ABSTRACT
UNLABELLED: Salmonella specifically localize to malignant tumors in vivo, a trait potentially exploitable as a delivery system for cancer therapeutics. To characterize mechanisms and genetic responses of Salmonella during interaction with living neoplastic cells, we custom-designed a promoterless transposon reporter containing bacterial luciferase. Analysis of a library containing 7,400 independent Salmonella transposon insertion mutants in coculture with melanoma or colon carcinoma cells identified five bacterial genes specifically activated by cancer cells: adiY, yohJ, STM1787, STM1791, and STM1793. Experiments linked acidic pH, a common characteristic of the tumor microenvironment, to a strong, specific, and reversible stimulus for activation of these Salmonella genes in vitro and in vivo. Indeed, a Salmonella reporter strain encoding a luciferase transgene regulated by the STM1787 promoter, which contains a tusp motif, showed tumor-induced bioluminescence in vivo. Furthermore, Salmonella expressing Shiga toxin from the STM1787 promoter provided potent and selective antitumor activity in vitro and in vivo, showing the potential for a conditional bacterial-based tumor-specific therapeutic. SIGNIFICANCE: Salmonella, which often encounter acidic environments during classical host infection, may co-opt evolutionarily conserved pathways for tumor colonization in response to the acidic tumor microenvironment. We identified specific promoter sequences that provide a platform for targeted Salmonella-based tumor therapy in vivo.
Subject(s)
DNA Transposable Elements/genetics , Luciferases/genetics , Neoplasms/therapy , Promoter Regions, Genetic/genetics , Salmonella typhimurium/genetics , Animals , Cell Line, Tumor , Gene Expression , Gene Transfer Techniques , Genes, Bacterial/genetics , Genetic Therapy/methods , HCT116 Cells , HeLa Cells , Humans , Luciferases/metabolism , Luminescent Measurements/methods , Mice , Mice, Nude , Neoplasms/genetics , Neoplasms/pathology , Tumor Microenvironment/genetics , Xenograft Model Antitumor Assays/methodsABSTRACT
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.
Subject(s)
Bacterial Proteins/genetics , Genes, Bacterial , Proton-Translocating ATPases/genetics , Salmonella Infections/genetics , Salmonella typhimurium/genetics , Adenocarcinoma/genetics , Anti-Bacterial Agents/pharmacology , Brain Neoplasms/genetics , Cell Line, Tumor , Colonic Neoplasms/genetics , Gene Deletion , Gene Expression Regulation, Bacterial , Gentamicins/pharmacology , Glioma/genetics , HT29 Cells , Humans , Kinetics , Luminescence , Luminescent Measurements , Melanoma/genetics , Photorhabdus/genetics , Salmonella Infections/microbiology , Salmonella Infections/pathology , Salmonella typhimurium/metabolism , Sensitivity and SpecificityABSTRACT
The type III secretion system involved in Salmonella enterica serovar Typhimurium invasion of host cells has been disrupted using inducibly expressed oligonucleotide external guide sequences (EGSs) complementary to invB or invC mRNA. These EGSs direct single site cleavage in these mRNAs by endogenous RNase P, and their expression in Salmonella results in invC mRNA and InvC protein depletion, decreased type III secretion and interference with host cell invasion. Comparison of these effects with those from studies of Salmonella invB and invC mutants suggests that invB EGSs have polar effects on invC mRNA.
