Biology subjects

Werts, C.

Publications and source records attributed to Werts, C..

3 recordsLinked to original sources

bioRxiv2020

Humanized TLR4 immunocompetent mice are more susceptible to kidney disease after infection with Leptospira interrogans than wild-type mice

Toll-Like Receptor (TLR) 4, the LPS receptor, plays a central role in the control of leptospirosis and absence of TLR4 results in lethal infection in mice. Because human TLR4 does not sense the atypical leptospiral-LPS, we hypothesized that TLR4/MD-2 humanized transgenic mice (huTLR4) may be more susceptible to leptospirosis than wild-type mice, and thus may constitute a model of acute human leptospirosis. Therefore, we infected huTLR4 mice, which express human TLR4 but not murine TLR4, with a high but sublethal dose of L. interrogans serovar Copenhageni FioCruz (Leptospira) in comparison to C57BL/6J wildtype (WT) and, as a control, a congenic strain in which the tlr4 coding sequences are deleted (muTLR4Lps-del). We show that the huTLR4 gene is fully functional in the murine background. We found that dissemination of Leptospira in blood, shedding in urine, colonization of the kidney and overall kinetics of leptospirosis progression is equivalent between WT and huTLR4 C57BL/6J mice. Furthermore, inflammation of the kidney appeared to be subdued in huTLR4 compared to WT mice in that we observed less infiltrates of mononuclear lymphocytes, less innate immune markers and no relevant differences in fibrosis markers. Contrary to our hypothesis, huTLR4 mice showed less inflammation and kidney pathology, and are not more susceptible to leptospirosis than WT mice. This study is significant as it indicates that one intact TLR4 gene, be it mouse or human, is necessary to control acute leptospirosis. Contribution to the fieldDifferences of recognition exist between mouse and human TLR4, in that the anchor of LPS in the outer membrane of Leptospira (LipidA) activates murine, but not human TLR4. We hypothesized that if human TLR4 does not sense leptospiral-LPS, then transgenic mice in which murine TLR4 was replaced with human TLR4, should be more susceptible to Leptospira dissemination as compared to congenic wild-type mice, which could result in a more robust inflammatory response and pathology in the kidney. However, we found that impaired sensing of leptospiral-LPS in huTLR4 mice did not affect overall infection in comparison to wild-type mice and does not result in increased pathology of the kidney. Our study indicates that rather than leptospiral-LPS sensing, the presence of a fully functional TLR4 receptor is necessary to control acute leptospirosis.

microbiology↗

bioRxiv2020

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

Leptospira interrogans are invasive bacteria responsible for leptospirosis, a worldwide zoonosis. They possess two periplasmic endoflagella that allow their motility. L. interrogans are stealth pathogens that escape the innate immune responses of the NOD-like receptors NOD1/2, and the human Toll-like receptor (TLR)4, sensing peptidoglycan and lipopolysaccharide (LPS), respectively. TLR5 is another receptor of bacterial cell wall components, recognizing flagellin subunits. To study the contribution of TLR5 in the host defense against leptospires, we infected WT and TLR5 deficient mice with pathogenic L. interrogans and tracked the infection by in vivo live imaging of bioluminescent bacteria or by q-PCR. We did not identify any protective or inflammatory role of murine TLR5 to control pathogenic Leptospira. Likewise, subsequent in vitro experiments showed that infections with different live strains of L. interrogans and L. biflexa did not trigger TLR5. However, unexpectedly, heat-killed bacteria stimulated human and bovine TLR5, although barely mouse TLR5. Abolition of TLR5 recognition required extensive boiling time of the bacteria or proteinase K treatment, showing an unusual high stability of the leptospiral flagellins. Interestingly, using antimicrobial peptides to destabilize live leptospires, we detected some TLR5 activity, suggesting that TLR5 could participate in the fight against leptospires in humans or cattle. Using different Leptospira strains with mutations in flagellin proteins, we further showed that neither FlaAs nor Fcps participated in the recognition by TLR5, suggesting a role for the FlaBs. These have structural homology to Salmonella FliC, and conserved residues important for TLR5 activation, as shown by in silico analyses. Accordingly, we found that leptospires regulate the expression of FlaB mRNA according to the growth phase in vitro, and that infection with L. interrogans in hamsters and in mice downregulated the expression of the FlaBs but not the FlaAs subunits. Altogether, in contrast to different bacteria that modify their flagellin sequences to escape TLR5 recognition, our study suggests that the peculiar central localization and stability of the FlaB monomers in the periplasmic endoflagella, associated with the downregulation of FlaB subunits in hosts, constitute an efficient strategy of leptospires to escape TLR5 recognition and the immune response.

immunology↗

bioRxiv2020

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

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

immunology↗