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

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.


Acid-base titration
Powder peptide samples (13.00-16.00 mg) were diluted in Milli-Q water 10.0 mL (final concentration of dendrimers is 1.00 mg/mL) and acidified to pH ~3 with 1 M HCl. Then, 0.1 M NaOH was added in step of 2 µL with a Dosimat plus (Metrohm, Zofingen, Switzerland) and pH was measured on a 692 pH/ion meter (Metrohm).  MD simulations were performed for dendrimers G3KL and XC1 using GROMACS software version 2020.4 and the gromos53a6 force field. The dendrimer topologies were built by combining topologies of two linear peptides with the same sequence, one with alpha and one with epsilon connectivity at the branching lysines, using in house scripts. The starting confirmation was built by hand in PyMol software by setting all the dihedral angles to α-helix conformation. A dodecahedral box was created around the peptide 1.0 nm from the edge of the system and filled with extended simple point charge water molecules. Sodium and chloride ions were added to produce an electroneutral solution at a final concentration of 0.15 M NaCl.
The energy was minimized using a steepest gradient method to remove any close contacts before the system was subjected to a two-phase position-restrained MD equilibration procedure.
The assay was performed in the biosafety level 2 lab and was repeated at least two times.

Quantification of bacterial binding of G3KL-Fluo
A single colony of E. coli, A. baumannii, Pseudomonas aeruginosa, K. pneumoniae and MRSA was grown overnight with shaking (180 rpm) in LB-broth (5 mL) at 37 °C. 100 μL of the overnight culture was regrown in 5 mL LB-broth to the exponential phase OD600 = 1.0 (1 x 10 9 CFU/mL). Bacteria (1 mL, OD600 = 1.0) were washed once with MH medium (at pH 7.4 or pH 8.0) and resuspended in 960 μL of MH medium (at pH 7.4 or pH 8.0). 100 mM NaCl, 200 mM NaCl and 300 mM NaCl was added when specified. 40 μL of 1 mg/mL G3KL-Fluo was then added to bacteria. After 2 hours, 180 μL of the sample were isolated and centrifuged at 12 000 rpm for 10 min. The supernatant was collected and added to a 96 well-plate (TPP, untreated, Faust Laborbedarf, AG, Schaffhausen) prior to fluorescence measurement with a Tecan instrument Infinite M1000. The plate was enabled to shake for 30 sec before measurement. The excitation wavelength used was 495 nm ± 5 nm and the emission wavelength 519 nm ± 5 nm.
The assay was performed in the biosafety level 2 lab was repeated at least two times.