A single mutation in the gatekeeper residue in TgMAPKL-1 restores the inhibitory effect of a bumped kinase inhibitor on the cell cycle.
Toxoplasma gondii is the causative pathogen for Toxoplasmosis. Bumped kinase inhibitor 1NM-PP1 inhibits the growth of T. gondii by targeting TgCDPK1. However, we recently reported that resistance to 1NM-PP1 can be acquired via a mutation in T. gondii mitogen-activated protein kinase like 1 (TgMAPKL-1). Further characterization of how this TgMAPKL-1 mutation restores the inhibitory effect of 1NM-PP1 would shed further light on the function of TgMAPKL-1 in the parasite life cycle. Therefore, we made parasite clones with TgMAPKL-1 mutated at the gatekeeper residue Ser 191, which is critical for 1NM-PP1 susceptibility. Host cell lysis of RH/ku80(-)/HA-TgMAPKL-1(S191A) was completely inhibited at 250 nM 1NM-PP1, whereas that of RH/ku80(-)/HA-TgMAPKL-1(S191Y) was not. By comparing 1NM-PP1-sensitive (RH/ku80(-)/HA-TgMAPKL-1(S191A)) and -resistant (RH/ku80(-)/HA-TgMAPKL-1(S191Y)) clones, we observed that inhibition of TgMAPKL-1 blocked cell cycle progression after DNA duplication. Morphological analysis revealed that TgMAPKL-1 inhibition caused enlarged parasite cells with many daughter cell scaffolds and imcomplete cytokinesis. We conclude that the mutation in TgMAPKL-1 restored the cell cycle-arresting effect of 1NM-PP1 on T. gondii endodyogeny. Given that endodyogeny is the primary mechanism of cell division for both the tachyzoite and bradyzoite stages of this parasite, TgMAPKL-1 may be a promising target for drug development. Exploration of the signals that regulate TgMAPKL-1 will provide further insights into the unique mode of T. gondii cell division.
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.
Tatsuki
T
Sugi
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.
Shin-Ichiro
S
Kawazu
Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
Taisuke
T
Horimoto
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan ; Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
Kentaro
K
Kato
D000818:Animals; D030801:Animals, Genetically Modified; D000981:Antiprotozoal Agents / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000469:parasitology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011720:Pyrazoles / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D014122:Toxoplasma / Q000235:genetics*
C479693:1-tert-butyl-3-naphthalen-1-ylmethyl-1H-pyrazolo(3,4-d)pyrimidin-4-ylemine; D000981:Antiprotozoal Agents; D004791:Enzyme Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; D020928:Mitogen-Activated Protein Kinases
BKI; Cytokinesis; MAPK; Toxoplasma gondii