Study On The Function Of Toxoplasma Gondii SM Family Proteins As Drug Targets

Toxoplasma gondii is an obligate intracellular parasitic protozoan parasite that can cause infections in humans and many animals.When the cysts of T.gondii are ingested by humans,the parastes will develop from bradyzoites to tachyzoites,and eventually infect humans through the blood.Traditional anti-toxoplasma drugs have defects such as large side effects,so there is an urgent need to develop novel efficient drugs.Recent research proves that T.gondii has a highly polarized secretion system,and uses it to secrete proteins outside or into host cells.These secreted proteins play a crucial role in the process of invasion and infection establishment of the parasites.The transporting vesicles distributed by the postGolgi network carry proteins targeting to the plasma membrane,the sub-plasma network and the apical secretory organelles.How T.gondii completes vesicle transportation is unclear.SNARE complex is a key complex for vesicle fusion,which drives membrane fusion by forming a zipper structure.Sec1/Munc18-like(SM)proteins are key element proteins that regulates SNARE complex.Bioinformatics analysis revealed that T.gondii encodes four SM proteins(TgVps45,TgSec1,TgVps33 and TgSly1).In this study,through CRISPR/Cas9 and plant auxin-induced protein degradation system(AID),we successfully constructed T.gondii TgVps45 and TgSec1-conditional knockout strains.Through indirect immunofluorescence assays,a part of TgVps45 of T.gondii was found in the compartment between TGN and ELC.The loss of TgVps45 led to the inability of the inner membrane complex to assemble properly,organization disorder of the microtubule structure,and morphological changes of the progeny parasites,which will affect the division and proliferation of T.gondii.At the same time,loss of TgVps45 affected the correct delivery of microneme proteins and dense granule proteins.Further research found that TgVps45 could interact with the amino-terminal domain of TgStx16 and positively regulate the function of SNARE complex.Deletion of TgStx16 by AID method can also inhibit the growth of parasites and similar phenotypes as described above.TgSec1 is diffusely distributed in the entire cytoplasm of the T.gondii.Its location did not change under the treatment of BFA,but its expression level reduced.The loss of TgSec1 did not affect the secretion of microneme proteins and dense granule proteins,but affects the biosynthesis of the plasma membrane,which in turn led to the death of the progeny T.gondii.This study revealed that TgVps45 and TgSec1 are involved in the transporting process of secreted proteins,and proved their potential as drug targets.The results of this study provided a theoretical basis for the development of new therapies for toxoplasmosis.