Live Imaging of Bioluminescent Leptospira interrogans in Mice Reveals Renal Colonization as a Stealth Escape from the Blood Defenses and Antibiotics

International audienceLeptospira (L.) interrogans are bacteria responsible for a worldwide reemerging zoonosis. Some animals asymptomatically carry L. interrogans in their kidneys and excrete bacteria in their urine, which contaminates the environment. Humans are infected through skin contact with leptospires and develop mild to severe leptospirosis. Previous attempts to construct fluorescent or bioluminescent leptospires, which would permit in vivo visualization and investigation of host defense mechanisms during infection, have been unsuccessful. Using a firefly luciferase cassette and random transposition tools, we constructed bioluminescent chromosomal transformants in saprophytic and pathogenic leptospires. The kinetics of leptospiral dissemination in mice, after intraperitoneal inoculation with a pathogenic transformant, was tracked by bioluminescence using live imaging. For infective doses of 10 ^6 to 10 ^7 bacteria, we observed dissemination and exponential growth of leptospires in the blood, followed by apparent clearance of bacteria. However, with 2X10 8 bacteria, the septicemia led to the death of mice within 3 days post-infection. In surviving mice, one week after infection, pathogenic leptospires reemerged only in the kidneys, where they multiplied and reached a steady state, leading to a sustained chronic renal infection. These experiments reveal that a fraction of the leptospiral population escapes the potent blood defense, and colonizes a defined number of niches in the kidneys, proportional to the infective dose. Antibiotic treatments failed to eradicate leptospires that colonized the kidneys, although they were effective against L. interrogans if administered before or early after infection. To conclude, mice infected with bioluminescent L. interrogans proved to be a novel model to study both acute and chronic leptospirosis, and revealed that, in the kidneys, leptospires are protected from antibiotics. These bioluminescent leptospires represent a powerful new tool to challenge mice treated with drugs or vaccines, and test the survival, dissemination, and transmission of leptospires between environment and hosts. Citation: Ratet G, Veyrier FJ, Fanton d'Andon M, Kammerscheit X, Nicola M-A, et al. (2014) Live Imaging of Bioluminescent Leptospira interrogans in Mice Reveals Renal Colonization as a Stealth Escape from the Blood Defenses and Antibiotics. PLoS Negl Trop Dis 8(12): e3359

Leptospira Interrogans Induces Fibrosis in the Mouse Kidney through Inos-Dependent, TLR- and NLR-Independent Signaling Pathways

International audienceLeptospira (L.) interrogans are bacteria responsible for a worldwide reemerging zoonosis. Rodents carry L. interrogans asymptomatically in their kidneys and excrete bacteria in the urine, contaminating the environment. Humans get infected through skin contact and develop a mild or severe leptospirosis that may lead to renal failure and fibrosis. L. interrogans provoke an interstitial nephritis, but the induction of fibrosis caused by L. interrogans has not been studied in murine models. Innate immune receptors from the TLR and NLR families have recently been shown to play a role in the development and progression of tissue fibrosis in the lung, liver and kidneys under different pathophysiological situations. We recently showed that TLR2, TLR4, and NLRP3 receptors were crucial in the defense against leptospirosis. Moreover, infection of a human cell line with L. interrogans was shown to induce TLR2-dependent production of fibronectin, a component of the extracellular matrix. Therefore, we thought to assess the presence of renal fibrosis in L. interrogans infected mice and to analyze the contribution of some innate immune pathways in this process

Downregulation of the Na/K-ATPase pump by leptospiral glycolipoprotein activates the NLRP3 inflammasome

International audienceLeptospira interrogans is responsible for a zoonotic disease known to induce severe kidney dysfunction and inflammation. In this work, we demonstrate that L. interrogans induces NLRP3 inflammasome-dependent secretion of IL-1 beta through the alteration of potassium transport in bone marrow-derived macrophages. Lysosome destabilization also contributed to the IL-1 beta production upon stimulation with live, but not dead, bacteria. Using bone marrow-derived macrophages from various TLRs and nucleotide-binding oligomerization domain-deficient mice, we further determined that IL-1 beta production was dependent on TLR2 and TLR4, suggesting a participation of the leptospiral LPS to this process. Hypokaliemia in leptospirosis has been linked to the presence of glycolipoprotein, a cell wall component of L. interrogans that is known to inhibit the expression and functions of the Na/K-ATPase pump. We show in this study that glycolipoprotein activates the inflammasome and synergizes with leptospiral LPS to produce IL-1 beta, mimicking the effect of whole bacteria. These results were confirmed in vivo, as wild-type mice expressed more IL-1 beta in the kidney than TLR2/4-deficient mice 3 d postinfection with L. interrogans. Collectively, these findings provide the first characterization, to our knowledge, of bacteria-induced activation of the NLRP3 inflammasome through the downregulation of a specific host potassium transporter

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

International audienceLeptospirosis is a widespread zoonosis, potentially severe in humans, caused by spiro-chetal bacteria, Leptospira interrogans (L. interrogans). Host defense mechanisms involved in leptospirosis are poorly understood. Recognition of lipopolysaccharide (LPS) and lipopro-teins 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 hydro-lases 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 spec-trometry 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 PLOS Pathogens | https://doi

Primer list for cloning.

<p>Boldface indicates restriction sites.</p><p>Primer list for cloning.</p

Model of acute and chronic leptospirosis in mice.

<p>This figure depicts the course of leptospirosis in mice following an IP infection with a lethal dose of bioluminescent MFlum1, leading to a septicemia or with a sub-lethal dose leading to a chronic leptospirosis, and the effects of different antibiotics administered at the acute (upper part of the figure) or chronic phase (lower part of the figure) of the leptospiral infection. Mice depicted without kidneys represent mice at the acute phase of infection. Mice depicted with kidneys represent mice at the chronic phase. Inside the kidneys schemed in longitudinal cross-section, the niches colonized by leptospires, presumably the proximal part of renal tubules (proximal tubules) are depicted by small circles. A grey color scale indicates the degree of leptospiral infection, where white means free of leptospires and black means a maximum of infection or colonization. The cross indicates that the mice died or were sacrificed because of acute leptospirosis. D1 to D15: days post-infection. T1-T7 duration of antibiotic treatments. PT15: 15 days post treatment. T-2 prophylaxis treatment 2 days prior to infection.</p

Survival test of C57BL/6J mice to the infection with <i>L. interrogans</i> Manilae and its mutant MFlum1.

<p>Survival test of C57BL/6J mice to the infection with <i>L. interrogans</i> Manilae and its mutant MFlum1.</p

Effect of antibiotic treatments at the chronic phase of leptospirosis.

<p>C57BL/6J mice chronically infected for 25 days (D25) with 10⁷ MFlum1 were (A) IP injected daily for 7 days with penicillin G (Pen) (brown) or Ciprofloxacin (Cipr) (blue) from 25 until 31 dpi (T1 to T7), (B) injected daily for 7 days with azithromycin (Azi) from 25 until 31 dpi, and from 112 until 118 dpi (T1 to T7). The arrows indicate the duration of the different treatments. All the bioluminescence analyses were done after IP administration of D-luciferin. Data are expressed as the mean ± SEM of average radiance of light measured in photons/second/cm² in n = 4 infected treated or untreated mice. <i>p</i> values (+<i>p</i><0.05, ++<i>p</i><0.01, +++<i>p</i><0.001) are indicated in corresponding colors for each group <i>versus</i> the uninfected group. Below are shown images of the tracking of one untreated, infected (MFlum1) mouse and an infected treated (Azi) mouse photographed at different crucial time points. The corresponding <i>p</i> values (*<i>p</i><0.05) were calculated between infected untreated group and treated group (Azi). Images depict photographs overlaid with color representations of luminescence intensity, measured in photons/second/cm² and indicated on the scales, where red is most intense and purple is least intense. PT, days post treatment.</p

Kinetics of dissemination of bioluminescent MFlum1 in mice.

<p>All the bioluminescence analyses were performed after IP administration or addition of D-luciferin. (A) Live imaging tracking of 10⁷ MFlum1 IP injected to albino C57BL/6J mice. Images below the graph show the tracking of one infected mouse, photographed at different crucial time points. Data are expressed as the mean ± SEM of average radiance of light measured in photons/second/cm² in n = 4 infected mice, imaged in the dorsal view, except for 30 min post-infection for which only imaging in the ventral view allows the visualization of the leptospiral dissemination in the peritoneal cavity. <i>p</i> values (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001) between infected and uninfected groups. Images depict photographs overlaid with color representations of luminescence intensity, measured in photons/second/cm² as indicated on the scales, where red is the most intense and purple the least intense. (B) Kinetics of leptospiral quantification by q-PCR in blood and urine of albino C57BL/6J mice infected with 10⁷ MFlum1. Below are shown corresponding images of the <i>ex vivo</i> live imaging of MFLum1 in blood, in the presence or absence of ATP. (C) Monitoring expressed as the percentage of weight loss of mice infected or not with 10⁷ MFlum1. Panels B and C are representative of 2 independent experiments with a total of n = 8 mice for each group. <i>p</i> values (*<i>p</i><0.05, **<i>p</i><0.01) between infected and uninfected groups.</p

Characterization of bioluminescent <i>Leptospira</i> transformants.

<p>(A) Growth curves of <i>L. interrogans</i> Manilae wild-type and MFlum1 strains in EMJH medium at 28°C (Left Y axis) and corresponding bioluminescence of MFlum1 (Right Y axis). (B) Comparison of bioluminescence according to known numbers of MFlum1, grown to mid-log phase or to old stationary phase (four months). Measures were done with the IVIS Spectrum machine after addition of D-luciferin, in the presence or absence of ATP. Panels A and B are representative of 6 and 2 experiments, respectively. (C) Live imaging tracking over time of 2×10⁸ MFlum1 or bioluminescent <i>L. biflexa</i> Patoc PFlum7 injected intra-peritoneally (IP) into albino C57BL/6J mice (Left Y axis) and corresponding weight losses (Right Y axis) of MFlum1 infected mice. All bioluminescence analyses were carried out after the IP administration of D-luciferin. Data are expressed as the mean ± SEM of average radiance of light measured in photons/second/cm² in mice and imaged in the ventral view. This panel represents 3 experiments with a total of n = 12 mice infected with MFlum1, n = 4 mice infected with PFlum7, and n = 8 naïve mice. <i>p</i> values (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001) between infected and uninfected group. The cross indicates that the mice died or were sacrificed because of acute leptospirosis. Below are shown images of the tracking of one mouse photographed at different time post infection. (D) Live imaging tracking over time of 50 µl of blood collected from the 2×10⁸ MFlum1 infected mice at different time points from the experiment shown in panel 1C (Left Y axis) and corresponding number of leptospires measured by q-PCR (Right Y axis). Data are expressed as the mean ± SEM of average radiance of light measured in photons/second/cm² in 50 µL of blood. <i>p</i> values (*<i>p</i><0.05), between groups. This panel represents 2 experiments with a total of n = 6 mice. Below are shown the corresponding images at different dpi in the presence or the absence of ATP. Below D0 are shown images of the MFLum1 in PBS imaged just before IP injection.</p