Structural and functional investigation revealed gut microbiota-derived peptidoglycan disaccharides as immunomodulatory molecules that maintain host gut homeostasis

• Main Author: Li, Chenyu
• Other Authors: Qiao Yuan
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2024
• Subjects: Chemistry; Medicine, Health and Life Sciences
• Online Access: https://hdl.handle.net/10356/181730
• Institution: Nanyang Technological University

Abundant microbes reside in host gastrointestinal (GI) tract and participate in food digestion and host immune regulation via diverse metabolites, including short-chain fatty acids, bile acids and peptidoglycan fragments (PGNs). In particular, PGNs as distinctive structures of bacteria, communicate with host innate immune system mainly via mammalian NOD1/2 signaling pathways. Despite many efforts in establishing the biological functions of PGNs using MDP and iE-DAP as representative model ligands for NOD2 and NOD1 respectively, the knowledge about the presence and the identities of soluble PGNs in host GI tract and circulatory system remains limited due to lack of detection methods. Considering the diversity of microflora in host gut, understanding the structures of main participants in the family of soluble PGNs in the host are important and elementary for the following functional investigation. In this thesis, I developed a HPLC-HRMS/MS-based workflow to reveal the total PGN loads and to identify the main players of PGN reservoirs in the host gut and blood. Exploiting the current protocol, I unraveled PGN levels in healthy hosts and their dynamic changes in the gut upon antibiotics perturbation. Furthermore, PGN-derived saccharides were identified as the predominant species in host gut and serum. To be specific, only monosaccharides were found in serum so far, whereas disaccharides, especially N-acetyl-glucosamine- -1,4-N-acetylmuramic acid (GM), were abundant in host feces. Following structural characterization, I also investigated the potential biological functions of GM. Although neither NOD1 nor NOD2 responds to GM treatment, I elucidated that GM could activate different types of immune cells, including macrophages, monocytes and dendritic cells (DCs), albeit showing weaker potency than canonical NOD2 ligand MDP. GM elicited a mild proinflammatory effect in macrophages and monocytes, reflected by elevated expression of proinflammatory cytokines and chemokines, suggesting its involvement in modulation of host innate immunity. In addition, GM promoted maturation of conventional DCs, supported by increased surface marker CD80/86 expression, implying its engagement in modulating adaptive immunity indirectly. Furthermore, I applied small-molecule inhibitors to immune cells before GM stimulation and screened out TLR4 as a potential receptor for GM recognition. To further validate the dependency of TLR4 in GM sensing, I compared the performance of GM in wild-type (WT) cells and in TLR4-deficient (TLR4—/—) cells. Multiple evidence, including western blots, ELISA, RNAseq and reporter assays, illustrated simultaneous NF- B activation and IRF activity, which are two pathways downstream of TLR4 signaling, after GM induction in WT cells but not in TLR4—/— cells. Impeded inducing activities by GM were also found in Myd88-deficient cells, suggesting the dependency of Myd88 in GM sensing. Besides, enrichment of TLR4 by GM-immobilized beads and endocytosis of TLR4 upon GM coincubation both highlighted the direct interactions between GM and TLR4. Furthermore, TLR4/MD-2 complex is crucial for GM recognition in immune cells by comparing NF- B activity of GM in cells transfecting different variants of TLR4/MD-2 combination. On the other hand, structure-activity relationship analysis underscored the specific recognition of GM and its regio-isomers (i.e. MG, G-1,6-M) by TLR4 but not other analogs or fungal cell wall fragments. Additionally, priming cells with GM could effectively suppress the NF- B activation induced by LPS, indicating competition between GM and LPS in TLR4 binding. Importantly, polymyxin B titration ruled out the possibility of LPS contaminants that could contribute to positive results of all the tests done. Collectively, it is safe to conclude GM as a natural PGN-derived saccharide which acts as a mild TLR4 stimulus and tune host immune system by activating various immune cells. Gratifyingly, I confirmed the activity of GM in vivo and found that GM supplementation on top of normal gut microbiota could mitigate colon inflammation during colitis progression. Despite elusive working mechanisms, GM shed the protective effects in a TLR4-dependent manner. In conclusion, this thesis builds up the robust analytical methods for detection and identification of soluble PGNs. It also reveals alternate pathways other than canonical NOD-pathways that PGN saccharide derivatives engage in, highlighting the importance of TLR4 in sensing variant glycan units and in host regulation. Most importantly, it broadcasts the protection of host gut from colitis progression by PGN-derived disaccharide GM, offering insights of natural microbial products as agents sustaining host health.