title: Quantitative assessment of cytosolic Salmonella in epithelial cells.
abstract: Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV). We have recently shown that wild type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1), but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol.
authors.affiliation: Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University, Pullman, Washington, United States of America ; Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
authors.forename: Leigh A
authors.initials: LA
authors.lastname: Knodler
authors.affiliation: Research Technologies Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
authors.forename: Vinod
authors.initials: V
authors.lastname: Nair
authors.affiliation: Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
authors.forename: Olivia
authors.initials: O
authors.lastname: Steele-Mortimer
mesh_terms: D058491:Bacterial Load; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D003600:Cytosol / Q000382:microbiology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology*; D006801:Humans; D012475:Salmonella / Q000187:drug effects / Q000502:physiology*; D012486:Salmonella typhimurium / Q000187:drug effects / Q000502:physiology; D014158:Transcription, Genetic; D014617:Vacuoles / Q000382:microbiology / Q000648:ultrastructure
chemical_list: D001426:Bacterial Proteins; C419942:Spi1 protein, Salmonella; D002738:Chloroquine