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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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Sequence alignment of the cytoplasmic domain of microneme proteins and the EBA-175 mutations used in this paper. Also shown is the EBA-175 allelic replacement and Southern blot analysis. (A) Comparison of the microneme proteins EBA-175, EBA-181 (JESEBL), EBA-140 (BAEBL), EBL1, AMA1, TRAP, EBA165 (PAEBL), and MAEBL. The COOH-terminal amino acids of the highly conserved transmembrane domain are in black italics, tyrosine motifs and tyrosine residues are indicated by red letters, acidic amino acids are highlighted in blue. (B) Sequences of the constructs used in this paper to modify the cytoplasmic domain of EBA-175. COOH-terminal amino acids of the transmembrane domain are in black italics, and amino acid substitutions are in red. In gray is the sequence of TRAP fused to EBA-175 backbone. (C) Schematic representation of the 3′ replacement of the EBA-175 gene by single crossover recombination of pHH1 constructs in the EBA-175 locus. The positive selection cassette (hDHFR) of the pHH1 vector is represented by the black box. An ∼1.1-kb fragment of the COOH terminus with the introduced mutation (indicated by asterisks) was cloned in the pHH1 vector including the 3′ cysteine rich region (purple), the transmembrane domain (black), and the mutated cytoplasmic domain (green). This fragment is flanked by the 3′ UTR of the P. berghei dihydrofolate reductase gene (gray) in the pHH1 vector. Crosses refer to the regions where recombination events were expected. The intron/exon structure of the endogenous EBA-175 gene is shown. The red boxes indicate the adhesive F1/F2 ectodomains of the endogenous EBA-175. The MfeI (M) restriction sites are marked and the position of the EBA-175 probe used for Southern analysis is indicated. (D) Southern blot analysis of genomic DNA (MfeI restricted) of W2mef and transgenic EBA-175 mutant parasites reveals that the plasmid has integrated into the EBA-175 gene. Variable numbers of plasmid copies have integrated into each transgenic parasite. The position of the probe used in the Southern hybridization leads to large fragments of 8.1 and 11 kb that differ in intensity depending on the number of plasmids integrated. The 2.4-kb band is indicative of the integration of the plasmid via single recombination into the 3′ end of the EBA-175 gene. Importantly, the endogenous EBA-175 hybridizing band is 10 kb, and is different in the parasite lines where the 3′ end of EBA-175 has been replaced. Sizes of the hybridizing bands are shown in kb.
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fig1: Sequence alignment of the cytoplasmic domain of microneme proteins and the EBA-175 mutations used in this paper. Also shown is the EBA-175 allelic replacement and Southern blot analysis. (A) Comparison of the microneme proteins EBA-175, EBA-181 (JESEBL), EBA-140 (BAEBL), EBL1, AMA1, TRAP, EBA165 (PAEBL), and MAEBL. The COOH-terminal amino acids of the highly conserved transmembrane domain are in black italics, tyrosine motifs and tyrosine residues are indicated by red letters, acidic amino acids are highlighted in blue. (B) Sequences of the constructs used in this paper to modify the cytoplasmic domain of EBA-175. COOH-terminal amino acids of the transmembrane domain are in black italics, and amino acid substitutions are in red. In gray is the sequence of TRAP fused to EBA-175 backbone. (C) Schematic representation of the 3′ replacement of the EBA-175 gene by single crossover recombination of pHH1 constructs in the EBA-175 locus. The positive selection cassette (hDHFR) of the pHH1 vector is represented by the black box. An ∼1.1-kb fragment of the COOH terminus with the introduced mutation (indicated by asterisks) was cloned in the pHH1 vector including the 3′ cysteine rich region (purple), the transmembrane domain (black), and the mutated cytoplasmic domain (green). This fragment is flanked by the 3′ UTR of the P. berghei dihydrofolate reductase gene (gray) in the pHH1 vector. Crosses refer to the regions where recombination events were expected. The intron/exon structure of the endogenous EBA-175 gene is shown. The red boxes indicate the adhesive F1/F2 ectodomains of the endogenous EBA-175. The MfeI (M) restriction sites are marked and the position of the EBA-175 probe used for Southern analysis is indicated. (D) Southern blot analysis of genomic DNA (MfeI restricted) of W2mef and transgenic EBA-175 mutant parasites reveals that the plasmid has integrated into the EBA-175 gene. Variable numbers of plasmid copies have integrated into each transgenic parasite. The position of the probe used in the Southern hybridization leads to large fragments of 8.1 and 11 kb that differ in intensity depending on the number of plasmids integrated. The 2.4-kb band is indicative of the integration of the plasmid via single recombination into the 3′ end of the EBA-175 gene. Importantly, the endogenous EBA-175 hybridizing band is 10 kb, and is different in the parasite lines where the 3′ end of EBA-175 has been replaced. Sizes of the hybridizing bands are shown in kb.

Mentions: Micronemal proteins identified so far in P. falciparum and other apicomplexa have an NH2-terminal signal peptide and a single transmembrane domain with a short cytoplasmic tail of ∼50 amino acids at the COOH terminus (Di Cristina et al., 2000; Adams et al., 2001). The cytoplasmic domains do not display any overall homology, but have common features in that they are rich in acidic amino acids (15–24%) and have tyrosine-based motifs that may function in trafficking of these proteins to the micronemes of the apical complex (Fig. 1 A). The TRAP protein is localized within the micronemes of the mosquito sporozoite stage (Rogers et al., 1992a), whereas EBA-175 is expressed in the blood-stage merozoites and is also localized in micronemes (Sim et al., 1992). Both of these proteins function in invasion; however, the target cell of the sporozoite is liver cells, whereas merozoites invade erythrocytes.


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)

Sequence alignment of the cytoplasmic domain of microneme proteins and the EBA-175 mutations used in this paper. Also shown is the EBA-175 allelic replacement and Southern blot analysis. (A) Comparison of the microneme proteins EBA-175, EBA-181 (JESEBL), EBA-140 (BAEBL), EBL1, AMA1, TRAP, EBA165 (PAEBL), and MAEBL. The COOH-terminal amino acids of the highly conserved transmembrane domain are in black italics, tyrosine motifs and tyrosine residues are indicated by red letters, acidic amino acids are highlighted in blue. (B) Sequences of the constructs used in this paper to modify the cytoplasmic domain of EBA-175. COOH-terminal amino acids of the transmembrane domain are in black italics, and amino acid substitutions are in red. In gray is the sequence of TRAP fused to EBA-175 backbone. (C) Schematic representation of the 3′ replacement of the EBA-175 gene by single crossover recombination of pHH1 constructs in the EBA-175 locus. The positive selection cassette (hDHFR) of the pHH1 vector is represented by the black box. An ∼1.1-kb fragment of the COOH terminus with the introduced mutation (indicated by asterisks) was cloned in the pHH1 vector including the 3′ cysteine rich region (purple), the transmembrane domain (black), and the mutated cytoplasmic domain (green). This fragment is flanked by the 3′ UTR of the P. berghei dihydrofolate reductase gene (gray) in the pHH1 vector. Crosses refer to the regions where recombination events were expected. The intron/exon structure of the endogenous EBA-175 gene is shown. The red boxes indicate the adhesive F1/F2 ectodomains of the endogenous EBA-175. The MfeI (M) restriction sites are marked and the position of the EBA-175 probe used for Southern analysis is indicated. (D) Southern blot analysis of genomic DNA (MfeI restricted) of W2mef and transgenic EBA-175 mutant parasites reveals that the plasmid has integrated into the EBA-175 gene. Variable numbers of plasmid copies have integrated into each transgenic parasite. The position of the probe used in the Southern hybridization leads to large fragments of 8.1 and 11 kb that differ in intensity depending on the number of plasmids integrated. The 2.4-kb band is indicative of the integration of the plasmid via single recombination into the 3′ end of the EBA-175 gene. Importantly, the endogenous EBA-175 hybridizing band is 10 kb, and is different in the parasite lines where the 3′ end of EBA-175 has been replaced. Sizes of the hybridizing bands are shown in kb.
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Related In: Results  -  Collection

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

fig1: Sequence alignment of the cytoplasmic domain of microneme proteins and the EBA-175 mutations used in this paper. Also shown is the EBA-175 allelic replacement and Southern blot analysis. (A) Comparison of the microneme proteins EBA-175, EBA-181 (JESEBL), EBA-140 (BAEBL), EBL1, AMA1, TRAP, EBA165 (PAEBL), and MAEBL. The COOH-terminal amino acids of the highly conserved transmembrane domain are in black italics, tyrosine motifs and tyrosine residues are indicated by red letters, acidic amino acids are highlighted in blue. (B) Sequences of the constructs used in this paper to modify the cytoplasmic domain of EBA-175. COOH-terminal amino acids of the transmembrane domain are in black italics, and amino acid substitutions are in red. In gray is the sequence of TRAP fused to EBA-175 backbone. (C) Schematic representation of the 3′ replacement of the EBA-175 gene by single crossover recombination of pHH1 constructs in the EBA-175 locus. The positive selection cassette (hDHFR) of the pHH1 vector is represented by the black box. An ∼1.1-kb fragment of the COOH terminus with the introduced mutation (indicated by asterisks) was cloned in the pHH1 vector including the 3′ cysteine rich region (purple), the transmembrane domain (black), and the mutated cytoplasmic domain (green). This fragment is flanked by the 3′ UTR of the P. berghei dihydrofolate reductase gene (gray) in the pHH1 vector. Crosses refer to the regions where recombination events were expected. The intron/exon structure of the endogenous EBA-175 gene is shown. The red boxes indicate the adhesive F1/F2 ectodomains of the endogenous EBA-175. The MfeI (M) restriction sites are marked and the position of the EBA-175 probe used for Southern analysis is indicated. (D) Southern blot analysis of genomic DNA (MfeI restricted) of W2mef and transgenic EBA-175 mutant parasites reveals that the plasmid has integrated into the EBA-175 gene. Variable numbers of plasmid copies have integrated into each transgenic parasite. The position of the probe used in the Southern hybridization leads to large fragments of 8.1 and 11 kb that differ in intensity depending on the number of plasmids integrated. The 2.4-kb band is indicative of the integration of the plasmid via single recombination into the 3′ end of the EBA-175 gene. Importantly, the endogenous EBA-175 hybridizing band is 10 kb, and is different in the parasite lines where the 3′ end of EBA-175 has been replaced. Sizes of the hybridizing bands are shown in kb.
Mentions: Micronemal proteins identified so far in P. falciparum and other apicomplexa have an NH2-terminal signal peptide and a single transmembrane domain with a short cytoplasmic tail of ∼50 amino acids at the COOH terminus (Di Cristina et al., 2000; Adams et al., 2001). The cytoplasmic domains do not display any overall homology, but have common features in that they are rich in acidic amino acids (15–24%) and have tyrosine-based motifs that may function in trafficking of these proteins to the micronemes of the apical complex (Fig. 1 A). The TRAP protein is localized within the micronemes of the mosquito sporozoite stage (Rogers et al., 1992a), whereas EBA-175 is expressed in the blood-stage merozoites and is also localized in micronemes (Sim et al., 1992). Both of these proteins function in invasion; however, the target cell of the sporozoite is liver cells, whereas merozoites invade erythrocytes.

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