N-phenyl-1-naphthylamine Research Articles

High-Throughput Fluorometric Assay For Quantifying Polysorbate In Biopharmaceutical Products Using Micelle Activated Fluorescence Probe N-Phenyl-1-Naphthylamine.

Polysorbates are among the most used surfactants in biopharmaceutical products containing proteins. Our work aims to develop a high-throughput fluorometric assay to further diversify the analytical toolbox for quantification of PSs. The assay leverages the micelle activated fluorescence signal from N-Phenyl-1-Naphthylamine (NPN). The development and optimization of assay parameters were guided by the pre-defined analytical target profile. Furthermore, NMR was used to probe the interaction between protein, PS80 and NPN in the measurement system and understand protein interference. All assay parameters including excitation and emission wavelengths, standard curve, NPN concentration, and incubation time have been optimized and adapted to a microplate format, making it compatible with automated solutions that will be pursued in the near future to drive consistency and efficiency in our workflows. The specificity, accuracy, and precision of the assay have been demonstrated through a case study. Furthermore, NMR results provided additional insight into the change of the interaction dynamics between PS80 and NPN as the protein concentration increases. The results indicate minimal interaction between the protein and PS80 at lower concentration. However, when the concentration exceeds 75mg/mL, there is a significant interaction between the protein and PS-80 micelle and monomer. A high-throughput fluorometric assay has been developed for quantification of polysorbates in biopharmaceutical samples including in-process samples, drug substance and drug product. The assay reported herein could serve as a powerful analytical tool for polysorbate quantification and control, complementing the widely used liquid chromatography with charged aerosol detection method.

Metastable Amorphous Dispersions of Hydrophobic Naphthalene Compounds Can Be Formed in Water without Stabilizing Agents via the "Ouzo Effect".

Hydrophobic molecules dissolved in water-miscible organic solvents are used in vitro for biological membrane studies and for testing of potential pharmaceuticals in high-throughput screenings. When these solutions are introduced into an aqueous environment, it is possible that metastable "ouzo-like" dispersions form from liquid-liquid phase separation. It is therefore hypothesized that when solutions of naphthalene compounds in water-miscible solvents are added to water, metastable dispersions will form. Millimolar solutions of naphthalene, N-phenyl-1-naphthylamine (NPN), 1-aminonaphthalene, 1-iodonaphthalene (INAP), 1,4-dimethoxynaphthalene, and 1-naphthol were prepared in either dimethyl sulfoxide, ethanol, or acetone at concentrations similar to those used in biological membrane studies. Each solution was diluted 10-fold in water. Particle formation was characterized by qualitative observations, dynamic light-scattering, nephelometry, and optical microscopy. It was discovered that two of the compounds tested made metastable dispersions: INAP and NPN. The initial particle sizes were ∼400 nm (radius), with turbidity ranging from 1,000 to 20,000 NTU, depending on the initial concentrations used. Fluorescence microscopy imaging showed spherical particles that do not aggregate while under observation. Slow-nucleating crystallization occurs over days, presumably from a heterogeneous nucleation process. The formation of these dispersions has implications for in vitro delivery of hydrophobic molecules to biological membranes.

Ligand Binding Properties of Odorant-Binding Protein OBP5 from Mus musculus.

Odorant-binding proteins (OBPs) are abundant soluble proteins secreted in the nasal mucus of a variety of species that are believed to be involved in the transport of odorants toward olfactory receptors. In this study, we report the functional characterization of mouse OBP5 (mOBP5). mOBP5 was recombinantly expressed as a hexahistidine-tagged protein in bacteria and purified using metal affinity chromatography. The oligomeric state and secondary structure composition of mOBP5 were investigated using gel filtration and circular dichroism spectroscopy. Fluorescent experiments revealed that mOBP5 interacts with the fluorescent probe N-phenyl naphthylamine (NPN) with micromolar affinity. Competitive binding experiments with 40 odorants indicated that mOBP5 binds a restricted number of odorants with good affinity. Isothermal titration calorimetry (ITC) confirmed that mOBP5 binds these compounds with association constants in the low micromolar range. Finally, protein homology modeling and molecular docking analysis indicated the amino acid residues of mOBP5 that determine its binding properties.

Expression, purification and characterisation of the recombinant possum lipocalin vulpeculin

BackgroundLipocalins are a large family of proteins, which possess a highly conserved eight-stranded antiparallel beta-barrel structure as distinctive trait. This family includes Major Urinary Proteins (MUPs) from rats and mouse, studied for their role in urinary protein-mediated chemosignalling. Vulpeculin has been identified as the most abundant protein in the urine of the common brushtail possum, Trichosurus vulpecula. On the basis of high similarity with other MUPS, we hypothesised that vulpeculin might have a role in possum chemosignalling and investigated its stability and binding ability. MethodsWe expressed and purified vulpeculin using an E.coli-based system and confirmed correct folding by circular dichroism (CD) spectroscopy. Thermal stability was studied by CD and binding properties were investigated using two optical probes N-phenyl-naphthylamine (NPN) and 8-anilino-1-naphthalene sulphonic acid (ANS). ResultsCD revealed a secondary structure typical of a predominantly β-sheet protein, consistent with the beta barrel structure of the lipocalin family. Vulpeculin showed a high level of thermostability, as assessed by CD, exhibiting a small shift in the secondary structure even at 95 °C. Binding assays indicated that vulpeculin cannot accommodate the NPN ligand but can bind ANS. ConclusionThe urinary secretion, high degree of sequence similarity with other lipocalins, its beta sheet structure assessed by CD and potential to bind hydrophobic ligands in the hydrophobic cavity or an external hydrophobic pocket, suggest vulpeculin may be involved in possum chemosignalling. General significanceThis work represents a first step towards the further investigation of the newly discovered lipocalin and its role in possum chemosignalling.

Colistin Interaction and Surface Changes Associated with mcr-1 Conferred Plasmid Mediated Resistance in E. coli and A. veronii Strains.

Colistin, a polycationic antimicrobial peptide, is one of the last-resort antibiotics for treating infections caused by carbapenem-resistant Gram-negative bacteria. The antibacterial activity of colistin occurs through electrostatic interaction between the polycationic peptide group of colistin and the negatively charged phosphate groups of lipid A membrane. This study investigated the interaction of colistin with the outer membrane and surface constituents of resistant and susceptible strains of Escherichia coli and Aeromonas veronii harboring mcr-1 resistance gene. Bacterial membrane and lipopolysaccharide used in this study were isolated from susceptible as well as colistin-resistant strains of E. coli and A. veronii. Interaction of colistin with the bacterial surface was studied by deoxycholate and lysozyme sensitivity test, N-phenyl-1-naphthylamine (NPN) uptake assay, Atomic force microscopy (AFM), Zeta potential measurements and 1H NMR. The binding affinity of colistin was found to be lower with outer membrane from resistant strains in comparison with the susceptible strains. Colistin exposure enhances the outer membrane permeability of the susceptible strains to deoxycholate and lysozyme. However, on the other hand, colistin dose of 256 µg/mL did not permeabilize the outer membrane of resistant bacteria. The NPN permeability in resistant strains was greater in comparison with susceptible strains. Atomic force microscopy images depicted smooth, featherless and deformed membranes in treated susceptible cells. Contrary to the above, resistant treated cells displayed surface roughness topography even at 256 µg/mL colistin concentration. Surface charge alterations were confirmed by Zeta potential measurements as a function of the growth phase. Mid-logarithmic phase susceptible strains showed a greater negative charge than resistant strains upon exposure to colistin. However, there was no statistical variation in the Zeta potential measurements between resistant and susceptible strains at the stationary phase. NMR analysis revealed line broadening in susceptible strains with increasing colistin: LPS aggregates mass ratio. Moreover, resistant strains did not show line broadening for the outer membrane, even at the highest mass ratio. The findings of this study suggest that the resistant strains of E. coli and A. veronii can block the electrostatic contact between the cationic peptide and anionic lipid A component that drives the first phase of colistin action, thereby preventing hydrophobically driven second-tier action of colistin on the outer lipopolysaccharide layer.

Influence of the Metabolic Activity of Microorganisms on Disinfection Efficiency of the Visible Light and P25 TiO2 Photocatalyst

The beneficial photocatalytic properties of UV light activated TiO2 powder are well-known and have been demonstrated with various pollutants and pathogens. However, traditionally observed photocatalytic activity of visible light activated pristine TiO2 is insignificant but there are a few studies which have reported that under some specific conditions commercially available TiO2 powder could at least partially disinfect microorganisms even under visible light. To better understand this phenomenon, in the current study we focused on bacteria response to the treatment by visible light and P25 TiO2 powder. More specifically, we analyzed the relationship between the bacteria viability, outer membrane permeability, metabolism, and its capacity to generate intracellular reactive oxygen species. During the study we assayed the viability of treated bacteria by the spread plate technique and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction method. Changes in bacterial outer membrane permeability were determined by measuring the fluorescence of N-phenyl-1-naphthylamine (NPN). To detect intracellular reactive oxygen species formation, the fluorescence of dichlorodihydrofluorescein diacetate (DCFH-DA) was assayed. Results of our study indicated that TiO2 and wide spectrum visible light irradiation damaged the integrity of the outer membrane and caused oxidative stress in the metabolizing bacteria. When favorable conditions were created, these effects added up and unexpectedly high bacterial inactivation was achieved.

Effects of lipids and surfactants on the fermentation production of echinocandin B by Aspergillus nidulans.

Echinocandin B (ECB) is a kind of lipopeptide antifungal antibiotic, as well as the key precursor of antifungal drug Anidulafungin. Its efficient bioproduction plays an important role in promoting the industrial production of Anidulafungin. In this study, methyl oleate and Tween 80 were firstly used to enhance the ECB fermentation by Aspergillus nidulans, the results showed that the ECB titre was significantly enhanced with the addition of methyl oleate and Tween 80. Among the lipids, methyl oleate was found to play a pivotal role in increasing the ECB titre to 2123 mg l-1 , which was more than five times higher than that of the control. The addition of Tween 80 in the medium resulted in ECB titre increased to 2584 mg l-1 . The scanning electron microscope (SEM) and N-phenyl-1-naphthylamine (NPN) assay indicated that Tween 80 could influence the cell membrane permeability of A. nidulans, and enhance the intracellular and extracellular substance exchange, therefore lead to the increasing of ECB titre. Methyl oleate and Tween 80 are optimal carbon sources and surfactants for efficient ECB biosynthesis respectively. Surfactant was used in ECB fermentation for the first time, which provided feasible ideas for optimizing the fermentation process of other fungi.

Loss of the lipopolysaccharide (LPS) inner core increases the electrocompetence of Escherichia coli.

Mutations that shorten the lipopolysaccharide (LPS) in Escherichia coli were found to significantly increase the number of transformants after electroporation. The loss of the LPS outer core increased the number of transformants with plasmid pAmCyan (3.3 kb) from 5.0 × 105 colony-forming units (CFU)/μg in the wild-type E. coli BW25113 to 3.3 × 107 CFU/μg in a ΔwaaG background, a 66.2-fold increase in efficiency. Truncation of the inner core improved this even further, with the ΔwaaF mutant exhibiting the best transformation efficiencies obtained, i.e., a 454.7-fold increase in the number of colonies over the wild-type strain. Similar results were obtained when a larger plasmid (pDA1; 11.3 kb) was used, with the ΔwaaF mutant once more giving the best transformation rates, i.e., a 73.7-fold increase. Subsequent tests proved that the enhanced transformabilities of these mutants were not due to a better survival or their surface charge properties, nor from preferential binding of these strains to the plasmid. Using N-phenyl-1-naphthylamine (NPN), we confirmed that the outer membranes of these mutant strains were more permeable. We also found that they leaked more ATP (3.4- and 2.0-fold higher for the ΔwaaF and ΔwaaG mutants, respectively, than wild-type E. coli BW25113), suggesting that the inner membrane stability is also reduced, helping to explain how the DNA enters these cells more easily. KEY POINTS: • LPS inner core gene knockouts increase the electrocompetence of E. coli. • No significant difference in survival, surface charge, or DNA binding was evident. • The LPS inner core mutants, however, exhibited higher outer membrane permeability. • Their inner membranes were also leaky, based on supernatant ATP concentrations.

Effect of AgWPA nanoparticles on the inhibition of Staphylococcus aureus growth in biofilms

Staphylococcus aureus (S. aureus) is known as a food-borne pathogenic bacteria that is difficult to deal with, especially in a biofilm. An appropriate and effective agent against S. aureus is urgently needed, especially due to the high pathogenicity of S. aureus within biofilms. This paper aims to synthesize Ag3PW12O40 nanoparticles (AgWPA-NPs) and investigate their antimicrobial activity and mechanism against S. aureus. N-phenyl-1-naphthylamine (NPN) uptake and membrane potential assays showed that AgWPA-NPs damaged the structure of the cell membrane. Furthermore, the results of crystal violet staining and fluorescence microscopy indicated that AgWPA-NPs were remarkably efficient at suppressing the formation of S. aureus biofilms. Furthermore, real-time PCR analysis showed that the expression of the biofilm-related genes, icaA, sarA and cidA, was downregulated. Taken together, these results suggest that AgWPA-NPs have the potential to be an anti-S. aureus biofilm agent.

Development of New Antimicrobial Agents from Cationic PG-Surfactants Containing Oligo-Lys Peptides

Peptide gemini-surfactant (PG-surfactant), a kind of lipopeptide, is composed of a short linker peptide (X) between two alkyl-chain-modified Cys residues and peripheral peptides at the N-terminal (Y) and the C-terminal (Z) sides, respectively, of the alkylated Cys residues. In this study, we developed and examined a series of PG-surfactants containing two C12 saturated alkanes and oligo-Lys, arranged at the X-, Y-, or Z-positions. To arrange oligo-Lys at the Y- or Z-positions, a repeat sequence of -Asp-Lys-Asp-Lys- was used at the X-position. All of the PG-surfactants exhibited high antimicrobial activity against both Gram-positive and -negative bacteria. In addition to high antimicrobial activity, a low hemolysis activity is prerequisite for efficient intravenous administration. Among the synthesized PG-surfactants, those having -(Lys)3- at the Y- or Z-positions, i.e. K3-DKDKC12 and DKDKC12K3, showed reasonably low hemolytic activities. This combination of high antimicrobial activity along with low hemolytic activity is an essential and unique property and has not been previously reported for the synthesized lipopeptides. Further, using scanning electron microscopy (SEM) and N-phenyl-1-naphthylamine (NPN) uptake assay we showed that the antimicrobial activity of these PG-surfactants may be attributed to membrane disruptive mechanisms. Although the PG-surfactants with low hemolytic activity could interact and localize onto red blood cell surfaces and cause slight expansion of cell morphologies, no subsequent penetration occurred. In summary, we describe here the successful development of PG-surfactants having high antibacterial and low hemolytic activity, thus providing a significant molecular platform to develop novel antimicrobial agents.

The role of the outer membrane protein gene ybjX in the pathogenicity of avian pathogenic Escherichia coli

ABSTRACT Avian pathogenic Escherichia coli (APEC) are important pathogens that cause serious economic losses to the poultry industry worldwide. The PhoP/Q system is essential for APEC pathogenicity, and its mutation leads to a decrease in ybjX gene expression, but whether ybjX regulated by PhoP/Q plays a role in pathogenicity remains unclear. The role of ybjX in APEC pathogenicity was investigated using a ybjX mutant strain constructed from APEC strain AE17 by the Red recombination method. The mutant strain displayed significantly reduced adherence to and invasion of chicken embryo fibroblast cell line (DF-1) cells. Moreover, N-phenyl-1-naphthylamine (NPN) assays revealed an increase in outer membrane permeabilization for the ybjX mutant, suggesting ybjX affected susceptibility to avian beta-defensin through this mechanism. The ybjX mutation increased susceptibility to avian beta-defensin 9 (AvBD9), and in vivo LD50 assays revealed reduced bacterial virulence in a chicken model. Taken together, these results indicated that mutation of ybjX may attenuate APEC pathogenicity, not only by affecting bacterial adhesion and invasion, but also via reducing resistance to avian beta-defensin by increasing outer membrane permeabilization of bacteria.

Antimicrobial peptides secreted by equine mesenchymal stromal cells inhibit the growth of bacteria commonly found in skin wounds

BackgroundThe prevalence of chronic skin wounds in humans is high, and treatment is often complicated by the presence of pathogenic bacteria. Therefore, safe and innovative treatments to reduce the bacterial load in cutaneous wounds are needed. Mesenchymal stromal cells (MSC) are known to provide paracrine signals that act on resident skin cells to promote wound healing, but their potential antibacterial activities are not well described. The present study was designed to examine the antibacterial properties of MSC from horses, as this animal model offers a readily translatable model for MSC therapies in humans. Specifically, we aimed to (i) evaluate the in vitro effects of equine MSC on the growth of representative gram-negative and gram-positive bacterial species commonly found in skin wounds and (ii) define the mechanisms by which MSC inhibit bacterial growth.MethodsMSC were isolated from the peripheral blood of healthy horses. Gram-negative E. coli and gram-positive S. aureus were cultured in the presence of MSC and MSC conditioned medium (CM), containing all factors secreted by MSC. Bacterial growth was measured by plating bacteria and counting viable colonies or by reading the absorbance of bacterial cultures. Bacterial membrane damage was detected by incorporation of N-phenyl-1-naphthylamine (NPN). Antimicrobial peptide (AMP) gene and protein expression by equine MSC were determined by RT-PCR and Western blot analysis, respectively. Blocking of AMP activity of MSC CM was achieved using AMP-specific antibodies.ResultsWe found that equine MSC and MSC CM inhibit the growth of E. coli and S. aureus, and that MSC CM depolarizes the cell membranes of these bacteria. In addition, we found that equine MSC CM contains AMPs, and blocking these AMPs with antibodies reduces the effects of MSC CM on bacteria.ConclusionsOur results demonstrate that equine MSC inhibit bacterial growth and secrete factors that compromise the membrane integrity of bacteria commonly found in skin wounds. We also identified four specific AMPs produced by equine MSC. The secretion of AMPs may contribute to the value of MSC as a therapy for cutaneous wounds in both horses and humans.

Enhanced toxicity to the cyanobacterium Microcystis aeruginosa by low-dosage repeated exposure to the allelochemical N-phenyl-1-naphthylamine

It has been puzzling whether and how a plant could exert a strong allelopathic inhibition to the target organisms by releasing low concentrations of allelochemicals. Plant allelochemicals have been proposed to be released continuously, however, direct evidence from specific allelochemicals is urgently required. In the present study, the toxicity of allelochemical N-phenyl-1-naphthylamine (NPN) towards the cyanobacterium Microcystis aeruginosa by two different exposure patterns was compared. One was low-dosage repeated exposure (LRE), in which 50 μg L−1 NPN was repeatedly dosed to simulate the continual release of allelochemicals, and the other one was high-dosage single exposure (HSE) as per the routine toxicity assay. The results showed a significant growth inhibition to M. aeruginosa in the LRE group, where the inhibition rate reached above 90% from day 6 to day 9. The cell-membrane damage ratio increased from 64.05% on day 5 up to 96.60% on day 9. PSII photosynthesis activity expressed as Fv/Fm, ΦPSII, NPQ and ETRmax was also thoroughly inhibited in this group. Whereas the growth and PSII photosynthesis activity of M. aeruginosa in the HSE group were inhibited initially, but recovered gradually from day 4 or 5, which was accompanied by a continuous reduction of NPN content in culture solutions. Although NPN content in the LRE group was relatively lower, it remained at a more stable level throughout the experiment. These results indicate that continual release of low-dosage allelochemicals by aquatic plants plays crucial roles in their potent inhibition against cyanobacteria. Low-dosage continual exposure pattern needs to be investigated further.

Molecular simulations of micellar aggregation of polysorbate 20 ester fractions and their interaction with N-phenyl-1-naphthylamine dye

Micellar aggregation behavior of polysorbate 20 (PS20) has generated significant interest because of the wide use of PS20 as a surfactant to minimize protein surface adsorption and mitigate protein aggregation. Thus, there is a need for better molecular understanding of what drives the biophysical behavior of PS20 in solution. We observe that a complex amphipathic PS20 molecule, which contains both hydrophobic tail and relatively large hydrophilic head, self-associates strongly within the course of a molecular dynamics simulation performed with a fully atomistic representation of the molecule and an explicit water solvent model. The in silico behavior is consistent with micellar models of PS20 in solution. The dynamics of this self-association is rather complex involving both internal reorganization of the molecule and diffusion to form stable micelle-like aggregates. The micellar aggregates of PS20 are long-lived and are formed by the balance between the large hydrophobic interactions associated with the aliphatic tail of PS20, and the steric repulsion of the hydrophilic sorbitan head structure. In the present work, molecular models of PS20 that represent naturally occurring PS20 fractions were produced and characterized in silico. The study investigated the monoester and diester fractions: PS20M, and PS20D. These fractions present differences in the strength of their hydrophobic effect, which influences the aggregation behavior. Adaptive biasing force (ABF) simulations were carried out with the PS20M and PS20D molecular constructs to calculate the free energy of their pairwise interaction. The free energy barrier for the dissociation is higher for PS20D compared with PS20M. The results show that hydrogen bonds can form when head groups are in close proximity, such as in the PS20 aggregate assembly, and the free energy of interaction can be used to predict the morphology of the micellar aggregate for the different PS20 fractions. We were also able to simulate PS20 in the presence of N-phenyl-1-naphthylamine (NPN) to study the solution behavior of the hydrophobic molecule and of the mechanism in which it is sequestered in the hydrophobic core of the PS20 micellar aggregate.

Researches on Improving Sensitivity of Luminous Bacteria Toxicity Test Using Chelating Agent

The biological toxicity test of luminous bacteria has been widely applied. However, sensitivity of this test needs to be further improved. Cell membranes are bacteria’s first barrier against outside invasions. In this research, permeability of cell membranes will be changed so as to improve toxicity detection sensitivity of vibrio qinghaiensis (Q67). During the cultivation process of Q67, the Ethylene Diamine Tetraacetic Acid (EDTA-Na2) will be added and the hydrophobic fluorescent probe N-Phenyl-1-Naphthylamine (NPN) will be used to test changes of Q67 cell permeability. In addition, the low temperature centrifuge method will be used to eliminate interferences of residual EDTA of the nutrient medium to the acute toxicity test. The microporous plate method of biological toxicity test will be used to test the acute toxicity of K2Cr2Q7, the phenol and the desmetryn on Q67 before and after the permeability is modified. The results show that: during the cultivation process of Q67, if 2.0 mmol/L EDTA-Na2 is added, 40.2% of its absorbability to NPN can be improved. What is more, normal growth and bioluminescent property of Q67 will not be affected. EC50 of the K2Cr2Q7, the phenol and the desmetryn to the modified Q67 is 4.39 mg/L, 128.75 mg/L and 145.89 mg/L respectively. Compared with those before the modification, their EC50 reduces 45.70%, 39.77% and 38.61% respectively. Thus, during the cultivation process of Q67, the addition of EDTA-Na2 can improve its permeability to poisonous substances and enhance the sensitivity of Q67 biological toxicity test.

N-phenyl-1-naphthylamine/β-cyclodextrin inclusion complex as a new fluorescent probe for rapid and visual detection of Pd2+

Inclusion complex between N-phenyl-1-naphthylamine (NPN) and β-cyclodextrin (β-CD) was studied by FT-IR, 1H and 2D NMR, XRD, FT-Raman, SEM and DSC techniques. The formation of 1:1 stoichiometric inclusion complex of NPN with β-CD was proposed based on the Nuclear magnetic resonance spectroscopy and Molecular docking study. The molecular encapsulation of host–guest inclusion complex based on simple chemosensor has high selectivity and sensitivity for the determination of Pd2+ ion. Host–guest inclusion complex as a spectroscopic probe is used for the detection of transition metal cation Pd2+. Coordination of this Pd2+ with (NPN/β-CD) inclusion complex exhibited a noticeable color change in the solution state it used for naked-eye detection.

Influence of Lipid A Acylation Pattern on Membrane Permeability and Innate Immune Stimulation

Lipid A, the hydrophobic anchor of lipopolysaccharide (LPS), is an essential component in the outer membrane of Gram-negative bacteria. It can stimulate the innate immune system via Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD2), leading to the release of inflammatory cytokines. In this study, six Escherichia coli strains which can produce lipid A with different acylation patterns were constructed; the influence of lipid A acylation pattern on the membrane permeability and innate immune stimulation has been systematically investigated. The lipid A species were isolated and identified by matrix assisted laser ionization desorption-time of flight/tandem mass spectrometry. N-Phenyl naphthylamine uptake assay and antibiotic susceptibility test showed that membrane permeability of these strains were different. The lower the number of acyl chains in lipid A, the stronger the membrane permeability. LPS purified from these strains were used to stimulate human or mouse macrophage cells, and different levels of cytokines were induced. Compared with wild type hexa-acylated LPS, penta-acylated, tetra-acylated and tri-acylated LPS induced lower levels of cytokines. These results suggest that the lipid A acylation pattern influences both the bacterial membrane permeability and innate immune stimulation. The results would be useful for redesigning the bacterial membrane structure and for developing lipid A vaccine adjuvant.

Surface changes and polymyxin interactions with a resistant strain of Klebsiella pneumoniae

This study examines the interaction of polymyxin B and colistin with the surface and outer membrane components of a susceptible and resistant strain of Klebsiella pneumoniae. The interaction between polymyxins and bacterial membrane and isolated LPS from paired wild type and polymyxin-resistant strains of K. pneumoniae were examined with N-phenyl-1-naphthylamine (NPN) uptake, fluorometric binding and thermal shift assays, lysozyme and deoxycholate sensitivity assays, and by (1)H NMR. LPS from the polymyxin-resistant strain displayed a reduced binding affinity for polymyxins B and colistin in comparison with the wild type LPS. The outer membrane NPN permeability of the resistant strain was greater compared with the susceptible strain. Polymyxin exposure enhanced the permeability of the outer membrane of the wild type strain to lysozyme and deoxycholate, whereas polymyxin concentrations up to 32 mg/ml failed to permeabilize the outer membrane of the resistant strain. Zeta potential measurements revealed that mid-logarithmic phase wild type cells exhibited a greater negative charge than the mid-logarithmic phase-resistant cells. Taken together, our findings suggest that the resistant derivative of K. pneumoniae can block the electrostatically driven first stage of polymyxin action, which thereby renders the hydrophobically driven second tier of polymyxin action on the outer membrane inconsequential.

Role of Burkholderia cenocepacia afcE and afcF genes in determining lipid-metabolism-associated phenotypes

Burkholderia cenocepacia is an opportunistic pathogen that primarily infects cystic fibrosis patients. Previously we have reported that mutations in shvR, a LysR-type transcriptional regulator, and ShvR-regulated genes BCAS0208 and BCAS0201 (designated afcE and afcF, respectively) affect colony morphotype, biofilm and pellicle formation and virulence in B. cenocepacia. In this study we investigated the role of afcE and afcF in influencing lipid-metabolism-associated phenotypes. As previously reported for K56-2ΔshvR, the Δ2afcE and afcF : : lux mutants had no antifungal activity against Fusarium and Rhizoctonia solani, suggesting that these genes are involved in synthesis of a membrane-associated antifungal lipopeptide. Strains Δ2afcE and afcF : : lux had reduced swarming motility and altered cell membrane morphology, both of which were restored to wild-type levels upon providing these genes in trans. Both K56-2ΔshvR and Δ2afcE showed increased uptake of the hydrophobic fluorescent probe N-phenylnaphthylamine (NPN), indicating altered outer membrane properties. Total lipid profiles determined by TLC revealed distinct differences in cellular lipid compositions of K56-2ΔshvR, Δ2afcE and afcF : : lux compared with K56-2. Taken together, these results indicate that afcE and afcF are involved in metabolic pathway(s) influencing lipid profiles and affect both cell surface and antifungal properties of B. cenocepacia.

Quantitative analysis of pheromone‐binding protein specificity

Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins, using β-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila odorant-binding protein that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in Escherichia coli was assessed by measuring N-phenyl-1-naphthylamine (NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 (W123) and NPN. Binding of cVA was measured from quenching of W123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between β-cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the β-cyclodextrin-cVA dissociation constant, gives the LUSH-cVA dissociation constant: ∼100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH-ligand dissociation constants were determined to be ∼200 nM for the silk moth pheromone bombykol and ∼90 nM for methyl oleate. The results indicate that the ligand-binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the Laughlin, Ha, Jones and Smith model of pheromone reception are discussed.