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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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Generation of a TgHMGB1a B box mutant strain.A. Schematic of the experimental design. TgHMGB1a is encoded by 4 exons, and the B box is located in the fourth exon (shown for the TgHMGB1a genomic locus). An “O-type” knock-in/knock-out vector (pTCR eGFP KO TgHMGB1a B box) was constructed to target and replace the B box domain. The vector included an approximately 2200 bp homologous arm upstream of the TgHMGB1a B box, and a monoclonal site of restriction endonuclease (BsiWI) throughout the whole vector sequence was not only needed, but also necessary in the homologous arm, and it is was best ranged around the middle of homologous frame [55]. If the targeted gene was successfully disrupted, eGFP should replace the TgHMGB1a B box domain and create a fusion protein of TgHMGB1a B box−/eGFP expressed under the control of its endogenous promoter. CAT-RFP was used as a selection marker. B. Genomic PCR analysis of the ΔKU80-TgHMGB1a B box−/eGFP strain. The position of the primers and the expected sizes are shown in panel A. C. Western blot performed with anti-TgHMGB1a 4E antibodies on total extracts from RHΔKU80 and ΔKU80-TgHMGB1a B box−/eGFP strains. TgIMP1 was used as an internal control. D. Observation of the TgHMGB1a B box−/eGFP and CAT-RFP strains by fluorescent confocal microscopy, and the result indicated that B box mutant TgHMGB1a was dispersed throughout the parasite cells. Scale bar, 3 µm.
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pone-0111993-g005: Generation of a TgHMGB1a B box mutant strain.A. Schematic of the experimental design. TgHMGB1a is encoded by 4 exons, and the B box is located in the fourth exon (shown for the TgHMGB1a genomic locus). An “O-type” knock-in/knock-out vector (pTCR eGFP KO TgHMGB1a B box) was constructed to target and replace the B box domain. The vector included an approximately 2200 bp homologous arm upstream of the TgHMGB1a B box, and a monoclonal site of restriction endonuclease (BsiWI) throughout the whole vector sequence was not only needed, but also necessary in the homologous arm, and it is was best ranged around the middle of homologous frame [55]. If the targeted gene was successfully disrupted, eGFP should replace the TgHMGB1a B box domain and create a fusion protein of TgHMGB1a B box−/eGFP expressed under the control of its endogenous promoter. CAT-RFP was used as a selection marker. B. Genomic PCR analysis of the ΔKU80-TgHMGB1a B box−/eGFP strain. The position of the primers and the expected sizes are shown in panel A. C. Western blot performed with anti-TgHMGB1a 4E antibodies on total extracts from RHΔKU80 and ΔKU80-TgHMGB1a B box−/eGFP strains. TgIMP1 was used as an internal control. D. Observation of the TgHMGB1a B box−/eGFP and CAT-RFP strains by fluorescent confocal microscopy, and the result indicated that B box mutant TgHMGB1a was dispersed throughout the parasite cells. Scale bar, 3 µm.

Mentions: To further characterize the biological role of TgHMGB1a, the B box of TgHMGB1a was targeted and replaced by eGFP in the RHΔKU80 strain using homologous recombination (Figure 5A). PCR was used to confirm both replacement of the B box and the insertion of a CAT-RFP cassette in the parasite genome (Figure 5B). Replacement of the B box of TgHMGB1a by eGFP was also confirmed using western blotting (Figure 5C). We also attempted to generate a complete knockout strain of TgHMGB1a, yet so far have been unsuccessful. Confocal microscopy showed that eGFP was distributed throughout parasite cells, but was not concentrated into the nucleus like integral TgHMGB1a (Figure 5D). Nevertheless, the absence of TgHMGB1a B box did not affect parasite growth in vitro, as TgHMGB1a B box−/eGFP parasites formed an equivalent number and size of plaques (Figure S8A) and had a replication rate similar to the RHΔKU80 strain (Figure S8B). These results suggested that the function of TgHMGB1a may be at least partially redundant, and other homologous proteins may play overlapping roles in parasites.


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)

Generation of a TgHMGB1a B box mutant strain.A. Schematic of the experimental design. TgHMGB1a is encoded by 4 exons, and the B box is located in the fourth exon (shown for the TgHMGB1a genomic locus). An “O-type” knock-in/knock-out vector (pTCR eGFP KO TgHMGB1a B box) was constructed to target and replace the B box domain. The vector included an approximately 2200 bp homologous arm upstream of the TgHMGB1a B box, and a monoclonal site of restriction endonuclease (BsiWI) throughout the whole vector sequence was not only needed, but also necessary in the homologous arm, and it is was best ranged around the middle of homologous frame [55]. If the targeted gene was successfully disrupted, eGFP should replace the TgHMGB1a B box domain and create a fusion protein of TgHMGB1a B box−/eGFP expressed under the control of its endogenous promoter. CAT-RFP was used as a selection marker. B. Genomic PCR analysis of the ΔKU80-TgHMGB1a B box−/eGFP strain. The position of the primers and the expected sizes are shown in panel A. C. Western blot performed with anti-TgHMGB1a 4E antibodies on total extracts from RHΔKU80 and ΔKU80-TgHMGB1a B box−/eGFP strains. TgIMP1 was used as an internal control. D. Observation of the TgHMGB1a B box−/eGFP and CAT-RFP strains by fluorescent confocal microscopy, and the result indicated that B box mutant TgHMGB1a was dispersed throughout the parasite cells. Scale bar, 3 µm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4219823&req=5

pone-0111993-g005: Generation of a TgHMGB1a B box mutant strain.A. Schematic of the experimental design. TgHMGB1a is encoded by 4 exons, and the B box is located in the fourth exon (shown for the TgHMGB1a genomic locus). An “O-type” knock-in/knock-out vector (pTCR eGFP KO TgHMGB1a B box) was constructed to target and replace the B box domain. The vector included an approximately 2200 bp homologous arm upstream of the TgHMGB1a B box, and a monoclonal site of restriction endonuclease (BsiWI) throughout the whole vector sequence was not only needed, but also necessary in the homologous arm, and it is was best ranged around the middle of homologous frame [55]. If the targeted gene was successfully disrupted, eGFP should replace the TgHMGB1a B box domain and create a fusion protein of TgHMGB1a B box−/eGFP expressed under the control of its endogenous promoter. CAT-RFP was used as a selection marker. B. Genomic PCR analysis of the ΔKU80-TgHMGB1a B box−/eGFP strain. The position of the primers and the expected sizes are shown in panel A. C. Western blot performed with anti-TgHMGB1a 4E antibodies on total extracts from RHΔKU80 and ΔKU80-TgHMGB1a B box−/eGFP strains. TgIMP1 was used as an internal control. D. Observation of the TgHMGB1a B box−/eGFP and CAT-RFP strains by fluorescent confocal microscopy, and the result indicated that B box mutant TgHMGB1a was dispersed throughout the parasite cells. Scale bar, 3 µm.
Mentions: To further characterize the biological role of TgHMGB1a, the B box of TgHMGB1a was targeted and replaced by eGFP in the RHΔKU80 strain using homologous recombination (Figure 5A). PCR was used to confirm both replacement of the B box and the insertion of a CAT-RFP cassette in the parasite genome (Figure 5B). Replacement of the B box of TgHMGB1a by eGFP was also confirmed using western blotting (Figure 5C). We also attempted to generate a complete knockout strain of TgHMGB1a, yet so far have been unsuccessful. Confocal microscopy showed that eGFP was distributed throughout parasite cells, but was not concentrated into the nucleus like integral TgHMGB1a (Figure 5D). Nevertheless, the absence of TgHMGB1a B box did not affect parasite growth in vitro, as TgHMGB1a B box−/eGFP parasites formed an equivalent number and size of plaques (Figure S8A) and had a replication rate similar to the RHΔKU80 strain (Figure S8B). These results suggested that the function of TgHMGB1a may be at least partially redundant, and other homologous proteins may play overlapping roles in parasites.

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