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The C-terminus of Toxoplasma RON2 provides the crucial link between AMA1 and the host-associated invasion complex.

Tyler JS, Boothroyd JC - PLoS Pathog. (2011)

Bottom Line: The Toxoplasma MJ is a secreted complex including TgAMA1, a transmembrane protein on the parasite surface, and a complex of rhoptry neck proteins (TgRON2/4/5/8) described as host cell-associated.How these proteins connect the parasite and host cell has not previously been described.Pre-incubation of parasites with D3 (fused to glutathione S-transferase) dramatically reduces invasion but does not prevent injection of rhoptry bulb proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
Host cell invasion by apicomplexan parasites requires formation of the moving junction (MJ), a ring-like apposition between the parasite and host plasma membranes that the parasite migrates through during entry. The Toxoplasma MJ is a secreted complex including TgAMA1, a transmembrane protein on the parasite surface, and a complex of rhoptry neck proteins (TgRON2/4/5/8) described as host cell-associated. How these proteins connect the parasite and host cell has not previously been described. Here we show that TgRON2 localizes to the MJ and that two short segments flanking a hydrophobic stretch near its C-terminus (D3 and D4) independently associate with the ectodomain of TgAMA1. Pre-incubation of parasites with D3 (fused to glutathione S-transferase) dramatically reduces invasion but does not prevent injection of rhoptry bulb proteins. Hence, the entire C-terminal region of TgRON2 forms the crucial bridge between TgAMA1 and the rest of the MJ complex but this association is not required for rhoptry protein injection.

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Alignment of TgRON2 with its orthologues reveals greatest sequence conservation in carboxy-terminal third of protein.The Toxoplasma RON2 polypeptide sequence was aligned with its orthologues in Neospora caninum and Plasmodium spp. (P. falciparum strain 3D7, P. berghei, P. knowlesi, and P. vivax) using ClustalX. The percent identity or percent similarity for 50 amino acid windows was calculated in 5 amino acid steps over the length of the alignment. The length of the alignment corresponds to the longest orthologue. Shown below is the relative region of TgRON2 that corresponds to the most conserved region of RON2 across the species. The regions between AA1293–1346 and AA1366–1479 were designated domain 3 (D3) and domain 4 (D4), respectively.
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ppat-1001282-g002: Alignment of TgRON2 with its orthologues reveals greatest sequence conservation in carboxy-terminal third of protein.The Toxoplasma RON2 polypeptide sequence was aligned with its orthologues in Neospora caninum and Plasmodium spp. (P. falciparum strain 3D7, P. berghei, P. knowlesi, and P. vivax) using ClustalX. The percent identity or percent similarity for 50 amino acid windows was calculated in 5 amino acid steps over the length of the alignment. The length of the alignment corresponds to the longest orthologue. Shown below is the relative region of TgRON2 that corresponds to the most conserved region of RON2 across the species. The regions between AA1293–1346 and AA1366–1479 were designated domain 3 (D3) and domain 4 (D4), respectively.

Mentions: Like most of the other identified MJ components, TgRON2 orthologues are present within all Apicomplexa that show MJ formation during invasion, suggesting a conserved function for this protein. To identify regions of TgRON2 that are crucial for interacting with the other members of the MJ complex, therefore, we generated a multiple sequence alignment of TgRON2 and its orthologues in Neospora caninum and several Plasmodium species. We observed that the greatest sequence conservation among these orthologues is in the C-terminal-most third of the protein (Figure 2). To determine if this conserved region of TgRON2 is important for interactions with the remaining members of the MJ complex we generated protein fusions with glutathione S-transferase (GST) and used these in co-affinity purification studies. We chose to use regions outside of the hydrophobic helices as we anticipated that fusion proteins including these regions would not be soluble in E. coli, a prediction that was subsequently confirmed experimentally (data not shown). GST fusions with portions of TgRON2 N-terminal to the second putative hydrophobic helix (HH2), which spans residues 1277 to 1296, were also refractory to purification under soluble conditions (TgRON2 residue numbers are from Genbank accession HQ110093 with residue 1 as the start methionine); however, fusion of GST with TgRON2 amino acids 1293 to 1346 and 1366 to 1479, generating recombinant proteins GST-domain 3 (D3) and GST-domain 4 (D4), respectively, were successfully expressed and purified from E. coli (Figure 2 and data not shown).


The C-terminus of Toxoplasma RON2 provides the crucial link between AMA1 and the host-associated invasion complex.

Tyler JS, Boothroyd JC - PLoS Pathog. (2011)

Alignment of TgRON2 with its orthologues reveals greatest sequence conservation in carboxy-terminal third of protein.The Toxoplasma RON2 polypeptide sequence was aligned with its orthologues in Neospora caninum and Plasmodium spp. (P. falciparum strain 3D7, P. berghei, P. knowlesi, and P. vivax) using ClustalX. The percent identity or percent similarity for 50 amino acid windows was calculated in 5 amino acid steps over the length of the alignment. The length of the alignment corresponds to the longest orthologue. Shown below is the relative region of TgRON2 that corresponds to the most conserved region of RON2 across the species. The regions between AA1293–1346 and AA1366–1479 were designated domain 3 (D3) and domain 4 (D4), respectively.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037364&req=5

ppat-1001282-g002: Alignment of TgRON2 with its orthologues reveals greatest sequence conservation in carboxy-terminal third of protein.The Toxoplasma RON2 polypeptide sequence was aligned with its orthologues in Neospora caninum and Plasmodium spp. (P. falciparum strain 3D7, P. berghei, P. knowlesi, and P. vivax) using ClustalX. The percent identity or percent similarity for 50 amino acid windows was calculated in 5 amino acid steps over the length of the alignment. The length of the alignment corresponds to the longest orthologue. Shown below is the relative region of TgRON2 that corresponds to the most conserved region of RON2 across the species. The regions between AA1293–1346 and AA1366–1479 were designated domain 3 (D3) and domain 4 (D4), respectively.
Mentions: Like most of the other identified MJ components, TgRON2 orthologues are present within all Apicomplexa that show MJ formation during invasion, suggesting a conserved function for this protein. To identify regions of TgRON2 that are crucial for interacting with the other members of the MJ complex, therefore, we generated a multiple sequence alignment of TgRON2 and its orthologues in Neospora caninum and several Plasmodium species. We observed that the greatest sequence conservation among these orthologues is in the C-terminal-most third of the protein (Figure 2). To determine if this conserved region of TgRON2 is important for interactions with the remaining members of the MJ complex we generated protein fusions with glutathione S-transferase (GST) and used these in co-affinity purification studies. We chose to use regions outside of the hydrophobic helices as we anticipated that fusion proteins including these regions would not be soluble in E. coli, a prediction that was subsequently confirmed experimentally (data not shown). GST fusions with portions of TgRON2 N-terminal to the second putative hydrophobic helix (HH2), which spans residues 1277 to 1296, were also refractory to purification under soluble conditions (TgRON2 residue numbers are from Genbank accession HQ110093 with residue 1 as the start methionine); however, fusion of GST with TgRON2 amino acids 1293 to 1346 and 1366 to 1479, generating recombinant proteins GST-domain 3 (D3) and GST-domain 4 (D4), respectively, were successfully expressed and purified from E. coli (Figure 2 and data not shown).

Bottom Line: The Toxoplasma MJ is a secreted complex including TgAMA1, a transmembrane protein on the parasite surface, and a complex of rhoptry neck proteins (TgRON2/4/5/8) described as host cell-associated.How these proteins connect the parasite and host cell has not previously been described.Pre-incubation of parasites with D3 (fused to glutathione S-transferase) dramatically reduces invasion but does not prevent injection of rhoptry bulb proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
Host cell invasion by apicomplexan parasites requires formation of the moving junction (MJ), a ring-like apposition between the parasite and host plasma membranes that the parasite migrates through during entry. The Toxoplasma MJ is a secreted complex including TgAMA1, a transmembrane protein on the parasite surface, and a complex of rhoptry neck proteins (TgRON2/4/5/8) described as host cell-associated. How these proteins connect the parasite and host cell has not previously been described. Here we show that TgRON2 localizes to the MJ and that two short segments flanking a hydrophobic stretch near its C-terminus (D3 and D4) independently associate with the ectodomain of TgAMA1. Pre-incubation of parasites with D3 (fused to glutathione S-transferase) dramatically reduces invasion but does not prevent injection of rhoptry bulb proteins. Hence, the entire C-terminal region of TgRON2 forms the crucial bridge between TgAMA1 and the rest of the MJ complex but this association is not required for rhoptry protein injection.

Show MeSH
Related in: MedlinePlus