LipL21 lipoprotein binding to peptidoglycan as one of the peculiar mechanisms of Leptospira interrogans to escape from NOD recognition

Leptospirosis is a widespread zoonosis caused by spirochetal bacteria, Leptospira interrogans (L. interrogans) that can be severe in humans. Host defense mechanisms involved in leptospirosis are little known. Recognition of lipopolysaccharide (LPS) and lipoproteins by Toll-Like Receptors (TLR)4 and TLR2 is crucial for the clearance of leptospires in mice, but the role of Nucleotide Oligomerisation Domain (NOD)-like receptors (NOD)1 and NOD2, recognizing peptidoglycan (PG) fragments has not yet been addressed.Here we show that pathogenic leptospires escape from NOD1 and NOD2 recognition both in vitro and in mice. We found that leptospiral PG is resistant to digestion by certain hydrolases and that a conserved outer membrane lipoprotein of unknown function, LipL21, specific of L. interrogans, is tightly bound to the PG. Leptospiral PG prepared from a mutant not expressing the LipL21 (lipL21-) was more easily digested than the parental or complemented strains. Muropeptides released from the PG of the lipL21- mutant, or prepared using a procedure to eliminate the LipL21 protein from the PG of the WT strain, were recognized in vitro by the human NOD1 (hNOD1) and NOD2 (hNOD2) receptors, suggesting that LipL21 protects the PG from degradation into muropeptides. LipL21 expressed in E. coli also resulted in impaired PG digestion and NOD signaling. We found that murine NOD1 (mNOD1) did not recognize the leptospiral PG. This result was confirmed by mass spectrometry showing that leptospiral PG was primarily composed of MurTriDAP, the natural agonist of hNOD1, and contained only trace amounts of the tetra muropeptide, the mNOD1 agonist. Finally, we showed an enhanced clearance of a lipL21- mutant in transgenic mice expressing the human NOD1 and deficient for the murine NOD1 (mNOD1ko) compared to NOD1ko mice, suggesting that the LipL21 protein may participate in the escape of immune responses in humans. These novel mechanisms allowing L. interrogans to escape recognition by the NOD receptors may be important in circumventing innate host responses.