Extremophile bacteria glycolipids: structure and biological activity

Abstract

Extremophile bacteria are able to survive in harsh life conditions, such as high or low temperatures (thermophiles and psychrophiles, respectively), high pressure (barophiles), high or low pH values (acidophiles or alkalophiles), environments characterized by high salt concentrations (halophiles). Structural features of the macromolecules belonging to the external layer are fundamental in adaptation mechanisms, e.g. it is well known that halophiles membrane phospholipids showed an increased negative charge density, while in psychrophiles these molecules display shorter acyl chains and higher unsaturation degree. In Gram-negative bacteria, 75% of the outer membrane is constituted by lipopolysaccharides (LPSs). Consequently they play a key role in the adaptation and survival in extreme life conditions. Nevertheless, very few LPSs isolated from extremophilic bacteria has been characterized so far. LPS are constituted by three covalently linked regions: - lipid A, which is the glycolipidic portion of the macromolecule. It is the most conservative region between bacteria belonging to the same genus and represents the minimal endotoxic structural motif; - core region, which is an oligosaccharidic portion where it is possible to find LPSs peculiar monosaccharides, such as heptoses and Kdo (3-deoxy oct-2-oulosonic acid); - O-chain, which is the polysaccharidic region, not always expressed by the bacterium. Moreover, O-chain is highly variable even among bacteria belonging to the same species. Beside the structural characterization of LPS, aimed at adaptation mechanisms comprehension, also their biological activity is worth being investigated. In fact extremophilic bacteria are rarely found to be pathogen, so they are source of lipid A with potential anti-inflammatory (antagonist) or adjuvant activity. During this phD work, the LPSs from three haloalkaliphilic and two psychrophilic bacteria has been investigated. Each LPS has been extracted from dried cells, then purified and analysed by chemical analysis, NMR spectroscopy and mass spectrometry. As for the haloalkaliphilic bacteria, the LPSs belonging to Halomonas alkaliantarctica strain CRSS, Halomonas stevensii strain S18214 and Salinivibrio sharmensis strain BAGT were completely characterized. By comparing the structures obtained, especially for core oligosaccharides, it is possible to speculate that they are all characterized by high negative charge density, due to phosphate groups, usually linked to Kdo and lipid A saccharidic residues, or to uronic acids. Such structural elements contribute to the tightness of the outer-membrane and decrease the ion permeability, due to the association of LPS molecules through divalent cations (Ca2+ and Mg2+). Moreover, lipid A structural characterization of lipid A from H. stevensii and S. sharmensis has been carried out. Both the psychrophilic bacteria Pseudoalteromonas haloplanktis strain TAB 23 and Colwellia psychrerythraea strain 34H expressed a rough-LPS. The core and lipid A structures were obtained. Moreover, biological assays on both lipid A were performed. The structural common features of these two bacteria are the high negative charge density and the lack of the O-chain. The first helps membrane permeability, allowing bacterial survival in marine environment, where these microorganisms are often isolated. The second characteristic was found in all known LPS from psychrophiles and can be explained as a consequence of cell economy: O-chain biosynthesis is an energy-demanding process avoided by the organisms in low-temperature life conditions. As for the lipid A structures, they both share the presence of short and unsaturated fatty acids chains, as already found in psychrophilic bacteria membrane phospholipids. Moreover, the TNFα production was not elicited in both cases, and for P. haloplanktis TAB 23 lipid A an inhibitory activity was found. These results led to a deeper knowledge of halo- and cold adaptation mechanisms in Gram-negative bacteria. Moreover, molecules with different and interesting physicochemical and biological properties has been isolated and characterized. It is evident that extremophilic bacteria are an important source of biomolecules of which probably nowadays only the peak of the iceberg is known.