Divergent kinase regulates membrane ultrastructure of the
            <i>Toxoplasma</i>
            parasitophorous vacuole

Beraki, Tsebaot; Hu, Xiaoyu; Broncel, Malgorzata; Young, Joanna C.; O’Shaughnessy, William; Borek, Dominika; Treeck, Moritz; Reese, Michael L.

doi:10.1073/pnas.1816161116

Cited by 47 publications

(75 citation statements)

References 72 publications

(103 reference statements)

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“…Recent reports have experimentally determined these two to be non-rhoptry-localizing proteins. This is consistent with our observation of discordance between their and known ROPs' expression profiles along the pseudotime 42 . Through analysis of pseudotime clustering, we also identified genes not annotated as ROPs within the ROP-dominated cluster 3, such as TGGT1_218270 and TGGT1_230350, that have recently been shown to encode bona fide rhoptry and rhoptry neck proteins, now designated as ROP48 and RON11, respectively 43,44 (left panel in Figure 2 -Supplementary Figure 3f).…”

Section: Technical Validation Of Single-parasite Sorting and Sequencingsupporting

confidence: 93%

A single-parasite transcriptional atlas of Toxoplasma Gondii reveals novel control of antigen expression

Xue

Theisen

Rastogi

et al. 2020

eLife

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Toxoplasma gondii, a protozoan parasite, undergoes a complex and poorly understood developmental process that is critical for establishing a chronic infection in its intermediate hosts. Here, we applied single-cell RNA-sequencing (scRNA-seq) on >5,400 Toxoplasma in both tachyzoite and bradyzoite stages using three widely studied strains to construct a comprehensive atlas of cell-cycle and asexual development, revealing hidden states and transcriptional factors associated with each developmental stage. Analysis of SAG1-related sequence (SRS) antigenic repertoire reveals a highly heterogeneous, sporadic expression pattern unexplained by measurement noise, cell cycle, or asexual development. Furthermore, we identified AP2IX-1 as a transcription factor that controls the switching from the ubiquitous SAG1 to rare surface antigens not previously observed in tachyzoites. In addition, comparative analysis between Toxoplasma and Plasmodium scRNA-seq results reveals concerted expression of gene sets, despite fundamental differences in cell division. Lastly, we built an interactive data-browser for visualization of our atlas resource.

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Section: Technical Validation Of Single-parasite Sorting and Sequencingsupporting

confidence: 93%

A single-parasite transcriptional atlas of Toxoplasma Gondii reveals novel control of antigen expression

Xue

Theisen

Rastogi

et al. 2020

eLife

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“…Similar to GRA4 and GRA9, deletion of GRA12 did not affect the normal ultrastructure of the IVN. Recently, a secreted kinase WNG1 was identified in T. gondii as an important regulator of tubular membrane biogenesis, and can phosphorylate GRA proteins involved in IVN biogenesis . WNG1‐dependent phosphorylation of the GRA proteins is important for the proper formation of the IVN.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2020

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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.

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“…This indicates that other proteins or posttranslational modifications are involved in SFP1 regulation in parasites upon stage conversion. Recently, the PV-localized kinase WNG1/ROP35 was shown to phosphorylate several GRAs, including GRA6, and to regulate their membrane association (21). As many GRAs form complexes during trafficking (34), WNG1 phosphorylation is hypothesized to release them from association with a chaperone, freeing them to associate with membranes.…”

Section: Downloaded Frommentioning

confidence: 99%

“…A previous phosphoproteome analysis revealed that a large number of Toxoplasma secreted proteins are phosphorylated after their release (20), raising the intriguing possibility that their function is dynamically regulated. Indeed, the PV-localized kinase WNG1 was shown to contribute to the formation of a functional IVN (21). Furthermore, it was recently shown that IVN-associated GRAs show dynamic localization patterns as the parasite remodels the PV to form the chronic-stage cyst (22).…”

mentioning

confidence: 99%

Phosphorylation of Toxoplasma gondii Secreted Proteins during Acute and Chronic Stages of Infection

Young

Broncel

Teague

et al. 2020

mSphere

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The intracellular parasite Toxoplasma gondii resides within a membrane-bound parasitophorous vacuole (PV) and secretes an array of proteins to establish this replicative niche. It has been shown previously that Toxoplasma secretes kinases and that numerous proteins are phosphorylated after secretion. Here, we assess the role of the phosphorylation of strand-forming protein 1 (SFP1) and the related protein GRA29, two secreted proteins with unknown function. We show that both proteins form stranded structures in the PV that are independent of the previously described intravacuolar network or actin. SFP1 and GRA29 can each form these structures independently of other Toxoplasma secreted proteins, although GRA29 appears to regulate SFP1 strands. We show that an unstructured region at the C termini of SFP1 and GRA29 is required for the formation of strands and that mimicking the phosphorylation of this domain of SFP1 negatively regulates strand development. When tachyzoites convert to chronic-stage bradyzoites, both proteins show a dispersed localization throughout the cyst matrix. Many secreted proteins are reported to dynamically redistribute as the cyst forms, and secreted kinases are known to play a role in cyst formation. Using quantitative phosphoproteome and proteome analyses comparing tachyzoite and early bradyzoite stages, we reveal widespread differential phosphorylation of secreted proteins. While we found no direct evidence for phosphorylation playing a dominant role for SFP1/GRA29 redistribution in the cyst, these data support a model in which secreted kinases and phosphatases contribute to the regulation of secreted proteins during stage conversion. IMPORTANCE Toxoplasma gondii is a common parasite that infects up to one-third of the human population. Initially, the parasite grows rapidly, infecting and destroying cells of the host, but subsequently switches to a slow-growing form and establishes chronic infection. In both stages, the parasite lives within a membrane-bound vacuole within the host cell, but in the chronic stage, a durable cyst wall is synthesized, which provides protection to the parasite during transmission to a new host. Toxoplasma secretes proteins into the vacuole to build its replicative niche, and previous studies identified many of these proteins as phosphorylated. We investigate two secreted proteins and show that a phosphorylated region plays an important role in their regulation in acute stages. We also observed widespread phosphorylation of secreted proteins when parasites convert from acute to chronic stages, providing new insight into how the cyst wall may be dynamically regulated.

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Divergent kinase regulates membrane ultrastructure of the Toxoplasma parasitophorous vacuole

Cited by 47 publications

References 72 publications

A single-parasite transcriptional atlas of Toxoplasma Gondii reveals novel control of antigen expression

A single-parasite transcriptional atlas of Toxoplasma Gondii reveals novel control of antigen expression

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

Phosphorylation of Toxoplasma gondii Secreted Proteins during Acute and Chronic Stages of Infection

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