Identification And Characterization Of High Mobility Group Box1Protein In Toxoplasma Gondii

High mobility group box1(HMGBl) is a nuclear factor that usually binds DNA and modulates gene expression in multicellular organisms. It plays variety roles while it released to the extracellular media, such as proinflammatory and immune regulatory, through interacted with different receptors on the cell surface, and blocking the HMGB1secrection pathway or neutralization of the extracellular HMGB1have been selected as the target to therapy of inflammatory diseases. Three HMGB1orthologs were predicted in the genome of Toxoplasmagondii, 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. To reveal the functions and mechanisms of TgHMGB1in the development of T.gondii, we cloned the TgHMGB1a, most homologlized to the mammalian HMGB1, and carry out the comparative study between TgHMGB1a and mice HMGB1. Furthermore, we are interesting to the possibility of the host HMGB1changes induced by T.gondii infection, and we explored the possible mechanisms.Firstly, we prepared the mouse anti-TgHMGBla abs, cloned TgHMGBla, western blot shown that TgHMGB1a codes a34kD protein in T.gondii, and immunofluorescence assay indicated TgHMGB1a concentrated in the nucleus of intracellular tachyzoites during the whole process of endogenesis in the three genotype parasites, but translocated into the cytoplasm while the parasites release to extracellular. We demonstrated that the TgHMGBla binds to cruciform DNA in electrophoretic mobility shift assays (EMSA). And the recombinant TgHMGB la B box can stimulates macrophages to release TNF-a, which is enhanced by it bound DNAs fragments from the E.coli genome.To reveal the functions of endogenous TgHMGB1a, we generated TgHMGB1a overexpress and mutated strains. The results showed that there were no significant phenotypic changes when the TgHMGBla B box was deleted, while transgenic parasites that overexpressed TgHMGB1a showed slower intracellular growth and caused delayed death in mouse, further quantitative RT-PCR and chromatin immunoprecipitation (ChIP) analyses suggested that TgHMGB1a regulated the transcription of many important genes, including ROP18、Toxofin、PLP1、GRA7、Profilin and so on, which were enhanced when TgHMGBla was overexpressed, but no significant changes in TgHMGBla B box-deficient parasites, while the transcription level of TgHMGB1b was increased. And these regulation probably dependent on that TgHMGB1a binds to the promoters of the indicated genes directly. Our findings demonstrated that TgHMGB1a is indeed a nuclear factor that maintains HMG box architectural functions which involved to transcription regulatory, most likely as an activator of transcription.To investigate the potential functions of host HMGB1in inflammatory responses during the T.gondii infection, we examined the transcription, expression and secretion of HMGB1in T.gondii infection, and finally, we found that the transcription and translation of miceHMGB1in the IFN-γ treated anal cells and mice peritoneal exudated cells were raised and then declined after T.gondii infection. Furthermore, HMGB1were released from mice intraperitoneal lavage macrophages after T.gondii infection, which can be enhanced by IFN-y, and the inhibition experiment suggested that this release most likely related to the NLRPl-Caspase1-inflammasome pathway. However, the functions of extracellular HMGB1and the mechanisms need to be more investigated in the toxopalsosis, probably especially in the encephalitis in chronic infection, and might provide new insight into host-parasite interactions for T. gondii infection.