The University of Georgia College of Veterinary Medicine

eorgia Veterinary Scholars Program

GVSP Summer 2007 Scholars

Georgia Veterinary Scholar	Faculty Mentor

Candice Bailey Tuskegee University Class of 2009	Dr. Roberto Docampo

Characterization of acidocalcisomes of Leishmania major after knock out of dihydroxyacetone phosphate acyltransferase, an enzyme involved in glycerolipid biosynthesis
Candice Bailey* , Juliany C. F. Rodrigues, Rachel Zufferey, Roberto Docampo
College of Veterinary Medicine, Nursing and Allied Sciences, Tuskegee University, Tuskegee, AL 36088, Department of Cellular Biology and Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA 30602, and Department of Biochemistry, Kansas State University, Manhattan, KS 66506

Abstract

Leishmaniasis is one of the most important tropical diseases in the World with different clinical manifestations and a high rate of mortality. Domestic dogs serve as reservoirs of the Leishmania parasite that is transmitted to humans by sand fly vectors. Glycerolipids are important components of the parasite membranes constituting 70% of the total lipids of Leishmania, and are essential for rapid growth, survival at high cell density, and virulence. Here, we characterized the acidocalcisomes, acidic compartments rich in calcium and polyphophate present in these parasites, in the null mutants for dihydroxyacetone phosphate acyltransferase (DAT), an important enzyme in the biosynthesis pathway of glycerolipids. For these studies we used Leishmania major Friendlin wild type, null mutant (Δdat), and complemented (Δdat/DAT clone 1, and Δdat/DAT clone 5) strains. To determine if the lack of DAT had an effect on growth rate, we monitored the number of cells in the culture medium during several days. Δdat cells grew slowly, independently of the number of cells present at the beginning of the growth curve. Complemented cells grew better than wild-type cells and completely recovered the morphologic phenotype. Immunofluorescence analysis with an antibody against the vacuolar H+-pyrophosphatase, a marker for acidocalcisomes, revealed that the null mutants had decreased numbers of these organelles, and in some cases total disappearance. These results were confirmed by staining with acridine orange, which stains acidic compartments, and also by energy-filtered transmission electron microscopy. In addition, experiments of proton-uptake and polyphosphate detection indicated that acidocalcisomes of the mutant cells have altered H+ transport and alterations in their polyphosphate composition, respectively. Taken together, these results suggest that glycerolipid synthesis is important for acidocalcisome biogenesis and function.