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

Abstract

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.

Keywords: Chemical degradations of glycoconjugates; Extraction of glycoconjugates; Gut microbiota; Lipopolysaccharides (LPS); Purification of glycoconjugates.