| Toxoplasmosis is an important zoonotic parasitic disease.Toxoplasmosis can cause serious or even fatal injury to fetuses and immunocompromised humans or animals.Toxoplasma gondii(T.gondii)is an obligate intracellular parasite that can infect nucleated cells in almost all warm-blooded animals,including humans.Vesicular transport is the main mode of macromolecular transport between various organelles,and soluble N-ethylmaleimide-sensitive factor attachment protein receptors(SNAREs)proteins pair with each other to form specific SNARE complexes that mediate the fusion of vesicular membranes with target membranes.The secretion system of T.gondii consists of organelles such as microneme,rhoptry,and apicoplast,as well as the pellicle structures composed of multilayer membranes.To investigate the vesicular transport pathway between organelles in the secretory system and the molecular mechanisms of exocytosis proteins,this study was conducted to identify SNARE proteins in the secretory system by localization analysis of T.gondii SNARE proteins.Secretory proteins play an essential role in host cell invasion and egress,and the maturation process of these proteins requires transport to endosome-like compartment(ELC)for further processing.In this study,we identified SNARE proteins Tg Stx12,Tg Stx10 and Tg VAMP4-2 in the ELC.Conditional knockout strains were constructed using the AID system,and phenotypic experiments revealed that the deletion of Tg Stx12 affected the ability of the parasites to invade and egress from the host cell.The apicoplast proteins are transported to the apicoplast to synthesize a large number of essential nutrients for the parasite,and the SNARE protein Tg SNAP29,which is localized in the apicoplast.Indirect immunofluorescence assay(IFA)demonstrated that deletion of Tg SNAP29 affected the correct localization of apicoplast proteins and caused the apicoplast loss;Tg ATG8 was transported to the apicoplast by vesicle transport mediated by Tg SNAP29-Tg VAMP4-2 complex as demonstrated by Coimmunoprecipitation(Co-IP)and IFA experiments.Membrane proteins are transported to the plasma membrane of the parasite to facilitate the exchange of material between the parasite and the external environment.In this study,SNARE protein Tg Stx1,Tg Stx20 and Tg Stx21 were found to be localized in the plasma membrane and apical annuli;Co-IP experiments demonstrated that Tg Stx20 and Tg Stx21 formed an unconventional SNARE complex with the conserved Tg Stx1;IFA experiments demonstrated that the absent of this complex affected the correct localization of membrane proteins in parasites.Metabolomics experiments proved that the absence of Tg Stx21 affected the central carbon source pathway of glycolysis;Living cell experiments dynamically observed that this complex was enriched on the apical annuli and participate in the vesicle fusion process in coordination with Tg Rab11 A targeting the apical annuli transport.This study analyzed the molecular mechanism of vesicle fusion in the secretion pathway of T.gondii by studying the function of vesicle fusion-related proteins in the secretion system of T.gondii,and enhanced the understanding of the molecular mechanism of vesicle fusion of T.gondii specific organelles.The discovery of SNARE proteins localized in ELC strengthened the understanding of the transport mechanism of secreted proteins in T.gondii.We identified key SNARE proteins involved in apicoplasttargeting transport,improved the understanding of the vesicle transport pathway of nuclear-encoded protein(NEAT)targeting to apicoplast;Discovered transmembrane proteins localized in the plasma membrane and apical annuli structure,which revealed that the apical annuli may be part of a closed membrane structure or a closed compartment,and breakthrough the understanding of the apical annuli structure. |
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