Synthesis of mature peptidoglycan fragments for innate immune programming

Abstract

The human body harbors trillions of microorganisms that collectively form the microbiome, a diverse community composed of both commensal and pathogenic bacteria. Commensal bacteria play essential roles in host health, including the synthesis of vitamins and the breakdown of otherwise indigestible nutrients. The innate immune system distinguishes between beneficial and harmful microbes through recognition of microbe-associated molecular patterns (MAMPs), such as peptidoglycan (PG), flagellin, and viral RNA. PG, a macromolecule composed of sugar chains crosslinked by peptides, is a major structural component of the bacterial cell wall. During bacterial turnover, fragments of PG are shed and detected by the host immune system, which mounts an inflammatory response to eliminate perceived threats. However, improper recognition of commensal-derived PG can lead to persistent inflammation, contributing to disorders such as Crohn’s disease and psoriasis. ☐ To investigate how PG fragments enter host cells, I collaborated with the Silverman Lab at UMass Chan Medical School to study the role of solute carrier (SLC) transporters in PG uptake. My work contributed to the identification of SLC46A2 as a transporter of meso-diaminopimelic acid (m-DAP)-containing PG fragments, and SLC46A3 as a transporter of muramyl dipeptide (MDP). I also found that methotrexate, a known SLC inhibitor, impedes PG transport—highlighting the potential of these carriers as therapeutic targets for inflammatory disease. To further study these transporters, I began designing and testing photoactivatable PG probes, with the goal of capturing and characterizing transporter-protein interactions. This work aims to uncover the substrate specificity and mechanistic roles of PG transporters in immune signaling. ☐ In a separate but related project, I focused on the bacterial pathogen Staphylococcus aureus, a Gram-positive organism known for its multi-drug resistance, including methicillin-resistant S. aureus (MRSA). A hallmark of S. aureus PG is the pentaglycine bridge, which crosslinks glycan chains and is essential for cell wall strength and survival. Using automated solid-phase peptide synthesis (SPPS), I synthesized mature PG fragments that incorporate this pentaglycine structure. These synthetic fragments provide a platform for probing the immunological impact of S. aureus-derived PG and offer a tool for studying host-pathogen interactions at a molecular level. ☐ Through my research—including the synthesis of meso-diaminopimelic acid (m-DAP) and the integration of automated synthetic methodologies—I have advanced the understanding of how specific peptidoglycan (PG) structures are transported by the host and established a foundation for the novel synthesis of muropeptides with potential therapeutic applications in treating bacterial infections and chronic inflammatory diseases.