ARCHAEABACTERIA: FROM THE NATIVE SALARS TO ARCHAEOSOMES IN THE LAB

GONZALEZ R; KLOSTER A; PRIETO MJ; MONTANARI J; PETRAY P; MORILLA MJ; ROMERO EL

Rosario, Argentina

Congreso; XX Reunión Anual de la Sociedad Argentina de Protozoología; 2004

Sociedad Argentina de Protozoología

Recent work form another groups had determined that the so-called archaeosomes (vesicles formed from polar lipids extracted from archeabacteria) are excellent adyuvants of humoral and cellular immune responses. In this work we will briefly describe the steps conducing to the optimisation of the growth conditions of micro-organisms extracted from the surface of a patagonic saline. First, we could isolate 3 morphological different colonies (from different strata: grey crystals GC, black mud BM and red crystals RC). The half of the gene RNA R16S for each of these colonies was sequenced using two primers for the Archaea Domain. The three colonies resulted to be extreme halophilic archaea, with high probability to belong to Halorubrum genera. In order to prepare the archaeosomes, the total polar lipids were extracted from batches from each type of colony and lipid films were obtained by evaporating the solvent under N2 stream. The lipid films were suspended in 10 mM Tris pH 7.4; the resulted vesicular suspensions were negatively stained and observed under electronic microscope. The micrographs resulted the confirmation of the obtainment of the first archaeosomes prepared from extreme halophilic archaea grown in native ground; the formed vesicles resulted to be multilamellar, with an average size smaller than 500 nm. Further experiments showed that the three types of archaeosomes were efficiently internalised (phagocytosed) with retention of their aqueous content in macrophages cells. No change of viability of Vero cells and low cytotoxicity on J774 cells was found for 1, 10, 50 and 100 mg archaeolipids vs. 50000 cells. These preliminary results showed that: a) It is possible to prepare in vitro structurally stable archaeosomes from material extracted from extreme halophilic archaea grown in native ground. b) The archaeosomes are not cytotoxic in vitro and c) they are efficiently phagocytosed by macrophages. Due to their inherent absence of toxicity, archaeolipids are good candidates to be used as vaccine adjuvants in future approaches.archaeosomes (vesicles formed from polar lipids extracted from archeabacteria) are excellent adyuvants of humoral and cellular immune responses. In this work we will briefly describe the steps conducing to the optimisation of the growth conditions of micro-organisms extracted from the surface of a patagonic saline. First, we could isolate 3 morphological different colonies (from different strata: grey crystals GC, black mud BM and red crystals RC). The half of the gene RNA R16S for each of these colonies was sequenced using two primers for the Archaea Domain. The three colonies resulted to be extreme halophilic archaea, with high probability to belong to Halorubrum genera. In order to prepare the archaeosomes, the total polar lipids were extracted from batches from each type of colony and lipid films were obtained by evaporating the solvent under N2 stream. The lipid films were suspended in 10 mM Tris pH 7.4; the resulted vesicular suspensions were negatively stained and observed under electronic microscope. The micrographs resulted the confirmation of the obtainment of the first archaeosomes prepared from extreme halophilic archaea grown in native ground; the formed vesicles resulted to be multilamellar, with an average size smaller than 500 nm. Further experiments showed that the three types of archaeosomes were efficiently internalised (phagocytosed) with retention of their aqueous content in macrophages cells. No change of viability of Vero cells and low cytotoxicity on J774 cells was found for 1, 10, 50 and 100 mg archaeolipids vs. 50000 cells. These preliminary results showed that: a) It is possible to prepare in vitro structurally stable archaeosomes from material extracted from extreme halophilic archaea grown in native ground. b) The archaeosomes are not cytotoxic in vitro and c) they are efficiently phagocytosed by macrophages. Due to their inherent absence of toxicity, archaeolipids are good candidates to be used as vaccine adjuvants in future approaches.