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The cytoplasmic domain of the Plasmodium falciparum ligand EBA-175 is essential for invasion but not protein trafficking.

Gilberger TW, Thompson JK, Reed MB, Good RT, Cowman AF - J. Cell Biol. (2003)

Bottom Line: The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein-protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process.In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain.These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Melbourne 3050, Australia.

ABSTRACT
The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein-protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process. The transmembrane erythrocyte binding protein-175 (EBA-175) and thrombospondin-related anonymous protein (TRAP) play central roles in this process. EBA-175 binds to glycophorin A on human erythrocytes during the invasion process, linking the parasite to the surface of the host cell. In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain. Further, we show that the cytoplasmic domain of TRAP, a protein that is not expressed in merozoites but is essential for invasion of liver cells by the sporozoite stage, can substitute for the cytoplasmic domain of EBA-175. These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.

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Immunolocalization of EBA-175 mutant proteins in transgenic parasites. The names on the left refer to the W2mef parasite lines expressing mutant EBA-175 proteins. The structure of EBA-175 and mutant proteins are schematically shown. Free merozoites were incubated with anti-EBA175 and anti-EBA181, followed by FITC-labeled anti–mouse and rhodamine-labeled anti–rabbit antibodies. To precisely visualize the localization of mutant EBA-175 with respect to the microneme protein EBA-181, the two fluorescence photomicrographs were merged. The function of the mutant EBA-175 was measured in each parasite line as shown in Fig. 4 A. + refers to absence of a switch in invasion demonstrating EBA-175 function is retained, whereas − signifies a switch in invasion phenotype and loss of function for EBA-175.
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fig3: Immunolocalization of EBA-175 mutant proteins in transgenic parasites. The names on the left refer to the W2mef parasite lines expressing mutant EBA-175 proteins. The structure of EBA-175 and mutant proteins are schematically shown. Free merozoites were incubated with anti-EBA175 and anti-EBA181, followed by FITC-labeled anti–mouse and rhodamine-labeled anti–rabbit antibodies. To precisely visualize the localization of mutant EBA-175 with respect to the microneme protein EBA-181, the two fluorescence photomicrographs were merged. The function of the mutant EBA-175 was measured in each parasite line as shown in Fig. 4 A. + refers to absence of a switch in invasion demonstrating EBA-175 function is retained, whereas − signifies a switch in invasion phenotype and loss of function for EBA-175.

Mentions: Previously, it has been shown that the cytoplasmic tail of type 1 transmembrane proteins in Apicomplexa can contain sorting signals that are essential for correct subcellular localization (for review see Joiner and Roos, 2002). To determine if the mutant EBA-175 proteins were correctly localized to the micronemes, we used immunofluorescence to test colocalization with other microneme proteins including EBA-181 (Gilberger et al., 2003) and EBA-140. Using the parasite line W2mefΔ230, in which EBA-175 is expressed without the 3′ cysteine-rich region, transmembrane, and cytoplasmic domain, we showed that this truncation leads to incorrect localization of truncated EBA-175 (Fig. 3). Although still detectable in schizonts (Reed et al., 2000a), it does not colocalize with the microneme marker EBA-181 and importantly is not detectable in merozoites, as it appears to be released into the supernatant on schizont rupture (Fig. 3). This was confirmed by colocalization with a second microneme marker EBA-140 (unpublished data). Previously, it has been suggested that the same truncated EBA-175 may be localized to micronemes in schizont stages; however, no additional microneme markers were available at that time to confirm this result, and immuno-localization in free merozoites was not performed (Kaneko et al., 2000; Reed et al., 2000a).


The cytoplasmic domain of the Plasmodium falciparum ligand EBA-175 is essential for invasion but not protein trafficking.

Gilberger TW, Thompson JK, Reed MB, Good RT, Cowman AF - J. Cell Biol. (2003)

Immunolocalization of EBA-175 mutant proteins in transgenic parasites. The names on the left refer to the W2mef parasite lines expressing mutant EBA-175 proteins. The structure of EBA-175 and mutant proteins are schematically shown. Free merozoites were incubated with anti-EBA175 and anti-EBA181, followed by FITC-labeled anti–mouse and rhodamine-labeled anti–rabbit antibodies. To precisely visualize the localization of mutant EBA-175 with respect to the microneme protein EBA-181, the two fluorescence photomicrographs were merged. The function of the mutant EBA-175 was measured in each parasite line as shown in Fig. 4 A. + refers to absence of a switch in invasion demonstrating EBA-175 function is retained, whereas − signifies a switch in invasion phenotype and loss of function for EBA-175.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172798&req=5

fig3: Immunolocalization of EBA-175 mutant proteins in transgenic parasites. The names on the left refer to the W2mef parasite lines expressing mutant EBA-175 proteins. The structure of EBA-175 and mutant proteins are schematically shown. Free merozoites were incubated with anti-EBA175 and anti-EBA181, followed by FITC-labeled anti–mouse and rhodamine-labeled anti–rabbit antibodies. To precisely visualize the localization of mutant EBA-175 with respect to the microneme protein EBA-181, the two fluorescence photomicrographs were merged. The function of the mutant EBA-175 was measured in each parasite line as shown in Fig. 4 A. + refers to absence of a switch in invasion demonstrating EBA-175 function is retained, whereas − signifies a switch in invasion phenotype and loss of function for EBA-175.
Mentions: Previously, it has been shown that the cytoplasmic tail of type 1 transmembrane proteins in Apicomplexa can contain sorting signals that are essential for correct subcellular localization (for review see Joiner and Roos, 2002). To determine if the mutant EBA-175 proteins were correctly localized to the micronemes, we used immunofluorescence to test colocalization with other microneme proteins including EBA-181 (Gilberger et al., 2003) and EBA-140. Using the parasite line W2mefΔ230, in which EBA-175 is expressed without the 3′ cysteine-rich region, transmembrane, and cytoplasmic domain, we showed that this truncation leads to incorrect localization of truncated EBA-175 (Fig. 3). Although still detectable in schizonts (Reed et al., 2000a), it does not colocalize with the microneme marker EBA-181 and importantly is not detectable in merozoites, as it appears to be released into the supernatant on schizont rupture (Fig. 3). This was confirmed by colocalization with a second microneme marker EBA-140 (unpublished data). Previously, it has been suggested that the same truncated EBA-175 may be localized to micronemes in schizont stages; however, no additional microneme markers were available at that time to confirm this result, and immuno-localization in free merozoites was not performed (Kaneko et al., 2000; Reed et al., 2000a).

Bottom Line: The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein-protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process.In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain.These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Melbourne 3050, Australia.

ABSTRACT
The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein-protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process. The transmembrane erythrocyte binding protein-175 (EBA-175) and thrombospondin-related anonymous protein (TRAP) play central roles in this process. EBA-175 binds to glycophorin A on human erythrocytes during the invasion process, linking the parasite to the surface of the host cell. In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain. Further, we show that the cytoplasmic domain of TRAP, a protein that is not expressed in merozoites but is essential for invasion of liver cells by the sporozoite stage, can substitute for the cytoplasmic domain of EBA-175. These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.

Show MeSH
Related in: MedlinePlus