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Molecular and biochemical characterization of key enzymes in the cysteine and serine metabolic pathways of Acanthamoeba castellanii

Author:
Wu, Duo, Feng, Meng, Wang, Zhi-xin, Qiao, Ke, Tachibana, Hiroshi, Cheng, Xun-jia
Source:
Parasites & vectors 2018 v.11 no.1 pp. 604
ISSN:
1756-3305
Subject:
Acanthamoeba castellanii, Escherichia coli, biochemical pathways, biosynthesis, catalytic activity, confocal laser scanning microscopy, cysteine, cysteine synthase, dose response, drugs, encephalitis, energy metabolism, enzyme activity, gene expression, genes, human diseases, isoelectric point, keratitis, mice, molecular weight, phosphoglycerate dehydrogenase, polyclonal antibodies, quantitative polymerase chain reaction, recombinant proteins, serine, sodium sulfide
Abstract:
BACKGROUND: 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. METHODS: 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. RESULTS: 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 Na₂S 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. CONCLUSIONS: 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.
Agid:
6231612