A Novel Method for Inducing Amastigote-To-Trypomastigote Transformation In Vitro in Trypanosoma cruzi Reveals the Importance of Inositol 1,4,5-Trisphosphate Receptor.
BACKGROUND
Trypanosoma cruzi is a parasitic protist that causes Chagas disease, which is prevalent in Latin America. Because of the unavailability of an effective drug or vaccine, and because about 8 million people are infected with the parasite worldwide, the development of novel drugs demands urgent attention. T. cruzi infects a wide variety of mammalian nucleated cells, with a preference for myocardial cells. Non-dividing trypomastigotes in the bloodstream infect host cells where they are transformed into replication-capable amastigotes. The amastigotes revert to trypomastigotes (trypomastigogenesis) before being shed out of the host cells. Although trypomastigote transformation is an essential process for the parasite, the molecular mechanisms underlying this process have not yet been clarified, mainly because of the lack of an assay system to induce trypomastigogenesis in vitro.


RESULTS
Cultivation of amastigotes in a transformation medium composed of 80% RPMI-1640 and 20% Grace's Insect Medium mediated their transformation into trypomastigotes. Grace's Insect Medium alone also induced trypomastigogenesis. Furthermore, trypomastigogenesis was induced more efficiently in the presence of fetal bovine serum. Trypomastigotes derived from in vitro trypomastigogenesis were able to infect mammalian host cells as efficiently as tissue-culture-derived trypomastigotes (TCT) and expressed a marker protein for TCT. Using this assay system, we demonstrated that T. cruzi inositol 1,4,5-trisphosphate receptor (TcIP3R)-an intracellular Ca2+ channel and a key molecule involved in Ca2+ signaling in the parasite-is important for the transformation process.


CONCLUSIONS
Our findings provide a new tool to identify the molecular mechanisms of the amastigote-to-trypomastigote transformation, leading to a new strategy for drug development against Chagas disease.
Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
Muneaki
M
Hashimoto
Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
Jorge
J
Morales
Department of Protozoology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
Haruki
H
Uemura
Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, Saitama 351-0198, Japan; Calcium Oscillation Project, International Cooperative Research Project and Solution-Oriented Research for Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan.
Katsuhiko
K
Mikoshiba
Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
Takeshi
T
Nara
D000818:Animals; D002460:Cell Line; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D051379:Mice; D014349:Trypanosoma cruzi / Q000166:cytology* / Q000378:metabolism* / Q000472:pathogenicity
D053496:Inositol 1,4,5-Trisphosphate Receptors