L'enkystement chez Acanthomoeba castellanii : profil métabolique étudié par analyses biochimiques et spectroscopie de résonance magnétique nucléaire.

Abstract

Acanthamoeba castellanii is a small living amoeba that assumes two states of differentiation: an amoeboid trophozoite form where cells can grow and divide, and a resting cyst where cells have suspended their life. The encystment process arises when the environmental conditions do not further support growth. In the laboratory, the process can be induced synchronously by a proper manipulation of the growth medium. Acanthamoeba castellanii is an interesting model for the study of cell differentiation since this organism possesses many features, like endocytosis and osmoregulation, that are found in higher organisms. In this thesis, studies of metabolic changes that occur during the encystment process are presented. During the last few years, NMR spectroscopy has been shown to be an outstanding technique to follow in a continuous and nondestructive way metabolic changes in living organisms. $\sp{31}$P NMR spectroscopy is used to assess the levels of metabolites related to the cell energetic process such as ATP and P$\sb{\rm i}$. It also provides information about intracellular pH. The $\sp $C NMR is used to monitor metabolites that are present in relatively high concentrations, such as some carbohydrates and amino acids. In order to study the encystment process by NMR, we have built a perfusion system that maintains under physiological conditions densely packed cells trapped in the observation part of an NMR coil. It is difficult to obtain quantitative measurements by NMR spectroscopy. In order to get more information about the encystment process, we have developed biochemical and chromatographic techniques to analyse mono- and disaccharides, amino acids, glycogen and cellulose, from only a few milligrams of cells. Our results have shown that, during the encystment process, an important part of the glycogen is transformed to trehalose. The missing source of glycosyl units seems to be generated by de novo synthesis. Lipid reserves are prime candidates as carbon sources for the synthesis. The use of $\sp $C labelled compounds have shown unusual gluconeogenic pathways in the amoeba. Trehalose and proline are found in large amounts in the cyst. These products seem to provide the cells the protection needed to survive dessication.