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Processing of Plasmodium falciparum Merozoite Surface Protein MSP1 Activates a Spectrin-Binding Function Enabling Parasite Egress from RBCs.

Das S, Hertrich N, Perrin AJ, Withers-Martinez C, Collins CR, Jones ML, Watermeyer JM, Fobes ET, Martin SR, Saibil HR, Wright GJ, Treeck M, Epp C, Blackman MJ - Cell Host Microbe (2015)

Bottom Line: The function of MSP1 and its processing are unknown.Here we show that SUB1-mediated processing of MSP1 is important for parasite viability.Parasites expressing an inefficiently processed MSP1 mutant show delayed egress, and merozoites lacking surface-bound MSP1 display a severe egress defect.

View Article: PubMed Central - PubMed

Affiliation: The Francis Crick Institute, Mill Hill Laboratory, Mill Hill, London, NW7 1AA, UK.

No MeSH data available.


Related in: MedlinePlus

Episomal Expression of Cleavage-Resistant MSP1 Inhibits P. falciparum Growth(A) Blasticidin-regulated co-selection episome. A bi-directional P. falciparum promoter (the intron of PlasmoDB: PFC0005w) drives expression of the blasticidin-S-deaminase gene (bsd) and msp1-f transgene. The hrp2 gene 3′ UTR controls transgene transcript termination and polyadenylation. Three variants were used, expressing wild-type msp-1f (pHBIMFwt, blue), or with mutations at all four known and putative 38/42 sites, (pHIBMFmut38/42, yellow; same mutations as Fmut38/42triple, Figure S1A) or mutations at all primary processing sites (pHBIMFmutall, red; same as mutant Fmutall, Figure S1A). All msp1-f sequences included the GPI anchor sequence. Increasing blasticidin concentration selects for parasites harboring multi-copy concatamers to maintain drug resistance, leading to increased msp1-f expression. A construct containing the Renilla luciferase gene (pHBIRH, green) was used as control.(B) Immunofluorescence analysis (IFA) of parental 3D7 and FCB1 schizonts, as well as 3D7 schizonts harboring the constructs in the indicated concentrations of blasticidin. Parasites were probed with MSP1 isoform-specific antibodies. Merged signals include that of the DNA dye 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 5 μm.(C) Quantification by FACS of parasite replication over a single erythrocytic cycle. Top: no significant differences between parental parasites and the transgenic 3D7pHBIMFwt and 3D7pHBIRH lines. Bottom: replication of the 3D7pHBIMFwt line compared to the 3D7pHIBMFmut38/42 and 3D7pHBIMFmutall lines expressing mutant MSP1-F, at similar blasticidin concentrations. Columns show mean values of >3 biological replicates. Error bars, SEM. Statistically different growth rates are indicated (∗p < 0.05; ∗∗p < 0.01. Kruskal-Wallis test).(D) Transgene RNA transcript levels measured by qRT-PCR, as a percentile of endogenous msp1-d transcript levels (100%). SEM values in all cases were <0.1%. See also Figure S2.
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fig2: Episomal Expression of Cleavage-Resistant MSP1 Inhibits P. falciparum Growth(A) Blasticidin-regulated co-selection episome. A bi-directional P. falciparum promoter (the intron of PlasmoDB: PFC0005w) drives expression of the blasticidin-S-deaminase gene (bsd) and msp1-f transgene. The hrp2 gene 3′ UTR controls transgene transcript termination and polyadenylation. Three variants were used, expressing wild-type msp-1f (pHBIMFwt, blue), or with mutations at all four known and putative 38/42 sites, (pHIBMFmut38/42, yellow; same mutations as Fmut38/42triple, Figure S1A) or mutations at all primary processing sites (pHBIMFmutall, red; same as mutant Fmutall, Figure S1A). All msp1-f sequences included the GPI anchor sequence. Increasing blasticidin concentration selects for parasites harboring multi-copy concatamers to maintain drug resistance, leading to increased msp1-f expression. A construct containing the Renilla luciferase gene (pHBIRH, green) was used as control.(B) Immunofluorescence analysis (IFA) of parental 3D7 and FCB1 schizonts, as well as 3D7 schizonts harboring the constructs in the indicated concentrations of blasticidin. Parasites were probed with MSP1 isoform-specific antibodies. Merged signals include that of the DNA dye 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 5 μm.(C) Quantification by FACS of parasite replication over a single erythrocytic cycle. Top: no significant differences between parental parasites and the transgenic 3D7pHBIMFwt and 3D7pHBIRH lines. Bottom: replication of the 3D7pHBIMFwt line compared to the 3D7pHIBMFmut38/42 and 3D7pHBIMFmutall lines expressing mutant MSP1-F, at similar blasticidin concentrations. Columns show mean values of >3 biological replicates. Error bars, SEM. Statistically different growth rates are indicated (∗p < 0.05; ∗∗p < 0.01. Kruskal-Wallis test).(D) Transgene RNA transcript levels measured by qRT-PCR, as a percentile of endogenous msp1-d transcript levels (100%). SEM values in all cases were <0.1%. See also Figure S2.

Mentions: To test whether mutations that prevent processing are tolerated by P. falciparum, we adopted two complementary strategies. First, we exploited an episomal transgene expression system (Epp et al., 2008) that allows blasticidin-regulated control of expression levels. Constructs for expression of three forms of MSP1-F (Figure 2A; Figures S2A, and S2B) were transfected into 3D7 P. falciparum, then antibodies specific for MSP1-F used to examine transgene expression on the background of endogenous MSP1-D. Parasites harboring a wild-type msp1-f transgene (3D7pHBIMFwt), or the same gene with mutations at all putative 38/42 sites (3D7pHBIMFmut38/42), or at all primary processing sites (3D7pHBIMFmutall), correctly expressed the transgene product on developing merozoites at all blasticidin concentrations tested (Figure 2B; Figure S2C). Varying blasticidin levels from 2–15 μg ml−1 did not affect growth of the 3D7pHBIMFwt line or parasites harboring a control plasmid, pHBIRH (Figure 2C, top). However, parasites harboring mutant constructs pHBIMFmut38/42 and pHBIMFmutall showed significantly lower growth rates than the 3D7pHBIMFwt line (Figure 2C, bottom). Whereas the 3D7pHBIMFwt line responded to increases in blasticidin concentration by substantially upregulating msp1-f transcript levels (Figure 2D; Figure S2D), likely via increases in episome copy number (confirmed by copy number estimation, data not shown), much less upregulation was seen in the mutant lines, indicating an inability to respond to elevated drug concentrations. Since the episomes differed only at the msp1-f cleavage sites, these results suggested that expression of cleavage-resistant MSP1, even in the presence of endogenous MSP1, is deleterious.


Processing of Plasmodium falciparum Merozoite Surface Protein MSP1 Activates a Spectrin-Binding Function Enabling Parasite Egress from RBCs.

Das S, Hertrich N, Perrin AJ, Withers-Martinez C, Collins CR, Jones ML, Watermeyer JM, Fobes ET, Martin SR, Saibil HR, Wright GJ, Treeck M, Epp C, Blackman MJ - Cell Host Microbe (2015)

Episomal Expression of Cleavage-Resistant MSP1 Inhibits P. falciparum Growth(A) Blasticidin-regulated co-selection episome. A bi-directional P. falciparum promoter (the intron of PlasmoDB: PFC0005w) drives expression of the blasticidin-S-deaminase gene (bsd) and msp1-f transgene. The hrp2 gene 3′ UTR controls transgene transcript termination and polyadenylation. Three variants were used, expressing wild-type msp-1f (pHBIMFwt, blue), or with mutations at all four known and putative 38/42 sites, (pHIBMFmut38/42, yellow; same mutations as Fmut38/42triple, Figure S1A) or mutations at all primary processing sites (pHBIMFmutall, red; same as mutant Fmutall, Figure S1A). All msp1-f sequences included the GPI anchor sequence. Increasing blasticidin concentration selects for parasites harboring multi-copy concatamers to maintain drug resistance, leading to increased msp1-f expression. A construct containing the Renilla luciferase gene (pHBIRH, green) was used as control.(B) Immunofluorescence analysis (IFA) of parental 3D7 and FCB1 schizonts, as well as 3D7 schizonts harboring the constructs in the indicated concentrations of blasticidin. Parasites were probed with MSP1 isoform-specific antibodies. Merged signals include that of the DNA dye 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 5 μm.(C) Quantification by FACS of parasite replication over a single erythrocytic cycle. Top: no significant differences between parental parasites and the transgenic 3D7pHBIMFwt and 3D7pHBIRH lines. Bottom: replication of the 3D7pHBIMFwt line compared to the 3D7pHIBMFmut38/42 and 3D7pHBIMFmutall lines expressing mutant MSP1-F, at similar blasticidin concentrations. Columns show mean values of >3 biological replicates. Error bars, SEM. Statistically different growth rates are indicated (∗p < 0.05; ∗∗p < 0.01. Kruskal-Wallis test).(D) Transgene RNA transcript levels measured by qRT-PCR, as a percentile of endogenous msp1-d transcript levels (100%). SEM values in all cases were <0.1%. See also Figure S2.
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Related In: Results  -  Collection

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fig2: Episomal Expression of Cleavage-Resistant MSP1 Inhibits P. falciparum Growth(A) Blasticidin-regulated co-selection episome. A bi-directional P. falciparum promoter (the intron of PlasmoDB: PFC0005w) drives expression of the blasticidin-S-deaminase gene (bsd) and msp1-f transgene. The hrp2 gene 3′ UTR controls transgene transcript termination and polyadenylation. Three variants were used, expressing wild-type msp-1f (pHBIMFwt, blue), or with mutations at all four known and putative 38/42 sites, (pHIBMFmut38/42, yellow; same mutations as Fmut38/42triple, Figure S1A) or mutations at all primary processing sites (pHBIMFmutall, red; same as mutant Fmutall, Figure S1A). All msp1-f sequences included the GPI anchor sequence. Increasing blasticidin concentration selects for parasites harboring multi-copy concatamers to maintain drug resistance, leading to increased msp1-f expression. A construct containing the Renilla luciferase gene (pHBIRH, green) was used as control.(B) Immunofluorescence analysis (IFA) of parental 3D7 and FCB1 schizonts, as well as 3D7 schizonts harboring the constructs in the indicated concentrations of blasticidin. Parasites were probed with MSP1 isoform-specific antibodies. Merged signals include that of the DNA dye 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 5 μm.(C) Quantification by FACS of parasite replication over a single erythrocytic cycle. Top: no significant differences between parental parasites and the transgenic 3D7pHBIMFwt and 3D7pHBIRH lines. Bottom: replication of the 3D7pHBIMFwt line compared to the 3D7pHIBMFmut38/42 and 3D7pHBIMFmutall lines expressing mutant MSP1-F, at similar blasticidin concentrations. Columns show mean values of >3 biological replicates. Error bars, SEM. Statistically different growth rates are indicated (∗p < 0.05; ∗∗p < 0.01. Kruskal-Wallis test).(D) Transgene RNA transcript levels measured by qRT-PCR, as a percentile of endogenous msp1-d transcript levels (100%). SEM values in all cases were <0.1%. See also Figure S2.
Mentions: To test whether mutations that prevent processing are tolerated by P. falciparum, we adopted two complementary strategies. First, we exploited an episomal transgene expression system (Epp et al., 2008) that allows blasticidin-regulated control of expression levels. Constructs for expression of three forms of MSP1-F (Figure 2A; Figures S2A, and S2B) were transfected into 3D7 P. falciparum, then antibodies specific for MSP1-F used to examine transgene expression on the background of endogenous MSP1-D. Parasites harboring a wild-type msp1-f transgene (3D7pHBIMFwt), or the same gene with mutations at all putative 38/42 sites (3D7pHBIMFmut38/42), or at all primary processing sites (3D7pHBIMFmutall), correctly expressed the transgene product on developing merozoites at all blasticidin concentrations tested (Figure 2B; Figure S2C). Varying blasticidin levels from 2–15 μg ml−1 did not affect growth of the 3D7pHBIMFwt line or parasites harboring a control plasmid, pHBIRH (Figure 2C, top). However, parasites harboring mutant constructs pHBIMFmut38/42 and pHBIMFmutall showed significantly lower growth rates than the 3D7pHBIMFwt line (Figure 2C, bottom). Whereas the 3D7pHBIMFwt line responded to increases in blasticidin concentration by substantially upregulating msp1-f transcript levels (Figure 2D; Figure S2D), likely via increases in episome copy number (confirmed by copy number estimation, data not shown), much less upregulation was seen in the mutant lines, indicating an inability to respond to elevated drug concentrations. Since the episomes differed only at the msp1-f cleavage sites, these results suggested that expression of cleavage-resistant MSP1, even in the presence of endogenous MSP1, is deleterious.

Bottom Line: The function of MSP1 and its processing are unknown.Here we show that SUB1-mediated processing of MSP1 is important for parasite viability.Parasites expressing an inefficiently processed MSP1 mutant show delayed egress, and merozoites lacking surface-bound MSP1 display a severe egress defect.

View Article: PubMed Central - PubMed

Affiliation: The Francis Crick Institute, Mill Hill Laboratory, Mill Hill, London, NW7 1AA, UK.

No MeSH data available.


Related in: MedlinePlus