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Start of funding 01.01.2009

Construction of lux expression cassettes for lactic acid bacteria

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Some probiotic bacteria share properties with bacterial pathogens, which may be considered as pathogenicity traits including adhesion to intestinal mucosal cells or influence on the human immune system. Enterococcus faecalis is a prominent model to study such opportunistic pathogenicity versus probiosis. In this project a functionally active lux expression cassette is constructed, which shall transform E. faecalis into a light emitting bacterium. It is further intended to get the lux gene expression controlled by pathogenicity factors known to affect chronic intestinal bowl disease. The obtained strains shall subsequently be used in a project of the German research foundation to monitor their in vivo behaviour in mice, namely distribution and virulence gene expression, to possibly delineate a probiotic bacterium from being an opportunistic pathogen.

Final report:
The aim of the project was to establish a reporter system for Enterococcus faecalis in cooperation with CaliperLS R&D Biology, Kevin P. Francis PhD. With this system we want to investigate the behaviour of pathogenic and probiotic strains in vivo. Such systems are already described for closely related species (1), but they cannot be used in E. faecalis. During an initial three months research stay at CaliperLS R&D Biology various reporter genes (for the production of luminescent and fluorescent E.faecalis) were adapted to the species and successfully tested for their function in E. faecalis. Additionally different vector-systems (plasmids, selection markers and promoters) were developed to express these genes under the control of constitutive or regulated promoters. These systems were and are further optimized at the TU München. During a second two week research stay at CaliperLS, one of the previously in this project constructed luminescence reporter systems was tested successfully in vivo in mice. Various conditions (different E.faecalis strains, applications of antibiotics etc.) were visualized with several IVIS Imagine Systems (standard, 3D and X-ray technology) at the manufacturer. Building upon that further reporter-plasmid-systems are developed.

References:
(1) Francis K.P. et al. (2000) Infect Immun.; 68(6), 3594-600.