Journal: Applied and Environmental Microbiology

Article Title: Multifunctional Acidocin 4356 Combats Pseudomonas aeruginosa through Membrane Perturbation and Virulence Attenuation: Experimental Results Confirm Molecular Dynamics Simulation

doi: 10.1128/AEM.00367-20

Figure Lengend Snippet: Antimicrobial activity of CFS and ACD from L. acidophilus ATCC 4356. (a) Inhibitory effect of CFS on bacterial growth. Bacterial growth assays were performed by measuring the absorbance at 600 nm after 12 h of the same CFS treatment. As shown, CFS has an inhibitory effect on the growth of all four bacterial strains tested. However, there was substantially more impact against P. aeruginosa (*, P < 0.01 [Student t test]). (b) Antimicrobial activity of ACD against P. aeruginosa. The growth rate of bacteria after 12 h of treatment with different concentrations of ACD was evaluated using a fluorescence-based method (FDA, arbitrary units [AU]) and spectrometry (optical density [OD600]). (c) Evaluation of antimicrobial activity of ACD under physiological conditions. (i) Comparison of inhibition of growth by ACD at its MIC50 levels in the presence of human serum (HS), human plasma (HP), fetal bovine serum (FBS), high salt concentrations, and acidic conditions versus control. (ii) Growth inhibition of P. aeruginosa by ACD before and after treatment with eukaryotic and prokaryotic proteases. Each assay was performed with at least two independent experiments and three repetitions. *, P < 0.01 (one-way analysis of variance [ANOVA], significant difference from the control).

Article Snippet: KEYWORDS: antimicrobial peptide, acidocin 4356, Lactobacillus acidophilus ATCC 4356, Pseudomonas aeruginosa , molecular dynamics simulations, mouse infection model, confocal laser scanning microscopy, CLSM, flow cytometry

Techniques: Activity Assay, Bacteria, Fluorescence, Comparison, Inhibition, Clinical Proteomics, Control

Journal: Applied and Environmental Microbiology

Article Title: Multifunctional Acidocin 4356 Combats Pseudomonas aeruginosa through Membrane Perturbation and Virulence Attenuation: Experimental Results Confirm Molecular Dynamics Simulation

doi: 10.1128/AEM.00367-20

Figure Lengend Snippet: Effect of ACD on the production of P. aeruginosa virulence factors. (a) Pyocyanin. The production of pyocyanin was measured at 695 nm after 24 h of treatment with concentrations of ACD that did not inhibit growth. The lack of blue-green color (right tube) indicates the inhibition of pyocyanin production at an ACD concentration of 20 μg ml−1. (b) Pyoverdine. The production of pyoverdine was estimated by fluorescence spectroscopy (excitation, 400 nm; emission, 470 nm). Measurements are averages of three independent experiments. (c) Elastase. Elastase activity was measured using elastin-Congo red substrate at 495 nm. The color changes of the samples show a decrease in soluble hydrolysis products due to a decrease in elastase activity. (d) Protease. Proteolytic activity is estimated using a qualitative halo on skim-milk plates. Halo size indicates the proteolytic activity of the treated sample compared to the control. *, P < 0.01 (one-way ANOVA, significant difference from the control).

Article Snippet: KEYWORDS: antimicrobial peptide, acidocin 4356, Lactobacillus acidophilus ATCC 4356, Pseudomonas aeruginosa , molecular dynamics simulations, mouse infection model, confocal laser scanning microscopy, CLSM, flow cytometry

Techniques: Inhibition, Concentration Assay, Fluorescence, Spectroscopy, Activity Assay, Control

Journal: Applied and Environmental Microbiology

Article Title: Multifunctional Acidocin 4356 Combats Pseudomonas aeruginosa through Membrane Perturbation and Virulence Attenuation: Experimental Results Confirm Molecular Dynamics Simulation

doi: 10.1128/AEM.00367-20

Figure Lengend Snippet: Effect of ACD peptide on P. aeruginosa biofilm. Images were taken from P. aeruginosa biofilms formed in TSB plus 0.2% glucose culture medium after 72 h without treatment and after an hour of treatment. (a) SEM images. (i) Control; treatment at MIC (ii) and at 2× MIC (iii). (b) Three-dimensional CLSM images from biofilms. (i) Control; (ii) treatment at its MIC level. The experiment was repeated three times; three regions were examined each time, and a representative image is shown for each of the conditions. The live cells are green, and dead cells are seen in red. A video of peptide effects on P. aeruginosa cells in various biofilm layers is accessible in the supplemental material (https://zenodo.org/record/3706572#.XmjKgKgzbIU). Images were taken with a 40× lens objective. (c) Subsets of dead and live cell populations in biofilms. The live/dead ratio was calculated using ImageJ software using six points. *, P < 0.01 (Student t test, significant difference from the control group). (d) Determination of detachment of P. aeruginosa biofilm after 1 h of treatment with various concentrations of peptide. Biofilm mass was measured by crystal violet staining. The results were derived from an average of three independent repetitions as ± the standard deviations. *, P < 0.01 (one-way ANOVA, significant difference from the untreated control). (e) Comparison of the detachment of P. aeruginosa biofilm with bacterial killing after ACD treatment. The pictures were taken from a mature biofilm after treatment with ACD. In the fluorescent images, green and red fluorescence shows live and dead cells, respectively (scale bar, 50 μm).

Article Snippet: KEYWORDS: antimicrobial peptide, acidocin 4356, Lactobacillus acidophilus ATCC 4356, Pseudomonas aeruginosa , molecular dynamics simulations, mouse infection model, confocal laser scanning microscopy, CLSM, flow cytometry

Techniques: Control, Software, Staining, Derivative Assay, Comparison, Fluorescence

Journal: Applied and Environmental Microbiology

Article Title: Multifunctional Acidocin 4356 Combats Pseudomonas aeruginosa through Membrane Perturbation and Virulence Attenuation: Experimental Results Confirm Molecular Dynamics Simulation

doi: 10.1128/AEM.00367-20

Figure Lengend Snippet: In vivo analysis of ACD activity. (a) Schematic illustration of the design of the in vivo experiment. (i) Mice were anesthetized and then infected with intranasal instillation with 20 μl of PBS buffer containing 107 CFU of P. aeruginosa/ml. One group was inoculated with a nonbacterial PBS buffer (control group). (ii) At 1 h after infection, intranasal administration of ACD was performed for two groups: the treated I group (12 mg kg−1) and the treated II group (6 mg kg−1). An untreated group was also included as infection group. (iii) At 24 h after treatment, the mice were euthanized, lung tissues were isolated, and slides of lung tissues were prepared to evaluate the histology of the specimens. (b) Comparison of mouse pulmonary tissue properties and histology. (i) Tissue sections were stained with H&E for comparison of the lung inflammation properties. (ii) PAS-stained lung tissue sections to measure goblet cells (arrowheads). Representative images are shown for n = 3 mice. (c) Histopathological tissue scoring of each group based on the characteristics of the lung pathology. (d) Goblet cell count. The data are based on two independent experiments, and at least two replicates were used for each variation. *, P < 0.01 (one-way ANOVA, significant difference from the control group).

Article Snippet: KEYWORDS: antimicrobial peptide, acidocin 4356, Lactobacillus acidophilus ATCC 4356, Pseudomonas aeruginosa , molecular dynamics simulations, mouse infection model, confocal laser scanning microscopy, CLSM, flow cytometry

Techniques: In Vivo, Activity Assay, Infection, Control, Isolation, Comparison, Staining, Cell Counting

Journal: Applied and Environmental Microbiology

Article Title: Multifunctional Acidocin 4356 Combats Pseudomonas aeruginosa through Membrane Perturbation and Virulence Attenuation: Experimental Results Confirm Molecular Dynamics Simulation

doi: 10.1128/AEM.00367-20

Figure Lengend Snippet: Mechanistic studies of action of ACD against P. aeruginosa. (a) Kinetics of ACD-induced membrane permeabilization. A flow cytometry correlogram (PI versus FDA) is shown at the specified time points after treatment with peptide at a concentration of 4× MIC. PI-positive and FDA-positive results are indicated in red and green, respectively. The population gating selection was determined based on live cells (control without treatment) and cells killed with isopropyl alcohol (dead cells). Two independent measurements were performed with two replications for each variable. (b) Comparison of the kinetics of antimicrobial activity of ACD at various concentrations by flow cytometry. The permeability of P. aeruginosa cell membrane was evaluated after treatment with different concentrations of ACD for 1 h in a linear fashion with time. Comparison of two concentrations at different times indicated that the antibacterial action of the ACD peptide is time dependent. On the dot plots, the x and y axes represent the fluorescent emissions of PI and FDA, respectively. (c) Evaluation of morphological changes of P. aeruginosa cells 1 h after treatment. TEM images of P. aeruginosa without treatment (i) and treated with ACD at its MIC (ii to iv) are shown. Untreated cells display a normal cell shape and undamaged membrane (i). Deformation of cell membrane and a loss of integrity (ii), detachment of the bacterial membrane from the bacterial cytoplasm (iii), and complete cell lysis (iv) occurred over time. (d) TEM micrographs of P. aeruginosa treated with ACD after 4 h. (i and ii) Cells without peptide treatment (control); (iii and iv) cells after treatment with an MIC level of ACD. (i) Cells have complete cell membrane and homogenous cytoplasm; (ii) cells are dividing without any damage; (iii) more damaged cells with signs of general lysis are observable; (iv) the membranes of the dividing cells are degraded, and leakage of the intracellular contents from the cytoplasm is visible. The regions of interest are indicated by red arrows.

Article Snippet: KEYWORDS: antimicrobial peptide, acidocin 4356, Lactobacillus acidophilus ATCC 4356, Pseudomonas aeruginosa , molecular dynamics simulations, mouse infection model, confocal laser scanning microscopy, CLSM, flow cytometry

Techniques: Membrane, Flow Cytometry, Concentration Assay, Selection, Control, Comparison, Activity Assay, Permeability, Lysis

Journal: Applied and Environmental Microbiology

Article Title: Multifunctional Acidocin 4356 Combats Pseudomonas aeruginosa through Membrane Perturbation and Virulence Attenuation: Experimental Results Confirm Molecular Dynamics Simulation

doi: 10.1128/AEM.00367-20

Figure Lengend Snippet: Equilibrated bilayer configuration containing ACD. (a) Representative structure of the end of simulation time and fluctuations of RMSD values during simulation time. In the inset image, the interactions of a single peptide and the peptide complex with P. aeruginosa membrane are shown. The RMSD value is seen to be constant after about 100 ns until the end of the simulation time period, and this indicates that this simulation was stable. (b) Hydropathy plot analysis of the ACD sequence using the ProtScale tool. Hydrophilic and hydrophobic regions in the secondary structure are indicated in red and green, respectively. The configuration of peptides in the membrane in complex form shows that their hydrophobic regions pass through the membrane and interact with the hydrophobic tails of the lipid in the inner part of bilayer.

Article Snippet: KEYWORDS: antimicrobial peptide, acidocin 4356, Lactobacillus acidophilus ATCC 4356, Pseudomonas aeruginosa , molecular dynamics simulations, mouse infection model, confocal laser scanning microscopy, CLSM, flow cytometry

Techniques: Membrane, Sequencing