An Extracellular Redox Signal Triggers Calcium Release and Impacts the Asexual Development of Toxoplasma gondii

Alves, Eduardo Rodrigues; Benns, Henry James; Magnus, Lilian; Dominicus, Caia; Dobai, Tamás; Blight, Joshua; Wincott, Ceire Joanna; Child, Matthew A.

doi:10.3389/fcimb.2021.728425

Cited by 14 publications

(15 citation statements)

References 89 publications

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“…We therefore chose peak intracellular calcium levels as a reference point to facilitate a direct comparison between BIPPO- and A23187-treated parasites. To this end, we generated a stable calcium sensor line that co-expresses, through use of a T2A ribosomal skip peptide, an internal GFP control and the genetically encoded ruby Ca 2+ biosensor jRCaMP1b (Alves et al, 2021) from a single promoter (Fig. 1A-B).…”

Section: Resultsmentioning

confidence: 99%

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A positive feedback loop mediates crosstalk between calcium, cyclic nucleotide and lipid signalling in Toxoplasma gondii

Dominicus

Nofal

Broncel

et al. 2021

Preprint

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Fundamental processes of obligate intracellular parasites, such as Toxoplasma gondii and Plasmodium falciparum, are controlled by a set of plant-like calcium dependent kinases (CDPKs), the conserved cAMP- and cGMP-dependent protein kinases (PKA and PKG), secondary messengers and lipid signalling. While some major components of the signalling networks have been identified, how these are connected remains largely unknown. Here, we compare the phospho-signalling networks during Toxoplasma egress from its host cell by artificially raising cGMP or calcium levels to activate PKG or CDPKs, respectively. We show that both these inducers trigger near identical signalling pathways and provide evidence for a positive feedback loop involving CDPK3. We measure phospho- and lipid signalling in parasites treated with the Ca2+ ionophore A23187 in a sub-minute timecourse and show CDPK3-dependent regulation of diacylglycerol levels and increased phosphorylation of four phosphodiesterases (PDEs), suggesting their function in the feedback loop. Disruption of CDPK3 leads to elevated cAMP levels and inhibition of PKA signalling rescues the egress defect of ΔCDPK3 parasites treated with A23187. Biochemical analysis of the four PDEs identifies PDE2 as the only cAMP-specific PDE among these candidates, while the other PDEs are cGMP specific, two of which are inhibited by the predicted PDE inhibitor BIPPO. Conditional deletion of the four PDEs supports an important, but non-essential role for PDE1 and PDE2 in growth, with PDE2 controlling A23187-mediated egress. In summary we uncover a positive feedback loop that enhances signalling during egress and links several signalling pathways together.

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Section: Resultsmentioning

confidence: 99%

“…Primers used throughout this study are listed in Table S6. The calcium sensor construct was generated as recently described (Alves et al, 2021). The construct was linearised using NaeI and transfected into RH Δku80Δhxgprt parasites as described previously (Soldati and Boothroyd, 1993) to generate the GFP-T2A-jRCaMP1b calcium sensor line.…”

Section: Methodsmentioning

confidence: 99%

A positive feedback loop mediates crosstalk between calcium, cyclic nucleotide and lipid signalling in Toxoplasma gondii

Dominicus

Nofal

Broncel

et al. 2021

Preprint

Self Cite

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“…ROS, particularly non-radical species such as hydrogen peroxide or singlet molecular oxygen at physiological levels, i.e. oxidative eustress, mediate various cellular processes through redox signaling ( Sies et al., 2017 ) and may be associated with calcium-signaling, parasite egress and replication of tachyzoites in fibroblasts ( Alves et al., 2021 ). We suggest that oxidative stress in myotubes may trigger bradyzoite formation, presumably through phosphorylation of the T. gondii homologue of the eukaryotic initiation factor 2 α subunit (TgIF2α) by the kinase TgIF2K-B and translational control of ApiAP2 transcription factors ( Narasimhan et al., 2008 ; Augusto et al., 2021 ).…”

Section: Discussionmentioning

confidence: 99%

The Redox Homeostasis of Skeletal Muscle Cells Regulates Stage Differentiation of Toxoplasma gondii

Rahman

Swierzy

Downie

et al. 2021

Front. Cell. Infect. Microbiol.

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Toxoplasma gondii is an obligatory intracellular parasite that causes persistent infections in birds and mammals including ~30% of the world’s human population. Differentiation from proliferative and metabolically active tachyzoites to largely dormant bradyzoites initiates the chronic phase of infection and occurs predominantly in brain and muscle tissues. Here we used murine skeletal muscle cells (SkMCs) to decipher host cellular factors that favor T. gondii bradyzoite formation in terminally differentiated and syncytial myotubes, but not in proliferating myoblast precursors. Genome-wide transcriptome analyses of T. gondii-infected SkMCs and non-infected controls identified ~6,500 genes which were differentially expressed (DEGs) in myotubes compared to myoblasts, largely irrespective of infection. On the other hand, genes related to central carbohydrate metabolism, to redox homeostasis, and to the Nrf2-dependent stress response pathway were enriched in both infected myoblast precursors and myotubes. Stable isotope-resolved metabolite profiling indicated increased fluxes into the oxidative branch of the pentose phosphate pathway (OxPPP) in infected myoblasts and into the TCA cycle in infected myotubes. High OxPPP activity in infected myoblasts was associated with increased NADPH/NADP+ ratio while myotubes exhibited higher ROS levels and lower expression of anti-oxidants and detoxification enzymes. Pharmacological reduction of ROS levels in SkMCs inhibited bradyzoite differentiation, while increased ROS induced bradyzoite formation. Thus, we identified a novel host cell-dependent mechanism that triggers stage conversion of T. gondii into persistent tissue cysts in its natural host cell type.

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“…The importance of iron storage and detoxification across the tree of life highlights the dangers of iron to almost all cells. In T. gondii, the response of the parasites to oxidative stress and its role in the lytic life cycle has of been of interest recently (Alves et al, 2021;Augusto et al, 2021) and how iron uptake and storage are linked with these pathways will be an interesting future area of research.…”

Section: Discussionmentioning

confidence: 99%

“…Although the ΔVIT parasites have a small growth defect in vitro, we were intrigued to see that there was no increase in ROS under normal conditions. The parasite has various lines of defense against ROS, including thioredoxins, several SODs and a cytosolically localised catalase (Alves et al, 2021;Kwok et al, 2004;Xue et al, 2017). Although the RNAseq did not show any changes in SOD, SOD2 (Fig.…”

Section: Iron Overload Leads To Ros Accumulation In the Absence Of Vitmentioning

confidence: 94%

The vacuolar iron transporter mediates iron detoxification inToxoplasma gondii

Aghabi

Sloan

Dou

et al. 2021

Preprint

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Iron is essential to living cells, acting as a cofactor in a number of essential enzymes in metabolism; however, iron requires proper storage or it can be dangerous to the cell. In both yeast and plants, iron is stored in a vacuole through the action of a vacuolar iron transporter (VIT). This transporter is conserved in the apicomplexan family of obligate intracellular parasites, including in Toxoplasma gondii, a pathogen of medical and veterinary importance. Here, we assess the role of VIT in T. gondii. We show that deletion of VIT causes a slight growth defect in vitro, however leads to hypersensitivity in the presence of excess iron, confirming its essential role in iron detoxification in the parasite. In the absence of VIT, parasites contain less iron and are at a growth disadvantage when moving into an iron-depleted environment. We show parasite VIT expression is regulated by environmental iron levels at both the transcript and protein level, and by altering the distribution of VIT within the cell. In the absence of VIT, we find that the T. gondii responds by altering expression of genes with a role in iron metabolism and by increasing the activity of the antioxidant protein catalase. We also show that iron detoxification has an important role both in parasite survival within macrophages and in pathogenesis in a mouse model. Together, by demonstrating a critical role for VIT during iron detoxification in T. gondii, we reveal the importance of iron storage in the parasite and provide the first insight into the machinery involved.

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An Extracellular Redox Signal Triggers Calcium Release and Impacts the Asexual Development of Toxoplasma gondii

Cited by 14 publications

References 89 publications

A positive feedback loop mediates crosstalk between calcium, cyclic nucleotide and lipid signalling in Toxoplasma gondii

A positive feedback loop mediates crosstalk between calcium, cyclic nucleotide and lipid signalling in Toxoplasma gondii

The Redox Homeostasis of Skeletal Muscle Cells Regulates Stage Differentiation of Toxoplasma gondii

The vacuolar iron transporter mediates iron detoxification inToxoplasma gondii

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