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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

Truncation of MSP1 Produces an Egress Defect(A) Predicted RAP-induced MSP1 truncation in the 3D7MSP1flox42C clones, showing loss of the GPI anchor and C-terminal domain containing the mAb X509 epitope.(B) MSP1 truncation confirmed by western blot of 3D7MSP1flox42C1 clone E3 schizonts, 44 hr following treatment ± RAP. The PV protein SERA5 was used as a loading control.(C) RAP treatment produces a loss of mAb X509 reactivity and a shift in the IFA pattern of MSP1 to one typical of PV proteins, consistent with the predicted truncation. Numbers of DAPI-stained nuclei did not differ between control and RAP-treated schizonts (mean values: 21.2 ± 3.4 and 20.6 ± 4.0 nuclei per schizont, respectively, n = 24).(D) IFA showing co-localization of truncated MSP1 with SERA5 indicating a PV location. The punctate localization of SUB1 and the microneme protein AMA1 indicates normal organelle biogenesis.(E) IFA showing lack of surface-bound MSP1 on merozoites of RAP-treated 3D7MSP1flox42C1 clone E3. Antibodies to AMA1 (which is expressed on free merozoites) were used as a control.(F) Stills from time-lapse DIC microscopic imaging of egress in control and RAP-treated 3D7MSP1flox42C1 clone E3. Scale bar, 10 μm.(G) Replication rates of RAP- or control-treated 3D7MSP1flox42C1 clone E3. Cultures were passaged at intervals by 10-fold dilution into fresh medium plus erythrocytes as described in Supplemental Experimental Procedures. Observed parasitaemia values were adjusted for these dilutions and are displayed as adjusted values. The plot shows mean values of three biological replicate experiments. Error bars, SEM. The RAP-treated cultures showed an ∼2.1-fold reduction in replication rate per cycle, but this was an over-estimate of mutant viability due to rapid expansion of the few (∼1%) non-excised parasites in the RAP-treated cultures. See also Figure S6 and Movies S3 and S4.
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fig6: Truncation of MSP1 Produces an Egress Defect(A) Predicted RAP-induced MSP1 truncation in the 3D7MSP1flox42C clones, showing loss of the GPI anchor and C-terminal domain containing the mAb X509 epitope.(B) MSP1 truncation confirmed by western blot of 3D7MSP1flox42C1 clone E3 schizonts, 44 hr following treatment ± RAP. The PV protein SERA5 was used as a loading control.(C) RAP treatment produces a loss of mAb X509 reactivity and a shift in the IFA pattern of MSP1 to one typical of PV proteins, consistent with the predicted truncation. Numbers of DAPI-stained nuclei did not differ between control and RAP-treated schizonts (mean values: 21.2 ± 3.4 and 20.6 ± 4.0 nuclei per schizont, respectively, n = 24).(D) IFA showing co-localization of truncated MSP1 with SERA5 indicating a PV location. The punctate localization of SUB1 and the microneme protein AMA1 indicates normal organelle biogenesis.(E) IFA showing lack of surface-bound MSP1 on merozoites of RAP-treated 3D7MSP1flox42C1 clone E3. Antibodies to AMA1 (which is expressed on free merozoites) were used as a control.(F) Stills from time-lapse DIC microscopic imaging of egress in control and RAP-treated 3D7MSP1flox42C1 clone E3. Scale bar, 10 μm.(G) Replication rates of RAP- or control-treated 3D7MSP1flox42C1 clone E3. Cultures were passaged at intervals by 10-fold dilution into fresh medium plus erythrocytes as described in Supplemental Experimental Procedures. Observed parasitaemia values were adjusted for these dilutions and are displayed as adjusted values. The plot shows mean values of three biological replicate experiments. Error bars, SEM. The RAP-treated cultures showed an ∼2.1-fold reduction in replication rate per cycle, but this was an over-estimate of mutant viability due to rapid expansion of the few (∼1%) non-excised parasites in the RAP-treated cultures. See also Figure S6 and Movies S3 and S4.

Mentions: MSP1 is tethered to the merozoite surface via a C-terminal GPI anchor. To further test our model that direct interactions between merozoite-bound MSP1 and the erythrocyte cytoskeleton facilitates egress, we used a recently published conditional strategy to generate P. falciparum transgenics in which a 3′ segment of the msp1 gene could be deleted by rapamycin (RAP)-inducible, Cre recombinase-mediated excision (Figure 6A; Figure S6A) (Collins et al., 2013a). This was predicted to generate a truncated MSP1 that lacked a GPI anchor and so would not be bound to the merozoite surface. Analysis of RAP-treated 3D7MSP1flox42C parasites showed highly efficient excision, resulting in exclusive expression of truncated MSP1 in mature schizonts at the end of the same erythrocytic cycle (Figures 6B and 6C). No effects on merozoite development were discernible. The modified MSP1 was trafficked to the PV as expected for a non-membrane-bound merozoite surface protein (Figures 6C–6D; Figure S6B) but was not present on the surface of free merozoites (Figure 6E). Video microscopy of the RAP-treated 3D7MSP1flox42C schizonts revealed a dramatic egress defect characterized by abortive erythrocyte membrane rupture and trapping of the merozoites in the partially ruptured cell (Figure 6F; Movies S3 and S4). Consistent with this, the RAP-treated mutants displayed a substantially reduced replication rate (Figure 6G and Figure S6C). These results show that MSP1 functions at egress and that this role requires it to be tethered to the merozoite surface. Collectively, our findings support the model that processing of MSP1 facilitates host cell membrane rupture, probably through interactions between the mature merozoite surface and the erythrocyte cytoskeleton.


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)

Truncation of MSP1 Produces an Egress Defect(A) Predicted RAP-induced MSP1 truncation in the 3D7MSP1flox42C clones, showing loss of the GPI anchor and C-terminal domain containing the mAb X509 epitope.(B) MSP1 truncation confirmed by western blot of 3D7MSP1flox42C1 clone E3 schizonts, 44 hr following treatment ± RAP. The PV protein SERA5 was used as a loading control.(C) RAP treatment produces a loss of mAb X509 reactivity and a shift in the IFA pattern of MSP1 to one typical of PV proteins, consistent with the predicted truncation. Numbers of DAPI-stained nuclei did not differ between control and RAP-treated schizonts (mean values: 21.2 ± 3.4 and 20.6 ± 4.0 nuclei per schizont, respectively, n = 24).(D) IFA showing co-localization of truncated MSP1 with SERA5 indicating a PV location. The punctate localization of SUB1 and the microneme protein AMA1 indicates normal organelle biogenesis.(E) IFA showing lack of surface-bound MSP1 on merozoites of RAP-treated 3D7MSP1flox42C1 clone E3. Antibodies to AMA1 (which is expressed on free merozoites) were used as a control.(F) Stills from time-lapse DIC microscopic imaging of egress in control and RAP-treated 3D7MSP1flox42C1 clone E3. Scale bar, 10 μm.(G) Replication rates of RAP- or control-treated 3D7MSP1flox42C1 clone E3. Cultures were passaged at intervals by 10-fold dilution into fresh medium plus erythrocytes as described in Supplemental Experimental Procedures. Observed parasitaemia values were adjusted for these dilutions and are displayed as adjusted values. The plot shows mean values of three biological replicate experiments. Error bars, SEM. The RAP-treated cultures showed an ∼2.1-fold reduction in replication rate per cycle, but this was an over-estimate of mutant viability due to rapid expansion of the few (∼1%) non-excised parasites in the RAP-treated cultures. See also Figure S6 and Movies S3 and S4.
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fig6: Truncation of MSP1 Produces an Egress Defect(A) Predicted RAP-induced MSP1 truncation in the 3D7MSP1flox42C clones, showing loss of the GPI anchor and C-terminal domain containing the mAb X509 epitope.(B) MSP1 truncation confirmed by western blot of 3D7MSP1flox42C1 clone E3 schizonts, 44 hr following treatment ± RAP. The PV protein SERA5 was used as a loading control.(C) RAP treatment produces a loss of mAb X509 reactivity and a shift in the IFA pattern of MSP1 to one typical of PV proteins, consistent with the predicted truncation. Numbers of DAPI-stained nuclei did not differ between control and RAP-treated schizonts (mean values: 21.2 ± 3.4 and 20.6 ± 4.0 nuclei per schizont, respectively, n = 24).(D) IFA showing co-localization of truncated MSP1 with SERA5 indicating a PV location. The punctate localization of SUB1 and the microneme protein AMA1 indicates normal organelle biogenesis.(E) IFA showing lack of surface-bound MSP1 on merozoites of RAP-treated 3D7MSP1flox42C1 clone E3. Antibodies to AMA1 (which is expressed on free merozoites) were used as a control.(F) Stills from time-lapse DIC microscopic imaging of egress in control and RAP-treated 3D7MSP1flox42C1 clone E3. Scale bar, 10 μm.(G) Replication rates of RAP- or control-treated 3D7MSP1flox42C1 clone E3. Cultures were passaged at intervals by 10-fold dilution into fresh medium plus erythrocytes as described in Supplemental Experimental Procedures. Observed parasitaemia values were adjusted for these dilutions and are displayed as adjusted values. The plot shows mean values of three biological replicate experiments. Error bars, SEM. The RAP-treated cultures showed an ∼2.1-fold reduction in replication rate per cycle, but this was an over-estimate of mutant viability due to rapid expansion of the few (∼1%) non-excised parasites in the RAP-treated cultures. See also Figure S6 and Movies S3 and S4.
Mentions: MSP1 is tethered to the merozoite surface via a C-terminal GPI anchor. To further test our model that direct interactions between merozoite-bound MSP1 and the erythrocyte cytoskeleton facilitates egress, we used a recently published conditional strategy to generate P. falciparum transgenics in which a 3′ segment of the msp1 gene could be deleted by rapamycin (RAP)-inducible, Cre recombinase-mediated excision (Figure 6A; Figure S6A) (Collins et al., 2013a). This was predicted to generate a truncated MSP1 that lacked a GPI anchor and so would not be bound to the merozoite surface. Analysis of RAP-treated 3D7MSP1flox42C parasites showed highly efficient excision, resulting in exclusive expression of truncated MSP1 in mature schizonts at the end of the same erythrocytic cycle (Figures 6B and 6C). No effects on merozoite development were discernible. The modified MSP1 was trafficked to the PV as expected for a non-membrane-bound merozoite surface protein (Figures 6C–6D; Figure S6B) but was not present on the surface of free merozoites (Figure 6E). Video microscopy of the RAP-treated 3D7MSP1flox42C schizonts revealed a dramatic egress defect characterized by abortive erythrocyte membrane rupture and trapping of the merozoites in the partially ruptured cell (Figure 6F; Movies S3 and S4). Consistent with this, the RAP-treated mutants displayed a substantially reduced replication rate (Figure 6G and Figure S6C). These results show that MSP1 functions at egress and that this role requires it to be tethered to the merozoite surface. Collectively, our findings support the model that processing of MSP1 facilitates host cell membrane rupture, probably through interactions between the mature merozoite surface and the erythrocyte cytoskeleton.

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