RESUMEN
Toxoplasma gondii possesses sphingolipid synthesis capabilities and is equipped to salvage lipids from its host. The contribution of these two routes of lipid acquisition during parasite development is unclear. As part of a complete ceramide synthesis pathway, T. gondii expresses two serine palmitoyltransferases (TgSPT1 and TgSPT2) and a dihydroceramide desaturase. After deletion of these genes, we determine their role in parasite development in vitro and in vivo during acute and chronic infection. Detailed phenotyping through lipidomic approaches reveal a perturbed sphingolipidome in these mutants, characterized by a drastic reduction in ceramides and ceramide phosphoethanolamines but not sphingomyelins. Critically, parasites lacking TgSPT1 display decreased fitness, marked by reduced growth rates and a selective defect in rhoptry discharge in the form of secretory vesicles, causing an invasion defect. Disruption of de novo ceramide synthesis modestly affects acute infection in vivo but severely reduces cyst burden in the brain of chronically infected mice.
Asunto(s)
Toxoplasma , Animales , Ceramidas/metabolismo , Ratones , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismoRESUMEN
Acetyl-CoA participates in post-translational modification of proteins and in central carbon and lipid metabolism in several cell compartments. In mammals, acetyl-CoA transporter 1 (AT1, also known as SLC33A1) facilitates the flux of cytosolic acetyl-CoA into the endoplasmic reticulum (ER), enabling the acetylation of proteins of the secretory pathway, in concert with the activity of dedicated acetyltransferases such as NAT8. However, the involvement of the ER acetyl-CoA pool in acetylation of ER-transiting proteins in Apicomplexa is unknown. Here, we identified homologs of AT1 and NAT8 in Toxoplasma gondii and Plasmodium berghei parasites. Proteome-wide analyses revealed widespread N-terminal acetylation of secreted proteins in both species. Such extensive acetylation of N-terminally processed proteins has not been observed previously in any other organism. Deletion of AT1 homologs in both T. gondii and P. berghei resulted in considerable reductions in parasite fitness. In P. berghei, AT1 was found to be important for growth of asexual blood stages, production of female gametocytes and male gametocytogenesis, implying its requirement for parasite transmission. In the absence of AT1, lysine acetylation and N-terminal acetylation in T. gondii remained globally unaltered, suggesting an uncoupling between the role of AT1 in development and active acetylation occurring along the secretory pathway.
Asunto(s)
Parásitos , Toxoplasma , Acetilcoenzima A/metabolismo , Acetilación , Animales , Retículo Endoplásmico/metabolismo , Femenino , Masculino , Mamíferos/metabolismo , Parásitos/metabolismo , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismoRESUMEN
Toxoplasma gondii infects virtually any nucleated cell and resides inside a non-phagocytic vacuole surrounded by a parasitophorous vacuolar membrane (PVM). Pivotal to the restriction of T. gondii dissemination upon infection in murine cells is the recruitment of immunity regulated GTPases (IRGs) and guanylate binding proteins (GBPs) to the PVM that leads to pathogen elimination. The virulent T. gondii type I RH strain secretes a handful of effectors including the dense granule protein GRA7, the serine-threonine kinases ROP17 and ROP18, and a pseudo-kinase ROP5, that synergistically inhibit the recruitment of IRGs to the PVM. Here, we characterise GRA60, a novel dense granule effector, which localises to the vacuolar space and PVM and contributes to virulence of RH in mice, suggesting a role in the subversion of host cell defence mechanisms. Members of the host cell IRG defence system Irgb10 and Irga6 are recruited to the PVM of RH parasites lacking GRA60 as observed previously for the avirulent RHΔrop5 mutant, with RH preventing such recruitment. Deletion of GRA60 in RHΔrop5 leads to a recruitment of IRGs comparable to the single knockouts. GRA60 therefore represents a novel parasite effector conferring resistance to IRGs in type I parasites, and found associated to ROP18, a member of the virulence complex.