Molecular and Biochemical Characterization of a Secreted Lipase in Leishmania (2007 - Present)

Investigator:  Alison Shakarian, Salve Regina University 

Abstract:  This subproject is designed to contribute to the understanding of mechanisms of Leishmania survival, growth and development. The disease spectrum of leishmaniasis ranges from mild self-limiting cutaneous ulcers to a potentially fatal visceral infection, each dependant on the species of parasite with which one is infected. Proteins secreted by Leishmania are of interest because it is through these molecules that the parasites are able to sense, respond to and alter their environments. Such secreted proteins could make unique suitable targets for the development of new compounds to prevent or treat these diseases as currently there is no vaccine available for the prevention of transmission of these parasites and toxic antimony compounds are often used unsuccessfully for its treatment. Lipases are lipolytic enzymes that hydrolyze the ester bond of triglycerides. In other systems, lipase activity is involved in the reorganization of membrane structure, cell signaling or nutrient acquisition. These observations suggest that lipase activity is essential for Leishmania although presently the biological significance of this enzyme has not been proven experimentally. The working hypothesis, that lipase is essential to Leishmania, will be tested. The following approaches to determine the role of this enzyme in the parasite life cycle are planned: 1) The full-length copy of the lipase gene will be identified and characterized; the expression of lipase will be examined though out the parasite developmental life cycle and the phylogenic conservation of lipase among kinetoplastid protozoa will be determined. 2) Null-mutants deficient in lipase activity will be generated and tested for viability. 3) The lipase gene will be expressed as a recombinant protein and assayed for substrate preference. In addition, various classes of organophosphate inhibitors of lipase will be tested to determine the sensitivity of the leishmanial lipase to these compounds.

Presentations

2008 - 2009

Shakarian, A.M., McGugan, G., Dwyer, D. 2008. LdLIP3 encodes a secreted lipase in the human pathogen Leishmania donovani. American Society of Tropical Medicine and Hygiene 57th Annual Meeting, New Orleans, LA, December 7-11.

Shakarian, A.M. 2008. Lipase activity secreted by the human pathogen Leishmania during growth in vitro is encoded by the LIP3 gene. Rhode Island Research Alliance Symposium, Providence, RI, June 3.

Shakarian, A.M., McGugan, G., Joshi, M., Dwyer, D.M. 2008. Identification and characterization of the LdLIP3 lipase from the human pathogen Leishmania donovani. Gordon Research Conference: The Biology of Host-Parasite Interactions, Newport, RI, June 22-27.

Ganim, C., Byrne, M., Shakarian, A.M. 2008. A biochemical characterization of recombinant lipases secreted by Leishmania. 2nd Biennial IDeA Symposium of Biomedical Research Excellence, Washington, D.C., August 6-8.

Dodson, B., McEwan, B., Shakarian, A.M. 2008. Identification and characterization of the gene encoding a secretory lipase in the human pathogen Leishmania donovani. 2nd Biennial IDeA Symposium of Biomedical Research Excellence, Washington, D.C., August 6-8.

Ganim, C., Shakarian, A.M. 2008. A biochemical characterization of lipases secreted by Leishmania. New England Association of Parasitologists 8th Annual Meeting, Boston, MA, October 18.

Byrne, M., Shakarian, A.M. 2008. Determining the copy number of LdLIP3 transcripts in Leishmania promastigotes using real-time PCR. New England Association of Parasitologists 8th Annual Meeting, Boston, MA, October 18.

Dodson, B., Shakarian, A.M. 2008. Identification and characterization of the gene encoding a secretory lipase in the human pathogen Leishmania donovani. New England Association of Parasitologists 8th Annual Meeting, Boston, MA, Oct 18.

Shakarian, A.M., McGugan, G., Dwyer, D. 2008. A lipase secreted by the human pathogen Leishmania donovani is encoded by LdLIP3. Keystone Symposium (S4): Pathogenesis and Control of Emerging Infections and Drug-Resistant Organisms, Bangkok, Thailand, October 22–27.

Dodson, B., Shakarian, A.M. 2008. Detection and hybridization of the secretory lipase gene LdLIP3 in Leishmania tarentolae. 3rd Annual BioNES Meeting, Bristol, RI, December 2.

Byrne, M., Shakarian, A.M. 2008. Determining the copy number of LdLIP3 transcripts using real-time RT- PCR. 3rd Annual BioNES Meeting, Bristol, RI, December 2.

Stromberg, M., Shakarian, A.M. 2008. Southern blot analysis of the LdLIP3 gene loci in the human pathogen Leishmania donovani. 3rd Annual BioNES Meeting, Bristol, RI, December 2.

DiLorenzo, J., Byrne, M., Shakarian, A.M. 2008. Cloning and sequencing of the beta (β) tubulin gene encoded in the human pathogen Leishmania donovani. 3rd Annual BioNES Meeting, Bristol, RI, December 2.

Ganim, C., Shakarian, A.M. 2008. A biochemical characterization of the enzyme activity encoded by the secretory lipase gene LdLIP3 in Leishmania donovani. 3rd Annual BioNES Meeting, Bristol, RI, December 2.

Langley, K., Shakarian, A.M. 2008. Northern analysis of L. tarenotale total RNA using a secreted lipase gene specific probe. 3rd Annual BioNES Meeting, Bristol, RI, December 2.

2007 - 2008

Ganim, C., Bowers, L., Byrne, M., Shakarian, A.M. 2007. Expression of a secretory lipase from Leishmania donovani. Biology New England South Undergraduate Research Symposium, Bristol, RI. Nov 30.

Goulette, R., Shakarian, A.M. 2007. Isolation and quantification of Leishmania lipase RNA using real-time PCR. 65th Eastern New England Biology Conference, Boston, MA, April 22.

Shakarian, A.M., McGugan, G., Dwyer, D.M. 2007. The human pathogen Leishmania secretes lipase activity during growth in vitro. Rhode Island COBRE/INBRE Symposium, Providence, RI. May 30.

Bowers, L., Shakarian, A.M. 2007. Secretion of episomally expressed LdLIP3 in Leishmania donovani. Biology New England South Undergraduate Research Symposium, Roger Williams University, Bristol, RI. Nov 30.

Bowers, L., Shakarian, A.M. 2007. Characterization of secretory lipases produced by Leishmania promastigotes. 65th Eastern New England Biology Conference, Boston, MA, April 22.

Bowers, L., Shakarian, A.M. 2007. Expression of a secretory lipase gene in Leishmania donovani. New England Association of Parasitologits 7th Annual Meeting, Boston, MA, October 20.

Ganim, C., Bowers, L., Shakarian, A.M. 2007. Expression of the secretory lipase gene LdLIP3 in Leishmania tarentolae. New England Association of Parasitologits 7th Annual Meeting, Boston, MA, October 20.

Greco, J., Shakarian, A.M. 2007. The expression of a Leishmania secretory lipase gene in E. coli. 65th Eastern New England Biology Conference, Boston, MA, April 22.

Shakarian, A.M. 2007. Identification and characterization of lipases secreted by the human pathogen Leishmania. 2nd Northeast Regional IDeA Meeting, Burlington, VT, August 15-17.

2005 - 2006

Shakarian, A. 2006. Leishmania: Why study a tropical parasite? Biology Undergraduate Research Programs Retreat, Providence, RI, December 1, 2006.

Kpea, L., Jamal, A., Shakarian, A. M.* A genomics approach to characterize a lipase gene in Leishmania tarentolae. 1st Biennial National IDeA Symposium of Biomedical Research Excellence (NISBRE), Washington, DC, July 20-22.

Shakarian, A.M., McGugan G., Joshi, M., Dwyer, D.M. 2006. Identification, biochemical and molecular characterization of secreted lipases in the human pathogen Leishmania. The 16^th International Congress on Cell Biology and 46th American Society for Cell Biology Annual Meeting, San Diego, CA, December 9-13.

Garrett, K., Jamal, A., Shakarian, A.M. 2006. Identification of the gene encoding a secretory lipase in the human pathogen Leishmania mexicana. 1st Biennial National IDeA Symposium of Biomedical Research Excellence (NISBRE), Washington, DC, July 20-22.

Greco, J., Shakarian, A.M. 2006. The expression of a Leishmania secretory lipase gene in E. coli. Biology Undergraduate Research Programs Retreat, Providence, RI, December 1.

Bowers, L., Zuccarelli, L., Shakarian, A.M. 2006. A biochemical characterization of Leishmania secreted lipases. 1st Biennial National IDeA Symposium of Biomedical Research Excellence (NISBRE), Washington, DC, July 20-22.

Bowers, L., Shakarian, A.M. 2006. Biochemical characterization of Leishmania secreted lipases using a fluorescent microplate assay. Biology Undergraduate Research Programs Retreat, Providence, RI, December 1.

Goulette, R., Langley, K., Shakarian, A.M. 2006. Isolation and quantification of Leishmania lipase RNA using real-time PCR. Biology Undergraduate Research Programs Retreat, Providence, RI, December 1.

2004 - 2005

McGugan, G., Shakarian, A., Joshi, M., Dwyer, D. 2005. Identification and characterization of Leishmania lipases. The 16th Annual International Molecular Parasitology Meeting, Woods Hole, MA, September 11-15.

McGugan, G., Dwyer, D., Shakarian, A. 2005. Characterization of three putative lipase genes in the human pathogen Leishmania donovani. Northeast Regional IDeA Meeting (INBRE and COBRE), Mount Desert Island Biological Laboratories, Bar Harbor, ME, July 17-19.

Medeiros, J., Shakarian, A. 2005. The identification and characterization of three lipase genes/proteins from Leishmania using a genomics approach. The 18th Annual NSF-EPSCoR, National Conference, Rio Grande, Puerto Rico, September 25-29.