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Atomic Force Microscopy Measurements of Type IV Pili and the Nanomechanical Response of Bacteria to Cationic Antimicrobial Peptides

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Title: Atomic Force Microscopy Measurements of Type IV Pili and the Nanomechanical Response of Bacteria to Cationic Antimicrobial Peptides
Author: Lu, Shun
Department: Department of Physics
Program: Physics
Advisor: Dutcher, John
Abstract: We have used an AFM-based creep deformation technique to measure changes to the viscoelastic properties of individual Pseudomonas aeruginosa PAO1 cells after exposure to two cationic peptides: polymyxin B (PMB), and polymyxin B nonapeptide (PMBN), which are structurally-related compounds. These measurements provide a distinctive signature for the loss of integrity of the bacterial cell envelope following exposure to the peptides. Measurements performed before and after sufficiently long exposure to the peptides, as well as time-resolved measurements following the introduction of the peptides, revealed large changes to the viscoelastic parameters that we can attribute to different components of the bacterial cells. Differences between the results for exposure to the two peptides are consistent with the difference between their membrane permeabilizing effects. Large differences were also observed for exposure to high and low concentrations of PMB. These measurements provide new, unique insight into the kinetics and mechanism of action of antimicrobial peptides on bacterial cells. We also applied AFM as a single molecule force microscopy (SMFM) to study the mechanical properties of type IV pili (T4P) filaments on P. aeruginosa bacterial cells as polar appendages. Force-separation curves, obtained during retraction of AFM tip away from underlying surfaces, show two different behaviours: stretching T4P with a fixed contact point (described by the Worm-like chain (WLC) model) and a forced desorption of T4P filaments off the underlying surfaces (indicated by a force plateau). The results can provide valuable insights on questions difficult to answer in previous studies. We showed that T4P filaments can bind to surfaces with multiple adhesins distributed along the pili instead of a single adhesin located at the distal end. Moreover, different rupture forces on gold and mica surfaces suggest that the breakage in force curves is due to the failure of adhesion between the pili filaments and underlying surfaces. A more accurate persistence length of 1.4 nm was measured for type IV pili filament, using a 600 nm colloidal probe to reduce AFM tip height effect in force curves. Our study can shed new light on bacterial pili adhesion mechanism and its role to sustain external forces in flows.
Date: 2014-01
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