A divergent kinase lacking the glycine-rich loop regulates membrane ultrastructure of the<i>Toxoplasma</i>parasitophorous vacuole

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

doi:10.1101/397489

Cited by 3 publications

(4 citation statements)

References 89 publications

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“…Further investigation into the available literatures confirmed the presence of many non-canonical kinase folds, one of which presents a 4 stranded antiparallel β-sheet flanked by three parallel and one perpendicular helix (Fig. 2A, left panel) [20]. Taken together, these results suggest that the NiRAN domain assumes a kinase like fold, possibly functioning either as a pseudokinase or a phosphotransferase.…”

Section: Prediction Of the Plausible Functions Of The Niran Domainmentioning

confidence: 58%

In silico characterization of the NiRAN domain of RNA-dependent RNA polymerase provides insights into a potential therapeutic target against SARS-CoV2

Dwivedy¹,

Mariadasse²,

Ahmed³

et al. 2020

Preprint

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Apart from the canonical fingers, palm and thumb domains, the RNA dependent RNA polymerases (RdRp) from the viral order Nidovirales possess two additional domains. Of these, the function of the Nidovirus RdRp associated nucleotidyl transferase domain (NiRAN) remains unanswered. The elucidation of the 3D structure of the RdRp from the novel coronavirus – SARS-CoV2, provided the first ever insights into the domain organisation and possible functional characteristics of the NiRAN domain. Using in silico tools, this study predicts that the NiRAN domain assumes a kinase or phosphotransferase like fold and binds GTP and UTP at its proposed active site. Additionally, using molecular docking this study predicts the binding of five well characterized anti-microbial compounds at the NiRAN domain active site and their drug-likeliness and DFT properties. In line with the current global COVID-19 pandemic urgency, this study provides a new target and potential lead compounds for drug repurposing against SARS-CoV2.

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Section: Prediction Of the Plausible Functions Of The Niran Domainmentioning

confidence: 58%

In silico characterization of the NiRAN domain of RNA-dependent RNA polymerase provides insights into a potential therapeutic target against SARS-CoV2

Dwivedy¹,

Mariadasse²,

Ahmed³

et al. 2020

Preprint

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“…It was recently shown that phosphorylation of GRAs by WNG1, a closely related family member of WNG2, after exocytosis from the dense granules is important for the correct localization of transmembrane-domain-containing GRAs (68). We have shown that GRA42 and GRA43 also play a role in the correct trafficking of PVM-localized GRAs and that in Δgra42 or Δgra43 parasites GRA23 and GRA35 are mislocalized (79).…”

Section: Discussionmentioning

confidence: 74%

“…GRAs with a transmembrane domain have peculiar trafficking and do not traffic to the parasite plasma membrane but instead traffic to the dense granules where they exist in a soluble state (67). What prevents their transmembrane domain from inserting into the ER membrane or the dense granule membrane is unknown but has been hypothesized to be mediated by a dense-granule-localized chaperone (67,68).…”

Section: Deletion Of Gra45 Leads To Aggregation Of Dense Granule Protmentioning

confidence: 99%

A genome-wide loss-of-function screen identifiesToxoplasma gondiigenes that determine fitness in interferon gamma-activated murine macrophages

Wang

Sangaré

Paredes-Santos

et al. 2019

Preprint

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Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identified ~130Toxoplasma genes that determine parasite fitness in naїve macrophages and ~466 genes that determine fitness in IFNγ-stimulated murine macrophages, seven of which we investigated and confirmed. We show that one of these genes encodes dense granule protein GRA45, which contains a putative chaperone-like domain, and which we show is critical in preventing other GRA effectors from aggregating. Parasites lacking GRA45 mislocalize GRA effectors upon secretion, are more susceptible to IFNγ-mediated growth inhibition, and have reduced virulence in mice. Our results provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγstimulated macrophages. IMPORTANCEThe intracellular parasite Toxoplasma gondii can cause congenital infections and severe disease in immunocompromised patients. The cytokine IFNγ can block parasite replication by upregulating a variety of toxoplasmacidal mechanisms in many cells, including macrophages. Toxoplasma preferentially infects macrophages. Therefore, the parasite has evolved mechanisms to survive in these cells in the presence of IFNγ. Here, we generated pools of Toxoplasma mutants for every gene and determined which mutants were specifically depleted in IFNγ-stimulated macrophages, thus identifying parasite genes determining fitness in these cells. We show that one of these genes encodes for a dense granule protein (GRA45) that plays an important role in preventing other GRA effectors from aggregating. Parasites without GRA45 mislocalize GRA effectors upon secretion, have enhanced susceptibility toIFNγ-mediated growth inhibition, and are avirulent in mice. Thus, our screen provides a resource to the Toxoplasma community to determine the function of Toxoplasma genes that affect its fitness in IFNγstimulated macrophages. INTRODUCTION:Toxoplasma gondii is an obligate intracellular parasite that causes disease in immunocompromised individuals, such as HIV/AIDS patients, and when contracted congenitally (1). It causes lifelong infections by converting from rapidly dividing tachyzoite stages into encysted slow-growing bradyzoites, which mainly localize to the brain and muscle tissues. Once established in the host cell, Toxoplasma resides in a unique replication niche called the parasitophorous vacuole (PV), which is separated from the host cytoplasm by the PV membrane (PVM) and does not fuse with the endolysosomal system (2,3).The cytokine interferon gamma (IFNγ) is essential for the control of Toxoplasma replication in host cells (4). Mice with macrophages that can no longer respond to IFNγ are extremely susceptible toToxoplasma demonstrating the crucial role of this cell type in IFNγ-mediated control of Toxoplasma (5).IFNγ-induced upregulation of im...

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“…including GRA6, and to regulate their membrane association (20). As many GRAs form 393 complexes during trafficking (31), WNG1 phosphorylation is hypothesised to release them 394 from association with a chaperone, freeing them to associate with membranes.…”

Section: Conversion 391mentioning

confidence: 99%

Differential protein phosphorylation affects the localisation of two secretedToxoplasmaproteins and is widespread during stage conversion

Young

Broncel

Teague

et al. 2020

Preprint

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cyst wall is synthesized that provides protection to the parasite during transmission to a new 46 host. Toxoplasma secretes proteins into the vacuole to build its replicative niche and previous 47 studies identified many of these proteins as phosphorylated. We investigate two secreted 48 proteins and show that phosphorylation plays an important role in their regulation. We also 49 observed widespread phosphorylation of secreted proteins when parasites convert from acute 50 to chronic stages, providing new insight into how the cyst wall may be dynamically regulated. 51During infection Toxoplasma secretes proteins that subvert host cell signalling and develops its 67 replicative niche within the PV (6, 7). The repertoire of secreted proteins is thought to include 68 up to 200 proteins (ToxoDB, LOPIT dataset) including 50 kinases/pseudokinases (8), although 69 the functions of many of these remain unknown. These proteins are secreted from the 70 rhoptries or dense granules and are called ROPs or GRAs, respectively. Secreted proteins 71 extensively modify the PV allowing selective passage of molecules and proteins across the PV 72 membrane. For example, GRA17 and GRA23 form pores in the PV membrane allowing the 73 passage of small molecules into the PV (9), while the MYR complex mediates the transport of 74 secreted proteins to the host cell (10, 11). Additionally, the parasite develops a complex set of 75 membrane structures within the PV including GRA7-lined invaginations (12) and a network of 76 tubules called the intravacuolar network (IVN) (13). The formation of this network is dependent 77 on GRA2 and the accessory protein GRA6, and is required for full virulence in mice (14,15). 78Both structures have been reported to play a role in scavenging nutrients from the host cell 79 with the uptake of host cell proteins (16), lipid droplets (17), and endolysosomal compartments 80 (18). 81Previous phosphoproteome analysis revealed that a large number of Toxoplasma secreted 82 proteins are phosphorylated after their release (19), raising the intriguing possibility that their 83 function is dynamically regulated. Indeed, the PV localised kinase WNG1 was shown to 84 contribute to formation of a functional IVN (20). Furthermore, it was recently shown that IVN 85 associated GRAs show dynamic localisation patterns as the parasite remodels the PV to form 86 the chronic stage cyst (21). Here we analyse two secreted, strand forming proteins that are 87 phosphorylated after secretion and show that phosphorylation regulates their localisation, 88 disrupting normal strand formation. Both proteins disperse in chronic stage cysts, so we 89

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A divergent kinase lacking the glycine-rich loop regulates membrane ultrastructure of theToxoplasmaparasitophorous vacuole

Cited by 3 publications

References 89 publications

In silico characterization of the NiRAN domain of RNA-dependent RNA polymerase provides insights into a potential therapeutic target against SARS-CoV2

In silico characterization of the NiRAN domain of RNA-dependent RNA polymerase provides insights into a potential therapeutic target against SARS-CoV2

A genome-wide loss-of-function screen identifiesToxoplasma gondiigenes that determine fitness in interferon gamma-activated murine macrophages

Differential protein phosphorylation affects the localisation of two secretedToxoplasmaproteins and is widespread during stage conversion

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