Dr fimbriae of uropathogenic Eschericha coli strains are an example of surface-located adhesive structures assembled via the chaperone-usher pathway. These structures are crucial for specific attachment of bacteria to host receptors. Dr fimbriae are linear associates of DraE proteins, the structure of which is determined by a donor strand complementation between the consecutive subunits. The biogenesis of these structures is dependent on a function of the specific periplasmic chaperone and outer membrane usher proteins. In a consequence of these structural and assembly properties the potential unfolding of a single subunit in a linear associate would cause a destruction of fimbrial adhesion function. This correlates with the observed high resistance of fimbrial structures for denaturation. In this paper we show that the mechanism of thermal denaturation of DraE-sc protein is well described by an irreversible two-state model which is the reduced form of a Lumry-Eyring protein denaturation model. In theory of this model the observed stability of DraE-sc protein is determined by the high activation barrier for the unfolding stage N-->U. The microcalorimetry experiments permit to determine kinetic parameters of the DraE-sc unfolding process: energy of activation of 463.5 +/- 20.8 kJ.mol(-1) and rate constant of order 10(-17) s(-1). This corresponds to the dissociation/unfolding half-life of Dr fimbriae of 10(8) years at 25 degrees C. The FT-IR experiments show that the high stability of DraE is determined by the cooperative rigid protein core. The presented mechanism of kinetic stability of Dr fimbriae is probably universal to adhesive structures of the chaperone-usher type.

Fimbrial adhesins of pathogenic bacteria are linear protein associates responsible for binding to the specific host cell receptors. They are assembled via the chaperone/usher pathway conserved in Gram-negative bacteria. These adhesive organelles are characterized by the high resistance to dissociation and unfolding caused by temperature or chemical denaturants. The self-complemented (SC) recombinant subunits of adhesive structures make up the minimal model used to analyze stability phenomena of these organelles. The SC subunits are both highly stabilized thermodynamically and kinetically. They are characterized by a standard free energy of unfolding of 70-80 kJ/mol and a rate constant of unfolding of 10(-17) s(-1) (half-life of unfolding of 10(8) years at 25 degrees C). The DraE subunit of Dr fimbriae is characterized by a disulfide bond that joins the beginning of the A1 strand with the end of the B strand. Such localization is unique and differentiates this protein from other proteins of the Ig-like family. Sequence analysis shows that many protein subunits of adhesive structures possess cysteines that may form a potential disulfide bond homologous to that of DraE. In this paper, we investigate the influence of this noncanonical disulfide bond on the stability of DraE-sc by constructing a DraE-sc-DeltaSS mutant protein (Cys/Ala mutant). This construct unfolds thermally at a T(m) of 65.4 degrees C, more than 20 degrees C lower than that of the native DraE-sc protein, and possesses a different unfolding mechanism. The calculated standard free energy of unfolding of DraE-sc-DeltaSS is equal to 30 +/- 5 kJ/mol. This allows us to suggest that the disulfide bond is an important stabilizing feature of many fimbrial subunits.

Urinary tract infections (UTIs) are regarded as one of the most common bacterial infections affecting millions of people, in all age groups, annually in the world. The major causative agent of complicated and uncomplicated UTIs are uropathogenic E. coli strains (UPECs). Huge problems with infections of this type are their chronicity and periodic recurrences. Other disadvantages that are associated with UTIs are accompanying complications and high costs of health care, systematically increasing resistance of uropathogens to routinely used antibiotics, as well as biofilm formation by them. This creates the need to develop new approaches for the prevention and treatment of UTIs, among which phage therapy has a dominant potential to eliminate uropathogens within urinary tract. Due to the growing interest in such therapy in the last decade, the bacteriophages (natural, genetically modified, engineered, or combined with antibiotics or disinfectants) represent an innovative antimicrobial alternative and a strategy for managing the resistance of uropathogenic microorganisms and controlling UTIs.

BackgroundThe global spread of bacterial resistance has given rise to a growing interest in new anti-bacterial agents with a new strategy of action. Pilicides are derivatives of ring-fused 2-pyridones which block the formation of the pili/fimbriae crucial to bacterial pathogenesis. They impair by means of a chaperone-usher pathway conserved in the Gram-negative bacteria of adhesive structures biogenesis. Pili/fimbriae of this type belong to two subfamilies, FGS and FGL, which differ in the details of their assembly mechanism. The data published to date have shown that pilicides inhibit biogenesis of type 1 and P pili of the FGS type which are encoded by uropathogenic E. coli strains.ResultsWe evaluated the anti-bacterial activity of literature pilicides as blockers of the assembly of a model example of FGL-type adhesive structures, – the Dr fimbriae encoded by a dra gene cluster of uropathogenic Escherichia coli strains. In comparison to the strain grown without pilicide, the Dr+ bacteria cultivated in the presence of the 3.5 mM concentration of pilicides resulted in a reduction of 75 to 87% in the adherence properties to CHO cells expressing Dr fimbrial DAF receptor protein. Using quantitative assays, we determined the amount of Dr fimbriae in the bacteria cultivated in the presence of 3.5 mM of pilicides to be reduced by 75 to 81%. The inhibition effect of pilicides is concentration dependent, which is a crucial property for their use as potential anti-bacterial agents. The data presented in this article indicate that pilicides in mM concentration effectively inhibit the adherence of Dr+ bacteria to the host cells, – the crucial, initial step in bacterial pathogenesis.ConclusionsStructural analysis of the DraB chaperone clearly showed it to be a model of the FGL subfamily of chaperones. This permits us to conclude that analyzed pilicides in mM concentration are effective inhibitors of the assembly of adhesins belonging to the Dr family, and more speculatively, of other FGL-type adhesive organelles. The presented data and those published so far permit to speculate that based on the conservation of chaperone-usher pathway in Gram-negative bacteria , the pilicides are potential anti-bacterial agents with activity against numerous pathogens, the virulence of which is dependent on the adhesive structures of the chaperone-usher type.

BackgroundSSB (single-stranded DNA-binding) proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis.ResultsThis paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein). This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity). The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7±1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100°C and melting temperature (Tm) is 100.2°C as shown by differential scanning calorimetry (DSC) analysis.Conclusion NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids.

Urinary tract infections (UTIs) are the most widespread and annoying infections affecting millions of people every year annually. The biggest problems of urinary diseases are recurrences, increasing resistance of uropathogens to commonly used antibiotics, as well as the high health care costs of afflicted persons. Uropathogenic Escherichia coli strains (UPECs) are the most dominant etiologic agents of community-acquired infections of this type. During UTI pathogenesis, UPECs utilize various virulence factors, especially mono- and polyadhesive appendages of the chaperone-usher secretion pathway (CUP) required for adhesion, invasion and biofilm formation. Commonly used antibiotics for UTI treatment are usually effective, but their long-term utility may affect gut microbiota of the treated individuals and cause selection of drug resistant uropathogenic variants. Due to increasing resistance of UPEC strains to antibiotics via the evolution of specific defense mechanisms, there is a need to develop alternative methods and therapeutic strategies to fight UTIs (vaccines, receptor analogues, pilicides and curlicides, bacterial interference or phagotherapy). Such therapeutic approaches usually target processes enabling uropathogens to survive within the urinary tract and cause recurrent infections.

Urinary tract infections caused by Escherichia coli are very common health problem in the developed countries. The virulence of the uropathogenic E. coli Dr(+) IH11128 is determined by Dr fimbriae, which are homopolymeric structures composed of DraE subunits with the DraD protein capping the fiber. In this study, we have analyzed the structural and biochemical properties of biofilms developed by E. coli strains expressing Dr fimbriae with or without the DraD tip subunit and the surface-exposed DraD protein. We have also demonstrated that these E. coli strains form biofilms on an abiotic surface in a nutrient-dependent fashion. We present evidence that Dr fimbriae are necessary during the first stage of bacterial interaction with the abiotic surface. In addition, we reveal that the DraD alone is also sufficient for the initial surface attachment at an even higher level than Dr fimbriae and that chloramphenicol is able to reduce the normal attachment of the analyzed E. coli. The action of chloramphenicol also shows that protein synthesis is required for the early events of biofilm formation. Additionally, we have identified reduced exopolysaccharide coverage in E. coli that express only Dr fimbrial polyadhesins at the cell surface with or without the DraD capping subunit.

The constantly growing number of people suffering from bacterial, viral, or fungal infections, parasitic diseases, and cancers prompts the search for innovative methods of disease prevention and treatment, especially based on vaccines and targeted therapy. An additional problem is the global threat to humanity resulting from the increasing resistance of bacteria to commonly used antibiotics. Conventional vaccines based on bacteria or viruses are common and are generally effective in preventing and controlling various infectious diseases in humans. However, there are problems with the stability of these vaccines, their transport, targeted delivery, safe use, and side effects. In this context, experimental phage therapy based on viruses replicating in bacterial cells currently offers a chance for a breakthrough in the treatment of bacterial infections. Phages are not infectious and pathogenic to eukaryotic cells and do not cause diseases in human body. Furthermore, bacterial viruses are sufficient immuno-stimulators with potential adjuvant abilities, easy to transport, and store. They can also be produced on a large scale with cost reduction. In recent years, they have also provided an ideal platform for the design and production of phage-based vaccines to induce protective host immune responses. The most promising in this group are phage-displayed vaccines, allowing for the display of immunogenic peptides or proteins on the phage surfaces, or phage DNA vaccines responsible for expression of target genes (encoding protective antigens) incorporated into the phage genome. Phage vaccines inducing the production of specific antibodies may in the future protect us against infectious diseases and constitute an effective immune tool to fight cancer. Moreover, personalized phage therapy can represent the greatest medical achievement that saves lives. This review demonstrates the latest advances and developments in the use of phage vaccines to prevent human infectious diseases; phage-based therapy, including clinical trials; and personalized treatment adapted to the patient’s needs and the type of bacterial infection. It highlights the advantages and disadvantages of experimental phage therapy and, at the same time, indicates its great potential in the treatment of various diseases, especially those resistant to commonly used antibiotics. All the analyses performed look at the rich history and development of phage therapy over the past 100 years.