The human innate immune system responds to both pathogen and commensal bacteria at the molecular level using bacterial peptidoglycan (PG) recognition elements. Traditionally, synthetic and commercially accessible PG monosaccharide units known as muramyl dipeptide (MDP) and N-glycolyl MDP (ng-MDP) have been used to probe the mechanism of innate immune activation of pattern recognition receptors, such as NOD-like receptors. However, bacterial PG is a dynamic and complex structure, with various chemical modifications and trimming mechanisms that result in the production of disaccharide-containing elements. These molecules pose as attractive targets for immunostimulatory screening; however, studies are limited because of their synthetic accessibility. Inspired by disaccharide-containing compounds produced from the gut microbe Lactobacillus acidophilus, a robust and scalable chemical synthesis of PG-based disaccharide ligands was implemented. Together with a monosaccharide PG library, compounds were screened for their ability to stimulate proinflammatory genes in bone-marrow-derived macrophages. The data reveal distinct gene induction patterns for monosaccharide and disaccharide PG units, suggesting that PG innate immune signaling is more complex than a one activator–one pathway program, as biologically relevant fragments induce transcriptional programs to different degrees. These disaccharide molecules will serve as critical immunostimulatory tools to more precisely define specialized innate immune regulatory mechanisms that distinguish between commensal and pathogenic bacteria residing in the microbiome.

Bacterial peptidoglycan (PG) is recognized by the human innate immune system to generate an appropriate response. To gain an appreciation of how this essential polymer is sensed, a surface plasmon resonance (SPR) assay using varied PG surface presentation was developed. PG derivatives were synthesized and immobilized on the surface at different positions on the molecule to assess effects of ligand orientation on the binding affinities of NOD-like receptors (NLRs). NLRP1 and NOD2 are cytosolic innate immune proteins known to generate an immune response to PG. Both possess conserved leucine rich repeat domains (LRR) as proposed sites of molecular recognition, though limited biochemical evidence exists regarding the mechanisms of PG recognition. Here direct biochemical evidence for the association of PG fragments to NOD2 and NLRP1 with nanomolar affinity is shown. The orientations in which the fragments were presented on the SPR surface influenced the strength of PG recognition by both NLRs. This assay displays fundamental differences in binding preferences for PG by innate immune receptors and reveals unique recognition mechanisms between the LRRs. Each receptor uses specific ligand structural features to achieve optimal binding, which will be critical information to manipulate these responses and combat diseases.

The human innate immune system responds to both pathogen and commensal bacteria at the molecular level using bacterial peptidoglycan (PG) recognition elements. Traditionally, synthetic and commercially accessible PG monosaccharide units known as muramyl dipeptide (MDP) and N-glycolyl MDP (ng-MDP) have been used to probe the mechanism of innate immune activation of pattern recognition receptors (PRRs) such as NOD-like receptors (NLRs). However, bacterial PG is a dynamic and complex structure, with various chemical modifications and trimming mechanisms that result in the production of disaccharide containing elements. These molecules pose as attractive targets for immunostimulatory screening; however, studies are limited due to their synthetic accessibility. Inspired by disaccharide containing compounds produced from the gut microbe, Lactobacillus acidophilus, a robust and scalable chemical synthesis of PG-based disaccharide ligands was implemented. Together with a monosaccharide PG library, compounds were screened for their ability to stimulate proinflammatory genes in bone marrow derived macrophages (BMDMs). The data reveal a diverse gene induction pattern between monosaccharide and disaccharide PG units, suggesting that PG innate immune signaling is more complex than a one-activator-one pathway program, as biologically relevant fragments induce distinct transcriptional programs. These disaccharide molecules will serve as critical immunostimulatory tools to more precisely define specialized innate immune regulatory mechanisms that distinguish between commensal and pathogenic bacteria residing in the microbiome.

Peptidoglycan (PG) is the core structural motif of the bacterial cell wall. Fragments released from the PG serve as fundamental recognition elements for the immune system. The structure of the PG, however, encompasses a variety of chemical modifications among different bacterial species. Here, the applicability of organic synthetic methods to address this chemical diversity is explored, and the synthesis of cross-linked PG fragments, carrying biologically relevant amino acid modifications and peptide cross-linkages, is presented using solution and solid phase approaches.