Institut Pasteur, Unité Biologie et Génétique de la Paroi Bactérienne, Paris, France.
INSERM, équipe Avenir, Paris, France.
PLoS Pathog. 2017 Dec 6;13(12):e1006725. doi: 10.1371/journal.ppat.1006725. eCollection 2017 Dec.
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.
钩端螺旋体病是一种广泛存在的人畜共患病，对人类可能很严重，由螺旋体细菌钩端螺旋体（L.interrogans）引起。宿主防御机制在钩端螺旋体病中的作用还不太清楚。Toll 样受体（TLR）4 和 TLR2 识别脂多糖（LPS）和脂蛋白对于清除小鼠中的钩端螺旋体至关重要，但核苷酸寡聚化结构域（NOD）样受体（NOD）1 和 NOD2 识别肽聚糖（PG）片段的作用尚未被研究。在这里，我们表明致病性钩端螺旋体在体外和体内均能逃避 NOD1 和 NOD2 的识别，在小鼠中也是如此。我们发现，钩端螺旋体 PG 抵抗某些水解酶的消化，并且一种保守的、功能未知的外膜脂蛋白 LipL21，是致病性钩端螺旋体的特异性脂蛋白，与 PG 紧密结合。与亲本株或互补株相比，不表达 LipL21 的突变株（lipl21-）制备的钩端螺旋体 PG 更容易被消化。从 lipl21-突变株 PG 释放的肽聚糖片段，或使用一种从亲本株 PG 中去除 LipL21 蛋白的程序制备的肽聚糖片段，在体外被人 NOD1（hNOD1）和 NOD2（hNOD2）受体识别，表明 LipL21 可保护 PG 免受降解为肽聚糖片段。在大肠杆菌中表达的 LipL21 也导致 PG 消化和 NOD 信号受损。我们发现，小鼠 NOD1（mNOD1）不能识别钩端螺旋体的 PG。这一结果通过质谱证实，结果表明钩端螺旋体 PG 主要由 MurTriDAP 组成，MurTriDAP 是 hNOD1 的天然激动剂，并且仅含有痕量的四肽聚糖，即 mNOD1 的激动剂。最后，在表达人 NOD1 且缺乏小鼠 NOD1 的转基因小鼠中，我们发现与互补株或 NOD1KO 小鼠观察到的相比，lipl21-突变株的清除率增加，这表明 LipL21 有助于逃避人类的免疫监视。这些使 L.interrogans 逃避 NOD 受体识别的新机制可能在规避先天宿主反应中很重要。
