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Sharma, Atin
Publications (2 of 2) Show all publications
Sharma, A. & Puhar, A. (2019). Gentamicin Protection Assay to Determine the Number of Intracellular Bacteria during Infection of Human TC7 Intestinal Epithelial Cells by Shigella flexneri. BIO-PROTOCOL, 9(13), Article ID UNSP e3292.
Open this publication in new window or tab >>Gentamicin Protection Assay to Determine the Number of Intracellular Bacteria during Infection of Human TC7 Intestinal Epithelial Cells by Shigella flexneri
2019 (English)In: BIO-PROTOCOL, ISSN 2331-8325, Vol. 9, no 13, article id UNSP e3292Article in journal (Refereed) Published
Abstract [en]

Shigella flexneri is an intracellular bacterial pathogen that gains access to the gut epithelium using a specialized Type III Secretion System (T3SS). Various determinants mediating this invasive infection have been experimentally verified using the classical gentamicin protection assay presented here. In this assay epithelial cell lines are infected by bacteria in vitro and the extracellular bacteria are killed by gentamicin. The internalized bacteria, which are protected from the bactericidal action of gentamicin, are recovered by lysing the epithelial cells and enumerated by determining the colonies formed on solid medium. Various techniques based on light microscopy, such as immunofluorescence and bacteria expressing fluorescent proteins, are also used for studying intracellular bacteria. However, these techniques are not only labor intensive and require sophisticated equipment, but mostly are also not quantitative. Despite being an easy quantitative method to study invasiveness of bacteria, the gentamicin protection assay cannot distinguish between the survival and multiplication of the internalized bacteria over longer incubation periods. To alleviate the complications created by multiplication and dissemination of internalized bacteria, complementary assays like plaque formation assays are required. This protocol presents an easy and cost-effective method to determine the invasiveness and the capacity to establish an infection of Shigella under different conditions.

Place, publisher, year, edition, pages
BIO-PROTOCOL, 2019
Keywords
Shigella, Intracellular pathogen, Invasion, Type III Secretion System, Intestinal epithelial cells
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-162003 (URN)10.21769/BioProtoc.3292 (DOI)000476647800009 ()
Available from: 2019-08-13 Created: 2019-08-13 Last updated: 2019-08-13Bibliographically approved
Sharma, A. & Puhar, A. (2019). Plaque Assay to Determine Invasion and Intercellular Dissemination of Shigella flexneri in TC7 Human Intestinal Epithelial Cells. BIO-PROTOCOL, 9(13), Article ID UNSP e3293.
Open this publication in new window or tab >>Plaque Assay to Determine Invasion and Intercellular Dissemination of Shigella flexneri in TC7 Human Intestinal Epithelial Cells
2019 (English)In: BIO-PROTOCOL, ISSN 2331-8325, Vol. 9, no 13, article id UNSP e3293Article in journal (Refereed) Published
Abstract [en]

Shigella flexneri invades the epithelial cells lining the gut lumen and replicates intracellularly. The specialized Type III Secretion System (T3SS) and its effector proteins, encoded on a large virulence plasmid, assist the bacterium to gain access to the cytosol. Thereafter Shigella disseminates to neighboring cells in an epithelial layer without further extracellular steps. Host cell lysis occurs when these bacteria have extensively replicated in the target cell cytosol. Here we describe a simple method to qualitatively as well as quantitatively study the capacity of Shigella to invade and disseminate within an epithelium by assessing the number and size of plaques representing the dead cells in a monolayer of TC7 cells. This classical protocol follows a simple approach of infecting the monolayers of epithelial cell lines with Shigella and visualizing the dead cells as plaques formed against a stained background.

Place, publisher, year, edition, pages
BIO-PROTOCOL, 2019
Keywords
Intracellular bacteria, Intestinal epithelial cells, Giemsa stain, Shigella, Invasion, Dissemination
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:umu:diva-162002 (URN)10.21769/BioProtoc.3293 (DOI)000476647800010 ()
Available from: 2019-08-13 Created: 2019-08-13 Last updated: 2019-08-13Bibliographically approved
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