Side-Chain Amino Acid-Based Cationic Antibacterial Polymers: Investigating the Morphological Switching of a Polymer-Treated Bacterial Cell

Abstract

Synthetic polymer-based antimicrobial materials destroy conventional antibiotic resistant microorganisms. Although these antibacterial polymers imitate the properties of antimicrobial peptides (AMPs), their effect on bacterial cell morphology has not been studied in detail. To investigate the morphology change of a bacterial cell in the presence of antimicrobial polymer, herein we have designed and synthesized side-chain amino acid-based cationic polymers, which showed efficient antibacterial activity against Gram-negative (Escherichia coli), as well as Gram-positive (Bacillus subtilis) bacteria. Morphological switching from a rod shape to a spherical shape of E. coli cells was observed by field emission-scanning electron microscopy analysis due to cell wall disruption, whereas the B. subtilis cell structure and size remained intact, but stacks of the cells formed after polymer treatment. The zone of inhibition experiment on an agar plate for E. coli cells exhibited drastic morphological changes at the vicinity of the polymer-treated portion and somewhat less of an effect at the periphery of the plate.

Scheme 1. Synthesis of Amino Acid-Based Homopolymers and Block Copolymers by RAFT Polymerization, Followed by Deprotection of Side-Chain Boc Groups

Figure 1

Zone of inhibition (circled portion) against E. coli treatment: (A) control (without polymer), (B) treated with P(Ala-HEMA)₁₄, (C) P(Leu-HEMA)₁₅, (D) P(Phe-HEMA)₁₀, (E) P(Ala-HEMA)₁₄-b-PPEGMA₆₀, and (F) P(Ala-HEMA)₁₄-b-PMMA₃₇ at (1) 50 μL, (2) 100 μL, and (3) 200 μL from 10 mg/mL stock solution and zoomed view of zone of inhibition treatment with (G) P(Ala-HEMA)₁₄, (H) P(Leu-HEMA)₁₅, and (I) P(Phe-HEMA)₁₀. Bacterial growth is not inhibited in control disk in the absence of polymer, but when the disk was loaded with polymer, the inhibition zone was prominent for three homopolymers and expanded with increasing concentration of the polymer solution, on the contrary, no clear inhibitory effect of the two block copolymers was observed. Each experiment was run in duplicate.

Figure 2

Optical microscope images of E. coli cells following Gram staining: (A) control (40× resolution), (B) control (100× resolution), (C) treated with P(Ala-HEMA)₁₄ within the zone of inhibition (100× resolution), (D) treated with P(Ala-HEMA)₁₄ away from the zone of inhibition (100× resolution), (E) treated with P(Leu-HEMA)₁₅ within the zone of inhibition (100× resolution), (F) treated with P(Leu-HEMA)₁₅ away from the zone of inhibition (100× resolution), (G) treated with P(Phe-HEMA)₁₀ within the zone of inhibition (100× resolution), and (H) treated with P(Phe-HEMA)₁₀ away from the zone of inhibition (100× resolution). Polymer-treated bacterial cells appear to be stacked and CV color is retained in the vicinity of the zone of inhibition and the effect becomes less prominent with increasing distance from the inhibitory zone.

Figure 3

FESEM images of E. coli cells: (A) control, where the smooth bacterial cell membrane was preserved, (B) treated with P(Ala-HEMA)₁₄ within the zone of inhibition, stacking of cells was observed, (C) treated with P(Ala-HEMA)₁₄ away from the zone of inhibition, presence of corrugated cell surface was found, (D) treated with P(Leu-HEMA)₁₅ within the zone of inhibition, the cells were stacked through leakage of cytoplast and a spherical morphology appeared, (E) treated with P(Leu-HEMA)₁₅ away from the zone of inhibition, spherical cells and cell debris were observed, (F) treated with P(Phe-HEMA)₁₀ within the zone of inhibition, cleavage of bacterial cell was found during treatment, (G) treated with P(Phe-HEMA)₁₀ away from the zone of inhibition, debris of polymer-treated cells appeared, and (H) treated with P(Leu-HEMA)₁₅ at the vicinity of polymer-treated region, bacterial cells appear as spherical.

Figure 4

Growth curve of E. coli cells in LB media in the presence and absence of P(Leu-HEMA)₁₅. For the control experiment, where the polymer was absent, exponential cell growth was observed, and this was absent in the presence of the polymer.

Figure 5

FESEM images of E. coli during bacterial growth in LB media: control (without P(Leu-HEMA)₁₅ polymer) images of bacterial cell from (A) congested cell area and (B) discrete cell area, where cell size and morphology were intact; P(Leu-HEMA)₁₅ treated cell images from (C) congested cell area (sheetlike structure) and (D) discrete cell area (stacking of cells) after 7 h incubation.

Figure 6

Step 1: positively charged polymer disrupts the OM of Gram-negative bacterial cell wall through electrostatic interactions. Step 2: polymer penetrates the intermediate peptidoglycan layer and interacts with the IM through cleavable intermediate morphological variation. Step 3: total morphological switching of bacterial cell from rod shape to spherical shape with destruction of inner cell membrane.

Figure 7

Zone of inhibition for B. subtilis treatment with P(Leu-HEMA)₁₅: (A) control (without polymer), (B) after polymer treatment, and zoomed view of zone of inhibition at (C) 50 μL, (D) 100 μL, and (E) 200 μL from 10 mg/mL stock solution. Inhibitory effect is localized and area of zone of inhibition increases with increasing concentration of P(Leu-HEMA)₁₅ solution. Each experiment was run in duplicate.

Figure 8

FESEM images of B. subtilis cells: (A) control (without polymer treatment), P(Leu-HEMA)₁₅ treated cells (B) near and (C) away from the zone of inhibition. During polymer treatment, stacking of cells was observed, although overall cell morphology and average cell length remain unchanged from control set.