A Nuclear Factor of High Mobility Group Box Protein in Toxoplasma gondii

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.

Rhoptry protein 5 (ROP5) Is a Key Virulence Factor in Neospora caninum

“…After digestion with NotI (NEB, Beijing), the linearized pTCR-NcROP5-CD KO plasmids were purified using ethanol precipitation and then resuspended using cytomix (Hui et al, 2014). Nc-1 tachyzoites (1 × 10 7 ), mixed with 50 μg of the above plasmids, were transferred to a 0.4 mm gap cuvette and electroporated with 2 kV at 25 μFd and 50 Ω with the Gene Pulser Xcell electroporation system (Bio-Rad, USA).…”

“…For complementation analysis, pDMG plasmid (Hui et al, 2014; Tao et al, 2014; Li et al, 2016) was used to express NcROP5 in the ΔNcROP5 strain. The complete coding sequence of NcROP5 was amplified using primers F3/R3 (Supplementary Table S1) that appended the flanking EcoRV and AvrII restriction endonuclease sites.…”

“…Nc-GFP strain (kindly provided by Professor Xuenan Xuan, Obihiro University of Agriculture and Veterinary Medicine, Japan), as a null vector constructing group, which is a transgenic parasite by transfecting Nc-1 wild-type strain with the pDMG plasmid, was propagated as tachyzoites by serial passages in HFF cells as previously described (Gaskell et al, 2009). The plaque assay was performed as previously described (Hui et al, 2014). Five hundred parasites were added to each well of a 6-well plate containing confluent host cell HFF monolayers.…”

“…cDNA was synthesized using the EasyScript First-Strand cDNA Synthesis SuperMix kit (TransGen, China). Specific primers were designed using Primer Premier 5.0 (Hui et al, 2014), including primers for rhoptry necks ( RON2 and RON4 ), rhoptrys ( ROP4, ROP5, ROP7, ROP16 , and ROP17 ), dense granules ( GRA2, GRA6 , and GRA7 ) and the endogenous reference gene NcActin (Supplementary Table S1). The specificity of these primers was evaluated using conventional quantitative real-time PCR (qRT-PCR).…”

“…We suspected that another rhoptry gene of N. caninum might play a crucial role in virulence. Transgenic strains of T. gondii and Plasmodium falciparum of Apicomplexa parasites have been widely used to study the functions of parasite proteins (Ghorbal et al, 2014; Hui et al, 2014; Shen et al, 2014; Hammoudi et al, 2015; Williams et al, 2015; Li et al, 2016). However, no laboratory strains and genome-editing techniques have been developed and no transgenic strains constructed for the study of protein functions of N. caninum .…”