MSP7 null phenotype of D10ΔMSP7 parasite clone. (A) Western blot analysis of late-stage D10 (lanes 1 and 3) or D10ΔMSP7 clone (lanes 2 and 4) with rabbit polyclonal antibodies to the MSP7 protein. The knockout parasite clone was cultured in the presence (lane 2) or absence (lane 4) of WR99210. Different forms of MSP7, such as the precursor (MSP7) and processed forms (MSP733, MSP722, and MSP719), are indicated. (B) Indirect immunofluorescence analysis of formaldehyde-fixed, acetone-permeabilized late-stage parasites from D10 and the D10ΔMSP7 clone with monoclonal anti-MSP1 (MSP1) and anti-MSP7 (MSP7) antibodies. DNA was visualized with Hoechst 33428. Colocalization was confirmed by color-merging (merge).
FIG. 3.
FIG. 3.
Immunoprecipitation analysis of D10 and D10ΔMSP7 35S-biosynthetically labeled parasite extracts. (A and B) NP-40- or SDS-solubilized parasites from D10 (odd-numbered lanes) and D10ΔMSP7 clone (even-numbered lanes) immunoprecipitated with nonimmune mouse (lanes 1 and 2) or rabbit antisera (lanes 3 and 4), MSP1-specific MAb 12.8 (lanes 5 and 6), or rabbit polyclonal antibodies to MSP7 (lanes 7 and 8). (C) MSP1 processing in D10 and D10ΔMSP7 parasites allowed to develop for 6 h after radiolabeling. The sizes of molecular markers are shown in kDa. The figure labels are the same as those in panels A and B, except that polyclonal rabbit antibodies to MSP1 were used (lanes 5 and 6). The MSP1 precursor and its processed fragments (MSP183, MSP142, MSP138, and MSP130) and the precursor MSP7 and its processed forms (MSP733, MSP722, and MSP719) are indicated. The band below MSP1 at about 160 kDa in NP-40 extracts results from detergent-promoted cleavage of the MSP1 precursor.
FIG. 4.
FIG. 4.
Analysis of the expression of peripheral merozoite surface and vacuolar proteins by immunoblotting and indirect immunofluorescence. (A) Immunoblot analysis of late-stage parasites (lanes 1 and 2) and purified merozoites (lanes 3 and 4) from D10 (lanes 1 and 3) or D10ΔMSP7 (lanes 2 and 4) cloned parasites with antisera to the MSP7, MSP1, MSP3, and MSP6 proteins. Antibodies to immunoglobulin binding protein (BiP) orthologue were used to ascertain that equal amounts of proteins were loaded. (B) Indirect immunofluorescence analysis of D10 and D10ΔMSP7 parasites. Fixed smears of late-stage parasites were probed with antibodies to MSP3, MSP6, MSP9, and SERA5 together with anti-MSP1 antibodies. Rabbit polyclonal antibodies are in red, while mouse antibodies are in green. Hoechst 33428 was used to visualize DNA (blue). Color-merging was carried out to analyze colocalization with MSP1 (merge).
FIG. 5.
FIG. 5.
Transcription at msp7 locus. Northern blot analysis of late-stage (33 to 36 h) parasites from D10 and D10ΔMSP7 clones using DNA probes specific to MSP7 and MSP7-related protein genes (MSRP1 to MSRP5). A calmodulin cDNA probe was used to confirm equal loading of RNA in each lane.
FIG. 6.
FIG. 6.
Effect of msp7 gene disruption on erythrocyte invasion by merozoites. (A) Invasion assay using O-positive erythrocytes and an initial parasitemia of 0.25 or 0.5%. The level of invasion was determined by flow cytometry and then normalized to that of the parental D10 parasite line. Values are represented as percent invasion compared to that of D10 parasites, with 95% CIs calculated from three independent assays as error bars. (B) Relative levels of invasion into enzyme-treated erythrocytes for the parental D10 line (▪) and the D10ΔMSP7 (□) clone. The erythrocytes in trophozoite cultures (1% parasitemia) were treated with neuraminidase (Neu), trypsin, chymotrypsin (Chymo), or trypsin and chymotrypsin (Try-Chy), and then the parasitemia was determined after 48 h. In control experiments, the cells were treated with buffer alone. An appropriate control for trypsin-chymotrypsin inhibitor treatment alone (TCInhibitor) was included. The levels of invasion were normalized to that in buffer-treated control cultures to yield percent invasion. The results are the means of three independent assays, and the error bars indicate 95% CIs.
FIG. 7.
FIG. 7.
Growth assay in a mixed culture of D10 and D10ΔMSP7 cloned parasites determined by DNA hybridization analysis. (A and B) Genomic DNA isolated from D10, the D10ΔMSP7 (ΔMSP7) clone, and a mixed culture of the two after 0, 2, 4, 6, or 10 generations was restricted with EcoRI and probed with DNA corresponding to the 5′ (A) or 3′ (B) target sequence, as explained in the legend to Fig. 1. Molecular sizes of msp7-hybridizing restriction fragments of D10 (4.8 kb) and D10ΔMSP7 (3.7 or 3.2 kb) are indicated. (C) Signal intensities determined by using ImageJ from each lane of panel A (solid line) or panel B (dashed line) were plotted as percent parasite population versus number of generations. ⧫, D10; ▴, D10ΔMSP7.
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