An Activatable Lanthanide Luminescent Probe for Time‐Gated Detection of Nitroreductase in Live Bacteria

Herein we report the development of a turn‐on lanthanide luminescent probe for time‐gated detection of nitroreductases (NTRs) in live bacteria. The probe is activated through NTR‐induced formation of the sensitizing carbostyril antenna and resulting energy transfer to the lanthanide center. This novel NTR‐responsive trigger is virtually non‐fluorescent in its inactivated form and features a large signal increase upon activation. We show that the probe is capable of selectively sensing NTR in lysates as well as in live bacteria of the ESKAPE family which are clinically highly relevant multiresistant pathogens responsible for the majority of hospital infections. The results suggest that our probe could be used to develop diagnostic tools for bacterial infections.     

The bifunctional role of Ag nanoparticles on bacteria--a MALDI-MS perspective

MALDI-MS is now replacing the conventional cumbersome bacterial identification techniques with its high efficiency, reliability and rapidity. One of the methodologies facilitating increased detection sensitivity of bacterial cells by MALDI-MS is through the use of nanoparticle (NP) based affinity probes. The present paper brings out the bifunctional property of Ag NPs in acting as affinity probes at ideal concentrations and bactericidal at higher concentrations. These interactions have been explained on the basis of the studies conducted with two model bacterial systems, Escherichia coli and Serratia marcescens. This work highlights the importance of NP concentration when used for affinity capture of bacteria. The results of the paper indicate a critical concentration specific to every bacteria and hence the need to identify the critical concentration of affinity probes (CCAP) for a specific NP with respect to individual bacterial species before considering its use as affinity probe for bacterial studies. The CCAP for Ag NPs was identified in the present study to be 1 mL L(-1) in the case of E. coli and was 0.5 mL L(-1) for S. marcescens. So far, MALDI-MS results were categorized as qualitative only; in this paper we have used a methodology for converting the mass data to yield quantitative results. The MYSTAT software based Tukey-Kramer multiple comparison tests proved that these threshold values (CCAP) were statistically significant. Above these concentrations Ag NPs were found to show pronounced bactericidal activity rather than affinity probe properties. The following study also demonstrates a modified protocol in that the NPs were incubated with the bacterial cultures at low concentrations, instead of the usual method of adding NPs at high concentrations to well grown bacterial cultures. A possible mechanism for the mode of action of NPs in enhancing bacterial detection sensitivity in the MALDI-MS is also proposed in this paper.     

Dual Fluorescent- and Isotopic-Labelled Self-Assembling Vancomycin for in vivo Imaging of Bacterial Infections

The increase of bacterial resistance demands rapid and accurate diagnosis of bacterial infections. Biosurface-induced supramolecular assembly for diagnosis and therapy has received little attention in detecting bacterial infections. Herein we present a dual fluorescent-nuclear probe based on self-assembly of vancomycin (Van) on Gram-positive bacteria for imaging bacterial infection. A Van- and rhodamine-modified peptide derivative (Rho-FF-Van), as the imaging agent, binds to the terminal peptide of the methicillin-resistant staphylococcus aureus (MRSA) and self-assembles to form nanoaggregates on the surface of MRSA. In an in vivo myositis model, Rho-FF-Van results in a significant increased fluorescence signal at the MRSA infected site. Radiolabeled with iodine-125, Rho-FF-Van shows strong radioactive signal in the MRSA-infected lungs in a murine model. This novel dual fluorescent and nuclear probe promises a new way for in vivo imaging of bacterial infections.     

Monitoring photodynamic therapy of localized infections by bioluminescence imaging of genetically engineered bacteria

The increasing occurrence of multi-antibiotic resistant microbes has led to the search for alternative methods of killing pathogens and treating infections. Photodynamic therapy (PDT) uses the combination of non-toxic dyes and harmless visible light to produce reactive oxygen species that can kill mammalian and microbial cells. Although the photodynamic inactivation of bacteria has been known for over a hundred years, its use to treat infections has not been much developed. This may be partly due to the difficulty of monitoring the effectiveness of PDT in animal models of infection. In order to facilitate this monitoring process, we have developed a procedure that uses bioluminescent genetically engineered bacteria and a light sensitive imaging system to allow real-time visualization of infections. When these bacteria are treated with PDT in vitro, the loss of luminescence parallels the loss of colony-forming ability. We have developed several models of infections in wounds and soft-tissue abscesses in mice that can be followed by bioluminescence imaging. The size and intensity of the infection can be sequentially monitored in a non-invasive fashion in individual mice in real-time. When photosensitizers are introduced into the infected tissue followed by illumination with red light, a light-dose dependent loss of luminescence is seen. If the bacterium is invasive, the loss of luminescence correlates with increased survival of the mice, whilst animals in control groups die of sepsis within five days. Healing of the PDT treated wounds is not impaired and may actually be improved. This approach can allow many animal models of localized infections to be accurately monitored for efficacy of treatment by PDT.     

Dual Fluorescent‐ and Isotopic‐Labelled Self‐Assembling Vancomycin for in vivo Imaging of Bacterial Infections

The increase of bacterial resistance demands rapid and accurate diagnosis of bacterial infections. Biosurface‐induced supramolecular assembly for diagnosis and therapy has received little attention in detecting bacterial infections. Herein we present a dual fluorescent‐nuclear probe based on self‐assembly of vancomycin (Van) on Gram‐positive bacteria for imaging bacterial infection. A Van‐ and rhodamine‐modified peptide derivative (Rho‐FF‐Van), as the imaging agent, binds to the terminal peptide of the methicillin‐resistant staphylococcus aureus (MRSA) and self‐assembles to form nanoaggregates on the surface of MRSA . In an in vivo myositis model, Rho‐FF‐Van results in a significant increased fluorescence signal at the MRSA infected site. Radiolabeled with iodine‐125, Rho‐FF‐Van shows strong radioactive signal in the MRSA ‐infected lungs in a murine model. This novel dual fluorescent and nuclear probe promises a new way for in vivo imaging of bacterial infections.     

A redesigned vancomycin engineered for dual D-Ala-D-ala And D-Ala-D-Lac binding exhibits potent antimicrobial activity against vancomycin-resistant bacteria

The emergence of bacteria resistant to vancomycin, often the antibiotic of last resort, poses a major health problem. Vancomycin-resistant bacteria sense a glycopeptide antibiotic challenge and remodel their cell wall precursor peptidoglycan terminus from d-Ala-d-Ala to d-Ala-d-Lac, reducing the binding of vancomycin to its target 1000-fold and accounting for the loss in antimicrobial activity. Here, we report [Ψ[C(═NH)NH]Tpg(4)]vancomycin aglycon designed to exhibit the dual binding to d-Ala-d-Ala and d-Ala-d-Lac needed to reinstate activity against vancomycin-resistant bacteria. Its binding to a model d-Ala-d-Ala ligand was found to be only 2-fold less than vancomycin aglycon and this affinity was maintained with a model d-Ala-d-Lac ligand, representing a 600-fold increase relative to vancomycin aglycon. Accurately reflecting these binding characteristics, it exhibits potent antimicrobial activity against vancomycin-resistant bacteria (MIC = 0.31 μg/mL, VanA VRE). Thus, a complementary single atom exchange in the vancomycin core structure (O → NH) to counter the single atom exchange in the cell wall precursors of resistant bacteria (NH → O) reinstates potent antimicrobial activity and charts a rational path forward for the development of antibiotics for the treatment of vancomycin-resistant bacterial infections.     

An AIEgen‐Peptide Conjugate as a Phototheranostic Agent for Phagosome‐Entrapped Bacteria

The detection and elimination of intracellular bacteria remain a major challenge. In this work, we report an aggregation‐induced emission (AIE) bioprobe that can detect bacterial infection and kill bacteria surviving inside macrophages through a dynamic process, notably specific molecular tailoring of the probe by caspase‐1 activation in infected macrophages and accumulation of the residue on phagosomes containing bacteria, leading to light‐up fluorescent signals. Moreover, the AIEgen can serve as a photosensitizer for generation of reactive oxygen species (ROS); and the average ROS indicator fluorescent signal intensity per unit area in the bacterial phagosomes is approximately 2.7‐fold higher than that in the cytoplasm. This, in turn, induces bacteria killing with high efficiency and minimal cytotoxicity towards macrophages. We envision that this specific light‐up bioprobe may provide a new approach for selective and sensitive detection and eradication of intracellular bacterial infections.     

Specific detection and effective inhibition of a single bacterial species in situ using peptide mineralized Au cluster probes

Increasingly serious microbial infections call for the development of new simpler methods for the precise diagnosis and specific inhibition of such pathogens. In this work, a peptide mineralized Au cluster probe was applied as a new simplified strategy to both recognize and inhibit a single bacteria species of Staphylococcus aureus(S. aureus) simultaneously. The probes are composed of peptides and Au clusters. Moreover, the peptides specifically target S. aureus cells and the Au clusters provide fluorescent imaging and have an antibacterial effect. These new probes enable the simultaneous specific detection and effective destruction S. aureus cells in situ.     

Protective effect of baker's yeast mannan against Listeria monocytogenes and Pseudomonas aeruginosa infection in mice

The neutral mannan (WNM) and the acidic mannan (WAM025) fractions from baker's yeast (Saccharomyces cerevisiae) were found to manifest significant protective effects against intraperitoneal and intravenous infections of Listeria monocytogenes and Pseudomonas aeruginosa in mice. A remarkable decrease in the number of microbial cells in spleen and liver was observed in mice inoculated with these microorganisms after administration of either mannan fraction. In order to clarify the mechanism of the protective effects, we investigated in vitro the bactericidal activity and lysosomal enzyme activities such as myeloperoxidase, acid phosphatase, and neutral protease, in Kupffer cells (KCs) from mice pretreated with either mannan fraction. KCs from mice administered with these mannan fractions showed an enhanced killing effect on these bacteria in vitro, and neutral protease activity was considered to be one of the important factors in the killing effect on both L. monocytogenes and P. aeruginosa.     

Evaluation of Relative Aptamer Binding to Campylobacter jejuni Bacteria Using Affinity Probe Capillary Electrophoresis

Abstract

A qualitative capillary electrophoresis immunoassay was developed for the first-time to evaluate aptamer binding to bacterial cells. Binding affinity of aptamers developed against a Campylobacter jejuni bacterial cell target, relative to other common food-borne pathogens was investigated and specific binding affinity was evidenced by pronounced mobility shift and peak broadening with increasing bacteria concentration for both aptamers. Little to no mobility shift was observed for food-borne pathogens, Salmonella typhirium and Escherichia coli, even when increasing concentrations 10-fold over target. These results suggest that affinity probe capillary electrophoresis could be useful for qualitative screening of aptamer candidates for bacterial cell targets.     

Evaluation of interaction forces between profilin and designed peptide probes by atomic force microscopy

We evaluated the binding affinity of peptide probes for profilin (protein) using force curve measurement techniques and atomic force microscopy (AFM). The peptide probes designed and synthesized for this investigation were H-A3GP5GP5GP5G-OH (1), H-A3GP5G-OH (2), H-A3G7-OH (3), and H-A3G-OH (4). Each peptide probe was immobilized on a cantilever tip, and the interaction force to profilin, immobilized on a mica substrate, was examined by force curve measurements. The retraction forces obtained showed a sequence-dependent affinity of the peptide probe for profilin. The retraction force for peptide probe 1 was the largest of the four probes examined, and it confirmed that peptide probe 1 has high affinity for profilin. The single molecular retraction force between peptide probe 1 and profilin was estimated to be 96 pN, as determined by Gaussian fitting to the histogram of the retraction forces.     

Wound infection kinetics probed by MALDI-MS: rapid profiling of Staphylococcus aureus in mice

Using direct matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), we were able to investigate the role of the clinically important bacteria, Staphylococcus aureus, in wound infections using mice. The infection kinetics of S. aureus at the wound site and the host immune response has been investigated using MALDI-MS. In this study, for the first time, we report the growth pattern of S. aureus infection at a wound site. Using mice wound infection models; the following study fingerprints the bacterial-host (mice) response at the wound site as a function of increasing wound infection in order to establish the infection pattern of Staphylococcus aureus in wounds. The current approach is extremely simple, rapid, highly selective, sensitive and established MALDI-MS as a versatile tool for detecting bacteria in clinical samples, such as those collected from wound sites.     

An Epitope‐Specific Respiratory Syncytial Virus Vaccine Based on an Antibody Scaffold

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in children. We have generated an epitope‐specific RSV vaccine by grafting a neutralizing epitope (F‐epitope) in its native conformation into an immunoglobulin scaffold. The resulting antibody fusion exhibited strong binding affinity to Motavizumab, an RSV neutralizing antibody, and effectively induced potent neutralizing antibodies in mice. This work illustrates the potential of the immunoglobulin molecule as a scaffold to present conformationally constrained B‐cell epitopes.     

High performance liquid chromatography coupled on-line to capillary electrophoresis with laser-induced fluorescence detection for detecting inhibitors of Src homology 2 domain-phosphopeptide binding in mixtures

Reversed-phase HPLC was coupled on-line to a rapid, competitive affinity probe capillary electrophoresis (APCE) assay to screen mixtures for compounds that inhibit protein-ligand interactions. The Fyn Src homology 2 (SH2) domain and its phosphopeptide binding partner were used as a model interaction for demonstration of this technique. In the method, mixtures containing possible inhibitors of binding were separated by HPLC at a flow rate of 0.3 mL/min. A small portion of effluent was directed to a fluidic tee where it was mixed on-line with Fyn SH2 domain and a fluorescent phosphopeptide ("affinity probe") known to bind selectively to Fyn SH2 domain. Electropherograms of the reaction mixture were collected on-line at approximately 6s intervals using a flow-gated interface to control injections onto the capillary electrophoresis with laser-induced fluorescence system. The resulting electropherograms contained two peaks, one corresponding to the free affinity probe and the other a complex of the affinity probe and Fyn SH2 domain. Compounds that bound the protein were detected as a decrease in the peak height of the complex and an increase in the peak height of affinity probe with relative standard deviations of <5%. The assay was shown to resolve multiple peptidergic inhibitors and selectively detect them within a complex mixture of peptides. Signals were dependent upon both concentration of active peptide and its potency in binding inhibition. Detection limits were in the range of 2-11 microM depending upon the peptide. Common organic solvents used in HPLC were shown to have minimal effect in the on-line measurement up to approximately 60% in the mobile phase.     

Membrane Disruption and Enhanced Inhibition of Cell‐Wall Biosynthesis: A Synergistic Approach to Tackle Vancomycin‐Resistant Bacteria

Resistance to glycopeptide antibiotics, the drugs of choice for life‐threatening bacterial infections, is on the rise. In order to counter the threat of glycopeptide‐resistant bacteria, we report development of a new class of semi‐synthetic glycopeptide antibiotics, which not only target the bacterial membrane but also display enhanced inhibition of cell‐wall biosynthesis through increased binding affinity to their target peptides. The combined effect of these two mechanisms resulted in improved in vitro activity of two to three orders of magnitude over vancomycin and no propensity to trigger drug resistance in bacteria. In murine model of kidney infection, the optimized compound was able to bring bacterial burden down by about 6 logs at 12 mg kg^?1 with no observed toxicity. The results furnished in this report emphasize the potential of this class of compounds as future antibiotics for drug‐resistant Gram‐positive infections.     

Single drop microextraction using silver nanoparticles as electrostatic probes for peptide analysis in atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry and comparison with gold electrostatic probes and silver hydrophobic probes

Single drop microextraction using tetraalkylammonium bromide coated silver nanoparticles (SDME-AgNPs) prepared in toluene has been successfully applied as electrostatic affinity probes to preconcentrate peptide mixtures in biological samples prior to atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry (AP-MALDI-MS) analysis. This approach is based on the isoelectric point (pI) of peptides and surface charge of AgNPs. Using the SDME-AgNPs technique, from a peptide mixture, Met- and Leu-enkephalins (Met-enk and Leu-enk) were extracted into a droplet of toluene containing AgNPs, but not the neutral peptides (gramicidins). The best peptide extraction efficiency for SDME-AgNPs was observed with the optimized parameters: extraction time 2 min, sample agitation rate 240 rpm, and sample pH 7. The limits of detection (LODs) of the SDME-AgNPs/AP-MALDI-MS technique for Met-enk and Leu-enk peptides were 160 and 210 nM, respectively. Furthermore, the application of the technique has been shown for the analysis of peptides from a sample containing high matrix interferences such as 1% Triton X-100 and 6 M urea. Finally, this approach has been compared with the SDME-AuNPs technique and the results have clearly revealed that the SDME-AgNP affinity probe exhibits higher affinity to extract the sulfur-bearing peptide (Met-enk). We also compared this electrostatic affinity probe of AgNPs with the previously demonstrated hydrophobic affinity probe of AgNPs and found that the electrostatic probe can greatly reduce the extraction time from 1.5 h to 2 min. This is due to the fact that electrostatic attraction forces are much stronger than the hydrophobic attraction forces. Therefore, we concluded that the electrostatic affinity probe based on SDME-AgNPs coupled with AP-MALDI-MS is a high-throughput technique for the analysis of low-abundance peptides from biological samples containing complex matrices. Copyright (c) 2008 John Wiley & Sons, Ltd.     

高温非平衡条件下氟原子亲电动力学的激波管研究

To study electron affinity kinetics, a shock tube method was applied, in which the test gas was ionized by a reflected shock wave and subsequently quenched by a strong rarefaction wave. As the quenching speed of 106 K/s was reached, a nonequilibrium ionization recombination process occurred, which was dominated by ion recombination with electrons. A Langmuir electrostatic probe was used to monitor variation in the ion number density at the reflection shock region. The working state of the probe was analyzed, and a correction was introduced for reduction of the probe current due to elastic scattering in the probe sheath. The three body electron affinity rate coefficient of the fluorine atom over the temperature range 1200 to 2200 K in an ambiance of argon gas was directly determined. The temperature dependence of electron affinity rate coefficient was discussed.     

A comparative study on the mode of interaction of different nanoparticles during MALDI‐MS of bacterial cells

We propose the benefits of preincubation during nanoparticle‐assisted bacterial analysis, where the bacteria are grown along with the nanoparticles. We were able to obtain a two to ten fold enhancement of bacterial signals in 3 h compared to the generally used methodology followed in previous literature. The previous literature method required a long time (18 h) to obtain such an enhancement. We probe the interactions of two bacteria, Staphylococcus aureus and Pseudomonas aeruginosa, with Ag, NiO, Pt TiO₂ and ZnO nanoparticles via transmission electron microscopy, ultraviolet spectroscopy and matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Based on these results, we propose a mechanism for interaction of these five nanoparticles with bacteria. Two mechanisms were observed for the interactions: (1) Mechanism A is proposed for the Pt and NiO NPs which functioned based on affinity for bacterial cells. (2) Mechanism B was proposed for the bactericidal NPs such as TiO₂, ZnO and Ag NPs. The results indicate that the success of the unmodified NPs in MALDI‐MS bacterial studies lies in following the ideal protocol for incubation at the ideal concentrations. Copyright © 2013 John Wiley & Sons, Ltd.     

Detection of Staphylococcus aureus cell walls by enzyme-linked immunoassay using antibodies prepared from a semi-synthetic peptidoglycan precursor

The peptidoglycan layer of Staphylococcus aureus contains a (Gly)(5) cross-link which is not found in other bacteria, and which could be used to develop a specific immunoassay for detection of S. aureus in MRSA infections. A semi-synthetic route was used to prepare the S. aureus peptidoglycan precursor UDPMurNAc-L-Ala-γ-D-Glu-L-Lys(Gly)(5)-D-Ala-D-Ala, which was covalently attached to carrier protein bovine serum albumin via the UDP nucleotide. Serum raised using this antigen showed specificity for chemically immobilised peptidoglycan monomer containing (Gly)(5), using an ELISA immunoassay. ELISA assays using 0.1 or 1.0 μg samples of cell walls prepared from two MRSA strains and one penicillin-sensitive S. aureus strain, and from three other bacteria, showed the highest response against cell walls containing (Gly)(5), with a particularly high response against cell walls from one MRSA strain. Competition assays to investigate antibody selectivity demonstrated that the antibody response could be most effectively antagonised using ligands containing (Gly)(5). These data demonstrate that it is possible to generate antibodies with high affinity and selectivity for the (Gly)(5) containing monomer in S. aureus peptidoglycan, that could be used to develop an immunoassay for S. aureus.