title: Identification of mutations in TgMAPK1 of Toxoplasma gondii conferring resistance to 1NM-PP1.
abstract: Toxoplasma gondii is an important food and waterborne pathogen that causes severe disease in immunocompromised patients. Bumped kinase inhibitors (BKIs) have an antiparasitic effect on T. gondii tachyzoite growth by targeting T. gondii calmodulin-domain protein kinase 1 (TgCDPK1). To identify mutations that confer resistance to BKIs, chemical mutagenesis was performed, followed by selection in media containing either 250 or 1000 nM 1NM-PP1. Whole-genome sequence analysis of resistant clones revealed single nucleotide mutations in T. gondii mitogen-activated protein kinase 1 (TgMAPK1) at amino acids 162 (L162Q) and 171 (I171N). Plasmid constructs having the TgMAPK1 L162Q mutant sequence successfully replaced native TgMAPK1 genome locus in the presence of 1000 nM 1NM-PP1. The inhibitory effect of 1NM-PP1 on cell division observed in the parent clone was decreased in 1NM-PP1-resistant clones; however, effects on parasite invasion and calcium-induced egress were similar in both parent and resistant clones. A plasmid construct expressing the full length TgMAPK1 splicing isoform with L162Q mutation successfully complemented TgMAPK1 function in the pressure of 250 nM 1NM-PP1 in plaque assay. 1NM-PP1-resistant clones showed resistance to other BKIs (3MB-PP1 and 3BrB-PP1) with different levels. Here we identify TgMAPK1 as a novel target for 1NM-PP1 activity. This inhibitory effect is mediated through inhibition of tachyzoite cell division, and can be overcome through mutations at multiple residues in TgMAPK1.
authors.affiliation: 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.
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Sugi
authors.affiliation: Department of Host-Parasite Interaction, Division of Microbiology and Immunology, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
authors.forename: Kyousuke
authors.initials: K
authors.lastname: Kobayashi
authors.affiliation: 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.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Takemae
authors.affiliation: 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.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Gong
authors.affiliation: 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.
authors.forename: Akiko
authors.initials: A
authors.lastname: Ishiwa
authors.affiliation: 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.
authors.forename: Fumi
authors.initials: F
authors.lastname: Murakoshi
authors.affiliation: 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.
authors.forename: Frances C
authors.initials: FC
authors.lastname: Recuenco
authors.affiliation: 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.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Iwanaga
authors.affiliation: 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.
authors.forename: Taisuke
authors.initials: T
authors.lastname: Horimoto
authors.affiliation: 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.
authors.forename: Hiroomi
authors.initials: H
authors.lastname: Akashi
authors.affiliation: 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 ; National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kato
keywords: 1NM-PP1; Bumped kinase inhibitor; Drug resistance; TgMAPK1; Toxoplasma gondii