Ceragenin CSA-13 displays high antibacterial efficiency in a mouse model of urinary tract infection

Abstract

Ceragenins (CSAs) are synthetic, lipid-based molecules that display activities of natural antimicrobial peptides. Previous studies demonstrated their high in vitro activity against pathogens causing urinary tract infections (UTIs), but their efficiency in vivo was not explored to date. In this study, we aimed to investigate the bactericidal efficiency of ceragenins against E. coli (Xen14 and clinical UPEC strains) isolates both in vitro and in vivo, as well to explore CSA-13 biodistribution and ability to modulate nanomechanical alterations of infected tissues using animal model of UTI. CSA-44, CSA-131 and particularly CSA-13 displayed potent bactericidal effect against tested E. coli strains, and this effect was mediated by induction of oxidative stress. Biodistribution studies indicated that CSA-13 accumulates in kidneys and liver and is eliminated with urine and bile acid. We also observed that ceragenin CSA-13 reverses infection-induced alterations in mechanical properties of mouse bladders tissue, which confirms the preventive role of CSA-13 against bacteria-induced tissue damage and potentially promote the restoration of microenvironment with biophysical features unfavorable for bacterial growth and spreading. These data justify the further work on employment of CSA-13 in the treatment of urinary tract infections.

Figure 1

Antibacterial activity of ceragenins (CSA-13, CSA-44 and CSA-131) against E. coli Xen14. Decrease of bacteria-derived luminescence signal upon 1 h treatment with tested ceragenins. Results are presented as mean ± SD from 3 replicates (A). Decrease of survival of E. coli Xen 14 planktonic bacteria when exposed to ceragenin CSA-13, CSA-44 and CSA-131 evaluated using the “killing assay” method. Results are presented as mean ± SD from 3 replicates (B). Induction of reactive oxygen species (ROS) generation by E. coli Xen14 was evaluated by DFCH-DA fluorometric assay. Formation of ROS upon treatment with CSA-13, CSA-44, and CSA-131 at a concentration of 1–100 μg/mL was presented. Results are presented as mean ± SD from 3 replicates; *indicates statistical significance ≤ 0.05, ** ≤ 0.01, and *** ≤ 0.001 (C). Anti-biofilm properties of CSA-13 (D), CSA-44 (E), CSA-131 (F) against E. coli Xen14. Ability of ceragenins to prevent the biofilm formation of E. coli was measured using crystal violet staining upon 24 (grey bars), 48 (green bars) and 72 h (white bars). Results are presented as mean ± SD from 3 replicates. Dashed horizontal line indicate untreated control (0 µg/mL of ceragenins).

Figure 2

Survival of human urinary bladder cancer cells T24 (HTB-4™) upon incubation with CSA-13, CSA-44 and CSA-131 at doses of 0–10 µg/mL for 24 h (A). Cytotoxic effect of cationic lipids against T24 cell line after cells incubation with both heat-inactivated (B) and life (C) E. coli.

Figure 3

Scheme description of CSA-13 labeling with IRDye 800CW (A). Biodistribution of intravenously administrated CSA-13 labeled with IRDye®800CW (CSA-13-IRDye800CW) estimated by fluorescence-based analysis of CSA-13-IRDye800CW-targeted fluorescence signal in healthy, non-infected Cby.Cg-Foxn1nu/cmdb mice (n = 5; group 8) 4, 8, 12 and 24 h post injection of CSA-13 IRDye800CW. Results from representative animals are shown (B). Representative scans of urine and feces collected from healthy mice (n = 5; group 8) 4, 8, 12 and 24 h after injection of CSA-13-IRDye800CW (C). Presence of labeled CSA-13 in urine and feces was estimated based on fluorescence intensity of collected excreta and presented as mean value ± SEM from all areas of each urine and feces (D). Organ uptake of CSA-13-IRDye800CW (group 8) and IRDye800CW (group 9) after 24 h post its administration was estimated based on fluorescence intensity of collected organs (1—stomach, 2—pancreas, 3—spleen, 4—liver, 5—lungs, 6—heart, 7—left kidney, 8—right kidney, 9—bladder) and presented as mean value ± SEM from all areas of each organs (E, F).

Figure 4

Decrease in E. coli Xen14 colonies in urine culture from mice post CSA-13 IRDye800CW treatment (grey columns) when compared to urinary tract infected untreated mice (green columns) (A) and decrease in E. coli clinical strains (S1, (B) and S2 (C), colonies in urine culture from mice post CSA-13 treatment (grey columns) when compared to urinary tract infected untreated mice (green columns). Histological analysis of mice bladder tissues: (1–3) normal murine bladder; (4–9) murine bladder infected with E. coli Xen14; tissue edema (black star), exfoliation of transitional epithelial cells (black arrow), invasion of inflammatory cells in the mucosa (red star), and bladder mucosa hyperplasia (red arrows) (10–12) Murine bladder infected with E. coli Xen14 and treated with CSA-13 (D).

Figure 5

The Young's modulus values measured for each bladder samples collected from healthy animals, both untreated (A) and treated with IRDye800CW (B) or CSA-13-IRDye800CW (C), as well as E. coli Xen14-infected animals, both untreated (D) and treated (E) with CSA-13-IRDye800CW, with the logarithm of the normal distribution of the adjusted probability function density. The mean values of Young’s modulus ± standard deviation (F).