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Microscopic and molecular assessment of chlorhexidine tolerance mechanisms in Delftia acidovorans biofilms2016 March 1900 (has links)
One of the most concerning characteristics of microbial biofilms is that of increased resistance to antimicrobial agents such as the commonly used biocide chlorhexidine (CHX). This can have huge impact on clinical, household and environmental settings. This is particularly alarming when it involves opportunistic pathogenic environmental organisms such as Delftia acidovorans as routine mitigation practices may fail to be effective.
This thesis examines tolerance mechanisms of D. acidovorans biofilms exposed to CHX at inhibitory and sub-inhibitory concentrations. To achieve the study goals and objectives, a CHX-tolerant D. acidovorans strain (WT15), (Minimum Inhibitory Concentration; MIC-15 μg ml-1) was compared to a CHX-sensitive strain (MT51, MIC-1 μg ml-1) that was obtained by mutating the wild type strain using transposon mutagenesis. Specific morphological, structural and chemical compositional differences between the CHX-treated and untreated biofilms of wild type and mutant strains were documented using microscopic techniques including confocal laser scanning microscopy (CLSM), scanning transmission x-ray microscopy (STXM), transmission electron microscopy (TEM) and infrared (IR) spectroscopy. Molecular level changes between biofilms formed by these two strains due to CHX treatment were compared using whole-cell proteomic analysis (determined using differential in-gel electrophoresis, or DIGE) along with fatty acid methyl ester (FAME) analysis.
The gene disrupted by transposon insertion that led to increased susceptibility to CHX in the mutant strain was identified as tolQ. CLSM revealed differences in biofilm architecture and thickness between the biofilms formed by strains WT15 and MT51. STXM analyses showed that WT15 biofilms contained two morpho-chemical cell variants; whereas, only one type was detected in MT51 biofilms. STXM and IR spectral analyses revealed that CHX-susceptible MT51 cells accumulated the highest levels of CHX, an observation supported by TEM wherein prominent changes in the cell envelope of CHX-susceptible MT51 cells were observed. DIGE analysis demonstrated that numerous changes in protein abundance occurred in biofilm cells following CHX exposure and that most of these proteins were associated with amino acid and lipid biosynthesis, protein translation, energy metabolism and stress-related functions.
Overall, these studies indicate the probable role of the cell membrane and TolQ protein in CHX tolerance in D. acidovorans biofilms, in association with various proteins that are differentially-expressed.
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The Effects Of Environmental Pollutants On Adipogenesis In The 3T3-L1 ModelWang, Jing 17 December 2015 (has links)
Humans are continuously exposed to mixtures of environmental pollutants. Polycyclic aromatic hydrocarbons (PAHs), such as 2-naphthol, and heavy metals, such as lead, are some of these pollutants. Results from epidemiological studies show associations between exposure to 2-naphthol, exposure to lead, and obesity. However, the individual and combined effects of 2-naphthol and lead on fat cell development (adipogenesis) have not been directly characterized in a biological system. In this study, we evaluated the effects of 2-naphthol and/or lead on adipogenesis using mouse 3T3-L1 cells.
Cells were exposed to different doses of 2-naphthol and/or lead. Induced terminal differentiation was evaluated by cell morphology, lipid production, and mRNA expression of marker genes characteristic of either early adipocyte differentiation: CCAAT-enhancer-binding protein β (C/EBPβ), insulin receptor substrate 2 (IRS2), and sterol responsive element binding protein 1 c (SREBP1c); or terminal differentiation: C/EBPα, peroxisome proliferator-activated receptor-γ (PPARγ), and fatty acid binding protein 4 (aP2). Production of antimicrobial peptide cathelicidin (Camp), which is produced by differentiating adipocytes and modulates inflammation and immunity, was also evaluated.
Cell morphology changes and increased lipid accumulation indicated that, individually, 2-naphthol and lead induced 3T3-L1 differentiation; however, the highest dose of lead (10 μM) showed the lowest induction level. During terminal differentiation, 2-naphthol and low doses of lead increased C/EBPα, PPARγ, and aP2 expression, whereas 10 μM lead suppressed PPARγ and aP2. During early differentiation, 2-naphthol stimulated C/EBPβ, IRS2, and SREBP1c expression, while lead upregulated C/EBPα and aP2. The 2-naphthol/10 μM lead mixture induced a counterbalancing effect on 3T3-L1 adipogenesis, where 10 μM lead suppressed 2-naphthol-induced adipogenesis. Moreover, 2-naphthol elevated Camp expression in a dose-dependent manner, whereas lead slightly increased Camp at lower doses but suppressed it at 10 μM. The 2-naphthol/10 μM lead mixture showed no effect on Camp expression.
In conclusion, 2-naphthol and low lead doses accelerate adipocyte differentiation and Camp production in 3T3-L1 cells; however, high doses of lead attenuate the induction. This effect of lead at high dose counterbalances the upregulation of adipocyte differentiation and Camp production by 2-naphthol. Together, these findings indicate that 2-naphthol and lead play potential roles in the development of inflammation and obesity.
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Study on lipid droplet dynamics in live cells and fluidity changes in model bacterial membranes using optical microscopy techniquesWong, Christine Shiang Yee January 2014 (has links)
In this thesis optical microscopy techniques are used to consider aspects of viral and bacterial infections. In part 1, the physical effects of cytomegalovirus on lipid droplet dynamics in live cells are studied; in part 2, the effects of an antimicrobial peptide on the fluidity of model bacterial membranes are studied. The optical microscopy techniques used to study the effects of murine-cytomegalovirus (mCMV) on lipid droplets in live NIH/3T3 fibroblast cells in real-time are coherent anti- Stokes Raman scattering (CARS), two-photon fluorescence (TPF) and differential interference contrast (DIC) microscopies. Using a multimodal CARS and TPF imaging system, the infection process was monitored by imaging the TPF signal caused by a green fluorescent protein (GFP)-expressing strain of mCMV, where the amount of TPF detected allowed distinct stages of infection to be identified. Meanwhile, changes to lipid droplet configuration were observed using CARS microscopy. Quantitative analysis of lipid droplet numbers and size distributions were obtained from live cells, which showed significant perturbations as the infection progressed. The CARS and TPF images were acquired simultaneously and the experimental design allowed incorporation of an environmental control chamber to maintain cell viability. Photodamage to the live cell population was also assessed, which indicated that alternative imaging methods must be adopted to study a single cell over longer periods of time. To this end, DIC microscopy was used to study the lipid droplet dynamics, allowing lipid droplet motion to be tracked during infection. In this way, the effects of viral infection on the mobility and arrangement of the lipid droplets were analysed and quantified. It was found that the diffusion coefficient of the lipid droplets undergoing diffusive motion increased, and the droplets undergoing directed motion tended to move at greater speeds as the infection progressed. In addition, the droplets were found to accumulate and cluster in infected cells. The second part of this thesis presents a study on the effects of an antimicrobial peptide on model bacterial membranes. Giant unilamellar vesicles (GUVs) were produced as a simple model of E. Coli membrane using a 3:1 mixture of DPPC and POPG lipids. Incorporating Laurdan fluorescent dye into the lipid membrane of the GUVs allowed the membrane fluidity to be probed and visualised using TPF microscopy, whereby the fluidity was quantified by determining the general polarization (GP) values. Studying GUVs comprising single lipid and mixed lipid compositions over a temperature range from 25 C to 55 C enabled the lipid phase bands to be identified on the basis of GP value as gel phase and liquid crystalline phase. As such, the changes in lipid phase as a result of interaction with AMP were quantified, and phase domains were identified. It was found that the amount of liquid crystalline phase domains increased significantly as a result of AMP interaction.
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Amphiphilic Peptide Interactions with Complex Biological Membranes : Effect of peptide properties on antimicrobial and anti-inflammatory effectsSingh, Shalini January 2016 (has links)
With increasing problem of resistance development in bacteria against conventional antibiotics, as well as problems associated with diseases either triggered or enhanced by infection, there is an urgent need to identify new types of effective therapeutics for the treatment of infectious diseases and its consequences. Antimicrobial and anti-inflammatory peptides have attracted considerable interest as potential new antibiotics in this context. While antimicrobial function of such peptides is being increasingly understood demonstrated to be due to bacterial membrane disruption, the mechanisms of their anti-inflammatory function are poorly understood. Since bacterial membrane component lipopolysaccharide triggers inflammation, this thesis aims at clarifying importance of lipopolysaccharide (LPS)-peptide interactions while investigating possible modes of action of peptides exhibiting anti-inflammatory effect. Furthermore, effect of poly(ethylene)glycol (PEG)-conjugation was investigated to increase performance of such peptides. Results presented in this thesis demonstrate that peptide-induced LPS- and lipid A binding/scavenging is necessary but not sufficient criterium for anti-inflammatory effects of peptides. Furthermore, preferential binding to LPS over lipid membrane, as well as higher binding affinity to the lipid A moiety within LPS, are seen for these peptides. In addition, results demonstrate that apart from direct LPS scavenging, membrane-localized peptide-induced LPS scavenging seem to contribute partially to anti-inflammatory effect. Furthermore, fragmentation and densification of LPS aggregates, in turn dependent on the peptide secondary structure on LPS binding, as well as aromatic packing interactions, correlate to the anti-inflammatory effect, thus promoting peptide-induced packing transition in LPS aggregates as key for anti-inflammatory functionality. Thus, peptide-induced LPS aggregate disruption together with reduction of the negative charge of LPS suggests the importance of phagocytosis as an alternative to the inflammatory pathway, which needs to be further investigated. Furthermore, PEG conjugation of peptide results in strongly reduced toxicity at a cost of reduced antimicrobial activity but markedly retained anti-inflammatory effect. Taken together, the results obtained in this work have demonstrated several key issues which need to be taken into consideration in the development of effective and selective anti-inflammatory peptide therapeutics for the treatment of severe Gram-negative bacterial infections.
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Materials modification strategies to improve praseodymium-doped visible-to-ultraviolet upconversion systems for environmental applicationsCates, Stephanie 27 May 2016 (has links)
UV radiation is utilized in a number of environmental technologies, most notably for the disinfection of water, air, and surfaces through the use of UVC fluorescent lamps. Recently, our group developed a luminescent material that could emit germicidal UVC simply by irradiating it with a household fluorescent lamp, thus introducing a new type of antimicrobial surface powered by low-intensity visible light. The materials were doped with praseodymium ions (Pr3+) which have the unique capability of converting visible light to higher energy UV using an optical mechanism called upconversion. While visible-to-UV upconversion materials appeared promising for environmental application—particularly because solar irradiation could be used for their activation—their practical application was thwarted by low light conversion efficiencies. Herein we discuss the pursuit of new material forms and modifications designed to improve the efficiency of Pr3+-based upconversion systems. These enabled successful enhancement of antimicrobial activity and led to a proof of concept for upconversion-sensitized TiO2 photocatalysis. Correlations between material properties and optical behavior will be presented, followed by commentary on how these strategies might be used to further advance upconversion systems toward environmental application.
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Qualitative structure-activity relationships of the major tyrocidines, cyclic decapeptides from Bacillus aneurinolyticusSpathelf, Barbara Marianne 03 1900 (has links)
Thesis (PhD (Biochemistry))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT:
The need for alternative or supplementary treatments due to the global problem of
microbial resistance towards conventional antimicrobials may be met by the
development of novel drugs based on antimicrobial peptides. The antimicrobial peptides
of interest to this study were the tyrocidines, cyclic decapeptides produced by Bacillus
aneurinolyticus. Although these antimicrobial peptides were the first natural antibiotic
to be discovered though a systematic search for antibacterial compounds, information
regarding their bioactivity, structure-activity relationships, determinants of bioactivity
and mode of action is limited. The aim of this study was to investigate the antibacterial
and antiplasmodial activity, as well as to identify determinants of bioactivity
modulation, of the natural tyrocidine library.
The study indicated that the tyrocidines exhibit significant activity toward
Gram-positive bacteria, notably Listeria monocytogenes, and the intraerythocytic
parasite, Plasmodium falciparum. Both the antilisterial and antiplasmodial activity was
found to be highly dependent on peptide identity and self-assembly. The antilisterial
activity of the tyrocidines was shown to be associated with increased self-assembly
within a membrane-like environment, which suggested that formation of lytic
complexes within the bacterial membrane may play a crucial role in tyrocidine activity.
In contrast to the observations for antilisterial activity, the antiplasmodial activity of the
tyrocidines was shown to be associated with reduced self-assembly within a
membrane-like environment, which suggested that the antiplasmodial activity of the
tyrocidines is mediated by a mechanism other than the formation of lytic complexes
within the target cell membrane.
In addition to the influence of peptide identity and self-assembly, the bioactivity of the
tyrocidines was found to be highly sensitive to environmental conditions, notably the
presence of calcium. The antilisterial activity, as well as the mode of action, of the
tyrocidines was also found to be highly sensitive to tyrocidine-Ca2+ complexation and
the concomitant induction of higher-order structures. Tyrocidine-Ca2+ complexation
was shown to greatly enhance antilisterial activity and change the mechanism of action
from a predominantly membranolytic to an alternative, non-lytic mode of action. The results of this investigation suggest that the alternative mode of tyrocidine activity
may be related to complexation with Ca2+. It is hypothesised that such complexation
may either (1) promote tyrocidine-DNA complexation, and thus inhibition of
transcription and/or replication; or (2) interfere with Ca2+ homeostasis, and thus
influence vital cell functions.
Overall, it may be hypothesised that tyrocidine activity and mode of action is modulated
by a critical play-off between self-assembly, cation-complexation and
membrane-interaction. As these modulators of activity are highly dependent on
tyrocidine sequence/structure, the wide variety of tyrocidines found in the natural
complex may allow for optimal interaction with and activity toward a variety of
microbes. / AFRIKAANSE OPSOMMING:
Die universele probleem van mikrobiese weerstand teen konvensionele antimikrobiese
middels en die wêreld-wye noodsaaklikheid vir alternatiewe of bykomende behandeling
mag deur die ontwikkeling van nuwe middels, gebasseer op antimikrobiese peptiede,
vervul word. Die antimikrobiese peptiede van belang tot hierdie studie is die tirosidiene,
sikliese dekapeptiede wat deur Bacillus aneurinolyticus geproduseer word. Informasie
ten opsigte van die tirosidiene se bioaktiwiteit, struktuur-funksieverwantskap,
determinante van bio-aktiwiteit en meganisme van aksie was beperk, alhoewel hierdie
peptiede die eerste antimikrobiese peptiede was wat ontdek is deur ‘n sistematiese
soektog vir antimikrobiese middels. Die doelwit van hierdie studie was die ondersoek
van antibakteriële and antiplasmodiese aktiwiteit, sowel as om die determinante van
bio-aktiwiteit modulering van die natuurlike tirosidienbiblioteek te ondersoek.
Hierdie studie het getoon dat die tirosidiene merkwaardige aktiwiteit teenoor
Gram-positiewe bakterië, in besonder Listeria monocytogenes het, asook teenoor die
intra-eritrositiese parasiet, Plasmodium falciparum. Daar is bevind dat beide die antilisteriese
en antiplasmodiese aktiwiteite hoogs afhanklik is van peptiedidentiteit en
self-verpakking. Daar is gewys dat die antilisteriese aktiwiteit van die tirosidiene
geassosieer is met verhoogde self-verpakking in ’n membraanagtige omgewing, wat ’n
aanduiding is dat die vorming van litiese komplekse in die bakteriële membraan ’n
kritiese rol in tirosidienaktiwiteit speel. Kontrasterend tot die waarnemings van
antilisteriese aktiwiteit, is getoon dat die antiplasmodiese aktiwiteit van die tirosidiene
geassosieer is met verlaagde self-verpakking in ’n membraanagtige omgewing. Dis ’n
aanduiding dat die antiplasmodiese aktiwiteit van die tirosidiene gemediëer word deur
‘n ander meganisme en nie die vorming van litiese komplekse in die teikenselmembraan
nie.
Bykomend tot die invloed van peptiedidentiteit en self-verpakking, is daar bevind dat
die bioaktiwiteit van die tirosidiene hoogs sensitief is vir die omgewing, in besonder die
teenwoordigheid van kalsium. Daar is ook bevind dat die antilisteriese aktiwiteit, sowel
as die meganisme van aksie, van tirosidiene hoogs sensitief is vir tirosidien-Ca2+
kompleksvorming en die gevolglike induksie van of hoër-orde strukture. Daar is gewys dat tirosidien-Ca2+ kompleksvorming die antilisteriese aktiwiteit drasties verhoog en dat
die meganisme van aksie verander van ’n oorwegende membranolitiese meganisme na
’n alternatiewe nie-litiese meganisme van aksie.
Die resultate van hierdie ondersoek het aangedui dat die alternatiewe meganisme van
aksie van tirosidienaktiwiteit moontlik verband kan hou met kompleksvorming met
Ca2+. Die hipotese is dat sodanige kompleksvorming moontlik of (1) tirosidien-DNA
komplekvorming aanmoedig, en dus transkripsie en/of replikasie inhibibeer of (2) met
Ca2+ homeostase inmeng, en sodoende lewensnoodsaaklike selfunksies beïnvloed.
Die algemene hipotese is dat tirosidienaktiwiteit en meganisme van aksie deur ’n
kritiese spel tussen self-verpakking, katioonkompleksvorming en membraaninteraksie
gemoduleer word. Die wye verskeidenheid van tirosidiene, wat in die natuurlike
kompleks gevind word, kan moontlik toelaat vir die optimale interaksie met, en
aktiwiteit teenoor ’n verskeidenheid van mikrobes, aangesien die aktiwiteitmoduleerders hoogs afhanklik is van tirosidien struktuur/volgorde.
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Signaling and transcriptional regulation of antimicrobial peptide genes in <i>Drosophila</i> melanogasterUvell, Hanna January 2006 (has links)
<p>Insects rely solely on innate immune reactions for protection against infect-ing microbes in their environment. In <i>Drosophila</i>, one major defense mechanism is the production of a battery of antimicrobial peptides (AMPs). The expression of AMPs is primarily regulated at the level of transcription and constitutes both constitutive expression in a tissue-specific manner and inducible systemic expression in response to infection. The aim of my thesis has been to investigate the regulation of AMP gene expression at different levels. I have studied a novel cis-regulatory element, Region 1 (R1) found in the proximal promoter of all Cecropin genes in Drosophila melanogaster, as well as in other species of <i>Drosophila.</i> We found that the R1 element was important for the expression of CecropinA1 (CecA1) both in vitro and in vivo. A signaling-dependent R1-binding activity (RBA) was identified in nuclear extracts from <i>Drosophila</i> cells and flies. The molecular nature of the RBA, has despite considerable effort, not yet been identified. I also have studied the role of the JNK pathway in transcriptional regulation of AMP genes. The role of the JNK pathway in the regulation of AMP genes has long been elusive, however, in this study we showed that the pathway is directly involved in the expression of AMP genes. Analysis of cells mutant for JNK pathway components showed severely reduced AMP gene expression. Fur-thermore, over-expression of a JNK pathway-inhibitor also inhibited AMP gene expression. Lastly, I have studied transcription factors that have not previously been implicated in transcriptional regulation of AMP genes. In a yeast screen, three members of the POU family of transcription factors were identified as regulators of CecA1. Two of them, Drifter (Dfr) and POU do-main protein 1 (Pdm1) were further characterized. We showed that Dfr was able to promote AMP gene expression in the absence of infection, suggest-ing it to play a role in constitutive expression of AMP genes. Indeed, down-regulation of Dfr expression using RNAi severely reduced the constitutive expression of AMP genes in the male ejaculatory duct. We also identified an enhancer element important for Dfr-mediated expression of CecA1. Pdm1, on the other hand, was shown to be important for the systemic expression of AMP genes. In Pdm1 mutant flies, several AMP genes are systemically expressed even in the absence of infection, suggesting that Pdm1 works as a repressor of those genes. However, at least on AMP gene, AttacinA (AttA) requires Pdm1 for its expression, suggesting that Pdm1 works as an activator for this gene. Upon infection, Pdm1 was rapidly degraded, but, regenerated shortly after infection. We propose that the degradation of Pdm1 is important for the activation of the Pdm1-repressed genes and that regeneration sup-ports the expression of AttA.</p>
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Relish and the Regulation of Antimicrobial Peptides in <i>Drosophila melanogaster</i>Hedengren Olcott, Marika January 2004 (has links)
<p>The fruit fly <i>Drosophila melanogaster</i> has been a powerful model system in which to study the immune response. When microorganisms breach the mechanical barrier of the insect, phagocytosing cells and a battery of induced antimicrobial molecules rapidly attack them. These antimicrobial peptides can reach micromolar concentrations within a few hours. This immediate response is reminiscent of the mammalian innate immune response and utilizes transcription factors of the NF-κB family. </p><p>We have generated loss-of-function mutants of the NF-κB-like transcription factor Relish in order to investigate Relish's role in the <i>Drosophila</i> immune response to microbes. Relish mutant flies have a severely impaired immune response to Gram-negative (G<sup>-</sup>) bacteria and some Gram-positive (G<sup>+</sup>) bacteria and fungi and succumb to an otherwise harmless infection. The main reason for the high susceptibility to infection is that these mutant flies fail to induce the antimicrobial peptide genes. The cellular responses appear to be normal. </p><p>Relish is retained in the cytoplasm in an inactive state. We designed a set of expression plasmids to investigate the requirements for activation of Relish in a hemocyte cell line after stimulation with bacterial lipopolysaccharide. Signal-induced phosphorylation of Relish followed by endoproteolytic processing at the caspase-like target motif in the linker region released the inhibitory ankyrin-repeat (ANK) domain from the DNA binding Rel homology domain (RHD). Separation from the ANK domain allowed the RHD to move into the nucleus and initiate transcription of target genes like those that encode the inducible antimicrobial peptides, likely by binding to κB-like sites in the promoter region. </p><p>By studying the immune response of the Relish mutant flies in combination with mutants for another NF-κB-like protein, Dorsal-related immunity factor (Dif), we found that the <i>Drosophila</i> immune system can distinguish between various microbes and generate a differential response by activating the Toll/Dif and Imd/Relish pathways. The recognition of foreign microorganisms is believed to occur through pattern recognition receptors (PRRs) that have affinity for selective pathogen-associated molecular patterns (PAMPs). We found that the <i>Drosophila</i> PRRs can recognize G<sup>-</sup> bacteria as a group. Interestingly, the PRRs are specific enough to distinguish between peptidoglycans from G<sup>+</sup> bacteria such as <i>Micrococcus luteus</i> and <i>Bacillus megaterium </i>and fungal PAMPs from <i>Beauveria bassiana</i> and <i>Geotrichum candidum</i>. </p><p>This thesis also investigates the expression of the antimicrobial peptide genes, <i>Diptericin B</i> and <i>Attacin C</i>, and the putative intracellular antimicrobial peptide gene <i>Attacin D</i>, and explores a potential evolutionary link between them.</p>
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Production and pharmacological analysis of microcultures of Pelargonium sidoides DC and Pelargonium reniforme CurtisKotze, Danelle 12 1900 (has links)
Thesis (MSc (Botany and Zoology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: See full text for abstract / AFRIKAANSE OPSOMMING: sien volteks vir opsomming
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Antimicrobial peptides : structure, function and resistanceVargues, Thomas January 2009 (has links)
Higher eukaryotes produce a vast range of antimicrobial peptides (AMPs) that play important roles in their defence against microbial infection. Beta defensins are small (3-5 kDa), cationic peptides that display broad, potent antimicrobial activity against a range of microbes and also act as chemoattractants of important immunomodulatory cells. To generate highly pure peptides for structural and functional studies, we developed a method to prepare recombinant human beta defensin-2 (HBD2). The HBD2 gene was synthesised by recursive PCR with codons optimised for expression in Escherichia coli. HBD2 was expressed as an insoluble fusion to a His-tagged ketosteroid isomerase. After cleavage from the fusion with cyanogen bromide, 1H NMR spectroscopy and mass spectrometry confirmed that the oxidised HBD2 was folded and possessed the correct b-defensin disulfide bond topology. The recombinant HBD2 was active against E. coli, P. aeruginosa, S. aureus and C. albicans and was also a chemoattractant against HEK293 cells expressing the chemokine receptor CCR6. 15N-labelled HBD2 was also prepared and was highly suitable for future structural studies. Since defensins are thought to interact with bacterial membranes we also tested the recombinant HBD2 in biophysical studies (surface plasmon resonance, SPR, Biacore). We observed different binding to artificial model membranes containing either E. coli Kdo2-lipid A or phospholipids. Bacterial resistance to AMPs has been linked to the covalent modification of the outer membrane lipid A by 4-amino-4-deoxy-L-arabinose (L-Ara4N). This neutralises the charge of the LPS, thereby decreasing the electrostatic attraction of cationic peptides to the bacterial membrane. The pathogen Burkholderia cenocepacia displays extremely high resistance to AMPs and other antibiotics and the Ara4N pathway appears to be essential. To explore this further we expressed recombinant forms of two enzymes (ArnB and ArnG) from the B. cenocepacia Ara4N pathway. Purified ArnB is a pyridoxal 5’-phosphate (PLP)-dependent transaminase and we tested its ability to bind amino acid substrates. We investigated the binding of inhibitors L- and D-cycloserine to ArnB and tested their antibiotic activity against Burkholderia strains. We also studied the B. cenocepacia ArnG – a proposed membrane protein undecaprenyl-L-Ara4N flippase – and showed that the protein behaved as a dimer by non-denaturing gel analysis. The B. cenocepacia ArnG failed to complement E. coli knock-out strains encoding the equivalent flippase proteins ArnE and ArnF, suggesting that ArnG is a Burkholderia-specific protein.
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