Bacterial Lipopolysaccharide, OPS, and Lipid A
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Lipopolysaccharides are amphiphilic molecules indispensable for viability and survival of Gram-negative bacteria, as they heavily contribute to the structural integrity of their outer membrane (OM) and to the protection of the bacterial cell envelope (Di Lorenzo et al. 2015). The highly ordered structure and low fluidity of the LPS layer, stabilized by electrostatic interactions between divalent cations (as Ca2+ and Mg2+) and negatively charged groups present on LPS molecules, are responsible for the increase of permeability to hydrophobic compounds and to higher molecular weight hydrophilic compounds but also for Gram-negative superior resistance to external stress factors. Indeed, only certain antibiotics directed against Gram-negative bacteria, such as polymyxin B, are able to destabilize abovementioned ionic interactions leading to the disruption of membrane integrity. In addition, since they are exposed toward the external environment, LPS molecules participate in crucial mechanisms of host-bacterium interactions as colonization, virulence in the case of pathogen and opportunistic bacteria, adhesion, and symbiosis. Among all these activities, LPS has been shown to be the most potent immunostimulant molecule playing a key role in the pathogenesis of Gram-negative infections triggering the immune system in a wide range of eukaryotic organisms ranging from insects to plants and to humans (De Castro et al. 2012).
Both polar head and acyl residue assortments in lipid A may vary in number, type, and distribution and determine the three-dimensional structure, i.e., the conical or cylindrical molecular shape, of lipid A which is correlated to its biological activity, i.e., the binding and recognition by proteins of the innate immune system of both animals and plants (Molinaro et al. 2015; Silipo et al. 2010).
Core oligosaccharide is a complex component of the LPS molecule since it can be characterized by up to 15 monosaccharides which can be organized giving either a linear or a branched structure. The inner part of core region is directly linked to the lipid A, is well conserved, and consists of characteristic monosaccharide residues such as heptoses (L-glycero-D-manno-heptose and D-glycero-D-manno-heptose) and Kdo (3-deoxy-D-manno-octulosonic acid); this latter is considered a diagnostic marker for all Gram-negative bacteria and covalently connects the core oligosaccharide to lipid A backbone with a α-configured ketosidic linkage in almost every LPS investigated to date.
The OPS is the most variable portion of the LPS also within bacteria belonging to the same genus, and for most Gram-negative bacteria, it consists of up to 50 identical repeating oligosaccharide units consisting of two to eight different glycosyl residues (heteroglycans) or of identical monosaccharides (homoglycans). A bacterial strain produces LPSs with O-chains characterized by a wide range of lengths; this different degree of polymerization is responsible for the ladderlike pattern, showed by SDS-PAGE, typical of a S-LPS. The high structural variability of the O-polysaccharide is ascribable to the large number of sugar residues, to their different arrangements (in pyranose or furanose rings, anomeric and absolute configurations) that can build up the repeating units, as well as to the glycosidic sequence and to the presence of noncarbohydrate substituents such as phosphate, amino acids, sulfate, and acyl groups, often present in a nonstoichiometric fashion.
Quali-/quantitative composition of monosaccharides and fatty acids (GLC-MS)
Determination of size of the rings of each sugar residue (GLC-MS)
Determination of attachment points of each sugar residue (GLC-MS, NMR)
Determination of the absolute configuration of each sugar residue (GLC-MS)
Determination of the anomeric configuration of each sugar residue (NMR)
Determination of the sequence of monosaccharides in the chain (MS, NMR)
Determination of primary and secondary fatty acid location (MS)
Determination of nature and location of noncarbohydrate/fatty acid components (MS, NMR, GLC)
- Di Lorenzo F, Silipo A, Lanzetta R, Parrilli M, Molinaro A (2015) Bacterial lipopolysaccharides: an overview of their structure, biosynthesis and immunological activity. In: Cipolla L (ed) Carbohydrates chemistry: state-of-the art and challenges for drug development. Imperial College Press, London, pp 57–89Google Scholar