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. 2017 Jul 31;7(1):6953.
doi: 10.1038/s41598-017-07440-0.

A short D-enantiomeric antimicrobial peptide with potent immunomodulatory and antibiofilm activity against multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii

Mohamed F Mohamed  1 , Anna Brezden  2 , Haroon Mohammad  1 , Jean Chmielewski  2   3 , Mohamed N Seleem  4   5
Affiliations

A short D-enantiomeric antimicrobial peptide with potent immunomodulatory and antibiofilm activity against multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii

Mohamed F Mohamed et al. Sci Rep. .

Abstract

Antimicrobial peptides (AMPs) represent a promising therapeutic alternative for the treatment of antibiotic-resistant bacterial infections. The present study investigates the antimicrobial activity of new, rationally-designed derivatives of a short α-helical peptide, RR. From the peptides designed, RR4 and its D-enantiomer, D-RR4, emerged as the most potent analogues with a more than 32-fold improvement in antimicrobial activity observed against multidrug-resistant strains of Pseudomonas aeruginosa and Acinetobacter baumannii. Remarkably, D-RR4 demonstrated potent activity against colistin-resistant strains of P. aeruginosa (isolated from cystic fibrosis patients) indicating a potential therapeutic advantage of this peptide over several AMPs. In contrast to many natural AMPs, D-RR4 retained its activity under challenging physiological conditions (high salts, serum, and acidic pH). Furthermore, D-RR4 was more capable of disrupting P. aeruginosa and A. baumannii biofilms when compared to conventional antibiotics. Of note, D-RR4 was able to bind to lipopolysaccharide to reduce the endotoxin-induced proinflammatory cytokine response in macrophages. Finally, D-RR4 protected Caenorhabditis elegans from lethal infections of P. aeruginosa and A. baumannii and enhanced the activity of colistin in vivo against colistin-resistant P. aeruginosa.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Efficacy of peptides on established biofilms of P. aeruginosa PAO1 and A. baumannii ATCC BAA-1605. Mature (24 hour) biofilms were treated with different concentrations of RR4, D-RR4, colistin, tobramycin and gentamicin for 24 hours at 37 °C. After incubation, wells were washed and biofilms were stained with 0.5% (w/v) crystal violet for 30 minutes. The dye was solubilized with ethanol (95%) and the optical density (595 nm) of the biofilm mass was measured. Experiments were repeated twice independently and the average values are reported.
Figure 2
Figure 2
Intracellular antibacterial activity of peptides in infected murine macrophage cells (J774A.1). The effect of RR4 and D-RR4, at 8 × MIC, to kill intracellular P. aeruginosa PAO1 and A. baumannii ATCC 19606 inside infected J774A.1 cells after treatment for 24 hours. Statistical analysis was calculated using one-way ANOVA, with post hoc Tukey’s multiple comparisons test. P < 0.05 was considered significant. One asterisk (*) indicates significance from the negative control. Results are expressed as means from three biological replicates ± standard deviation.
Figure 3
Figure 3
Ability of peptides to neutralize LPS and reduce inflammatory cytokines expression. (A) LPS binding activity of peptides. Peptides at different concentrations were incubated with one endotoxin unit (EU) of LPS at 37 °C for 30 minutes. Colistin was used as a positive control due to its high binding affinity for LPS. The binding of peptides with LPS is expressed as percent change relative to the untreated samples. (B) Anti-inflammatory effect of peptides on LPS-stimulated macrophages. J774A.1 cells were stimulated with LPS (150 ng/mL final concentration) in the presence of different concentrations of peptides. Cells stimulated with LPS alone and untreated cells served as controls. Cells were incubated for six hours at 37 °C before the supernatant from each treatment group was collected. Detection of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in supernatants was determined using ELISA. Cytokine levels are expressed as percent change relative to the LPS-stimulated control. Statistical analysis was calculated using one-way ANOVA, with post hoc Tukey’s multiple comparisons test. P < 0.05 was considered significant. One asterisk (*) indicates significant difference with the negative control.
Figure 4
Figure 4
Antibacterial activity of D-RR4 and colistin in C. elegans models of bacterial infection. (A) Evaluation of toxicity of peptides in Caenorhabditis elegans. C. elegans strain glp-4;sek 1 was grown for four days in the presence of peptides at different concentrations. Worms were monitored daily and the live worms were counted. Results are expressed as a Kaplan-Meier survival curve. (B) Evaluation of antimicrobial activity of peptides to reduce the bacterial CFU inside infected Caenorhabditis elegans. C. elegans strain glp-4; sek 1 were infected with P. aeruginosa PAO1, colistin-resistant P. aeruginosa 1109, or A. baumannii ATCC BAA-1605. After infection, worms were treated with D-RR4 (at 8 × MIC), colistin (at 8 × MIC for all strains except colistin-resistant P. aeruginosa 1109 (tested at 1 × MIC)), or a combination of both agents. After treatment, worms were lysed and bacteria were counted. One asterisk (*) indicates significant difference with the negative control. Two asterisks (**) indicates significant difference with the monotherapy treatment (P < 0.05). Results are expressed as means from three biological replicates ± standard deviation. (C) Efficacy of peptides in a Caenorhabditis elegans model of bacterial infection. C. elegans strain glp-4; sek 1 were infected with P. aeruginosa PAO1, colistin resistant P. aeruginosa 1109, or A. baumannii ATCC BAA-1605. After infection, worms were treated with D-RR4 (at 8 × MIC), colistin (at 8 × MIC for all strains except colistin-resistant P. aeruginosa 1109 (tested at 1 × MIC)), or a combination of both. Worms were monitored daily and the live worms were counted. Results are expressed as a Kaplan-Meier survival curve. C. elegans receiving no treatment served as a negative control.
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