Galectin-3-mediated adherence of Proteus mirabilis to Madin-Darby canine kidney cells.

Proteus mirabilis is an important cause of urinary tract infections (UTIs) and can result in acute pyelonephritis. Proteus mirabilis expresses several, morphologically distinct, fimbrial species, and previous studies have shown that the nonagglutinating fimbriae (NAF) mediate bacterial adherence to a number of cell lines, including Madin-Darby canine kidney (MDCK) cells. Immunoblot overlay analysis of the plasma membrane fraction from MDCK cells with purified NAF revealed a 34-kDa band, which has been analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Database search identified galectin-3 as a potential protein candidate. Immunocytochemical assay of MDCK cells with a galectin-3-specific monoclonal antibody, anti-Mac-2, confirmed its presence on the plasma membrane extracellular surface. Preincubation of P. mirabilis with anti-Mac-2 monoclonal antibodies, specific for galectin-3, resulted in the inhibition of bacterial binding to MDCK cells. These data suggest a role for galectin-3, interacting with appropriately glycosylated surface receptors and P. mirabilis fimbriae, as a mediator of bacterial adherence in vitro.

Differential expression of nonagglutinating fimbriae and MR/P pili in swarming colonies of Proteus mirabilis.

The expression of nonagglutinating fimbriae (NAF) and mannose-resistant/Proteus-like (MR/P) pili in swarming colonies of Proteus mirabilis was investigated. Elongated swarmer cells do not express pili, and the relative number of bacteria expressing NAF during swarming and early consolidation phases was very low (<5%). Relative expression of NAF in a terrace increased to approximately 30% at 48 h. We also determined the expression of NAF and MR/P pili in two phenotypically distinguishable regions of each terrace. The expression of both NAF and MR/P pili was always higher in the region closer (proximal) to the middle of the colony than in the distal region of the terrace. The relative numbers of bacteria expressing NAF or MR/P pili in the proximal region were between 39.1 and 63% and between 5.9 and 7.7%, respectively. In the distal region, expression levels were between 20.8 and 27.3% and between 3.7 and 5. 6%, respectively. A time course experiment testing NAF expression in both the proximal and distal regions of a terrace indicated that NAF expression in the proximal regions was always higher than in the distal regions and increased to a plateau 40 to 50 h after the start of the swarming phase for any given terrace. These results indicate that expression of NAF or MR/P pili in swarming colonies of P. mirabilis is highly organized, spatially and temporally. The significance of this controlled differentiation remains to be uncovered.

Phosphatidyl choline-mediated inhibition of Streptococcus pneumoniae adherence to type II pneumocytes in vitro.

Nearly 80% of the adherence of several strains of Streptococcus pneumoniae to A549 lung cells was inhibited by dimyristoylphosphatidylcholine (DMPC), as well as by the following mixtures of lipids: DMPC/globoside, DMPC/asialo GM-1 and DMPC/asialo GM-1/globoside liposomes. Control phosphatidylserine liposomes were ineffective at inhibiting bacterial adherence demonstrating the specificity of the interaction between bacteria and liposomes. FITC-labelled bacteria were shown to adhere directly to silica beads coated with DMPC. The proportion of S. pneumoniae bacteria binding to DMPC-coated beads did not exceed 20% of the bacterial population as shown by the binding isotherm. This clearly demonstrates that only a fraction of the bacterial population (a subpopulation) was capable of binding to the beads. The specificity of bacterial binding to DMPC was further demonstrated by surface plasmon resonance. By this latter technique, the affinity between DMPC and bacteria was shown to be high and substantially non-reversible. Finally, we established that in order to be efficient at inhibiting bacterial binding to A549 cells the average liposome diameter must be greater than approximately 200 nm suggesting that a multivalent attachment of the bacterium to a liposome is required for high affinity binding.

The effect of growth conditions on in vitro adherence, invasion, and NAF expression by Proteus mirabilis 7570.

Proteus mirabilis is a common cause of upper urinary tract infections. Fimbriae-mediated adherence of this organism to urinary tract epithelium and invasion of host cells are factors thought to be important in its pathogenesis. We have assessed the effect of growth in serum, blood, and urine on the ability of P. mirabilis 7570 to adhere to and invade in vitro the cell line EJ/28, derived from a human urinary tract tumour, and to express nonagglutinating fimbriae (NAF). Proteus mirabilis was capable of adhering to EJ/28 cells to varying degrees depending upon the growth conditions used. It was invasive under all conditions, except when grown in urine, and was found to be particularly so when serum or blood was present in the media. Expression of NAF occurred under all growth conditions examined and was limited only by a decrease in temperature.

The expression of nonagglutinating fimbriae and its role in Proteus mirabilis adherence to epithelial cells.

Proteus mirabilis is a common causative agent of human urinary tract infections, especially in catheterized patients and in those patients with structural abnormalities of the urinary tract. In addition to the production of hemolysin and urease, fimbriae-mediated adherence to uroepithelial cells and kidney epithelium may be essential for virulence of P. mirabilis. A single P. mirabilis strain is capable of expressing several morphologically distinct fimbrial species, which can each be favoured by specific in vitro growth conditions. The fimbrial species reported to date include mannose-resistant/Proteus-like fimbriae, ambient temperature fimbriae, P. mirabilis fimbriae, and nonagglutinating fimbriae (NAF). Here, using intact bacteria or purified NAF as immunogens, we have generated the first reported NAF-specific monoclonal antibodies (mAbs). Bacteria expressing NAF as their only fimbrial species adhered strongly to a number of cell lines in vitro, including uroepithelial cell lines. Binding of P. mirabilis was markedly reduced following preincubation with NAF-specific mAbs and Fab fragments. The presence of NAF with highly conserved N-terminal sequences on all P. mirabilis strains so far examined, combined with the ability of both anti-NAF mAbs and purified NAF molecules to inhibit P. mirabilis adherence in vitro, suggests that NAF may contribute to the pathogenesis of P. mirabilis.

Bile acid inhibition of xenobiotic-metabolizing enzymes is a factor in the mechanism of colon carcinogenesis: tests of aspects of the concept with glucuronosyltransferase.

A factor in colon carcinogenesis might be the partial defeat in colon epithelial cells of the protective enzymic barrier against xenobiotics, via bile acid inhibition of enzymes that detoxify mutagens. The applicability of aspects of this concept to glucuronosyltransferase, a phenol detoxification enzyme, was tested in a colon cancer cell line. Inhibition of glucuronidation of the test substrate, 4-methylumbelliferone, occurred at bile acid concentrations found in faecal water, and depended on pH for some bile acids. Lithocholate was the most inhibitory: the concentration causing 50% inhibition of the initial rate of glucuronidation (IC50) was about 3 microM at pH 7.4 and at pH 6.2. The inhibitory potency of deoxycholate and chenodeoxycholate increased when pH decreased, but still remained less than that of lithocholate: the IC50 for deoxycholate was 88.5 microM at pH 7.4, and 14.8 microM at pH 6.2, and for chenodeoxycholate the IC50 was 67.4 microM at pH 7.4, and 21.7 microM at pH 6.2. Cholate did not cause appreciable inhibition. The inhibitory effects were additive when lithocholate was present together with either deoxycholate or chenodeoxycholate. The results provide a mechanism for the comutagenicity of bile acids, a feature of which is the inter-relation of bile acid comutagenicity specifically with mutagens that are inactivated by a bile acid-inhibitable enzyme. The results are also in accord with the view that high concentrations of bile acids in solution in faecal water, especially lithocholate, are a risk factor for colon cancer.

Toxicity of bile acids to colon cancer cell lines.

Quantitative aspects of bile acid cytotoxicity to colon cancer cell lines were investigated because of the etiological role in colon carcinogenesis attributed to the toxic effects of bile acids on colon mucosal cells. The cytotoxicity of major colonic bile acids differed. Lithocholate was the most toxic, followed by chenodeoxycholate and deoxycholate, with cholate being non-toxic over the concentration range studied. Cytotoxicity increased with time of exposure. Values for IC50 for some of the acids were determined to be in the physiological range, as estimated from their concentrations in fecal water. The results suggest dietary factors that contribute to bile acid mucosal damage. They also identify factors of possible importance in the association of high concentrations of bile acids in fecal water with risk for colon cancer.

Some Properties of Extracellular Acetylxylan Esterase Produced by the Yeast Rhodotorula mucilaginosa.

The red yeast Rhodotorula mucilaginosa produced an esterase that accumulated in the culture supernatant on induction with triacetin. The enzyme was specific for substrates bearing an O-acetyl group, but was relatively nonspecific for the rest of the molecule, which could consist of a phenol, a monosaccharide, a polysaccharide, or an aliphatic alcohol. The esterase was more active against acetylxylan and glucose beta-d-pentaacetate than were a number of esterases from plant and animal sources, when activities on 4-nitrophenyl acetate were compared. The enzyme exhibited Michaelis-Menten kinetics and was active over a broad pH range (5.5 to 9.2), with an optimum between pH 8 and 10. In addition, the enzyme retained its activity for 2 h at 55 degrees C. The yeast that produced the enzyme did not produce xylanase and, hence, is of interest for the production of acetylxylan esterase that is free of xylanolytic activity.

Utilization of Xylan by Yeasts and Its Conversion to Ethanol by Pichia stipitis Strains.

Yeasts able to grow on d-xylose were screened for the ability to hydrolyze xylan. Xylanase activity was found to be rare; a total of only 19 of more than 250 strains yielded a positive test result. The activity was localized largely in the genus Cryptococcus and in Pichia stipitis and its anamorph Candida shehatae. The ability to hydrolyze xylan was generally uncoupled from that to hydrolyze cellulose; only three of the xylan-positive strains also yielded a positive test for cellulolytic activity. Of the 19 xylanolytic strains, 2, P. stipitis CBS 5773 and CBS 5775, converted xylan into ethanol, with about 60% of a theoretical yield computed on the basis of the amount of d-xylose present originally that could be released by acid hydrolysis.