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Background and Aims: The density of epithelial cell extrusion zones in the intestinal lining, also known as gap density (number of gaps/1000 epithelial cells counted), can be quantitated using probe-based confocal laser endomicroscopy (pCLE). Gap density has been reported to be higher than normal in both inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) patients. Epithelial cells destined for extrusion from the intestinal surface would stain positive for either activated caspase-1 or caspase-3 on mucosal biopsy samples. The aim of this study was to determine whether epithelial gap density on pCLE correlates with quantitative analysis of activated caspase staining of mucosal biopsy samples from patients.Methods: We obtained pCLE images and biopsy samples of the terminal ileum during colonoscopies of healthy controls and patients with either IBD or IBS. The pCLE images and biopsy samples were blindly analyzed for gap density and for cells staining positive for activated caspases, respectively. The degree of correlation was determined using nonparametric statistical tests.Results: The median results were 10 gaps/1000 cells counted for controls versus 33 gaps/1000 cells counted for chronic intestinal disorder patients (p = 0.02). Activated caspase staining showed 13 positive cells/1000 epithelial cells counted versus 26 positive cells/1000 epithelial cells counted, respectively (p = 0.02), thus showing a strong correlation with a Spearman's coefficient ρ of 0.61 (strong correlation for ρ = 0.4-0.75, p = 0.01).Conclusions: Intestinal epithelial gap density via pCLE correlated strongly with quantitative analysis of immunohistochemical staining of mucosal biopsy samples. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa (PA) is a common bacterial pathogen, responsible for a high incidence of nosocomial and respiratory infections. KatA is the major catalase of PA that detoxifies hydrogen peroxide (H2O2), a reactive oxygen intermediate generated during aerobic respiration. Paradoxically, PA displays elevated KatA activity under anaerobic growth conditions where the substrate of KatA, H2O2, is not produced. The aim of the present study is to elucidate the mechanism underlying this phenomenon and define the role of KatA in PA during anaerobiosis using genetic, biochemical and biophysical approaches. We demonstrated that anaerobic wild-type PAO1 cells yielded higher levels of katA transcription and expression than aerobic cells, whereas a nitrite reductase mutant ΔnirS produced ∼50% the KatA activity of PAO1, suggesting that a basal NO level was required for the increased KatA activity. We also found that transcription of the katA gene was controlled, in part, by the master anaerobic regulator, ANR. A ΔkatA mutant and a mucoid mucA22 ΔkatA bacteria demonstrated increased sensitivity to acidified nitrite (an NO generator) in anaerobic planktonic and biofilm cultures. EPR spectra of anaerobic bacteria showed that levels of dinitrosyl iron complexes (DNIC), indicators of NO stress, were increased significantly in the ΔkatA mutant, and dramatically in a ΔnorCB mutant compared to basal levels of DNIC in PAO1 and ΔnirS mutant. Expression of KatA dramatically reduced the DNIC levels in ΔnorCB mutant. We further revealed direct NO-KatA interactions in vitro using EPR, optical spectroscopy and X-ray crystallography. KatA has a 5-coordinate high spin ferric heme that binds NO without prior reduction of the heme iron (Kd ∼6 μM). Collectively, we conclude that KatA is expressed to protect PA against NO generated during anaerobic respiration. We proposed that such protective effects of KatA may involve buffering of free NO when potentially toxic concentrations of NO are approached. [ABSTRACT FROM AUTHOR]

Surfactant protein-A (SP-A) is an important antimicrobial protein that opsonizes and permeabilizes membranes of microbial pathogens in mammalian lungs. Previously, we have shown that Pseudomonas aeruginosa flagellum-deficient mutants are preferentially cleared in the lungs of wild-type mice by SP-A-mediated membrane permeabilization, and not by opsonization. In this study, we report a flagellum-mediated mechanism of P. aeruginosa resistance to SP-A. We discovered that flagellum-deficient (Δ fliC) bacteria are unable to produce adequate amounts of exoproteases to degrade SP-A in vitro and in vivo, leading to its preferential clearance in the lungs of SP-A mice. In addition, Δ fliC bacteria failed to degrade another important lung antimicrobial protein lysozyme. Detailed analyses showed that Δ fliC bacteria are unable to upregulate the transcription of lasI and rhlI genes, impairing the production of homoserine lactones necessary for quorum-sensing, an important virulence process that regulates the production of multiple exoproteases. Thus, reduced ability of Δ fliC bacteria to quorum-sense attenuates production of exoproteases and limits degradation of SP-A, thereby conferring susceptibility to this major pulmonary host defence protein. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa readily binds to stainless steel and other abiotic surfaces, causing major problems in both the medical and food industries. In this study, we show that P. aeruginosa binds to abiotic surfaces in a concentration-dependent, saturable manner during the initial stages of biofilm formation. P. aeruginosa type IV pili mediate binding to stainless steel as a pilus-deficient strain does not bind to steel, purified type IV pili bound in a concentration-dependent, saturable manner, and purified pili competitively inhibited whole cell binding. PAK pili can also bind polystyrene and polyvinylchloride in a concentration-dependant and saturable manner. As an antibody specific for the C-terminal pilin receptor binding domain inhibited adherence to abiotic surfaces, the role of the C-terminal receptor binding domain in mediating binding to steel surfaces was examined. A synthetic peptide of the PAK pilin epithelial cell receptor binding domain [PAK(128–144)ox] bound directly to steel with high affinity. The interaction of pili with steel was specifically inhibited by this peptide with an apparent Ki of ∼0.2 nM and effectively inhibited the binding of viable homologous and heterologous P. aeruginosa strains to steel with an apparent Ki of ∼4 nM. A single point mutation (K130I) in the PAO receptor binding domain was observed to abolish binding to stainless steel while binding to human buccal epithelial cells was enhanced. Therefore, the C-terminal receptor binding domain appears to have evolved for binding a variety of surfaces. [ABSTRACT FROM AUTHOR]

The article presents information on cryocrystallography. Several technical developments in the past decade have led to improvements in resolution, in particular the construction of third-generation synchrotrons and the adoption of cryocooling of crystals. The availability of the brilliant synchrotron X-ray sources is making it possible to derive high-resolution protein structures more easily. However, to withstand the increased X-ray dose the crystals must be held at cryogenic temperatures. The goal of macromolecular crystallography is to determine the physiological three-dimensional structure of the macromolecule of interest as accurately as possible.

The backbone dynamics of a 15N-labeled recombinant PAK pilin peptide spanning residues 128–144 in the C-terminal receptor binding domain of Pseudomonas aeruginosa pilin protein strain PAK (Lys128-Cys-Thr-Ser-Asp-Gln-Asp-Glu-Gln-Phe-Ile-Pro-Lys-Gly-Cys-Ser-Lys144) were probed by measurements of 15N NMR relaxation. This PAK(128–144) sequence is a target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. The 15N longitudinal (T1) and transverse (T2) relaxation rates and the steady-state heteronuclear {1H}-15N NOE were measured at three fields (7.04, 11.74 and 14.1 Tesla), five temperatures (5, 10, 15, 20, and 25 °C ) and at pH 4.5 and 7.2. Relaxation data was analyzed using both the `model-free' formalism [Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546–4559 and 4559–4570] and the reduced spectral density mapping approach [Farrow, N.A., Szabo, A., Torchia, D.A. and Kay, L.E. (1995) J. Biomol. NMR, 6, 153–162]. The relaxation data, spectral densities and order parameters suggest that the type I and type II β-turns spanning residues Asp134-Glu-Gln-Phe137 and Pro139-Lys-Gly-Cys142, respectively, are the most ordered and structured regions of the peptide. The biological implications of these results will be discussed in relation to the role that backbone motions play in PAK pilin peptide immunogenicity, and within the framework of developing a pilin peptide vaccine capable of conferring broad immunity across P. aeruginosa strains. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa PAK pili and Candida albicans fimbriae are adhesins present on the microbial cell surfaces which mediate binding to epithelial cell-surface receptors. The receptor-binding domain (adhesintope) of the PAK pilus adhesin has been shown previously to reside in the carboxy-terminal disulphide-bonded region of P. aeruginosa pilin (PAK128-144). The delineation of the C. albicans fimbrial adhesintope was investigated in these studies using synthetic peptides which correspond to the whole (PAK128-144) or part of (PAK134-140) adhesintope of the PAK pilus and their respective anti-peptide antisera and biotinylated PAK pili (Bt-PAK pili), fimbriae (Bt-fimbriae), P. aeruginosa whole cells (Bt-P. aeruginosa) and C. albicans whole cells (Bt-C. albicans). The results from these studies confirmed that a structurally conserved motif akin to the PAK(126144) peptide sequence is present in C. albicans fimbrial adhesin and that the seven-amino-acid residue PAK(134-140) sequence plays an important role in forming the adhesintope for both P. aeruginosa PAK pilus and C. albicans fimbrial adhesins. [ABSTRACT FROM AUTHOR]

The monomeric pilin from Pseudomonas aeruginosa strain K1224 has been crystallized and preliminary X-ray diffraction data have been collected. Pilin is the monomeric subunit of the type IV pilus, the dominant adhesin of the opportunistic pathogen P. aeruginosa. The K1224 pilin crystallizes as a dimer in space group P1, with unitcell parameters a=40,19, b=38.93, c=37.22 Å, α=66.38, β=111.12, γ=93.74°. Diffraction data were collected using a synchrotron, radiation source and were processed to 1.54 Å d-spacing. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor referencing 15N resonances of the Pseudomonas aeruginosa K122-4 pilin monomer, published in the March 2001 issue of the "Journal of Biomolecular NMR."

A correction to the article "The Pseudomonas Aeruginosa Type IV Pilin Receptor Binding Domain Functions As An Adhesin for Both Biotic and Abiotic Surfaces," by Carmen L. Giltner,Erin J. van Schaik, Gerald F. Auclette, Dan Kao, Robert S. Hodges, Daniel J. Hassett and Randall T. Irvin in the 2006 issue is presented.

Discusses the employment of an antiinfective strategy to block colonization of human teeth by the pathogen Streptococcus mutans. Role of Streptococcus mutans in the occurrence of dental caries; Efficacy of anti-adhesin therapeutic strategies to control dental caries; Shortcomings of anti-adhesin therapeutic strategies.