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. 2022 Mar 23;13:794779. doi: 10.3389/fimmu.2022.794779

Figure 6.

Figure 6

Function and mechanism of Tatritin in antibacterial process. (A) Localization of Tatritin peptide on the bacteria. Approximate 1–5 × 108 CFU of E. coli ATCC35218, and S. aureus ATCC6538 were incubated with FITC-labeled Tatritin (2.5 μM) for 1 h. The bacteria were washed, fixed, and stained with DAPI (blue). Images were taken using confocal microscope. The cells in the red and yellow boxes were enlarged. Scale bar is 5 μm. (B) PI labeling of bacterial cells treated with Tatritin peptide. Approximately 5 × 107 cells in 50 µl PBS were added to an EP tube containing 150 µl of Tatritin (10 µM). Then PI was added, and the influx of PI into bacterial cells was investigated by flow cytometry. All bacteria were gated, and the cell-penetrating efficiency was determined using the data from three tests. (C) Scanning electron microscope of E. coli and S. aureus cultured with or without Tatritin (20 µM) for 2 h at 37˚C. (D) Evaluation of the LPS-binding activity of Tatritin. The LPS-binding activities of Tatritin were investigated by incubating 0.05–0.2 µg of Tatritin in the LPS-immobilized 96-well microtiter plates (0.2 µg LPS/well) for 1 h at 37°C in 50 µl of RPMI 1640. The bound peptides were detected by TMB reaction using rabbit anti-Tatritin and HRP-conjugated goat anti-rabbit IgG. Tatritin (0.2 µg/well) was incubated in the LPS-immobilized 96-well microtiter plates for 1 h at 37°C in the absence or the presence of LPS (0.5–2.5 µg/well) in 50 µl of RPMI 1640, and the bound peptide was detected as described above. Binding of Tatritin to the LPS-immobilized plates was expressed as a percentage of that incubated with 0.2 µg of each peptide in the absence of added LPS. Data are the mean ± SD (n = 3). (E) Gel shift assay of bacterial gDNA of E. coli and S. aureus mixed with increasing concentrations of Tatritin. The abscissa represents the peptide/gDNA ratio. ImageJ was used to analyze the intensity of nucleic acid bands in gel shift assay. The abscissa represents the peptide/gDNA ratio and the ordinate represents the gDNA mobility shift, *P<0.05.