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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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TgHMGB1a involves to gene transcription regulatory and overexpress but not disrupt TgHMGB1a can delayed the death of mice.A and B. Quantitative RT-PCR was used to analyze the transcription levels of the indicated genes in TgHMGB1a overexpression (A) and B box-deficient (B) parasites compared with their parental strains. Each bar indicates the relative quantity (RQ) ± SD. RQs of the transgenic parasites were calibrated using their parental strains (i.e., ΔRQtransgenic parasite = RQtransgenic parasite – RQparental strain( = 1)). Data presented are representative of three independent experiments, each done in triplicate. Statistical significance was determined using Student's t-test (*P<0.1, **P<0.05, ***P<0.005). C and D. Analysis of TgHMGB1a binds to promoters of indicated genes through chromatin immunoprecipitation (ChIP). C. Regular PCR was performed on the ChIP DNAs from the TgHMGB1a overexpress and RH strains using the promoter-specific primers of indicated genes, normal mice sera, input DNA and TgHMGB1a B box−/eGFP strain were used as ChIP controls, ROP18 coding region was also tested as negative control. PCR products were run on 1.5% agarose gels. D. Quantitative real-time PCR was carried out and the pull-down promoters in RH and TgHMGB1a overexpress strains were normalized against the corresponding input DNA, the promoters level in TgHMGB1a overexpress parasites were represented as log2 functions of relative ratios to RH strain. Data presented are representative of three independent experiments, each done in triplicate. Analysis was carried out using Student's t-test. *P<0.1, **P<0.01, ***P<0.001. E and F. Mouse survival curves for parental and TgHMGB1a transgenic lines. Balb/c mice were separately injected i.p. with the indicated parasites and doses. Mean values shown per group (n = 5), representative of 3 experiments with similar outcomes. Mice infected with the TgHMGB1a overexpression showed a significantly delayed time to death (3 to 5 days) in a low doses (102 and 103) infection compared to its parental RH or RH-GFP strains (P<0.0001 was considered as statistically significant difference, see the Data S1).
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pone-0111993-g007: TgHMGB1a involves to gene transcription regulatory and overexpress but not disrupt TgHMGB1a can delayed the death of mice.A and B. Quantitative RT-PCR was used to analyze the transcription levels of the indicated genes in TgHMGB1a overexpression (A) and B box-deficient (B) parasites compared with their parental strains. Each bar indicates the relative quantity (RQ) ± SD. RQs of the transgenic parasites were calibrated using their parental strains (i.e., ΔRQtransgenic parasite = RQtransgenic parasite – RQparental strain( = 1)). Data presented are representative of three independent experiments, each done in triplicate. Statistical significance was determined using Student's t-test (*P<0.1, **P<0.05, ***P<0.005). C and D. Analysis of TgHMGB1a binds to promoters of indicated genes through chromatin immunoprecipitation (ChIP). C. Regular PCR was performed on the ChIP DNAs from the TgHMGB1a overexpress and RH strains using the promoter-specific primers of indicated genes, normal mice sera, input DNA and TgHMGB1a B box−/eGFP strain were used as ChIP controls, ROP18 coding region was also tested as negative control. PCR products were run on 1.5% agarose gels. D. Quantitative real-time PCR was carried out and the pull-down promoters in RH and TgHMGB1a overexpress strains were normalized against the corresponding input DNA, the promoters level in TgHMGB1a overexpress parasites were represented as log2 functions of relative ratios to RH strain. Data presented are representative of three independent experiments, each done in triplicate. Analysis was carried out using Student's t-test. *P<0.1, **P<0.01, ***P<0.001. E and F. Mouse survival curves for parental and TgHMGB1a transgenic lines. Balb/c mice were separately injected i.p. with the indicated parasites and doses. Mean values shown per group (n = 5), representative of 3 experiments with similar outcomes. Mice infected with the TgHMGB1a overexpression showed a significantly delayed time to death (3 to 5 days) in a low doses (102 and 103) infection compared to its parental RH or RH-GFP strains (P<0.0001 was considered as statistically significant difference, see the Data S1).

Mentions: Quantitative RT-PCR analysis of 1×107 transgenic and their parental parasites were conducted to determine the potential involvement of TgHMGB1a in transcriptional regulation. Our quantitative RT-PCR analysis indicated that ROP18, toxofilin, PLP, MIC3, profilin, and GRA7, but not ROP16, were up regulated at least 1.5-fold in TgHMGB1a overexpression parasites (Figure 7A), yet there were no obvious changes observed in the TgHMGB1a B box−/eGFP strain. However, a dramatic increase of ROP16 expression was detected in the TgHMGB1a B box−/eGFP strain (Figure 7B), even though profilin expression was reduced. Expression of the TgHMGB1a homologues, TgHMGB1b and TgHMGB1c, were also measured. TgHMGB1b and TgHMGB1c were almost undetectable and similarly expressed in transgenic and parental strains, while TgHMGB1a was overexpressed (Figure 7A). However, in the TgHMGB1a B box−/eGFP parasites, TgHMGB1b showed a significant increase, whereas TgHMGB1c showed a nonsignificant trend towards reduced expression (Figure 7B). Furthermore, ChIP-qPCR analysis showed that promoter levels of PLP1, profilin, ROP16, ROP18 and Toxofilin in the pull down of TgHMGB1a overexpress strain are significant higher than RH strain, although MIC3 and GRA7 are not obvious (Figure 7C and D). Meanwhile, there are almost no signals in TgHMGB1a B box−/eGFP parasites and in the negative controls (Figure 7C). Moreover, we also tested the coding regions through the regular and real-time PCR, and results showed no significant difference between the wild type and TgHMGB1a overexpress strain (Figure 7C bottom panel and data not shown). These results suggested that TgHMGB1a overexpress enhanced the promoters binding ability, that is generally consistent with the transcription assays of these indicated genes (Figure 7A), demonstrating that TgHMGB1a plays role in regulating gene expression, and which is most like an activator of transcription and its roles might be somewhat redundant among HMG family members.


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 involves to gene transcription regulatory and overexpress but not disrupt TgHMGB1a can delayed the death of mice.A and B. Quantitative RT-PCR was used to analyze the transcription levels of the indicated genes in TgHMGB1a overexpression (A) and B box-deficient (B) parasites compared with their parental strains. Each bar indicates the relative quantity (RQ) ± SD. RQs of the transgenic parasites were calibrated using their parental strains (i.e., ΔRQtransgenic parasite = RQtransgenic parasite – RQparental strain( = 1)). Data presented are representative of three independent experiments, each done in triplicate. Statistical significance was determined using Student's t-test (*P<0.1, **P<0.05, ***P<0.005). C and D. Analysis of TgHMGB1a binds to promoters of indicated genes through chromatin immunoprecipitation (ChIP). C. Regular PCR was performed on the ChIP DNAs from the TgHMGB1a overexpress and RH strains using the promoter-specific primers of indicated genes, normal mice sera, input DNA and TgHMGB1a B box−/eGFP strain were used as ChIP controls, ROP18 coding region was also tested as negative control. PCR products were run on 1.5% agarose gels. D. Quantitative real-time PCR was carried out and the pull-down promoters in RH and TgHMGB1a overexpress strains were normalized against the corresponding input DNA, the promoters level in TgHMGB1a overexpress parasites were represented as log2 functions of relative ratios to RH strain. Data presented are representative of three independent experiments, each done in triplicate. Analysis was carried out using Student's t-test. *P<0.1, **P<0.01, ***P<0.001. E and F. Mouse survival curves for parental and TgHMGB1a transgenic lines. Balb/c mice were separately injected i.p. with the indicated parasites and doses. Mean values shown per group (n = 5), representative of 3 experiments with similar outcomes. Mice infected with the TgHMGB1a overexpression showed a significantly delayed time to death (3 to 5 days) in a low doses (102 and 103) infection compared to its parental RH or RH-GFP strains (P<0.0001 was considered as statistically significant difference, see the Data S1).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4219823&req=5

pone-0111993-g007: TgHMGB1a involves to gene transcription regulatory and overexpress but not disrupt TgHMGB1a can delayed the death of mice.A and B. Quantitative RT-PCR was used to analyze the transcription levels of the indicated genes in TgHMGB1a overexpression (A) and B box-deficient (B) parasites compared with their parental strains. Each bar indicates the relative quantity (RQ) ± SD. RQs of the transgenic parasites were calibrated using their parental strains (i.e., ΔRQtransgenic parasite = RQtransgenic parasite – RQparental strain( = 1)). Data presented are representative of three independent experiments, each done in triplicate. Statistical significance was determined using Student's t-test (*P<0.1, **P<0.05, ***P<0.005). C and D. Analysis of TgHMGB1a binds to promoters of indicated genes through chromatin immunoprecipitation (ChIP). C. Regular PCR was performed on the ChIP DNAs from the TgHMGB1a overexpress and RH strains using the promoter-specific primers of indicated genes, normal mice sera, input DNA and TgHMGB1a B box−/eGFP strain were used as ChIP controls, ROP18 coding region was also tested as negative control. PCR products were run on 1.5% agarose gels. D. Quantitative real-time PCR was carried out and the pull-down promoters in RH and TgHMGB1a overexpress strains were normalized against the corresponding input DNA, the promoters level in TgHMGB1a overexpress parasites were represented as log2 functions of relative ratios to RH strain. Data presented are representative of three independent experiments, each done in triplicate. Analysis was carried out using Student's t-test. *P<0.1, **P<0.01, ***P<0.001. E and F. Mouse survival curves for parental and TgHMGB1a transgenic lines. Balb/c mice were separately injected i.p. with the indicated parasites and doses. Mean values shown per group (n = 5), representative of 3 experiments with similar outcomes. Mice infected with the TgHMGB1a overexpression showed a significantly delayed time to death (3 to 5 days) in a low doses (102 and 103) infection compared to its parental RH or RH-GFP strains (P<0.0001 was considered as statistically significant difference, see the Data S1).
Mentions: Quantitative RT-PCR analysis of 1×107 transgenic and their parental parasites were conducted to determine the potential involvement of TgHMGB1a in transcriptional regulation. Our quantitative RT-PCR analysis indicated that ROP18, toxofilin, PLP, MIC3, profilin, and GRA7, but not ROP16, were up regulated at least 1.5-fold in TgHMGB1a overexpression parasites (Figure 7A), yet there were no obvious changes observed in the TgHMGB1a B box−/eGFP strain. However, a dramatic increase of ROP16 expression was detected in the TgHMGB1a B box−/eGFP strain (Figure 7B), even though profilin expression was reduced. Expression of the TgHMGB1a homologues, TgHMGB1b and TgHMGB1c, were also measured. TgHMGB1b and TgHMGB1c were almost undetectable and similarly expressed in transgenic and parental strains, while TgHMGB1a was overexpressed (Figure 7A). However, in the TgHMGB1a B box−/eGFP parasites, TgHMGB1b showed a significant increase, whereas TgHMGB1c showed a nonsignificant trend towards reduced expression (Figure 7B). Furthermore, ChIP-qPCR analysis showed that promoter levels of PLP1, profilin, ROP16, ROP18 and Toxofilin in the pull down of TgHMGB1a overexpress strain are significant higher than RH strain, although MIC3 and GRA7 are not obvious (Figure 7C and D). Meanwhile, there are almost no signals in TgHMGB1a B box−/eGFP parasites and in the negative controls (Figure 7C). Moreover, we also tested the coding regions through the regular and real-time PCR, and results showed no significant difference between the wild type and TgHMGB1a overexpress strain (Figure 7C bottom panel and data not shown). These results suggested that TgHMGB1a overexpress enhanced the promoters binding ability, that is generally consistent with the transcription assays of these indicated genes (Figure 7A), demonstrating that TgHMGB1a plays role in regulating gene expression, and which is most like an activator of transcription and its roles might be somewhat redundant among HMG family members.

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