ABSTRACT
Muropeptides are fragments of peptidoglycan that trigger innate immune responses by activating nucleotide-binding oligomerization domain (NOD) 1 and NOD2. Muropeptides from Gram-negative bacteria contain a meso-diaminopimelic acid (meso-DAP) residue in either a terminal or a non-terminal position. While the former ones are known to be recognized by NOD1, much less is known about recognition of muropeptides with non-terminal meso-DAP, which are most abundant moieties of Gram-negative peptidoglycans. Here, we developed a novel system to assess biological activity of muropeptides, based on CRISPR/Cas9-mediated knockout (KO) of NOD1 and NOD2 genes in modified HEK293T cells. Using NOD1/NOD2 knockout and overexpression systems, as well as human monocytes and macrophages, we refine the current view of muropeptide recognition. We show that NOD2 can recognize different natural muropeptides containing a meso-DAP residue (preferably in a non-terminal position), provided they are present at micromolar concentrations. NOD2 accepts muropeptides with long and branched peptide chains and requires an intact N-acetylmuramyl residue. Muropeptides with non-terminal meso-DAP can activate NOD1 as well, but, in this case, probably require peptidase pre-processing to expose the meso-DAP residue. Depending on NOD1/NOD2 ratio in specific cell types, meso-DAP-containing muropeptides can be recognized either primarily via NOD2 (in monocytes) or via NOD1 (in monocyte-derived macrophages and HEK293T-derived cells). The dual NOD1/NOD2 agonism of meso-DAP-containing muropeptides should be taken into account when assessing cellular responses to muropeptides and designing muropeptide immunostimulants and vaccine adjuvants.
Subject(s)
Diaminopimelic Acid/pharmacology , Immunity, Innate/drug effects , Macrophages/immunology , Monocytes/immunology , Nod1 Signaling Adaptor Protein/agonists , Nod2 Signaling Adaptor Protein/agonists , Adjuvants, Immunologic/pharmacology , Cells, Cultured , Cytokines/metabolism , HEK293 Cells , Humans , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Monocytes/cytology , Monocytes/drug effects , Monocytes/metabolism , Nod1 Signaling Adaptor Protein/metabolism , Nod2 Signaling Adaptor Protein/metabolismABSTRACT
This is the first report to have identified an O-linked repetitive glycan in bacterial flagellin, a structural protein of the flagellum. Studies by sugar analysis, Smith degradation, (1)H and (13)C NMR spectroscopy, and mass spectrometry showed that the glycan chains of the polar flagellum flagellin of the plant-growth-promoting rhizobacterium Azospirillum brasilense Sp7 are represented by a polysaccharide with a molecular mass of 7.7 kDa, which has a branched tetrasaccharide repeating unit of the following structure:
