Journal: The Febs Journal

Article Title: Structure‐based design of peptides that trigger Streptococcus pneumoniae cell death

doi: 10.1111/febs.15514

Figure Lengend Snippet: Protein complex disruption and subsequent in vivo cell feasibility assays using binding region‐mimicking peptides. (A) In vitro ribonuclease activity assays using binding region‐mimicking peptides. Each experiment was performed in triplicate. Each mimicking peptide (10 μm) was added to 2 μm HigBA. Forty units of RiboLock TM (Thermo Scientific) RNase inhibitor was used to prevent ribonuclease contamination. (B) Size‐exclusion chromatography of HigBA, HigA, HigB, and HigB with the HigB α2 helix‐mimicking peptide added. Absorption at 280 nm is plotted as a function of the elution volume. The volume of injected protein was 100 μL, and the concentrations of proteins and HigB α2‐mimicking peptide were 200 μm and 1 mm, respectively. (C) Effect of peptides on the growth of S. pneumoniae TIGR4 was measured based on the OD 600 . Error bars represent the standard error of the mean across three biological replicates. (D–I) Cell viability confirmation using flow cytometry and confocal imaging. (D) S. pneumoniae TIGR4 was treated with various concentrations of HigB α2 mimicking peptide selected by the results of the MIC test. Cells were labeled with LIVE/DEAD BacLight stains (Syto 9; PI) and analyzed by fluorescence‐activated cell sorting (FACS). The results of the untreated peptide cells are also presented for comparison. Live cell/dead cell areas were established using this TIGR4 control. (E) FACS data of 6.3 μm peptide‐treated S. pneumoniae TIGR4 after 1 h of incubation. (F) Flow cytometry of S. pneumoniae TIGR4 cells obtained from untreated (cyan) and 6.3 μm peptide‐treated (purple) cells stained with the fluorescent dye PI. Detection of cell‐penetrating peptides in living cells by confocal laser scanning microscopy using (G) S. pneumoniae TIGR4 and (H) S. pneumoniae D39 containing active site‐mutated higBA (shown data are images of R73A). (G,H) Shown data are representative maximum intensity Z projection images of three independent experiments with scale bar, 25 μm. (I) FACS data of S. pneumoniae D39 that was treated with 6.3 μm peptide and contained the active site‐mutated higBA

Article Snippet: The activity of the peptides against Gram‐positive S. pneumoniae TIGR4 (ATCC ® BAA‐334 TM ) was tested.

Techniques: Disruption, In Vivo, Binding Assay, In Vitro, Activity Assay, Size-exclusion Chromatography, Injection, Flow Cytometry, Imaging, Labeling, Fluorescence, FACS, Comparison, Control, Incubation, Staining, Confocal Laser Scanning Microscopy