Antimicrobial peptides (AMPs) are broad spectrum antibiotics that selectively target bacteria. Here we investigate the activity of human AMP LL37 against Escherichia coli by integrating quantitative, population and single-cell level experiments with theoretical modeling. We observe an unexpected, rapid absorption and retention of a large number of LL37 peptides by E. coli cells upon the inhibition of their growth, which increases population survivability. This transition occurs more likely in the late stage of cell division cycles. Cultures with high cell density exhibit two distinct subpopulations: a non-growing population that absorb peptides and a growing population that survive owing to the sequestration of the AMPs by others. A mathematical model based on this binary picture reproduces the rather surprising observations, including the increase of the minimum inhibitory concentration with cell density (even in dilute cultures) and the extensive lag in growth introduced by sub-lethal dosages of LL37 peptides.

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Antimicrobial peptides (AMPs) are broad spectrum antibiotics that selectively target 14 bacteria. Here we investigate the activity of human AMP LL37 against Escherichia coli by integrating 15 quantitative, population and single-cell level experiments with theoretical modeling. Our data 16 indicate an unexpected, rapid absorption and retention of a large number of LL37 by E. coli cells 17 upon the inhibition of their growth, which increases the chance of survival for the rest of 18 population. Cultures with high-enough cell density exhibit two distinct subpopulations: a 19 non-growing population that absorb peptides and a growing population that survive owing to the 20 sequestration of the AMPs by others. A mathematical model based on this binary picture 21 reproduces the rather surprising behaviors of E. coli cultures in the presence of LL37, including the 22 increase of the minimum inhibitory concentration with cell density (even in dilute cultures) and the 23 extensive lag in growth introduced by sub-lethal dosages of LL37. 24 25 34 (2006). 35 A hallmark of the AMPs antibacterial mechanism is the role of physical interactions. AMP's 36 structures exhibit two common motifs: cationic charge and amphiphilic form Zasloff, M (2002); 37 Brogden (2005). The cationic charge enables them to attack bacteria, enclosed in negatively charged 38 membranes, rather than mammalian cells, which possess electrically neutral membranes. The 39 1 of 12 Manuscript submitted to eLife amphiphilic structure allows AMPs to penetrate into the lipid membrane structures Matsuzaki et al. Despite our detailed knowledge on AMP's interactions with membranes, we lack a compre-43 hensive picture of the dynamics of AMPs in a population of cells. We are yet to determine the 44 extent to which the AMP's physical interactions disrupt biological processes in bacteria and the 45 degree to which electrostatic forces govern the diffusion and partitioning of AMPs among various 46 cells. Specifically, it was suggested by Matsuzaki and Castanho et al. that the density of cells in a 47 65 (∼1×1× µ ) Taheri-Araghi et al. (2015). With no direct interactions among the cells and nutrients 66 in excess for all, this dependence suggests that the effective peptide concentration is somehow 67 compromised in a cell density dependent manner. 68 By tracking a dye-tagged version of LL37 peptide, we found that the inhibition of growth of E. 69 coli cells was followed by the translocation of a large number of AMPs into the cells cytoplasm, thus 70 reducing the peptide concentration in the culture, which works in favor of other cells. In the sense 71 of such dynamics, MIC refers to a sufficient concentration of AMPs for absorption into all the cells. 72 Below the MIC, peptide is absorbed by only a fraction of cells, leaving an inadequate amount of 73 AMPs to inhibit the growth of remaining cells. We have directly observed that cultures with sub-MIC 74 concentrations of dye-tagged LL37 exhibit a heterogenous population combining non-growing cells 75 containing m...