DOI: 10.5061/dryad.x3ffbg7vs
title: Data from: Antimicrobial peptide LL37 is potent against non-growing Escherichia coli cells despite a slower action rate

description: Antimicrobial peptides (AMPs) have long been considered as potential
 agents against non-growing, dormant cells due to their membrane-targeted
 action, which is largely independent of the cell’s growth state. However,
 the relationship between the action of AMPs and the physiological state of
 their target cells has been unclear, with recent reports offering
 conflicting views on the efficacy of AMPs against bacteria in a stationary
 phase. In this study, we employ single-cell approaches combined with
 population-level experiments to examine the action of human LL37 peptides
 against Escherichia coli cells in different growth phases. Time-lapse,
 single-cell data from our experiments reveal that LL37 peptides act faster
 on large, dividing cells than on small, newborn cells. We extend this
 investigation to non-growing E. coli cells in a stationary phase, where we
 observe that the action of LL37 peptides is slower on non-growing cells
 compared to exponentially growing cells. This slower action rate is,
 however, not mirrored in the minimum bactericidal concentration (MBC)
 measurements. Notably, we find that the MBC for non-growing cells is lower
 than for exponentially growing cells, indicating that, given sufficient
 time, LL37 peptides exhibit strong potency against non-growing cells. We
 propose that the enhanced potency of LL37 peptides against non-growing
 cells, despite their slower action, can be attributed to a continuous
 absorption of AMPs on the cell membrane over time.
description: # Data and supplementary information and video for article
 "Antimicrobial Peptide LL37 is Potent Against Non-Growing
 *Escherichia coli* Cells Despite a Slower Action Rate" by Mohammadi,
 Saucedo and Taheri-Araghi
 [https://doi.org/10.5061/dryad.x3ffbg7vs](https://doi.org/10.5061/dryad.x3ffbg7vs) ## Description of the data and file structure **A. Description of the CSV data files.** **Fig_1A_Size_vs_Time_Until_Death.csv**: This file contains data for cell size (in micrometers) as a function of time (minutes) for each cell until cell death. Each line of data represents one cell.  **Fig_1C_InitialCellSize_vs_DeathTime.csv**: This file contains data on the average of initial cell size (in micrometers) as a function of death time (minutes). X, Y, and standard deviation values are labeled for each column of the data.   **Fig_3C_DeathRate_vs_CellLength_Stationary.csv**: This file contains data on the average death rate (per minute) as a function of cell size (in micrometers) for cells in stationary phase. X, Y, and standard deviation values are labeled for each column of the data.   **Fig_3C_DeathRate_vs_CellLength_Exponential.csv**: This file contains data on the average death rate (per minute) as a function of cell size (in micrometers) for cells in exponential phase. X, Y, and standard deviation values are labeled for each column of the data.   **B. Description of the supplementary PDF file.** The content of this PDF file presents detailed of the experimental protocols and imaging method for the study presented in the manuscript "Antimicrobial Peptide LL37 is Potent Against Non-Growing *Escherichia coli* Cells Despite a Slower Action Rate" **C. Description of the experiment related to the Video S1.** To explore the relationship between the physiological state of the target cells and their susceptibility to LL37 peptides, we devised and tested a controlled starvation condition for *E. coli* cells. Our aim was to effectively suppress growth and cell division without compromising cell viability. This was achieved by using the buffer component of the growth media (rich defined media, RDM) without including any carbon, nitrogen, sugar or supplements. When inoculated in this buffer, the cells displayed no detectable population growth using a spectrophotometer (Genesys 2000, Fisher Scientific). To monitor the transition of cells from the exponential phase to the buffer, we used the microfluidic "mother machine." This device enables continuous and rapid environmental control along with single-cell resolution microscopy. Approximately 400 individual cells over tens of generations were monitored as they transitioned from RDM to the buffer and back. Specifically, cells were initially grown in RDM within the mother machine for approximately ten hours, then we switched to the buffer for a duration of three hours. The rapid infusion of the media into the microfluidics system allows us to make a swift transition to the nutrient-deprived buffer. We observed that after switching the media to the buffer, the elongation rate of individual cells quickly dropped to zero, and the division rate followed after roughly a 60-minute delay. Over the three-hour buffer period, growth was largely arrested. Lastly, we switched back to RDM to assess cell viability. We observed that all cells resumed growth shortly after the infusion of RDM, with the cell division rate recovering after a brief delay (Video S1). In general, the loss of viability in nutrient-deprived buffers has been reported to occur very slowly, typically following a prolonged lag phase of several hours. Therefore, consistent with our observations, it can be reasonably concluded that no significant viability loss due to starvation occurred during the 3-hour buffer exposure period. This provided a platform to investigate the action of LL37 peptides on non-growing cells. Note that the buffer composition match the ionic strength of the original RDM, ensuring that the absence of growth was due solely to the lack of carbon and nitrogen sources rather than any changes in salt concentration. This consistency in ionic environment is critical, as fluctuations in ionic strength could alter the electrostatic interactions between LL37 peptides and the bacterial membrane, potentially influencing the peptide's efficacy.