Differential recognition of members of the carcinoembryonic antigen family by Afa/Dr adhesins of diffusely adhering Escherichia coli (Afa/Dr DAEC)

Little is known about the molecular bases underlying the virulence of diffusely adhering Escherichia coli (DAEC) harbouring the Afa/Dr family of adhesins. These adhesins recognize as receptors the GPI-anchored proteins CD55 (decay-accelerating factor, DAF) and CD66e (carcinoembryonic antigen, CEA). CD66e is a member of the CEA-related cell adhesion molecules (CEACAM) family, comprising seven members. We analysed the interactions of Afa/Dr DAEC with the CEACAMs using CEACAM-expressing CHO and HeLa cells. The results demonstrate that only E. coli expressing a subfamily of Afa/Dr adhesins, named here Afa/Dr-I, including Dr, F1845 and AfaE-III adhesins, bound onto CHO cells expressing CEACAM1, CEA or CEACAM6. Whereas all the Afa/Dr adhesins elicit recruitment of CD55 around adhering bacteria, only the Afa/Dr-I subfamily elicits the recruitment of CEACAM1, CEA and CEACAM6. In addition, although CEACAM3 is not recognized as a receptor by the subfamily of Afa/Dr adhesins, it is recruited around bacteria in HeLa cells. The recruited CEACAM1, CEA and CEACAM6 around adhering bacteria resist totally or in part a detergent extraction, whereas the recruited CEACAM3 does not. Finally, the results show that recognition of CEA and CEACAM6, but not CEACAM1, is accompanied by tight attachment to bacteria of cell surface microvilli-like extensions, which are elongated. Moreover, recognition of CEA is accompanied by an activation of the Rho GTPase Cdc42 and by a phosphorylation of ERM, which in turn elicit the observed cell surface microvilli-like extensions.     

The solution structure of the invasive tip complex from Afa/Dr fibrils

Afa/Dr family of adhesins are produced by pathogenic Escherichia coli strains that are especially prevalent in chronic diarrhoeal and recurrent urinary tract infections. Most notably, they are found in up to 50% of cystitis cases in children and 30% of pyelonephritis in pregnant women. Afa/Dr adhesins are capped surface fibrils that mediate recognition of the host and subsequent bacterial internalization. Using the newly solved three-dimensional structure of the minimal invasive complex (AfaDE) combined with biochemical and cellular assays, we reveal the architecture of the fibrillar cap and identify a novel mode of synergistic integrin recognition.     

Up-regulation of intestinal vascular endothelial growth factor by Afa/Dr diffusely adhering Escherichia coli

Background

Angiogenesis has been recently described as a novel component of inflammatory bowel disease pathogenesis. The level of vascular endothelial growth factor (VEGF) has been found increased in Crohn''s disease and ulcerative colitis mucosa. To question whether a pro-inflammatory Escherichia coli could regulate the expression of VEGF in human intestinal epithelial cells, we examine the response of cultured human colonic T84 cells to infection by E. coli strain C1845 that belongs to the typical Afa/Dr diffusely adhering E. coli family (Afa/Dr DAEC).

Methodology

VEGF mRNA expression was examined by Northern blotting and q-PCR. VEGF protein levels were assayed by ELISA and its bioactivity was analysed in endothelial cells. The bacterial factor involved in VEGF induction was identified using recombinant E. coli expressing Dr adhesin, purified Dr adhesin and lipopolysaccharide. The signaling pathway activated for the up-regulation of VEGF was identified using a blocking monoclonal anti-DAF antibody, Western blot analysis and specific pharmacological inhibitors.

Principal Findings

C1845 bacteria induce the production of VEGF protein which is bioactive. VEGF is induced by adhering C1845 in both a time- and bacteria concentration-dependent manner. This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells. Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways.

Conclusions

Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells. Thus, these results suggest a link between an entero-adherent, pro-inflammatory E. coli strain and angiogenesis which appeared recently as a novel component of IBD pathogenesis.     

High resolution characterization of formamidopyrimidine-DNA glycosylase interaction with its substrate by chemical cross-linking and mass spectrometry using substrate analogs

Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and human 8-oxoguanine-DNA glycosylase (hOgg1) initiate the base excision repair pathway for 7,8-dihydro-8-oxoguanine (8-oxoG) residues present in DNA. Recent structural and biochemical studies of Fpg-DNA and hOgg1-DNA complexes point to the existence of extensive interactions between phosphate groups and amino acids. However, the role of these contacts and their physiological relevance remains unclear. In the present study, we combined chemical cross-linking and electrospray ionization mass spectrometry (ESI/MS/MS) approaches to identify interacting residues in the Fpg-DNA and hOgg1-DNA complexes. The active centers of Fpg and hOgg1 were cross-linked with a series of reactive oligonucleotide duplexes containing both a single 8-oxoG residue and an O-ethyl-substituted pyrophosphate internucleotide (SPI) group at different positions in duplex DNA. The cross-linking efficiency reached 50% for Fpg and 30% for hOgg1. We have identified seven phosphate groups on both strands of the DNA duplex specifically interacting with nucleophilic amino acids in Fpg, and eight in hOgg1. MS/MS analysis of the purified proteolytic fragments suggests that lysine 56 of Fpg and lysine 249 of hOgg1 cross-link to the phosphate located 3' to the 8-oxoG residue. Site-specific mutagenesis analysis of Fpg binding to DNA substrate confirms the conclusions of our approach. Our results are consistent with crystallographic data on the Fpg-DNA complex and provide new data on the hOgg1-DNA interaction. The approach developed in this work provides a useful tool to study pro- and eukaryotic homologues of Fpg as well as other repair enzymes.     

NMR studies on puerarin and its interaction with beta-cyclodextrin

The interaction between puerarin and β-cyclodextrin (CD) has been studied in D₂O, H₂O/acetone-d ₆, acetone-d ₆ and DMSO-d ₆ solutions by ¹H NMR spectroscopy. The NMR data obtained from hydroxy protons indicate that the formation of the inclusion complex between the two molecules is not stabilized by strong hydrogen bond interactions. The sugar part of puerarin as well as the A ring are outside the β-CD cavity while the B and C rings are located inside the cavity and the interaction is mainly stabilized by hydrophobic interactions. In DMSO at 30°C and in acetone-d ₆/H₂O at temperature below −5°C, doubling of some signals indicated that, in these solvent systems, free rotation of the C-glycosyl bond was restricted due to the steric hindrance between the phenolic hydroxy group at C-7 and the bulky sugar moiety at C-8. In acetone, fast exchange of phenolic protons on the NMR timescale was observed, showing the effect of the solvent on the hindered rotation.     

High resolution deuterium NMR studies of bacterial metabolism

High resolution deuterium NMR spectra were obtained from suspensions of five bacterial strains: Escherichia coli, Clostridium perfringens, Klebsiella pneumoniae, Proteus mirabilis, and Staphylococcus aureus. Deuterium-labeled D-glucose at C-1, C-2, and C-6 was used to monitor dynamically anaerobic metabolism. The flux of glucose through the various bacterial metabolic pathways could be determined by following the disappearance of glucose and the appearance of the major end products in the 2H NMR spectrum. The presence of both labeled and unlabeled metabolites could be detected using 1H NMR spectroscopy since the proton resonances in the labeled species are shifted upfield due to an isotopic chemical shift effect. The 1H-1H scalar coupling observed in both the 2H and 1H NMR spectra was used to assign definitively the resonances of labeled species. An increase in the intensity of natural abundance deuterium signal of water can be used to monitor pathways in which a deuteron is lost from the labeled metabolite. The steps in which label loss can occur are outlined, and the influence these processes have on the ability of 2H NMR spectroscopy to monitor metabolism are assessed.     

High resolution nuclear magnetic resonance studies of nigeran

Polymer motion in solution can be studied by ¹³CNMR relaxation methods, which provide information about the correlation time for C-H vectors. ¹³C-Relaxation and Nuclear Overhauser Enhancement (NOE) data may frequently be combined to determine the dipole-dipole relaxation contribution. An alternative method is proposed based on a comparison of the proton spin-lattice relaxation rates of the centre proton resonances of an unlabelled molecule with the relaxation rates of the ¹³C satellites (from ¹³C labelled molecules).Selectively labelled nigeran which is an alternating $\text{1 → 3 and 1 → 4 α-}\text{d}\text{-glucan}$ has been investigated. The discussion in terms of the occurrence of different motions for each of the two units of the polymer requires an unambiguous assignment of the two anomeric carbons. For this reason a detailed assignment of the ¹H and ¹³C Nuclear Magnetic Resonance (NMR) spectra of nigeran in dimethylsulphoxide-d₆ is described, based on T₁ and NOE measurements in addition to selective homonuclear and heteronuclear spin decoupling experiments. These values are correlated with a conformation estimated by HSEA hard-spheres calculation. The measurements of the relaxation parameters for labelled and unlabelled compounds which provide an alternative determination of the ¹³C-¹H dipole-dipole relaxation contribution in a macromolecule agree well with ¹³C-{¹H} NOE experiments.     

真菌黑色素及其与巨噬细胞免疫研究进展

真菌黑色素在真菌抵抗外界理化损伤及宿主的免疫杀伤过程中起重要作用,是真菌重要的毒力因子。巨噬细胞是抗真菌固有免疫的一线细胞,探讨真菌黑色素与巨噬细胞的相互作用,可为阐明产黑色素真菌感染的发病机制奠定基础。本文重点从黑色素的生物学特性、真菌黑色素对巨噬细胞免疫的影响两个方面进行阐述。     

Epithelial cells secrete interleukin-8 in response to adhesion and invasion of diffusely adhering Escherichia coli lacking Afa/Dr genes

Escherichia coli that sparsely adhere to human epithelial cells are known as diffusely adherent E. coli (DAEC), and the role of the Afa/Dr family of adhesins is now understood. Strains that do not possess Afa/Dr, however, comprise another group of DAEC, of which the pathogenicity remains unknown. The ability to induce interleukin-8 (IL-8) secretion from intestinal epithelial cells might be a feature of enterovirulent bacteria. We previously found that some Afa/Dr DAEC strains induce IL-8 by stimulating epithelial cells with flagella. The present study examines whether non-Afa/Dr DAEC can induce IL-8 in epithelial cells (HEp-2, INT407, and T84). Among 21 strains, 11 (52%; 11/21) induced as much IL-8 as high inducer strains of Afa/Dr DAEC. Adhesion did not significantly differ between high and low inducers; therefore diffuse adhesion alone is probably insufficient to induce IL-8. It was shown that IL-8 induction and the number of intracellular bacteria directly correlated. Wortmannin, an inhibitor of the phosphatidylinositol-3-phosphate kinase, reduced both intracellular bacteria and IL-8 secretion. Motile strains were significantly more prevalent among high (10/11) than low (4/10) inducers. However, 4 low invasive strains hardly induced IL-8 despite their motility. In conclusion, some non-Afa/Dr DAEC invoke the induction of high levels of inflammatory cytokines. Unlike Afa/Dr DAEC, however, non-Afa/Dr strains may require invasion to cause strong induction. These non-Afa/Dr high inducers can be enteropathogenic for the cytokine-inducing properties.     

High resolution q-space imaging studies of water in elastin

We report on the direct measurement of the molecular diffusion coefficients of water confined to purified bovine nuchal ligament elastin by high resolution q-space NMR imaging. The experimental data indicate that water trapped within an elastin fiber has two distinguishable molecular diffusion coefficients. The component with the slowest mobility has a diffusion coefficient on the order of 10(-6) cm(2)/s that varies inversely with the diffusion time and is seen to reduce near 37 degrees C. The component with higher mobility has a diffusion coefficient reminiscent of free water but is observed to also behave similarly at 37 degrees C. From our experimental data we extract the surface-to-volume ratio of pores within elastin and associated changes as a function of temperature.     

Structural basis of the interaction of the pyelonephritic E. coli adhesin to its human kidney receptor.

PapG is the adhesin at the tip of the P pilus that mediates attachment of uropathogenic Escherichia coli to the uroepithelium of the human kidney. The human specific allele of PapG binds to globoside (GbO4), which consists of the tetrasaccharide GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc linked to ceramide. Here, we present the crystal structures of a binary complex of the PapG receptor binding domain bound to GbO4 as well as the unbound form of the adhesin. The biological importance of each of the residues involved in binding was investigated by site-directed mutagenesis. These studies provide a molecular snapshot of a host-pathogen interaction that determines the tropism of uropathogenic E. coli for the human kidney and is critical to the pathogenesis of pyelonephritis.     

Kinetic studies of the interaction of synthetic RNAs with quinacrine and its analogs.

Temperature-jump relaxation method has been used to study the interaction of synthetic RNAs with quinacrine (QAC) and its analog. Two relaxation times were observed. The dependence of relaxation times on the RNA concentration and optical properties of the RNA-dye complexes suggests that (1) QAC binds to poly-(rA).poly(rU) through two bimolecular reactions including isomerization from one complex form to another and (2) the 2-methoxy group of the acridine ring plays a significant role in the isomerization.     

Spectroscopic studies of the interaction of synthetic RNAs with quinacrine and its analogs.

The interaction of synthetic RNAs with quinacrine (QAC) and its analogs has been studied by absorption, fluorescence and circular dichroism (CD) measurements. The results indicate that the 2-methoxy group of the acridine ring plays an important role in the appearance of the new absorption band upon binding of QAC to poly-(rA).poly(rU).     

Electrochemical studies of DNA immobilization onto the azide-terminated monolayers and its interaction with taxol

A surface modification procedure for the creation of self-assembled monolayers (SAMs) that can be used as a scaffold for double-stranded DNA (dsDNA) incorporation onto the gold surfaces is described. The SAMs of an azidohexane thiol derivative were prepared on the Au electrode and then used for the immobilization of dsDNA. The electrochemical characteristics of dsDNA onto the SAM-modified gold electrode were investigated by cyclic voltammetry and electrochemical impedance spectroscopy, and the surface concentration of dsDNA onto the SAMs surface was estimated. The interaction of dsDNA with the anticancer drug, taxol (paclitaxel), was also studied on the surface of DNA/SAM/Au electrode. The observed decrease in the guanine oxidation peak current was used to monitor the interaction of taxol with DNA. The resulting Langmuir isotherm for taxol binding to DNA at the modified electrode was used to evaluate the binding constant of taxol-DNA. The results obtained supported the groove binding interaction of taxol with DNA. The modified electrode was used as a sensitive sensor for quantification of taxol in human serum sample.     

Theoretical studies on the interaction of guanine riboswitch with guanine and its closest analogues

Experimental studies (M. Mandal, B. Boese, J.E. Barrick, W.C. Winkler and R.R. Breaker, Riboswitches control fundamental biochemical pathways in bacillus subtilis and other bacteria, Cell 113 (2003), pp. 577–586) demonstrated that, besides recognising guanine with high specificity, guanine riboswitch could also bind guanine analogues, but the alteration of every functionalised position on the guanine heterocycle could cause a substantial loss of binding affinity. To investigate the nature of guanine riboswitch recognising metabolites, molecular docking and molecular dynamics simulation were carried out on diverse guanine analogues. The calculation results reveal that (1) most guanine analogues could bind to guanine riboswitch at the same binding pocket, with identical orientations and dissimilar binding energies, which is related to the positions of the functional groups; (2) the two tautomers of xanthine adopt different binding modes, and the enol-tautomer shows similar binding mode and affinity of hypoxanthine, which agrees well with the experimental results and (3) the riboswitch could form stable complexes with guanine analogues by hydrogen bonding contacts with U51 and C74. Particularly, U51 plays an important role in stabilising the complexes.     

Fluorescence quenching studies on the interaction of riboflavin with tryptophan and its analytical application

The ineraction between riboflavin (RBF) and tryptophan (Trp) was investigated using fluorescence spectroscopy and UV–vis absorption spectroscopy under physiological conditions. The fluorescence of Trp was quenched by RBF via dynamic quenching, which was analyzed using the Stern–Volmer relation. The value of the Forster distance R₀ (2.31 nm) was obtained according to the Forster's theory of nonradiative energy transfer. Under physiological conditions, a linear relationship could be established between the quenched fluorescence intensity of Trp and the concentration of RBF in the range of 5.8 × 10^‐7–2.0 × 10^‐5 mol/L. The detection limit was 1.8 × 10^‐7 mol/L. The method was successfully applied to determine riboflavin concentrations in pharmaceutical samples. Copyright © 2012 John Wiley & Sons, Ltd.     

High resolution studies on the pattern of induced exchanges in the human karyotype

The pattern of breakage and exchange induced by X-irradiation of human lymphocytes at G₁ has been analysed in PHA transformed cultures at X₁ metaphase in cells treated with trypsin. All the events observed occurred in interband segments. Moreover, as far as the autosomes were concerned, these events appear to be at random in relation to trypsin interband length but give some indication of non-randomness when total chromosome length is considered. The X and Y chromosomes, on the other hand, show far fewer breaks than would be expected whichever criterion is adopted, and in particular appear to be isolated from the autosomes with respect to the occurrence of exchange events. — The analysis of specific break points in relation to trypsin banding sequences, makes it clear that conclusions regarding chromosome rearrangements based solely on conventional preparations may be misleading.