• DNA microarrays and global gene expression patterns
• Genomic indexing of Salmonella and E.coli
• Salmonella virulence gene expression during infectionLag Phase
• Salmonella Infection of Chicken oviduct (SafeHouse)
• Influence of Commensal flora, immune response and behaviour on Salmonella infection (VTRI104)
• Environmental regulation of the invasion and intracellular virulence gene programmes of Salmonella: a ppGpp dependent switch

• Regulation of Salmonella SPI1 & SPI2
• Bacterial nitrate metabolism in colonisation of the mammalian gastrointestinal tract
• Analysis of factors affecting Salmonella contamination of meat during the slaughter process (Biotracer)

• "Non genetic variation" gene expression within bacterial populations (Finished)

• Nucleoid associated proteins: DNA topology and gene expression.
• Acambis project on enterotoxigenic E.coli (Finished)

In vivo expression of Salmonella virulence genes during infection

Main researcher: Isabelle Hautefort

Transcriptomic analysis of bacterial gene expression has extended its complexity level and now allows us to look at expression of all genes of a pathogen inside cultivated mammalian cells (Eriksson et al., 2003; Lucchini et al., 2005). A plethora of virulence genes had already been identified as involved in the virulence of Salmonella, many of them located within big clusters called Salmonella Pathogenicity Islands (SPI) (Galan and Curtiss, 1989; Murray and Lee, 2000; Shea et al., 1999; Beuzon et al., 2000; Bumann 2002). We have recently established the expression of profile of Salmonella Typhimurium inside human epithelial cells and have identified 1000s of genes whose expression changes intracellularly.

Figure 1: Gene expression profiles of key S. Typhimurium virulence determinants The expression profiles of virulence genes inside J774 and HeLa cells are shown. Each horizontal bar represents the expression level of a single gene. Blue shows that the gene expression is down regulated, yellow that it is similar and red that it is up regulated compared with expression in our reference sample. .

Figure 2: Expression of a SPI2-gfp+ transcriptional fusion within cultured MDCK epithelial cells is shown in green. The red fluorescence shows the Actin cytoskeleton of the epithelial cells and the blue fluorescence indicates the nuclei of all cells.

Figure 3: Expression of a SPI2-gfp+ transcriptional fusion (green fluorescence) in Salmonella cells (red-orange fluorescence) located in phagocytes (blue fluorescence) in infected murine spleen tissue

We are interested in determining the degree of specificity of Salmonella gene expression during infection. We combine the use of microarray technology with In Vivo-Induced Fluorescence (IVIF) technology to look at gene expression patterns in infected mammalian cells or more complex samples such as infected animal tissue.
Following infection of cultivated mammalian cells Salmonella total RNA is extracted and hybridised to microarrays in order to identify the genes whose expression is altered during the process of infection (Figure 1)

IVIF system consists in constructing chromosomally-integrated single copy transcriptional fusions of promoters of interest with the gfp+ reporter gene. These fusions allow us to ask questions about Salmonella gene expression in specific organs and cell types, using a combination of immunohistochemistry and fluorescence microscopy (Figures 2, 3).

An accurate measure of the level of GFP expression per bacterial cell is obtained with flow cytometry.

The level of expression of GFP is measured in individual bacterial, allowing direct comparison of virulence gene expression between different samples

Figure 3A:Anti-Salmonella labelling allows discrimination between Salmonella cells released from infected spleen and tissue debris as shown in the hexagonal region drawn here Figure 3B:Comparison by flow cytometry of the level of expression of a gfp+ fusion in individual Salmonella cells released from an infected spleen (green) and the same bacterial strain grown in culture medium. Here we see a down-regulation of gene expression.

• Beuzon, C.R., Meresse, S., Unsworth, K.E., Ruiz-Albert, J., Garvis, S., Waterman, S.R., Ryder, T.A., Boucrot, E., and Holden, D.W. (2000) Embo J 19, 3235-3249.
• Bumann, D. (2002), Mol. Microbiol., 43:1269-1283.
• Eriksson, S., Lucchini, S., Thompson, A., Rhen, M., and Hinton, J.C. (2003) Mol. Microbiol. 47: 103-118.
• Galan, J.E., and Curtiss, R.D. (1989) Proc Natl Acad Sci U S A 86, 6383-6387.
• Hautefort, I., and Hinton, J.C.D. (2002) Methods in Microbiology 31, 55-90.
• Hautefort, I., Proença, M., and Hinton, J.C.D. (2003) Appl. Environ. Microbiol. 69(12): 7480-7491.
• Lucchini, S., Liu, H., Jin, Q., Hinton, J.C., Yu, J. (2005) Infect. Immun. 73: 88-102.
• Murray, R.A., and Lee, C.A. (2000) Infect Immun 68, 5050-5055.
• Shea, J.E., Beuzon, C.R., Gleeson, C., Mundy, R., and Holden, D.W. (1999) Infect Immun 67, 213-219