Abstract
BackgroundExisting virulence models are often difficult to apply for quantitative comparison of invasion potentials of Listeria monocytogenes. Well-to-well variation between cell-line based in vitro assays is practically unavoidable, and variation between individual animals is the cause of large deviations in the observed capacity for infection when animal models are used.One way to circumvent this problem is to carry out virulence studies as competition assays between 2 or more strains. This, however, requires invasion-neutral markers that enable easy discrimination between the different strains.ResultsA fluorescent marker system, allowing visualization and identification of single L. monocytogenes cells as well as colonies in a non-destructive manner, was developed. Five different fluorescent labels are available, and allowed simultaneous visual discrimination between three differently labelled strains at the single cell level by use of fluorescence microscopy. More than 90% of the L. monocytogenes host cells maintained the fluorescence tags for 40 generations.The fluorescence tags did not alter the invasive capacity of the L. monocytogenes cells in a traditional Caco-2 cell invasion assay, and visual discrimination between invaded bacteria carrying different fluorescent labels inside the cells was possible.ConclusionThe constructed fluorescent marker system is stable, easy to use, does not affect the virulence of L. monocytogenes in Caco-2 cell assays, and allows discrimination between differently labelled bacteria after internalization in these cells.
Highlights
Existing virulence models are often difficult to apply for quantitative comparison of invasion potentials of Listeria monocytogenes
The fluorescence tags did not alter the invasive capacity of the L. monocytogenes cells in a traditional Caco-2 cell invasion assay, and visual discrimination between invaded bacteria carrying different fluorescent labels inside the cells was possible
A multicolour tagging system based on the synthesis of Cyan Fluorescent Protein (CFP), Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP), DsRedExpress or HcRed Fluorescent microscopy at 16× magnification revealed that L. monocytogenes ScottA harbouring either pNF8, pJEBAN2, pJEBAN3, pJEBAN4 and pJEBAN6 all gave rise to detectable fluorescent colonies of the expected colour, demonstrating that a heterolog expression of Green Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP), Yellow Fluorescent Protein (YFP), or the red fluorescent markers HcRed and DsRedexpress had successfully been established in L. monocytogenes ScottA
Summary
Existing virulence models are often difficult to apply for quantitative comparison of invasion potentials of Listeria monocytogenes. A solution to this problem is to carry out virulence studies as competition assays between two or more strains This approach has been applied with success by use of Listeria carrying resistance markers to various antibiotics [7]. The Green Fluorescent Protein (GFP) was introduced approximately ten years ago as a convenient tool for studies of the pathogenesis of e.g. Salmonella typhimurium and Yersenia paratuberculosis [8] This protein has been applied in studies of Grampositive bacteria including Streptococcus suis [9], and Listeria monocytogenes [10,11]. The advantage of fluorescent markers, when compared to resistance gene markers, is that they allow direct visualization and real-time studies of tagged bacteria. This disadvantage may be overcome by combining fluorescent markers with antibiotic resistance markers
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