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Abstract
BackgroundAcanthamoeba 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.MethodsThe 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.ResultsThe 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.ConclusionsThis 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.
Highlights
Acanthamoeba spp. can cause serious human infections, including Acanthamoeba keratitis, granulomatous amoebic encephalitis and cutaneous acanthamoebiasis
This study demonstrated that AcCS participates in cysteine biosynthesis and serine degradation via the nonphosphorylation serine metabolic pathway, providing a molecular basis for the discovery of novel antiAcanthamoeba drugs
Reactivity of mouse polyclonal antibodies to AcCS, AcGDH and AcSPAT The reactivities of polyclonal antibodies raised against AcCS, AcGDH and AcSPAT were assessed by a dot blot analysis (Fig. 1)
Summary
Acanthamoeba spp. can cause serious human infections, including Acanthamoeba keratitis, granulomatous amoebic encephalitis and cutaneous acanthamoebiasis. L-cysteine is a sulphurcontaining amino acid that plays an important role in the structure, stability, catalytic activity and regulation of numerous proteins [5,6,7,8,9]. Cysteine can be generated from methionine through the transsulphuration pathway in mammals [5] and fungi [10] or from serine and inorganic sulphide. The latter pathway is known as sulphur assimilation and occurs in bacteria [11], plants [12] and a few protozoa, such as Entamoeba histolytica [13] and Trypanosoma cruzi [14]. OAS reacts with sulphide to generate cysteine, and this step is catalysed by cysteine synthase (CS, OAS thiolyase) [15]
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