scholarly journals Fluorescent Ion Efflux Screening Assay for Determining Membrane-Active Peptides

2017 ◽  
Vol 70 (2) ◽  
pp. 220
Author(s):  
Neil M. O'Brien-Simpson ◽  
Wenyi Li ◽  
Namfon Pantarat ◽  
Mohammed Akhter Hossain ◽  
Frances Separovic ◽  
...  
Keyword(s):  
Inhibitory Concentration ◽  
Solid Phase ◽  
Bacterial Species ◽  
Membrane Integrity ◽  
Membrane Model ◽  
Bacterial Membrane ◽  
Membrane Composition ◽  
Screening Assay ◽  
Ion Efflux ◽  
Membrane Interactive

A major global health threat is the emergence of antibiotic-resistant microbes. Coupled with a lack of development of modified antibiotics, there is a need to develop new antimicrobial molecules and screening assays for them. In this study, we provide proof of concept that a large unilamellar vesicle (LUV) method used to study chloride ion efflux facilitated by ionophores and surfactant-like molecules that disrupt membrane integrity can be adapted to identify membrane-interactive antimicrobial peptides (AMPs) and to screen relative activity of AMPs. Lucigenin was encapsulated in LUVs in the presence of Cl– ion (NaCl), which quenches fluorescence, and then incubated with AMPs in 100 mM NaNO3 buffer. Upon AMP membrane interaction or disruption, the Cl– ion is exchanged with the NO3– ion, and the resultant lucigenin fluorescence is indicative of relative AMP activity. Seven AMPs were synthesized by solid-phase peptide chemistry and incubated with LUVs of different phospholipid compositions. Each AMP resulted in lucigenin fluorescence, which was dose dependent, and the relative fluorescence correlated with the minimum inhibitory concentration and minimum bactericidal concentration values for the corresponding peptide. Furthermore, using mammalian model phospholipid LUVs, lucigenin-induced fluorescence also correlated with the AMP cytotoxicity half-maximal inhibitory concentration values. The proline-rich AMP, Chex1-Arg20, which is non-lytic but interacts with the bacterial membrane resulted in lucigenin fluorescence of bacterial membrane model LUVs but not of mammalian membrane model LUVs. The fluorescent ion efflux assay developed here should have applicability for most AMPs and could be tailored to target particular bacterial species membrane composition, potentially leading to the identification of novel membrane-interactive AMPs. The rapid high-throughput method also allows for screening of relative AMP activity and toxicity before biological testing.

scholarly journals HJH-1, a Broad-Spectrum Antimicrobial Activity and Low Cytotoxicity Antimicrobial Peptide

Molecules ◽  
2018 ◽  
Vol 23 (8) ◽  
pp. 2026 ◽  
Author(s):  
Qing Wang ◽  
Yanzhao Xu ◽  
Mengmeng Dong ◽  
Bolin Hang ◽  
Yawei Sun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Broad Spectrum ◽  
Ph Value ◽  
Bacterial Species ◽  
Membrane Integrity ◽  
Resistant Bacteria ◽  
Bacterial Membrane ◽  
Α Subunit ◽  
Promising Alternative

With the overuse of antibiotics, multidrug-resistant bacteria pose a significant threat to human health. Antimicrobial peptides (AMPs) are a promising alternative to conventional antibiotics. This study examines the antimicrobial and membrane activity of HJH-1, a cationic peptide derived from the hemoglobin α-subunit of bovine erythrocytes P3. HJH-1 shows potent antimicrobial activity against different bacterial species associated with infection and causes weaker hemolysis of erythrocytes, at least five times the minimum inhibitory concentration (MIC). HJH-1 has good stability to tolerance temperature, pH value, and ionic strength. The anionic membrane potential probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] and propidium iodide are used as indicators of membrane integrity. In the presence of HJH-1 (1× MIC), Escherichia coli membranes rapidly depolarise, whereas red blood cells show gradual hyperpolarisation. Scanning electron microscopy and transmission electron micrographs show that HJH-1 (1× MIC) damaged the membranes of Escherichia coli, Staphylococcus aureus, and Candida albicans. In conclusion, HJH-1 damages the integrity of the bacterial membrane, preventing the growth of bacteria. HJH-1 has broad-spectrum antibacterial activity, and these activities are performed by changing the normal cell transmembrane potential and disrupting the integrity of the bacterial membrane.

scholarly journals Microbial Photoinactivation by Visible Light Results in Limited Loss of Membrane Integrity

Antibiotics ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 341
Author(s):  
Katharina Hoenes ◽  
Richard Bauer ◽  
Barbara Spellerberg ◽  
Martin Hessling
Keyword(s):  
Visible Light ◽  
Pseudomonas Fluorescens ◽  
Bacterial Cell ◽  
Bacterial Species ◽  
Membrane Integrity ◽  
Cell Lysis ◽  
Microbial Inactivation ◽  
Bacterial Cells ◽  
Resistance Development ◽  
Transmission Electron

Interest in visible light irradiation as a microbial inactivation method has widely increased due to multiple possible applications. Resistance development is considered unlikely, because of the multi-target mechanism, based on the induction of reactive oxygen species by wavelength specific photosensitizers. However, the affected targets are still not completely identified. We investigated membrane integrity with the fluorescence staining kit LIVE/DEAD® BacLight™ on a Gram positive and a Gram negative bacterial species, irradiating Staphylococcus carnosus and Pseudomonas fluorescens with 405 nm and 450 nm. To exclude the generation of viable but nonculturable (VBNC) bacterial cells, we applied an ATP test, measuring the loss of vitality. Pronounced uptake of propidium iodide was only observed in Pseudomonas fluorescens at 405 nm. Transmission electron micrographs revealed no obvious differences between irradiated samples and controls, especially no indication of an increased bacterial cell lysis could be observed. Based on our results and previous literature, we suggest that visible light photoinactivation does not lead to rapid bacterial cell lysis or disruption. However, functional loss of membrane integrity due to depolarization or inactivation of membrane proteins may occur. Decomposition of the bacterial envelope following cell death might be responsible for observations of intracellular component leakage.

Comparative analysis of the transferrin and lactoferrin binding proteins in the family Neisseriaceae

1989 ◽  
Vol 35 (3) ◽  
pp. 409-415 ◽  
Author(s):  
Anthony B. Schryvers ◽  
B. Craig Lee
Keyword(s):  
Binding Proteins ◽  
Solid Phase ◽  
Bacterial Species ◽  
Binding Activity ◽  
Intact Cells ◽  
Human Transferrin ◽  
Affinity Isolation ◽  
Lactoferrin Receptor ◽  
Solid Phase Binding ◽  
Branhamella Catarrhalis

Intact cells of several bacterial species were tested for their ability to bind human transferrin and lactoferrin by a solid-phase binding assay using horseradish peroxidase conjugated transferrin and lactoferrin. The ability to bind lactoferrin was detected in all isolates of Neisseria and Branhamella catarrhalis but not in isolates of Escherichia coli or Pseudomonas aeruginosa. Transferrin-binding activity was similarly detected in most isolates of Neisseria and Branhamella but not in E. coli or P. aeruginosa. The expression of transferrin- and lactoferrin-binding activity was induced by addition of ethylenediamine di-o-phenylacetic acid and reversed by excess FeCl3, indicating regulation by the level of available iron in the medium. The transferrin receptor was specific for human transferrin and the lactoferrin receptor had a high degree of specificity for human lactoferrin in all species tested. The transferrin- and lactoferrin-binding proteins were identified after affinity isolation using biotinylated human transferrin or lactoferrin and streptavidin–agarose. The lactoferrin-binding protein was identified as a 105-kilodalton protein in all species tested. Affinity isolation with biotinylated transferrin yielded two or more proteins in all species tested. A high molecular mass protein was observed in all isolates, and was of similar size (approximately 98 kilodaltons) in all species of Neisseria but was larger (105 kilodaltons) in B. catarrhalis.Key words: iron, Neisseria, transferrin, lactoferrin, receptor.

Toxicity of the First Ten MEIC Chemicals to Bacteria

1993 ◽  
Vol 21 (2) ◽  
pp. 151-155
Author(s):  
Gustaw Kerszman
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Linear Regression ◽  
Minimal Inhibitory Concentration ◽  
Blood Concentration ◽  
Inhibitory Concentration ◽  
Human Lymphocytes ◽  
Bacterial Species ◽  
E Coli ◽  
Mild Toxicity

The toxicity of the first ten MEIC chemicals to Escherichia coli and Bacillus subtilis was examined. Nine of the chemicals were toxic to the bacteria, with the minimal inhibitory concentration (MIC) ranging from 10-3 to 4.4M. The sensitivities of both organisms were similar, but the effect on E. coli was often bactericidal, while it was bacteriostatic for B. subtilis. Digoxin was not detectably toxic to either bacterial species. Amitriptyline and FeSO4 were relatively less toxic to the bacteria than to human cells. For seven chemicals, a highly significant linear regression was established between log MIC in bacteria and log of blood concentration, giving lethal and moderate/mild toxicity in humans, as well as with toxicity to human lymphocytes.

scholarly journals Polyunsaturated Fatty Acids Mediated Regulation of Membrane Biochemistry and Tumor Cell Membrane Integrity

Membranes ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 479
Author(s):  
Souvik Mukerjee ◽  
Abdulaziz S. Saeedan ◽  
Mohd. Nazam Ansari ◽  
Manjari Singh
Keyword(s):  
Fatty Acids ◽  
Polyunsaturated Fatty Acids ◽  
Human Health ◽  
Biological Membrane ◽  
Membrane Integrity ◽  
Dietary Fatty Acids ◽  
Body System ◽  
Omega 3 ◽  
Membrane Composition ◽  
Tumor Cell Membrane

Particular dramatic macromolecule proteins are responsible for various cellular events in our body system. Lipids have recently recognized a lot more attention of scientists for understanding the relationship between lipid and cellular function and human health However, a biological membrane is formed with a lipid bilayer, which is called a P–L–P design. Our body system is balanced through various communicative signaling pathways derived from biological membrane proteins and lipids. In the case of any fatal disease such as cancer, the biological membrane compositions are altered. To repair the biological membrane composition and prevent cancer, dietary fatty acids, such as omega-3 polyunsaturated fatty acids, are essential in human health but are not directly synthesized in our body system. In this review, we will discuss the alteration of the biological membrane composition in breast cancer. We will highlight the role of dietary fatty acids in altering cellular composition in the P–L–P bilayer. We will also address the importance of omega-3 polyunsaturated fatty acids to regulate the membrane fluidity of cancer cells.

scholarly journals Characterization of Epigallocatechin-Gallate-Grafted Chitosan Nanoparticles and Evaluation of Their Antibacterial and Antioxidant Potential

Polymers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1375
Author(s):  
María J. Moreno-Vásquez ◽  
Maribel Plascencia-Jatomea ◽  
Saúl Sánchez-Valdes ◽  
Judith C. Tanori-Córdova ◽  
Francisco J. Castillo-Yañez ◽  
...  
Keyword(s):  
Biomedical Applications ◽  
Food Packaging ◽  
Inhibitory Concentration ◽  
Antioxidant Activities ◽  
Bacterial Species ◽  
Chitosan Nanoparticles ◽  
Epigallocatechin Gallate ◽  
Antioxidant Power ◽  
Ferric Reducing Antioxidant Power

Nanoparticles based on chitosan modified with epigallocatechin gallate (EGCG) were synthetized by nanoprecipitation (EGCG-g-chitosan-P). Chitosan was modified by free-radical-induced grafting, which was verified by Fourier transform infrared (FTIR). Furthermore, the morphology, particle size, polydispersity index, and zeta potential of the nanoparticles were investigated. The grafting degree of EGCG, reactive oxygen species (ROS) production, antibacterial and antioxidant activities of EGCG-g-chitosan-P were evaluated and compared with those of pure EGCG and chitosan nanoparticles (Chitosan-P). FTIR results confirmed the modification of the chitosan with EGCG. The EGCG-g-chitosan-P showed spherical shapes and smoother surfaces than those of Chitosan-P. EGCG content of the grafted chitosan nanoparticles was 330 μg/g. Minimal inhibitory concentration (MIC) of EGCG-g-chitosan-P (15.6 μg/mL) was lower than Chitosan-P (31.2 μg/mL) and EGCG (500 μg/mL) against Pseudomonas fluorescens (p < 0.05). Additionally, EGCG-g-chitosan-P and Chitosan-P presented higher Staphylococcus aureus growth inhibition (100%) than EGCG at the lowest concentration tested. The nanoparticles produced an increase of ROS (p < 0.05) in both bacterial species assayed. Furthermore, EGCG-g-chitosan-P exhibited higher antioxidant activity than that of Chitosan-P (p < 0.05) in 2,2′-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and ferric-reducing antioxidant power assays. Based on the above results, EGCG-g-chitosan-P shows the potential for food packaging and biomedical applications.

MEMBRANE-ACTIVE METHANOLIC CRUDE EXTRACT OF THERMOTOLERANT, Aspergillus fumigatus SSH01 AND ITS MODE OF ACTION AGAINST GRAM-POSITIVE PATHOGENS

2017 ◽  
Vol 80 (1) ◽  
Author(s):  
Mohamad Khairil Radzali ◽  
Akmal Hayat Abdul Karim ◽  
Syahida Ahmad ◽  
Wan Zuhainis Saad
Keyword(s):  
Aspergillus Fumigatus ◽  
Crude Extract ◽  
Inhibitory Concentration ◽  
Minimum Bactericidal Concentration ◽  
Antibacterial Properties ◽  
Bacterial Membrane ◽  
Antibacterial Effects ◽  
Gram Positive ◽  
Bacillus Subtilis Atcc ◽  
Time Kill

This study was undertaken to investigate the antibacterial properties and the mode of actions of crude extract of Aspergillus fumigatus SSH01. Antibacterial properties was observed against Gram-positive pathogens and showed inhibition against Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538, methicillin-resistant S. aureus S547 (MRSA) and Listeria monocytogenes L10 with minimum inhibitory concentration (MIC, 0.097- 12.5 mg/ml) and minimum bactericidal concentration (MBC, 0.195 – 25 mg/ml). No surviving cells were detected after 15 h of treatment with the 2MIC of extracts for time-kill assay. Leakage of cellular contents of the treated test pathogens were identified and increased as the concentrations of the extracts increased. The study of morphological surface has shown the bacterial membrane was disrupted and caused loss of viability. This implies the antibacterial effects of A. fumigatus SSH01 extract may serve as the potential antibiotic. 

scholarly journals Screening Of Antibacterial Activity Of Nepeta Ciliaris Benth. Against Respiratory Tract Pathogens

1970 ◽  
Vol 8 (1) ◽  
pp. 100-103 ◽  
Author(s):  
Gautam Shiv Shankar ◽  
M Navneet ◽  
Kumar Sanjay ◽  
M Prabhat
Keyword(s):  
Respiratory Tract ◽  
Antimicrobial Agents ◽  
Inhibitory Concentration ◽  
Methanol Extract ◽  
Bacterial Species ◽  
Positive Control ◽  
Maximum Activity ◽  
Diffusion Method ◽  
Aerial Parts ◽  
Antibacterial Potential

The aim of present study was to evaluate the antibacterial potential of various extracts (petroleum ether, acetone, methanol and aqueous) of Nepeta ciliaris against selected respiratory tract pathogens. The extracts from the aerial parts of N. ciliaris at concentration of 200 mg/ml were screened against three gram-positive (Staphylococcus aureus MTCC 1144, Streptococcus pneumoniae MTCC 655 and Streptococcus pyogenes MTCC 442) and one gram-negative (Pseudomonas aeruginosa MTCC 2474) bacterial pathogens. The agar well diffusion method was adopted to examine antibacterial and minimum inhibitory concentration (MIC) values of most effective extracts against the susceptible bacteria. Erythromycin was used as positive control to determine the sensitivity of the strains. Out of the four bacterial species tested, S. pneumoniae was the most susceptible. The acetone extract exhibited maximum activity against all the tested microorganisms while methanol extract showed activity against P. aeruginosa. The MIC values ranged from 40 to 50 mg/ml for all the organisms. The N. ciliaris is potentially a good source of antimicrobial agents. DOI: http://dx.doi.org/10.3126/kuset.v8i1.6049 KUSET 2012; 8(1): 100-103

scholarly journals Cytotoxic Potential of the Novel Horseshoe Crab Peptide Polyphemusin III

Marine Drugs ◽  
2018 ◽  
Vol 16 (12) ◽  
pp. 466 ◽  
Author(s):  
Mariana Marggraf ◽  
Pavel Panteleev ◽  
Anna Emelianova ◽  
Maxim Sorokin ◽  
Ilia Bolosov ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Horseshoe Crab ◽  
Inhibitory Concentration ◽  
Human Cancer ◽  
Membrane Integrity ◽  
Antimicrobial Effect ◽  
Human Leukemia ◽  
Bacterial Strains ◽  
Trypan Blue Exclusion

Biological activity of the new antimicrobial peptide polyphemusin III from the horseshoe crab Limulus polyphemus was examined against bacterial strains and human cancer, transformed, and normal cell cultures. Polyphemusin III has the amino acid sequence RRGCFRVCYRGFCFQRCR and is homologous to other β-hairpin peptides from the horseshoe crab. Antimicrobial activity of the peptide was evaluated and MIC (minimal inhibitory concentration) values were determined. IC50 (half-maximal inhibitory concentration) values measured toward human cells revealed that polyphemusin III showed a potent cytotoxic activity at concentrations of <10 μM. Polyphemusin III caused fast permeabilization of the cytoplasmic membrane of human leukemia cells HL-60, which was measured with trypan blue exclusion assay and lactate dehydrogenase-release assay. Flow cytometry experiments for annexin V-FITC/ propidium iodide double staining revealed that the caspase inhibitor, Z-VAD-FMK, did not abrogate disruption of the plasma membrane by polyphemusin III. Our data suggest that polyphemusin III disrupts the plasma membrane integrity and induces cell death that is apparently not related to apoptosis. In comparison to known polyphemusins and tachyplesins, polyphemusin III demonstrates a similar or lower antimicrobial effect, but significantly higher cytotoxicity against human cancer and transformed cells in vitro.

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