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It is estimated that more than 500 different bacterial species colonize the human gut, and they are collectively known as the gut microbiota. Such a massive bacterial presence is now considered an additional organ of the human body, thus becoming the object of an intense and daily growing research activity. Gram-negative bacteria represent a large percentage of the gut microbiota strains. The main constituent of the outer membrane of Gram-negatives is the lipopolysaccharide (LPS). Since its first discovery, LPS has been extensively studied for its structure-dependent capability to elicit a potent immune inflammatory reaction when perceived by specific immune receptors present in our body. Therefore, traditionally, LPS, due to its peculiar chemistry, has been associated with pathogenic bacteria, and it has been extensively studied for its dangerous effects on human health. However, LPS is also expressed on the cell surface of harmless and beneficial bacteria that colonize our intestines. This necessarily implies that the LPS from harmless gut microbes is "chemically different" from that owned by pathogenic ones, hence enabling successful colonization of the intestinal tract without creating a threat to the host immune system. Deciphering the structural features of LPS from these gut bacteria is essential to improve our still scarce knowledge of how the human host lives in a harmonious relationship with its own microbiota. To this end, LPS extraction and purification are essential steps in this field of research. Yet working with gut bacteria is extremely complex for a number of reasons, one being related to the fact that they produce an array of other glycans and glycoconjugates, such as capsular polysaccharides and/or exopolysaccharides, which render the isolation and characterization of the sole LPS not at all trivial. Here, we provide a protocol that might help when dealing with LPS from gut microbial species. We describe the preliminary manipulations and checks, extraction, and purification approaches, as well as the necessary chemical manipulations that should be performed to enable the characterization of the structure of an LPS by means of techniques like nuclear magnetic resonance spectroscopy and mass spectrometry.

It is estimated that more than 500 different bacterial species colonize the human gut, and they are collectively known as the gut microbiota. Such a massive bacterial presence is now considered an additional organ of the human body, thus becoming the object of an intense and daily growing research activity. Gram-negative bacteria represent a large percentage of the gut microbiota strains. The main constituent of the outer membrane of Gram-negatives is the lipopolysaccharide (LPS). Since its first discovery, LPS has been extensively studied for its structure-dependent capability to elicit a potent immune inflammatory reaction when perceived by specific immune receptors present in our body. Therefore, traditionally, LPS, due to its peculiar chemistry, has been associated with pathogenic bacteria, and it has been extensively studied for its dangerous effects on human health. However, LPS is also expressed on the cell surface of harmless and beneficial bacteria that colonize our intestines. This necessarily implies that the LPS from harmless gut microbes is "chemically different" from that owned by pathogenic ones, hence enabling successful colonization of the intestinal tract without creating a threat to the host immune system. Deciphering the structural features of LPS from these gut bacteria is essential to improve our still scarce knowledge of how the human host lives in a harmonious relationship with its own microbiota. To this end, LPS extraction and purification are essential steps in this field of research. Yet working with gut bacteria is extremely complex for a number of reasons, one being related to the fact that they produce an array of other glycans and glycoconjugates, such as capsular polysaccharides and/or exopolysaccharides, which render the isolation and characterization of the sole LPS not at all trivial. Here, we provide a protocol that might help when dealing with LPS from gut microbial species. We describe the preliminary manipulations and checks, extraction, and purification approaches, as well as the necessary chemical manipulations that should be performed to enable the characterization of the structure of an LPS by means of techniques like nuclear magnetic resonance spectroscopy and mass spectrometry.

Bacteroides thetaiotaomicron is one of the most extensively studied symbionts of the human gut. Despite its widespread distribution among human populations, still very little is known about the role of its cell envelope in the crosstalk with the immune system. Due to the extraordinary characteristic of B. thetaiotaomicron to express multiple capsular polysaccharides on its surface, research activities focused on defining how these polymers affect immune responses. This resulted in the drawback of neglecting another immunostimulatory cell surface glycoconjugate, the lipopolysaccharide (LPS). By taking advantage of an acapsular mutant of B. thetaiotaomicron, here we describe the characterization of the structure of the rough-type LPS produced by this gut mutualist. This was made up of a mono-phosphorylated and hypoacylated lipid A and of a highly charged core oligosaccharide. In vitro studies revealed a weak ability to engage the MD-2/TLR4 pathway, while it was able to promote TLR2-mediated response.

Zunongwangia profundaSM-A87 is a deep-sea sedimentary bacterium from the phylum Bacteroidetes, representing a new genus ofFlavobacteriaceae. It was previously investigated for its capability of yielding high quantities of capsular polysaccharides (CPS) with interesting rheological properties, including high viscosity and tolerance to high salinities and temperatures. However, as a Gram-negative,Z. profundaSM-A87 also expresses lipopolysaccharides (LPS) as the main components of the external leaflet of its outer membrane. Here, we describe the isolation and characterization of the glycolipid part of this LPS, i.e. the lipid A, which was achieved by-passing the extraction procedure of the full LPS and by working on the ethanol precipitation product, which contained both the CPS fraction and bacterial cells. To this aim a dual approach was adopted and all analyses confirmed the isolation ofZ. profundaSM-A87 lipid A that turned out to be a blend of species with high levels of heterogeneity both in the acylation and phosphorylation pattern, as well as in the hydrophilic backbone composition.Mono-phosphorylated tetra- and penta-acylated lipid A species were identified and characterized by a high content of branched, odd-numbered, and unsaturated fatty acid chains as well as, for some species, by the presence of a hybrid disaccharide backbone.