LEISHMANIA SPP - NITRIC OXIDE-MEDIATED METABOLIC INHIBITION OF PROMASTIGOTE AND AXENICALLY GROWN AMASTIGOTE FORMS

The antimicrobial effect of activated macrophages on parasites involve s nitric oxide (NO). NO induces intracellular parasite killing in muri ne leishmaniasis. Nevertheless. the mechanisms of action of NO as a fi nal effector molecule on intracellular forms of Leishmania are unknown . The recent development of axenically grown amastigote forms of diffe rent Leishmania species allowed direct investigation of NO activity on active and dividing populations of the mammalian stage of various Lei shmania species, which normally are only found intracellularly. Authen tic NO gas, which reproduced the antimicrobial effect elaborated by ac tivated macrophages, was flushed on promastigote and axenically cultur ed amastigote forms of L. mexicana, L. amazonensis, and L. chagasi sus pended in degassed phosphate-buffered saline (PBS). After NO treatment , the viability of parasites gradually decreased as a function of time postflushing when compared to controls. Interestingly NO killing was more effective on promastigote forms than on amastigote forms. After 1 2-hr postflushing incubation in PBS, cultures of NO-treated parasites, contrary to controls (N-2-treated), failed to proliferate whatever th e species and the developmental stage considered. Addition of both FeS O4 and L-cysteine to PBS immediately after NO treatment reversed the c apacity of authentic NO gas to inhibit the multiplication of both para site stages of Leishmania. Supplementation of PBS with alpha-ketogluta rate and cis-aconitate (citric acid cycle substrates) also reversed th e leishmanicidal activity of NO, whereas addition of citrate was less effective. The course of the developmental life cycle in vitro was als o inhibited by NO gas treatment. Enzymatic analysis showed that aconit ase activity was dramatically reduced by NO gas, whereas glucose phosp hate isomerase, aspartate transferase, and phosphoglucomutase activiti es were unchanged. In accordance, promastigote and amastigote forms of Leishmania were shown to be killed by antimycin A, an inhibitor of mi trochondrial respiration. All these data demonstrated that NO action l ed to lethal metabolic inhibition in both developmental parasite stage s by, at least in part, triggering iron loss from enzyme(s) with iron- sulfur prosthetic groups, in particular aconitase. (C) 1997 Academic P ress.