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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

Rate of MSP1 Processing Regulates the Kinetics of Egress(A) Top: the 38/42 region in chim_Δ+can and chim_wt parasites and the MSP1 processing pathway (Child et al., 2010). Bottom: time course comparing processing of MSP1 from chim_Δ+can and chim_wt clones by western blot. Schizonts were sampled at the indicated times following removal of a C1 block. Note the slightly smaller full-length chim_Δ+can MSP1 due to the 69-residue deletion.(B) Left: stills from time-lapse microscopy of chim_Δ+can and chim_wt clones (imaging started 4 min 20 s after C1 removal). Right: box plot comparison of time to egress after C1 removal. Data are from 4 independent experiments each assessing 12–24 egress events per clone. Whiskers, range. A single outlier point (>1.5× the interquartile range) indicated. The chim_Δ+can clones showed a mean egress delay of 7.5 ± 1.4 min (p < 0.005, Student’s t test). Similar results were obtained with two other chim_Δ+can and chim_wt clones (data not shown). See also Figure S5 and Movies S1 and S2.
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fig5: Rate of MSP1 Processing Regulates the Kinetics of Egress(A) Top: the 38/42 region in chim_Δ+can and chim_wt parasites and the MSP1 processing pathway (Child et al., 2010). Bottom: time course comparing processing of MSP1 from chim_Δ+can and chim_wt clones by western blot. Schizonts were sampled at the indicated times following removal of a C1 block. Note the slightly smaller full-length chim_Δ+can MSP1 due to the 69-residue deletion.(B) Left: stills from time-lapse microscopy of chim_Δ+can and chim_wt clones (imaging started 4 min 20 s after C1 removal). Right: box plot comparison of time to egress after C1 removal. Data are from 4 independent experiments each assessing 12–24 egress events per clone. Whiskers, range. A single outlier point (>1.5× the interquartile range) indicated. The chim_Δ+can clones showed a mean egress delay of 7.5 ± 1.4 min (p < 0.005, Student’s t test). Similar results were obtained with two other chim_Δ+can and chim_wt clones (data not shown). See also Figure S5 and Movies S1 and S2.

Mentions: The erythrocyte cytoskeleton lies beneath the cell membrane so merozoites are unlikely to contact it during invasion. However, intracellular merozoites impinge on the inner face of the host cell membrane in the brief period between PVM rupture and egress (e.g., Glushakova et al., 2009), so we explored the possibility that direct interactions between processed merozoite surface MSP1 and host cell spectrin might play a part in egress. For this, we returned to the chim_Δ+can mutant (Figure 3) in which an MSP1 segment had been deleted to remove the 38/42alt1 and 38/42alt2 sites entirely, leaving just the canonical 38/42 cleavage site. Since this is a relatively poor substrate for PfSUB1 (Figure 1C), we predicted that cleavage within the 38/42 region in the chim_Δ+can mutant should be less efficient than in wild-type parasites. To test this, we compared the kinetics of processing in chim_Δ+can parasites with that in chim_wt parasites, which expressed the same chimeric MSP1 but retained all three 38/42 cleavage sites. For these experiments, schizonts were treated with the reversible PKG inhibitor compound 1 (C1), which prevents PfSUB1 discharge, stalling schizont development at the final stage of maturation. MSP1 processing and egress occur within minutes of washing away the inhibitor (Collins et al., 2013b). As shown in Figure 5A, processing of MSP1 in the chim_Δ+can mutant was delayed relative to chim_wt parasites and was characterized by an unusually prominent MSP138+42 processing intermediate. Comparison of the kinetics of chim_Δ+can and chim_wt egress by time-lapse microscopy showed a reproducible delay in egress in the chim_Δ+can parasites following C1 removal (Figure 5B; Movie S1), mirroring the delay in MSP1 processing. This was confirmed in further experiments in which the clones were imaged simultaneously following fluorescent labeling of one population to identify it (Movie S2; Figure S5). Since the chim_Δ+can and chim_wt parasites differed only by the presence or absence of an MSP1 segment encompassing the 38/42alt1 and alt2 cleavage sites, these results showed that processing of MSP1 regulates the kinetics of egress.


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)

Rate of MSP1 Processing Regulates the Kinetics of Egress(A) Top: the 38/42 region in chim_Δ+can and chim_wt parasites and the MSP1 processing pathway (Child et al., 2010). Bottom: time course comparing processing of MSP1 from chim_Δ+can and chim_wt clones by western blot. Schizonts were sampled at the indicated times following removal of a C1 block. Note the slightly smaller full-length chim_Δ+can MSP1 due to the 69-residue deletion.(B) Left: stills from time-lapse microscopy of chim_Δ+can and chim_wt clones (imaging started 4 min 20 s after C1 removal). Right: box plot comparison of time to egress after C1 removal. Data are from 4 independent experiments each assessing 12–24 egress events per clone. Whiskers, range. A single outlier point (>1.5× the interquartile range) indicated. The chim_Δ+can clones showed a mean egress delay of 7.5 ± 1.4 min (p < 0.005, Student’s t test). Similar results were obtained with two other chim_Δ+can and chim_wt clones (data not shown). See also Figure S5 and Movies S1 and S2.
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Related In: Results  -  Collection

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fig5: Rate of MSP1 Processing Regulates the Kinetics of Egress(A) Top: the 38/42 region in chim_Δ+can and chim_wt parasites and the MSP1 processing pathway (Child et al., 2010). Bottom: time course comparing processing of MSP1 from chim_Δ+can and chim_wt clones by western blot. Schizonts were sampled at the indicated times following removal of a C1 block. Note the slightly smaller full-length chim_Δ+can MSP1 due to the 69-residue deletion.(B) Left: stills from time-lapse microscopy of chim_Δ+can and chim_wt clones (imaging started 4 min 20 s after C1 removal). Right: box plot comparison of time to egress after C1 removal. Data are from 4 independent experiments each assessing 12–24 egress events per clone. Whiskers, range. A single outlier point (>1.5× the interquartile range) indicated. The chim_Δ+can clones showed a mean egress delay of 7.5 ± 1.4 min (p < 0.005, Student’s t test). Similar results were obtained with two other chim_Δ+can and chim_wt clones (data not shown). See also Figure S5 and Movies S1 and S2.
Mentions: The erythrocyte cytoskeleton lies beneath the cell membrane so merozoites are unlikely to contact it during invasion. However, intracellular merozoites impinge on the inner face of the host cell membrane in the brief period between PVM rupture and egress (e.g., Glushakova et al., 2009), so we explored the possibility that direct interactions between processed merozoite surface MSP1 and host cell spectrin might play a part in egress. For this, we returned to the chim_Δ+can mutant (Figure 3) in which an MSP1 segment had been deleted to remove the 38/42alt1 and 38/42alt2 sites entirely, leaving just the canonical 38/42 cleavage site. Since this is a relatively poor substrate for PfSUB1 (Figure 1C), we predicted that cleavage within the 38/42 region in the chim_Δ+can mutant should be less efficient than in wild-type parasites. To test this, we compared the kinetics of processing in chim_Δ+can parasites with that in chim_wt parasites, which expressed the same chimeric MSP1 but retained all three 38/42 cleavage sites. For these experiments, schizonts were treated with the reversible PKG inhibitor compound 1 (C1), which prevents PfSUB1 discharge, stalling schizont development at the final stage of maturation. MSP1 processing and egress occur within minutes of washing away the inhibitor (Collins et al., 2013b). As shown in Figure 5A, processing of MSP1 in the chim_Δ+can mutant was delayed relative to chim_wt parasites and was characterized by an unusually prominent MSP138+42 processing intermediate. Comparison of the kinetics of chim_Δ+can and chim_wt egress by time-lapse microscopy showed a reproducible delay in egress in the chim_Δ+can parasites following C1 removal (Figure 5B; Movie S1), mirroring the delay in MSP1 processing. This was confirmed in further experiments in which the clones were imaged simultaneously following fluorescent labeling of one population to identify it (Movie S2; Figure S5). Since the chim_Δ+can and chim_wt parasites differed only by the presence or absence of an MSP1 segment encompassing the 38/42alt1 and alt2 cleavage sites, these results showed that processing of MSP1 regulates the kinetics of egress.

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