Real‐Time Monitoring of Multitarget Antimicrobial Mechanisms of Peptoids Using Label‐Free Imaging with Optical Diffraction Tomography

Abstract

Antimicrobial peptides (AMPs) are promising therapeutics in the fight against multidrug‐resistant bacteria. As a mimic of AMPs, peptoids with N‐substituted glycine backbone have been utilized for antimicrobials with resistance against proteolytic degradation. Antimicrobial peptoids are known to kill bacteria by membrane disruption; however, the nonspecific aggregation of intracellular contents is also suggested as an important bactericidal mechanism. Here,structure‐activity relationship (SAR) of a library of indole side chain‐containing peptoids resulting in peptoid 29 as a hit compound is investigated. Then, quantitative morphological analyses of live bacteria treated with AMPs and peptoid 29 in a label‐free manner using optical diffraction tomography (ODT) are performed. It is unambiguously demonstrated that both membrane disruption and intracellular biomass flocculation are primary mechanisms of bacterial killing by monitoring real‐time morphological changes of bacteria. These multitarget mechanisms and rapid action can be a merit for the discovery of a resistance‐breaking novel antibiotic drug.

Structural Patterns Enhancing the Antibacterial Activity of Metallacarborane-Based Antibiotics

“…Through the use of holotomographic microscopy, the morphology of bacterial cells can be described in more detail through refractive index measurements (RI) in all three dimensions (3D). 49 Analysis of 3D holotomographs allows the measurement of various parameters related to the size and shape of the cell (volume, surface area, sphericity) as well as their contents (dry mass, protein concentration, mean RI). In this experiment, we measured two populations (n = 65) of treated and untreated S. aureus ATCC 6538 cells and compared them to each other.…”

“…These morphological changes were significantly less abundant in untreated bacterial cells (Figure A–D). Through the use of holotomographic microscopy, the morphology of bacterial cells can be described in more detail through refractive index measurements (RI) in all three dimensions (3D) . Analysis of 3D holotomographs allows the measurement of various parameters related to the size and shape of the cell (volume, surface area, sphericity) as well as their contents (dry mass, protein concentration, mean RI).…”

Antimicrobial Peptide–Peptoid Hybrids with and without Membrane Disruption

“…Indeed, TEM micrographs of bacterial cells exposed to EB9 showed the aggregation of intracellular components as the major effect, without visible membrane disruption. This intracellular action is comparable to that reported for several nonmembrane-disruptive peptoids. − …”

“…Although lacking hydrogen-bonding amide NH groups and therefore unable to form canonical peptide secondary structures, peptoids designed to fold into amphiphilic polyproline-like type I helices by introducing chiral side chains have been shown to display membrane-disruptive antibacterial activities and tunable helicity and toxicity. − Interestingly however, polycationic and amphiphilic antimicrobial peptoids were also reported which appear not to act as membrane disruptors but rather on intracellular targets. − Such intracellular targeting is reminiscent of proline-rich antimicrobial peptides (PrAMPs) such as the nonadecapeptide oncocin (VDKPPYLPRPRPPRRIYNR), which have a reduced number of amide NH groups and act on intracellular targets including various sites on the ribosome as well as the heat shock protein DnaK (Hsp70). − This analogy suggests that gradually substituting amino acids with their peptoid equivalents in an AMP sequence, and thereby reducing the number of backbone amide NH groups, might decrease membrane-disruptive effects and at some point enable intracellular targeting. Although introducing peptoid building blocks has been previously investigated as a method to tune AMP activity, − a systematic study of the effect of peptoid building blocks on AMP antibacterial activity and mechanism has not been previously reported.…”

Improving Specificity and Axial Spatial Resolution of Refractive Index Imaging by Exploiting Uncorrelated Subcellular Dynamics

“…HT’s inverse scattering approach enables rapid and accurate reconstruction of the 3D RI distribution of individual cells. Its label-free and quantitative imaging prowess has quickly propelled its application across diverse biological domains, from cell biology and microbiology, − to the niches of phase separation in biology, − lipid imaging, − immunology, preclinical research, and regenerative medicine …”