Kinetic and spectroscopic studies on the quercetin 2,3-dioxygenase from Bacillus subtilis

Quercetin 2,3-dioxygenase from Bacillus subtilis (QueD) converts the flavonol quercetin and molecular oxygen to 2-protocatechuoylphloroglucinolcarboxylic acid and carbon monoxide. QueD, the only known quercetin 2,3-dioxygenase from a prokaryotic organism, has been described as an Fe2+-dependent bicupin dioxygenase. Metal-substituted QueDs were generated by expressing the enzyme in Escherichia coli grown on minimal media in the presence of a number of divalent metals. The addition of Mn2+, Co2+, and Cu2+ generated active enzymes, but the addition of Zn2+, Fe2+, and Cd2+ did not increase quercetinase activity to any significant level over a control in which no divalent ions were added to the media. The Mn2+- and Co2+-containing QueDs were purified, characterized by metal analysis and EPR spectroscopy, and studied by steady-state kinetics. Mn2+ was found to be incorporated nearly stoichiometrically to the two cupin motifs. The hyperfine coupling constant of the g = 2 signal in the EPR spectra of the Mn2+-containing enzyme showed that the two Mn2+ ions are ligated in an octahedral coordination. The turnover number of this enzyme was found to be in the order of 25 s(-1), nearly 40-fold higher than that of the Fe2+-containing enzyme and similar in magnitude to that of the Cu2+-containing quercertin 2,3-dioxygenase from Aspergillus japonicus. In addition, kinetic and spectroscopic data suggest that the catalytic mechanism of QueD is different from that of the Aspergillus quercetinases but similar to that proposed for the extradiol catechol dioxygenases. This study provides evidence that Mn2+ might be the preferred cofactor for this enzyme and identifies QueD as a new member of the manganese dioxygenase family.     

The modular nature of the β‐barrel assembly machinery,illustrated in Borrelia burgdorferi

Diderm bacteria have an outer membrane that provides defense against environmental factors including antibiotics. Understanding the process of outer membrane biogenesis is, therefore, of critical importance in order to envisage new treatments of these bacterial pathogens. Borrelia burgdorferi is the pathogen responsible for Lyme disease. Its outer membrane contains integral, β‐barrel proteins as well as swathes of externally exposed lipoproteins. Previous work has demonstrated that the β‐barrel assembly machine (BAM complex) in B. burgdorferi and other Spirochetes shares several similarities with the BAM complex in other bacterial lineages, such as the Proteobacteria that includes Escherichia coli. However, Iqbal et al. ( 2016 ) have identified the inner membrane protein TamB as a subunit of the BAM complex in Spirochetes. This latest study highlights the modular nature of the BAM complex, and suggests that in some bacterial lineages the BAM complex and translocation and assembly module (the TAM) function as a single unit.     

Vaccination with Human Papillomavirus Pseudovirus-Encapsidated Plasmids Targeted to Skin Using Microneedles

Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation.     

Diacylglycerol Enrichment of Endoplasmic Reticulum or Lipid Droplets Recruits Perilipin 3/TIP47 during Lipid Storage and Mobilization

Fatty acid-induced triacylglycerol synthesis produces triacylglycerol droplets with a protein coat that includes perilipin 3/TIP47 and perilipin 4/S3-12. This study addresses the following two questions. Where do lipid droplets emerge, and how are their coat proteins recruited? We show that perilipin 3- and perilipin 4-coated lipid droplets emerge along the endoplasmic reticulum (ER). Blocking membrane trafficking with AlF₄⁻ during fatty acid-induced triacylglycerol synthesis drove perilipin 3 to the tubular ER. Forskolin, which like AlF₄⁻ activates adenylate cyclase, did not redistribute perilipin 3, but when added together with AlF₄⁻ perilipin 3 was recruited to lipid droplets rather than the ER. Thus inhibiting trafficking with AlF₄⁻ redistributed perilipin 3 differently under conditions of triacylglycerol synthesis (fatty acid addition) versus hydrolysis (forskolin) suggesting a shared acylglycerol-mediated mechanism. We tested whether diacylglycerol (DG), the immediate precursor of triacylglycerol and its first hydrolytic product, affects the distribution of perilipin 3. Stabilizing DG with the DG lipase inhibitor {"type":"entrez-protein","attrs":{"text":"RHC80267","term_id":"1470879788","term_text":"RHC80267"}}RHC80267 enhanced the perilipin 3 recruited to lipid droplets and raised DG levels in this fraction. Treating cells with a membrane-permeable DG recruited perilipin 3 to the ER. Stabilizing DG, by blocking its hydrolysis with {"type":"entrez-protein","attrs":{"text":"RHC80267","term_id":"1470879788","term_text":"RHC80267"}}RHC80267 or its acylation with triacsin C, enhanced recruitment of perilipin 3 to the ER. Expressing the ER enzyme DGAT1, which removes DG by converting it to triacylglycerol, attenuated perilipin 3 DG-induced ER recruitment. Membrane-permeable DG also drove perilipin 4 and 5 onto the ER. Together the data suggest that these lipid droplet proteins are recruited to DG-enriched membranes thereby linking lipid coat proteins to the metabolic state of the cell.     

Two Horizontally Transferred Xenobiotic Resistance Gene Clusters Associated with Detoxification of Benzoxazolinones by Fusarium Species

Microbes encounter a broad spectrum of antimicrobial compounds in their environments and often possess metabolic strategies to detoxify such xenobiotics. We have previously shown that Fusarium verticillioides, a fungal pathogen of maize known for its production of fumonisin mycotoxins, possesses two unlinked loci, FDB1 and FDB2, necessary for detoxification of antimicrobial compounds produced by maize, including the γ-lactam 2-benzoxazolinone (BOA). In support of these earlier studies, microarray analysis of F. verticillioides exposed to BOA identified the induction of multiple genes at FDB1 and FDB2, indicating the loci consist of gene clusters. One of the FDB1 cluster genes encoded a protein having domain homology to the metallo-β-lactamase (MBL) superfamily. Deletion of this gene (MBL1) rendered F. verticillioides incapable of metabolizing BOA and thus unable to grow on BOA-amended media. Deletion of other FDB1 cluster genes, in particular AMD1 and DLH1, did not affect BOA degradation. Phylogenetic analyses and topology testing of the FDB1 and FDB2 cluster genes suggested two horizontal transfer events among fungi, one being transfer of FDB1 from Fusarium to Colletotrichum, and the second being transfer of the FDB2 cluster from Fusarium to Aspergillus. Together, the results suggest that plant-derived xenobiotics have exerted evolutionary pressure on these fungi, leading to horizontal transfer of genes that enhance fitness or virulence.     

Detoxication of Benzo[a]pyrene-7,8-dione by Sulfotransferases (SULTs) in Human Lung Cells

Polycyclic aromatic hydrocarbons (PAH) are environmental and tobacco carcinogens. Human aldo-keto reductases catalyze the metabolic activation of proximate carcinogenic PAH trans-dihydrodiols to yield electrophilic and redox-active o-quinones. Benzo[a]pyrene-7,8-dione a representative PAH o-quinone is reduced back to the corresponding catechol to generate a futile redox-cycle. We investigated whether sulfonation of PAH catechols by human sulfotransferases (SULT) could intercept the catechol in human lung cells. RT-PCR identified SULT1A1, -1A3, and -1E1 as the isozymes expressed in four human lung cell lines. The corresponding recombinant SULTs were examined for their substrate specificity. Benzo[a]pyrene-7,8-dione was reduced to benzo[a]pyrene-7,8-catechol by dithiothreitol under anaerobic conditions and then further sulfonated by the SULTs in the presence of 3'-[(35)S]phosphoadenosine 5'-phosphosulfate as the sulfonate group donor. The human SULTs catalyzed the sulfonation of benzo[a]pyrene-7,8-catechol and generated two isomeric benzo[a]pyrene-7,8-catechol O-monosulfate products that were identified by reversed phase HPLC and by LC-MS/MS. The various SULT isoforms produced the two isomers in different proportions. Two-dimensional (1)H and (13)C NMR assigned the two regioisomers of benzo[a]pyrene-7,8-catechol monosulfate as 8-hydroxy-benzo[a]pyrene-7-O-sulfate (M1) and 7-hydroxy-benzo[a]pyrene-8-O-sulfate (M2), respectively. The kinetic profiles of three SULTs were different. SULT1A1 gave the highest catalytic efficiency (k(cat)/K(m)) and yielded a single isomeric product corresponding to M1. By contrast, SULT1E1 showed distinct substrate inhibition and formed both M1 and M2. Based on expression levels, catalytic efficiency, and the fact that the lung cells only produce M1, it is concluded that the major isoform that can intercept benzo[a]pyrene-7,8-catechol is SULT1A1.     

Mode of formation of cholesta-5,7-dien-3β-ol from cholest-5-en-3β-ol by larvae of Calliphora erythrocephala

1. The conversion of cholest-5-en-3beta-ol (cholesterol) into cholesta-5,7-dien-3beta-ol by axenic Calliphora erythrocephala larvae was demonstrated. 2. The transformation is probably direct (Delta(5)-->Delta(5,7)) and does not involve a Delta(0) intermediate (Delta(5)-->Delta(0)-->Delta(7)--> Delta(5,7)). 3. Delta(7)-bond formation involves the stereospecific elimination of the 7beta hydrogen atom. 4. The relative amounts of free and esterified sterols were determined in larvae grown on cholesterol as sole sterol source and on 5alpha-cholestan-3beta-ol supplemented with minimal amounts of cholesterol. 5. The significance of the results is assessed in relation to the probable role of cholesta-5,7-dien-3beta-ol as an intermediate in the biosynthesis of ecdysones.     

Shoot-bud regeneration in subcultured callus of engelmann spruce

Summary Callus cultures ofPicea engelmannii (Parry, Engelmann spruce) were initiated and established from mature embryos cultured on von Arnold and Eriksson’s medium (AE) supplemented with N⁶-benzyladenine (10μM) and naphthalene acetic acid (10 μM). Cultures were maintained by subculture at 3-to-4-wk intervals. After three subcultures, callus was transferred to AE medium with only N⁶-benzyladenine (25 μM). Adventitious buds appeared on the surface of the callus after 2-to 4-wk and grew to adventitious shoots on AE medium without growth hormones or on AE medium with kinetin (0.1 μM). Shoot-forming capacity was maintained through 7 further subcultures. This study was supported by the Natural Sciences and Engineering Research Council of Canada grant G1438 to T. A. Thorpe and D. I. Dunstan.     

Nasal Lipopolysaccharide Challenge and Cytokine Measurement Reflects Innate Mucosal Immune Responsiveness

Background

Practical methods of monitoring innate immune mucosal responsiveness are lacking. Lipopolysaccharide (LPS) is a component of the cell wall of Gram negative bacteria and a potent activator of Toll-like receptor (TLR)-4. To measure LPS responsiveness of the nasal mucosa, we administered LPS as a nasal spray and quantified chemokine and cytokine levels in mucosal lining fluid (MLF).

Methods

We performed a 5-way cross-over, single blind, placebo-controlled study in 15 healthy non-atopic subjects (n = 14 per protocol). Doses of ultrapure LPS (1, 10, 30 or 100μg/100μl) or placebo were administered by a single nasal spray to each nostril. Using the recently developed method of nasosorption with synthetic adsorptive matrices (SAM), a series of samples were taken. A panel of seven cytokines/chemokines were measured by multiplex immunoassay in MLF. mRNA for intercellular cell adhesion molecule-1 (ICAM-1) was quantified from nasal epithelial curettage samples taken before and after challenge.

Results

Topical nasal LPS was well tolerated, causing no symptoms and no visible changes to the nasal mucosa. LPS induced dose-related increases in MLF levels of IL-1β, IL-6, CXCL8 (IL-8) and CCL3 (MIP-1α) (AUC at 0.5 to 10h, compared to placebo, p<0.05 at 30 and 100μg LPS). At 100μg LPS, IL-10, IFN-α and TNF-α were also increased (p<0.05). Dose-related changes in mucosal ICAM-1 mRNA were also seen after challenge, and neutrophils appeared to peak in MLF at 8h. However, 2 subjects with high baseline cytokine levels showed prominent cytokine and chemokine responses to relatively low LPS doses (10μg and 30μg LPS).

Conclusions

Topical nasal LPS causes dose-dependent increases in cytokines, chemokines, mRNA and cells. However, responsiveness can show unpredictable variations, possibly because baseline innate tone is affected by environmental factors. We believe that this new technique will have wide application in the study of the innate immune responses of the respiratory mucosa.

Key Messages

Ultrapure LPS was used as innate immune stimulus in a human nasal challenge model, with serial sampling of nasal mucosal lining fluid (MLF) by nasosorption using a synthetic absorptive matrix (SAM), and nasal curettage of mucosal cells. A dose response could be demonstrated in terms of levels of IL-1β, IL-6, CXCL8 and CCL3 in MLF, as well as ICAM-1 mRNA in nasal curettage specimens, and levels of neutrophils in nasal lavage. Depending on higher baseline levels of inflammation, there were occasional magnified innate inflammatory responses to LPS.

Trial Registration

Clinical Trials.gov NCT02284074