LipL21 lipoprotein binding to peptidoglycan enables Leptospira interrogans to escape NOD1 and NOD2 recognition

Abstract

Leptospirosis is a widespread zoonosis, potentially severe in humans, caused by spirochetal bacteria, Leptospira interrogans (L. interrogans). Host defense mechanisms involved in leptospirosis are poorly understood. Recognition of lipopolysaccharide (LPS) and lipoproteins by Toll-Like Receptors (TLR)4 and TLR2 is crucial for clearance of leptospires in mice, yet the role of Nucleotide Oligomerization Domain (NOD)-like receptors (NOD)1 and NOD2, recognizing peptidoglycan (PG) fragments has not previously been examined. Here, we show that pathogenic leptospires escape from NOD1 and NOD2 recognition both in vitro and in vivo, 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 for pathogenic leptospires, is tightly bound to the PG. Leptospiral PG prepared from a mutant not expressing LipL21 (lipl21-) was more readily 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 parental strain, were recognized in vitro by the human NOD1 (hNOD1) and NOD2 (hNOD2) receptors, suggesting that LipL21 protects 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 PG of L. interrogans. 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, in transgenic mice expressing human NOD1 and deficient for the murine NOD1, we showed enhanced clearance of a lipl21- mutant compared to the complemented strain, or to what was observed in NOD1KO mice, suggesting that LipL21 facilitates escape from immune surveillance in humans. These novel mechanisms allowing L. interrogans to escape recognition by the NOD receptors may be important in circumventing innate host responses.

Recent findings related to immune responses against leptospirosis and novel strategies to prevent infection

“…1). This protective role of LipL21 seems to be unique to Leptospira spp., since comparison of sequences did not show homology of LipL21 with any other bacterial protein [43]. In addition, we also found a peculiarity of the leptospiral PG composition that influences the species recognition by the NOD1 receptor.…”

“…Our recent work showed that L. interrogans escape recognition by the cytosolic innate NOD1 and NOD2 receptors, which recognize peptidoglycan fragments called muropeptides [43]. Indeed, we showed that these receptors are not important for the clearance of L. interrogans in mice.…”

Leptospiral LPS escapes mouse TLR4 internalization and TRIF-associated antimicrobial responses through O antigen and associated lipoproteins

“…In the case of L. interrogans , over 170 putative lipoproteins have been described, but their function remains mostly undetermined. We showed that LipL21 contributes to the escape of the immune system via very tight binding to peptidoglycan (PG) that required 4 hours boiling in SDS to be detached from the PG [33], hence preventing its recognition by the PRRs of the NOD family NOD1 and NOD2 [33]. Therefore, L. interrogans in which the abundance and variety of the lipoproteins is unusual, allowed us to highlight once more a potential new role for bacterial lipoproteins in innate immune escape.…”

“…Even though most of the functions of these lipoproteins remain a mystery, previous publications showed that some leptospiral lipoproteins play a role in the innate immune escape. Indeed, LipL21 binds tightly to the leptospiral peptidoglycan and hence prevents its recognition by the PRRs NOD1 and NOD2 [33]. Additionally, LipL21 and other lipoproteins have also been described as inhibitors of neutrophil myeloperoxidase [34].…”

Murine Models for Leptospirosis Kidney Disease

“…We are only at the infancy of understanding the complexity in kidney disease due to pathogenic Leptospira and the positive or detrimental role of pattern recognition receptors. Although some pattern recognition receptors such as TLR4 and NOD1 display some host species specificities and induce differential recognition of leptospires between mice and human [38,39] , availability of mouse models, including transgenic and humanized mice, will be instrumental to boost further research and decipher the pathophysiology of renal leptospirosis and the mechanisms of pathogenic leptospires to escape the innate immune defenses.…”