Toxoplasma gondii Cyclic AMP-Dependent Protein Kinase Subunit 3 Is Involved in the Switch from Tachyzoite to Bradyzoite Development

Sugi, Tatsuki; Fen, Yan; Tomita, Tadakimi; Murakoshi, Fumi; Eaton, Michael; Yakubu, Rama R.; Han, Bing; Tu, Vincent; Kato, Kentaro; Kawazu, Shin Ichiro; Gupta, Nishith; Suvorova, Elena S.; White, Michael; Kim, Kami; Weiss, Louis M.

doi:10.1128/mbio.00755-16

Cited by 62 publications

(89 citation statements)

References 34 publications

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“…PruΔku80Δhxgprt LDH2-GFP parasites (18) were continuously passaged in human foreskin fibroblast (HFF : ATCC:CRL-1634; Hs27) host cells, as previously described (32). TgIST-KO parasites were obtained as a kind gift from the laboratory of Dr. David Sibley (Washington University School of Medicine, St. Louis, MO).…”

Section: Methodsmentioning

confidence: 99%

In vitrocharacterization of protein effector export in the bradyzoite stage ofToxoplasma gondii

Mayoral

Shamamian

Weiss

2020

Preprint

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The ubiquitous parasite Toxoplasma gondii exhibits an impressive ability to maintain a chronic infection of its host for prolonged periods. Despite this, little is known regarding if and how T. gondii bradyzoites, a quasi-dormant life-stage residing within intracellular cysts, manipulate the host cell so as to maintain a persistent infection. A previous proteomic study of the cyst wall, an amorphous layer of proteins that forms underneath the cyst membrane, identified MYR1 as a putative cyst wall protein in vitro. As MYR1 is known to be involved in the translocation of parasite derived effector proteins into the host cell, we sought to determine whether parasites transitioning toward the bradyzoite life stage retain the capacity to translocate proteins via this pathway. By epitope tagging the endogenous loci of four known effectors that translocate from the parasitophorous vacuole into the host cell nucleus, we show by immunofluorescence that most effectors accumulate in the host nucleus at early but not late timepoints post-infection during the tachyzoite to bradyzoite transition and when parasites farther along the bradyzoite differentiation continuum invade a new host cell. We demonstrate that the suppression of interferon-gamma (IFN-γ) signaling, previously shown to be mediated by the effector TgIST, also occurs in the context of prolonged infection with bradyzoites, and that TgIST export is a process that occurs beyond the early stages of host cell infection. These findings have important implications as to how this highly successful parasite maintains a persistent infection of its host.IMPORTANCEToxoplasma bradyzoites persist within tissue cysts and are refractory to current treatments, serving as a reservoir for acute complications in settings of compromised immunity. Much remains to be understood regarding how this life-stage successfully establishes and maintains a persistent infection. In this study, we investigated whether the export of parasite effector proteins into the host cell occurs during the development of in vitro tissue cysts. We quantified the presence of four previously described effectors in host cell nuclei at different timepoints post-bradyzoite differentiation and found that they accumulate largely during the early stages of infection. Despite a decline in nuclear accumulation, we found that one of these effectors still mediates its function after prolonged infection with bradyzoites and provide evidence that this effector is exported beyond early infection stages. These findings suggest that effector export from within developing tissue cysts provides one potential mechanism by which this parasite achieves chronic infection.

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Section: Methodsmentioning

confidence: 99%

In vitrocharacterization of protein effector export in the bradyzoite stage ofToxoplasma gondii

Mayoral

Shamamian

Weiss

2020

Preprint

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“…PKAr typically possesses two cAMP‐binding domains, and the association with cAMP provokes the release of active PKAc that can then phosphorylate downstream targets to propagate cAMP signalling events. In T. gondii , PKAc1 and PKAc2 localise to the parasite periphery whereas PKAc3 is cytosolic (Sugi et al , ) indicating that the spatial control of cAMP levels and PKAc isoforms is crucial to differentially regulate PKA activity. Both PKAc1 and PKAc3 are expressed in tachyzoites and bradyzoites, while PKAc2 is highly expressed during the sexual stages in felids.…”

Section: Introductionmentioning

confidence: 99%

“…Use of H89, an ATP competitive inhibitor showing high affinity for a wide range of PKAc isoforms (Lochner & Moolman, ), previously suggested that PKAc activity is important to regulate the rate of T. gondii division (Kurokawa et al , ) while transient elevation of cAMP by forskolin induced bradyzoite differentiation (Kirkman et al , ). Functional analysis of PKAc3 recently identified this isoform as a key cAMP effector in the regulation of stage conversion between tachyzoites and bradyzoites (Sugi et al , ).…”

Section: Introductionmentioning

confidence: 99%

Crosstalk between PKA and PKG controls pH ‐dependent host cell egress of Toxoplasma gondii

et al. 2017

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Toxoplasma gondii encodes three protein kinase A catalytic (PKAc1-3) and one regulatory (PKAr) subunits to integrate cAMP-dependent signals. Here, we show that inactive PKAc1 is maintained at the parasite pellicle by interacting with acylated PKAr. Either a conditional knockdown of PKAr or the overexpression of PKAc1 blocks parasite division. Conversely, down-regulation of PKAc1 or stabilisation of a dominant-negative PKAr isoform that does not bind cAMP triggers premature parasite egress from infected cells followed by serial invasion attempts leading to host cell lysis. This untimely egress depends on host cell acidification. A phosphoproteome analysis suggested the interplay between cAMP and cGMP signalling as PKAc1 inactivation changes the phosphorylation profile of a putative cGMP-phosphodiesterase. Concordantly, inhibition of the cGMP-dependent protein kinase G (PKG) blocks egress induced by PKAc1 inactivation or environmental acidification, while a cGMP-phosphodiesterase inhibitor circumvents egress repression by PKAc1 or pH neutralisation. This indicates that pH and PKAc1 act as balancing regulators of cGMP metabolism to control egress. These results reveal a crosstalk between PKA and PKG pathways to govern egress in T. gondii.

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“…In agreement with the observation that PKA is conserved in apicomplexans [23], two PKAs (SRCN_5610 and SRCN_3990) were identified in S. neurona. In T. gondii , increases in cytosolic cAMP levels activate PKA to trigger the developmental switch from the rapidly proliferating tachyzoites to the quiescent bradyzoites [66]. Additionally, two other S. neurona AGCs (SRCN_5165 and SRCN_3339) were putative PKAs given that they contained the characteristic GxGxxG motif found in PKA [51].…”

Section: Discussionmentioning

confidence: 99%

Genome-Wide Identification and Evolutionary Analysis of Sarcocystis neurona Protein Kinases

Murungi

Kariithi

2017

Pathogens

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The apicomplexan parasite Sarcocystis neurona causes equine protozoal myeloencephalitis (EPM), a degenerative neurological disease of horses. Due to its host range expansion, S. neurona is an emerging threat that requires close monitoring. In apicomplexans, protein kinases (PKs) have been implicated in a myriad of critical functions, such as host cell invasion, cell cycle progression and host immune response evasion. Here, we used various bioinformatics methods to define the kinome of S. neurona and phylogenetic relatedness of its PKs to other apicomplexans. We identified 97 putative PKs clustering within the various eukaryotic kinase groups. Although containing the universally-conserved PKA (AGC group), S. neurona kinome was devoid of PKB and PKC. Moreover, the kinome contains the six-conserved apicomplexan CDPKs (CAMK group). Several OPK atypical kinases, including ROPKs 19A, 27, 30, 33, 35 and 37 were identified. Notably, S. neurona is devoid of the virulence-associated ROPKs 5, 6, 18 and 38, as well as the Alpha and RIO kinases. Two out of the three S. neurona CK1 enzymes had high sequence similarities to Toxoplasma gondii TgCK1-α and TgCK1-β and the Plasmodium PfCK1. Further experimental studies on the S. neurona putative PKs identified in this study are required to validate the functional roles of the PKs and to understand their involvement in mechanisms that regulate various cellular processes and host-parasite interactions. Given the essentiality of apicomplexan PKs in the survival of apicomplexans, the current study offers a platform for future development of novel therapeutics for EPM, for instance via application of PK inhibitors to block parasite invasion and development in their host.

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Toxoplasma gondii Cyclic AMP-Dependent Protein Kinase Subunit 3 Is Involved in the Switch from Tachyzoite to Bradyzoite Development

Cited by 62 publications

References 34 publications

In vitrocharacterization of protein effector export in the bradyzoite stage ofToxoplasma gondii

In vitrocharacterization of protein effector export in the bradyzoite stage ofToxoplasma gondii

Crosstalk between PKA and PKG controls pH ‐dependent host cell egress of Toxoplasma gondii

Genome-Wide Identification and Evolutionary Analysis of Sarcocystis neurona Protein Kinases

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