SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

Reviewer #1 (Public Review):

Summary:
Herneisen et al characterise the Toxoplasma PDK1 orthologue SPARK and an associated protein SPARKEL in controlling important fate decisions in Toxoplasma. Over recent years this group and others have characterised the role of cAMP and cGMP signalling in negatively and positively regulating egress, motility, and invasion, respectively. This manuscript furthers this work by showing that SPARK and SPARKEL likely act upstream, or at least control the levels of the cAMP and cGMP-dependent kinases PKA and PKG, respectively, thus controlling the transition of intracellular replicating parasites into extracellular motile forms (and back again).

The authors use quantitative (phospho)proteomic techniques to elegantly demonstrate the upstream role of SPARK in controlling cAMP and cGMP pathways. They use sophisticated analysis techniques (at least for parasitology) to show the functional association between cGMP and cAMP signalling pathways. They therefore begin to unify our understanding of the complicated signalling pathways used by Toxoplasma to control key regulatory processes that control the activation and suppression of motility. The authors then use molecular and cellular assays on a range of generated transgenic lines to back up their observations made by quantitative proteomics that are clear in their design and approach.

The authors then extend their work by showing that SPARK/SPARKEL also control PKAc3 function. PKAc3 has previously been shown to negatively regulate differentiation into bradyzoite forms and this work backs up and extends this finding to show that SPARK also controls this. The authors conclude that SPARK could act as a central node of regulation of the asexual stage, keeping parasites in their lytic cell growth and preventing differentiation. Whether this is true is beyond the scope of this paper and will have to be determined at a later date.

Strengths:
This is an exceptional body of work. It is elegantly performed, with state-of-the-art proteomic methodologies carefully being applied to Toxoplasma. Observations from the proteomic datasets are masterfully backed up with validation using quantitative molecular and cellular biology assays.

The paper is carefully and concisely written and is not overreaching in its conclusions. This work and its analysis set a new benchmark for the use of proteomics and molecular genetics in apicomplexan parasites.

Weaknesses:
This reviewer did not identify any weaknesses.

Reviewer #2 (Public Review):

Summary:
The manuscript by Herneisen et al. examines the Toxoplasma SPARK kinase orthologous to mammalian PDK1 kinase. The extracellular signals trigger cascades of the second messengers and play a central role in the apicomplexan parasites' survival. In Toxoplasma, these cascades regulate active replication of the tachyzoites, which manifests as acute toxoplasmosis, or the development into drug-resilient bradyzoites characteristic of the chronic stage of the disease. This study focuses on the poorly understood signaling mechanisms acting upstream of such second messenger kinases as PKA and PKG. The authors showed that similar to PDK1, Toxoplasma SPARK appears to regulate several AGC kinases.

Strengths:
The study demonstrated a strong association of the SPARK kinase with an elongin-like SPARKEL factor and an uncharacterized AGC kinase. Using a set of standard assays, the authors determined the SPARK/SPARKEL role in parasite egress and invasion. Finally, the study presented evidence of the SPARK/SPARKEL involvement in the bradyzoite differentiation.

Weaknesses:
Although the study can potentially uncover essential sensing mechanisms operating in Toxoplasma, the evidence of the SPARK/SPARKEL mechanisms is weak. Specifically, due to incomplete data analysis, the SPARK/SPARKEL-dependent phosphoregulation of AGC kinases cannot be evaluated. The manuscript requires better organization and lacks guidance on the described experiments. Although the study is built on advanced genetics, at times, it is unnecessarily complicated, raising doubts rather than benefiting the study.

Reviewer #3 (Public Review):

Summary:
This paper focuses on the roles of a toxoplasma protein (SPARKEL) with homology to an elongin C and the kinase SPARK that it interacts with. They demonstrate that the two proteins regulate the abundance of PKA and PKG, and that depletion of SPARKEL reduces invasion and egress (previously shown with SPARK), and that their loss also triggers spontaneous bradyzoite differentiation. The data are overall very convincing and will be of high interest to those who study Toxoplasma and related apicomplexan parasites.

Strengths:
The study is very well executed with appropriate controls. The manuscript is also very well and clearly written. Overall, the work clearly demonstrates that SPARK/SPARKEL regulate invasion and egress and that their loss triggers differentiation.

Weaknesses:
1. The authors fail to discriminate between SPARK/SPARKEL acting as negative regulators of differentiation as a result of an active role in regulating stage-specific transcription/translation or as a consequence of a stress response activated when either is depleted.

2. The function of SPARKEL has not been addressed. In mammalian cells, Elongin C is part of an E3 ubiquitin ligase complex that regulates transcription and other processes. From what I can tell from the proteomic data, homologs of the Elongin B/C complex were not identified. This is an important issue as the authors find that PKG and PKA protein levels are reduced in the knockdown strains