Identification Of Alkyl Hydroperoxide Reductase 1 AHP1 Gene Of Toxoplasma Gondii And Its Protein Function

Toxoplasma gondii is an obligate intracellular parasitic protozoan,which can parasitize the nucleated cells of almost all warm-blooded animals and humans,and is a serious zoonotic pathogen.Therefore,it is very necessary to study the pathogenic mechanism of T.gondii and provide effective prevention and control methods.The process of invading the host must adapt to the oxidative erosion of the host cell.To cope with this challenge,T.gondii have developed a variety of antioxidant strategies.The use of peroxidase system to participate in the regulation of cell redox reactions is one of the important ways.At present,a variety of peroxidases have been found in T.gondii.They have different catalytic mechanisms.They catalyze the degradation of H₂O₂ to generate H₂O,making T.gondii possess a certain ability to resist oxidative stress.The results of bioinformatics analysis in the early stage of this study showed that Alkyl Hydroperoxide reductase 1（Ahp1）is a member of the Peroxiredoxins（Prxs）peroxidase family and belongs to the Prx5 subfamily.Therefore,this study aims to use gene editing and other methods to identify the Ahp1 gene of T.gondii;to clarify the oxidoreductase activity of TgAhp1;to clarify the influence of Ahp1-mediated oxidative stress on the growth,development and pathogenicity of T.gondii.The specific work includes the following aspects:Use bioinformatics analysis software to systematically analyze the gene structure,amino acid sequence characteristics,conservative motifs and spatial structure of TgAhp1,identify the Ahp1 gene and preliminarily explain the function of TgAhp1;prokaryotic expression of TgAhp1 protein,and in vitro enzyme activity The determination of AHP1 protein had catalase activity.The conservative motif of the Prx family was two cysteines,with point mutations Cys¹⁶⁶ and Cys¹⁹¹ respectively.After prokaryotic expression and in vitro enzyme activity determination,it was found that Cys¹⁶⁶ is the key enzyme active site of the enzyme,indicating that the enzyme followed the cysteine-dependent redox process.Real-time qPCR was used to screen sensitive peroxides that affect the transcription of TgAhp1.Using CRISPR/Cas9 technology,we successfully screened a monoclonal strain that knocked out the TgAhp1 gene.Invasion,proliferation and plaque experiments showed that knocking out the Ahp1 gene had no significant changes in the invasion and proliferation ability of T.gondii;mouse pathogenicity experiments showed that knocking out the Ahp1 gene had no significant effect on the pathogenicity of mice.The results of subsequent oxidative stress experiments showed that the△Ahp1 strain was more sensitive to tert-butyl hydrogen peroxide（tBOOH）than hydrogen peroxide（H₂O₂）,indicating that tBOOH may be a substrate of AHP1.Under tBOOH culture conditions,the invasion,proliferation,and pathogenicity of the△Ahp1strain to mice were significantly lower than that of the RH△Ku80 strain.This was mainly due to the induction of tBOOH,which caused the knockout strain to lose the Ahp1 gene.Makes the level of active oxygen in the insects rise,and finally leads to the apoptosis of the insects.In order to clarify the location of AHP1 in T.gondii,we used CRISPR/Cas9 technology-assisted site-directed insertion method.We fused and expressed 10 HA tags at the C-terminus of TgAhp1,and successfully constructed an endogenous marker Ahp1 strain.The results showed that the Ahp1 protein was localized in the cytoplasm of the parasite.This study identified the Ahp1 gene of Toxoplasma gondii and made it clear that TgAHP1is a peroxisome protein with cysteine-dependent peroxidase activity.The effects of Ahp1protein-mediated oxidative stress on the growth,development and pathogenicity of T.gondii were clarified.