The Role of GTP Sensing and the CodY Regulon in the Adaptation of Listeria Monocytogenes to Intracellular Growth

Listeria monocytogenes has become an important model for studying intracellular parasitism as well as being a paradigm for studying immune response to intracellular parasites. Much is known about the virulence of L. monocytogenes and certain aspects of the host response to infection are also well documented. Although invasion and cell-cell spread are well characterised, events surrounding the adaptation and subsequent proliferation of intracellular cytosolic listeriae are not well defined. There is growing evidence that the cytosol of a host cell may be a nutritionally challenging environment for intracellular bacteria to which they have to adapt to prior to the onset of bacterial replication. As a consequence the ability of L monocytogenes to adapt to these changes in its nutrient availability would be essential for the successful intracellular replication and spread of L monocytogenes. Studies of the physiological adaptation to intracellular growth represent a fascinating over-looked area of research in looking at microbe host interactions.<P> The overall aims of the work are to elucidate the mechanisms by which L monocytogenes adapts and responds to the nutrient stress that is encountered upon entry into the host cytosol. Specifically the project will look at the role of the CodY regulon, which is expressed upon entry into stationery phase in response to nutrient stress, on intracellular growth. In addition, the relationship between CodY, and two other nutrient stress regulators RelA and sigma B will also be investigated. There are four specific objectives within these overall aims.<OL> <LI> To demonstrate that in L monocytogenes CodY acts as a repressor of transcription and determine if GTP and BCAA can act as effectors. <LI> Establish the extent of the CodY regulon via array analysis and identify candidate genes for further investigation. <LI> Demonstrate expression of the CodY regulon during intracellular growth and determine the kinetics of this. <LI> To study the interaction between RelA, CodY and sigmaB.</ol> The project will use molecular microbiology in combination with cell biology. Aside from routine techniques, specifically the project will exploit DNase 1 foot printing, array analysis, allelic exchange, cellular invasion assays and confocal microscopy to study Gfp expression.