Detection of Anaerobic Bacteria in High Numbers in Sputum from Patients with Cystic Fibrosis

Rationale: Pulmonary infection in cystic ?brosis (CF) is polymicrobial and it is possible that anaerobic bacteria, not detected by routine aerobic culture methods, reside within infected anaerobic airway
mucus.
Objectives: To determine whether anaerobic bacteria are present in the sputum of patients with CF.
Methods: Sputum samples were collected from clinically stable adults with CF and bronchoalveolar lavage ?uid (BALF) samples from children with CF. Induced sputum samples were collected from healthy volunteers who did not have CF. All samples were processed using anaerobic bacteriologic techniques and bacteria within the samples were quanti?ed and identi?ed.
Measurements and Main Results: Anaerobic species primarily within the genera Prevotella,Veillonella, Propionibacterium, andActinomyces were isolated in high numbers from 42 of 66 (64%) sputum samples from adult patients with CF. Colonization with Pseudomonas aeruginosa signi?cantly increased the likelihood that anaerobic bacteria would be present in the sputum. Similar anaerobic species were identi?ed in BALF from pediatric patients with CF. Although anaerobes were detected in induced sputum samples from 16 of 20 volunteers, they were present in much lower numbers and were
generally different species compared with those detected in CF sputum. Species-dependent differences in the susceptibility of the anaerobes to antibiotics with known activity against anaerobes were apparent with all isolates susceptible to meropenem.
Conclusions: A range of anaerobic species are present in large numbers in the lungs of patients with CF. If these anaerobic bacteria are contributing signi?cantly to infection and in?ammation in the CF
lung, informed alterations to antibiotic treatment to target anaerobes, in addition to the primary infecting pathogens, may improve management.

Tandem enzyme-catalysed reduction/cis-dihydroxylation of 2,2,2-trifluoroacetophenone: chemoenzymatic routes to new enantiopure phenol and benzylic alcohol reagents

Factors that control the competition between toluene dioxgenase-catalysed arene cis-dihydroxylation and dehydrogenase-catalysed ketone reduction have been studied, using whole cells of Pseudomonas putida UV and three alkylaryl ketones. The triol metabolite, obtained from 2,2,2-trifluoroacetophenone, has been used in the synthesis of single enantiomer chiral phenols and benzylic alcohols. Potential applications of the methylether derivatives of the chiral phenols and benzylic alcohols, as resolving agents, have been found. (c) 2007 Society of Chemical Industry.

Dioxygenase-catalysed oxidation of alkylaryl sulfides: sulfoxidation versus cis-dihydrodiol formation

Toluene- and naphthalene-dioxygenase-catalysed sulfoxidation of nine disubstituted methylphenyl sulfides, using whole cells of Pseudomonas putida, consistently gave the corresponding enantioenriched sulfoxides. Using the P. putida UV4 mutant strain, and these substrates, differing proportions of the corresponding cis-dihydrodiol sulfides were also isolated. Evidence was found for the concomitant dioxygenase-catalysed cis-dihydroxylation and sulfoxidation of methyl paratolyl sulfide. A simultaneous stereoselective reductase-catalysed deoxygenation of (S)-methyl para-tolyl sulfoxide, led to an increase in the proportion of the corresponding cis-dihydrodiol sulfide. The enantiopurity values and absolute configurations of the corresponding cis-dihydrodiol metabolites from methyl ortho-and para-substituted phenyl sulfides were determined by different methods, including chemoenzymatic syntheses from the cis-dihydrodiol metabolites of para-substituted iodobenzenes. Further evidence was provided to support the validity of an empirical model to predict, (i) the stereochemistry of cis-dihydroxylation of para-substituted benzene substrates, and (ii) the regiochemistry of cis-dihydroxylation reactions of ortho-substituted benzenes, each using toluene dioxygenase as biocatalyst.

Stereoselective reductase-catalysed deoxygenation of sulfoxides in aerobic and anaerobic bacteria

Direct and indirect evidence, Of unexpected stereoselective reductase-catalysed deoxygenations of sulfoxides, was found. The deoxygenations proceeded simultaneously, with the expected dioxygenase-catalysed asymmetric sulfoxidation of sulfides, during some biotransformations with the aerobic bacterium Pseudomonas putida UV4. Stereoselective reductase-catalysed asymmetric deoxygenation of racemic alkylaryl, dialkyl and phenolic sulfoxides was observed, without evidence of the reverse sulfoxidation reaction, using anaerobic bacterial strains. A purified dimethyl sulfoxide reductase, obtained from the intact cells of the anaerobic bacterium Citrobacter braakii DMSO 11, yielded, from the corresponding racemates, enantiopure alkylaryl sulfoxide and thiosulfinate samples.

Dioxygenase-catalysed sulfoxidation of bicyclic alkylaryl sulfides and chemoenzymatic synthesis of acyclic disulfoxides

Toluene- and naphthalene-dioxygenase-catalysed oxidation of six bicyclic disulfide substrates, using whole cells of Pseudomonas putida, gave the corresponding monosulfoxides with high ee values and enantiocomplementarity, in most cases. Two alcohol-sulfoxide diastereoisomers, formed from the reaction of the (R)-1,3-benzodithiole-1-oxide metabolite with n-butyllithium and benzaldehyde, were separated and stereochemically assigned. Treatment, of enantiopure (1R,3R)-benzo-1,3-dithiole-1,3-dioxide, obtained by chemoenzymatic synthesis, with alkyllithium reagents, resulted in a novel ring-opening reaction which proceeded with inversion of configuration to yield a series of acyclic disulfoxides. (C) 2003 Elsevier Ltd. All rights reserved.

Crystallization and preliminary X-ray diffraction analysis of naphthalene dioxygenase from Rhodococcus sp strain NCIMB 12038

The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene by Rhodococcus sp. strain NCIMB 12038. The terminal oxygenase component (naphthalene 1,2-dioxygenase) that catalyzes this reaction belongs to the aromatic ring hydroxylating dioxygenase family and has been crystallized. These enzymes utilize a mononuclear nonheme iron centre to catalyze the addition of dioxygen to their respective substrates. In this reaction, two electrons, two protons and a dioxygen molecule are consumed. The Rhodococcus enzyme has only 33 and 29% sequence identity to the corresponding alpha- and beta-subunits of the NDO system of Pseudomonas putida NCIMB 9816-4, for which the tertiary structure has been reported. In order to determine the three-dimensional structure of the Rhodococcus NDO, diffraction-quality crystals have been prepared by the hanging-drop method. The crystals belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 87.5, b = 144, c = 185.6 Angstrom, alpha = beta = gamma = 90degrees, and diffract to 2.3 Angstrom resolution.

Dioxygenase-catalyzed cis-dihydroxylation of pyridine-ring systems

Toluene dioxygenase-catalyzed dihydroxylation, in the carbocyclic rings of quinoline, 2-chloroquinoline, 2-methoxyquinoline, and 3-bromoquinoline, was found to yield the corresponding enantiopure cis-5,6- and -7,8-dihydrodiol metabolites using whole cells of Pseudomonas putida UV4. cis-Dihydroxylation at the 3,4-bond of 2-chloroquinoline, 2-methoxyquinoline, and 2-quinolone was also found to yield the heterocyclic cis-dihydrodiol metabolite, (+)-cis-(3S,4S)-3,4-dihydroxy-3,4-dihydro-2-quinolone. Heterocyclic cis-dihydrodiol metabolites, resulting from dihydroxylation at the 5,6- and 3,4-bonds of 1-methyl 2-pyridone, were isolated from bacteria containing toluene, naphthalene, and biphenyl dioxygenases. The enantiomeric excess (ee) values (>98%) and the absolute configurations of the carbocyclic cis-dihydrodiol metabolites of quinoline substrates (benzylic R) and of the heterocyclic cis-diols from quinoline, 2-quinolone, and 2-pyridone substrates (allylic S) were found to be in accord with earlier models for dioxygenase-catalyzed cis-dihydroxylation of carbocyclic arenes. Evidence favouring the dioxygenase-catalyzed cis-dihydroxylation of pyridine-ring systems is presented.

Dioxygenase-catalysed mono-, di- and tri-oxygenation of dialkyl sulfides and thioacetals: chemoenzymatic synthesis of enantiopure cis-diol sulfoxides

Toluene dioxygenase (TDO)-catalysed monooxygenation of methylsulfanylmethyl phenyl sulfide 1 and methylsulfanylmethyl 2-pyridyl sulfide 4, using whole cells of Pseudomonas putida UV4, occurred exclusively at the alkyl aryl sulfur centre to yield the alkyl aryl sulfoxides 2 and 5 respectively. These sulfoxides, accompanied by the dialkyl sulfoxides 3 and 6, were also obtained from naphthalene dioxygenase (NDO)-catalysed sulfoxidation of thioacetals 1 and 4 using intact cells of P. putida NCIMB 8859. Enzymatic oxidation of methyl benzyl sulfide 7, 2-phenyl-1,3-dithiane 19, and 2-phenyl-1,3-dithiolane 23, using TDO, gave the corresponding dialkyl sulfoxides 8, 20 and 24 as minor bioproducts. TDO-catalysed dioxygenation of the alkyl benzyl sulfides 7, 15 and 17 and the thioacetals 19 and 23, with P. putida UV4, yielded the corresponding enantiopure cis-dihydrodiols 9, 16, 18, 21 and 25 as major metabolites and cis-dihydrodiol sulfoxides 14, 22 and 26 as minor metabolites, resulting from a tandem trioxygenation of substrates 7, 19 and 23 respectively. Chemical oxidation, of the enantiopure cis-dihydrodiol sulfides 9, 16, 18 and 21 with dimethyldioxirane (DMD), gave separable mixtures of the corresponding pairs of cis-dihydrodiol sulfoxide diastereoisomers 14 and 27, 28 and 29, 30 and 31, 22 and 32. While dialkyl sulfoxide bioproducts 3, 6, 20 and 24 were of variable enantiopurity (27-greater than or equal to 98% ee), alkyl aryl monosulfoxides 2 and 5, cis-dihydrodiols 9, 16, 18, 21 and 25 and cis-dihydrodiol sulfoxide bioproducts 14, 22 and 26 were all single enantiomers (greater than or equal to 98% ee). The absolute configurations of the products, obtained from enzyme-catalysed (TDO and NDO) and chemical (DMD) oxidation methods, were determined by stereochemical correlation, circular dichroism, and X-ray crystallographic methods.

Enzyme-catalysed synthesis and absolute configuration assignments of cis-dihydrodiol metabolites from 1,4-disubstituted benzenes

A series of ten cis-dihydro-diol metabolites has been obtained by bacterial biotransformation of the corresponding 1,4-disubstituted benzene substrates using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). Their enantiomeric excess (ee) values have been established using chiral stationary phase HPLC and H-1 NMR spectroscopy. Absolute configurations of the majority of cis-dihydrodiols have been established using stereochemical correlation and X-ray crystallography and the remainder have been tentatively assigned using NMR spectroscopic methods but finally confirmed by circular dichroism (CD) spectroscopy. These configurational assignments support and extend the validity of an empirical model, previously used to predict the preferred stereochemistry of TDO-catalysed cis-dihydroxylation of ten 1,4-disubstituted benzene substrates, to more than twenty-five examples.

Stereochemical and mechanistic aspects of dioxygenase-catalysed benzylic hydroxylation of indene and chromane substrates

Toluene dioxygenase (TDO)-catalysed benzylic hydroxylation of indene substrates (8, 16 and 17), using whole cell cultures of Pseudomonas putida UV4, was found to yield inden-1-ol (14 and 22) and indan-1-one bioproducts (15 and 23). The formation of these bioproducts is consistent with the involvement of carbon-centred radical intermediates. TDO-catalysed oxidation of indenes 8 and 16 also gave cis-diols 13 and 18 respectively. TDO and naphthalene dioxygenase (NDO), used as both whole-cell preparations and as purified enzymes, were found to catalyse the benzylic hydroxylation of chromane 30, deuteriated (+/-)-chromane 30(D) and enantiomers (4S)-30(D) and (4R)-30(D) to yield (4R)- and (4S)-chroman-4-ols 31/31(D) respectively. The mechanism of benzylic hydroxylation of chromane 30/30(D) involves the stereoselective abstraction of a pro-R (with TDO) or a pro-S (with NDO) hydrogen atom at C-4 and a marked preference for retention of configuration.

Biodegradation and biosorption of acid anthraquinone dye

The acid anthraquinone dye Tectilon Blue (TB4R) is a major coloured component from the aqueous effluent of a carpet printing plant in Northern Ireland. The aerobic biodegradation of TB4R has been investigated experimentally in batch systems, using three strains of bacteria, namely, Bacillus gordonae (NCIMB 12553), Bacillus benzeovorans (NCIMB 12555) and Pseudomonas putida (NCIMB 9776). All three strains successfully decolourised the dye, and results were correlated using Michaelis-Menten kinetic theory. A recalculation of the reaction rate constants, to account for biosorption, gave an accurate simulation of the colour removal over a 24-h period. Up to 19% of the decolorisation was found to be caused by biosorption of the dye onto the biomass, with the majority of the decolorisation caused by utilisation of the dye by the bacteria. The reaction rate was found to be intermediate between zero and first order at dye concentrations of 200-1000 mg/l. (C) 2000 Elsevier Science Ltd. All rights reserved.

Cloning and characterization of a novel cis-naphthalene dihydrodiol dehydrogenase gene (narB) from Rhodococcus sp NCIMB12038

Rhodococcus sp. NCIMB112038 can utilize naphthalene as its sole carbon and energy source. The gene encoding cis-naphthalene dihydrodiol dehydrogenase (narB) of this strain has been cloned and sequenced. Expression of NCIMB12038 cis-naphthalene dihydrodiol dehydrogenase was demonstrated in Escherichia coli cells. narB encodes a putative protein of 271 amino acids and shares 39% amino acid identity with the cis-naphthalene dihydrodiol dehydrogenase from Pseudomonas putida G7. Comparison of NarB with some putative cis-dihydrodiol dehydrogenases from Rhodococcus species revealed significant differences between these proteins. NarB together with two other proteins forms a new group of cis-dihydrodiol dehydrogenases. (C) 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Antimicrobial activity of neuropeptides against a range of micro-organisms from skin, oral, respiratory and gastrointestinal tract sites

Many neuropeptides are similar in size, amino acid composition and charge to antimicrobial peptides. This study aimed to determine whether the neuropeptides substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), displayed antimicrobial activity against Streptococcus mutans, Lactobacillus acidophilus, Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. SP, NPY, VIP and CGRP displayed variable degrees of antimicrobial activity against all the pathogens tested with the exception of S. aureus. These antimicrobial activities add a further dimension to the immunomodulatory roles for neuropeptides in the inflammatory and immune responses. (c) 2008 Elsevier B.V. All rights reserved.

Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway

Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFa and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NF?B was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.

Elafin, an Elastase-specific Inhibitor, Is Cleaved by Its Cognate Enzyme Neutrophil Elastase in Sputum from Individuals with Cystic Fibrosis

Elafin is a neutrophil serine protease inhibitor expressed in lung and displaying anti-inflammatory and anti-bacterial properties. Previous studies demonstrated that some innate host defense molecules of the cystic fibrosis (CF) and chronic obstructive pulmonary disease airways are impaired due to increased proteolytic degradation observed during lung inflammation. In light of these findings, we thus focused on the status of elafin in CF lung. We showed in the present study that elafin is cleaved in sputum from individuals with CF. Pseudomonas aeruginosa-positive CF sputum, which was found to contain lower elafin levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective in cleaving recombinant elafin. NE plays a pivotal role in the process as only NE inhibitors are able to inhibit elafin degradation. Further in vitro studies demonstrated that incubation of recombinant elafin with excess of NE leads to the rapid cleavage of the inhibitor. Two cleavage sites were identified at the N-terminal extremity of elafin (Val-5—Lys-6 and Val-9—Ser-10). Interestingly, purified fragments of the inhibitor (Lys-6—Gln-57 and Ser-10—Gln-57) were shown to still be active for inhibiting NE. However, NE in excess was shown to strongly diminish the ability of elafin to bind lipopolysaccharide (LPS) and its capacity to be immobilized by transglutamination. In conclusion, this study provides evidence that elafin is cleaved by its cognate enzyme NE present at excessive concentration in CF sputum and that P. aeruginosa infection promotes this effect. Such cleavage may have repercussions on the innate immune function of elafin.