Stably IntegratedluxCDABEfor Assessment ofSalmonellaInvasion Kinetics

Kelly N Flentie,David Piwnica-Worms,Aashish Manglik,Yasmin Razia,Min Qi,Seth T Gammon,Felix Lui,Luciano Marpegan,Jeffrey S Mckinney

doi:10.2310/7290.2008.00024

Kelly N Flentie, David Piwnica-Worms + Show 7 more

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https://doi.org/10.2310/7290.2008.00024

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Abstract

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.

Highlights

Salmonella Typhimurium is a common cause of gastroenteritis in humans and localizes to neoplastic tumors in animals
EUKARYOTIC CELL INVASION is used by Salmonella during the initial steps of pathogenesis[1] and leads to enteric symptoms and disseminated infection
Construction of Salmonella Strains with Stably Integrated luxCDABE Conjugative mating was performed between donor strain Escherichia coli S17-1 and recipient Salmonella enterica serovar typhimurium strain SB300A1.11 Mating was performed as described[12] in Luria-Bertani (LB) broth, plated onto

Summary

Introduction

Salmonella Typhimurium is a common cause of gastroenteritis in humans and localizes to neoplastic tumors in animals. The standard technique to assess Salmonella invasion into cultured cells is the gentamicin protection assay,[3] which exploits the poor penetration of this antibiotic into eukaryotic cells.[4] gentamicin is postulated to kill susceptible extracellular bacteria but not “protected” bacteria that have invaded. Such selective killing permits the preferential recovery of intracellular bacteria on subsequent culture of lysed cells. The invC gene in Salmonella encodes an adenosine triphosphatase (ATPase) that powers a type III secretion system, triggering eukaryotic actin reorganization and Salmonella invasion of some eukaryotic cell lines.[6,7]

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