Stably integrated luxCDABE for assessment of Salmonella invasion kinetics.
Salmonella Typhimurium is a common cause of gastroenteritis in humans and also localizes to neoplastic tumors in animals. Invasion of specific eukaryotic cells is a key mechanism of Salmonella interactions with host tissues. Early stages of gastrointestinal cell invasion are mediated by a Salmonella type III secretion system, powered by the adenosine triphosphatase invC. The aim of this work was to characterize the invC dependence of invasion kinetics into disparate eukaryotic cells traditionally used as models of gut epithelium or neoplasms. Thus, a nondestructive real-time assay was developed to report eukaryotic cell invasion kinetics using lux+ Salmonella that contain chromosomally integrated luxCDABE genes. Bioluminescence-based invasion assays using lux+ Salmonella exhibited inoculum dose-response correlation, distinguished invasion-competent from invasion-incompetent Salmonella, and discriminated relative Salmonella invasiveness in accordance with environmental conditions that induce invasion gene expression. In standard gentamicin protection assays, bioluminescence from lux+ Salmonella correlated with recovery of colony-forming units of internalized bacteria and could be visualized by bioluminescence microscopy. Furthermore, this assay distinguished invasion-competent from invasion-incompetent bacteria independent of gentamicin treatment in real time. Bioluminescence reported Salmonella invasion of disparate eukaryotic cell lines, including neoplastic melanoma, colon adenocarcinoma, and glioma cell lines used in animal models of malignancy. In each case, Salmonella invasion of eukaryotic cells was invC dependent.
Flentie
Kelly N
KN
Department of Molecular Microbiology, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Qi
Min
M
Gammon
Seth T
ST
Razia
Yasmin
Y
Lui
Felix
F
Marpegan
Luciano
L
Manglik
Aashish
A
Piwnica-Worms
David
D
McKinney
Jeffrey S
JS
D000230:Adenocarcinoma / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D001932:Brain Neoplasms / Q000235:genetics; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005839:Gentamicins / Q000494:pharmacology; D005910:Glioma / Q000235:genetics; D019073:HT29 Cells; D006801:Humans; D007700:Kinetics; D049449:Luminescence; D008163:Luminescent Measurements; D008545:Melanoma / Q000235:genetics; D020637:Photorhabdus / Q000235:genetics; D006180:Proton-Translocating ATPases / Q000235:genetics*; D012480:Salmonella Infections / Q000235:genetics* / Q000382:microbiology / Q000473:pathology; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005839:Gentamicins; C088598:invC protein, Salmonella typhimurium; D006180:Proton-Translocating ATPases