Extraction, Purification, and Chemical Degradation of LPS from Gut Microbiota Strains

: 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

Structural determination of the lipid A from the deep-sea bacterium Zunongwangia profunda SM-A87: a small-scale approach

: Zunongwangia profunda SM-A87 is a deep-sea sedimentary bacterium from the phylum Bacteroidetes, representing a new genus of Flavobacteriaceae. 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. profunda SM-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 of Z. profunda SM-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

The Unusual Lipid A Structure and Immunoinhibitory Activity of LPS from Marine Bacteria Echinicola pacifica KMM 6172T and Echinicola vietnamensis KMM 6221T

Gram-negative bacteria experiencing marine habitats are constantly exposed to stressful conditions dictating their survival and proliferation. In response to these selective pressures, marine microorganisms adapt their membrane system to ensure protection and dynamicity in order to face the highly mutable sea environments. As an integral part of the Gram-negative outer membrane, structural modifications are commonly observed in the lipopolysaccharide (LPS) molecule; these mainly involve its glycolipid portion, i.e., the lipid A, mostly with regard to fatty acid content, to counterbalance the alterations caused by chemical and physical agents. As a consequence, unusual structural chemical features are frequently encountered in the lipid A of marine bacteria. By a combination of data attained from chemical, MALDI-TOF mass spectrometry (MS), and MS/MS analyses, here, we describe the structural characterization of the lipid A isolated from two marine bacteria of the Echinicola genus, i.e., E. pacifica KMM 6172T and E. vietnamensis KMM 6221T. This study showed for both strains a complex blend of mono-phosphorylated tri- and tetra-acylated lipid A species carrying an additional sugar moiety, a d-galacturonic acid, on the glucosamine backbone. The unusual chemical structures are reflected in a molecule that only scantly activates the immune response upon its binding to the LPS innate immunity receptor, the TLR4-MD-2 complex. Strikingly, both LPS potently inhibited the toxic effects of proinflammatory Salmonella LPS on human TLR4/MD-2

Bacteroides thetaiotaomicron rough-type lipopolysaccharide: The chemical structure and the immunological activity

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

DataSheet1_The chemistry of gut microbiome-derived lipopolysaccharides impacts on the occurrence of food allergy in the pediatric age.PDF

Introduction: Food allergy (FA) in children is a major health concern. A better definition of the pathogenesis of the disease could facilitate effective preventive and therapeutic measures. Gut microbiome alterations could modulate the occurrence of FA, although the mechanisms involved in this phenomenon are poorly characterized. Gut bacteria release signaling byproducts from their cell wall, such as lipopolysaccharides (LPSs), which can act locally and systemically, modulating the immune system function.Methods: In the current study gut microbiome-derived LPS isolated from fecal samples of FA and healthy children was chemically characterized providing insights into the carbohydrate and lipid composition as well as into the LPS macromolecular nature. In addition, by means of a chemical/MALDI-TOF MS and MS/MS approach we elucidated the gut microbiome-derived lipid A mass spectral profile directly on fecal samples. Finally, we evaluated the pro-allergic and pro-tolerogenic potential of these fecal LPS and lipid A by harnessing peripheral blood mononuclear cells from healthy donors.Results: By analyzing fecal samples, we have identified different gut microbiome-derived LPS chemical features comparing FA children and healthy controls. We also have provided evidence on a different immunoregulatory action elicited by LPS on peripheral blood mononuclear cells collected from healthy donors suggesting that LPS from healthy individuals could be able to protect against the occurrence of FA, while LPS from children affected by FA could promote the allergic response.Discussion: Altogether these data highlight the relevance of gut microbiome-derived LPSs as potential biomarkers for FA and as a target of intervention to limit the disease burden.</p