title: The effect of reactive nitrogen intermediates on Leishmania mexicana mexicana
Creators: name: Reece, William Hugh Hathorn
Publisher: name: University of Glasgow
Year: 1997
Subjects: subject: Microbiology, Leishmania, nitrogen compounds.

description: Leishmania mexicana mexicana is a protozoan parasite that causes local and diffuse cutaneous leishmaniasis. There are no effective current vaccines, and current drugs have serious side effects. There is a wealth of evidence, both in vitro and in vivo, to suggest that the protective murine immune response involves the production of a set of partially oxidised products of nitrogen called Reactive Nitrogen Intermediates (RNI). RNI are synthesised during a protective murine immune response by the enzyme inducible Nitric Oxide Synthase (iNOS) and are extremely diverse both in their possible chemical targets and in their biological effects. Inhibition of RNI production in murine cutaneous leishmaniasis allows the parasite to survive in normally resistant hosts, and it is believed that they are the final effector mechanism in the protective Thl-type immune response. Very little was known about the targets of RNI in Leishmania. This thesis aims to investigate the processes by which RNI kill L. m. mexicana. This species was chosen because it has recently become possible to grow it as amastigotes axenically in vitro. [3H]- thymidine uptake and transformation efficiency viability assays were optimised for use with these axenic amastigotes. In addition, because of the unusual culture conditions for amastigotes - Schneider's Drosophila Medium, pH 5.5, with 20% (v/v) foetal calf serum - it was necessary to develop suitable methods for RNI production and detection. Production of RNI by S-nitroso-N-acetyl penicillamine and by acidified nitrite was used. Little work has been done on the role of RNI in the immune response to L. m. mexicana. Therefore, the susceptibility of this species to RNI produced by murine macrophages was determined. The kinetics of toxicity in axenic culture showed that RNI were cytotoxic rather than cytostatic to this species, and that RNI can take up to several hours to kill the parasite. RNI did not affect, and were not affected by various other components of the macrophage defence mechanism, namely beta-glucuronidase, cathepsin D and hydrogen peroxide. L. m. mexicana amastigotes were shown not to be particularly resistant to RNI compared to promastigotes, Escherichia coli, and the murine macrophage cell line J774. Low levels of RNI were also shown to be insufficient to induce a resistant phenotype in the parasite. There have been many suggestions of the possible antileishmanial targets of RNI, based on the action of RNI in other cell types. These include inhibition of the mitochondrial electron transport chain and other iron-containing proteins, damage to lipids, damage to DNA, damage of membrane components held on to the membrane by glycosyl-phosphatidylinositol (GPI) anchors, and inhibition of proteins that contain sulphydryl groups or tyrosine residues. The effect of RNI on the GPI-anchored glycoinositolphospholipids (GIPLs), DNA and mitochondrion of L. m. mexicana was examined. Of these, only the mitochondrion was affected by RNI at concentrations which approximated to the toxic concentrations, though DNA and GIPLs could be damaged by much higher concentrations of RNI. Inhibition of the mitochondrion was sufficient to account for all the toxicity of RNI to the parasite, since uncoupling the mitochondrial membrane with 2,4-dinitrophenol (DNP) caused similar kinetics of toxicity to RNI. It was also possible to inhibit the DNase activity of serum, and the implications of this to other biological systems are discussed. The involvement of the mitochondrion in the toxicity of RNI to amastigotes is not surprising given the evolutionary conservation of the enzymes in the mitochondrial electron transport chain, and given the involvement of these enzymes in RNI toxicity to E. coli and mammalian cells. However, since L. m. mexicana normally reside in low oxygen tensions, one might have expected that they would not be so reliant on their mitochondrion.