Toxoplasma gondii infections are spread over nearly one-third of the global population. One out of five Americans is seropositive, but infections stay predominantly subclinical in the immunocompetent host. However, during immunodeficiencies, e.g. organ transplantation, cancer or AIDS, the reactivated parasites can cause severe symptoms, which may be lethal. Infection of the heart results in pericarditis and myocarditis and can affect all patients, independent of their immune status. This inflammation results from the parasites lytic division cycle, destroying host tissue in the process. Toxoplasma divides by a unique internal budding mechanism and cell division is finalized by a specific structure, the basal complex (BC). The BC is the parasite's functional equivalent of the cytokinetic contractile ring, yet constricts differently form the known actin-myosin powered motor of the mammalian cell. We have recently established that assembly of the BC relies on differential phosphorylation. In addition, I have identified a novel kinase, MNK-L1, which is critically involved in these processes. Parasites lacking MNK-L1 are severely fitness impaired, exhibit strong segregation defects and form an unusual amount of daughter cells. Characterization of this kinase will provide us with deep insights into regulation and execution of budding, which contributes to our overarching goal: deciphering the signaling process of Toxoplasma cell division to identify novel drug targets. As part of this goal, the current proposal focuses on the identification of the MNK-L1 signaling pathway and comprises two specific aims: 1) In order to understand how MNK-L1 functions, I will identify kinase substrates by a chemical genetic approach to construct its signaling network. I have already engineered the endogenous kinase to accept ATP-analogs, which allows substrate identification via specific thiophosphate-labeling. 2) To elucidate the severe fitness defect of MNK-L1-depeleted parasites, I will use a recently published genome-wide loss-of-function screen mediated by CRISPR/Cas9. Revealing genes that act synergistically with MNK-L1 will further advance our understanding of parasite division. The correlation of MNK-L1 in concert with its substrates will establish mechanical insights into how budding is regulated and executed during cytokinesis. These insights will highlight specific targets to be exploited toward future drug candidates to treat Toxoplasma-induced heart disease.
|Program type||Postdoctoral Fellowship|
|Effective start/end date||07/01/2017 → 06/30/2019|