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.
Highlights
High mobility group box 1 (HMGB1) was first discovered in calf thymi as a nuclear protein that contained a unique DNA-binding domain and showed rapid migration in polyacrylamide gels, a property of the HMG superfamily
Phylogenetic analysis showed that TgHMGB1a, b and c could be classified into three significant clusters through both the neighbor-joining (NJ) and maximum likelihood (ML) algorithms (Figure S1), which most because for different conservation of HMG domain in their C-terminal
The three-dimensional (3D) structures of the HMG box domains of TgHMGB1a, b and c were homology modeled with the SWISS-MODEL server using M. musculus 2gzkA as a template structure
Summary
High mobility group box 1 (HMGB1) was first discovered in calf thymi as a nuclear protein that contained a unique DNA-binding domain and showed rapid migration in polyacrylamide gels, a property of the HMG superfamily. Mammalian HMGB1 encodes 219 amino acids (aa) and contains two DNA-binding motifs (A-box and B-box) that are arranged in tandem, following a long negatively charged C-terminus that is rich in aspartic and glutamic acids, which differ in length (HMGB1–3) or are absent (HMGB4) [2]. Interactions between DNA and HMGs are mediated by basic amino acid residues of the protein. Structural studies using nuclear magnetic resonance spectroscopy established that the DNA binding domain of HMG has an L-shaped structure made of three a-helices that provide surfaces for potential interactions with both DNA and protein [2,6]
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