LipL21 lipoprotein binding to peptidoglycan enables Leptospira interrogans to escape NOD1 and NOD2 recognition
(A) HPLC separation profiles of leptospiral muropeptides. Peptidoglycan (PG) extraction protocol influences the digestion of leptospiral PG into muropeptides. PG obtained from L. interrogans Manilae L495 and Fiocruz L1-130, extracted with 2 different protocols, called respectively 0.5 h and 4 h were digested by mutanolysin before HPLC. Commercial E. coli PG (Sigma) was used as the positive control for the mutanolysin digestion. Mutanolysin without PG (mutanolysin alone) was included as a control. (B) Leptospiral muropeptides signal through human NOD1 (hNOD1) and NOD2 (hNOD2). Six μg of PG 0.5 h and 4 h of L. interrogans Manilae (left panel) and Fiocruz (right panel), the NF-κB-luciferase reporter and NOD1 or NOD2 plasmids were co-transfected in HEK 293T cells. MurTriDAP (MTP) and MDP (100 nM) were used as positive controls (agonists) for NOD1 and NOD2 activation, respectively. Luciferase activity was measured 24 h after transfection. Data are expressed as the mean ± SEM of triplicates of relative light units, representing luciferase activity normalized with respect to β-galactosidase activity and are representative of 5 experiments. For each transfection, the unpaired t test was used to compare each condition to the negative control (water). A p value < 0.05 was considered significant. p values: *p < 0.05, ***p < 0.001. NS: non significant. (C) PG of pathogenic strains (Manilae L495 / Fiocruz L1-130) does not signal through murine NOD1. HEK293T cells were co-transfected with 10 μg of PG from Manilae L495 Fiocruz L1-130 strains or L. biflexa strain Patoc, prepared with the 4h SDS boiling protocol, along with the NF-κB-luciferase reporter (None) and human (h)NOD1, hNOD2 or murine NOD1 (mNOD1) plasmids. For each transfection, cells were stimulated with water as negative control, or with 100 nM of MurTriDAP, FK156, or MDP (agonists) as positive controls for hNOD1, mNOD1 and hNOD2 activation, respectively. Luciferase activity was measured 24 h after transfecting the cells. Data are expressed as the mean ± SEM of triplicates of relative light units representing luciferase activity normalized with respect to β-galactosidase activity and are representative of 3 experiments. The unpaired t test was used to compare the recognition of each PG by mNOD1 transfected cells (in red) to the negative control (water). A p value < 0.05 was considered significant. p values: ***p < 0.001. NS: non significant. For clarity, statistics relative to hNOD1 and hNOD2, already showed in panel (A), are not indicated. (D) HPLC analysis of the PG of L. interrogans strain Manilae prepared with the 4 h protocol and treated with chemotrypsin before digestion with mutanolysin. The numbered peaks were collected, and analyzed by mass spectrometry. The corresponding composition is in Table 1. The peak numbered 3 in red corresponds to GM4, the murine NOD1 agonist.