In vitro studies of epithelium-associated crystallization caused by uropathogens during urinary calculi development.

Infectious urinary stones account for about 10% of all urinary stones. In 50% of cases urolithiasis is a recurrent illness, which can lead to the loss of a kidney if not properly treated. One of the reasons for recurrence of the disease may be the ability of bacteria to invade urothelial cells, persist in the host cells and serve as potential reservoirs for infection. Various uropathogens are associated with the formation of bacteria-induced urinary stones but Proteus mirabilis is the most commonly isolated (70%). An in vitro model was used in this study to analyze intracellular growth and crystallization in the presence of P. mirabilis, Klebsiella pneumoniae and Escherichia coli. Human ureter (Hu 609) and bladder (HCV 29) epithelial cell lines were infected with bacteria and incubated (3-72 h) in the presence of synthetic urine and amikacin to prevent extracellular bacterial growth. During the incubation the number of bacteria (CFU/ml) inside epithelial cells and the intensity of crystallization were established. Crystallization was determined as an amount of a calcium radioisotope. The chosen strains of uropathogens were able to invade both types of epithelial cells but the Hu 609 cells were invaded to a higher extent. However, crystallization occurred only in the presence of P. mirabilis strains which were invasive and urease-positive. The highest intensity of cell-associated crystallization was observed when the number of bacteria within the urothelium remained stable during the time of incubation. These results show that P. mirabilis has an ability to form crystals inside the host cells. Under these conditions bacteria are protected from antibiotic killing, which leads to persistent and recurrent infections. We also suspect that this phenomenon may be an important stage of kidney stones formation.

Structural, serological, and genetic characterization of the O-antigen of Providencia alcalifaciens O40.

The O-polysaccharide chain of the lipopolysaccharide (O-antigen) on the bacterial cell surface is one of the most structurally variable cell components and serves as a basis for serotyping of Gram-negative bacteria, including human opportunistic pathogens of the genus Providencia. In this work, the O-antigen of Providencia alcalifaciens O40 was obtained by mild acid degradation of the isolated lipopolysaccharide and studied by chemical methods and high-resolution NMR spectroscopy. The following structure of the O-polysaccharide was established: →4)-ß-D-Quip3NFo-(1→3)-α-D-Galp-(1→3)-ß-D-GlcpA-(1→3)-ß-D-GalpNAc-(1→, where GlcA stands for glucuronic acid and Qui3NFo for 3,6-dideoxy-3-formamidoglucose. The O40-antigen was found to be structurally and serologically related to the O-antigens of P. alcalifaciens O5 and Providencia stuartii O18. The O40-antigen gene cluster between cpxA and yibK was sequenced, and the gene functions were predicted in silico. In agreement with the O-polysaccharide structure established, the genes for the synthesis of dTDP-D-Qui3NFo, UDP-D-Gal, UDP-D-GlcA, and UDP-D-GalNAc as well as those encoding three glycosyltransferases, flippase (Wzx), and O-antigen polymerase (Wzy) were recognized. In addition, homologues of wza, wzb, and wzc genes, which are required for the surface expression of capsular polysaccharides, were found within the gene cluster, suggesting that the O-polysaccharide studied is a part of the capsule-related form of the lipopolysaccharide called K(LPS).

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O28.

An O-polysaccharide and oligosaccharides were isolated by GPC following mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O28. The O-polysaccharide was studied by sugar and methylation analyses, (1)H and (13)C NMR spectroscopy, including 2D ROESY and H-detected (1)H,(13)C HSQC and HMBC experiments, and the following structure of the branched pentasaccharide repeating unit was established: [see formula in text]. This structure was confirmed by ESI MS of the isolated tridecasaccharide consisting of the lipopolysaccharide core and one O-polysaccharide repeat. The ESI mass spectrum also enabled inferring the composition of the core oligosaccharide.

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O60.

An O-polysaccharide (O-antigen) was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O60 and studied by sugar and methylation analyses as well as (1)H and (13)C NMR spectroscopy, including 2D ROESY and (1)H,(13)C HMBC experiments in D(2)O and a ROESY experiment in a 9:1 H(2)O-D(2)O mixture to reveal correlations for NH protons. It was found that the polysaccharide is built up of linear pentasaccharide repeating units containing an amide of d-glucuronic acid with l-serine and has the following structure: The O-antigen studied is structurally and serologically closely related to the O-antigen of Proteus vulgaris O44.

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O12.

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O12. Its structure was studied by sugar analysis using GLC of the alditol acetates and (S)-2-octyl glycosides, methylation analysis, Smith degradation, and (1)H and (13)C NMR spectroscopy, including 2D (1)H-(1)H COSY, TOCSY, ROESY, (1)H-(13)C HSQC, and HMBC experiments. It was found that the polymer is a neutral heteropolysaccharide and has a branched heptasaccharide repeating unit with the following structure:

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O31 containing an ether of D-mannose with (2R,4R)-2,4-dihydroxypentanoic acid.

Recently, ether-linked diastereomeric 2,4-dihydroxypentanoic acids have been reported as new components of bacterial glycans [Shashkov, A. S. et al.Nat. Prod. Commun.2008, 3, 1625-1630]. In this work, an ether of (2R,4R)-2,4-dihydroxypentanoic acid (Dhpa) with d-mannose was identified in the O-polysaccharide of Providencia alcalifaciens O31, and the polysaccharide structure was elucidated. Studies by NMR spectroscopy confirmed the ether linkage between O-2 of Dhpa and O-4 of Man, and the absolute configuration of Man was determined after ether cleavage with boron trichloride. In the polysaccharide, Dhpa was found to exist partially in the form of 1,4-lactone. Using sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, H-detected (1)H,(13)C HSQC, and gHMBC experiments, the following structure of the tetrasaccharide repeating unit of the polysaccharide was established: [carbohydrate structure: see text]