Novel roles of dense granule protein 12 (GRA12) inToxoplasma gondiiinfection

Abstract

are contributed equally to this work. AbstractDense granule protein 12 (GRA12) is implicated in a range of processes related to the establishment of Toxoplasma gondii infection, such as the formation of the intravacuolar network (IVN) within the parasitophorous vacuole (PV). This protein is also thought to be important for T. gondii-host interaction, pathogenesis, and immune evasion, but their exact roles remain unknown. In this study, the contributions of GRA12 to the molecular pathogenesis of T. gondii infection were examined in vitro and in vivo. Deletion of GRA12 in type I RH and type II Pru T. gondii strains did not affect the parasite growth and replication in vitro, however, it caused a significant reduction in the parasite virulence and tissue cyst burden in vivo. T. gondii Δgra12 mutants were more vulnerable to be eliminated by host immunity, without the accumulation of immunity-related GTPase a6 (Irga6) onto the PV membrane.The ultrastructure of IVN in Δgra12 mutants appeared normal, suggesting that GRA12 is not required for biogenesis of the IVN. Combined deletion of GRA12 and ROP18 induced more severe attenuation of virulence compared to single Δgra12 or Δrop18 mutant strains. These data suggest a functional association between GRA12 and ROP18 that is revealed by the severe attenuation of virulence in a double mutant relative to the single individual mutations. Future studies are needed to define the molecular basis of this putative association. Collectively these findings indicate that although GRA12 is not essential for the parasite growth and replication in vitro, it contributes to the virulence and growth of T. gondii in mice. K E Y W O R D Sdense granule protein 12, immunity-related GTPases, intravacuolar network, rhoptry protein 18, Toxoplasma gondii, virulence 3166 | WANG et Al. SUPPORTING INFORMATIONAdditional supporting information may be found online in the Supporting Information section. How to cite this article: Wang J-L, Bai M-J, Elsheikha HM, et al. Novel roles of dense granule protein 12 (GRA12) in Toxoplasma gondii infection.

iTRAQ-Based Phosphoproteomic Analysis of Toxoplasma gondii Tachyzoites Provides Insight Into the Role of Phosphorylation for its Invasion and Egress

“…Intriguingly, the phosphorylation level of GRA2 was detected to be up-regulated significantly in the comparison group of PE vs. JI, which suggested that phosphorylation may be one of the important mechanisms in mediating the function of GRA2. Surprisingly, both GRA3 and GRA12 were identified with enhanced phosphorylation level in this study yet were shown to be dispensable for the lytic cycle of T. gondii in vitro, however, they play essential roles in acute infection of T. gondii in vivo (Craver and Knoll, 2007;Rommereim et al, 2016;Fox et al, 2019;Wang et al, 2020). The explanation of this discrepancy may be that GRA3 and GRA12 do not play their roles independently in the lytic cycle process of tachyzoites but interdependently, just as other GRA proteins (Rommereim et al, 2016).…”

RHΔgra17Δnpt1 Strain of Toxoplasma gondii Elicits Protective Immunity Against Acute, Chronic and Congenital Toxoplasmosis in Mice

“…To achieve this, each mouse was infected i.p. with 10 3 tachyzoites (five mice/strain), and five days later, the parasite burden in the peritoneal fluid was examined by plaque assay as described previously [22]. Briefly, mice were humanely sacrificed, and peritoneal cells were harvested by lavage with phosphate buffered saline (PBS) containing 1% FBS.…”

“…Then 100 µL freshly liberated parasites were used to infect confluent HFF monolayers maintained in RPMI1640 medium for seven days. Then, T. gondii-infected HFFs were fixed and stained, and the sizes and numbers of plaques formed by the growing tachyzoites were determined using the plaque assay as described previously [22].…”

Toxoplasma gondii GRA60 is an effector protein that modulates host cell autonomous immunity and contributes to virulence

“…Corticosteroid (dexamethasone) immune suppressed or IFN‐γ −/− mice infected with parasites lacking GRA12 I/II succumb to infection. Despite its implication in modulation of IFN‐γ mediated immune responses, the GTPase effectors do not assemble on the vacuoles of parasites without GRA12 (Fox et al, 2019; Wang et al, 2020). GRA7/II together with other PVM associated GRAs were found to make up the cyst wall and membrane and shown to be important for cyst formation (Guevara, Fox, & Bzik, 2020).…”

“…Once phosphorylated, IRGs are unable to hydrolyse GTP and cannot form oligomers, hence IRG loading onto the PVM is disabled (Steinfeldt et al, 2010). GRA12, on the other hand, resides within the PV and contributes to both parasite virulence and establishment of chronic infection through modulation of IFN‐γ mediated immune responses without involving the GTPase effectors (Fox et al, 2019; Wang et al, 2020).…”