Cationic π-Conjugated Polyelectrolyte Shows Antimicrobial Activity by Causing Lipid Loss and Lowering Elastic Modulus of Bacteria

Abstract

Cationic, π-conjugated oligo-/polyelectrolytes (CCOEs/CCPEs) have shown great potential as antimicrobial materials to fight against antibiotic resistance. In this work, we treated wild-type and ampicillin-resistant (amp-resistant) Escherichia coli (E. coli) with a promising cationic, π-conjugated polyelectrolyte (P1) with a phenylene-based backbone and investigated the resulting morphological, mechanical, and compositional changes of the outer membrane of bacteria in great detail. The cationic quaternary amine groups of P1 led to electrostatic interactions with negatively charged moieties within the outer membrane of bacteria. Using atomic force microscopy (AFM), high-resolution transmission electron microscopy (TEM), we showed that due to this treatment, the bacterial outer membrane became rougher, decreased in stiffness/elastic modulus (AFM nanoindentation), formed blebs, and released vesicles near the cells. These evidences, in addition to increased staining of the P1-treated cell membrane by lipophilic dye Nile Red (confocal laser scanning microscopy (CLSM)), suggested loosening/disruption of packing of the outer cell envelope and release and exposure of lipid-based components. Lipidomics and fatty acid analysis confirmed a significant loss of phosphate-based outer membrane lipids and fatty acids, some of which are critically needed to maintain cell wall integrity and mechanical strength. Lipidomics and UV-vis analysis also confirmed that the extracellular vesicles released upon treatment (AFM) are composed of lipids and cationic P1. Such surface alterations (vesicle/bleb formation) and release of lipids/fatty acids upon treatment were effective enough to inhibit further growth of E. coli cells without completely disintegrating the cells and have been known as a defense mechanism of the cells against cationic antimicrobial agents.

Keywords: antibiotic-resistant; antimicrobial; cationic conjugated polyelectrolytes; elastic modulus; lipid loss; outer membrane.

Figure 1.

Structure of cationic π-conjugated polyelectrolyte (P1) used in this work.

Figure 2.

AFM height images of wild-type (a-c) and amp-resistant (d-f) E. coli cells treated with 5 0 (a, d), 30 (b, e), and 100 (c, f) μM P1.

Figure 3.

The average surface roughness values (with standard deviation) of untreated wild-type and amp-resistant E. coli cells (blue), treated with 30 μM (green), and 100 μM (red) P1. In the AFM height images (after first order flattening process), 30 (200×200 nm²) regions were randomly selected on at least 10 cells and used for roughness measurement. An unpaired t-test was used for calculating the p-value. The symbol ** represents p < 0.05 and *** represents p < 0.0005 when compared with untreated cells.

Figure 4.

AFM amplitude images of wild-type (a-c) and amp-resistant (d-f) E. coli cells treated 15 with 0 (a, d), 30 (b, e), and 100 (c, f) μM P1.

Figure 5.

TEM micrographs of untreated (a, b) and 100 μM P1-treated (c, d) amp-resistant E. coli cells. The cells were stained with uranyl acetate and lead acetate. The elliptical and circular shapes seen for intact cells were due to the orientation of the cells during the cross-section of the samples using the ultramicrotome. Red arrows indicate the formation of blebs, while the yellow arrow indicates a treated E. coli cell from which cytoplasmic content was released.

Figure 6.

Histograms (with Gaussian distributions (red lines)) of Young’s modulus (a-c) and force maps (d-f) for wild-type E. coli treated with 0 (a, d), 30 (b, e), and 100 (c, f) μM P1.

Figure 7.

Histograms (with Gaussian distributions (red lines)) of Young’s modulus (a-c) and force maps (d-f) for amp-resistant E. coli treated with 0 (a, d), 30 (b, e), and 100 (c, f) μM P1.

Figure 8.

Bright-field (a, b) and fluorescence (c, d) CLSM images of untreated (a, c), and amp-resistant E. coli cells treated with 100 μM P1 for 1 h (b, d). The scale bar is 20 μm.

Figure 9.

Lipid (a) and fatty acid (b) contents (nmol/cell) in untreated and P1-treated E. coli cells. The symbol ** represents p < 0.05 and shows a significant difference between the results.

Figure 10.

Proposed mechanism of action of P1 on wild-type and amp-resistant E. coli cells.