Article Figures & Data
Figures
- Fig. 1.
RT-PCR analysis of msrp expression. Total RNA isolated from synchronized 3D7 parasites collected at 3-h intervals (2 to 47 hpi) over one generation was used in a reverse transcription-PCR to amplify msp7, msrp1 to msrp5, PF13_0192 (0192), and msp1 mRNA.
- Fig. 2.
Expression and distribution of MSRP2. (A) MSRP2 is synthesized late in the cycle and processed; a time course immunoblot analysis of 3D7 parasite extracts is shown. The protein extracts prepared from Percoll-sorbitol-synchronized parasites were electrophoresed on SDS-12% PAGE and probed with rabbit polyclonal anti-MSRP2b antibodies. Molecular weight sizes (in thousands) of the reacting proteins are shown. Hours postinvasion are indicated for each lane. (B) MSRP2 proteins in laboratory parasite lines derived from various geographical isolates have identical patterns of mobility and processing. Protein extracts from Percoll-enriched late-stage parasites were immunoblotted with rabbit polyclonal anti-MSRP2b antibodies. Molecular weight markers (in thousands) are indicated. (C) MSRP2 is present in schizonts but not merozoites, and a processed form is released into culture supernatant. Immunoblot analysis of MSRP2 in trophozoite/schizonts (S), merozoites (M), and culture supernatant (CS). MSP3, MSP7, and BiP antibodies served as control reagents. (D) MSRP2 protein is a soluble protein. Total proteins (T) sequentially extracted by hypotonic- (H), salt-containing (S), and alkaline carbonate (C) buffers or carbonate-insoluble (P) fractions were immunoblotted with MSRP2-, MSP2-, MSP7-, and SERA5-specific antibodies. (E) MSRP2 protein is partially solubilized by saponin treatment. Late-stage parasites were solubilized by saponin; total (T), soluble (S), and insoluble (P) fractions were immunoblotted with MSRP2-, MSP3-, MSP7-, SERA5-, and BiP-specific antibodies. Uninfected RBC (R) extract was included in the blotting. (F and G) Brefeldin A inhibits MSRP2 processing. (F) The immunoprecipitates with antibodies to MSP1, MSP7, and MSRP2 from biosynthetically labeled cultures in the absence of BFA are shown. (G) Magnet-purified P. falciparum schizonts radiolabeled in the presence (+) or absence (−) of BFA were solubilized in Nonidet P40-containing buffer, and proteins were immunoprecipitated with either nonimmune rabbit serum (NRS) or rabbit polyclonal antibodies to MSP1, MSP7, and MSRP2. Antigen-antibody complexes were resolved in 8 to 15% SDS-PAGE, and labeled proteins were detected by fluorography. Major immunoprecipitated proteins are marked. Molecular masses of the markers are indicated in kDa. (H) Colocalization of MSP7 and MSRP2. Late-stage parasites were formaldehyde fixed, acetone permeabilized, and reacted with MAb 2G10 (mouse monoclonal anti-MSP7 antibody) and rabbit polyclonal anti-MSRP2 antibodies in an immunofluorescence assay.
- Fig. 3.
(A) Schematic of the msp7 gene family locus of P. falciparum 3D7 and knockout parasites. Nomenclature of MSP7-related genes is adapted from reference 29. Pseudogenes (black boxes) and the unrelated gene PF13_0192 are also shown. Gray boxes represent parental ORFs, and checkered and hashed boxes are from the transfecting plasmid. Human DHFR cassette is indicated by an arrow. Relevant restriction sites used in this investigation are marked (B, BglII; E, EcoRI; P, PacI; S, SacII; X, XbaI). DNA fragments generated in the Southern analysis are shown as lines below or above the physical map, with molecular masses indicated. (B to G) Southern hybridization analysis of genomic DNA from parental 3D7 and knockout parasites. Molecular weight sizes (in thousands) of the hybridizing restriction fragment of the resident locus, episome (Ep), and altered locus in the knockout parasites are marked. Hybridization probes are indicated below each panel. In panels B to F, EcoRI-restricted genomic DNA from 3D7 and transfected parasites (c0 for 3D7 Δmsrp1), two clones each from 3D7 Δmsrp1 (Δ1–1 and Δ1–2), 3D7 Δmsrp3 (Δ3–1 and Δ3–2), and 3D7 Δmsrp3-4 (Δ34–1 and Δ34–2) probed with 5′- and 3′-gene-specific DNA probes. For clones of 3D7 Δmsrp4 (Δ4–1 and Δ4–2) and 3D7 Δmsrp5 (Δ5–1 and Δ5–2) parasites, XbaI-restricted genomic DNA was used. In panel G, genomic DNA from 3D7 parasites, transfected parasites after 4 drug cyclings (c4), and two clones of 3D7 Δmsrp2 (Δ2–1 and Δ2– 2) restricted with BglII and hybridized with 5′-msrp2 and 3′-msrp2 DNA probes.
- Fig. 4.
Analysis of transcription of targeted genes. (A) Up to 3 μg of total RNA from parental line and two clones each from transfectant lines were hybridized with PCR-amplified ORFs of msp7, msrp2, msrp3, and msrp5. Calmodulin (cam) was used as a distant locus control for transcription. Weak nonspecific signals in 3D7 Δmsrp2 and 3D7 Δmsrp5 lanes are indicated by arrowheads. (B) cDNA synthesized from total RNA by priming with random hexamers was used to amplify msp7, msrp1, and msrp4 from parental and genetic deletion clones. Amplification of MSP7 in the presence (+) or absence (−) of reverse transcriptase (RT) served as a control. Lanes for 3D7 and deletion mutants are labeled as described in the legend for Fig. 3B to G. (C) Immunoblot analysis of protein extracts from parental (3D7) and msrp2 deletion clones (Δ2-1 and Δ2-2) with rabbit polyclonal antibodies to MSP7 and MSRP2B. Different forms of MSP7 and MSRP2 are identified. The precursor and proteolytic products of MSRP2 are absent in the deletion clones.
- Fig. 5.
Invasion and growth of deletion mutant parasite lines. (A) Single-cycle invasion assay of 3D7 and disruption mutants 3D7 Δmsrp1 (Δ1–1, Δ1–2), 3D7 Δmsrp2 (Δ2–1, Δ2–2), 3D7 Δmsrp3 (Δ3–1, Δ3-2), 3D7 Δmsrp4 (Δ4–1, Δ4-2), 3D7 Δmsrp3-4 (Δ34–1, Δ34–2), and 3D7 Δmsrp5 (Δ5–1, Δ5–2). The mean parasitemia compared to that of parental 3D7 parasites from three independent assays is presented. Error bars represent 95% CI. (B) Growth of parental (3D7, ▪) and cloned transfectant lines (3D7 Δmsrp1, •; 3D7 Δmsrp2, ○; 3D7 Δmsrp3, ▴; 3D7 Δmsrp4, ⧫; 3D7 Δmsrp3-4, □; 3D7 Δmsrp5, ⋄) over a 10-day period. Each point corresponds to mean parasitemia of two individual clones cultured in duplicates with 95% CI as error bars. Parasitemia was determined by FACS analysis of hydroethidine-labeled parasites. The results indicated that the knockout parasite lines grew at the same rate as 3D7 in vitro (P > 0.05).
Additional Files
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Oligonucleotide primers used for expression of MSP7-related protein in E. coli (Table S1); oligonucleotide primers used to construct msrp knockout plasmids (Table S2); test for cross-reactivity (Figure S1); reverse transcription-PCR analysis (Figure S2).
PDF file, 197K.
- Supplemental file 1 -
Oligonucleotide primers used for expression of MSP7-related protein in E. coli (Table S1); oligonucleotide primers used to construct msrp knockout plasmids (Table S2); test for cross-reactivity (Figure S1); reverse transcription-PCR analysis (Figure S2).
