Barrau, Clara (2018) Structural characterisation of endotoxins from marine and halophilic bacteria. [Tesi di dottorato]

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Gram-negative bacteria cell envelope is a complex structure that is constantly exposed to its environment. It is composed of an Inner-membrane (IM), a thin peptidoglycan layer and an Outer-membrane (OM). The main component of the OM are Lipopolysaccharides (LPS), also termed as endotoxins. Those molecules are composed of three main parts: a polysaccharide named the O-antigen, a core oligosaccharide and a Lipid A. Endotoxins lacking the polysaccharide are termed Lipooligosaccharides (LOS). LPS are known to interact with mammal’s innate immunity through the Toll-like receptor 4 (TLR4) and Myeloid Differentiation factor 2 (MD-2) receptorial complex. Depending on their structure, and in particular on their Lipid A, LPS can either have an agonist or an antagonist activity. Discovering new LPS structure is hence necessary in order to develop new therapies, since agonist LPS can be used as vaccines adjuvant and antagonist as drugs against sepsis and septic shock. In this context, LPS structures from various bacterial sources are currently under study. This project present the characterization of LPS and LOS extracted from marine and halophilic bacteria. As those organisms live in a particular environment, they developed specific strategies to adapt themselves and were hence investigated, as their LPS structure can be shaped by the adaptation to their environment. In particular, the study of LPS from the following strains is here reported. Pseudoalteromonas sp1A1 is a sponge-pathogen bacterium isolated from Suberites domuncula. The full structure of its LPS was resolved using NMR spectroscopy and Matrix assisted laser desorption (MALDI) Mass spectrometry (MS). Its O-antigen is a branched polysaccharide that have two remarkable features: (i) it possesses a pyruvate linked at 4,6-position of a Glucosamine and (ii) it possesses a 9-carbon ulosonic acid that is the 3-deoxy-D-glycero-D-galacto-nonulosonic acid (KDN). The Lipid A from Pseudoalteromonas sp1A1 was studied by MALDI MS. It is constituted of a mixture of tri- to penta-acylated species, among the penta-acylated Lipid A species, at m/z 1474.6 and 1446.6 were bis-phosphorylated species composed respectively of four C12:0 (3-OH) and one C12:0 and two C12:0 (3-OH), one C10:0 (3-OH), one C11:0 (3-OH) and one C13:0. The structure of the core oligosaccharide from Pseudoalteromonas sp1A1 LPS was also resolved and turned out to be composed of a pentasaccharide containing one Kdo, one heptose, two galactoses and one glucose. Pseudoalteromonas sp1A1 biological activity was assessed using ELISA and Quanti-blue assays. It was found that its LPS does not possess any significant immunostimulant activity on human and murine cells. The Outer-Membrane properties of Pseudoalteromonas sp1A1 were also studied through Molecular Dynamic (MD) simulation, that showed how the asymmetric repartition of Pseudoalteromonas sp1A1 Lipid A influenced the properties of the phospholipid bilayer, increasing its flexibility. Finally, MD simulation was also performed with Pseudoalteromonas sp1A1 LOS in water and in 0.5 M of NaCl, that is closed to the natural marine environment of the bacterium. Results showed that the presence of salts influenced the conformational behavior of the Kdo-Lipid A region. The second bacterial strain studied was Spiribacter salinus M19-40T, a halophile isolated from an intermediate salinity pound of a marine saltern in Spain, the structure of the Lipid A was resolved using MALDI MS and MS2 experiment. It is a mono-phosphorylated and penta-acylated species bearing two C10:0 (3-OH), one C12:0, one C14:0 (3-OH) and one C14:0 (3-oxo). This structure possesses two interesting structural features: (i) the 2+3 symmetry that is unusual - as most penta-acylated Lipid A have a 3+3 symmetry and (ii) the occurrence of the C14:0 (3-oxo). Halopeptonella vilamensis is a halophilic bacterium that have been isolated from a saline lagoon in Argentina whose Lipid A was characterized using MALDI MS and MS2 experiments. Results showed that H. vilamensis has a highly heterogeneous mixture of Lipid A species, mono-phosphorylated and hexa-acylated, that differ for the length and saturation of their acyl chains. H. vilamensis main Lipid A species possesses two C10:0 (3-OH), two C12:0 (3-OH), one C12:0 and one C12:1. Another major species possesses only saturated C12:0. Immunological assays were performed on HEK293 hTLR4/CD14/MD2 murine cell line and on Monocytes-derived Macrophages (MoMs) and Bone marrow-derived macrophages (BMDMs) human cell lines with H. vilamensis LOS and demonstrated its slight immunopotency. Finally, the characterization of cell envelope components of Halomonas smyrnensis was also attempted. H. smyrnensis is an Exopolysaccharide (EPS) producing halophile isolated from a Turkish salt lake. It is known to be a high levan producer and two novel EPS were isolated. The first one is formed by α-(1→4)-Glc polymer and the second one by α-(1→3)-GlcNAc units.

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