Fast uncoiling kinetics of F1C pili expressed by uropathogenic Escherichia coli are revealed on a single pilus level using force-measuring optical tweezers.

Uropathogenic Escherichia coli (UPEC) express various kinds of organelles, so-called pili or fimbriae, that mediate adhesion to host tissue in the urinary tract through specific receptor-adhesin interactions. The biomechanical properties of these pili have been considered important for the ability of bacteria to withstand shear forces from rinsing urine flows. Force-measuring optical tweezers have been used to characterize individual organelles of F1C type expressed by UPEC bacteria with respect to such properties. Qualitatively, the force-versus-elongation response was found to be similar to that of other types of helix-like pili expressed by UPEC, i.e., type 1, P, and S, with force-induced elongation in three regions, one of which represents the important uncoiling mechanism of the helix-like quaternary structure. Quantitatively, the steady-state uncoiling force was assessed as 26.4 ±1.4 pN, which is similar to those of other pili (which range from 21 pN for S(I) to 30 pN for type 1). The corner velocity for dynamic response (1,400 nm/s) was found to be larger than those of the other pili (400-700 nm/s for S and P pili, and 6 nm/s for type 1). The kinetics were found to be faster, with a thermal opening rate of 17 Hz, a few times higher than S and P pili, and three orders of magnitude higher than type 1. These data suggest that F1C pili are, like P and S pili, evolutionarily selected to primarily withstand the conditions expressed in the upper urinary tract.

Stabilization of HIF-1alpha is critical to improve wound healing in diabetic mice.

Relative hypoxia is essential in wound healing since it normally plays a pivotal role in regulation of all the critical processes involved in tissue repair. Hypoxia-inducible factor (HIF) 1alpha is the critical transcription factor that regulates adaptive responses to hypoxia. HIF-1alpha stability and function is regulated by oxygen-dependent soluble hydroxylases targeting critical proline and asparaginyl residues. Here we show that hyperglycemia complexly affects both HIF-1alpha stability and activation, resulting in suppression of expression of HIF-1 target genes essential for wound healing both in vitro and in vivo. However, by blocking HIF-1alpha hydroxylation through chemical inhibition, it is possible to reverse this negative effect of hyperglycemia and to improve the wound healing process (i.e., granulation, vascularization, epidermal regeneration, and recruitment of endothelial precursors). Local adenovirus-mediated transfer of two stable HIF constructs demonstrated that stabilization of HIF-1alpha is necessary and sufficient for promoting wound healing in a diabetic environment. Our findings outline the necessity to develop specific hydroxylase inhibitors as therapeutic agents for chronic diabetes wounds. In conclusion, we demonstrate that impaired regulation of HIF-1alpha is essential for the development of diabetic wounds, and we provide evidence that stabilization of HIF-1alpha is critical to reverse the pathological process.

Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coli.

The transcription factor FocB belongs to a family of regulators encoded by several different fimbriae gene clusters in uropathogenic Escherichia coli. Recent findings suggest that FocB-family proteins may form different protein-protein complexes and that they may exert both positive and negative effects on the transcription of fimbriae genes. However, little is known about the actual role and mode of action when these proteins interact with the fimbriae operons. The 109-amino-acid FocB transcription factor from the foc gene cluster in E. coli strain J96 has been cloned, expressed and purified. The His(6)-tagged fusion protein was captured by Ni(2+)-affinity chromatography, cleaved with tobacco etch virus protease and purified by gel filtration. The purified protein is oligomeric, most likely in the form of dimers. NMR analysis guided the crystallization attempts by showing that probable conformational exchange or oligomerization is reduced at temperatures above 293 K and that removal of the highly flexible His(6) tag is advantageous. The protein was crystallized using the hanging-drop vapour-diffusion method at 295 K. A native data set to 2.0 A resolution was collected at 100 K using synchrotron radiation.

Regulatory Interactions among adhesin gene systems of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli strain J96 carries multiple determinants for fimbrial adhesins. The regulatory protein PapB of P fimbriae has previously been implicated in potential coregulatory events. The focB gene of the F1C fimbria determinant is highly homologous to papB; the translated sequences share 81% identity. In this study we investigated the role of PapB and FocB in regulation of the F1C fimbriae. By using gel mobility shift assays, we showed that FocB binds to sequences in both the pap and foc operons in a somewhat different manner than PapB. The results of both in vitro cross-linking and in vivo oligomerization tests indicated that FocB could function in an oligomeric fashion. Furthermore, our results suggest that PapB and FocB can form heterodimers and that these complexes can repress expression of the foc operon. The effect of FocB on expression of type 1 fimbriae was also tested. Taken together, the results that we present expand our knowledge about a regulatory network for different adhesin gene systems in uropathogenic E. coli and suggest a hierarchy for expression of the fimbrial adhesins.

Increasing the permeability of Escherichia coli using MAC13243.

The outer membrane of gram-negative bacteria is a permeability barrier that prevents the efficient uptake of molecules with large scaffolds. As a consequence, a number of antibiotic classes are ineffective against gram-negative strains. Herein we carried out a high throughput screen for small molecules that make the outer membrane of Escherichia coli more permeable. We identified MAC13243, an inhibitor of the periplasmic chaperone LolA that traffics lipoproteins from the inner to the outer membrane. We observed that cells were (1) more permeable to the fluorescent probe 1-N-phenylnapthylamine, and (2) more susceptible to large-scaffold antibiotics when sub-inhibitory concentrations of MAC13243 were used. To exclude the possibility that the permeability was caused by an off-target effect, we genetically reconstructed the MAC13243-phenotype by depleting LolA levels using the CRISPRi system.

Auxotrophy-based High Throughput Screening assay for the identification of Bacillus subtilis stringent response inhibitors.

The stringent response is a central adaptation mechanism that allows bacteria to adjust their growth and metabolism according to environmental conditions. The functionality of the stringent response is crucial for bacterial virulence, survival during host invasion as well as antibiotic resistance and tolerance. Therefore, specific inhibitors of the stringent response hold great promise as molecular tools for disarming and pacifying bacterial pathogens. By taking advantage of the valine amino acid auxotrophy of the Bacillus subtilis stringent response-deficient strain, we have set up a High Throughput Screening assay for the identification of stringent response inhibitors. By screening 17,500 compounds, we have identified a novel class of antibacterials based on the 4-(6-(phenoxy)alkyl)-3,5-dimethyl-1H-pyrazole core. Detailed characterization of the hit compounds as well as two previously identified promising stringent response inhibitors - a ppGpp-mimic nucleotide Relacin and cationic peptide 1018 - showed that neither of the compounds is sufficiently specific, thus motivating future application of our screening assay to larger and more diverse molecular libraries.

Structure of FocB--a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli.

In uropathogenic Escherichia coli, UPEC, different types of fimbriae are expressed to mediate interactions with host tissue. FocB belongs to the PapB family of transcription factors involved in the regulation of fimbriae gene clusters. Recent findings suggest that members from this family of proteins may form homomeric or heteromeric complexes and exert both positive and negative effects on the transcription of fimbriae genes. To elucidate the detailed function of FocB, we have determined its crystal structure at 1.4 A resolution. FocB is an all alpha-helical protein with a helix-turn-helix motif. Interestingly, conserved residues important for DNA-binding are located not in the postulated recognition helix of the motif, but in the preceding helix. Results from protein-DNA-binding studies suggest that FocB interacts with the minor groove of its cognate DNA target, which is indicative of a DNA interaction that is unusual for this motif. FocB crystallizes in the form of dimers. Packing interactions in the crystals give two plausible dimerization interfaces. Conserved residues, known to be important for protein oligomerization, are present at both interfaces, suggesting that both sites could play a role in a functional FocB protein.