Figure 1.
Amino acid sequences of various HBc ARD peptides tested for bactericidal activity in this study.
The lower panel presents various phosphorylated peptides and an R-to-A mutant peptide with a total of four arg-to-ala substitutions in ARD-III and ARD-IV of HBc147-183.
Table 1.
Antimicrobial activity of HBC ARD peptides.
Table 2.
Antimicrobial activity of ARD peptide HBc147-183 against colistin-resistant and sensitive P. aeruginosa and A. baumannii.
Figure 2.
The killing kinetics of HBc147-183 against P. aeruginosa (circle), K. pneumoniae (diamond), E. coli (square) and S. aureus (triangle).
Bacteria were treated with HBc147-183 (1×MBC). The viability of bacteria was measured at indicated time point. Samples were measured in triplicates.
Figure 3.
Localization of FITC-HBc147-183 peptide on the bacteria.
Approximate 107 CFU of P. aeruginosa ATCC9027, ATCC27853 (A and B), K. pneumoniae ATCC13884 (C), E. coli ATCC25922 (D), and S.aureus ATCC19636, ATCC25923 and ATCC 29213 (E, F and G) were incubation with HBc147-183 (0.5×MBC) for 1 hour. The bacteria were washed, fixed and stained with DAPI (blue). Images were taken using confocal microscopy.
Figure 4.
Possible bactericidal mechanism of HBc147-183.
(A) SYTOX Green uptake of P. aeruginosa (circle), K. pneumoniae (cross) E. coli (square), and S. aureus (triangle) by HBc147-183. Measurements of the fluorescence were recorded every minute. (B) Dose-dependent curves of membrane permeabilization of P. aeruginosa by HBc147-183 (black) and HBc153-176 (grey) at 0.5, 1 and 2 µM (dots, dashes and solid line). 2 µM melittin (diamond) was used as a positive control. Samples were measured in triplicates. (C) DNA-binding activity of HBc147-183. HBc147-183 was mixed with pSUPER plasmid DNA at indicated N/P ratio for 30 minutes. The mobility of DNA was determined by gel retardation assay.
Figure 5.
Dose response effects of LPS and LPS antibody (triangle) on the bactericidal activity of HBc147-183.
LPS from P. aeruginosa (square) and E. coli (diamond), and LPS antibody (triangle) were mixed with P. aeruginosa and HBc147-183 (1×MBC) for 3 hours. The bacteria were then plated on MH agar for the measurement of viability. Samples were measured in triplicates.
Figure 6.
The ARD peptide HBc147-183 was shown to be capable of binding to LPS and Lipid A in several different in vitro binding assays.
For each assay, samples were always measured in triplicates. (A) The cartoon illustrates the in vitro assays of peptide-LPS and peptide-Lipid A binding as well as LPS/Lipid A competition. (B) Constant amount of LPS was incubated with increasing concentrations of biotinylated ARD HBc 147-183 peptide on the streptavidine-conjugated beads (0, 0.004, 0.02, 0.1, 0.5 and 2.5 µM). Unbound LPS in the supernatant was measured with the LAL ELISA assay (Materials and Methods). The EU values were normalized with a control without peptide treatment. HBc147-183 8p (containing 8 phosphorylated amino acids) was also included as a control peptide due to its poor binding with LPS. (C) Beads-bound LPS was released into the supernatant by overnight digestion with trypsin agarose. Free LPS in the supernatant was analyzed with the LAL ELISA assay. Released LPS in the supernatant appeared to be in proportion to the amount of ARD peptide HBc147-183 on the beads. (D) Constant amount of Lipid A was incubated with increasing concentrations of HBc147-183 and HBc147-183 8P, respectively. The supernatant was also detected with LAL ELISA reagent. The result here is consistent with the notion that Lipid A can bind to HBc147-183 directly. (E) LPS/Lipid A competition assay. Constant amount of LPS (1 µg) was coated on each well on the ELISA plate, and then incubated with a reaction mixture containing constant amount of 10 nM HBc147-183 and increasing concentrations of Lipid A. The gradual increase of Lipid A reduced the amount of plate-bound ARD peptide HBc147-183 in a dose dependent manner.
Figure 7.
Cytotoxicity assays of ARD peptide HBc147-183.
% human red blood cells (RBC). Compared to melittin, HBc147-183 showed no hemolytic activity. (B) Huh7, HepG2, Vero and HEK293 cells were incubated with varying concentrations (0 to 100 µM) of HBc147-183 (black) and melittin (white) for 1 hour at 37°C. The effects on cell viability were determined by MTT assay. Melittin was used as a positive control. HBc147-183 showed no detectable effect on cell viability, while melittin exhibited strong toxicity. (C) Kidney cells Vero and HEK293 were stained with CFSE and seeded at day 0 (Materials and Methods). At day 1, cells were incubated with varying concentrations (0 to 100 µM) of HBc147-183 for 1 hour. Cell proliferation at day 1 and day 3 were determined by flow cytometry. Similar to the mock control experiment, no significant effect on Vero and HEK293 cells was detected. Samples assayed in Figure 7A–C were always measured in triplicates. (D) In vivo toxicity of ARD peptide HBc147-183 was determined using three-week old male ICR mice. The mice were injected intraperitoneally with peptide (10 and 20 mg/kg of body weight). All mice were alive after 7 days.
Figure 8.
In vivo studies of the protection activity of ARD peptide HBc147-183 against S. aureus.
(A) Three-week old male ICR mice were challenged with a lethal dose of S. aureus ATCC 19636 and then divided into five separate groups for five different time points. At each indicated time point (n = 5), blood samples were collected, diluted and plated on BHI agar. The number of bacteria was counted the following day. A maximal bacterial load in the blood was observed at 2 h post-inoculation. The data were shown in mean ± SD. (B) ICR mice inoculated with a lethal dose of S. aureus as described above were treated by intraperitoneal injection with ARD peptide (10 mg/kg) at 1, 1.5 or 2 h post-inoculation, respectively. Each group contained 10 mice. All mice (100%) treated with the PBS control died at day 1, while treatment of ARD peptide at 1, 1.5 or 2 h post-inoculation protected the mice with survival rates of 100%, 70% and 40% after 7 days, respectively. (C) As described above, ICR mice were i.p. inoculated with S. aureus, followed by i.p. injection with PBS (n = 5) or 10 mg/kg ARD peptide (n = 5) at 1 h post-inoculation. At 4 h post-inoculation, blood, liver and spleen were collected. Liver and spleen samples were homogenized, diluted and, together with blood samples, plated on BHI agar. The number of bacteria was counted the following day. In comparison to mice treated with PBS, treatment of ARD peptide effectively reduced the bacterial load in blood, liver and spleen. (D) Quantitative comparison of bacterial loads in blood, liver and spleen samples of mice treated with PBS (open circle, diamond and square) versus ARD peptide HBc147-183 (solid circle, diamond and square). The line indicated the mean of bacterial load. **P<0.01 (Mann-Whitney U test) for PBS and ARD peptide HBc147-183.
Figure 9.
IVIS analysis of in vivo antimicrobial activity of ARD peptide against K. pneumoniae.
(A) K. pneumoniae-infected mice were treated with either PBS (n = 5) or 10 mg/kg ARD peptide (n = 5) at 1 hour post-inoculation. Four hours post-inoculation, mice were anesthetized and imaged. Bacterial load was displayed in the photographic image with an overlay of bioluminescence. False color imaging represents intense luminescence in red, moderate luminescence in green and low luminescence in blue and purple. (B) Total flux was quantified by IVIS imaging software. **P<0.01 (Mann-Whitney U test) for PBS and ARD peptide HBc147-183.