Identification of mutations in TgMAPK1 of Toxoplasma gondii conferring resistance to 1NM-PP1.
Identification of mutations in TgMAPK1 of Toxoplasma gondii conferring resistance to 1NM-PP1.
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DOI：
10.1016/j.ijpddr.2013.04.001
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发表时间：
2013-12
影响因子：
4
通讯作者：
Kato, Kentaro
中科院分区：
文献类型：
--
作者：
Sugi, Tatsuki;Kobayashi, Kyousuke;Takemae, Hitoshi;Gong, Haiyan;Ishiwa, Akiko;Murakoshi, Fumi;Recuenco, Frances C.;Iwanaga, Tatsuya;Horimoto, Taisuke;Akashi, Hiroomi;Kato, Kentaro
从化学随机突变的寄生虫中建立了1 nm-PP1抗性克隆。抗性克隆的TgMAPK1基因发生突变。抗性克隆在细胞分裂时对1 nm-PP1的敏感性较低。在看门人残留物上没有发现突变。弓形虫是一种重要的食物和水传播病原体，可在免疫功能低下的患者中引起严重疾病。BKIs通过靶向弓形虫钙调素结构域蛋白激酶1(TgCDPK1)对弓形虫速殖子的生长具有抗寄生性作用。为了鉴定对BKIs产生抗性的突变，进行了化学诱变，然后在含有250或1000nm1 nm-PP1的培养基中进行选择。对抗性克隆的全基因组序列分析显示，弓形虫有丝分裂原激活蛋白激酶1(TgMAPK1)的162(L162Q)和171(I171N)氨基酸发生单核苷酸突变。在1000nM 1 nm-PP1存在下，具有TgMAPK1 L162Q突变序列的质粒构建体成功地取代了天然的TgMAPK1基因组位点。在1 nm-PP1抗性克隆中，1 nm-PP1对亲本克隆细胞分裂的抑制作用减弱，但对寄生虫入侵和钙诱导的外泄的影响在亲本克隆和抗性克隆中相似。构建了表达TgMAPK1全长剪接异构体的L162Q突变表达载体，在250nm1 nm-PP1压力下成功地互补了TgMAPK1的功能。1 nm-PP1抗性克隆对其他BKI(3MB-PP1和3BrB-PP1)表现出不同程度的抗性。在这里，我们将TgMAPK1确定为1 nm-PP1活性的新靶点。这种抑制作用是通过抑制速殖子细胞分裂来实现的，并可通过TgMAPK1多个残基的突变来克服。
1NM-PP1 resistant clones were established from chemically random mutated parasites. Resistant clones had a mutation in TgMAPK1. Resistant clones had less susceptibility to 1NM-PP1 at cell division. The mutation was not found at the gatekeeper residue. 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.
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DOI：
10.1083/jcb.200309097
发表时间：
2004-06-07
期刊：
The Journal of cell biology
影响因子：
--
作者：
Arrizabalaga G;Ruiz F;Moreno S;Boothroyd JC
通讯作者：
Boothroyd JC
影响因子：
3.6
作者：
Khan, F;Tang, JZ;Kim, K
通讯作者：
Kim, K
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
6.7
作者：
Gubbels MJ;Lehmann M;Muthalagi M;Jerome ME;Brooks CF;Szatanek T;Flynn J;Parrot B;Radke J;Striepen B;White MW
通讯作者：
White MW
影响因子：
3
作者：
Pettersen, EF;Goddard, TD;Ferrin, TE
通讯作者：
Ferrin, TE