Measurement of Salmonella enterica Internalization and Vacuole Lysis in Epithelial Cells.
Establishment of an intracellular niche within mammalian cells is key to the pathogenesis of the gastrointestinal bacterium, Salmonella enterica serovar Typhimurium (S. Typhimurium). Here we will describe how to study the internalization of S. Typhimurium into human epithelial cells using the gentamicin protection assay. The assay takes advantage of the relatively poor penetration of gentamicin into mammalian cells; internalized bacteria are effectively protected from its antibacterial actions. A second assay, the chloroquine (CHQ) resistance assay, can be used to determine the proportion of internalized bacteria that have lysed or damaged their Salmonella-containing vacuole and are therefore residing within the cytosol. Its application to the quantification of cytosolic S. Typhimurium in epithelial cells will also be presented. Together, these protocols provide an inexpensive, rapid and sensitive quantitative measure of bacterial internalization and vacuole lysis by S. Typhimurium.
Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University, 647090, Pullman, WA, 99164-7090, USA.
Jessica A
JA
Klein
Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University, 647090, Pullman, WA, 99164-7090, USA.
TuShun R
TR
Powers
Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University, 647090, Pullman, WA, 99164-7090, USA. lknodler@vetmed.wsu.edu.
Leigh A
LA
Knodler
D001681:Biological Assay / Q000379:methods*; D002738:Chloroquine / Q000494:pharmacology; D004352:Drug Resistance, Microbial / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000382:microbiology*; D005839:Gentamicins / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D019779:Salmonella enterica / Q000187:drug effects / Q000502:physiology*; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism*
D005839:Gentamicins; D002738:Chloroquine
Chloroquine resistance assay; Colony forming units; Epithelial cells; Gentamicin protection assay; Salmonella enterica; Salmonella-containing vacuole; Type III secretion system; Vacuole lysis