ABSTRACTl-Cysteine is essential for virtually all living organisms, from bacteria to higher eukaryotes. Besides having a role in the synthesis of virtually all proteins and of taurine, cysteamine, glutathione, and other redox-regulating proteins,l-cysteine has important functions under anaerobic/microaerophilic conditions. In anaerobic or microaerophilic protozoan parasites, such asEntamoeba histolytica,l-cysteine has been implicated in growth, attachment, survival, and protection from oxidative stress. However, a specific role of this amino acid or related metabolic intermediates is not well understood. In this study, using stable-isotope-labeledl-cysteine and capillary electrophoresis-time of flight mass spectrometry, we investigated the metabolism ofl-cysteine inE. histolytica. [U-13C3,15N]l-cysteine was rapidly metabolized into three unknown metabolites, besidesl-cystine andl-alanine. These metabolites were identified as thiazolidine-4-carboxylic acid (T4C), 2-methyl thiazolidine-4-carboxylic acid (MT4C), and 2-ethyl-thiazolidine-4-carboxylic acid (ET4C), the condensation products ofl-cysteine with aldehydes. We demonstrated that these 2-(R)-thiazolidine-4-carboxylic acids serve for storage ofl-cysteine. Liberation ofl-cysteine occurred when T4C was incubated with amebic lysates, suggesting enzymatic degradation of thesel-cysteine derivatives. Furthermore, T4C and MT4C significantly enhanced trophozoite growth and reduced intracellular reactive oxygen species (ROS) levels when it was added to cultures, suggesting that 2-(R)-thiazolidine-4-carboxylic acids are involved in the defense against oxidative stress.IMPORTANCEAmebiasis is a human parasitic disease caused by the protozoan parasiteEntamoeba histolytica. In this parasite,l-cysteine is the principal low-molecular-weight thiol and is assumed to play a significant role in supplying the amino acid during trophozoite invasion, particularly when the parasites move from the anaerobic intestinal lumen to highly oxygenated tissues in the intestine and the liver. It is well known thatE. histolyticaneeds a comparatively high concentration ofl-cysteine for its axenic cultivation. However, the reason for and the metabolic fate ofl-cysteine in this parasite are not well understood. Here, using a metabolomic and stable-isotope-labeled approach, we investigated the metabolic fate of this amino acid in these parasites. We found thatl-cysteine inside the cell rapidly reacts with aldehydes to form 2-(R)-thiazolidine-4-carboxylic acid. We showed that these 2-(R)-thiazolidine-4-carboxylic derivatives serve as anl-cysteine source, promote growth, and protect cells against oxidative stress by scavenging aldehydes and reducing the ROS level. Our findings represent the first demonstration of 2-(R)-thiazolidine-4-carboxylic acids and their roles in protozoan parasites.
Mass Spectrometric Analysis of l-Cysteine Metabolism: Physiological Role and Fate of l-Cysteine in the Enteric Protozoan Parasite Entamoeba histolytica
