Digestion of Peptidoglycan and Analysis of Soluble Fragments

Background

Peptidoglycan is composed of a sugar backbone linked together by peptide stems that creates a mesh-like structure important for cell shape, and turgor pressure of bacterial cells. The macromolecular peptidoglycan is assembled from monomeric units that are synthesized in the cytoplasm and consist of an N-acetylglucosamine and N-acetylmuramic acid disaccharide with a five amino acid stem. When the monomer is flipped into the periplasm, it is added to the glycan chain by transglycosylation and a portion of the peptide stems are linked together by transpeptidation.

The amino acids comprising the peptide stem can vary by species but are generally attached to muramic acid in the order L-alanine, D-glutamic acid, meso-diaminopimelic acid, D-alanine, D-alanine, with L-lysine taking the place of diaminopimelic acid in some Gram-positives. Cross-linking occurs through the free amine of the third amino acid linking either the third or fourth amino acid directly or through linker amino acids (Schleifer and Kandler, 1972). Other common modifications include amidation of amino acids (Kato and Strominger, 1968) and O-acetylation (Clarke and Dupont, 1992) or N-deacetylation (Araki et al., 1971) of sugars.

A variety of enzymes act on peptidoglycan during growth and cell division. Classes of enzymes known as lytic transglycosylases cleave glycan chains between disaccharide units at the same position as lysozyme. The important difference is that lytic transglycosylases create a 1,6-anhydro bond, in contrast to a reducing end created by lysozyme and mutanolysin. Thus the relative abundance of 1,6-anhydro bonds can be used as an approximation of glycan chain length (Ward, 1973). Different classes of peptidases act at different bonds of the peptide stem and cross-links. For example, D,D-carboxypeptidases will cleave between the fourth and fifth amino acid, while, L,D-carboxypeptidases will cleave between the third and fourth amino acid (Holtje and Tuomanen, 1991).

Muropeptide analysis can resolve the different modifications and cross-linking to give a model of the overall structure of the macromolecular peptidoglycan. One of the first observations using HPLC-based peptidoglycan analysis was the discovery that Caulobacter crescentus lacks D,D-carboxypeptidase activity (Markiewicz et al., 1983). The first comprehensive peptidoglycan analysis was done on Escherichia coli with 80 different muropeptides species identified (Glauner et al., 1988). This method has also been used to show penicillin-resistance in Neisseria meningitidis is correlated with differences in peptidoglycan structure (Antignac et al., 2003a).

Interest in peptidoglycan has seen an increase in recent years. Continued bacterial resistance to peptidoglycan-targeting antibiotics created a need for a more complete understanding of peptidoglycan metabolism. The discovery of the human peptidoglycan-recognizing proteins, NOD1 and NOD2 have also led to increased investigations into how host cells recognize peptidoglycan and how they are able to differentiate between commensal and pathogenic bacteria (Clarke and Weiser, 2011).

The following method has a number of advantages over other types of peptidoglycan analysis, including ultra-performance liquid chromatography (UPLC)-based methods. The first advantage is that nearly all of the equipment and materials are a standard part of most laboratories, so a large investment is not needed. Second, the almost 30 year history of this protocol allows comparisons with similar chromatograms to be made, allowing for preliminary identification of peptidoglycan fragments to be made quickly, then using mass spectrometry to positively identify fragments that change or are of particular interest. The third advantage is the scale of this method, which yields enough separated material for additional analysis by mass spectrometry or enzymatic reactions. More information on the history and uses of HPLC-based peptidoglycan analysis can be found in this review (Desmarais et al., 2013)