Non-electrostatic interactions associated with aggregate formation between polyallylamine and Escherichia coli

Abstract

Bacterial aggregation by mixing with polymers is applied as pretreatment to identify pathogens in patients with infectious diseases. However, the detailed interaction between polymers and bacteria has yet to be fully understood. Here, we investigate the interaction between polyallylamine and Escherichia coli by isothermal titration calorimetry. Aggregation was observed at pH 10 and the binding was driven by favorable enthalpic gain such as the electrostatic interaction. Neither aggregation nor the apparent heat of binding was observed at pH 4.0, despite the strong positive charge of polyallylamine. These results suggest that intermolecular repulsive forces of the abundant positive charge of polyallylamine cause an increased loss of conformational entropy by binding. Non-electrostatic interaction plays a critical role for aggregation.

Figure 1

ITC measurements of PAA binding with E. coli at (A) pH 10 and (B) pH 4.0. Thermograms for the titration of PAA into E. coli are shown in the upper panels. Normalized changes in heat after subtraction of the heat of dilution are plotted against the molar ratio ([PAA]/[E. coli]) in the lower panels. The one-set of independent binding sites model was used to fit the binding isotherms, and the fitting curves are shown as continuous lines. The lower images show the solutions in ITC cells after the measurements. (C) ITC measurements of PAA binding with E. coli in 150 mM NaCl at pH 10. (D) ITC measurements of 19.7% G-PAA (left), 37.7% G-PAA (middle), and 59.0% (right) G-PAA binding with E. coli at pH 10.

Figure 2

Fluorescence microscopic images of GFP-expressed E. coli incubated with PAA. (A) GFP-expressed E. coli was incubated with PAA (0, 0.0025, 0.025, and 0.25% w/v) at pH 10 or 4.0 for 3 min. Scale bar: 200 μm. The aggregates were visually observed after weak centrifugation at 2000×g for 10 s. (B) GFP-expressed E. coli was incubated with 0.25% PAA or Cy5.5-labeled PAA at pH 10 for 3 min. The fluorescent signals of GFP show the co-localization with Cy5.5-labeled PAA. Left: GFP. Middle: Cy5.5. Right: Merged image. Scale bar: 200 μm.

Figure 3

Size distribution of PAA at pH 10 (blue) and 4.0 (red) from DLS measurements.

Figure 4

Proposed binding model of PAA to E. coli. (A) The pH-dependent conformational change of PAA. At pH 10, PAA shows compact structure as compared with that at pH 4.0. (B) At pH 10, the positive charge of the amine group in PAA binds to the negatively charged cell wall in E. coli through electrostatic interactions, and the structure of PAA becomes more compact. (C) At pH 4.0, PAA did not bind to E. coli, because the intermolecular repulsive forces of the abundant positive charge cause an increased loss of entropy.