wt1 against e coli atcc 11775  (ATCC)

Journal: Cell reports. Physical science

Article Title: Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom

doi: 10.1016/j.xcrp.2023.101459

Figure Lengend Snippet: Amino acid sequence, theoretical values of normalized hydrophobicity, normalized hydrophobic moment, net charge, ratio of polar/non-polar amino acid residues, propensity to in vitro aggregation, amphiphilicity index, and values of helical fraction in water and in a mixture 3:2 of TFE and water, for WT1, WT2, and their corresponding Ala-Scan analogs

Article Snippet: As previously discussed, when Gly at position 12 of WT1 was replaced by Ala, the resulting analog, A 12 -1, was more active than WT1 against E. coli ATCC 11775 and S. aureus ATCC 12600 (MIC = 2 μmol L −1 ) but showed no significant improvement in activity against P. aeruginosa strains PAO1 and PA14 compared with WT1 ( and ).

Techniques: Sequencing, In Vitro

Journal: Cell reports. Physical science

Article Title: Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom

doi: 10.1016/j.xcrp.2023.101459

Figure Lengend Snippet: (A) Schematic representation of the structure-function relationship studies, from the selection of the templates (EMP-EM1 [WT1] and EMP-EM2 [WT2]), isolated from the venom of the solitary wasp Eumenes micado , to the design of an optimized second-generation peptide. (B) Antimicrobial activity of WT1 and WT2 and Ala-Scan analogs for the four pathogenic bacterial strains tested in this study. The red color represents bacterial growth inhibition, and the blue color represents bacterial growth. (C and D) CD spectra of WT1 (C) and WT2 (D) and their respective Ala-Scan derivatives at 50 μmol L −1 in TFE:water 3:2 v/v and water showing the conformational transition of the peptides from random coil in water to α helix in TFE:water. CD spectra were recorded in three replicates at 25°C, using a quartz cuvette with 1-mm path length, between 260 and 190 nm at 50 nm min −1 , with a bandwidth of 0.5 nm. (E) Bidimensional helical wheel representations of the wild-type peptides WT1 and WT2, indicating positions where Ala-substitution decreased (blue arrows) or enhanced (red arrows) activity.

Article Snippet: As previously discussed, when Gly at position 12 of WT1 was replaced by Ala, the resulting analog, A 12 -1, was more active than WT1 against E. coli ATCC 11775 and S. aureus ATCC 12600 (MIC = 2 μmol L −1 ) but showed no significant improvement in activity against P. aeruginosa strains PAO1 and PA14 compared with WT1 ( and ).

Techniques: Selection, Isolation, Activity Assay, Inhibition, Circular Dichroism

Journal: Cell reports. Physical science

Article Title: Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom

doi: 10.1016/j.xcrp.2023.101459

Figure Lengend Snippet: Properties of peptides WT1, WT2, and their corresponding second-generation analogs

Article Snippet: As previously discussed, when Gly at position 12 of WT1 was replaced by Ala, the resulting analog, A 12 -1, was more active than WT1 against E. coli ATCC 11775 and S. aureus ATCC 12600 (MIC = 2 μmol L −1 ) but showed no significant improvement in activity against P. aeruginosa strains PAO1 and PA14 compared with WT1 ( and ).

Techniques: Sequencing, In Vitro

Journal: Cell reports. Physical science

Article Title: Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom

doi: 10.1016/j.xcrp.2023.101459

Figure Lengend Snippet: (A) Antimicrobial activity of WT1, WT2, and second-generation analogs for all tested pathogenic bacteria. The red color represents bacterial growth inhibition, and the blue color represents bacterial growth. Heat maps obtained directly from OD measurements of 96-well plates after treatment are shown in . (B) CD spectra of WT1 and WT2 and their respective second-generation derivatives at 50 μmol L −1 in TFE:water 3:2 v/v, showing α helix conformation, and in water, showing random-coil conformation. CD spectra were recorded in three replicates at 25°C, using a quartz cuvette with 1-mm path length, between 260 and 190 nm at 50 nm min −1 , with a bandwidth of 0.5 nm.

Article Snippet: As previously discussed, when Gly at position 12 of WT1 was replaced by Ala, the resulting analog, A 12 -1, was more active than WT1 against E. coli ATCC 11775 and S. aureus ATCC 12600 (MIC = 2 μmol L −1 ) but showed no significant improvement in activity against P. aeruginosa strains PAO1 and PA14 compared with WT1 ( and ).

Techniques: Activity Assay, Bacteria, Inhibition, Circular Dichroism

Journal: Cell reports. Physical science

Article Title: Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom

doi: 10.1016/j.xcrp.2023.101459

Figure Lengend Snippet: (A) Schematic representation of the NPN assay, in which molecules of NPN (represented by gray spheres) present weak fluorescence emission intensity in an aqueous environment. When the outer membranes are permeabilized by peptides, the NPN molecules interact with the lipidic environment of damaged outer membranes and the intensity of blue fluorescence emission increases (represented by blue spheres). (B) NPN graph for outer membrane permeabilization of Pseudomonas aeruginosa PAO1 by polymyxin B (PMB), WT1, K 12 -1, K 13 -1, WT2, and K 13 -2 peptides. Profiles with a rapid increase in fluorescence emission intensity, followed by a slow decay, were obtained after measurement of white 96-well plates on a Thermo Scientific Varioskan LUX fluorescence spectrophotometer, with the excitation wavelength set to 350 nm and the emission wavelength set to 420 nm, according to the experimental procedure described in the section “ .” All NPN assays were done in three replicates, including the controls, which consisted of only HEPES solution, HEPES solution and NPN (not shown), HEPES solution and P. aeruginosa PAO1 (not shown), and HEPES solution with both P. aeruginosa PAO1 and NPN. Data are represented as mean ± SD. (C) Schematic representation of the DiSC 3 -5 assay, in which molecules of DiSC 3 -5 (represented by gray spheres) accumulate in cytoplasmic membranes and aggregate at high concentrations, causing fluorescence quenching. When the cytoplasmic membrane is destabilized by peptides, DiSC 3 -5 migrates to the cytoplasm or to the external environment, and red fluorescence emission intensity (represented by red spheres) increases. (D) DiSC 3 -5 graph for cytoplasmic membrane depolarization of P. aeruginosa PAO1 by PMB, WT1, K 12 -1, K 13 -1, WT2, and K 13 -2 peptides. Profiles with increases and decreases in fluorescence emission intensity were obtained after measurement of black 96-well plates on a Thermo Scientific Varioskan LUX fluorescence spectrophotometer, with the excitation wavelength set to 622 nm and emission wavelength set to 670 nm as described in the section “ .” DiSC 3 -5 graph obtained after the addition of triton solution is shown in . All DiSC 3 -5 assays were done in three replicates, including the controls, which consisted of only HEPES solution, and HEPES solution containing PAO1 and DiSC 3 -5. Data are represented as mean ± SD. (E) Synergy assay for activity against P. aeruginosa PAO1 between ciprofloxacin, ofloxacin, gentamicin, polymyxin B, or erythromycin, and each of four peptides: WT1, WT2, K 12 -1, and K 13 -1. The Fractional Inhibitory Concentration Index (FICI) values, which indicate the degree of synergy between two antimicrobial agents against a target microorganism, were calculated based on the MICs of WT1, WT2, K 12 -1, and K 13 -1 and the commercial antibiotics used alone and in combination. FICI values <0.5 indicate synergy; 0.5 < FICI < 1 indicates additive effects; 1 < FICI < 4 indicates indifference; and FICI > 4 indicates antagonism (not represented in the graph). (F) Resistance assay: development of resistance to ciprofloxacin, PMB, WT1, K 12 -1, and K 13 -1 in Escherichia coli Δ mutS . The experiment was performed for 20 days as described in detail in the section “ .” Data are represented as mean ± SD.

Article Snippet: As previously discussed, when Gly at position 12 of WT1 was replaced by Ala, the resulting analog, A 12 -1, was more active than WT1 against E. coli ATCC 11775 and S. aureus ATCC 12600 (MIC = 2 μmol L −1 ) but showed no significant improvement in activity against P. aeruginosa strains PAO1 and PA14 compared with WT1 ( and ).

Techniques: NPN Assay, Fluorescence, Membrane, Spectrophotometry, Activity Assay, Concentration Assay

Journal: Cell reports. Physical science

Article Title: Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom

doi: 10.1016/j.xcrp.2023.101459

Figure Lengend Snippet: (A and B) Cytotoxic activity of WT1, A 14 -1, K 12 -1, K 13 -1, WT2, K 10 -2, and K 13 -2 against (A) human embryonic kidney cells (HEK293T) and (B) primary human keratinocytes. (C) Schematic representation of the in vivo assay procedure. The mice were anesthetized with isoflurane and weighed; their backs were shaved, and a superficial linear skin abrasion was made using a needle to damage the stratum corneum and upper layer of the epidermis. Then 50 μL of 10 7 CFU mL −1 in phosphate-buffered saline (PBS) of P. aeruginosa PAO1 was inoculated over the scratch in the back of the mice. After 1 h, peptide solutions in PBS at 32 μmol L −1 for K 13 -2 and 16 μmol L −1 for K 12 -1 and K 13 -1 were added to the infected area. This procedure was done for four mice per peptide tested. After 2 days, mice from each group were killed and weighed, and the area of scarified skin was cut, homogenized using a bead beater for 20 min (25 Hz), and serially diluted for CFU quantification. This procedure was repeated after 4 days with the mice from each group. Two technical replicates were performed for each sample to ensure accuracy. (D) Mice weight monitoring for potential in vivo toxicity assessment. The body weight of infected mice was normalized by the body weight of uninfected mice. Data are represented as mean ± SD. (E) Anti-infective activity of K 12 -1, K 13 -1, and K 13 -2 in vivo compared with control groups. Statistical significance was determined using one-way ANOVA followed by Dunnett’s test; p values are shown in the graph.

Article Snippet: As previously discussed, when Gly at position 12 of WT1 was replaced by Ala, the resulting analog, A 12 -1, was more active than WT1 against E. coli ATCC 11775 and S. aureus ATCC 12600 (MIC = 2 μmol L −1 ) but showed no significant improvement in activity against P. aeruginosa strains PAO1 and PA14 compared with WT1 ( and ).

Techniques: Activity Assay, In Vivo, Saline, Infection, Control