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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

Mutations that Prevent All Processing in the 38/42 Region Are Deleterious(A) Top: modification of the P. falciparum msp1-d locus by single-crossover homologous recombination. The targeting region (gray) incorporated into the integration constructs was fused just upstream of the 38/42 region to recodonized sequence (red/green, syn) encoding the rest of the ORF. In all except pHH1MSP1chim_wt, this incorporated mutations and/or deletions to block processing at one or more 38/42 cleavage sites (asterisk). The 3′ end of the recodonized sequence (green) contained the MSP1-F-specific mAb 111.4 epitope. Positions of hybridization of primers used for diagnostic PCR are indicated (blue and red half arrows). Integration replaced the msp1 3′ UTR with that of the P. berghei dihydrofolate reductase gene (pbdt). The hdhfr cassette confers resistance to WR99210. Integration produces a modified locus encoding a chimeric MSP1 recognized by both mAb X509 and mAb 111.4. Bottom: substitutions or deletions (hyphens) introduced by the constructs are indicated (red). Green tick, successful integration. Red cross, no integration detected.(B) IFA of schizonts of the parental and transgenic P. falciparum clones. All transgenics reacted with mAb 111.4. Scale bar, 5 μm.(C) Top: schematic of the MSP142 fragment (GPI anchored) and the slightly larger MSP142∗ and MSP142∗∗ species predicted to result from ablation of cleavage at the canonical and canonical plus 38/42alt1 sites, respectively. The MSP133 and modified MSP133∗ and MSP133∗∗ fragments, derived from cleavage by SUB2 within MSP142, MSP142∗, and MSP142∗∗, respectively, are also depicted. Bottom: western blot shows differences (highlighted, dotted lines) in migration of the wild-type and modified MSP142 forms.(D) Western blot of culture supernatants shows differences in migration of MSP133, MSP133∗, and MSP133∗∗ (highlighted as above). As controls, supernatants were probed with mAb 89.1, which recognizes MSP183, or antibodies to an irrelevant shed parasite protein, AMA1. See also Figure S3.
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fig3: Mutations that Prevent All Processing in the 38/42 Region Are Deleterious(A) Top: modification of the P. falciparum msp1-d locus by single-crossover homologous recombination. The targeting region (gray) incorporated into the integration constructs was fused just upstream of the 38/42 region to recodonized sequence (red/green, syn) encoding the rest of the ORF. In all except pHH1MSP1chim_wt, this incorporated mutations and/or deletions to block processing at one or more 38/42 cleavage sites (asterisk). The 3′ end of the recodonized sequence (green) contained the MSP1-F-specific mAb 111.4 epitope. Positions of hybridization of primers used for diagnostic PCR are indicated (blue and red half arrows). Integration replaced the msp1 3′ UTR with that of the P. berghei dihydrofolate reductase gene (pbdt). The hdhfr cassette confers resistance to WR99210. Integration produces a modified locus encoding a chimeric MSP1 recognized by both mAb X509 and mAb 111.4. Bottom: substitutions or deletions (hyphens) introduced by the constructs are indicated (red). Green tick, successful integration. Red cross, no integration detected.(B) IFA of schizonts of the parental and transgenic P. falciparum clones. All transgenics reacted with mAb 111.4. Scale bar, 5 μm.(C) Top: schematic of the MSP142 fragment (GPI anchored) and the slightly larger MSP142∗ and MSP142∗∗ species predicted to result from ablation of cleavage at the canonical and canonical plus 38/42alt1 sites, respectively. The MSP133 and modified MSP133∗ and MSP133∗∗ fragments, derived from cleavage by SUB2 within MSP142, MSP142∗, and MSP142∗∗, respectively, are also depicted. Bottom: western blot shows differences (highlighted, dotted lines) in migration of the wild-type and modified MSP142 forms.(D) Western blot of culture supernatants shows differences in migration of MSP133, MSP133∗, and MSP133∗∗ (highlighted as above). As controls, supernatants were probed with mAb 89.1, which recognizes MSP183, or antibodies to an irrelevant shed parasite protein, AMA1. See also Figure S3.

Mentions: In a second approach to evaluating the importance of PfSUB1-mediated MSP1 processing, we sought to modify the endogenous msp1 locus using homologous recombination to introduce mutations that prevent processing within the 38/42 region (the importance of processing at the 83/30 and 30/38 sites was not further examined). Our approach used a previously described strategy (Child et al., 2010) in which we transfected 3D7 parasites with constructs containing targeting sequence fused to synthetic “recodonized” sequence encoding a chimeric MSP1 C-terminal domain (Figure 3A). Integration produces a chimeric gene, the product of which can be distinguished from unmodified MSP1-D by its reactivity with the MSP1-F-specific monoclonal antibody (mAb) 111.4. Integration thus epitope tags the gene.


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)

Mutations that Prevent All Processing in the 38/42 Region Are Deleterious(A) Top: modification of the P. falciparum msp1-d locus by single-crossover homologous recombination. The targeting region (gray) incorporated into the integration constructs was fused just upstream of the 38/42 region to recodonized sequence (red/green, syn) encoding the rest of the ORF. In all except pHH1MSP1chim_wt, this incorporated mutations and/or deletions to block processing at one or more 38/42 cleavage sites (asterisk). The 3′ end of the recodonized sequence (green) contained the MSP1-F-specific mAb 111.4 epitope. Positions of hybridization of primers used for diagnostic PCR are indicated (blue and red half arrows). Integration replaced the msp1 3′ UTR with that of the P. berghei dihydrofolate reductase gene (pbdt). The hdhfr cassette confers resistance to WR99210. Integration produces a modified locus encoding a chimeric MSP1 recognized by both mAb X509 and mAb 111.4. Bottom: substitutions or deletions (hyphens) introduced by the constructs are indicated (red). Green tick, successful integration. Red cross, no integration detected.(B) IFA of schizonts of the parental and transgenic P. falciparum clones. All transgenics reacted with mAb 111.4. Scale bar, 5 μm.(C) Top: schematic of the MSP142 fragment (GPI anchored) and the slightly larger MSP142∗ and MSP142∗∗ species predicted to result from ablation of cleavage at the canonical and canonical plus 38/42alt1 sites, respectively. The MSP133 and modified MSP133∗ and MSP133∗∗ fragments, derived from cleavage by SUB2 within MSP142, MSP142∗, and MSP142∗∗, respectively, are also depicted. Bottom: western blot shows differences (highlighted, dotted lines) in migration of the wild-type and modified MSP142 forms.(D) Western blot of culture supernatants shows differences in migration of MSP133, MSP133∗, and MSP133∗∗ (highlighted as above). As controls, supernatants were probed with mAb 89.1, which recognizes MSP183, or antibodies to an irrelevant shed parasite protein, AMA1. See also Figure S3.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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Show All Figures
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fig3: Mutations that Prevent All Processing in the 38/42 Region Are Deleterious(A) Top: modification of the P. falciparum msp1-d locus by single-crossover homologous recombination. The targeting region (gray) incorporated into the integration constructs was fused just upstream of the 38/42 region to recodonized sequence (red/green, syn) encoding the rest of the ORF. In all except pHH1MSP1chim_wt, this incorporated mutations and/or deletions to block processing at one or more 38/42 cleavage sites (asterisk). The 3′ end of the recodonized sequence (green) contained the MSP1-F-specific mAb 111.4 epitope. Positions of hybridization of primers used for diagnostic PCR are indicated (blue and red half arrows). Integration replaced the msp1 3′ UTR with that of the P. berghei dihydrofolate reductase gene (pbdt). The hdhfr cassette confers resistance to WR99210. Integration produces a modified locus encoding a chimeric MSP1 recognized by both mAb X509 and mAb 111.4. Bottom: substitutions or deletions (hyphens) introduced by the constructs are indicated (red). Green tick, successful integration. Red cross, no integration detected.(B) IFA of schizonts of the parental and transgenic P. falciparum clones. All transgenics reacted with mAb 111.4. Scale bar, 5 μm.(C) Top: schematic of the MSP142 fragment (GPI anchored) and the slightly larger MSP142∗ and MSP142∗∗ species predicted to result from ablation of cleavage at the canonical and canonical plus 38/42alt1 sites, respectively. The MSP133 and modified MSP133∗ and MSP133∗∗ fragments, derived from cleavage by SUB2 within MSP142, MSP142∗, and MSP142∗∗, respectively, are also depicted. Bottom: western blot shows differences (highlighted, dotted lines) in migration of the wild-type and modified MSP142 forms.(D) Western blot of culture supernatants shows differences in migration of MSP133, MSP133∗, and MSP133∗∗ (highlighted as above). As controls, supernatants were probed with mAb 89.1, which recognizes MSP183, or antibodies to an irrelevant shed parasite protein, AMA1. See also Figure S3.
Mentions: In a second approach to evaluating the importance of PfSUB1-mediated MSP1 processing, we sought to modify the endogenous msp1 locus using homologous recombination to introduce mutations that prevent processing within the 38/42 region (the importance of processing at the 83/30 and 30/38 sites was not further examined). Our approach used a previously described strategy (Child et al., 2010) in which we transfected 3D7 parasites with constructs containing targeting sequence fused to synthetic “recodonized” sequence encoding a chimeric MSP1 C-terminal domain (Figure 3A). Integration produces a chimeric gene, the product of which can be distinguished from unmodified MSP1-D by its reactivity with the MSP1-F-specific monoclonal antibody (mAb) 111.4. Integration thus epitope tags the gene.

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