PP2Acα-B′/PR61 Holoenzyme of Toxoplasma gondii Is Required for the Amylopectin Metabolism and Proliferation of Tachyzoites

Zhao, Mingxiu; Yang, Yi; Shi, Yanfen; Chen, Xueqiu; Yang, Yimin; Pan, Lingtao; Du, Zhendong; Sun, Hongchao; Yao, Chaoqun; Ma, Guangxu; Du, Aifang

doi:10.1128/spectrum.00104-23

Cited by 5 publications

References 68 publications

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Cytokinetic abscission in Toxoplasma gondii is governed by protein phosphatase 2A and the daughter cell scaffold complex

Marq,

Gosetto,

Altenried

et al. 2024

EMBO J

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Cytokinetic abscission marks the final stage of cell division, during which the daughter cells physically separate through the generation of new barriers, such as the plasma membrane or cell wall. While the contractile ring plays a central role during cytokinesis in bacteria, fungi and animal cells, the process diverges in Apicomplexa. In Toxoplasma gondii, two daughter cells are formed within the mother cell by endodyogeny. The mechanism by which the progeny cells acquire their plasma membrane during the disassembly of the mother cell, allowing daughter cells to emerge, remains unknown. Here we identify and characterize five T. gondii proteins, including three protein phosphatase 2A subunits, which exhibit a distinct and dynamic localization pattern during parasite division. Individual downregulation of these proteins prevents the accumulation of plasma membrane at the division plane, preventing the completion of cellular abscission. Remarkably, the absence of cytokinetic abscission does not hinder the completion of subsequent division cycles. The resulting progeny are able to egress from the infected cells but fail to glide and invade, except in cases of conjoined twin parasites.

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Cytokinetic abscission in Toxoplasma gondii is governed by protein phosphatase 2A and the daughter cell scaffold complex

Marq,

Gosetto,

Altenried

et al. 2024

EMBO J

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The PP1 phosphatase exhibits pleiotropic roles controlling both the tachyzoite cell cycle and amylopectin-steady state levels inToxoplasma gondii

Khelifa,

Boissavy,

Mouveaux

et al. 2023

Preprint

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Virulence of apicomplexan parasites is based on their ability to divide rapidly to produce significant biomass. The regulation of their cell cycle is therefore key to their pathogenesis. Phosphorylation is a crucial post-transcriptional modification that regulates many aspects of the eucaryotic cell cycle. The phosphatase PP1 is known to play a major role in the phosphorylation balance in eukaryotes. We explored the role of TgPP1 during the cell cycle of the tachyzoite form of the apicomplexan parasite Toxoplasma gondii. Using a conditional mutant strain, we show that TgPP1 regulates many aspects of the cell cycle including the proper assembly of the daughter cells inner membrane complex (IMC), the segregation of organelles, and nuclear division. Unexpectedly, depletion of TgPP1 also results in the accumulation of amylopectin, a storage polysaccharide that is normally found in the latent bradyzoite form of the parasite. Using transcriptomics and phosphoproteomics, we show that TgPP1 mainly acts through post-transcriptional mechanisms by dephosphorylating target proteins including IMC proteins. TgPP1 also dephosphorylate a protein bearing a starch binding domain. Mutagenesis analysis reveals that the targeted phospho-sites are linked to the ability of the parasite to regulate amylopectin steady-state levels. Therefore, we show that TgPP1 has pleiotropic roles during the tachyzoite cell cycle regulation, but also regulates amylopectin accumulation.

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TgLaforin, a glucan phosphatase, reveals the dynamic role of storage polysaccharides inToxoplasma gondiitachyzoites and bradyzoites

Murphy,

Troublefield,

Miracle

et al. 2023

Preprint

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The asexual stages ofToxoplasma gondiiare defined by the rapidly growing tachyzoite during the acute infection and by the slow growing bradyzoite housed within tissue cysts during the chronic infection. These stages represent unique physiological states, each with distinct glucans reflecting differing metabolic needs. A defining feature ofT. gondiibradyzoites is the presence of insoluble storage glucans known as amylopectin granules (AGs) that are believed to play a role in reactivation, but their functions during the chronic infection remain largely unexplored. More recently, the presence of storage glucans has been recognized in tachyzoites where their precise function and architecture have yet to be fully defined. Importantly, theT. gondiigenome encodes activities needed for glucan turnover: a glucan phosphatase (TgLaforin; TGME49_205290) and a glucan kinase (TgGWD; TGME49_214260) that catalyze a cycle of reversible glucan phosphorylation required for glucan degradation by amylases. The expression of these enzymes in tachyzoites supports the existence of a storage glucan, evidence that is corroborated by specific labeling with the anti-glycogen antibody IV58B6. Disruption of reversible glucan phosphorylation via a CRISPR/Cas9 knockout (KO) of TgLaforin revealed no growth defects under nutrient-replete conditions in tachyzoites. However, the growth of TgLaforin-KO tachyzoites was severely stunted when starved of glutamine, even under glucose replete conditions. The loss of TgLaforin also resulted in the attenuation of acute virulence in mice accompanied by a lower cyst burden. Defective cyst formation due to profound changes in AG morphology was also observed in TgLaforin-KO parasites, bothin vitroandin vivo. Together, these data demonstrate the importance of glucan turnover across theT. gondiiasexual cycle. These findings, alongside our previously identified class of small molecules that inhibit TgLaforin, implicate reversible glucan phosphorylation as a legitimate target for the development of new drugs against chronicT. gondiiinfections.

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PP2Acα-B′/PR61 Holoenzyme of Toxoplasma gondii Is Required for the Amylopectin Metabolism and Proliferation of Tachyzoites

Cited by 5 publications

References 68 publications

Cytokinetic abscission in Toxoplasma gondii is governed by protein phosphatase 2A and the daughter cell scaffold complex

Cytokinetic abscission in Toxoplasma gondii is governed by protein phosphatase 2A and the daughter cell scaffold complex

The PP1 phosphatase exhibits pleiotropic roles controlling both the tachyzoite cell cycle and amylopectin-steady state levels inToxoplasma gondii

TgLaforin, a glucan phosphatase, reveals the dynamic role of storage polysaccharides inToxoplasma gondiitachyzoites and bradyzoites

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