Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains

Ruiz JC, D'Afonseca V, Silva A, et al. Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains. PLoS ONE. 2011;6(4): e18551.Background: Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. Methodology and Findings: We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. Conclusions: These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829

Rôle et mode d'action du régulateur traductionnel hnRNP A1 dans les cellules tumorales mammaires

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

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

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

List of bioluminescent leptospires transformants obtained in this study.

a<p>as annotated in MicroScope (<a href="http://www.genoscope.cns.fr/agc/microscope/home/index.php" target="_blank">http://www.genoscope.cns.fr/agc/microscope/home/index.php</a>).</p><p>italics: insertion in an intergenic region.</p><p>List of bioluminescent leptospires transformants obtained in this study.</p

Threshold of infective dose of MFlum1 to obtain renal colonization.

<p>(A) Comparison of the dissemination kinetics for MFlum1 injected to albino C57BL/6J at different doses. 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. On the right side are shown images of one representative mouse at 30 min post infection as a control of infection and on the left side, images of one representative mouse at the end of the kinetics. <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. <i>In vivo</i> (B) and <i>ex vivo</i> (C) live imaging and quantification of albino (W) and black (BL) C57BL/6J mice one month post infection with (10⁷) or without (NI) 10⁷ MFlum1. Bioluminescence imaging in dorsal view was carried out after dorsal shaving of the black mice. Below are shown corresponding images of live mice gated on the kidneys (B) and <i>ex vivo</i> (C) tracking of half-kidneys after sacrifice and addition of D-luciferin. <i>p</i> value (*<i>p</i><0.05) between infected and uninfected groups n = 4 mice <i>per</i> group. (D) Correlation between renal imaging and q-PCR. Bioluminescence imaging at 25 dpi in dorsal view of live chronically infected C57BL/6J mice (from 10⁶ or 10⁷ experiments), gated on the kidneys. Subsequently, mice were euthanized and kidneys were harvested and further processed for determination of the leptospiral load by q-PCR. Each cross represents an individual mouse. (E) <i>Ex vivo</i> imaging in presence or absence of ATP of half-kidneys from 10⁷ MFlum1 infected mice one month post infection. Data are expressed as the mean ± SEM of average radiance of light measured in photons/second/cm² for 6 half-kidneys for each group. Below are shown corresponding images of the tracking of half-kidney after sacrifice and addition of D-luciferin. On the left is shown the schematic representation of an infected kidney in longitudinal cross section. (F) <i>Ex vivo</i> live imaging (Left Y axis) and the corresponding number of leptospires measured by q-PCR (Right Y axis) of different organs from 10⁷ MFlum1 infected mice, one month post infection. Data are expressed as the mean ± SEM of average radiance of light measured in photons/second/cm². This panel represents 2 experiments with a total of n = 6 mice infected with MFlum1, and n = 5 naïve mice. Below are shown corresponding images of the live imaging of one representative organ or fluid and a mouse before the sacrifice. For imaging, urine was pooled from several mice, to obtain a minimum volume of 50 µL.</p

Azithromycin prophylactic treatment against leptospirosis.

<p>8 C57BL/6J mice were infected with 2×10⁸ MFlum1 and 4 of them were injected IP two days before infection (T-2) with azithromycin (Azi). As controls, 4 uninfected mice were treated with azithromycin. All bioluminescence analyses were performed 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 mice imaged in the dorsal view, except for 30 min post-infection (D0) 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) are indicated in corresponding colors for each group <i>versus</i> the uninfected treated group. The cross indicates that the mice died or were sacrificed because of acute leptospirosis. On the right side are shown images of the last tracking point (24 dpi) of one treated-infected mouse compared to an uninfected treated mouse.</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