LipL21 lipoprotein binding to peptidoglycan enables Leptospira interrogans to escape NOD1 and NOD2 recognition
(A) Color phenotypes of strains expressing alkaline phosphatase (phoA) derivatives and grown as colonies on agar with chloramphenicol and 5-bromo-4-chloro-3-indoyl-phosphate (XP). FL-phoA, Δ(2–22)phoA, FL-lipl21-phoA and ΔN-lipl21-phoA correspond respectively to E. coli (BTH101) expressing the full length phoA (positive control), E. coli expressing phoA without signal peptide (negative control), E. coli expressing the full length LipL21 fused with Δ(2–22)phoA and E. coli expressing the LipL21 without signal peptide fused with Δ(2–22)phoA. (B) Growth curves of BL-21 Rosetta-2 E. coli expressing the LipL21 lipoprotein (pLipL21) or not (pEmpty) in the pRSFDuet-1 vector, without IPTG induction. (C) Crude bacterial extracts of BL-21 Rosetta-2 E. coli with empty pASK-IBA6 vector (pEmpty) or LipL21 expressing vector (pLipL21), induced by anhydrous-Tetracyclin or not (NI) and migrated on 12% acrylamide gel. Stain free coloration (left) and immunodetection (right) using a LipL21 antiserum. The Rainbow marker ladder gives an indication for the LipL21size.(D) HPLC separation profiles of muropeptides after digestion with mutanolysin. Each peptidoglycan was extracted with both the 0.5 h and 4 h protocols from BL-21 Rosetta-2 E. coli expressing the LipL21 lipoprotein (pLipL21) or not (pEmpty), after induction with anhydrous-Tetracyclin. (E) and (F) E. coli PGs, extracted from the 0.5 h protocol, were used to stimulate HEK 293T cells expressing human NOD1 (E) or NOD2 (F). HEK 293T cells were co-transfected with 6 μg of PG or 100 nM of muropeptides controls (MTP for NOD1 and MDP for NOD2, along with the reporter constructions and NOD1 or NOD2 expression plasmids. Luciferase activity was measured 24 h after transfection. Cells left untreated with muropeptides were used as negative control (water). Data are expressed as the mean of triplicates ± SEM of relative light units representing luciferase activity normalized with respect to β-galactosidase activity. The graph shown is representative of 3 equivalent experiments. The unpaired t test was used to compare the recognition of PGs prepared from E. coli with the empty vector (pEmpty) to the PG prepared from E. coli expressing LipL21 (pLipL21). A p value < 0.05 was considered significant. p values: *** p < 0.001. For clarity, statistics comparing each PG or muropeptide control to the water treated cells have not been indicated but are all significant with at least p<0,05.