Ann LeFurgey

Protozoan parasites of the genus Leishmania are responsible for a spectrum of diseases ranging from mild cutaneous leishmaniasis to often lethal visceral forms. Leishmania parasites alternate between two major developmental stages, the flagellated promastigote which lives in the midgut of the sandfly vector and the obligatory intracellular nonmotile a flagellated amastigote which lives in the phagolysosome of mammalian macrophages. Our goal has been to begin to understand the processes by which these infective forms maintain ionic homeostasis in distinctly different and extreme environmental milieus. Our findings to date show that, similarly to the promastigote, the amastigote responds to hypo-osmotic stress by releasing ninhydrin-positive amino acids. The amount released is maximal between 1 and 4 minutes after perturbation. Cell swelling occurs at these same time points of exposure to 200 or 100 milliosmolar buffer. Immunostains sensitive to membrane potential changes in mitochondria and acidocalcisomes are currently being employed to monitor compartmental functional changes during osmotic stress. Parallel immunolabeling of membrane lipophosphoglycan (LPG) authenticated that other developmentally regulated activities, e.g., down-regulation of surface membrane LPG, did occur in these cultured amastigotes. These results suggest that volume regulatory mechanisms for instantaneous adaptation to osmotic stress are conserved in both developmental stages, in contrast to other characteristics. Our aim, to determine whether the response of amastigotes to osmotic stress entails alterations in amounts and/or subcellular distributions of Na, K and Cl, is a primary focus of on going experiments. Another aim is to determine changes, if any, in these same parameters when either form is exposed to heavy metal-containing agents, including a drug used frequently in treatment of leishmaniasis, sodium stibogluconate. During the past year, we determined that cryogenically prepared amastigotes, treated for up to 24 hours with this antimony-containing agent and evaluated with energy dispersive x-ray microanalysis, accumulate antimony in cytoplasmic regions and exhibit cytoplasmic changes in ion homeostasis (Na, K) consistent with active transport inhibition. We will continue to explore the hypotheses that an acidic subcellular compartment, the acidocalcisome, plays a critical role in maintenance of cellular osmotic balance and that the antileishmanial agents containing antimony act not only at the plasma membrane but also at the acidocalcisome membrane to disrupt transport mechanisms.