Characterization of the phytochelatin synthase from the human parasitic nematode Ancylostoma ceylanicum

Abstract

Hookworm disease is a debilitating worm infection that affects hundreds of millions of people. Despite the existence of anthelmintic drugs, reports have testified of a decrease in efficacy of these drugs. Therefore, it is imperative to find new drugs and drug targets for hookworm disease treatment. In this study we identify the gene encoding the phytochelatin synthase in the human hookworm, Ancylostoma ceylanicum (AcePCS). Phytochelatin synthase catalyzes the production of metal chelating peptides, the phytochelatins, from glutathione (GSH). In plants, algae, and fungi phytochelatin production is important for metal tolerance and detoxification. Phytochelatin synthase proteins also function in the elimination of xenobiotics by processing GSH S-conjugates. We found that in vitro AcePCS could both synthesize phytochelatins and hydrolyze a GSH S-conjugate. Interestingly, the enzyme works through a thiol-dependent and, notably, metal-independent mechanism for both transpeptidase (phytochelatin synthesis) and peptidase (hydrolysis of GSH S-conjugates) activities. AcePCS mRNAs are expressed in vivo throughout the life cycle of A. ceylanicum. Mature adult male hookworms isolated from the small intestines of their hosts displayed significantly enhanced expression of AcePCS with transcript levels 5-fold greater than other developmental forms. Although the role of AcePCS in A. ceylanicum biology has yet to be fully investigated the results reported here provide encouraging evidence of the potential that this enzyme holds as a target for new chemotherapeutic intervention.

Keywords: 2-mercaptoethanol; AcePCS; Ancylostoma ceylanicum phytochelatin synthase; EST; GSH; Glutathione; Hookworm; Metal toxicity; NTD; Neglected tropical disease; PCS; Phytochelatin; STNs; Xenobiotic metabolism; expressed sequence tag; glutathione; neglected tropical disease; phytochelatin synthase; qPCR; real-time quantitative PCR; soil-transmitted nematodes; β-ME.

Figure 1. Ancylostoma ceylanicum

phytochelatin synthase (PCS) displays high amino acid sequence similarity to phytochelatin synthases from other parasitic helminths. An alignment of the N-terminal domain containing the conserved active sites of PCS proteins from parasitic helminths was created using ClustalW and produced with Boxshade. PCS amino acid sequences included in the alignment are Ace, Ancylostoma ceylanicum (KC914882); Bma, Brugia malayi (XP_001902100); Llo, Loa loa (XP_003139039); Asu, Ascaris suum (CDS_Asuu|GS_24101 from Nematode net plus ADY48387); Sma, Schistosoma mansoni (XP_002569764); and Egr, Echinococcus granulosus (EgrG_000255400 from GeneDB at The Sanger Institute). The predicted active site Cys, His, and Asp (Glu in Egr) residues are highlighted with circles (•). Conserved residues are shown in reverse font; conservative changes are shown in grey. The C-terminal regions of these proteins are less well conserved and were not shown in the alignment.

Figure 2. Ancylostoma ceylanicum

phytochelatin synthase (AcePCS) mRNA is expressed throughout the hookworm life cycle and at significantly higher levels in adult male hookworms. AcePCS mRNA transcript levels were quantified in cDNA samples obtained from mixed populations of A. ceylanicum eggs and first stage larvae (Eggs/L1), infectious third stage larvae (L3), and adult male and female worms by qPCR. The 60S RNA gene from A ceylanicum was used as a control transcript. Fold differences in AcePCS mRNA abundance were calculated using the 2^−ΔΔCt method for the purposes of graphical representation. Normalized qPCR data was statistically analyzed using a Student’s t-Test.

Figure 3. Ancylostoma ceylanicum

PCS (AcePCS) is capable of synthesizing PC₂, PC₃, and PC₄in vitro. The kinetics of phytochelatin polymer (PC₂, PC₃, and PC₄) synthesis was measured in vitro as described in Materials and methods. The reaction mixture contained 200 mM Tris-HCl (pH 8), 10 mM β-Me, 10 mM GSH, 0.1 mM CdCl₂, and 4 μg of purified AcePCS. AcePCS-mediated synthesis of PC₂ (◆), PC₃ (■), and PC₄ (▲) are expressed in μmol per mg enzyme, as quantified by LC-MS.

Figure 4. Ancylostoma ceylanicum

PCS (AcePCS) activity is metal independent. PC₂ synthesis (black bars), S-methyl-PC₂ (grey bars), and glutathione S-bimane hydrolysis (open bars). The incubation mixture contained 200 mM Tris-HCl (pH 8), 10 mM GSH, 10 mM S-methyl-glutathione, or 1 mM glutathione S-bimane, and 3μg of purified AcePCS, with 0.1 mM CdCl₂ and 10 mM β-mercaptoethanol (β-Me) as specified. PC₂ and S-methyl-PC₂ synthesis and glutathione S-bimane hydrolysis were quantified by LC-MS. Mean value of three separate assays expressed as activity as a percentage of the reaction with 100 μM CdCl₂ and 10 mM β-Me added ± SE.