Rigidification of the Escherichia coli cytoplasm by the human antimicrobial peptide LL-37 revealed by superresolution fluorescence microscopy

Abstract

Superresolution, single-particle tracking reveals effects of the cationic antimicrobial peptide LL-37 on the Escherichia coli cytoplasm. Seconds after LL-37 penetrates the cytoplasmic membrane, the chromosomal DNA becomes rigidified on a length scale of ∼30 nm, evidenced by the loss of jiggling motion of specific DNA markers. The diffusive motion of a subset of ribosomes is also frozen. The mean diffusion coefficients of the DNA-binding protein HU and the nonendogenous protein Kaede decrease twofold. Roughly 108 LL-37 copies flood the cell (mean concentration ∼90 mM). Much of the LL-37 remains bound within the cell after extensive rinsing with fresh growth medium. Growth never recovers. The results suggest that the high concentration of adsorbed polycationic peptides forms a dense network of noncovalent, electrostatic linkages within the chromosomal DNA and among 70S-polysomes. The bacterial cytoplasm comprises a concentrated collection of biopolymers that are predominantly polyanionic (e.g., DNA, ribosomes, RNA, and most globular proteins). In normal cells, this provides a kind of electrostatic lubrication, enabling facile diffusion despite high biopolymer volume fraction. However, this same polyanionic nature renders the cytoplasm susceptible to massive adsorption of polycationic agents once penetration of the membranes occurs. If this phenomenon proves widespread across cationic agents and bacterial species, it will help explain why resistance to antimicrobial peptides develops only slowly. The results suggest two design criteria for polycationic peptides that efficiently kill gram-negative bacteria: facile penetration of the outer membrane and the ability to alter the cytoplasm by electrostatically linking double-stranded DNA and 70S-polysomes.

Parietal Structures of Escherichia coli Can Impact the D-Cateslytin Antibacterial Activity

“…In the absence of Type 1 fimbriae, a stiffening of about 30% is observed over 24 hour-treatment whereas a dramatic softening of about 85% when Type 1 fimbriae are expressed.The mechanical behavior of E2152 under D-Ctl treatment seemed counterintuitive in respect with the membrane permeabilization supported by the BacLight™ assays. However, mechanical stiffening under AMP has been already reported for huge peptides accumulation at bacterial envelope concomitantly to its permeabilization 31,[41][42]. The mechanical behavior of E2146 under D-Ctl treatment is in good agreement with previous observations24 that have evidenced a reduction of bacterial stiffness by a factor of 3 with respect to the untreated bacteria.…”

Direct visualization of four diffusive LexA states controlling SOS response strength during antibiotic treatment

“…The nonspecifically DNA bound state is supported by previous reports that prior to finding the target sequence and firmly binding to it, repressors often associate with the DNA in a sequence non-specific manner and perform a 1D target search, as previously shown for the lac repressor (47,48). Tight binding to operator sites results in an apparent diffusion coefficient " * close to zero, defined by minimal movement of DNA within bacterial cells (49,50). The number of tightly bound repressors quickly decreased already after 10 min of CIP treatment (Fig.…”

Mechanisms of action of ionic liquids on living cells: the state of the art

“…The cytoplasm is a very crowded environment which comprises a huge variety of biopolymers that are predominantly anionic (e.g., DNA, ribosomes, RNA, and most proteins). The presence of ILs (cations) could alter the electrostatic equilibrium of the cytoplasm and change its viscosity, affecting the rate of diffusion of the biopolymers, as shown for antimicrobial peptides (Zhu et al 2019 ). In this respect, it could be interesting to investigate how ILs affect protein mobility in cells and bacteria by tracking single molecules, e.g., Kapanidis et al ( 2018 ).…”