Overexpression of the rhamnose catabolism regulatory protein, RhaR: a novel mechanism for metronidazole resistance in Bacteroides thetaiotaomicron

Objectives: The aim of the investigation was to use in vitro transposon mutagenesis to generate metronidazole resistance in the obligately anaerobic pathogenic bacterium Bacteroides thetaiotaomicron, and to identify the genes involved to enable investigation of potential mechanisms for the generation of metronidazole resistance.
Methods: The genes affected by the transposon insertion were identified by plasmid rescue and sequencing. Expression levels of the relevant genes were determined by semi-quantitative RNA hybridization and catabolic activity by lactate dehydrogenase/pyruvate oxidoreductase assays.
Results: A metronidazole-resistant mutant was isolated and the transposon insertion site was identified in an intergenic region between the rhaO and rhaR genes of the gene cluster involved in the uptake and catabolism of rhamnose. Metronidazole resistance was observed during growth in defined medium containing either rhamnose or glucose. The metronidazole-resistant mutant showed improved growth in the presence of rhamnose as compared with the wild-type parent. There was increased transcription of all genes of the rhamnose gene cluster in the presence of rhamnose and glucose, likely due to the transposon providing an additional promoter for the rhaR gene, encoding the positive transcriptional regulator of the rhamnose operon. The B. thetaiotaomicron metronidazole resistance phenotype was recreated by overexpressing the rhaR gene in the B. thetaiotaomicron wild-type parent. Both the metronidazole-resistant transposon mutant and RhaR overexpression strains displayed a phenotype of higher lactate dehydrogenase and lower pyruvate oxidoreductase activity in comparison with the parent strain during growth in rhamnose.
Conclusions: These data indicate that overexpression of the rhaR gene generates metronidazole resistance in B. thetaiotaomicron

Vaginal delivery of the recombinant HIV-1 clade-C trimeric gp140 envelope protein CN54gp140 within novel rheologically structured vehicles elicits specific immune responses

Rheologically structured vehicle (RSV) gels were developed as delivery systems for vaginal mucosal vaccination with an HIV-1 envelope glycoprotein (CN54gp140). RSVs comprised a mucoadhesive matrix forming and vaginal fluid absorbing polymer. The mucoadhesive and rheological properties of the RSVs were evaluated in vitro, and the distribution, antigenicity and release of CN54gp140 were analysed by ELISA. CN54gp140 was uniformly distributed within the RSVs and continuously released in vitro in an antigenically intact form over 24 h. Vaginal administration to rabbits induced specific serum IgG, and IgG and IgA in genital tract secretions. The RSVs are a viable delivery modality for vaginal immunization.

PdT-2: a novel myotropic Type-2 tryptophyllin from the skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor.

Amphibian skin secretions represent a unique resource for the discovery of new bioactive peptides. Here we report the isolation, structural and functional characterization of a novel heptapeptide amide, DMSPPWHamide, from the defensive skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor. This peptide is of unique primary structure and has been classified as a member of the rather heterogenous tryptophyllin-2 (T-2) family of amphibian skin peptides and named P. dacnicolor Tryptophyllin-2 (PdT-2) in accordance. PdT-2 is the first Type 2-tryptophyllin to possess discrete bioactivity. Both natural and synthetic replicates of the peptide were found to contract the smooth muscle of rat urinary bladder, the latter displaying an EC50 of 4 nM.

Amolopkinins W1 and W2 – novel bradykinin-related peptides (BRPs) from the skin of the Chinese torrent frog, Amolops wuyiensis: antagonists of bradykinin-induced smooth muscle contraction of the rat ileum.

Bradykinin-related peptides (BRPs) represent one of the most widespread and closely studied families of amphibian defensive skin secretion peptides. Apart from canonical bradykinin (RPPGFSPFR) that was first reported in skin extracts of the European brown frog, Rana temporaria, many additional site-substituted, N- and/or C-terminally extended peptides have been isolated from skin extracts and secretions from representative species of the families Ranidae, Hylidae, Bombinatoridae and Leiopelmatidae. The most diverse range of BRPs has been found in ranid frog skin secretions and this probably reflects the diversity and number of species studied and their associated life histories within this taxon. Amolops (torrent or cascade frogs) is a genus within the Ranidae that has been poorly studied. Here we report the presence of two novel BRPs in the skin secretions of the Chinese Wuyi Mountain torrent frog (Amolops wuyiensis). Amolopkinins W1 and W2 are dodecapeptides differing in only one amino acid residue at position 2 (Val/Ala) that are essentially (Leu1, Thr6)-bradykinins extended at the N-terminus by either RVAL (W1) or RAAL (W2). Amolopkinins W1 and W2 are structurally similar to amolopkinin L1 from Amolops loloensis and the major BRP (Leu1, Thr6, Trp8)-bradykinin from the skin of the Japanese frog, Rana sakuraii. A. wuyiensis amolopkinins were separately encoded as single copies within discrete precursors of 61 amino acid residues as deduced from cloned skin cDNA. Synthetic replicates of both peptides were found to potently antagonize the contractile effects of canonical bradykinin on isolated rat ileum smooth muscle preparations. Amolopkinins thus appear to represent a novel sub-family of ranid frog skin secretion BRPs.

Sauvatide - a novel amidated myotropic decapeptide from the skin secretion of the waxy monkey frog, Phyllomedusa sauvagei.

Here we describe the structural and functional characterization of a novel myotropic peptide, sauvatide, from the skin secretion of the waxy monkey frog, Phyllomedusa sauvagei. Sauvatide is a C-terminally amidated decapeptide with the following primary structure – LRPAILVRTKamide – monoisotopic mass 1164.77 Da, which was found to contract the smooth muscle of rat urinary bladder with an EC50 of 2.2 nM. The sauvatide precursor, deduced from cloned skin cDNA, consists of 62 amino acid residues with a single copy of sauvatide located near the C-terminus. The mature peptide is generated from the precursor by cleavage at a classical –KR-cleavage site located proximal to the N-terminus and by removal of a –GKGK sequence at the C-terminus, the first glycyl residue acting as amide donor. Amphibian skin secretions thus continue to be a source of novel and potent biologically active peptides acting through functional targets in mammalian tissues.

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.

Development of a novel PCR assay for the identification of the black yeast, Exophiala (Wangiella) dermatitidis from adult patients with cystic fibrosis (CF)

Cystic fibrosis (CF) patients may suffer increased morbidity and mortality through colonisation, allergy and invasive infection from fungi. The black yeast, Exophiala dermatitidis (synonym Wangiella dermatitidis) has been found with increasing frequency in sputum specimens of CF patients, with reported isolation rates ranging from 1.1 to 15.7%. At present, no diagnostic PCR exists to aid with the clinical laboratory detection and identification of this organism. A novel species-specific PCR-based assay was developed for the detection of E. dermatitidis, based on employment of rDNA operons and interspacer (ITS) regions between these rDNA operons. Two novel primers, (designated ExdF & ExdR) were designed in silico with the aid of computer-aided alignment software and with the alignment of multiple species of Exophiala, as well as with other commonly described yeasts and filamentous fungi within CF sputum, including Candida. Aspergillus and Scedosporium. An amplicon of approximately 455 by was generated, spanning the partial ITS I region - the complete 5.8S rDNA region - partial ITS2 region, employing ExdF (forward primer [16-mer], 5'-CCG CCT ATT CAG GTC C-3' and ExdR (reverse primer [16-mer], 5'-TCT CTC CCA CTC CCG C-3', was employed and optimised on extracted genomic DNA from a well characterised culture of E. dermatitidis, as well as with high quality genomic DNA template from a further 16 unrelated fungi, including Candida albicans, C. dubliniensis, C. parapsilosis, C. glabrata, Scedosporium apiospermum, Penicillium sp., Aspergillus fumigatus, Aspergillus versicolor, Pichia guilliermondii, Rhodotorula sp., Trichosporon sp., Aureobasidium pullulans, Fusarium sp., Mucor hiemalis, Bionectria ochroleuca, Gibberella pulicaris. Results demonstrated that only DNA from E. dermatitidis gave an amplification product of the expected sire, whilst none of the other fungi were amplifiable. Subsequent employment of this primer pair detected this yeast from mycological cultures from 2/50 (4%) adult CF patients. These two patients were the only patients who were previously shown to have a cultural history of E. dermatitidis from their sputum. E. dermatitidis is a slow-growing fungus, which usually takes up to two weeks to culture in the microbiology laboratory and therefore is slow to detect conventionally, with the risk of bacterial overgrowth from common co-habiting pan- and multiresistant bacterial pathogens from sputum. namely Pseudomonas aeruginosa and Burkholderia cepacia complex organisms, hence this species-specific PCR assay may help detect this organism from CF sputum more specifically and rapidly. Overall, employment of this novel assay nay help in the understanding of the occurrence. aetiology and epidemiology of E. dermatitidis, as an emerging fungal agent in patients with CF. (C) 2008 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Comparison of a novel spray congealing procedure with emulsion-based methods for the micro-encapsulation of water-soluble drugs in low melting point triglycerides

The particle size characteristics and encapsulation efficiency of microparticles prepared using triglyceride materials and loaded with two model water-soluble drugs were evaluated. Two emulsification procedures based on o/w and w/o/w methodologies were compared to a novel spray congealing procedure. After extensive modification of both emulsification methods, encapsulation efficiencies of 13.04% tetracycline HCl and 11.27% lidocaine HCl were achievable in a Witepsol (R)-based microparticle. This compares to much improved encapsulation efficiencies close to 100% for the spray congealing method, which was shown to produce spherical particles of similar to 58 mu m. Drug release studies from a Witepsol (R) formulation loaded with lidocaine HCl showed a temperature-dependent release mechanism, which displayed diffusion-controlled kinetics at temperatures similar to 25 degrees C, but exhibited almost immediate release when triggered using temperatures close to that of skin. Therefore, such a system may find application in topical semi-solid formulations, where a temperature-induced burst release is preferred.

Peptide DV-28 amide: Prototype of a novel class of bradykinin receptor antagonist isolated from the defensive skin secretion of bombinid toads

Kinestatin, isolated from the skin of the Chinese toad, Bombina maxima, was the first bradykinin B2 receptor antagonist identified in amphibians. Molecular cloning established that it is co-encoded with the bradykinin-related peptide, maximakinin, within one of several skin kininogens. To examine other species within the genus Bombina for the presence of structural homologues of kinestatin, we subjected skin secretion of the toad, Bombina orientalis, to HPLC fractionation with subsequent bioassay of fractions for antagonism of bradykinin activity using an isolated rat tail artery smooth muscle preparation. A single fraction was located that inhibited bradykinin-induced relaxation of rat arterial smooth muscle and MALDI-TOF analysis of this fraction revealed that it contained a single peptide of molecular mass 3198.5 Da. Further primary structural analysis of this peptide showed that it was a 28-mer with an N-terminal Asp (D) residue and a C-terminal Val (V) residue that was amidated. The peptide was named DV-28 amide in accordance with these primary structural attributes. Synthetic DV-28 amide replicated the observed bradykinin antagonistic effect within the smooth muscle bioassay in a dose-dependent manner. In addition, it was observed to inhibit the proliferation of human microvessel endothelial cells (HMECs) as assessed by MTT assay. Bioinformatic analysis revealed that DV-28 amide was, like kinestatin, co-encoded with a bradykinin receptor agonist on one of two skin kininogens identified in B. orientalis. DV-28 amide thus represents a novel class of bradykinin antagonist from skin secretions of bombinid toads that appear to be a rich source of such novel peptides.

Molecular cloning of the helokinestatin/CNP precursor from a venom-derived cDNA library of the Mexican beaded lizard, Heloderma horridum, and identification of a novel encoded bradykinin-antagonist peptide, helokinestatin-6

Helokinestatins 1–5 represent a novel family of bradykinin antagonist peptides originally isolated from the venom of the Gila Monster, Heloderma suspectum. We found that they were encoded in tandem along with a single copy of C-type natriuretic peptide (CNP), by two different but almost identical biosynthetic precursors that were cloned from a venom-derived cDNA library. Here we have applied the same strategy to the venom of a related species, the Mexican beaded lizard, Heloderma horridum. Lyophilised venom was used as a surrogate tissue to generate a cDNA library that was interrogated with primers from the previous study and for reverse phase HPLC fractionation. The structure of a single helokinestatin precursor was obtained following sequencing of 20 different clones. The open-reading frame contained 196 amino acid residues, somewhat greater than the 177–178 residues of the corresponding helokinestatin precursors in H. suspectum. The reason for this difference in size was the insertion of an additional domain of 18 amino acid residues encoding an additional copy of helokinestatin-3. Helokinestatin-6 (GPPFNPPPFVDYEPR) was a novel peptide from this precursor identified in venom HPLC fractions. A synthetic replicate of this peptide antagonised the relaxation effect of bradykinin on rat arterial smooth muscle. The novel peptide family, the helokinestatins, have been shown to be present in the venom of H. horridum and to be encoded by a single precursor of different structure to those from H. suspectum. Studies such as this reveal the naturally-selected structures of bioactive peptides that have been optimised for purpose and provide the scientist with a natural analogue library for pharmacological investigation.