Date Published: December 4, 2014
Publisher: Public Library of Science
Author(s): Gwenn Ratet, Frédéric J. Veyrier, Martine Fanton d’Andon, Xavier Kammerscheit, Marie-Anne Nicola, Mathieu Picardeau, Ivo G. Boneca, Catherine Werts, Jenifer Coburn. http://doi.org/10.1371/journal.pntd.0003359
Abstract: Leptospira (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 106 to 107 bacteria, we observed dissemination and exponential growth of leptospires in the blood, followed by apparent clearance of bacteria. However, with 2×108 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.
Partial Text: Leptospirosis is a worldwide zoonosis transmitted by chronically infected animals, through excretion of bacteria in their urine, contaminating the soil and water. Pathogenic Leptospira interrogans (L. interrogans) are spiral motile bacteria that enter a diverse range of hosts through broken skin and mucosa. Infected humans develop either a flu-like, usually mild illness, or severe disease with renal, liver and heart failure, and eventually pulmonary hemorrhages that may be fatal. Leptospirosis is a reemerging and under-diagnosed neglected zoonotic disease, that may increase worldwide because of global warming and uncontrolled urbanization . In East Asia, leptospirosis is a serious health issue affecting paddy field workers, and people living in regions often flooded after typhoons. Frequent outbreaks of leptospirosis occur in developing countries, where a large part of the population lives in slums with poor urban sanitation, in close contact with infected rats. In Europe, leptospirosis affects people in contact with animals, or taking part in outdoors activities in water environments –. The pathophysiology of leptospirosis remains poorly understood. Leptospirosis has been studied in animal models by immunohistochemistry to assess pulmonary, hepatic and renal lesions , . Different techniques, such as direct isolation and subsequent culture of leptospires from organs and body fluids, electron microscopy and more recently, quantitative real time polymerase chain reaction (q-PCR) of leptospiral DNA, have been very useful to better understand the course and burden of leptospires in their hosts –. Nowadays, in vivo imaging of small live animals is easily performed, allowing non-invasive longitudinal monitoring of disease progression using luminescent or fluorescently labeled microbes . One such study has recently been performed with L. interrogans in transparent Zebrafish embryos, demonstrating fast uptake of leptospires by phagocytic cells . However to our knowledge, no dynamic study of the in vivo dissemination of leptospires has been undertaken in mammals. Two teams have succeeded in constructing genetically modified leptospires species expressing either fluorescent GFP or mRFP proteins  or luciferase from a luxCDABE cassette from Photorhabdus luminescens. These labeled strains proved to be useful tools to easily enumerate the bacteria or for use in in vitro assays. However, these constructs did not allow the monitoring of infection with L. interrogans in cells or in animals. In contrast, a recent study showed the feasibility of using the firefly luciferase gene under the control of a strong promoter to obtain luminescent Borrelia species and track the bacteria in vivo in mice . Therefore, we chose to construct bioluminescent leptospires, introducing a cassette expressing the firefly luciferase gene under the control of a strong leptospiral promoter, by transposon insertion in the chromosome of leptospires . Since the reaction catalyzed by the firefly luciferase in the presence of its substrate, D-luciferin, requires ATP to emit photons, only metabolically active and therefore live bacteria expressing the firefly luciferase are bioluminescent in the presence of D-luciferin.
In this study, bioluminescent strains of pathogenic L. interrogans serovar Manilae were constructed and successfully used in vivo to determine and characterize the kinetics of dissemination of L. interrogans in mice. This new model proved to reproduce both acute and chronic leptospirosis and was further used to test the efficacy of antibiotics.