Structural Characterization and Immune Activation Capacity of Peptidoglycan from Corynebacterium glutamicum in RAW264.7 Cells

Peptidoglycan (PGN) is a unique component of prokaryotic cell walls with immune-enhancing capacities. Here, we extracted PGN from <i>Corynebacterium glutamicum</i>, a by-product of amino acid fermentation, using the trichloroacetic acid (TCA) method. SDS-PAGE analysis confirmed the presence of PGN, with a band of approximately 28 kDa. Further analysis was conducted through amino acid analysis, FTIR, and MALDI-TOF/TOF MS, and the results showed that the chemical structural monomer of PGN is NAG-(β-1,4-)-NAM-l-Ala-d-Glu-l-Lis-d-Ala. The immune activation effects of PGN were evaluated in a RAW264.7 cell model. Our results showed that PGN could increase the secretion level of NO, ROS, and immune regulatory substances, including TNF-α and IL-1β, and up-regulated the mRNA expression of <i>TNF-α</i> and <i>iNOS</i>. In addition, PGN stimulated the expression of <i>ERK2</i>, <i>MyD88</i>, <i>RIP2</i>, and the related receptor <i>NOD1</i> in the NF-κB and MAPK pathways. Comparative RNA sequencing was conducted to analyze the gene expression profiles in RAW264.7 cells. KEGG analysis indicated that most of the genes were enriched in the NF-κB, MAPK, and TNF signaling pathways. Taken together, these findings suggest that PGN may have immune-activating potential for the development and application of immune adjuvants. Importantly, the application of PGN also provides a new way to utilize amino acid fermentation by-products.