Wieslaw Kaca

Bacterial Urease and its Role in Long-Lasting Human Diseases

Urease is a virulence factor found in various pathogenic bacteria. It is essential in colonization of a host organism and in maintenance of bacterial cells in tissues. Due to its enzymatic activity, urease has a toxic effect on human cells. The presence of ureolytic activity is an important marker of a number of bacterial infections. Urease is also an immunogenic protein and is recognized by antibodies present in human sera. The presence of such antibodies is connected with progress of several long-lasting diseases, like rheumatoid arthritis, atherosclerosis or urinary tract infections. In bacterial ureases, motives with a sequence and/or structure similar to human proteins may occur. This phenomenon, known as molecular mimicry, leads to the appearance of autoantibodies, which take part in host molecules destruction. Detection of antibodies-binding motives (epitopes) in bacterial proteins is a complex process. However, organic chemistry tools, such as synthetic peptide libraries, are helpful in both, epitope mapping as well as in serologic investigations. In this review, we present a synthetic report on a molecular organization of bacterial ureases - genetic as well as structural. We characterize methods used in detecting urease and ureolytic activity, including techniques applied in disease diagnostic processes and in chemical synthesis of urease epitopes. The review also provides a summary of knowledge about a toxic effect of bacterial ureases on human body and about occurrence of anti-urease antibodies in long-lasting diseases.

Structural Study of O-Specific Polysaccharides of Proteus

Abstract Proteus bacteria are important human opportunistic pathogens which frequently cause urinary tract infections. According to Bergeys Manual of Systematic Bacteriology,1 this genus includes three species: P. mirabilis, P. vulgaris, and P. myxofaciens. A novel species of P. penneri has been recently proposed2,3 for strains formerly called P. vulgaris biogroup I. Proteus is an antigenically heterogeneous group of bacteria, and this is mainly associated with diverse composition and structures of O-specific polysaccharide chains of outer-membrane lipopolysaccharides (O-antigens). The Kauffman-Perch serological classification4 of P. mirabilis and P. vulgaris includes 49 O-serogroups. However, a number of S-strains remain unclassified,5 including strains of P. penneri.

Structure and serology of O-antigens as the basis for classification of Proteus strains:

This review is devoted to structural and serological characteristics of the O-antigens (O-polysaccharides) of the lipopolysaccharides of various Proteus species, which provide the basis for classifying Proteus strains to Oserogroups. The antigenic relationships of Proteus strains within and beyond the genus as well as their O-antigenrelated bioactivities are also discussed.

Developing an international Pseudomonas aeruginosa reference panel

Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis (CF) patients and causes a wide range of infections among other susceptible populations. Its inherent resistance to many antimicrobials also makes it difficult to treat infections with this pathogen. Recent evidence has highlighted the diversity of this species, yet despite this, the majority of studies on virulence and pathogenesis focus on a small number of strains. There is a pressing need for a P. aeruginosa reference panel to harmonize and coordinate the collective efforts of the P. aeruginosa research community. We have collated a panel of 43 P. aeruginosa strains that reflects the organisms diversity. In addition to the commonly studied clones, this panel includes transmissible strains, sequential CF isolates, strains with specific virulence characteristics, and strains that represent serotype, genotype or geographic diversity. This focussed panel of P. aeruginosa isolates will help accelerate and consolidate the discovery of virulence determinants, improve our understanding of the pathogenesis of infections caused by this pathogen, and provide the community with a valuable resource for the testing of novel therapeutic agents.

Laser interferometric and cultivation methods for measurement of colistin/ampicilin and saponin interactions with smooth and rough of Proteus mirabilis lipopolysaccharides and cells

Laser interferometry is commonly used in permeability studies of soluble substances. In this study a modification that allowed testing partially insoluble mixtures is presented. The modification relies on the measurement of diffusion from 1% agarose gel. As a model for this study, two Proteus mirabilis strains were used that differ in polysaccharide content: smooth P. mirabilis S1959 strain and its Re-type mutant, strain R45. By laser interferometry and precipitation it is shown that R45 lipopolysaccharide is more effective in binding colistin. It has been shown with the laser interferometric method that saponins, which are detergent-like substances of plant origin, partially enhance the interaction of colistin with the S and Re types of P. mirabilis. These results were confirmed with whole cell Proteus studies. The saponin partially inhibited the growth of the S and Re P. mirabilis strains at doses of 31-500 microg/ml. A sub-inhibitory dose--15 microg/ml of saponins alone do not reduced the numbers of P. mirabilis S1959 and R45 cells. However, the presence of colistin or amipicillin and 15 microg/ml of saponins reduced the amount of P. mirabilis S1959 and R45 cells. The saponins enhanced sensitivities of S and R P. mirabilis cells towards colistin and amipicillin. One may proposed that saponins binds to lipid A part of LPS may resulted on an increase in bacterial cell wall outer-membrane permeabilities and by that facilitated antibiotics penetration into the bacterial cells. In conclusion, the laser interferometric method is a useful tool for studies of lipopolysaccharide-antibiotic interactions even if the tested substances are not fully soluble in water.

Phenotypic characterization of an international Pseudomonas aeruginosa reference panel: strains of cystic fibrosis (CF) origin show less in vivo virulence than non-CF strains

Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF) and acute opportunistic infections in people without CF. Forty-two P. aeruginosa strains from a range of clinical and environmental sources were collated into a single reference strain panel to harmonise research on this diverse opportunistic pathogen. To facilitate further harmonized and comparable research on P. aeruginosa, we characterized the panel strains for growth rates, motility, virulence in the Galleria mellonella infection model, pyocyanin and alginate production, mucoid phenotype, LPS pattern, biofilm formation, urease activity, and antimicrobial and phage susceptibilities. Phenotypic diversity across the P. aeruginosa panel was apparent for all phenotypes examined, agreeing with the marked variability seen in this species. However, except for growth rate, the phenotypic diversity among strains from CF versus non-CF sources was comparable. CF strains were less virulent in the G. mellonella model than non-CF strains (P = 0.037). Transmissible CF strains generally lacked O-antigen, produced less pyocyanin and had low virulence in G. mellonella. Furthermore, in the three sets of sequential CF strains, virulence, O-antigen expression and pyocyanin production were higher in the earlier isolate compared to the isolate obtained later in infection. Overall, this full phenotypic characterization of the defined panel of P. aeruginosa strains increases our understanding of the virulence and pathogenesis of P. aeruginosa and may provide a valuable resource for the testing of novel therapies against this problematic pathogen.

Effects of Saponins against Clinical E. coli Strains and Eukaryotic Cell Line

Saponins are detergent-like substances showing antibacterial as well as anticancer potential. In this study, the effects of saponins from Quillaja saponaria were analyzed against prokaryotic and eukaryotic cells. Multidrug-resistant clinical E. coli strains were isolated from human urine. As eukaryotic cells, the CHO-K1 cell lines were applied. Antibacterial effect of ampicillin, streptomycin, and ciprofloxacin in the presence of saponins was measured by cultivation methods. Properties of saponins against CHO-K1 cells were measured by the MTT test, hemolysis assay and flow cytometry. Saponin from Quillaja saponaria has a cytotoxic effect at concentrations higher than 25 μg/mL and in the range of 12–50 μg/mL significantly increases the level of early apoptotic cells. Saponin at dose of 12 μg/mL enhances the six E. coli strains growth. We postulate that saponins increase the influx of nutrients from the medium into E. coli cells. Saponins do not have synergetic effects on antibacterial action of tested antibiotics. In contrary, in the presence of saponins and antibiotics, more CFU/mL E. coli cells were observed. This effect was similar to saponins action alone towards E. coli cells. In conclusion, saponins was cytotoxic against CHO-K1 cells, whereas against E. coli cells this effect was not observed.

New structures of the o-specific polysaccharides of Proteus. 4. Polysaccharides containing unusual acidic N-acyl derivatives of 4-amino-4,6-dideoxy-D-glucose

The structures of the O-polysaccharides of the lipopolysaccharides of Proteus mirabilis O7 and O49 were determined by chemical methods, mass spectrometry, including MS/MS, and NMR spectroscopy, including experiments run in an H2O/D2O mixture to reveal correlations for NH protons. The O-polysaccharides were found to contain N-carboxyacetyl (malonyl) and N-(3-carboxypropanoyl) (succinyl) derivatives of 4-amino-4,6-dideoxyglucose (4-amino-4-deoxyquinovose, Qui4N), respectively. The behavior of Qui4N derivatives with the dicarboxylic acids under conditions of acid hydrolysis and methanolysis was studied using GLC-MS.

Structure and serological characterization of the O-antigen of Proteus mirabilis O18 with a phosphocholine-containing oligosaccharide phosphate repeating unit.

A phosphorylated, choline-containing polysaccharide was obtained by O-deacylation of the lipopolysaccharide (LPS) of Proteus mirabilis O18 by treatment with aqueous 12% ammonia, whereas hydrolysis with dilute acetic acid resulted in depolymerisation of the polysaccharide chain by the glycosyl phosphate linkage. Treatment of the O-deacylated LPS with aqueous 48% hydrofluoric acid cleaved the glycosyl phosphate group but, unexpectedly, did not affect the choline phosphate group. The polysaccharide and the derived oligosaccharides were studied by NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HMQC and HMQC-TOSCY experiments, along with chemical methods, and the following structure of the pentasaccharide phosphate repeating unit was established: [carbohydrate structure in text] Where ChoP=Phosphocoline Immunochemical studies of the LPS, O-deacylated LPS and partially dephosphorylated pentasaccharide using rabbit polyclonal anti-P. mirabilis O18 serum showed the importance of the glycosyl phosphate group in manifesting the serological specificity of the O18-antigen.

The Generation of Superoxide Anion in Blood Platelets in Response to Different Forms of Proteus mirabilis Lipopolysaccharide: Effects of Staurosporin, Wortmannin, and Indomethacin

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria may activate blood platelets. The aim of our study was to evaluate the effects of different forms of Proteus mirabilis LPS and isolated lipid A and polysaccharide part on the production of superoxide radicals in blood platelets and to estimate the role staurosporin, wortmannin and indomethacin on this process. We compared the generation of superoxide radicals in platelets treated with LPS after preincubation with inhibitors of the signal transduction pathways, namely staurosporin (inhibitor of protein kinase C), wortmannin (inhibitor of phosphoinositide 3-kinase), and indomethacin (inhibitor of cycloxygenase). Our results demonstrate that all LPS molecules and their fragments caused a stimulation of O2- generation in platelets (P<.5). LPSS1959 had the strongest stimulatory effect. Straurosporin and wortmannin, but not indomethacin inhibited O2- production in LPS-stimulated platelets. Staurosporin (8 nM) and wortmannin (50 nM) caused about 50% inhibition of thrombin-induced O2- generation in platelets, while indomethacin (10 microM) had only a slight inhibitory effect on this process. Our results provide support that in LPS- and thrombin-activated platelets, at least part of O2- generation in platelets, while indomethacin (10 microM) had only a slight inhibitory effect on this process. Our results provide support that in LPS- and thrombin-activated platelets, at least part of O2- is generated due to the activation of the enzymes (protein kinase C and phosphoinositide 3-kinase) involved in signal transduction pathway. Cycloxygenase seems to be not involved in this process.