Abstract
Listeria monocytogenes causes listeriosis, a human foodborne infection leading to gastroenteritis, meningoencephalitis and maternofetal infections. InlA and InlB, two L. monocytogenes surface proteins, interact with their respective receptors E-cadherin and Met and mediate bacterial entry into human cultured cells. Here, we present protocols for studying listeriosis in three complementary animal models: (i) the human E-cadherin (hEcad) transgenic mouse line; (ii) the knock-in E16P mouse line; and (iii) the gerbil, in which both InlA–E-cadherin and InlB–Met species-specific interactions occur as in humans. Two routes of infection are described: oral inoculation, the natural route for infection; and intravenous inoculation that bypasses the intestinal barrier. We describe how to monitor L. monocytogenes infection, both qualitatively by imaging techniques and quantitatively by bacterial enumeration. The advantage of these methods over the classical intravenous inoculation of L. monocytogenes in wild-type mice (in which the InlA–E-cadherin interaction does not occur) is that it allows the pathophysiology of listeriosis to be studied in animal models relevant to humans, as they are permissive to the interactions that are thought to mediate L. monocytogenes crossing of human host barriers. The whole procedure (inoculation, in vivo imaging, bacterial enumeration, histopathology) takes one full week to complete, including 3 d of actual experiments.
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Acknowledgements
We thank E. Huillet, F. Langa, S. Vandormael-Pornin and C. Babinet for their respective contributions in the transgenesis and KI mouse projects; C. Hill for gift of the pPL2lux-PhlyA plasmid; M.-A. Nahori for her expertise in animal experiments; H. Khun for immunohistochemistry techniques; and V. Masse for help in setting up the competitive assay. M.L. thanks Olivier Lortholary for his support. Marc Lecuit and Pascale Cossart laboratories received financial support from the Institut Pasteur, Inserm, INRA, FRM and ANR.
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Supplementary Video 1: Intravenous infection of an anesthetized gerbil with L. monocytogenes
(see manuscript for details) (MOV 10588 kb)
Supplementary Video 2: Confocal 3D reconstruction of a gerbil placenta infected with EGD 42h pi
A 200 µm-thick section of a gerbil placenta was cut with a vibratome. The syncytiotrophoblast, placental capillaries and L. monocytogenes were stained in red (mouse anti-Ecad antibody), cyan (Isolectin B4) and green (rabbit anti-Listeria antibody), respectively. One hundred and fifty 200µmx200µm optical sections at 200 nm intervals were then imaged with a Leica TCS SP5 confocal microscope and a 3D-reconstruction was performed using the Imaris® software. (MOV 16375 kb)
Supplementary Video 3: Confocal 3D reconstruction of a gerbil placenta infected with EGDΔinlAB 42h pi
A 200 µm-thick section of a gerbil placenta was cut with a vibratome. The syncytiotrophoblast, placental capillaries and L. monocytogenes were stained in red (mouse anti-Ecad antibody), cyan (Isolectin B4) and green (rabbit anti-Listeria antibody), respectively. One hundred and fifty 250µmx250µm optical sections at 200 nm intervals were then imaged with a Leica TCS SP5 confocal microscope and a 3D-reconstruction was performed using the Imaris® software. (MOV 14209 kb)
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Disson, O., Nikitas, G., Grayo, S. et al. Modeling human listeriosis in natural and genetically engineered animals. Nat Protoc 4, 799–810 (2009). https://doi.org/10.1038/nprot.2009.66
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DOI: https://doi.org/10.1038/nprot.2009.66
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