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Title: Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm

Abstract

Highlights: Black-Right-Pointing-Pointer We established Trypanosoma cruzi lacking the gene for carbamoyl phosphate synthetase II. Black-Right-Pointing-Pointer Disruption of the cpsII gene significantly reduced the growth of epimastigotes. Black-Right-Pointing-Pointer In particular, the CPSII-null mutant severely retarded intracellular growth. Black-Right-Pointing-Pointer The de novo pyrimidine pathway is critical for the parasite growth in the host cell. -- Abstract: The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes-an insect form-possess both activities, amastigotes-an intracellular replicating form of T. cruzi-are unable to mediate the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzicpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm andmore » represents a promising target for chemotherapy against Chagas disease.« less

Authors:
 [1]; ; ; ; ; ;  [1];  [2];  [3];  [4];  [5];  [1];  [1]
  1. Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan)
  2. Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)
  3. Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)
  4. Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan)
  5. Systems and Structural Biology Center, RIKEN, Tsurumi, Yokohama 230-0045 (Japan)
Publication Date:
OSTI Identifier:
22207660
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 417; Journal Issue: 3; Other Information: Copyright (c) 2011 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; BIOSYNTHESIS; CELL PROLIFERATION; CHEMOTHERAPY; CYTOPLASM; DISEASES; GENES; INFUSION; INSECTS; LIGASES; LIVER; MUTANTS; NUCLEOTIDES; PHOSPHATES; PYRIMIDINES; TRYPANOSOMA; TUMOR CELLS

Citation Formats

Hashimoto, Muneaki, E-mail: muneaki@juntendo.ac.jp, Morales, Jorge, Fukai, Yoshihisa, Suzuki, Shigeo, Takamiya, Shinzaburo, Tsubouchi, Akiko, Inoue, Syou, Inoue, Masayuki, Kita, Kiyoshi, Harada, Shigeharu, Tanaka, Akiko, Aoki, Takashi, and Nara, Takeshi, E-mail: tnara@juntendo.ac.jp. Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm. United States: N. p., 2012. Web. doi:10.1016/J.BBRC.2011.12.073.
Hashimoto, Muneaki, E-mail: muneaki@juntendo.ac.jp, Morales, Jorge, Fukai, Yoshihisa, Suzuki, Shigeo, Takamiya, Shinzaburo, Tsubouchi, Akiko, Inoue, Syou, Inoue, Masayuki, Kita, Kiyoshi, Harada, Shigeharu, Tanaka, Akiko, Aoki, Takashi, & Nara, Takeshi, E-mail: tnara@juntendo.ac.jp. Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm. United States. doi:10.1016/J.BBRC.2011.12.073.
Hashimoto, Muneaki, E-mail: muneaki@juntendo.ac.jp, Morales, Jorge, Fukai, Yoshihisa, Suzuki, Shigeo, Takamiya, Shinzaburo, Tsubouchi, Akiko, Inoue, Syou, Inoue, Masayuki, Kita, Kiyoshi, Harada, Shigeharu, Tanaka, Akiko, Aoki, Takashi, and Nara, Takeshi, E-mail: tnara@juntendo.ac.jp. 2012. "Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm". United States. doi:10.1016/J.BBRC.2011.12.073.
@article{osti_22207660,
title = {Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm},
author = {Hashimoto, Muneaki, E-mail: muneaki@juntendo.ac.jp and Morales, Jorge and Fukai, Yoshihisa and Suzuki, Shigeo and Takamiya, Shinzaburo and Tsubouchi, Akiko and Inoue, Syou and Inoue, Masayuki and Kita, Kiyoshi and Harada, Shigeharu and Tanaka, Akiko and Aoki, Takashi and Nara, Takeshi, E-mail: tnara@juntendo.ac.jp},
abstractNote = {Highlights: Black-Right-Pointing-Pointer We established Trypanosoma cruzi lacking the gene for carbamoyl phosphate synthetase II. Black-Right-Pointing-Pointer Disruption of the cpsII gene significantly reduced the growth of epimastigotes. Black-Right-Pointing-Pointer In particular, the CPSII-null mutant severely retarded intracellular growth. Black-Right-Pointing-Pointer The de novo pyrimidine pathway is critical for the parasite growth in the host cell. -- Abstract: The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes-an insect form-possess both activities, amastigotes-an intracellular replicating form of T. cruzi-are unable to mediate the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzicpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm and represents a promising target for chemotherapy against Chagas disease.},
doi = {10.1016/J.BBRC.2011.12.073},
journal = {Biochemical and Biophysical Research Communications},
number = 3,
volume = 417,
place = {United States},
year = 2012,
month = 1
}
  • Highlights: Black-Right-Pointing-Pointer An Escherichia coli strain co-expressing CPSII, ATC, and DHO of Trypanosoma cruzi was constructed. Black-Right-Pointing-Pointer Molecular interactions between CPSII, ATC, and DHO of T. cruzi were demonstrated. Black-Right-Pointing-Pointer CPSII bound with both ATC and DHO. Black-Right-Pointing-Pointer ATC bound with both CPSII and DHO. Black-Right-Pointing-Pointer A functional tri-enzyme complex might precede the establishment of the fused enzyme. -- Abstract: The first 3 reaction steps of the de novo pyrimidine biosynthetic pathway are catalyzed by carbamoyl-phosphate synthetase II (CPSII), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), respectively. In eukaryotes, these enzymes are structurally classified into 2 types: (1) a CPSII-DHO-ATC fusionmore » enzyme (CAD) found in animals, fungi, and amoebozoa, and (2) stand-alone enzymes found in plants and the protist groups. In the present study, we demonstrate direct intermolecular interactions between CPSII, ATC, and DHO of the parasitic protist Trypanosoma cruzi, which is the causative agent of Chagas disease. The 3 enzymes were expressed in a bacterial expression system and their interactions were examined. Immunoprecipitation using an antibody specific for each enzyme coupled with Western blotting-based detection using antibodies for the counterpart enzymes showed co-precipitation of all 3 enzymes. From an evolutionary viewpoint, the formation of a functional tri-enzyme complex may have preceded-and led to-gene fusion to produce the CAD protein. This is the first report to demonstrate the structural basis of these 3 enzymes as a model of CAD. Moreover, in conjunction with the essentiality of de novo pyrimidine biosynthesis in the parasite, our findings provide a rationale for new strategies for developing drugs for Chagas disease, which target the intermolecular interactions of these 3 enzymes.« less
  • Cytotoxic CD8{sup +} T cells are particularly important to the development of protective immunity against the intracellular protozoan parasite, Trypanosoma cruzi, the etiological agent of Chagas disease. We have developed a new effective strategy of genetic immunization by activating CD8{sup +} T cells through the ubiquitin-fusion degradation (UFD) pathway. We constructed expression plasmids encoding the amastigote surface protein-2 (ASP-2) of T. cruzi. To induce the UFD pathway, a chimeric gene encoding ubiquitin fused to ASP-2 (pUB-ASP-2) was constructed. Mice immunized with pUB-ASP-2 presented lower parasitemia and longer survival period, compared with mice immunized with pASP-2 alone. Depletion of CD8{sup +}more » T cells abolished protection against T. cruzi in mice immunized with pUB-ASP-2 while depletion of CD4{sup +} T cells did not influence the effective immunity. Mice deficient in LMP2 or LMP7, subunits of immunoproteasomes, were not able to develop protective immunity induced. These results suggest that ubiquitin-fused antigens expressed in antigen-presenting cells were effectively degraded via the UFD pathway, and subsequently activated CD8{sup +} T cells. Consequently, immunization with pUB-ASP-2 was able to induce potent protective immunity against infection of T. cruzi.« less
  • Results are reported from a study of the influence gamma interferon (GIFN) and interleukin 2 (IL2) have on the capability of P388D1 cells and mouse resident peritoneal macrophages (MPM) to attach to the blood-resident parasites Trypanosoma cruzi and kill them. Cultures of trypomastigote forms of the Tulahuen strain of T. cruzi grown in bovine serum were introduced into peritoneal cells of mice, along with P388D1 cells incubated with GIFN, IL2 and both. Control cells were also maintained. Statistical analysis were then performed on data on counts of the number of dead T. Cruzi cells. The GIFN enhanced the interaction ofmore » MPM and P388D1 cells with the surface of T. Cruzi, provided the interaction was given over 12 hr to take place. A depression of the cytotoxicity of P388D1 cells was attributed to mediation by H2O2, an effect partially offset by incubation with the lymphokine GIFN. 23 references.« less