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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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TgRON2 fusions GST-D3 and GST-D4 independently and specifically interact with the shed ectodomain of TgAMA1.Extracellular parasites were incubated under conditions to induce shedding of secreted TgAMA1 into culture supernatants. Immunoblotting analysis of pelleted parasites (lane 1), cleared culture supernatants (lane 2), or material from supernatants co-precipitated with molar equivalents of GST (lane 3), GST-D3 (lane 4) or GST-D4 (lane 5) was conducted using the monoclonal antibodies B3.90 (A), specific for the TgAMA1 N-terminus, or CL22 (B), specific for the TgAMA1 C-terminus. The former detects the intact, integral membrane form (∼65 kDa) and the shed ectodomain (∼53 kDa) while CL22 detects only the intact form. TgSAG1 serves as a negative control. Parentheses indicate loaded parasite equivalents. Size markers are indicated in kDa.
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ppat-1001282-g004: TgRON2 fusions GST-D3 and GST-D4 independently and specifically interact with the shed ectodomain of TgAMA1.Extracellular parasites were incubated under conditions to induce shedding of secreted TgAMA1 into culture supernatants. Immunoblotting analysis of pelleted parasites (lane 1), cleared culture supernatants (lane 2), or material from supernatants co-precipitated with molar equivalents of GST (lane 3), GST-D3 (lane 4) or GST-D4 (lane 5) was conducted using the monoclonal antibodies B3.90 (A), specific for the TgAMA1 N-terminus, or CL22 (B), specific for the TgAMA1 C-terminus. The former detects the intact, integral membrane form (∼65 kDa) and the shed ectodomain (∼53 kDa) while CL22 detects only the intact form. TgSAG1 serves as a negative control. Parentheses indicate loaded parasite equivalents. Size markers are indicated in kDa.

Mentions: Current models of the assembly of the MJ complex predict that TgRON2 is associated with the host cell where it acts as a receptor for the ectodomain of TgAMA1 [12], [13]. To test this hypothesis and determine whether D3 and/or D4 are the domains of TgRON2 that are functioning in this association we used GST-D3 and GST-D4 in GST pull-down experiments with parasite culture supernatants containing the shed, N-terminal ectodomain of TgAMA1 [15], [26]. To discriminate between the shed and the intact, full-length forms of TgAMA1, immunoblotting was performed using monoclonal antibodies specific for either the C-terminal intracellular domain of TgAMA1 (CL22; [15]) or the N-terminal extracellular domain (B3.90; [26]). The results showed that both GST-D3 and GST-D4 but not GST alone efficiently co-precipitate the more rapidly migrating (shed) form of TgAMA1 (Figure 4A). The identity of this as the shed form was confirmed by its failure to react to CL22 (Figure 4B). A trace amount of contaminating, intact, full-length TgAMA1 in the supernatant material was also co-precipitated, as expected, since this also includes the entire ectodomain (Figure 4 A and B, lanes 4 and 5). The specificity of these co-purification studies was confirmed by a complete lack of enrichment for the abundant surface antigen TgSAG1 (Figure 4). These results demonstrate that both GST-D3 and GST-D4 independently and specifically interact with the ectodomain of TgAMA1.


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)

TgRON2 fusions GST-D3 and GST-D4 independently and specifically interact with the shed ectodomain of TgAMA1.Extracellular parasites were incubated under conditions to induce shedding of secreted TgAMA1 into culture supernatants. Immunoblotting analysis of pelleted parasites (lane 1), cleared culture supernatants (lane 2), or material from supernatants co-precipitated with molar equivalents of GST (lane 3), GST-D3 (lane 4) or GST-D4 (lane 5) was conducted using the monoclonal antibodies B3.90 (A), specific for the TgAMA1 N-terminus, or CL22 (B), specific for the TgAMA1 C-terminus. The former detects the intact, integral membrane form (∼65 kDa) and the shed ectodomain (∼53 kDa) while CL22 detects only the intact form. TgSAG1 serves as a negative control. Parentheses indicate loaded parasite equivalents. Size markers are indicated in kDa.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037364&req=5

ppat-1001282-g004: TgRON2 fusions GST-D3 and GST-D4 independently and specifically interact with the shed ectodomain of TgAMA1.Extracellular parasites were incubated under conditions to induce shedding of secreted TgAMA1 into culture supernatants. Immunoblotting analysis of pelleted parasites (lane 1), cleared culture supernatants (lane 2), or material from supernatants co-precipitated with molar equivalents of GST (lane 3), GST-D3 (lane 4) or GST-D4 (lane 5) was conducted using the monoclonal antibodies B3.90 (A), specific for the TgAMA1 N-terminus, or CL22 (B), specific for the TgAMA1 C-terminus. The former detects the intact, integral membrane form (∼65 kDa) and the shed ectodomain (∼53 kDa) while CL22 detects only the intact form. TgSAG1 serves as a negative control. Parentheses indicate loaded parasite equivalents. Size markers are indicated in kDa.
Mentions: Current models of the assembly of the MJ complex predict that TgRON2 is associated with the host cell where it acts as a receptor for the ectodomain of TgAMA1 [12], [13]. To test this hypothesis and determine whether D3 and/or D4 are the domains of TgRON2 that are functioning in this association we used GST-D3 and GST-D4 in GST pull-down experiments with parasite culture supernatants containing the shed, N-terminal ectodomain of TgAMA1 [15], [26]. To discriminate between the shed and the intact, full-length forms of TgAMA1, immunoblotting was performed using monoclonal antibodies specific for either the C-terminal intracellular domain of TgAMA1 (CL22; [15]) or the N-terminal extracellular domain (B3.90; [26]). The results showed that both GST-D3 and GST-D4 but not GST alone efficiently co-precipitate the more rapidly migrating (shed) form of TgAMA1 (Figure 4A). The identity of this as the shed form was confirmed by its failure to react to CL22 (Figure 4B). A trace amount of contaminating, intact, full-length TgAMA1 in the supernatant material was also co-precipitated, as expected, since this also includes the entire ectodomain (Figure 4 A and B, lanes 4 and 5). The specificity of these co-purification studies was confirmed by a complete lack of enrichment for the abundant surface antigen TgSAG1 (Figure 4). These results demonstrate that both GST-D3 and GST-D4 independently and specifically interact with the ectodomain of TgAMA1.

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