The Dual NOD1/NOD2 Agonism of Muropeptides Containing a Meso-Diaminopimelic Acid Residue

• Published in: PloS one 2016-08, Vol.11 (8), p.e0160784-e0160784
• Main Authors: Dagil, Yulia A, Arbatsky, Nikolai P, Alkhazova, Biana I, L'vov, Vyacheslav L, Mazurov, Dmitriy V, Pashenkov, Mikhail V
• Format: Article
• Language: English
• Subjects: Acids; Adjuvants; Adjuvants, Immunologic - pharmacology; Analysis; Bacteria; Biochemistry; Biological activity; Biology and Life Sciences; Cell culture; Cells, Cultured; Chain branching; CRISPR; Cytokines - metabolism; Diaminopimelic Acid - pharmacology; Genomes; Gram-negative bacteria; HEK293 Cells; Humans; Immune response; Immunity, Innate - drug effects; Immunology; Immunostimulants; Innate immunity; Kinases; Laboratories; Macrophages; Macrophages - cytology; Macrophages - drug effects; Macrophages - immunology; Macrophages - metabolism; Medicine and Health Sciences; Monocytes; Monocytes - cytology; Monocytes - drug effects; Monocytes - immunology; Monocytes - metabolism; Mutation; Nod1 protein; Nod1 Signaling Adaptor Protein - agonists; Nod1 Signaling Adaptor Protein - metabolism; NOD2 protein; Nod2 Signaling Adaptor Protein - agonists; Nod2 Signaling Adaptor Protein - metabolism; Oligomerization; Peptidase; Peptides; Peptidoglycans; Properties; Recognition; Research and analysis methods
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0160784

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.