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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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Pre-incubation of RH and RHΔama1/AMA1-myc parasites with GST-D3 decreases invasion efficiency.(A, B) Extracellular RH (A) or RHΔama1/AMA1-myc (B) parasites were pre-treated with a buffer control or indicated molar equivalents of GST alone, GST-D3, or GST-D3scramble (B only) and then permitted to infect HFF monolayers for 15 minutes using temperature-based synchronized invasion conditions. The number of intracellular parasites was determined by differential staining of the extracellular vs. total parasites before and after detergent permeabilization. The number of intracellular parasites was determined for 15 (A) or 20 (B) randomly-selected fields from three coverslips for each condition tested. The invasion levels for each condition are shown relative to the buffer-treated control (shown are means with standard deviation). An asterisk indicates a statistically significant reduction in invasion relative to the GST controls (unpaired Student's t-test), with p<0.0099 (A) or p<0.0002 (B). (C) To determine if GST-D3 treatment affects attachment, RHΔama1/AMA1-myc parasites were pre-treated with molar equivalents of GST alone, GST-D3, or a buffer control as described above and then permitted to attach to formaldehyde-fixed HFF monolayers. The number of stained, attached parasites was counted in 15 randomly-selected fields from three coverslips for each condition tested. The attachment levels for each condition are shown relative to the buffer-treated control (shown are means with standard deviation).
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ppat-1001282-g006: Pre-incubation of RH and RHΔama1/AMA1-myc parasites with GST-D3 decreases invasion efficiency.(A, B) Extracellular RH (A) or RHΔama1/AMA1-myc (B) parasites were pre-treated with a buffer control or indicated molar equivalents of GST alone, GST-D3, or GST-D3scramble (B only) and then permitted to infect HFF monolayers for 15 minutes using temperature-based synchronized invasion conditions. The number of intracellular parasites was determined by differential staining of the extracellular vs. total parasites before and after detergent permeabilization. The number of intracellular parasites was determined for 15 (A) or 20 (B) randomly-selected fields from three coverslips for each condition tested. The invasion levels for each condition are shown relative to the buffer-treated control (shown are means with standard deviation). An asterisk indicates a statistically significant reduction in invasion relative to the GST controls (unpaired Student's t-test), with p<0.0099 (A) or p<0.0002 (B). (C) To determine if GST-D3 treatment affects attachment, RHΔama1/AMA1-myc parasites were pre-treated with molar equivalents of GST alone, GST-D3, or a buffer control as described above and then permitted to attach to formaldehyde-fixed HFF monolayers. The number of stained, attached parasites was counted in 15 randomly-selected fields from three coverslips for each condition tested. The attachment levels for each condition are shown relative to the buffer-treated control (shown are means with standard deviation).

Mentions: Given that GST-D3 associates with the ectodomain of TgAMA1 on the surface of intact parasites, we predicted that pre-incubation of extracellular parasites with GST-D3 would result in an invasion-inhibitory phenotype. To test this hypothesis, equivalent numbers of freshly prepared, extracellular RH parasites were pre-incubated in medium supplemented with molar equivalents of GST-D3, GST alone, or a buffer control and then allowed to invade host cells using a temperature-shift assay to synchronize the process. Following ∼15 minutes at an invasion-permissive temperature, infected monolayers were fixed and analyzed by IFA. Extracellular vs. intracellular parasites were identified by sequential staining for TgSAG1, before and after detergent-permeabilization of the host cells. While treatment of RH parasites with GST alone did not significantly alter the number of intracellular parasites compared to the buffer-treated control parasites, treatment with GST-D3 resulted in a dose-dependent decrease of up to ∼55% (Figure 6A and Supplemental Figure S1A). This level of inhibition is similar to that previously reported with anti-TgAMA1 antibodies [15] and is consistent with disruption of TgAMA1 function through binding of GST-D3.


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)

Pre-incubation of RH and RHΔama1/AMA1-myc parasites with GST-D3 decreases invasion efficiency.(A, B) Extracellular RH (A) or RHΔama1/AMA1-myc (B) parasites were pre-treated with a buffer control or indicated molar equivalents of GST alone, GST-D3, or GST-D3scramble (B only) and then permitted to infect HFF monolayers for 15 minutes using temperature-based synchronized invasion conditions. The number of intracellular parasites was determined by differential staining of the extracellular vs. total parasites before and after detergent permeabilization. The number of intracellular parasites was determined for 15 (A) or 20 (B) randomly-selected fields from three coverslips for each condition tested. The invasion levels for each condition are shown relative to the buffer-treated control (shown are means with standard deviation). An asterisk indicates a statistically significant reduction in invasion relative to the GST controls (unpaired Student's t-test), with p<0.0099 (A) or p<0.0002 (B). (C) To determine if GST-D3 treatment affects attachment, RHΔama1/AMA1-myc parasites were pre-treated with molar equivalents of GST alone, GST-D3, or a buffer control as described above and then permitted to attach to formaldehyde-fixed HFF monolayers. The number of stained, attached parasites was counted in 15 randomly-selected fields from three coverslips for each condition tested. The attachment levels for each condition are shown relative to the buffer-treated control (shown are means with standard deviation).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037364&req=5

ppat-1001282-g006: Pre-incubation of RH and RHΔama1/AMA1-myc parasites with GST-D3 decreases invasion efficiency.(A, B) Extracellular RH (A) or RHΔama1/AMA1-myc (B) parasites were pre-treated with a buffer control or indicated molar equivalents of GST alone, GST-D3, or GST-D3scramble (B only) and then permitted to infect HFF monolayers for 15 minutes using temperature-based synchronized invasion conditions. The number of intracellular parasites was determined by differential staining of the extracellular vs. total parasites before and after detergent permeabilization. The number of intracellular parasites was determined for 15 (A) or 20 (B) randomly-selected fields from three coverslips for each condition tested. The invasion levels for each condition are shown relative to the buffer-treated control (shown are means with standard deviation). An asterisk indicates a statistically significant reduction in invasion relative to the GST controls (unpaired Student's t-test), with p<0.0099 (A) or p<0.0002 (B). (C) To determine if GST-D3 treatment affects attachment, RHΔama1/AMA1-myc parasites were pre-treated with molar equivalents of GST alone, GST-D3, or a buffer control as described above and then permitted to attach to formaldehyde-fixed HFF monolayers. The number of stained, attached parasites was counted in 15 randomly-selected fields from three coverslips for each condition tested. The attachment levels for each condition are shown relative to the buffer-treated control (shown are means with standard deviation).
Mentions: Given that GST-D3 associates with the ectodomain of TgAMA1 on the surface of intact parasites, we predicted that pre-incubation of extracellular parasites with GST-D3 would result in an invasion-inhibitory phenotype. To test this hypothesis, equivalent numbers of freshly prepared, extracellular RH parasites were pre-incubated in medium supplemented with molar equivalents of GST-D3, GST alone, or a buffer control and then allowed to invade host cells using a temperature-shift assay to synchronize the process. Following ∼15 minutes at an invasion-permissive temperature, infected monolayers were fixed and analyzed by IFA. Extracellular vs. intracellular parasites were identified by sequential staining for TgSAG1, before and after detergent-permeabilization of the host cells. While treatment of RH parasites with GST alone did not significantly alter the number of intracellular parasites compared to the buffer-treated control parasites, treatment with GST-D3 resulted in a dose-dependent decrease of up to ∼55% (Figure 6A and Supplemental Figure S1A). This level of inhibition is similar to that previously reported with anti-TgAMA1 antibodies [15] and is consistent with disruption of TgAMA1 function through binding of GST-D3.

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