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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.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.

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.
© Copyright Policy
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.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.

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