Title:
In vitro and in vivo expression of virulence genes of pathogenic Listeria species and functions of new virulence factors
Project management at the University of Würzburg:
Abstract:
Our work aims at the understanding on the molecular level of the pathogenicity mechanisms of Listeria monocytogenes. This gram-positive, facultative intracellular bacterium can cause systemic infections (listeriosis) in man and animals.
The work performed during the reporting period dealt with the following aspects_
- Uptake of L. monocytogenes into different animal and human cell types
- Requirements for bacterial growth in the cytosol of eukaryotic cells
- Differential regulation of virulence genes
We could demonstrate that human bone marrow stem cells, the progenitors of dendritic cells and macrophages (the most relevant host cells for L. monocytogenes), are initially resistant. Only during differentiation, by sequential formation of at first the mannose receptor and afterwards the complement and Fc receptors, they become susceptive for L. monocytogenes (1-3). The uptake by non-phagocytic cells (epithelial, endothelial cells and hepatocytes have been tested here) is induced by several Listeria-specific surface proteins (internalins). We could demonstrate that internalin B is sufficient for the uptake of L. monocytogenes into some host cells (endothelial cells and certain hepatocytes) whereas the internalinA-dependent uptake into other host cell types requires the assistance of other internalins which have been found and characterized by us for the first time (4).
Using direct microinjection of the bacteria into the cytosol of epithelial cells we could show that this cellular compartment is not generally a suitable medium for the multiplication of heterotrophic bacteria. Rather particular physiological adaptations of the bacteria are necessary (5). In the case of L. monocytogenes a specifically regulated gene (hpt) could be found, which encodes a transporter for phosphorylated hexoses (6). Certain additional, yet poorly understood adaptations are also required (7,8).
The majority of the virulence genes of L. monocytogenes known to date, including hpt, is activated on the transcriptional level by a central regulator, PrfA. This activation takes place differentially and results in elevated levels of dedicated virulence factors during defined phases of the infection (9). By establishing an in vitro trancription assay we now could develop a tool (10, 11) which should enable us to identify the additional factors which are required for differential gene expression. Our current investigations indicate that the activity of PrfA is controlled also by catabolite repression.
Key words:
pathogenesis
bacteria
infection
Listeria
intracellular
internalization
adaptation
transcriptional regulation
Projekt period: from 01.1999 to 12.2002