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A nuclear factor of high mobility group box protein in Toxoplasma gondii.

Wang H, Lei T, Liu J, Li M, Nan H, Liu Q - PLoS ONE (2014)

Bottom Line: We cloned TgHMGB1a, a 33.9 kDa protein that can stimulates macrophages to release TNF-α, and, we demonstrated that the TgHMGB1a binds distorted DNA structures such as cruciform DNA in electrophoretic mobility shift assays (EMSA).Immunofluorescence assay indicated TgHMGB1a concentrated in the nucleus of intracellular tachyzoites but translocated into the cytoplasm while the parasites release to extracellular.There were no significant phenotypic changes when the TgHMGB1a B box was deleted, while transgenic parasites that overexpressed TgHMGB1a showed slower intracellular growth and caused delayed death in mouse, further quantitative RT-PCR analyses showed that the expression levels of many important genes, including virulence factors, increased when TgHMGB1a was overexpressed, but no significant changes were observed in TgHMGB1a B box-deficient parasites.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, China.

ABSTRACT
High mobility group box 1 (HMGB1) is a nuclear factor that usually binds DNA and modulates gene expression in multicellular organisms. Three HMGB1 orthologs were predicted in the genome of Toxoplasma gondii, an obligate intracellular protozoan pathogen, termed TgHMGB1a, b and c. Phylogenetic and bioinformatic analyses indicated that these proteins all contain a single HMG box and which shared in three genotypes. We cloned TgHMGB1a, a 33.9 kDa protein that can stimulates macrophages to release TNF-α, and, we demonstrated that the TgHMGB1a binds distorted DNA structures such as cruciform DNA in electrophoretic mobility shift assays (EMSA). Immunofluorescence assay indicated TgHMGB1a concentrated in the nucleus of intracellular tachyzoites but translocated into the cytoplasm while the parasites release to extracellular. There were no significant phenotypic changes when the TgHMGB1a B box was deleted, while transgenic parasites that overexpressed TgHMGB1a showed slower intracellular growth and caused delayed death in mouse, further quantitative RT-PCR analyses showed that the expression levels of many important genes, including virulence factors, increased when TgHMGB1a was overexpressed, but no significant changes were observed in TgHMGB1a B box-deficient parasites. Our findings demonstrated that TgHMGB1a is indeed a nuclear protein that maintains HMG box architectural functions and is a potential proinflammatory factor during the T.gondii infection. Further studies that clarify the functions of TgHMGB1s will increase our knowledge of transcriptional regulation and parasite virulence, and might provide new insight into host-parasite interactions for T. gondii infection.

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Related in: MedlinePlus

TgHMGB1a binds to cruciform DNA.A. Oligonucleotides used to form the different synthetic DNA structures. Identical text formats (italic, bold, dotted/solid underlines) represent complementary sequences. All of the primers were labeled using EMSA Probe Biotin Labeling Kit as described in methods. Cruciform 4H structures were obtained by annealing equal amounts of 4H-a, -b, -c and -d primers; 2H hairpin-like structures, by annealing of 4H-a and -b, -b and -c, -c and -d, -d and -a, respectively; double stranded (ds) linear DNA structures were obtained by annealing of 4H-a or 4H-c with its fully complementary sequence (compl 4H-a and compl 4H-c). B. EMSA with cruciform DNA (4H) and recombinant TgHMGB1a 4E. Increasing concentrations of TgHMGB1a 4E protein (0.01–1.5 mg/mL) were incubated with biotinlabeled 4H DNA for 30 min at 25°C. DNA–protein complexes formed were resolved on 6% non-denaturing polyacrylamide gel and transferred to nylon membrane and then detected by Chemiluminescent EMSA Kit. Lane 1, free labeled 4H; lanes 2–8, 4H DNA–protein complexes formed with increasing concentrations of TgHMGB1a 4E. C. Competition EMSA experiments were performed with different cold DNA structures. After incubation of TgHMGB1a 4E (1 mg/mL) with biotinlabeled 4H, cold complete 4H (in a 100-fold excess), hairpin-like 2H structures (in a 500- fold excess) or ds linear DNA (in a 500-fold excess) were added, and incubated for an additional 20 min. Complex formation was analyzed as in B. Lane 1, free labeled 4H; lane 2, 4H DNA–protein complexes formed with TgHMGB1a 4E without competitor DNA; lane 3, competition assay with 100-fold excess cold 4H; lanes 4–7 competition assay with 500-fold excess cold 2H hairpin-like structures; lanes 8–9, competition assay with 500-fold excess cold linear ds DNA.
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pone-0111993-g004: TgHMGB1a binds to cruciform DNA.A. Oligonucleotides used to form the different synthetic DNA structures. Identical text formats (italic, bold, dotted/solid underlines) represent complementary sequences. All of the primers were labeled using EMSA Probe Biotin Labeling Kit as described in methods. Cruciform 4H structures were obtained by annealing equal amounts of 4H-a, -b, -c and -d primers; 2H hairpin-like structures, by annealing of 4H-a and -b, -b and -c, -c and -d, -d and -a, respectively; double stranded (ds) linear DNA structures were obtained by annealing of 4H-a or 4H-c with its fully complementary sequence (compl 4H-a and compl 4H-c). B. EMSA with cruciform DNA (4H) and recombinant TgHMGB1a 4E. Increasing concentrations of TgHMGB1a 4E protein (0.01–1.5 mg/mL) were incubated with biotinlabeled 4H DNA for 30 min at 25°C. DNA–protein complexes formed were resolved on 6% non-denaturing polyacrylamide gel and transferred to nylon membrane and then detected by Chemiluminescent EMSA Kit. Lane 1, free labeled 4H; lanes 2–8, 4H DNA–protein complexes formed with increasing concentrations of TgHMGB1a 4E. C. Competition EMSA experiments were performed with different cold DNA structures. After incubation of TgHMGB1a 4E (1 mg/mL) with biotinlabeled 4H, cold complete 4H (in a 100-fold excess), hairpin-like 2H structures (in a 500- fold excess) or ds linear DNA (in a 500-fold excess) were added, and incubated for an additional 20 min. Complex formation was analyzed as in B. Lane 1, free labeled 4H; lane 2, 4H DNA–protein complexes formed with TgHMGB1a 4E without competitor DNA; lane 3, competition assay with 100-fold excess cold 4H; lanes 4–7 competition assay with 500-fold excess cold 2H hairpin-like structures; lanes 8–9, competition assay with 500-fold excess cold linear ds DNA.

Mentions: DNA–protein complexes can be analyzed by EMSAs with four-way junction (4H) DNA, preferentially bound by HMGB proteins and a synthetic substrate commonly used to study proteins involved in recognizing and resolving Holliday-type junctions formed during in vivo genetic recombination events (Figure 4A). At least two different 4H DNA–TgHMGB1a 4E complexes formed with increasing of the protein concentration, resulting in two discrete retarded bands (Figure 4B). These two bands presumably correspond to complexes formed upon binding of one or more TgHMGB1a 4E molecules per 4H DNA. Binding competition with 2H “hairpin like” structures (Figure 4A and C, lanes 4–7) demonstrated that TgHMGB1a 4E preferentially binds to complete 4H DNA, since 500-fold excess of these 2H structures does not affect the 4H DNA–TgHMGB1a 4E complex migration. Simultaneously, competition experiments with linear dsDNA derived from the 4H arms (Figure 4A) indicated that TgHMGB1a 4E preferentially binds to the DNA cross-over of the cruciform DNA, since 500-fold excess of these duplex DNA molecules were not able to inhibit the formation of the cruciform DNA–TgHMGB1a 4E complexes. As expected, the retarded bands almost disappeared when 100-fold cold 4H was added (Figure 4C, lane 3), suggested that there is competition between labeled 4H DNA and excess cold 4H DNA. Purified TgGRA1 was used in similar EMSAs, but it didn't show mobility shift to the cruciform DNA, demonstrating that binding of TgHMGB1a 4E protein is specific and independent of the His-tag fusion (Figure S7).


A nuclear factor of high mobility group box protein in Toxoplasma gondii.

Wang H, Lei T, Liu J, Li M, Nan H, Liu Q - PLoS ONE (2014)

TgHMGB1a binds to cruciform DNA.A. Oligonucleotides used to form the different synthetic DNA structures. Identical text formats (italic, bold, dotted/solid underlines) represent complementary sequences. All of the primers were labeled using EMSA Probe Biotin Labeling Kit as described in methods. Cruciform 4H structures were obtained by annealing equal amounts of 4H-a, -b, -c and -d primers; 2H hairpin-like structures, by annealing of 4H-a and -b, -b and -c, -c and -d, -d and -a, respectively; double stranded (ds) linear DNA structures were obtained by annealing of 4H-a or 4H-c with its fully complementary sequence (compl 4H-a and compl 4H-c). B. EMSA with cruciform DNA (4H) and recombinant TgHMGB1a 4E. Increasing concentrations of TgHMGB1a 4E protein (0.01–1.5 mg/mL) were incubated with biotinlabeled 4H DNA for 30 min at 25°C. DNA–protein complexes formed were resolved on 6% non-denaturing polyacrylamide gel and transferred to nylon membrane and then detected by Chemiluminescent EMSA Kit. Lane 1, free labeled 4H; lanes 2–8, 4H DNA–protein complexes formed with increasing concentrations of TgHMGB1a 4E. C. Competition EMSA experiments were performed with different cold DNA structures. After incubation of TgHMGB1a 4E (1 mg/mL) with biotinlabeled 4H, cold complete 4H (in a 100-fold excess), hairpin-like 2H structures (in a 500- fold excess) or ds linear DNA (in a 500-fold excess) were added, and incubated for an additional 20 min. Complex formation was analyzed as in B. Lane 1, free labeled 4H; lane 2, 4H DNA–protein complexes formed with TgHMGB1a 4E without competitor DNA; lane 3, competition assay with 100-fold excess cold 4H; lanes 4–7 competition assay with 500-fold excess cold 2H hairpin-like structures; lanes 8–9, competition assay with 500-fold excess cold linear ds DNA.
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Related In: Results  -  Collection

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pone-0111993-g004: TgHMGB1a binds to cruciform DNA.A. Oligonucleotides used to form the different synthetic DNA structures. Identical text formats (italic, bold, dotted/solid underlines) represent complementary sequences. All of the primers were labeled using EMSA Probe Biotin Labeling Kit as described in methods. Cruciform 4H structures were obtained by annealing equal amounts of 4H-a, -b, -c and -d primers; 2H hairpin-like structures, by annealing of 4H-a and -b, -b and -c, -c and -d, -d and -a, respectively; double stranded (ds) linear DNA structures were obtained by annealing of 4H-a or 4H-c with its fully complementary sequence (compl 4H-a and compl 4H-c). B. EMSA with cruciform DNA (4H) and recombinant TgHMGB1a 4E. Increasing concentrations of TgHMGB1a 4E protein (0.01–1.5 mg/mL) were incubated with biotinlabeled 4H DNA for 30 min at 25°C. DNA–protein complexes formed were resolved on 6% non-denaturing polyacrylamide gel and transferred to nylon membrane and then detected by Chemiluminescent EMSA Kit. Lane 1, free labeled 4H; lanes 2–8, 4H DNA–protein complexes formed with increasing concentrations of TgHMGB1a 4E. C. Competition EMSA experiments were performed with different cold DNA structures. After incubation of TgHMGB1a 4E (1 mg/mL) with biotinlabeled 4H, cold complete 4H (in a 100-fold excess), hairpin-like 2H structures (in a 500- fold excess) or ds linear DNA (in a 500-fold excess) were added, and incubated for an additional 20 min. Complex formation was analyzed as in B. Lane 1, free labeled 4H; lane 2, 4H DNA–protein complexes formed with TgHMGB1a 4E without competitor DNA; lane 3, competition assay with 100-fold excess cold 4H; lanes 4–7 competition assay with 500-fold excess cold 2H hairpin-like structures; lanes 8–9, competition assay with 500-fold excess cold linear ds DNA.
Mentions: DNA–protein complexes can be analyzed by EMSAs with four-way junction (4H) DNA, preferentially bound by HMGB proteins and a synthetic substrate commonly used to study proteins involved in recognizing and resolving Holliday-type junctions formed during in vivo genetic recombination events (Figure 4A). At least two different 4H DNA–TgHMGB1a 4E complexes formed with increasing of the protein concentration, resulting in two discrete retarded bands (Figure 4B). These two bands presumably correspond to complexes formed upon binding of one or more TgHMGB1a 4E molecules per 4H DNA. Binding competition with 2H “hairpin like” structures (Figure 4A and C, lanes 4–7) demonstrated that TgHMGB1a 4E preferentially binds to complete 4H DNA, since 500-fold excess of these 2H structures does not affect the 4H DNA–TgHMGB1a 4E complex migration. Simultaneously, competition experiments with linear dsDNA derived from the 4H arms (Figure 4A) indicated that TgHMGB1a 4E preferentially binds to the DNA cross-over of the cruciform DNA, since 500-fold excess of these duplex DNA molecules were not able to inhibit the formation of the cruciform DNA–TgHMGB1a 4E complexes. As expected, the retarded bands almost disappeared when 100-fold cold 4H was added (Figure 4C, lane 3), suggested that there is competition between labeled 4H DNA and excess cold 4H DNA. Purified TgGRA1 was used in similar EMSAs, but it didn't show mobility shift to the cruciform DNA, demonstrating that binding of TgHMGB1a 4E protein is specific and independent of the His-tag fusion (Figure S7).

Bottom Line: We cloned TgHMGB1a, a 33.9 kDa protein that can stimulates macrophages to release TNF-α, and, we demonstrated that the TgHMGB1a binds distorted DNA structures such as cruciform DNA in electrophoretic mobility shift assays (EMSA).Immunofluorescence assay indicated TgHMGB1a concentrated in the nucleus of intracellular tachyzoites but translocated into the cytoplasm while the parasites release to extracellular.There were no significant phenotypic changes when the TgHMGB1a B box was deleted, while transgenic parasites that overexpressed TgHMGB1a showed slower intracellular growth and caused delayed death in mouse, further quantitative RT-PCR analyses showed that the expression levels of many important genes, including virulence factors, increased when TgHMGB1a was overexpressed, but no significant changes were observed in TgHMGB1a B box-deficient parasites.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, China.

ABSTRACT
High mobility group box 1 (HMGB1) is a nuclear factor that usually binds DNA and modulates gene expression in multicellular organisms. Three HMGB1 orthologs were predicted in the genome of Toxoplasma gondii, an obligate intracellular protozoan pathogen, termed TgHMGB1a, b and c. Phylogenetic and bioinformatic analyses indicated that these proteins all contain a single HMG box and which shared in three genotypes. We cloned TgHMGB1a, a 33.9 kDa protein that can stimulates macrophages to release TNF-α, and, we demonstrated that the TgHMGB1a binds distorted DNA structures such as cruciform DNA in electrophoretic mobility shift assays (EMSA). Immunofluorescence assay indicated TgHMGB1a concentrated in the nucleus of intracellular tachyzoites but translocated into the cytoplasm while the parasites release to extracellular. There were no significant phenotypic changes when the TgHMGB1a B box was deleted, while transgenic parasites that overexpressed TgHMGB1a showed slower intracellular growth and caused delayed death in mouse, further quantitative RT-PCR analyses showed that the expression levels of many important genes, including virulence factors, increased when TgHMGB1a was overexpressed, but no significant changes were observed in TgHMGB1a B box-deficient parasites. Our findings demonstrated that TgHMGB1a is indeed a nuclear protein that maintains HMG box architectural functions and is a potential proinflammatory factor during the T.gondii infection. Further studies that clarify the functions of TgHMGB1s will increase our knowledge of transcriptional regulation and parasite virulence, and might provide new insight into host-parasite interactions for T. gondii infection.

Show MeSH
Related in: MedlinePlus