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Involvement of a Toxoplasma gondii chromatin remodeling complex ortholog in developmental regulation.

Rooney PJ, Neal LM, Knoll LJ - PLoS ONE (2011)

Bottom Line: In this study we characterized C9, an insertional mutant showing reduced expression of bradyzoite differentiation marker BAG1, in cultured human fibroblasts.The C9 mutant displayed reduced steady state transcript levels of bradyzoite-induced genes BAG1, LDH2, SUSA1, and ENO1, all of which were significantly increased with addition of TgRSC8 to the mutant.Thus chromatin remodeling, by both ATP-independent and dependent mechanisms, is an important mode of gene regulation during stage differentiation in parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.

ABSTRACT
The asexual cycle of the parasite Toxoplasma gondii has two developmental stages: a rapidly replicating form called a tachyzoite and a slow growing cyst form called a bradyzoite. While the importance of ATP-independent histone modifications for gene regulation in T. gondii have been demonstrated, ATP-dependent chromatin remodeling pathways have not been examined. In this study we characterized C9, an insertional mutant showing reduced expression of bradyzoite differentiation marker BAG1, in cultured human fibroblasts. This mutant contains an insertion in the gene encoding TgRSC8, which is homologous to the Saccharomyces cerevisiae proteins Rsc8p (remodel the structure of chromatin complex subunit 8) and Swi3p (switch/sucrose non-fermentable [SWI/SNF]) of ATP-dependent chromatin-remodeling complexes. In the C9 mutant, TgRSC8 is the downstream open reading frame on a dicistronic transcript. Though protein was expressed from the downstream gene of the dicistron, TgRSC8 levels were decreased in C9 from those of wild-type parasites, as determined by western immunoblot and flow cytometry. As TgRSC8 localized to the parasite nucleus, we postulated a role in gene regulation. Transcript levels of several markers were assessed by quantitative PCR to test this hypothesis. The C9 mutant displayed reduced steady state transcript levels of bradyzoite-induced genes BAG1, LDH2, SUSA1, and ENO1, all of which were significantly increased with addition of TgRSC8 to the mutant. Transcript levels of some bradyzoite markers were unaltered in C9, or unable to be increased by complementation with TgRSC8, indicating multiple pathways control bradyzoite-upregulated genes. Together, these data suggest a role for TgRSC8 in control of bradyzoite-upregulated gene expression. Thus chromatin remodeling, by both ATP-independent and dependent mechanisms, is an important mode of gene regulation during stage differentiation in parasites.

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Expression of BAG1 is reduced in strain C9 bradyzoites.A. BAG1 expression and reactivity with Dolichos biflorus agglutinin (DbA) was analyzed in bradyzoites of strains PruΔHPT (WT), C9 and an amino-terminally tagged, genetically complemented strain (compN) by immunofluorescence microscopy. The left column contains differential interference contrast (DIC) images. The rightmost column contains merged images including DAPI-detection of DNA (blue). All vacuoles show surface reactivity with DbA (green) indicating bradyzoite conversion, but BAG1 expression (red) of C9 is absent, or on only some bradzoites within. B. Quantitation of BAG1 expression by fluorescence microscopy. Vacuoles reacting with mouse anti-T. gondii (Tg) serum were assessed for BAG1 expression. Those showing anti-BAG1 reactivity of all DAPI-detected bradyzoites within were scored as complete (black bars), while BAG1-negative vacuoles and those containing only some bradyzoites were scored as incomplete (gray bars), displayed as a percentage of anti-Tg reactive vacuoles. Strain C9 shows a reduction in complete BAG1 reactivity relative to WT that is increased on genetic complementation of disrupted locus TgRSC8 by plasmid pTPR17 (comp1-3). Addition of an amino-terminal HA-tag to TgRSC8 also complemented the BAG1 phenotype (compN), but placement of the tag at the carboxy-terminus does not (compC). Shown is the average and standard deviation of three independent experiments. C. Quantitation of BAG1 expression by flow cytometry. Reactivity of C9 to anti-BAG1 antiserum (blue) was compared to that of a vector control strain (VC2, green), and C9 complemented strain compN (red). Background reactivity to fluorescent-conjugated secondary antiserum in the absence of anti-BAG1 antiserum exposure is shown for strain VC2 (gray). BAG1 reactivity is indicated on the x-axis in arbitrary units, and events counted (counts) are on the y-axis. Strains were analyzed in three independent experiments, showing similar outcomes. Results of a representative experiment are shown.
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pone-0019570-g003: Expression of BAG1 is reduced in strain C9 bradyzoites.A. BAG1 expression and reactivity with Dolichos biflorus agglutinin (DbA) was analyzed in bradyzoites of strains PruΔHPT (WT), C9 and an amino-terminally tagged, genetically complemented strain (compN) by immunofluorescence microscopy. The left column contains differential interference contrast (DIC) images. The rightmost column contains merged images including DAPI-detection of DNA (blue). All vacuoles show surface reactivity with DbA (green) indicating bradyzoite conversion, but BAG1 expression (red) of C9 is absent, or on only some bradzoites within. B. Quantitation of BAG1 expression by fluorescence microscopy. Vacuoles reacting with mouse anti-T. gondii (Tg) serum were assessed for BAG1 expression. Those showing anti-BAG1 reactivity of all DAPI-detected bradyzoites within were scored as complete (black bars), while BAG1-negative vacuoles and those containing only some bradyzoites were scored as incomplete (gray bars), displayed as a percentage of anti-Tg reactive vacuoles. Strain C9 shows a reduction in complete BAG1 reactivity relative to WT that is increased on genetic complementation of disrupted locus TgRSC8 by plasmid pTPR17 (comp1-3). Addition of an amino-terminal HA-tag to TgRSC8 also complemented the BAG1 phenotype (compN), but placement of the tag at the carboxy-terminus does not (compC). Shown is the average and standard deviation of three independent experiments. C. Quantitation of BAG1 expression by flow cytometry. Reactivity of C9 to anti-BAG1 antiserum (blue) was compared to that of a vector control strain (VC2, green), and C9 complemented strain compN (red). Background reactivity to fluorescent-conjugated secondary antiserum in the absence of anti-BAG1 antiserum exposure is shown for strain VC2 (gray). BAG1 reactivity is indicated on the x-axis in arbitrary units, and events counted (counts) are on the y-axis. Strains were analyzed in three independent experiments, showing similar outcomes. Results of a representative experiment are shown.

Mentions: Strain C9 demonstrated a defect in expression of BAG1, but not of cyst wall components reacting with Dolichos biflorus agglutinin, both of which are markers traditionally used to identify bradyzoites [27]. To quantify the BAG1 expression phenotype, PruΔHPT and C9 parasites were subjected to bradyzoite induction conditions. Three days post-induction, infected monolayers were fixed and reacted with mouse anti-T. gondii antiserum to detect all parasitophorous vacuoles, rabbit anti-BAG1, and DAPI to identify parasite nuclei. BAG1 expression was scored as complete if all DAPI-reactive parasites within the vacuole reacted with anti-BAG1 antiserum. While BAG1 was readily detected in most vacuolar parasites of parental strain PruΔHPT, over 80% of vacuoles of identically treated C9 parasites display incomplete BAG1 expression (Fig. 3A,B). Flow cytometric quantitation of BAG1 in parasites exposed to bradyzoite induction conditions also demonstrated reduced reactivity in the C9 mutant in comparison to vector control-containing parasites (Fig. 3C).


Involvement of a Toxoplasma gondii chromatin remodeling complex ortholog in developmental regulation.

Rooney PJ, Neal LM, Knoll LJ - PLoS ONE (2011)

Expression of BAG1 is reduced in strain C9 bradyzoites.A. BAG1 expression and reactivity with Dolichos biflorus agglutinin (DbA) was analyzed in bradyzoites of strains PruΔHPT (WT), C9 and an amino-terminally tagged, genetically complemented strain (compN) by immunofluorescence microscopy. The left column contains differential interference contrast (DIC) images. The rightmost column contains merged images including DAPI-detection of DNA (blue). All vacuoles show surface reactivity with DbA (green) indicating bradyzoite conversion, but BAG1 expression (red) of C9 is absent, or on only some bradzoites within. B. Quantitation of BAG1 expression by fluorescence microscopy. Vacuoles reacting with mouse anti-T. gondii (Tg) serum were assessed for BAG1 expression. Those showing anti-BAG1 reactivity of all DAPI-detected bradyzoites within were scored as complete (black bars), while BAG1-negative vacuoles and those containing only some bradyzoites were scored as incomplete (gray bars), displayed as a percentage of anti-Tg reactive vacuoles. Strain C9 shows a reduction in complete BAG1 reactivity relative to WT that is increased on genetic complementation of disrupted locus TgRSC8 by plasmid pTPR17 (comp1-3). Addition of an amino-terminal HA-tag to TgRSC8 also complemented the BAG1 phenotype (compN), but placement of the tag at the carboxy-terminus does not (compC). Shown is the average and standard deviation of three independent experiments. C. Quantitation of BAG1 expression by flow cytometry. Reactivity of C9 to anti-BAG1 antiserum (blue) was compared to that of a vector control strain (VC2, green), and C9 complemented strain compN (red). Background reactivity to fluorescent-conjugated secondary antiserum in the absence of anti-BAG1 antiserum exposure is shown for strain VC2 (gray). BAG1 reactivity is indicated on the x-axis in arbitrary units, and events counted (counts) are on the y-axis. Strains were analyzed in three independent experiments, showing similar outcomes. Results of a representative experiment are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3104990&req=5

pone-0019570-g003: Expression of BAG1 is reduced in strain C9 bradyzoites.A. BAG1 expression and reactivity with Dolichos biflorus agglutinin (DbA) was analyzed in bradyzoites of strains PruΔHPT (WT), C9 and an amino-terminally tagged, genetically complemented strain (compN) by immunofluorescence microscopy. The left column contains differential interference contrast (DIC) images. The rightmost column contains merged images including DAPI-detection of DNA (blue). All vacuoles show surface reactivity with DbA (green) indicating bradyzoite conversion, but BAG1 expression (red) of C9 is absent, or on only some bradzoites within. B. Quantitation of BAG1 expression by fluorescence microscopy. Vacuoles reacting with mouse anti-T. gondii (Tg) serum were assessed for BAG1 expression. Those showing anti-BAG1 reactivity of all DAPI-detected bradyzoites within were scored as complete (black bars), while BAG1-negative vacuoles and those containing only some bradyzoites were scored as incomplete (gray bars), displayed as a percentage of anti-Tg reactive vacuoles. Strain C9 shows a reduction in complete BAG1 reactivity relative to WT that is increased on genetic complementation of disrupted locus TgRSC8 by plasmid pTPR17 (comp1-3). Addition of an amino-terminal HA-tag to TgRSC8 also complemented the BAG1 phenotype (compN), but placement of the tag at the carboxy-terminus does not (compC). Shown is the average and standard deviation of three independent experiments. C. Quantitation of BAG1 expression by flow cytometry. Reactivity of C9 to anti-BAG1 antiserum (blue) was compared to that of a vector control strain (VC2, green), and C9 complemented strain compN (red). Background reactivity to fluorescent-conjugated secondary antiserum in the absence of anti-BAG1 antiserum exposure is shown for strain VC2 (gray). BAG1 reactivity is indicated on the x-axis in arbitrary units, and events counted (counts) are on the y-axis. Strains were analyzed in three independent experiments, showing similar outcomes. Results of a representative experiment are shown.
Mentions: Strain C9 demonstrated a defect in expression of BAG1, but not of cyst wall components reacting with Dolichos biflorus agglutinin, both of which are markers traditionally used to identify bradyzoites [27]. To quantify the BAG1 expression phenotype, PruΔHPT and C9 parasites were subjected to bradyzoite induction conditions. Three days post-induction, infected monolayers were fixed and reacted with mouse anti-T. gondii antiserum to detect all parasitophorous vacuoles, rabbit anti-BAG1, and DAPI to identify parasite nuclei. BAG1 expression was scored as complete if all DAPI-reactive parasites within the vacuole reacted with anti-BAG1 antiserum. While BAG1 was readily detected in most vacuolar parasites of parental strain PruΔHPT, over 80% of vacuoles of identically treated C9 parasites display incomplete BAG1 expression (Fig. 3A,B). Flow cytometric quantitation of BAG1 in parasites exposed to bradyzoite induction conditions also demonstrated reduced reactivity in the C9 mutant in comparison to vector control-containing parasites (Fig. 3C).

Bottom Line: In this study we characterized C9, an insertional mutant showing reduced expression of bradyzoite differentiation marker BAG1, in cultured human fibroblasts.The C9 mutant displayed reduced steady state transcript levels of bradyzoite-induced genes BAG1, LDH2, SUSA1, and ENO1, all of which were significantly increased with addition of TgRSC8 to the mutant.Thus chromatin remodeling, by both ATP-independent and dependent mechanisms, is an important mode of gene regulation during stage differentiation in parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.

ABSTRACT
The asexual cycle of the parasite Toxoplasma gondii has two developmental stages: a rapidly replicating form called a tachyzoite and a slow growing cyst form called a bradyzoite. While the importance of ATP-independent histone modifications for gene regulation in T. gondii have been demonstrated, ATP-dependent chromatin remodeling pathways have not been examined. In this study we characterized C9, an insertional mutant showing reduced expression of bradyzoite differentiation marker BAG1, in cultured human fibroblasts. This mutant contains an insertion in the gene encoding TgRSC8, which is homologous to the Saccharomyces cerevisiae proteins Rsc8p (remodel the structure of chromatin complex subunit 8) and Swi3p (switch/sucrose non-fermentable [SWI/SNF]) of ATP-dependent chromatin-remodeling complexes. In the C9 mutant, TgRSC8 is the downstream open reading frame on a dicistronic transcript. Though protein was expressed from the downstream gene of the dicistron, TgRSC8 levels were decreased in C9 from those of wild-type parasites, as determined by western immunoblot and flow cytometry. As TgRSC8 localized to the parasite nucleus, we postulated a role in gene regulation. Transcript levels of several markers were assessed by quantitative PCR to test this hypothesis. The C9 mutant displayed reduced steady state transcript levels of bradyzoite-induced genes BAG1, LDH2, SUSA1, and ENO1, all of which were significantly increased with addition of TgRSC8 to the mutant. Transcript levels of some bradyzoite markers were unaltered in C9, or unable to be increased by complementation with TgRSC8, indicating multiple pathways control bradyzoite-upregulated genes. Together, these data suggest a role for TgRSC8 in control of bradyzoite-upregulated gene expression. Thus chromatin remodeling, by both ATP-independent and dependent mechanisms, is an important mode of gene regulation during stage differentiation in parasites.

Show MeSH
Related in: MedlinePlus