A single mutation in the gatekeeper residue in TgMAPKL-1 restores the inhibitory effect of a bumped kinase inhibitor on the cell cycle

Int J Parasitol Drugs Drug Resist. 2014 Dec 19;5(1):1-8. doi: 10.1016/j.ijpddr.2014.12.001. eCollection 2015 Apr.

Abstract

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.

Keywords: BKI; Cytokinesis; MAPK; Toxoplasma gondii.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Antiprotozoal Agents / pharmacology
  • Cell Cycle / drug effects*
  • Enzyme Inhibitors / pharmacology*
  • Fibroblasts / parasitology
  • Gene Expression Regulation, Enzymologic / physiology*
  • Humans
  • Mitogen-Activated Protein Kinases / genetics
  • Mitogen-Activated Protein Kinases / metabolism*
  • Mutation
  • Pyrazoles / pharmacology*
  • Pyrimidines / pharmacology*
  • Toxoplasma / genetics*

Substances

  • 1-tert-butyl-3-naphthalen-1-ylmethyl-1H-pyrazolo(3,4-d)pyrimidin-4-ylemine
  • Antiprotozoal Agents
  • Enzyme Inhibitors
  • Pyrazoles
  • Pyrimidines
  • Mitogen-Activated Protein Kinases