Qualitative and Quantitative Changes to Escherichia coli during Treatment with Magainin 2 Observed in Native Conditions by Atomic Force Microscopy

Abstract

The bacterial membrane has been suggested as a good target for future antibiotics, so it is important to understand how naturally occurring antibiotics like antimicrobial peptides (AMPs) disrupt those membranes. The interaction of the AMP magainin 2 (MAG2) with the bacterial cell membrane has been well characterized using supported lipid substrates, unilamellar vesicles, and spheroplasts created from bacterial cells. However, to fully understand how MAG2 kills bacteria, we must consider its effect on the outer membrane found in Gram-negative bacteria. Here, we use atomic force microscopy (AFM) to directly investigate MAG2 interaction with the outer membrane of Escherichia coli and characterize the biophysical consequences of MAG2 treatment under native conditions. While propidium iodide penetration indicates that MAG2 permeabilizes cells within seconds, a corresponding decrease in cellular turgor pressure is not observed until minutes after MAG2 application, suggesting that cellular homeostasis machinery may be responsible for helping the cell maintain turgor pressure despite a loss of membrane integrity. AFM imaging and force measurement modes applied in tandem reveal that the outer membrane becomes pitted, more flexible, and more adhesive after MAG2 treatment. MAG2 appears to have a highly disruptive effect on the outer membrane, extending the known mechanism of MAG2 to the Gram-negative outer membrane.

Extracellular Polymeric Substance Protects Some Cells in an Escherichia coli Biofilm from the Biomechanical Consequences of Treatment with Magainin 2

“…The minimum inhibitory concentration for biofilm formation (MICB) was defined as the lowest concentration of MAG2 that prevented visible biofilm formation in the wells of a 48-well plate. The MICB of MAG2 was determined using a previously published protocol altered for investigating biofilm growth rather than planktonic cell growth [ 25 , 31 ]. After preparing E. coli ZK1056 (from M. O. Martin and R. Kolter) in the same manner described in [ 25 ], 100 µL of bacteria was transferred to each well in a row.…”

“…We previously used atomic force microscopy (AFM) to better understand the cellular biophysics of bacteria [ 25 , 26 , 27 ]. AFM is a scanning probe technique which produces high resolution images and force-distance (FD) curves, and can be used to monitor individual cells during treatment [ 28 ].…”

“…AFM is a scanning probe technique which produces high resolution images and force-distance (FD) curves, and can be used to monitor individual cells during treatment [ 28 ]. We recently used AFM to investigate planktonic Escherichia coli ( E. coli ) cells treated with MAG2, demonstrating significant biophysical changes occurring over 30 min of treatment that directly correspond to the known mechanism of action for MAG2 [ 25 ]. Cells treated with MAG2 had decreased turgor pressure and increased surface roughness after treatment, illustrating that MAG2 had successfully formed pores in the inner and outer membranes of the E. coli , and that these cells could no longer maintain membrane integrity.…”

Physics Comes to the Aid of Medicine—Clinically-Relevant Microorganisms through the Eyes of Atomic Force Microscope

“…While melittin causes big gashes in the cell envelope and noticeable leakage of cytosolic fluid, indicating the damage to the inner membrane, Mag 2a was reported to cause pronounced vesiculation of the outer membrane [ 28 ]. Most recently, Overton et al extended the knowledge on Mag 2a-induced killing and investigated the biophysical consequences of magainin 2 treatment using AFM combined with fluorescence observations [ 63 ]. The authors demonstrated that upon treatment, E. coli cells are able to maintain the stable cellular turgor pressure, despite the permeabilization of cellular membranes by Mag 2a, which is governed by the bacterial homeostasis machinery.…”

“…Further exposition of bacteria to magainin 2 treatment eventually leads to a decrease of turgor pressure accompanied by remodeling of the outer membrane, resulting in its increased elasticity and greater adhesion properties. This provided new insights into the resistance of some gram-negative bacteria to the membrane-destructive effects of antimicrobial peptides [ 63 ].…”