Screening And Identification Of Interaction Proteins In Mice Brain Injuries Caused By Toxoplasmagondii Chronic Infection

Toxoplasma gondii(T.gondii)is an opportunistic protozoan that causes the cosmopolitan zoonosis Toxoplamosis,mainly through oral infections,blood infections and diaplacental infections.The fast replicating tachyzoites could infect a broad spectrum of nucleated cells with a preference to neurons,and lead to toxoplasma encephalitis(TE).Around 30% of the world population is considered to have latent infection with T.gondii,more than 90% patients can die of TE,and the burden of secondary paralysis is high.In recent years,TE has become a public health issue of concern.Patients of TE can develop neurological symptoms with both focal and diffuse neurological lesions,while mental symptoms and behavior disorders are frequently accompanied,like Alzheimer's disease(AD).All the three genotypes(I,II,III)of T.gondii cause worldwide toxoplasmosis,but the genotype II is predominant in human toxoplasmosis.T.gondii prugniaud(PRU)strain is one of genotype II attenuated strain isolated from human.It is neurotropic and can enter the central nervous system(CNS)through the blood-brain barrier(BBB),then damage the discrimination and memory function of host by invading nerve cells.There are several reasons behind these alterations,for instance,the neurotropism of tachyzoites,the distribution of cysts in neurons and the brain lesions associated with the parasites.Though we have obtained substantial information from these studies,the research on the interaction mechanism between T.gondii and the host CNS is largely confined to serological detection,knowledge about the exact proteins in host brains interfered by T.gondii and how these notable proteins induce CNS injuries is very limited.Isobaric tagging for relative and absolute quantitation(iTRAQ?Applied Biosystems,AB Sciex,Foster C ity,CA)combined with LC-MS /MS can accurately detect hydrophobic and low-abundancy proteins in cells and organe lles under various physiological or environmental conditions,which breaks most limitations of gel-based methods.In this study,we established mice models of T.gondii chronic infection and perform a global unbiased quantitative proteomic analysis of brain tissues from infected mice and healthy mice by i TRAQ labeling and LC-MS/MS.In addition,we validated part of the significant proteins tested with i TRAQ methods using q RT-PCR and WB.We detected 4,983 proteins,out of which 461 proteins were differentially expressed(>/=2.0-fold,p< 0.05)when the infected brains compared to control brain tissues.Pathway analysis indicated the deregulation of several pathways re lated to metabolism and neurological processes.Afterwards,taking Drebrin as the research object,we conducted an immunofluorescence experiment to validate the significant downregulation of Drebrin in murine neuroblastoma cells(N2a)infected with T.gondii PRU trachyzoits.We screened 6T.gondii proteins that interact with Drebrin by co-immunoprecipitation(Co-IP)and GST Pull-down,such as heat shock protein HSP60,Tubulin alpha chain,small GTPase Rab2.The study can contribute to a better understanding o f molecular mechanisms underlying the host–parasite relationship in chronic infection of T.gondii.