Loss of the conserved Alveolate kinase MAPK2 decouplesToxoplasmacell growth from the cell cycle

Abstract

Mitogen-activated protein kinases (MAPKs) are a conserved family of protein kinases that regulate signal transduction, proliferation, and development throughout eukaryotes. The Apicomplexan parasite Toxoplasma gondii expresses three MAPKs. Two of these, ERK7 and MAPKL1, have been respectively implicated in the regulation of conoid biogenesis and centrosome duplication. The third kinase, MAPK2, is specific to and conserved throughout Alveolata, though its function is unknown. We used the auxin-inducible degron system to determine phenotypes associated with MAPK2 loss-offunction in Toxoplasma. We found that parasites lacking MAPK2 were unable to replicate and arrested early in mitosis. While the parasites continued to replicate their mitochondria, apicoplasts, and Golgi apparatus, they failed to initiate daughter-cell budding, which ultimately led to parasite death. These phenotypes appear to be the result of a failure in centrosome duplication that is distinct from the MAPKL1 phenotype. As we did not observe MAPK2 localization at the centrosome at any point in the cell cycle, our data suggest MAPK2 regulates a process at a distal site that is required for completion of centrosome duplication. ImportanceToxoplasma gondii is a ubiquitous intracellular protozoan parasite that can cause severe and fatal disease in immunocompromised patients and the developing fetus. Rapid parasite replication is critical for establishing a productive infection. Here, we demonstrate that a Toxoplasma protein kinase called MAPK2 is conserved throughout Alveolata and essential for parasite replication. We found that parasites lacking MAPK2 protein were defective in the initiation of daughter cell budding and were rendered inviable. Specifically, TgMAPK2 appears required for centrosome replication, and its loss causes arrest early in the cell cycle. MAPK2 is unique to Alveolata and not found in metazoa, and likely is a critical component of an essential parasite-specific signalling network. 2 25 30 35 40

Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress

“…Other phenotypes included abnormalities in parasite morphology. Multiple genes displayed defects similar to those previously observed for TgMAPK-L1 and TgMAPK2 57 – 60 . Aberrant morphologies allude to failures in daughter cell assembly or cytokinesis.…”

“…TgMAPK-L1 displayed cell cycle–dependent localization to diffuse puncta, consistent with its localization to the pericentrosomal matrix. In addition to the reported cytosolic localization 60 , TgMAPK2 was observed at paired puncta in a subset of vacuoles, shedding additional light into the function of this kinase.…”

“…Indeed, our knockdown of TgCrk5 mirrored the phenotype of a temperature-sensitive allele of ECR1 56 . Knockdown of the centrosome-associated kinases NIMA-related kinase 1 (TgNek1; TGGT1_292140), MAPK-like protein 1 (TgMAPK-L1; TGGT1_312570), and TgMAPK2 (TGGT1_207820) phenocopied previously characterized conditional mutants 57 – 60 . TgMAPK-L1 displayed cell cycle–dependent localization to diffuse puncta, consistent with its localization to the pericentrosomal matrix.…”

A toolbox for conditional control of gene expression in apicomplexan parasites

“…A major advantage of this system is the rapid degradation of the targeted protein and its reversibility. Indeed, several studies have shown that phenotypes caused by the depletion of a targeted protein can be reversed by simply washing out auxin from the culture media (Hu et al, 2020;Khelifa et al, 2021). The AID system can also be used in vivo in mice, although in this case auxin needs to be provided by oral gavage and in the drinking water .…”

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

“…The proper division of the centrosome is regulated by TgCDPK7 6 , TgMAPK-L1 7 and TgMAPK2 8 . Moreover, cell division of the T. gondii tachyzoite is regulated by five cyclically expressed kinases denoted as TgCrks; these kinases control several cell cycle checkpoints and ensure the (which was not certified by peer review) is the author/funder.…”