FIGURE 5.

Cationic residues in the cell binding segment of Ang4 are crucial for the bacterial cell binding and killing. (A) RNase activity of Ang4 and its mutants was measured by absorbance at 260 nm. (B) Growth curve of bacteria after incubation with or without Ang4 or its mutants for 2 h. (C) Killing assay of bacteria performed 2 h post-incubation with Ang4 or its mutants estimated by colony count. PBS was used as a control. (D) Binding of Ang4 and its mutants to the bacterial cell membrane was estimated by ELISA at 450 nm. (E) NPN uptake assay to detect outer membrane disruption of bacteria after the addition of Ang4 or its mutants evaluated by increased fluorescence intensity. (F,G) PI staining to evaluate the alteration of bacterial cell membrane after 2 h post incubation at 37°C with Ang4 or its mutants. All data are presented as the mean ± SD from three independent experiments. P-values were determined by one-way ANOVA with Tukey’s multiple comparisons test; ns > 0.05, *P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. WT Ang4.