Phosphate modifications in lipid A with glycochemistry tools

The mammalian immune system possesses a remarkable ability to discern self from non-self, a critical function in safeguarding against infections. At the molecular level, this discrimination is facilitated by pattern recognition receptors present on eukaryotic cells, which can identify conserved non-self molecules characteristic of microorganisms. Among these molecules, lipopolysaccharide (LPS) stands out as a complex glycolipid abundantly present in bacterial cell wall, playing a pivotal role in host-pathogen interactions and universally recognised by specific proteins of the host`s innate immune system. LPS-recognising receptors trigger a beneficial pro-inflammatory defense response to infections while maintaining immune homeostasis. Bacterial pathogens, however, possess various mechanisms to adapt their cell membranes in response to transmission between environment, vectors, and human hosts, often altering LPS composition to modulate the host immune response. In particular, modifications to the phosphate groups of lipid A, the major immunostimulatory component of LPS, can shield bacteria from recognition by host cationic antimicrobial peptides. Yet, the impact of such modifications on LPS- specific pattern recognition receptors of the host innate immune system remains largely unexplored, particularly with regard to the recently identified cytosolic LPS-sensing proteins crucial for anti-tumor immunity. Given the high heterogeneity of bacterial glycans and the inherent instability of modified phosphate groups, the isolation of structurally defined intact LPS fragments from bacterial sources is unfeasible. Chemical synthesis however stands as the reliable method for providing molecularly defined immunomodulatory LPS motifs for studying the effects of unique phosphate group modifications on interaction with host immune receptors involved in antitumor defense. Carbohydrate chemistry, or glycochemistry, offers versatile tools for the synthesis of complex glycans, providing structurally defined, homogeneous molecules of high purity suitable for biological studies. Leveraging the glycochemistry toolbox, our project aims to develop innovative synthetic strategies for the assembly of complex phosphorylated glycans, culminating in a library of bacterial LPS motifs with phosphate group modifications reflecting those found in different bacterial species. In collaboration with international research groups in immunology and structural biology, we will investigate the immunobiological activity and interaction of our synthetic phosphorylated glycolipid-glycan library with corresponding proteins. By developing a collection of synthetic bacterial lipid A variants and LPS epitopes with uniquely modified phosphate groups, our research aims to elucidate the structural and molecular basis of their interaction with host innate immune receptors, thereby advancing our understanding of LPS-induced antibacterial defense and antitumor immunity mechanisms.