scholarly journals Genetically Encoded Photosensitizers as Light-Triggered Antimicrobial Agents

2019 ◽  
Vol 20 (18) ◽  
pp. 4608 ◽  
Author(s):  
Fabienne Hilgers ◽  
Nora Lisa Bitzenhofer ◽  
Yannic Ackermann ◽  
Alina Burmeister ◽  
Alexander Grünberger ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Fluorescent Protein ◽  
Photophysical Properties ◽  
Type I ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Intracellular Expression ◽  
Global Concern ◽  
Green Fluorescent

Diseases caused by multi-drug resistant pathogens have become a global concern. Therefore, new approaches suitable for treating these bacteria are urgently needed. In this study, we analyzed genetically encoded photosensitizers (PS) related to the green fluorescent protein (GFP) or light-oxygen-voltage (LOV) photoreceptors for their exogenous applicability as light-triggered antimicrobial agents. Depending on their specific photophysical properties and photochemistry, these PSs can produce different toxic ROS (reactive oxygen species) such as O2•− and H2O2 via type-I, as well as 1O2 via type-II reaction in response to light. By using cell viability assays and microfluidics, we could demonstrate differences in the intracellular and extracellular phototoxicity of the applied PS. While intracellular expression and exogenous supply of GFP-related PSs resulted in a slow inactivation of E. coli and pathogenic Gram-negative and Gram-positive bacteria, illumination of LOV-based PSs such as the singlet oxygen photosensitizing protein SOPP3 resulted in a fast and homogeneous killing of these microbes. Furthermore, our data indicate that the ROS type and yield as well as the localization of the applied PS protein can strongly influence the antibacterial spectrum and efficacy. These findings open up new opportunities for photodynamic inactivation of pathogenic bacteria.

Efficacy of Antimicrobial Agents in Lettuce Leaf Processing Water for Control of Escherichia coli O157:H7

2009 ◽  
Vol 72 (7) ◽  
pp. 1392-1397 ◽  
Author(s):  
GUODONG ZHANG ◽  
LI MA ◽  
VANESSA H. PHELAN ◽  
MICHAEL P. DOYLE
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Agents ◽  
Fluorescent Protein ◽  
Organic Load ◽  
Escherichia Coli O157 ◽  
Peroxyacetic Acid ◽  
E Coli ◽  
Processing Water ◽  
Green Fluorescent ◽  
The Impact

The objectives of this research were to study transfer and control of Escherichia coli O157:H7 during simultaneous washing of inoculated and uninoculated lettuce pieces and to determine the efficacy of antimicrobial agents (peroxyacetic acid, mixed peracid, and sodium hypochlorite) on reducing the transfer of E. coli O157:H7 through processing water with or without organic load. Lettuce leaf pieces (5 by 5 cm) were inoculated with a five-strain mixture of green fluorescent protein–labeled E. coli O157:H7 at 5.6 log CFU per piece. One inoculated lettuce piece was added to five uninoculated leaves during washing. Peroxyacetic acid and mixed peracid were tested at 10, 20, and 30 ppm, and chlorine was tested at 30 and 50 ppm. No organic load (liquefied lettuce leaves) and 10% organic load in processing water were compared. Without organic load, peroxyacetic acid at 30 ppm, mixed peracid at 10, 20, and 30 ppm, and chlorine at 30 and 50 ppm all significantly reduced E. coli O157: H7 in processing water by 1.83, 1.73, 1.50, 1.83, 1.34, and 1.83 log CFU/ml, respectively, compared with washing with water alone. These antimicrobials at all concentrations tested also significantly reduced transfer of the bacteria from an inoculated leaf to uninoculated leaves in the processing water by 0.96 to 2.57 log CFU per piece. A 10% organic load in the processing water reduced efficacy of antimicrobial agents. In this contaminated water, peroxyacetic acid at 10 and 20 ppm and chlorine at 30 ppm produced effects not significantly different from those of water alone. Therefore, it is important to understand the impact of organic load when validating the effectiveness of antimicrobial treatments.

A Unified Model for Photophysical and Electro-Optical Properties of Green Fluorescent Proteins

2019 ◽  
Author(s):  
Chi-Yun Lin ◽  
Matthew Romei ◽  
Luke Oltrogge ◽  
Irimpan Mathews ◽  
Steven Boxer
Keyword(s):  
Driving Force ◽  
Fluorescent Protein ◽  
Charge Transfer Band ◽  
Photophysical Properties ◽  
Polymethine Dyes ◽  
Emission Rates ◽  
Unified Framework ◽  
Underlying Factor ◽  
Green Fluorescent ◽  
Protein Environment

Green fluorescent protein (GFPs) have become indispensable imaging and optogenetic tools. Their absorption and emission properties can be optimized for specific applications. Currently, no unified framework exists to comprehensively describe these photophysical properties, namely the absorption maxima, emission maxima, Stokes shifts, vibronic progressions, extinction coefficients, Stark tuning rates, and spontaneous emission rates, especially one that includes the effects of the protein environment. In this work, we study the correlations among these properties from systematically tuned GFP environmental mutants and chromophore variants. Correlation plots reveal monotonic trends, suggesting all these properties are governed by one underlying factor dependent on the chromophore's environment. By treating the anionic GFP chromophore as a mixed-valence compound existing as a superposition of two resonance forms, we argue that this underlying factor is defined as the difference in energy between the two forms, or the driving force, which is tuned by the environment. We then introduce a Marcus-Hush model with the bond length alternation vibrational mode, treating the GFP absorption band as an intervalence charge transfer band. This model explains all the observed strong correlations among photophysical properties; related subtopics are extensively discussed in Supporting Information. Finally, we demonstrate the model's predictive power by utilizing the additivity of the driving force. The model described here elucidates the role of the protein environment in modulating photophysical properties of the chromophore, providing insights and limitations for designing new GFPs with desired phenotypes. We argue this model should also be generally applicable to both biological and non-biological polymethine dyes.<br>

scholarly journals Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an E. coli Based Cell-Free Protein Synthesis System

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1325 ◽  
Author(s):  
Ke Yue ◽  
Tran Nam Trung ◽  
Yiyong Zhu ◽  
Ralf Kaldenhoff ◽  
Lei Kai
Keyword(s):  
Functional Analysis ◽  
Membrane Proteins ◽  
Fluorescent Protein ◽  
Water Channel ◽  
New Approach ◽  
E Coli ◽  
Straightforward Method ◽  
Lipid Environment ◽  
Green Fluorescent ◽  
Eukaryotic Organisms

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from Escherichia coli and Nicotiana tabacum using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and NtPIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

Evaluation of antibacterial, teratogenicity and antibiofilm effect of sulfated chitosans extracted from marine waste against microorganism

2021 ◽  
pp. 088391152110142
Author(s):  
Velu Gomathy ◽  
Venkatesan Manigandan ◽  
Narasimman Vignesh ◽  
Aavula Thabitha ◽  
Ramachandran Saravanan
Keyword(s):  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Inhibitory Effect ◽  
Diffusion Method ◽  
Marine Litter ◽  
Ionization Time ◽  
Gram Positive Bacteria ◽  
Mineral Components ◽  
Ft Ir

Biofilms play a key role in infectious diseases, as they may form on the surface and persist after treatment with various antimicrobial agents. The Staphylococcus aureus, Klebsiella pneumoniae, S. typhimurium, P. aeruginosa, and Escherichia coli most frequently associated with medical devices. Chitosan sulphate from marine litter (SCH-MW) was extracted and the mineral components were determined using atomic absorption spectroscopy (AAS). The degree of deacetylation (DA) of SCH was predicted 50% and 33.3% in crab and shrimp waste respectively. The elucidation of the structure of the SCH-MW was portrayed using FT-IR and 1H-NMR spectroscopy. The molecular mass of SCH-MW was determined with Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF). The teratogenicity of SCH-MW was characterized by the zebrafish embryo (ZFE) model. Antimicrobial activity of SCH-MW was tested with the agar well diffusion method; the inhibitory effect of SCH-MW on biofilm formation was assessed in 96 flat well polystyrene plates. The result revealed that a low concentration of crab-sulfated chitosan inhibited bacterial growth and significantly reduced the anti-biofilm activity of gram-negative and gram-positive bacteria relatively to shrimp. It is potentially against the biofilm formation of pathogenic bacteria.

scholarly journals The Regularity of Sustained Firing Reveals Two Populations of Slowly Adapting Touch Receptors in Mouse Hairy Skin

2010 ◽  
Vol 103 (6) ◽  
pp. 3378-3388 ◽  
Author(s):  
Scott A. Wellnitz ◽  
Daine R. Lesniak ◽  
Gregory J. Gerling ◽  
Ellen A. Lumpkin
Keyword(s):  
Fluorescent Protein ◽  
Ex Vivo ◽  
Receptive Fields ◽  
Receptor Subtypes ◽  
Type I ◽  
Genetic Dissection ◽  
Merkel Cells ◽  
Simple Method ◽  
Green Fluorescent ◽  
Nerve Recordings

Touch is initiated by diverse somatosensory afferents that innervate the skin. The ability to manipulate and classify receptor subtypes is prerequisite for elucidating sensory mechanisms. Merkel cell–neurite complexes, which distinguish shapes and textures, are experimentally tractable mammalian touch receptors that mediate slowly adapting type I (SAI) responses. The assessment of SAI function in mutant mice has been hindered because previous studies did not distinguish SAI responses from slowly adapting type II (SAII) responses, which are thought to arise from different end organs, such as Ruffini endings. Thus we sought methods to discriminate these afferent types. We developed an epidermis-up ex vivo skin–nerve chamber to record action potentials from afferents while imaging Merkel cells in intact receptive fields. Using model-based cluster analysis, we found that two types of slowly adapting receptors were readily distinguished based on the regularity of touch-evoked firing patterns. We identified these clusters as SAI (coefficient of variation = 0.78 ± 0.09) and SAII responses (0.21 ± 0.09). The identity of SAI afferents was confirmed by recording from transgenic mice with green fluorescent protein–expressing Merkel cells. SAI receptive fields always contained fluorescent Merkel cells ( n = 10), whereas SAII receptive fields lacked these cells ( n = 5). Consistent with reports from other vertebrates, mouse SAI and SAII responses arise from afferents exhibiting similar conduction velocities, receptive field sizes, mechanical thresholds, and firing rates. These results demonstrate that mice, like other vertebrates, have two classes of slowly adapting light-touch receptors, identify a simple method to distinguish these populations, and extend the utility of skin–nerve recordings for genetic dissection of touch receptor mechanisms.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

scholarly journals Vaccinia Virus F13L Protein with a Conserved Phospholipase Catalytic Motif Induces Colocalization of the B5R Envelope Glycoprotein in Post-Golgi Vesicles

2001 ◽  
Vol 75 (16) ◽  
pp. 7528-7542 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Recombinant Vaccinia Virus ◽  
Open Reading Frames ◽  
Type I ◽  
Golgi Vesicles ◽  
Infected Cells ◽  
Catalytic Motif ◽  
Green Fluorescent

ABSTRACT The wrapping of intracellular mature vaccinia virions by modifiedtrans-Golgi or endosomal cisternae to form intracellular enveloped virions is dependent on at least two viral proteins encoded by the B5R and F13L open reading frames. B5R is a type I integral membrane glycoprotein, whereas F13L is an unglycosylated, palmitylated protein with a motif that is conserved in a superfamily of phospholipid-metabolizing enzymes. Microscopic visualization of the F13L protein was achieved by fusing it to the enhanced green fluorescent protein (GFP). F13L-GFP was functional when expressed by a recombinant vaccinia virus in which it replaced the wild-type F13L gene or by transfection of uninfected cells with a plasmid vector followed by infection with an F13L deletion mutant. In uninfected or infected cells, F13L-GFP was associated with Golgi cisternae and post-Golgi vesicles containing the LAMP 2 late endosomal-lysosomal marker. Association of F13L-GFP with vesicles was dependent on an intact phospholipase catalytic motif and sites of palmitylation. The B5R protein was also associated with LAMP2-containing vesicles when F13L-GFP was coexpressed, but was largely restricted to Golgi cisternae in the absence of F13L-GFP or when the F13L moiety was mutated. We suggest that the F13L protein, like its human phospholipase D homolog, regulates vesicle formation and that this process is involved in intracellular enveloped virion membrane formation.

scholarly journals Droplet- Rather than Aerosol-Mediated Dispersion Is the Primary Mechanism of Bacterial Transmission from Contaminated Hand-Washing Sink Traps

2018 ◽  
Vol 85 (2) ◽  
Author(s):  
Shireen M. Kotay ◽  
Rodney M. Donlan ◽  
Christine Ganim ◽  
Katie Barry ◽  
Bryan E. Christensen ◽  
...  
Keyword(s):  
Patient Care ◽  
Sampling Methods ◽  
Fluorescent Protein ◽  
Model Organism ◽  
Air Sampling ◽  
Multidrug Resistant ◽  
Hand Washing ◽  
Content Type ◽  
E Coli ◽  
Green Fluorescent

ABSTRACT An alarming rise in hospital outbreaks implicating hand-washing sinks has led to widespread acknowledgment that sinks are a major reservoir of antibiotic-resistant pathogens in patient care areas. An earlier study using green fluorescent protein (GFP)-expressing Escherichia coli (GFP-E. coli) as a model organism demonstrated dispersal from drain biofilms in contaminated sinks. The present study further characterizes the dispersal of microorganisms from contaminated sinks. Replicate hand-washing sinks were inoculated with GFP-E. coli, and dispersion was measured using qualitative (settle plates) and quantitative (air sampling) methods. Dispersal caused by faucet water was captured with settle plates and air sampling methods when bacteria were present on the drain. In contrast, no dispersal was captured without or in between faucet events, amending an earlier theory that bacteria aerosolize from the P-trap and disperse. Numbers of dispersed GFP-E. coli cells diminished substantially within 30 minutes after faucet usage, suggesting that the organisms were associated with larger droplet-sized particles that are not suspended in the air for long periods. IMPORTANCE Among the possible environmental reservoirs in a patient care environment, sink drains are increasingly recognized as a potential reservoir to hospitalized patients of multidrug-resistant health care-associated pathogens. With increasing antimicrobial resistance limiting therapeutic options for patients, a better understanding of how pathogens disseminate from sink drains is urgently needed. Once this knowledge gap has decreased, interventions can be engineered to decrease or eliminate transmission from hospital sink drains to patients. The current study further defines the mechanisms of transmission for bacteria that colonize sink drains.

scholarly journals Colonization of Arabidopsis thaliana with Salmonella enterica and Enterohemorrhagic Escherichia coli O157:H7 and Competition by Enterobacter asburiae

2003 ◽  
Vol 69 (8) ◽  
pp. 4915-4926 ◽  
Author(s):  
Michael B. Cooley ◽  
William G. Miller ◽  
Robert E. Mandrell
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Enterohemorrhagic Escherichia Coli ◽  
Plant Responses ◽  
Human Pathogens ◽  
E Coli ◽  
Green Fluorescent ◽  
Enterobacter Asburiae

ABSTRACT Enteric pathogens, such as Salmonella enterica and Escherichia coli O157:H7, have been shown to contaminate fresh produce. Under appropriate conditions, these bacteria will grow on and invade the plant tissue. We have developed Arabidopsis thaliana (thale cress) as a model system with the intention of studying plant responses to human pathogens. Under sterile conditions and at 100% humidity, S. enterica serovar Newport and E. coli O157:H7 grew to 109 CFU g−1 on A. thaliana roots and to 2 × 107 CFU g−1 on shoots. Furthermore, root inoculation led to contamination of the entire plant, indicating that the pathogens are capable of moving on or within the plant in the absence of competition. Inoculation with green fluorescent protein-labeled S. enterica and E. coli O157:H7 showed invasion of the roots at lateral root junctions. Movement was eliminated and invasion decreased when nonmotile mutants of S. enterica were used. Survival of S. enterica serovar Newport and E. coli O157:H7 on soil-grown plants declined as the plants matured, but both pathogens were detectable for at least 21 days. Survival of the pathogen was reduced in unautoclaved soil and amended soil, suggesting competition from indigenous epiphytes from the soil. Enterobacter asburiae was isolated from soil-grown A. thaliana and shown to be effective at suppressing epiphytic growth of both pathogens under gnotobiotic conditions. Seed and chaff harvested from contaminated plants were occasionally contaminated. The rate of recovery of S. enterica and E. coli O157:H7 from seed varied from undetectable to 19% of the seed pools tested, depending on the method of inoculation. Seed contamination by these pathogens was undetectable in the presence of the competitor, Enterobacter asburiae. Sampling of 74 pools of chaff indicated a strong correlation between contamination of the chaff and seed (P = 0.025). This suggested that contamination of the seed occurred directly from contaminated chaff or by invasion of the flower or silique. However, contaminated seeds were not sanitized by extensive washing and chlorine treatment, indicating that some of the bacteria reside in a protected niche on the seed surface or under the seed coat.

Sign in / Sign up

Export Citation Format

Share Document