(a) HEK-Blue cells were treated with iE-DAP (50 μM, NOD1 ligand, light grey), MurNAc-L-Ala-D-isoGln (5 μM, MDP, NOD2 ligand, dark grey), synthetic GlcNAc-MurNAc-L-Ala-D-isoGln (5 μM, syn GlcNAc-MDP, cyan), crosslinked peptidoglycan fragment (disaccharide-tetrapeptide-disaccharide-tripeptide, purple) and products (GlcNAc-MurNAc-L-Ala-D-isoGln: GlcNAc-MDP or GlcNAc-MurNAc-L-Ala-D-isoGln-L-Lys-L-Lys-D-Ala: GlcNAc-M7P) at 5 μM for 12 hr. The measured firefly luciferase activity was divided by Renilla luciferase activity. The plotted values are relative ratios normalized to cells without ligand treatment, valued as 1. Data are shown as the mean ±SD from triplicate values. Data was analyzed using two-tailed t-test. *p≤0.05; **p≤0.01; ***p≤0.005; ****p≤0.001. (b) NOD2 activity in HEK-Blue cells with NF-κB activation after stimulation of cells with the MurNAc-L-Ala-D-isoGln (MDP), synthetic GlcNAc-MurNAc-L-Ala-D-isoGln, purified GlcNAc-MurNAc-L-Ala-D-isoGln, purified GlcNAc-MurNAc-L-Ala-D-isoGln-L-Lys-L-Lys-D-Ala, and purified crosslinked peptidoglycan fragment at different concentrations (0.05, 0.1, 0.5, 1, 2.5, 5 μM). Two-way ANOVA with a Sidak’s posttest comparing buffer groups to MDP, syn GlcNAc-MDP, GlcNAc-MDP and GlcNAc-MDP to GlcNAc-M7P, crosslinked peptidoglycan fragment groups. Buffer shown as a negative control. *p≤0.05; **p≤0.01; ***p≤0.005; ****p≤0.001 for all analyses. (c) Expression of wild-type SagA and mutants in L. plantarum cell pellet and supernatant by anti-His6 western blot. (d) Mice were given antibiotics (ampicillin, metronidazole, neomycin, vancomycin (AMNV) for 7 days and colonized with 108 CFU of indicated bacteria 36 hr prior to oral infection with 106 C. difficile. Pooled data from three independent experiments, n = 9–10 mice/group. Survival curve of C. difficile infected mice. Log-rank analysis, p-value shown comparing L. plantarum expressing SagA, compared to vector control, C443A, signal sequence mutant (ΔSS), respectively. *p≤0.05, **p≤0.01, ***p≤0.001 for all analyses.