Iwona Konieczna

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.

The use of lysozyme modified with fluorescein for the detection of Gram-positive bacteria.

Lysozyme (1,4-β-N-acetylmuramidase) is commonly applied in the food, medical, and pharmaceutical industries. In this study, we tested a novel application of fluorescein-modified lysozyme (using carboxyfluorescein with a triazine-based coupling reagent) as a new tool for the detection of Gram-positive soil bacteria. The results, obtained by cultivation methods, fluorescence analysis, and laser interferometry, showed that, after optimization, fluorescein-modified lysozyme could be used to evaluate the prevalence of Gram-positive bacteria essential in bioremediation of soils with low pH, such as those degraded by sulfur.

Are anti-Helicobacter pylori urease antibodies involved in atherosclerotic diseases?

OBJECTIVES The ureB subunit of urease is a major target recognized by the antibodies of Helicobacter pylori-infected patients. The minimal epitope was determined to be an 8-mer peptide (H-SIKEDVQF-OH). DESIGN AND METHODS The aim of this study was to discover whether this synthetic 8-mer peptide (BK-61A) directly recognizes the anti-ureB subunit antibodies of H. pylori-infected and atherosclerotic patients. To achieve a better presentation of the epitopes to antibodies, a new isocyanuric linker was designed and used for to immobilize the peptides on a cellulose support. RESULTS In this study a new peptide synthesis method is presented. Anti-ureB antibodies were evaluated by the dot blot technique in 26 H. pylori-infected donors and the sera of 20 H. pylori-infected patients with atherosclerosis using the 8-mer peptide. CONCLUSIONS The results reveal that the BK-61A peptide could be used for diagnosing the presence of anti-ureB antibodies that may be involved in the initiation of atherosclerosis.

Novel tool in rheumatoid arthritis diagnosis-The usage of urease flap region peptidomimetics

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease. Early diagnosis can prevent joint erosion. However, available biomarkers do not always allow for clear distinction between RA and non‐RA individuals. It has become known that bacteria/viruses are among the environmental triggers that initiate RA via multiple molecular mechanisms.

Cross-Reactivity of Polyclonal Antibodies against Canavalia ensiformis (Jack Bean) Urease and Helicobacter pylori Urease Subunit A Fragments

Overlapping decapeptide fragments of H. pylori urease subunit A (UreA) were synthesized and tested with polyclonal antibodies against Canavalia ensiformis (Jack bean) urease. The linear epitopes of UreA identified using the dot blot method were then examined using epitope mapping. For this purpose, series of overlapping fragments of UreA, frameshifted ± four amino acid residues were synthesized. Most of the UreA epitopes which reacted with the Jack bean urease polyclonal antibodies had been recognized in previous studies by monoclonal antibodies against H. pylori urease. Fragments 11 – 24, 21 – 33, and 31 – 42 were able to interact with the Jack bean urease antibodies, giving stable immunological complexes. However, the lack of recognition by these antibodies of all the components in the peptide map strongly suggests that a non‐continuous (nonlinear) epitope is located on the N‐terminal domain of UreA.

Characterization of Microbial Communities in Acidified, Sulfur Containing Soils

Over a period of three years, microbial communities in acidified soil with high sulfur content were analyzed. In soil water extracts ureolytic, proteolytic, oxidoreductive, and lipolytic activity were detected. The presented results indicate that the enzymatic activity of soil microbial communities varied considerably over time. Isolated 26 (80%) bacterial strains belonged to genus Bacillus sp. and were identified by cultivation and 16S rRNA methods. The commercially available procedures for bacterial DNA isolation from acidified soil failed, therefore a new, specific DNA isolation method was established. Ureolytic activity, detected in soil extracts as well as in isolated Bacillus sp. strains may be considered as a tool for the bioremediation of acidified soils with high sulfate content.