Programme
18th | 19th | 20th | 21st | 22nd | 23rd | 24th | Week Two
During the first week of the course, we will work with two of the best well-known models of intracellular parasitism, Listeria monocytogenes and Shigella flexneri (Cossart P. & Sansonetti P.J. 2004. Bacterial invasion: the paradigms of enteroinvasive pathogens. Science 304: 242; Pizarro-Cerda J. & Cossart P. 2006. Bacterial adhesion and entry into host cells. Cell124: 715).
We will overview the different steps of the intracellular cycle of these bacterial pathogens, allowing us to present some of the basic techniques used in the field of cellular microbiology including RNA interference, cellular transfection, pathogen manipulation, cellular infection with pathogens, immuno-fluorescence, cellular stimulation with bacterial effectors, protein electrophoresis, plaque assay, transfer to nitrocellulose membranes, western blot, and gentamicin invasion assays.
Practical 1 “The First Contact Between the Pathogen and the Host Cell”: Study of early signaling events -protein tyrosine phosphorylation and PI3-kinase recruitment upon cellular stimulation by the protein InlB of Listeria and protein tyrosine phosphorylation upon cellular infection by Shigella flexneri (cell stimulation, cell lysate, immuno-precipitation, protein electrophoresis and transfer, Western Blot). |
Practical 2 “The Invasion Step”: Study of the involvement of the endocytic/phagocytic and ubiquitination machineries during the entry of Shigella flexneri and Listeria monocytogenes within host cells (RNAi, gentamicin invasion assay). |
Practical 3 “Life Within the Intracellular Environment”: Study of the role of the host cell cytoskeleton during the actin-based motility of Shigella flexneri and Listeria monocytogenes (transfection, immuno-fluorescence). |
Practical 4 “Escaping the Primary Infected Cells”: Study of the cell-to-cell spread of Shigella flexneri and Listeria monocytogenes (plaque formation assay).
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Practical 5 “The Animal Model”: Study in real time of the In Vivo infection of mice by Listeria monocytogenes (bioluminescence imaging). |
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Detailed Description of the First Week Practicals: “The Basics”
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09:30 |
General Introduction to the Course |
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10:00 |
Lecture: Listeria monocytogenes Part I |
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11:00 |
Coffee Break |
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11:30 |
Lecture: Listeria monocytogenes Part II |
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12:30 |
Lunch |
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14:00 |
Overview of Basic Techniques |
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14:30 |
Cell Seeding (Practical 1 to 4) |
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15:00 |
Students Presentations Part I |
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16:30 |
Coffee Break |
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17:00 |
Students Presentations Part II |
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19:30 |
Dinner |
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21:00 |
Summary of Practicals and blackboard sessions |
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09:00 |
DNA and RNAi transfection (Practical 3) |
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10:00 |
Lecture: Shigella flexneri Part I |
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11:00 |
Coffee Break |
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11:30 |
Lecture: Shigella flexneri Part II |
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12:30 |
Lunch |
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14:00 |
Cellular Stimulation with InlB |
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16:00 |
Animal Inoculation (Practical 5)/Coffee Break |
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17:30 |
Bacterial Seeding (Practicals 1, 3 and 4) |
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18:00 |
Gel Assembly |
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19:30 |
Dinner |
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21:00 |
Summary of Practicals and blackboard session |
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09:00 |
Cell Infection (Practical 3 and 4) |
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10:00 |
Cell Infection |
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11:00 |
Cell Washing and Gentamicin Incubation (Practical 3 and 4) |
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11:30 |
Immunoprecipitation Part II (Practical 1) |
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12:30 |
Protein Electrophoresis (Practical 1) |
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13:00 |
Lunch |
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14:30 |
First Agar Overlay (Practical 4) |
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15:00 |
Protein Transfer (Practical 1) |
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15:30 |
Cell Fixation (Practical 3) |
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16:00 |
Western Blot: Overnight First Antibody Incubation (Practical 1) |
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16:30 |
Animal Observation (Practical 5)/Coffee Break |
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17:00 |
Lecture from In-House Speaker |
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19:00 |
Dinner |
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21:00 |
Summary of Practicals and blackboard session |
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09:30 |
Western Blot: Washes and Secondary Antibody Incubation (Practical 1) |
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10:00 |
Lecture from In-House Speaker |
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11:00 |
Enhanced Chemiluminescence Development (Practical 1) |
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12:00 |
Lunch |
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13:30 |
Immunofluorescence Part I (Practical 3) |
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14:00 |
Animal Observation (Practical 5)/Coffee Break |
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15:00 |
Immunofluorescence Part II (Practical 3) |
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15:30 |
Second Agar Overlay (Practical 4) |
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16:00 |
Samples observation (Practical 3) |
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19:00 |
Dinner |
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21:00 |
Summary of Practicals and blackboard session |
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09:30 |
Bacterial dilution (Practical 2) |
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10:00 |
Preparation of cell lysates (Practical 2) |
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10:30 |
Protein electrophoresis (Practical 2) |
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11:00 |
Bacterial infection (Practical 2) |
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12:00 |
Cell washing and chase (Practical 2) |
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12:30 |
Protein transfer (Practical 2 |
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13:00 |
Lunch |
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14:00 |
Western Blot |
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15:00 |
Animal Observation/Coffee Break (Practical 5) |
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16:00 |
Western Blot: continuation |
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17:00 |
Enhanced Chemiluminescence Development (Practical 1) |
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19:30 |
Dinner |
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09:00 |
Lecture from In-House Speaker |
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10:30 |
Coffee Break |
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11:00 |
Lecture from In-House Speaker |
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12:30 |
Lunch |
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14:00 |
Bacterial counting (Practical 2) |
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15:00 |
Plaque observation (Practical 4) |
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16:00 |
Discussion of Results |
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19:00 |
Dinner |
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Free Day |
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