In Vivo Imaging of Bioluminescent Leptospires

International audienceThe study of pathological processes is often limited to in vitro or ex vivo assays, while understanding pathogenesis of an infectious disease requires in vivo analysis. The use of pathogens, genetically modified to express with luminescent enzymes, combined to charge-coupled device (CCD) cameras, constitutes a major technological advance for assessing the course of infection in an intact, living host in real time and in a noninvasive way. This technology, also called bioluminescence imaging, detects the photons emitted from biological sources of light through animal tissues. Here, we describe the method we developed to monitor leptospirosis in a mouse model, by following in a spatiotemporal scale, the dissemination and spread of leptospires. These bacteria have been genetically modified to express the firefly luciferase, which produces light in the presence of the substrate D-luciferin. This useful and accessible technology facilitates the study of the kinetics of blood and tissue dissemination of live leptospires, and the pharmacological impact of treatments and host directed therapeutics

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

Achievements from GLIP functional genomics platforms

  Format du poster : N° W212absen

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

IPD3 Controls the Formation of Nitrogen-Fixing Symbiosomes in Pea and Medicago Spp.

A successful nitrogen-fixing symbiosis requires the accommodation of rhizobial bacteria as new organelle-like structures, called symbiosomes, inside the cells of their legume hosts. Two legume mutants that are most strongly impaired in their ability to form symbiosomes are sym1/TE7 in Medicago truncatula and sym33 in Pisum sativum. We have cloned both MtSYM1 and PsSYM33 and show that both encode the recently identified interacting protein of DMI3 (IPD3), an ortholog of Lotus japonicus (Lotus) CYCLOPS. IPD3 and CYCLOPS were shown to interact with DMI3/CCaMK, which encodes a calcium- and calmodulin-dependent kinase that is an essential component of the common symbiotic signaling pathway for both rhizobial and mycorrhizal symbioses. Our data reveal a novel, key role for IPD3 in symbiosome formation and development. We show that MtIPD3 participates in but is not essential for infection thread formation and that MtIPD3 also affects DMI3-induced spontaneous nodule formation upstream of cytokinin signaling. Further, MtIPD3 appears to be required for the expression of a nodule-specific remorin, which controls proper infection thread growth and is essential for symbiosome formation

Medicago truncatula IPD3 is a member of the common symbiotic signaling pathway required for rhizobial and mycorrhizal symbioses

Legumes form endosymbiotic associations with nitrogen-fixing bacteria and arbuscular mycorrhizal (AM) fungi which facilitate nutrient uptake. Both symbiotic interactions require a molecular signal exchange between the plant and the symbiont, and this involves a conserved symbiosis (Sym) signaling pathway. In order to identify plant genes required for intracellular accommodation of nitrogen-fixing bacteria and AM fungi, we characterized Medicago truncatula symbiotic mutants defective for rhizobial infection of nodule cells and colonization of root cells by AM hyphae. Here, we describe mutants impaired in the interacting protein of DMI3 (IPD3) gene, which has been identified earlier as an interacting partner of the calcium/calmodulin-dependent protein, a member of the Sym pathway. The ipd3 mutants are impaired in both rhizobial and mycorrhizal colonization and we show that IPD3 is necessary for appropriate Nod-factor-induced gene expression. This indicates that IPD3 is a member of the common Sym pathway. We observed differences in the severity of ipd3 mutants that appear to be the result of the genetic background. This supports the hypothesis that IPD3 function is partially redundant and, thus, additional genetic components must exist that have analogous functions to IPD3. This explains why mutations in an essential component of the Sym pathway have defects at late stages of the symbiotic interactions

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

Primer list for cloning.

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