The antibacterial activity of peptide dendrimers and polymyxin B increases sharply above pH 7.4

Abstract

pH-activity profiling reveals that antimicrobial peptide dendrimers (AMPDs) kill Klebsiella pneumoniae and Methicillin-resistant Staphylococcus aureus (MRSA) at pH = 8.0, against which they are inactive at pH = 7.4, due to stronger electrostatic binding to bacterial cells at higher pH. A similar effect occurs with polymyxin B and might be general for polycationic antimicrobials.

Fig. 1. Structure of peptide dendrimers investigated in this study. Ahx = aminohexanoic acid.

Fig. 2. CD spectroscopy and MD simulations with AMPDs at different pH values. (a) CD spectra of XC1 and G3KL (0.100 mg mL⁻¹ of dendrimer) in aq. buffer (10 mM acetate pH 5.0, 10 mM phosphate pH 7.4 or pH 8.0) with or without 5 mM DPC. See methods for details. (b) RMSD of the central α-helix over the course of the MD simulations in water for XC1, G3KL with neutral N-termini (pH 7.4) or with protonated N-termini (pH 5.0). (c) MD simulation of XC1 with a DPC micelle after 150 ns. Lys and Ahx in blue, Leu, branching Lys in red, DPC in surface representation with C in grey, O in red, N in blue and P in orange.

Fig. 3. (a) Bacteria killing assay at pH 5.0 against E. coli, A. baumannii, P. aeruginosa PAO1 and K. pneumoniae at a concentration of 4 × MIC. (b) Bacteria killing assay at pH 7.4 and pH 8.0 against K. pneumoniae and MRSA. (c) TEM images of K. pneumoniae, 2 h after treatment of XC1, G3KL and PMB in MH medium at pH 8.0. Scale bar is 200 nm. (d) MIC of XC1 against K. pneumoniae and MRSA at different pH, MIC of PMB against MRSA at different pH. Growth of bacteria was not observed above the shown pH. (e) MIC of XC1 against K. pneumoniae and MRSA at pH 8.0 in presence of NaCl, MIC of PMB against MRSA at pH 8.0 in presence of NaCl. (f) Quantification of unbound G3KL-Fluo (40 μg mL⁻¹) in the presence of 10⁹ CFU mL⁻¹ (OD₆₀₀ = 1) of E. coli, A. baumannii, P. aeruginosa PAO1, K. pneumoniae and MRSA for 2 hours. 300 mM NaCl was added when specified. In figures d and e, each result represents two independent experiments performed in duplicate.