Invasion by Toxoplasma gondii establishes a moving junction that selectively excludes host cell plasma membrane proteins on the basis of their membrane anchoring

Abstract

The protozoan parasite Toxoplasma gondii actively penetrates its host cell by squeezing through a moving junction that forms between the host cell plasma membrane and the parasite. During invasion, this junction selectively controls internalization of host cell plasma membrane components into the parasite-containing vacuole. Membrane lipids flowed past the junction, as shown by the presence of the glycosphingolipid G(M1) and the cationic lipid label 1. 1'-dihexadecyl-3-3'-3-3'-tetramethylindocarbocyanine (DiIC(16)). Glycosylphosphatidylinositol (GPI)-anchored surface proteins, such as Sca-1 and CD55, were also readily incorporated into the parasitophorous vacuole (PV). In contrast, host cell transmembrane proteins, including CD44, Na(+)/K(+) ATPase, and beta1-integrin, were excluded from the vacuole. To eliminate potential differences in sorting due to the extracellular domains, parasite invasion was examined in host cells transfected with recombinant forms of intercellular adhesion molecule 1 (ICAM-1, CD54) that differed in their mechanism of membrane anchoring. Wild-type ICAM-1, which contains a transmembrane domain, was excluded from the PV, whereas both GPI-anchored ICAM-1 and a mutant of ICAM-1 missing the cytoplasmic tail (ICAM-1-Cyt(-)) were readily incorporated into the PV membrane. Our results demonstrate that during host cell invasion, Toxoplasma selectively excludes host cell transmembrane proteins at the moving junction by a mechanism that depends on their anchoring in the membrane, thereby creating a nonfusigenic compartment.

Figure 1

Confocal localization of cell surface DiIC₁₆-lipids versus biotinylated proteins within Toxoplasma- versus zymosan-containing vacuoles in HF cells (0.5-μm section). (A) Biotinylated surface proteins (green channel) were efficiently excluded from PVs (top), but were found in phagosomes containing zymosan (bottom). In contrast, surface DiIC₁₆-lipids (red channel) were internalized into both Toxoplasma- and zymosan-containing vacuoles. HF cells were surface labeled with DiIC₁₆ and sulfo-biotin, exposed to zymosan or parasites for 5 min, fixed, and stained. Parasites and zymosan were visualized with rabbit anti-p30 or antizymosan, respectively, followed by Cy5-conjugated goat anti–rabbit IgG. Biotinylated proteins were detected with Oregon green conjugated to streptavidin. Arrowheads mark the position of Toxoplasma- or zymosan-containing vacuole. (B) Kinetic analysis of the presence of host cell lipids versus proteins within Toxoplasma-containing PVs versus zymosan-containing phagosomes. Values represent mean and SD from a representative experiment. (C) Fluorescence localization of DiIC₁₆-lipids in mechanically isolated PVs (top) versus liberated parasites (Tg, bottom). DiIC₁₆ was present in intact PVs, but not in liberated parasites.

Figure 1

Confocal localization of cell surface DiIC₁₆-lipids versus biotinylated proteins within Toxoplasma- versus zymosan-containing vacuoles in HF cells (0.5-μm section). (A) Biotinylated surface proteins (green channel) were efficiently excluded from PVs (top), but were found in phagosomes containing zymosan (bottom). In contrast, surface DiIC₁₆-lipids (red channel) were internalized into both Toxoplasma- and zymosan-containing vacuoles. HF cells were surface labeled with DiIC₁₆ and sulfo-biotin, exposed to zymosan or parasites for 5 min, fixed, and stained. Parasites and zymosan were visualized with rabbit anti-p30 or antizymosan, respectively, followed by Cy5-conjugated goat anti–rabbit IgG. Biotinylated proteins were detected with Oregon green conjugated to streptavidin. Arrowheads mark the position of Toxoplasma- or zymosan-containing vacuole. (B) Kinetic analysis of the presence of host cell lipids versus proteins within Toxoplasma-containing PVs versus zymosan-containing phagosomes. Values represent mean and SD from a representative experiment. (C) Fluorescence localization of DiIC₁₆-lipids in mechanically isolated PVs (top) versus liberated parasites (Tg, bottom). DiIC₁₆ was present in intact PVs, but not in liberated parasites.

Figure 1

Confocal localization of cell surface DiIC₁₆-lipids versus biotinylated proteins within Toxoplasma- versus zymosan-containing vacuoles in HF cells (0.5-μm section). (A) Biotinylated surface proteins (green channel) were efficiently excluded from PVs (top), but were found in phagosomes containing zymosan (bottom). In contrast, surface DiIC₁₆-lipids (red channel) were internalized into both Toxoplasma- and zymosan-containing vacuoles. HF cells were surface labeled with DiIC₁₆ and sulfo-biotin, exposed to zymosan or parasites for 5 min, fixed, and stained. Parasites and zymosan were visualized with rabbit anti-p30 or antizymosan, respectively, followed by Cy5-conjugated goat anti–rabbit IgG. Biotinylated proteins were detected with Oregon green conjugated to streptavidin. Arrowheads mark the position of Toxoplasma- or zymosan-containing vacuole. (B) Kinetic analysis of the presence of host cell lipids versus proteins within Toxoplasma-containing PVs versus zymosan-containing phagosomes. Values represent mean and SD from a representative experiment. (C) Fluorescence localization of DiIC₁₆-lipids in mechanically isolated PVs (top) versus liberated parasites (Tg, bottom). DiIC₁₆ was present in intact PVs, but not in liberated parasites.

Figure 2

Confocal localization of surface CTB bound to G_M1 ganglioside versus CD44 protein within Toxoplasma- versus zymosan-containing vacuoles in 3T3 cells (0.5-μm section). (A) Surface CD44 (red channel) was efficiently excluded from PVs (top), but was incorporated into phagosomes containing zymosan (bottom). In contrast, G_M1 (green channel) was internalized into both Toxoplasma- and zymosan-containing vacuoles. 3T3 cells were prelabeled with biotinylated CTB, incubated with zymosan or parasites for 5 min, and fixed. Biotinylated CTB was detected with Oregon green streptavidin. CD44 was stained with the mAb IM7.8.1, followed by Texas red goat anti–rat IgG. Parasites and zymosan were visualized with rabbit anti-p30 or antizymosan, respectively, followed by Cy5-conjugated goat anti–rabbit IgG. Arrowheads mark the position of Toxoplasma- or zymosan-containing vacuole. (B) Kinetic analysis of CTB-G_M1 versus CD44 in Toxoplasma- versus zymosan-containing vacuoles. Values represent mean and SD from a representative experiment.

Figure 2

Confocal localization of surface CTB bound to G_M1 ganglioside versus CD44 protein within Toxoplasma- versus zymosan-containing vacuoles in 3T3 cells (0.5-μm section). (A) Surface CD44 (red channel) was efficiently excluded from PVs (top), but was incorporated into phagosomes containing zymosan (bottom). In contrast, G_M1 (green channel) was internalized into both Toxoplasma- and zymosan-containing vacuoles. 3T3 cells were prelabeled with biotinylated CTB, incubated with zymosan or parasites for 5 min, and fixed. Biotinylated CTB was detected with Oregon green streptavidin. CD44 was stained with the mAb IM7.8.1, followed by Texas red goat anti–rat IgG. Parasites and zymosan were visualized with rabbit anti-p30 or antizymosan, respectively, followed by Cy5-conjugated goat anti–rabbit IgG. Arrowheads mark the position of Toxoplasma- or zymosan-containing vacuole. (B) Kinetic analysis of CTB-G_M1 versus CD44 in Toxoplasma- versus zymosan-containing vacuoles. Values represent mean and SD from a representative experiment.

Figure 3

CryoimmunoEM localization of CD44 in 3T3 cells infected with Toxoplasma. (A) CD44 was found along the host cell plasma membrane (arrowheads), but not within newly formed PVs. T, Toxoplasma cell. (B) Quantitative analysis indicated an absence of CD44 within a majority of PVs while the plasma membrane was uniformly labeled (T = 2-min pulse, no chase). Results are shown as the percentage of cells or vacuoles versus the density of immunogold staining.

Figure 3

CryoimmunoEM localization of CD44 in 3T3 cells infected with Toxoplasma. (A) CD44 was found along the host cell plasma membrane (arrowheads), but not within newly formed PVs. T, Toxoplasma cell. (B) Quantitative analysis indicated an absence of CD44 within a majority of PVs while the plasma membrane was uniformly labeled (T = 2-min pulse, no chase). Results are shown as the percentage of cells or vacuoles versus the density of immunogold staining.

Figure 5

Confocal localization of wild-type ICAM-1 versus ICAM-1–GPI, and ICAM-1–Cyt⁻ in BHK cells infected with Toxoplasma (5-min pulse; 0.5-μm section). (A) Wild type ICAM-1 (top, green) was excluded from PVs, whereas ICAM-1–GPI (middle, green) and ICAM-1–Cyt⁻ (bottom, green) were internalized into PVs. Parasites were detected with rabbit anti-p30, followed by Texas red–conjugated goat anti–rabbit IgG. ICAM-1 was detected with mAb RR1/1, followed by bodipy-conjugated goat anti–mouse IgG. Arrowheads mark the position of PVs. Red lines indicate the transects used for densitometry analysis as plotted to the right. The location of the PV along the line is shown by a cartoon of the parasite above the plot. (B) Confocal quantitative analysis of the percentage of PVs that internalized various forms of ICAM-1 after a 5-min challenge with parasites. Results shown are the mean and SE from two experiments. (C) Diagram of the ICAM-1, ICAM-1–GPI, and ICAM-1–Cyt⁻ constructs anchored in the membrane.

Figure 5

Confocal localization of wild-type ICAM-1 versus ICAM-1–GPI, and ICAM-1–Cyt⁻ in BHK cells infected with Toxoplasma (5-min pulse; 0.5-μm section). (A) Wild type ICAM-1 (top, green) was excluded from PVs, whereas ICAM-1–GPI (middle, green) and ICAM-1–Cyt⁻ (bottom, green) were internalized into PVs. Parasites were detected with rabbit anti-p30, followed by Texas red–conjugated goat anti–rabbit IgG. ICAM-1 was detected with mAb RR1/1, followed by bodipy-conjugated goat anti–mouse IgG. Arrowheads mark the position of PVs. Red lines indicate the transects used for densitometry analysis as plotted to the right. The location of the PV along the line is shown by a cartoon of the parasite above the plot. (B) Confocal quantitative analysis of the percentage of PVs that internalized various forms of ICAM-1 after a 5-min challenge with parasites. Results shown are the mean and SE from two experiments. (C) Diagram of the ICAM-1, ICAM-1–GPI, and ICAM-1–Cyt⁻ constructs anchored in the membrane.

Figure 5

Confocal localization of wild-type ICAM-1 versus ICAM-1–GPI, and ICAM-1–Cyt⁻ in BHK cells infected with Toxoplasma (5-min pulse; 0.5-μm section). (A) Wild type ICAM-1 (top, green) was excluded from PVs, whereas ICAM-1–GPI (middle, green) and ICAM-1–Cyt⁻ (bottom, green) were internalized into PVs. Parasites were detected with rabbit anti-p30, followed by Texas red–conjugated goat anti–rabbit IgG. ICAM-1 was detected with mAb RR1/1, followed by bodipy-conjugated goat anti–mouse IgG. Arrowheads mark the position of PVs. Red lines indicate the transects used for densitometry analysis as plotted to the right. The location of the PV along the line is shown by a cartoon of the parasite above the plot. (B) Confocal quantitative analysis of the percentage of PVs that internalized various forms of ICAM-1 after a 5-min challenge with parasites. Results shown are the mean and SE from two experiments. (C) Diagram of the ICAM-1, ICAM-1–GPI, and ICAM-1–Cyt⁻ constructs anchored in the membrane.

Figure 4

Confocal localization of GPI-linked proteins Sca-1 and CD55 versus caveolin-1 in Toxoplasma PVs (0.5-μm section). (A) Sca-1 (top, green) and CD55 (middle, green) were incorporated into PVs, whereas caveolin-1 (bottom, green) was excluded (5-min pulse infection). Parasites were detected with rabbit anti-p30 (top and middle) or rabbit anti-ACT1 (bottom), followed by Texas red–conjugated goat anti–rabbit IgG. Sca-1 was detected with mAb E13 161-7, followed by bodipy-conjugated goat anti–rat IgG. CD55 and caveolin-1 were detected with mAb IA10 and mAb C37120, respectively, followed by Oregon green–conjugated goat anti–mouse IgG. Sca-1 was detected in 3T3 cells, CD55 in HeLa cells, and caveolin-1 in HF cells. Arrowheads mark the position of PVs. (B) The percentage of PVs that internalized Sca-1, CD55, and caveolin-1 after a 5-min challenge with parasites. Values represent the mean and SE from two experiments.

Figure 4

Confocal localization of GPI-linked proteins Sca-1 and CD55 versus caveolin-1 in Toxoplasma PVs (0.5-μm section). (A) Sca-1 (top, green) and CD55 (middle, green) were incorporated into PVs, whereas caveolin-1 (bottom, green) was excluded (5-min pulse infection). Parasites were detected with rabbit anti-p30 (top and middle) or rabbit anti-ACT1 (bottom), followed by Texas red–conjugated goat anti–rabbit IgG. Sca-1 was detected with mAb E13 161-7, followed by bodipy-conjugated goat anti–rat IgG. CD55 and caveolin-1 were detected with mAb IA10 and mAb C37120, respectively, followed by Oregon green–conjugated goat anti–mouse IgG. Sca-1 was detected in 3T3 cells, CD55 in HeLa cells, and caveolin-1 in HF cells. Arrowheads mark the position of PVs. (B) The percentage of PVs that internalized Sca-1, CD55, and caveolin-1 after a 5-min challenge with parasites. Values represent the mean and SE from two experiments.