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Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole.

Khaminets A, Hunn JP, Könen-Waisman S, Zhao YO, Preukschat D, Coers J, Boyle JP, Ong YC, Boothroyd JC, Reichmann G, Howard JC - Cell. Microbiol. (2010)

Bottom Line: Loading of IRG proteins onto the vacuoles of virulent Toxoplasma strains is attenuated and the two pioneer IRGs are the most affected.The polymorphic rhoptry kinases, ROP16, ROP18 and the catalytically inactive proteins, ROP5A-D, are not individually responsible for this effect.The complex cooperative behaviour of IRG proteins in resisting Toxoplasma may hint at undiscovered complexity also in virulence mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, University of Cologne, Zülpicher Strasse, Cologne 50674, Germany.

ABSTRACT
The immunity-related GTPases (IRGs) constitute an interferon-induced intracellular resistance mechanism in mice against Toxoplasma gondii. IRG proteins accumulate on the parasitophorous vacuole membrane (PVM), leading to its disruption and to death of the parasite. How IRGs target the PVM is unknown. We show that accumulation of IRGs on the PVM begins minutes after parasite invasion and increases for about 1 h. Targeting occurs independently of several signalling pathways and the microtubule network, suggesting that IRG transport is diffusion-driven. The intensity of IRG accumulation on the PVM, however, is reduced in absence of the autophagy regulator, Atg5. In wild-type cells IRG proteins accumulate cooperatively on PVMs in a definite order reflecting a temporal hierarchy, with Irgb6 and Irgb10 apparently acting as pioneers. Loading of IRG proteins onto the vacuoles of virulent Toxoplasma strains is attenuated and the two pioneer IRGs are the most affected. The polymorphic rhoptry kinases, ROP16, ROP18 and the catalytically inactive proteins, ROP5A-D, are not individually responsible for this effect. Thus IRG proteins protect mice against avirulent strains of Toxoplasma but fail against virulent strains. The complex cooperative behaviour of IRG proteins in resisting Toxoplasma may hint at undiscovered complexity also in virulence mechanisms.

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Related in: MedlinePlus

Loading of individual vacuoles by Irga6-ctag1-EGFP or Irgb6-FLAG-EGFP observed by time-lapse microscopy. C57BL/6 MEFs were transfected with the expression plasmid pEGFP-N3-Irga6-ctag1 or pEGFP-N3-Irgb6-FLAG and simultaneously induced with IFNγ. After 24 h, the cells were infected with T. gondii ME49 strain in microscope slide chambers as described in Zhao et al. (2009a). Cells were observed continuously in order to document the entry of individual parasites and the subsequent accumulation of Irga6-ctag1-EGFP or Irgb6-FLAG-EGFP on the PV. A. and B. Selected frames of two time-lapse videos of Irga6-ctag1-EGFP loading on ME49 T. gondii PV. Arrowheads indicate the location of the analysed T. gondii PVs. The arrow in (B) indicates a T. gondii vacuole already loaded with Irga6-ctag1-EGFP before the initiation of the movie (see also text). [Note that the frames shown in (B) are not a regular time series as some frames were out of focus and have not been included]. The videos from which frames in (A and B) were extracted are presented as Videos S1 and S2 respectively. pi, post inoculation. C. Mean pixel intensities of Irga6 and Irgb6 at the PVM were measured from the vacuoles shown in (A and B) (Irga6 I and Irga6 II respectively) and from two further videos of Irgb6 (Irgb6 I and Irgb6 II; these frames are shown in Fig. S1D), and plotted as percentage of the maximum intensity. The origin on the time axis is the time of addition of T. gondii to the cells. The first symbol of each plot gives the time when the observed parasite was seen to enter the cell. In the case of the Irgb6 I movie the protein signal slightly decreased after 13 min because of focus drift on the 15 and 17 min frames and resumed its rise after correction.
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fig02: Loading of individual vacuoles by Irga6-ctag1-EGFP or Irgb6-FLAG-EGFP observed by time-lapse microscopy. C57BL/6 MEFs were transfected with the expression plasmid pEGFP-N3-Irga6-ctag1 or pEGFP-N3-Irgb6-FLAG and simultaneously induced with IFNγ. After 24 h, the cells were infected with T. gondii ME49 strain in microscope slide chambers as described in Zhao et al. (2009a). Cells were observed continuously in order to document the entry of individual parasites and the subsequent accumulation of Irga6-ctag1-EGFP or Irgb6-FLAG-EGFP on the PV. A. and B. Selected frames of two time-lapse videos of Irga6-ctag1-EGFP loading on ME49 T. gondii PV. Arrowheads indicate the location of the analysed T. gondii PVs. The arrow in (B) indicates a T. gondii vacuole already loaded with Irga6-ctag1-EGFP before the initiation of the movie (see also text). [Note that the frames shown in (B) are not a regular time series as some frames were out of focus and have not been included]. The videos from which frames in (A and B) were extracted are presented as Videos S1 and S2 respectively. pi, post inoculation. C. Mean pixel intensities of Irga6 and Irgb6 at the PVM were measured from the vacuoles shown in (A and B) (Irga6 I and Irga6 II respectively) and from two further videos of Irgb6 (Irgb6 I and Irgb6 II; these frames are shown in Fig. S1D), and plotted as percentage of the maximum intensity. The origin on the time axis is the time of addition of T. gondii to the cells. The first symbol of each plot gives the time when the observed parasite was seen to enter the cell. In the case of the Irgb6 I movie the protein signal slightly decreased after 13 min because of focus drift on the 15 and 17 min frames and resumed its rise after correction.

Mentions: The rising phase of both the frequency and intensity data may be attributed to variation in the delay associated with loading of individual vacuoles and indeed to some residual asynchrony in the infection. We therefore used time-lapse photography to observe the loading of a transfected EGFP-tagged version of Irga6 (Irga6-ctag1-EGFP) (Zhao et al., 2009a) on to individual PVMs. The time delays before detecting loading were, respectively, 28 min and 9 min in the two frame series shown in Fig. 2A and B (see also Videos S1 and S2). The signal intensities of Irga6-ctag1-EGFP at the PVM in these videos were quantified in consecutive frames (Fig. 2C). Frames from two further videos showing the accumulation of Irgb6-FLAG-EGFP (Fig. S1D) were also quantified (Fig. 2C, Irgb6 I and II). The four data sets illustrate two independent contributions to the rise in loaded vacuoles shown in Fig. 1. First, there is variation in the time of entry of the T. gondii into the cell, which can be as early as the first minute or two in the Irga6 I series (Fig. 2A) but also as late as 60 min in the Irga6 II series (Fig. 2B). Second, there is heterogeneity in the length of the delay after infection before loading begins, which can be as short as 2.5 min in rare vacuoles seen in the fixed preparations (Fig. 1), and in one of the quantified Irgb6 videos (Fig. 2C, Irgb6 I), 9 min in Irga6 II (Fig. 2B and C) and as much as 28 min in Irga6 I (Fig. 2A and C). Once IRG accumulation is initiated it rises roughly linearly for 30–60 min.


Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole.

Khaminets A, Hunn JP, Könen-Waisman S, Zhao YO, Preukschat D, Coers J, Boyle JP, Ong YC, Boothroyd JC, Reichmann G, Howard JC - Cell. Microbiol. (2010)

Loading of individual vacuoles by Irga6-ctag1-EGFP or Irgb6-FLAG-EGFP observed by time-lapse microscopy. C57BL/6 MEFs were transfected with the expression plasmid pEGFP-N3-Irga6-ctag1 or pEGFP-N3-Irgb6-FLAG and simultaneously induced with IFNγ. After 24 h, the cells were infected with T. gondii ME49 strain in microscope slide chambers as described in Zhao et al. (2009a). Cells were observed continuously in order to document the entry of individual parasites and the subsequent accumulation of Irga6-ctag1-EGFP or Irgb6-FLAG-EGFP on the PV. A. and B. Selected frames of two time-lapse videos of Irga6-ctag1-EGFP loading on ME49 T. gondii PV. Arrowheads indicate the location of the analysed T. gondii PVs. The arrow in (B) indicates a T. gondii vacuole already loaded with Irga6-ctag1-EGFP before the initiation of the movie (see also text). [Note that the frames shown in (B) are not a regular time series as some frames were out of focus and have not been included]. The videos from which frames in (A and B) were extracted are presented as Videos S1 and S2 respectively. pi, post inoculation. C. Mean pixel intensities of Irga6 and Irgb6 at the PVM were measured from the vacuoles shown in (A and B) (Irga6 I and Irga6 II respectively) and from two further videos of Irgb6 (Irgb6 I and Irgb6 II; these frames are shown in Fig. S1D), and plotted as percentage of the maximum intensity. The origin on the time axis is the time of addition of T. gondii to the cells. The first symbol of each plot gives the time when the observed parasite was seen to enter the cell. In the case of the Irgb6 I movie the protein signal slightly decreased after 13 min because of focus drift on the 15 and 17 min frames and resumed its rise after correction.
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fig02: Loading of individual vacuoles by Irga6-ctag1-EGFP or Irgb6-FLAG-EGFP observed by time-lapse microscopy. C57BL/6 MEFs were transfected with the expression plasmid pEGFP-N3-Irga6-ctag1 or pEGFP-N3-Irgb6-FLAG and simultaneously induced with IFNγ. After 24 h, the cells were infected with T. gondii ME49 strain in microscope slide chambers as described in Zhao et al. (2009a). Cells were observed continuously in order to document the entry of individual parasites and the subsequent accumulation of Irga6-ctag1-EGFP or Irgb6-FLAG-EGFP on the PV. A. and B. Selected frames of two time-lapse videos of Irga6-ctag1-EGFP loading on ME49 T. gondii PV. Arrowheads indicate the location of the analysed T. gondii PVs. The arrow in (B) indicates a T. gondii vacuole already loaded with Irga6-ctag1-EGFP before the initiation of the movie (see also text). [Note that the frames shown in (B) are not a regular time series as some frames were out of focus and have not been included]. The videos from which frames in (A and B) were extracted are presented as Videos S1 and S2 respectively. pi, post inoculation. C. Mean pixel intensities of Irga6 and Irgb6 at the PVM were measured from the vacuoles shown in (A and B) (Irga6 I and Irga6 II respectively) and from two further videos of Irgb6 (Irgb6 I and Irgb6 II; these frames are shown in Fig. S1D), and plotted as percentage of the maximum intensity. The origin on the time axis is the time of addition of T. gondii to the cells. The first symbol of each plot gives the time when the observed parasite was seen to enter the cell. In the case of the Irgb6 I movie the protein signal slightly decreased after 13 min because of focus drift on the 15 and 17 min frames and resumed its rise after correction.
Mentions: The rising phase of both the frequency and intensity data may be attributed to variation in the delay associated with loading of individual vacuoles and indeed to some residual asynchrony in the infection. We therefore used time-lapse photography to observe the loading of a transfected EGFP-tagged version of Irga6 (Irga6-ctag1-EGFP) (Zhao et al., 2009a) on to individual PVMs. The time delays before detecting loading were, respectively, 28 min and 9 min in the two frame series shown in Fig. 2A and B (see also Videos S1 and S2). The signal intensities of Irga6-ctag1-EGFP at the PVM in these videos were quantified in consecutive frames (Fig. 2C). Frames from two further videos showing the accumulation of Irgb6-FLAG-EGFP (Fig. S1D) were also quantified (Fig. 2C, Irgb6 I and II). The four data sets illustrate two independent contributions to the rise in loaded vacuoles shown in Fig. 1. First, there is variation in the time of entry of the T. gondii into the cell, which can be as early as the first minute or two in the Irga6 I series (Fig. 2A) but also as late as 60 min in the Irga6 II series (Fig. 2B). Second, there is heterogeneity in the length of the delay after infection before loading begins, which can be as short as 2.5 min in rare vacuoles seen in the fixed preparations (Fig. 1), and in one of the quantified Irgb6 videos (Fig. 2C, Irgb6 I), 9 min in Irga6 II (Fig. 2B and C) and as much as 28 min in Irga6 I (Fig. 2A and C). Once IRG accumulation is initiated it rises roughly linearly for 30–60 min.

Bottom Line: Loading of IRG proteins onto the vacuoles of virulent Toxoplasma strains is attenuated and the two pioneer IRGs are the most affected.The polymorphic rhoptry kinases, ROP16, ROP18 and the catalytically inactive proteins, ROP5A-D, are not individually responsible for this effect.The complex cooperative behaviour of IRG proteins in resisting Toxoplasma may hint at undiscovered complexity also in virulence mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, University of Cologne, Zülpicher Strasse, Cologne 50674, Germany.

ABSTRACT
The immunity-related GTPases (IRGs) constitute an interferon-induced intracellular resistance mechanism in mice against Toxoplasma gondii. IRG proteins accumulate on the parasitophorous vacuole membrane (PVM), leading to its disruption and to death of the parasite. How IRGs target the PVM is unknown. We show that accumulation of IRGs on the PVM begins minutes after parasite invasion and increases for about 1 h. Targeting occurs independently of several signalling pathways and the microtubule network, suggesting that IRG transport is diffusion-driven. The intensity of IRG accumulation on the PVM, however, is reduced in absence of the autophagy regulator, Atg5. In wild-type cells IRG proteins accumulate cooperatively on PVMs in a definite order reflecting a temporal hierarchy, with Irgb6 and Irgb10 apparently acting as pioneers. Loading of IRG proteins onto the vacuoles of virulent Toxoplasma strains is attenuated and the two pioneer IRGs are the most affected. The polymorphic rhoptry kinases, ROP16, ROP18 and the catalytically inactive proteins, ROP5A-D, are not individually responsible for this effect. Thus IRG proteins protect mice against avirulent strains of Toxoplasma but fail against virulent strains. The complex cooperative behaviour of IRG proteins in resisting Toxoplasma may hint at undiscovered complexity also in virulence mechanisms.

Show MeSH
Related in: MedlinePlus