Bacterial Peptidoglycan Fragments Deferentially Regulate Innate Immune Signaling

Abstract

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.