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Mentor: Mary Wilson, M.D.
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Expression of major surface proteases (MSPs) in L. chagasi

The major surface protease (MSP) is an abundant surface metalloprotease of Leishmania spp. encoded by three distinct gene classes in Leishmania chagasi (MSPS, MSPL, MSPC). Although MSP was discovered and initially characterized in extracellular promastigotes, MSP is also expressed by obligate intracellular amastigotes. We compared MSP expression in the L. chagasi LcJ line that cycles between axenic amastigotes and promastigotes. Promastigotes express MSPS, MSPL, and two forms of MSPC RNA, whereas amastigotes only express MSPL and one MSPC transcript. Using two-dimensional immunoblots, amastigotes were found to express more than 10 MSP isoforms, similar to promastigotes. However, promastigote MSPs focus between pI 5.2 and 6.1, whereas amastigote MSP isoforms migrate at a more acidic pI. Promastigote MSP isoforms are N-glycosylated, whereas most amastigote MSPs are not. Immuno-electron microscopy showed 2/3 of total promastigote MSP is localized on the surface and 1/3 is intracellular. In contrast, most amastigote MSP is intracellular and localized at the flagellar pocket, the major site of endocytosis/exocytosis in leishmania. Alkaline carbonate treatment and high-salt washing indicated that the majority of amastigote MSP is cytosolic, whereas promastigote MSP is mostly membrane-associated. Furthermore, promastigote MSP is shed extracellularly whereas amastigote MSP is not despite the fact that MSP acts as a most active metalloprotease in both forms of the parasites. Moreover, immunoelectron microscopy indicated that MSP concentrates at this site of contact between parasite and parasitophorous vacuole (PV). We conclude that intracellular L. chagasi amastigotes express multiple MSP isoforms similar to promastigotes, but amastigote MSPs differ from promastigote MSPs biochemically, localize differently in the parasite cell. These observations suggest the abundant MSP protease plays different roles in the extracellular and intracellular environments.

Hsiao CH, Ueno N, Shao JQ, Schroeder KR, Moore KC, Donelson JE, Wilson ME. The effects of macrophage source on the mechanism of phagocytosis and intracellular survival of Leishmania. Microbes Infect. 2011 Nov;13(12-13):1033-44. Epub 2011 Jun 30. PubMed PMID: 21723411; PubMed Central PMCID: PMC3185139.

Hsiao CH, Yao C, Storlie P, Donelson JE, Wilson ME. The major surface protease (MSP or GP63) in the intracellular amastigote stage of Leishmania chagasi. Mol Biochem Parasitol. 2008 Feb;157(2):148-59. Epub 2007 Oct 30. PubMed PMID:18067978.

Yao C, Luo J, Hsiao CH, Donelson JE, Wilson ME. Leishmania chagasi: a tetracycline-inducible cell line driven by T7 RNA polymerase. Exp Parasitol. 2007 Jul;116(3):205-13. Epub 2007 Jan 19. PubMed PMID: 17320870; PubMed Central PMCID:PMC2231517.

Yao C, Luo J, Hsiao C, Donelson JE, Wilson ME. Internal and surface subpopulations of the major surface protease (MSP) of Leishmania chagasi. Mol Biochem Parasitol. 2005 Feb;139(2):173-83. PubMed PMID: 15664652.

Abstracts

Hsiao, C.-H. C., Yao C., Donelson J.E., and Wilson M.E. The major surface protease (MSP or GP63) on different life stages of Leishmania chagasi. The American Society of Tropical Medicine and Hygiene, Washington, DC, December 11-15, 2005.

Storlie, P. A., Hsiao, C.-H. C., Yao C. Miller M. A., Donelson J. E., and Wilson M. E. Design and characterization of monoclonal antibodies to distinguish between MSP isoforms found on the surface of Leishmania chagasi. The American Society of Tropical Medicine and Hygiene, Atlanta, Georgia, November 12-16, 2006.

Hsiao, C.-H. C., Yao C., Donelson J.E., and Wilson M.E. Characterization of major surface protease (MSP) in axenic cultured amastigotes of Leishmania chagasi. American Society for Biochemistry and Molecular Biology, Washington, DC, April 28- May 2, 2007.

Hsiao, C.-H. C., Yao C., Donelson J.E., and Wilson M.E. The major surface protease (MSP) in the obligate intracellular amastigote form of Leishmania chagasi. Annual Midwestern Conference of Parasitologists, Oshkosh, Wisconsin, June 14- 16, 2007.