Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China.
Department of Infectious Diseases, Tokai University School of Medicine, Isehara, Kanagawa, 259-1193, Japan.
Parasit Vectors. 2018 Nov 26;11(1):604. doi: 10.1186/s13071-018-3188-7.
Acanthamoeba spp. can cause serious human infections, including Acanthamoeba keratitis, granulomatous amoebic encephalitis and cutaneous acanthamoebiasis. Cysteine biosynthesis and the L-serine metabolic pathway play important roles in the energy metabolism of Acanthamoeba spp. However, no study has confirmed the functions of cysteine synthase (AcCS) in the cysteine pathway and phosphoglycerate dehydrogenase (AcGDH) or phosphoserine aminotransferase (AcSPAT) in the non-phosphorylation serine metabolic pathway of Acanthamoeba.
The AcCS, AcGDH and AcSPAT genes were amplified by PCR, and their recombinant proteins were expressed in Escherichia coli. Polyclonal antibodies against the recombinant proteins were prepared in mice and used to determine the subcellular localisation of each native protein by confocal laser scanning microscopy. The enzymatic activity of each recombinant protein was also analysed. Furthermore, each gene expression level was analysed by quantitative PCR after treatment with different concentrations of cysteine or L-serine.
The AcCS gene encodes a 382-amino acid protein with a predicted molecular mass of 43.1 kDa and an isoelectric point (pI) of 8.11. The AcGDH gene encodes a 350-amino acid protein with a predicted molecular mass of 39.1 kDa and a pI of 5.51. The AcSPAT gene encodes a 354-amino acid protein with a predicted molecular mass of 38.3 kDa and a pI of 6.26. Recombinant AcCS exhibited a high cysteine synthesis activity using O-acetylserine and NaS as substrates. Both GDH and SPAT catalysed degradation, rather than synthesis, of serine. Exogenous L-serine or cysteine inhibited the expression of all three enzymes in a time- and dose-dependent manner.
This study demonstrated that AcCS participates in cysteine biosynthesis and serine degradation via the non-phosphorylation serine metabolic pathway, providing a molecular basis for the discovery of novel anti-Acanthamoeba drugs.
棘阿米巴属可引起严重的人类感染，包括棘阿米巴角膜炎、肉芽肿性阿米巴脑炎和皮肤棘阿米巴病。半胱氨酸生物合成和 L-丝氨酸代谢途径在棘阿米巴属的能量代谢中发挥重要作用。然而，尚无研究证实半胱氨酸途径中的半胱氨酸合酶（AcCS）以及非磷酸化丝氨酸代谢途径中的磷酸甘油酸脱氢酶（AcGDH）或磷酸丝氨酸转氨酶（AcSPAT）在棘阿米巴中的功能。
通过 PCR 扩增 AcCS、AcGDH 和 AcSPAT 基因，并在大肠杆菌中表达其重组蛋白。用重组蛋白免疫小鼠制备多克隆抗体，并通过共聚焦激光扫描显微镜确定每种天然蛋白的亚细胞定位。还分析了每种重组蛋白的酶活性。此外，在用不同浓度半胱氨酸或 L-丝氨酸处理后，通过定量 PCR 分析每个基因的表达水平。
AcCS 基因编码一个 382 个氨基酸的蛋白质，预测分子量为 43.1 kDa，等电点（pI）为 8.11。AcGDH 基因编码一个 350 个氨基酸的蛋白质，预测分子量为 39.1 kDa，pI 为 5.51。AcSPAT 基因编码一个 354 个氨基酸的蛋白质，预测分子量为 38.3 kDa，pI 为 6.26。重组 AcCS 以 O-乙酰丝氨酸和 NaS 为底物表现出高半胱氨酸合成活性。GDH 和 SPAT 均催化丝氨酸的降解而不是合成。外源性 L-丝氨酸或半胱氨酸以时间和剂量依赖的方式抑制三种酶的表达。
本研究表明 AcCS 通过非磷酸化丝氨酸代谢途径参与半胱氨酸生物合成和丝氨酸降解，为发现新型抗棘阿米巴药物提供了分子基础。
