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

Abstract

16 Leptospirosis is a worldwide re-emerging zoonosis caused by pathogenic Leptospira spp. All 17 vertebrate species can be infected; humans are sensitive hosts whereas other species, such as rodents, 18 may become long-term renal carrier reservoirs. Upon infection, innate immune responses are initiated 19 by recognition of Microbial Associated Molecular Patterns (MAMPs) by Pattern Recognition 20Receptors (PRRs). Among MAMPs, the lipopolysaccharide (LPS) is recognized by the 21 Toll-Like-Receptor 4 (TLR4) and activates both the MyD88-dependent pathway at the plasma 22 membrane and the TRIF-dependent pathway after TLR4 internalization. We previously showed that 23 leptospiral LPS is not recognized by the human TLR4, whereas it signals through murine TLR4, 24 which mediates mouse resistance to acute leptospirosis. However, leptospiral LPS has low 25 endotoxicity in mouse cells and is an agonist of TLR2, the sensor for bacterial lipoproteins. Here, 26 using confocal microscopy and flow cytometry, we showed that the LPS of L. interrogans did not 27 induce internalization of TLR4 in mouse macrophages, unlike the LPS of Escherichia coli. 28Consequently, the LPS failed to induce the production of the TRIF-dependent nitric oxide and 29 RANTES, both important antimicrobial responses. Using shorter O antigen LPS and repurified 30 leptospiral LPS with reporter HEK cells, we further found this TLR4-TRIF escape to be dependent on 31 both the co-purifying lipoproteins and the full-length O antigen. Furthermore, our data suggest that the 32 O antigen could alter the binding of the leptospiral LPS to the co-receptor CD14 that is essential for 33 TLR4-TRIF activation. Overall, we describe here a novel immune escape mechanism linked to 34 leptospiral LPS. We hypothesize that the LPS, already known as a virulence factor, plays a major role 35 in the innate immune evasion of the leptospires, thereby contributing to their stealthiness and 36 chronicity in mice. Author summary 38Leptospira interrogans is a bacterial pathogen, responsible for leptospirosis, a worldwide neglected 39 reemerging disease. L. interrogans may cause an acute severe disease in humans, whereas rodents and 40 other animals asymptomatically carry the leptospires in their kidneys. They can therefore excrete live 41 bacteria in urine and contaminate the environment. Leptospires are stealth pathogens known to escape 42 the innate immune defenses of their hosts. They are covered in lipopolysaccharide (LPS), a bacterial 43 motif recognized in mammals through the Toll-like receptor 4 (TLR4), which triggers two different 44 signaling pathways. We showed previously that pathogenic leptospires escape TLR4 recognition in 45 humans. Here we show in mice that the leptospiral LPS triggers only one arm of the TLR4 pathway 46 and escapes the other, hence avoiding production of antimicrobial compounds. Removing the 47 lipoproteins that always co-purify with the leptospiral LPS, or using shorter LPS, restores the 48 stimulation of both pathways. This suggests a novel escape mechan...

Escape of TLR5 Recognition by Leptospira spp.: A Rationale for Atypical Endoflagella

“…The production of the murine chemokine KC (CXCL1) was measured by ELISA 24 h p.i in the cellular supernatants. This chemokine was chosen as it was recently shown to be fully dependent on the MyD88-dependent signaling pathway ( 34 ), with MyD88 being the first main adaptor involved in TLR5-induced signaling ( 35 ). We therefore considered changes in KC secretion to be a good readout for analyzing TLR5 contribution to the leptospiral-induced signaling.…”

“…Next, we assessed whether the lack of signaling through or escape of recognition by murine TLR5 is a species-specific phenomenon. Indeed, we previously highlighted PRR species-specificities of leptospiral MAMPs recognition, such as murine TLR4 receptor that only partially recognizes the leptospiral LPS ( 34 ), whereas human TLR4 does not. Conversely, human, but not the murine NOD1 is able to sense leptospiral muropeptides ( 17 , 36 ).…”

AAA+ Molecular Chaperone ClpB in Leptospira interrogans: Its Role and Significance in Leptospiral Virulence and Pathogenesis of Leptospirosis

“…Thus, the fact that the human TLR4 does not significantly participate in leptospiral recognition could indicate a bacterial mechanism of avoiding recognition by the human immune system [37,39]. Interestingly, according to the recent in vitro study of Bonhomme et al [40], although leptospiral LPS is recognized by the murine TLR4, it avoids the TRIF adaptor arm of the TLR4 response. This may prove that pathogenic leptospires have evolved yet another immune evasion mechanism linked to their LPS.…”

“…This may prove that pathogenic leptospires have evolved yet another immune evasion mechanism linked to their LPS. Further, Bonhomme and colleagues [40] suggest that the limited LPS recognition in mice could be the key to the chronicity of leptospires in rodents. In addition, LPS enables leptospires to adapt to temperature changes [41].…”

Anti-Leptospira immunoglobulin profiling in mice reveals strain specific IgG and persistent IgM responses associated with virulence and renal colonization

“…Because the humoral response is believed to be directed mostly at the O antigen part of the LPS, we wondered whether the Icterohaemorrhagiae Verdun strain would have a shorter LPS like the M895 Manilae strain that also did not elicit strong responses(47). However our recent study showed that the Icterohaemorrhagiae Verdun LPS was similar in size to those of the Copenhageni and Manilae strains(60). Since the transient IgM and low IgG responses induced by the Verdun Cl3 strain mimicked either the heat-inactivated Manilae L495 strain, the L495 at low dose or the Patoc strain, and since mice showed no clinical signs upon Icterohaemorrhagiae infection, we also surmised that the Verdun Cl3 might have lost its virulence.…”