The insect antimicrobial peptide cecropin A disrupts uropathogenic Escherichia coli biofilms

Abstract

Current antibiotics cannot eradicate uropathogenic Escherichia coli (UPEC) biofilms, leading to recurrent urinary tract infections. Here, we show that the insect antimicrobial peptide cecropin A (CecA) can destroy planktonic and sessile biofilm-forming UPEC cells, either alone or when combined with the antibiotic nalidixic acid (NAL), synergistically clearing infection in vivo without off-target cytotoxicity. The multi-target mechanism of action involves outer membrane permeabilization followed by biofilm disruption triggered by the inhibition of efflux pump activity and interactions with extracellular and intracellular nucleic acids. These diverse targets ensure that resistance to the CecA + NAL combination emerges slowly. The antimicrobial mechanisms of CecA, thus, extend beyond pore-forming activity to include an unanticipated biofilm-eradication process, offering an alternative approach to combat antibiotic-resistant UPEC infections.

Fig. 1. Effect of synergy between cecropin A (CecA) and nalidixic acid (NAL) on planktonic and biofilm-forming uropathogenic Escherichia coli cells.

a Time-kill curves of CecA (50 μg/ml), NAL (0.5 ng/ml), and their combination against strain CFT073. Cells were treated with CecA 1 (50 μg/ml), CecA 2 (75 μg/ml), NAL (0.5 ng/ml), or CecA (50 μg/ml) + NAL (0.5 ng/ml) to determine b hemolysis of porcine erythrocytes in comparison to 10% Triton X-100 (control), and c the viability of BHK-21 fibroblast in comparison to untreated control. Kaplan-Meier plots of CFT073-infected G. mellonella survival after treatment with d CecA (50 μg/ml), e NAL (0.5 ng/ml), and f their combination +/− protease inhibitor (PI). g–j Scanning and k–n transmission electronic microscopy analysis of biofilm structures formed by CFT073 following treatment with CecA (50 μg/ml) ± NAL (0.5 ng/ml). g–n The biofilm-containing discs were supplemented with h, l NAL, i, m CecA, or j, n their combination in fresh LB medium and incubated for another 24 h. Biofilm-forming discs grown in LB medium without supplements were used as controls (g, k). Representative images are shown (scale bar = 2 μm). White arrow heads show intercellular filaments (g–j), black short arrow heads indicate cytoplasm density (k–n), and black arrows show cell debris (m, n). o Percentage inhibition of CFT073 biofilms by NAL (0.5 ng/ml) or CecA (50 μg/ml) compared to their combination after 48 h. p Percentage eradication of pre-formed CFT073 biofilms by NAL (0.5 ng/ml) or CecA (50 μg/ml) compared to their combination. q CFT073 adaptation to NAL (20 μg/ml) or the CecA (20 μg/ml) + NAL (0.5 ng/ml) combination was determined by analyzing bacterial growth after repeated exposure to antimicrobials for 5 days. The figure represents bacterial growth at day 5 with reference to day 1 after antimicrobial exposure. Values are means with standard errors: n = 4 (panels a and q), n = 3 (panels b–f and o–p). Significance was determined by one-way ANOVA, Dunnett’s multiple comparison test (b, c, o, p), Holm-Šídák correction (q) (*P < 0.05; **P < 0.005; ***P < 0.0005).

Fig. 2. Molecular targets of cecropin A (CecA) in uropathogenic Escherichia coli cells.

a Steady-state levels of H33342 (2.5 µM) accumulating in CFT073 biofilm cells with and without exposure to CecA (10 µg/ml) or nalidixic acid (NAL; 0.5 ng/ml). b Inhibition of Nile red efflux by CecA (10 µg/ml) and NAL (0.5 ng/ml). Efflux was triggered at 100 s by the addition of 20 mM glucose. The intensity of fluorescence emission from Nile red was presented to show the effects of CecA, NAL or the absence of treatment in CFT073 biofilm cells. c Decreasing intensities of the fluorescent dye BOBO-3 that cannot penetrate intact membranes and consequently only stains external nucleic acids were measured following incubation with untreated CFT073 cells or those treated with NAL (0.5 ng/ml), CecA (50 μg/ml) or their combination. d Inhibition of E. coli DNA gyrase ATPase activity by CecA and NAL. Decreasing intensities of eDNA and f RNA bands from CFT073 cells treated with a high concentration of CecA were visualized by gel electrophoresis to highlight the DNA/RNA-binding activity of CecA. Values are means and standard errors: n = 3 (panels a, b, and d). Significance was determined by one-way ANOVA and Holm-Šídák correction (a, b), Dunnett’s multiple comparison test (d) (*P < 0.05; **P < 0.005; ***P < 0.0005; ns—not significant).