RESEARCH SUMMARY

Biology of non-proliferative state of Salmonella inside fibroblasts

In our aim of understanding how the intracellular bacterial pathogen Salmonella controls the proliferation rate inside eukaryotic cells, we are analyzing how bacteria modulate expression of small regulatory RNA (sRNAs) upon infection of cultured fibroblasts.

Our data demonstrate that some sRNAs are up-regulated in non-growing intracellular bacteria at early post-infection times while others respond only a late times. Such distinct expression patterns suggest that a subset of sRNAs might be dedicated to direct the adaptation of intracellular bacteria to limited nutrient availability and stress encountered in the phagosome. A novel sRNA has also been identified in the virulence plasmid pSLT. This sRNA is expressed at high levels by non-growing intracellular bacteria.

Current effort is focused in defining the targets of these sRNAs and the underlying regulatory mechanisms. We also interested in defining at the molecular level the entry process of Salmonella into fibroblasts. This process differs from the previously described for epithelial cells. A representative feature is the dispensability of the GTPase RhoG for the actin cytoskeleton remodelling that promotes bacterial entry into in fibroblasts.

Listeria monocytogenes surface proteins associated to the cell wall

In our effort to characterize the biological role of the L. monocytogenes surface protein family containing a C-terminal LPXTG motif, we are performing proteomic analysis of cell wall material isolated from bacteria growing in different conditions.

For the first time, we are obtaining enough material from bacteria growing inside eukaryotic cells to identify members of this large LPXTG protein family that are differentially expressed by intracellular bacteria.

In collaboration with other European laboratories, we are also addressing the function of these proteins by generating isogenic mutants deficient in each of the 41 LPXTG proteins encoded in the genome of the L. monocytogenes strain EGD-e. Cell wall proteome studies in each of these mutants are revealing that the lack of certain LPXTG proteins impairs the activity of the sigma-B (SigB)-dependent regulon. These data connect the cell wall integrity with the phosphorylation cascade that dictates the physiological state of the SigB protein. Current efforts are directed to dissect the exact mechanisms connecting the function of this group of LPXTG proteins to the activity of the SigB regulon.