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Metabolome Analysis Revealed Increase in S-Methylcysteine and Phosphatidylisopropanolamine Synthesis upon l-Cysteine Deprivation in the Anaerobic Protozoan Parasite Entamoeba histolytica*

  • Afzal Husain
    Footnotes
    Affiliations
    Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan

    Department of Parasitology, Gunma University, Graduate School of Medicine, Maebashi 371-8511, Japan
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  • Dan Sato
    Affiliations
    Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0052, Japan
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  • Ghulam Jeelani
    Footnotes
    Affiliations
    Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan

    Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, Shinjuku, Tokyo 160-8582, Japan
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  • Fumika Mi-ichi
    Footnotes
    Affiliations
    Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
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  • Vahab Ali
    Footnotes
    Affiliations
    Department of Parasitology, Gunma University, Graduate School of Medicine, Maebashi 371-8511, Japan
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  • Makoto Suematsu
    Affiliations
    Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, Shinjuku, Tokyo 160-8582, Japan
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  • Tomoyoshi Soga
    Affiliations
    Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0052, Japan
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  • Tomoyoshi Nozaki
    Correspondence
    To whom correspondence should be addressed: Dept. of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Tel.: 81-3-5285-1111, Ext. 2600; Fax: 81-3-5285-1219
    Footnotes
    Affiliations
    Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
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  • Author Footnotes
    * This work was supported by Grants-in-Aid for Scientific Research 18GS0314, 18050006, and 18073001 (to T. N.) and 20590429 (to D. S.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grant H20-Shinkosaiko-016 for research on emerging and re-emerging infectious diseases from the Ministry of Health, Labour and Welfare of Japan, and a grant for research to promote the development of anti-AIDS pharmaceuticals from the Japan Health Sciences Foundation (to T. N.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.
    1 Supported by the Monbukagakusho Scholarship from the Ministry of Education, Culture, Sports, Science and Technology.
    2 Supported by the Global Center of Excellence Program for Human Metabolomic System Biology of the Ministry of Education Culture, Sports, Science and Technology.
    3 Present address: Division of Molecular and Cellular Immunoscience, Dept. of Biomolecular Sciences, Saga University, Saga 849-8581, Japan.
    4 Present address: Dept. of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna-800007, India.
Open AccessPublished:October 05, 2010DOI:https://doi.org/10.1074/jbc.M110.167304
      l-Cysteine is ubiquitous in all living organisms and is involved in a variety of functions, including the synthesis of iron-sulfur clusters and glutathione and the regulation of the structure, stability, and catalysis of proteins. In the protozoan parasite Entamoeba histolytica, the causative agent of amebiasis, l-cysteine plays an essential role in proliferation, adherence, and defense against oxidative stress; however, the essentiality of this amino acid in the pathways it regulates is not well understood. In the present study, we applied capillary electrophoresis time-of-flight mass spectrometry to quantitate charged metabolites modulated in response to l-cysteine deprivation in E. histolytica, which was selected as a model for examining the biological roles of l-cysteine. l-Cysteine deprivation had profound effects on glycolysis, amino acid, and phospholipid metabolism, with sharp decreases in the levels of l-cysteine, l-cystine, and S-adenosylmethionine and a dramatic accumulation of O-acetylserine and S-methylcysteine. We further demonstrated that S-methylcysteine is synthesized from methanethiol and O-acetylserine by cysteine synthase, which was previously considered to be involved in sulfur-assimilatory l-cysteine biosynthesis. In addition, l-cysteine depletion repressed glycolysis and energy generation, as it reduced acetyl-CoA, ethanol, and the major nucleotide di- and triphosphates, and led to the accumulation of glycolytic intermediates. Interestingly, l-cysteine depletion increased the synthesis of isopropanolamine and phosphatidylisopropanolamine, and it was confirmed that their increment was not a result of oxidative stress but was a specific response to l-cysteine depletion. We also identified a pathway in which isopropanolamine is synthesized from methylglyoxal via aminoacetone. To date, this study represents the first case where l-cysteine deprivation leads to drastic changes in core metabolic pathways, including energy, amino acid, and phospholipid metabolism.

      Introduction

      Sulfur-containing amino acids are essential for all living organisms from bacteria to higher eukaryotes and play indispensable roles in various cellular processes, such as methylation and the generation of polyamines, iron-sulfur clusters, and antioxidants. l-Cysteine in particular is essential for the structure, stability, and various protein functions, including catalysis, electron transfer, redox regulation, nitrogen fixation, and sensing for regulatory processes (
      • Beinert H.
      • Holm R.H.
      • Münck E.
      ).
      Entamoeba histolytica is an enteric protozoan parasite that causes hemorrhagic dysentery and extraintestinal abscesses in millions of inhabitants of endemic areas (
      • Stanley Jr., S.L.
      ). This parasite is generally considered as anaerobic/microaerophilic and has been shown to consume oxygen and tolerate low levels of oxygen pressure but lacks most of the components of antioxidant defense mechanisms, such as catalase, peroxidase, glutathione, and the glutathione-recycling enzymes glutathione peroxidase and glutathione reductase (
      • Weinbach E.C.
      • Diamond L.S.
      ,
      • Mehlotra R.K.
      ). l-Cysteine, which replaces glutathione as a major thiol in E. histolytica, is synthesized via a sulfur assimilatory de novo cysteine biosynthetic pathway (
      • Nozaki T.
      • Asai T.
      • Kobayashi S.
      • Ikegami F.
      • Noji M.
      • Saito K.
      • Takeuchi T.
      ,
      • Clark C.G.
      • Alsmark U.C.
      • Tazreiter M.
      • Saito-Nakano Y.
      • Ali V.
      • Marion S.
      • Weber C.
      • Mukherjee C.
      • Bruchhaus I.
      • Tannich E.
      • Leippe M.
      • Sicheritz-Ponten T.
      • Foster P.G.
      • Samuelson J.
      • Noël C.J.
      • Hirt R.P.
      • Embley T.M.
      • Gilchrist C.A.
      • Mann B.J.
      • Singh U.
      • Ackers J.P.
      • Bhattacharya S.
      • Bhattacharya A.
      • Lohia A.
      • Guillén N.
      • Duchêne M.
      • Nozaki T.
      • Hall N.
      ,
      • Nozaki T.
      • Asai T.
      • Sanchez L.B.
      • Kobayashi S.
      • Nakazawa M.
      • Takeuchi T.
      ,
      • Nozaki T.
      • Ali V.
      • Tokoro M.
      ,
      • Hussain S.
      • Ali V.
      • Jeelani G.
      • Nozaki T.
      ) that is typically present in bacteria and plants. This pathway consists of two steps that are catalyzed by serine acetyltransferase (SAT, EC 2.3.1.30)
      The abbreviations used are: SAT
      serine acetyltransferase
      CS
      cysteine synthase
      CE-TOFMS
      capillary electrophoresis time-of-flight mass spectrometry
      2′,7′-DCF-DA
      2′,7′-dichlorodihydrofluorescein di-acetate
      ESI
      electrospray ionization
      Cho
      choline
      Cho-P
      choline phosphate
      OAS
      O-acetylserine
      SMC
      S-methylcysteine
      SAM
      S-adenosylmethionine
      Ispn
      isopropanolamine
      Ispn-P
      isopropanolamine phosphate
      PtdIspn
      phosphatidylisopropanolamine
      Etn
      ethanolamine
      Etn-P
      ethanolamine phosphate
      PtdEtn
      phosphatidylethanolamine.
      (
      • Nozaki T.
      • Asai T.
      • Sanchez L.B.
      • Kobayashi S.
      • Nakazawa M.
      • Takeuchi T.
      ,
      • Nozaki T.
      • Ali V.
      • Tokoro M.
      ) and cysteine synthase (CS; OAS (thiol) lyase; EC 4.2.99.8) (
      • Nozaki T.
      • Asai T.
      • Kobayashi S.
      • Ikegami F.
      • Noji M.
      • Saito K.
      • Takeuchi T.
      ). In addition to the presence of prokaryotic/plant-like l-cysteine biosynthesis, E. histolytica is also unique because the forward and reverse trans-sulfuration pathways are absent and interrupted, respectively. Furthermore, through lateral gene transfer from archaea, E. histolytica has acquired methionine γ-lyase (EC 4.4.1.11), an enzyme that degrades l-methionine, l-homocysteine, and l-cysteine (
      • Loftus B.
      • Anderson I.
      • Davies R.
      • Alsmark U.C.
      • Samuelson J.
      • Amedeo P.
      • Roncaglia P.
      • Berriman M.
      • Hirt R.P.
      • Mann B.J.
      • Nozaki T.
      • Suh B.
      • Pop M.
      • Duchene M.
      • Ackers J.
      • Tannich E.
      • Leippe M.
      • Hofer M.
      • Bruchhaus I.
      • Willhoeft U.
      • Bhattacharya A.
      • Chillingworth T.
      • Churcher C.
      • Hance Z.
      • Harris B.
      • Harris D.
      • Jagels K.
      • Moule S.
      • Mungall K.
      • Ormond D.
      • Squares R.
      • Whitehead S.
      • Quail M.A.
      • Rabbinowitsch E.
      • Norbertczak H.
      • Price C.
      • Wang Z.
      • Guillén N.
      • Gilchrist C.
      • Stroup S.E.
      • Bhattacharya S.
      • Lohia A.
      • Foster P.G.
      • Sicheritz-Ponten T.
      • Weber C.
      • Singh U.
      • Mukherjee C.
      • El-Sayed N.M.
      • Petri Jr., W.A.
      • Clark C.G.
      • Embley T.M.
      • Barrell B.
      • Fraser C.M.
      • Hall N.
      ,
      • Tokoro M.
      • Asai T.
      • Kobayashi S.
      • Takeuchi T.
      • Nozaki T.
      ,
      • Sato D.
      • Yamagata W.
      • Harada S.
      • Nozaki T.
      ). Thus, although typical parasitic protists show degenerated amino acid metabolic pathways, particularly those associated with catabolism, because of the parasitic lifestyle, sulfur-containing amino acid metabolism appears to have uniquely evolved in E. histolytica. However, the specific role of this pathway in this organism remains unclear.
      l-Cysteine is the principal low molecular weight thiol in E. histolytica and is involved in the survival, growth, attachment, elongation, motility, gene regulation, and antioxidative stress defense of this organism (
      • Fahey R.C.
      • Newton G.L.
      • Arrick B.
      • Overdank-Bogart T.
      • Aley S.B.
      ,
      • Gillin F.D.
      • Diamond L.S.
      ,
      • Gillin F.D.
      • Diamond L.S.
      ,
      • Gillin F.D.
      • Diamond L.S.
      ,
      • Jeelani G.
      • Husain A.
      • Sato D.
      • Ali V.
      • Suematsu M.
      • Soga T.
      • Nozaki T.
      ). Because sulfur-containing amino acid metabolism differs significantly between E. histolytica and its mammalian host, the molecular dissection and characterization of this pathway may lead to the development of new chemotherapeutics against this parasite (
      • Ali V.
      • Nozaki T.
      ).
      Here, to gain further insight into the roles and regulatory mechanisms of sulfur-containing amino acid metabolism and individual metabolites in E. histolytica, we utilized capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) (
      • Soga T.
      • Baran R.
      • Suematsu M.
      • Ueno Y.
      • Ikeda S.
      • Sakurakawa T.
      • Kakazu Y.
      • Ishikawa T.
      • Robert M.
      • Nishioka T.
      • Tomita M.
      ,
      • Sato S.
      • Soga T.
      • Nishioka T.
      • Tomita M.
      ,
      • Soga T.
      • Ohashi Y.
      • Ueno Y.
      • Naraoka H.
      • Tomita M.
      • Nishioka T.
      ) for the metabolomic profiling of this parasite. We observed drastic changes in the metabolome as a result of l-cysteine depletion, which led to the discovery of novel l-cysteine-mediated regulation of several metabolic pathways in E. histolytica.

      Acknowledgments

      We thank Takako Hishiki (Keio University) for the initial acquisition and analysis of CE-MS data and helpful discussions, Masahiro Sugimoto and Akiyoshi Hirayama (Keio University) for the use of CE-MS data analysis software (MasterHands), and all of the members of our laboratory for technical assistance and valuable discussions.

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