The virulence of the uropathogenic Escherichia coli Dr(+) IH11128 strain is associated with the presence of Dr fimbrial structures and a DraD invasin which can act as a fimbrial capping domain at the bacterial cell surface. However, a recent study suggests that the DraD protein is surface exposed in two forms: fimbria associated and fimbria nonassociated (prone to interaction with the N-terminal extension of the DraE protein located on the fimbrial tip). The actual mechanism of DraD surface secretion is presently unknown. We identified a previously unrecognized type II secretory pathway (secreton) in the uropathogenic E. coli Dr(+) strain which is well conserved among gram-negative bacteria and used mainly for secretion of virulence determinants. An active secreton is composed of 12 to 15 different proteins, among which GspD functions as an outer-membrane channel to permit extrusion of proteins in a folded state. Therefore, we inactivated the pathway by inserting the group II intron into a gspD gene of the type II secretion machinery by site-specific recombination. DraD secretion by the E. coli Dr(+) and gspD mutant strains was determined by immunofluorescence microscopy (with antibodies raised against DraD) and an assay of cell binding between bacteria and HeLa cells. The specificity of DraD-mediated bacterial binding for the integrin receptor was confirmed by examination of the adhesion of DraD-coated beads to HeLa cells in the presence and absence of alpha(5)beta(1) monoclonal antibodies. The investigations that we performed showed that type II secretion in E. coli Dr(+) strains leads to DraD translocation at the bacterial cell surfaces.

The enhancing effect on the water structure has been confirmed for most of the osmolytes exhibiting both stabilizing and destabilizing properties in regard to proteins. The presented work concerns osmolytes, which should be classified as “structure breaking” solutes: taurine and N,N,N-trimethyltaurine (TMT). Here, we combine FTIR spectroscopy, DSC calorimetry and DFT calculations to gain an insight into the interactions between osmolytes and two proteins: lysozyme and ubiquitin. Despite high structural similarity, both osmolytes exert different influence on protein stability: taurine is a stabilizer, TMT is a denaturant. We show also that taurine amino group interacts directly with the side chains of proteins, whereas TMT does not interact with proteins at all. Although two solutes weaken on average the structure of the surrounding water, their hydration spheres are different. Taurine is surrounded by two populations of water molecules: bonded with weak H-bonds around sulfonate group, and strongly bonded around amino group. The strong hydrogen-bonded network of water molecules around the amino group of taurine further improves properties of enhanced protein hydration sphere and stabilizes the native protein form. Direct interactions of this group with surface side chains provide a proper orientation of taurine and prevents the \({\text{SO}}_{3}^{ - }\) group from negative influence. The weakened \({\text{SO}}_{3}^{ - }\) hydration sphere of TMT breaks up the hydrogen-bonded network of water around the protein and destabilizes it. However, TMT at low concentration stabilize both proteins to a small extent. This effect can be attributed to an actual osmophobic effect which is overcome if the concentration increases.

Interactions between osmolytes and hen egg white lysozyme in aqueous solutions were studied by means of FTIR spectroscopy and molecular dynamics. A combination of difference spectra method and chemometric analysis of spectroscopic data was used to determine the number of osmolyte molecules interacting with the protein, and the preferential interaction coefficient in presented systems. Both osmolytes – L-proline and trimethylamine-N-oxide (TMAO) – belong to a group of stabilizing osmolytes, and according to the preferential exclusion/hydration hypothesis, both should be excluded from the vicinity of the protein backbone and surface. We provide experimental and computational evidence that although TMAO behaves according to the hypothesis, proline does not. Our results suggest that preferential exclusion is not a universal property of stabilizing osmolytes.