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. 2019 Feb 6;16(151):20180701. doi: 10.1098/rsif.2018.0701

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© 2019 The Author(s)

Published by the Royal Society. All rights reserved.

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Figure 3. — LL37 is absorbed by permeabilized bacterial cells, and the absorption causes depletion of LL37 in bacterial cultures. (a) Flow cytometry results demonstrate the transition of bacterial states over time during Rh-LL37 treatment. E. coli constitutively expressing green fluorescent proteins (GFP) is treated by Rh-LL37. Green dashed line separates populations with intact (high GFP) and permeabilized (low GFP) membrane. Rh negative (Rh−) represents bacterial population without Rh-LL37 association (see electronic supplementary material, figure S10, for negative controls). Bacterial cells can transit from ‘high GFP, Rh positive (Rh+)’ to ‘high GFP, Rh double positive (Rh++)’ over time, and the permeabilization (transition from high GFP to low GFP) only occurs in Rh++ bacteria. See electronic supplementary material, method S16. (b) Structured illumination microscopy (SIM) reveals co-localization of Rh-LL37, bacterial membrane and intracellular space. Bacterial populations are sorted to collect subpopulations under Rh-LL37 treatment (①, ②, and ③ from figure 3a). SIM images (left) and intensity heat maps (right) show that the Rh-LL37+ population (①) has Rh-LL37 co-localized at the perimeter of the cell membrane, whereas the Rh-LL37++ population (② and ③) has Rh-LL37 co-localized in the intracellular space of the bacteria. See electronic supplementary material, figure S20, for more SIM images. Scale bar represents 2 µm. See also electronic supplementary material, method S17. (c) Proposed model for the transition of bacterial states under Rh-LL37 treatment. We propose three bacterial states to explain the population dynamics: growing population, binding population (① from figure 3a) and absorbing population (② and ③ from figure 3a). Kinetics of the transitions between states are governed by three reaction rate constants (k_1f, k_1r and k_2f). The depletion rate of Rh-LL37 from the medium by the absorbing population is governed by k_ab. Intrinsic bacterial growth has a rate constant of k_g. See equation (4.1) and Methods (§4.3). (Online version in colour.)