A Toxoplasma gondii O-glycosyltransferase that modulates bradyzoite cyst wall rigidity is structurally and functionally distinct from host homologues

Infection with the Apicomplexan protozoan Toxoplasma gondii can be life-threatening in immunocompromised hosts. Transmission frequently occurs through the oral ingestion of T. gondii bradyzoite cysts, which transition to tachyzoites, disseminate, and then form cysts containing bradyzoites in the central nervous system, resulting in latent infection. There are currently no effective treatments to cure latent infection. Bradyzoites are encapsulated by a cyst wall that is critical for immune evasion, survival, and transmission. Cyst wall rigidity is influenced by the O-glycosylation of the mucin domain of the cyst wall protein CST1 by mucin-type O-glycosyltransferases (GalNAc-Ts). Here, we report the first structures of a protozoan GalNAc-T, T.gondii-GalNAc-T3 in the apo state and in complex with glycopeptide substrates. The structures reveal features that are strictly conserved in Apicomplexan homologues of T.gondii-GalNAc-T3, including a unique 2nd metal binding site that is coupled to substrate binding and enzymatic activity in vitro and cyst wall O-glycosylation in T. gondii. Additional structural features illustrate the divergence of GalNAc-Ts from parasite to host and highlight multiple druggable sites in T.gondii-GalNAc-T3 and its homologues in Apicomplexa that are responsible for a wide range of parasitic diseases.

顶复门原虫刚地弓形虫感染在免疫功能低下的宿主中可能危及生命。传播通常通过口服摄入刚地弓形虫缓殖子包囊发生，这些包囊转变为速殖子，扩散，然后在中枢神经系统中形成含有缓殖子的包囊，导致潜伏感染。目前没有有效的治疗方法来治愈潜伏感染。缓殖子被包囊壁包裹，这对免疫逃避、生存和传播至关重要。包囊壁的刚性受粘蛋白型O -糖基转移酶（GalNAc - Ts）对包囊壁蛋白CST1的粘蛋白结构域进行O -糖基化的影响。在此，我们报道了原虫GalNAc - T（刚地弓形虫 - GalNAc - T3）在无底物状态以及与糖肽底物复合时的首批结构。这些结构揭示了在刚地弓形虫 - GalNAc - T3的顶复门同源物中严格保守的特征，包括一个独特的第二金属结合位点，该位点在体外与底物结合和酶活性以及刚地弓形虫的包囊壁O -糖基化相关。其他结构特征说明了GalNAc - Ts从寄生虫到宿主的差异，并突出了刚地弓形虫 - GalNAc - T3及其在顶复门中的同源物中多个可成药位点，这些位点与多种寄生虫疾病有关。