A wide variety of naturally expressed anti-microbial peptides (AMPs) have been discovered in plants, animals, and humans. AMPs are remarkably for their general ability to halt growth of both Gram negative and Gram positive bacteria. Cationic AMPs bind to negatively charged bacterial cell membranes and evidently degrade their barrier function, eventually leading to the halting of growth and cell death. The relative inabilit of bacteria to resist this general mode of action makes AMPs and their mimics interesting drug candidates against antibiotic-resistant strains. Almost all mechanistic studies thus far have focused either on synthetic lipid vesicles in vitro or on long-time, bulk effects of AMPs on bacteria. The detailed mechanisms of AMP attack on bacterial membranes are not well understood. This work develops novel fluorescence microscopy assays in order to directly observe the attack of AMPs on bacterial membranes for individual cells in real time. On attack of ?-helical peptides on E. coli, we observe such events as: binding to and diffusion within the outer membrane (OM), translocation across the OM, permeabilization of the OM to periplasmic GFP, abrupt cell shrinkage and the halting of cell growth, permeabilization of the cytoplasmic membrane (CM) to the dye Sytox Green, and the onset of oxidative damage within the cytoplasm. We plan to extend our work from ?-helical AMPs to defensins, the other important class of human AMPs. In addition to studies of the model species E. coli and B. subtilis, we will observe AMP effects on non-pathogenic strains of P. aeruginosa and S. aureus. We will initiate studies of AMP activity against bacteria living in model biofilms. Our results will test the relevance of previous studies on synthetic lipid bilayers to growth-halting mechanisms in real bacteria. It is critical that we develop new means to kill drug-resistant pathogens. By dissecting the steps by which natural AMPs kill bacteria, we will provide new design criteria for synthetic mimics of AMPs that may prove clinically useful. The novel methods developed here will be widely applicable to mechanistic studies of natural AMPs, synthetic copolymers designed to mimic AMPS, and synthetic drugs.

Public Health Relevance

Especially in hospital settings, bacteria are increasingly resistant to drugs. Antimicrobial peptides are the front-line defense against pathogens throughout the animal kingdom, but we do not really understand how they work to kill bacterial cells. This work develops novel imaging methods that enable direct observation of the mechanisms of bacterial killing by antimicrobials with better spatial and time resolution than ever before.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Nie, Zhongzhen
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University of Wisconsin Madison
Schools of Arts and Sciences
United States
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Agrawal, Anurag; Weisshaar, James C (2018) Effects of alterations of the E. coli lipopolysaccharide layer on membrane permeabilization events induced by Cecropin A. Biochim Biophys Acta Biomembr 1860:1470-1479
Yang, Zhilin; Weisshaar, James C (2018) HaloTag Assay Suggests Common Mechanism of E. coli Membrane Permeabilization Induced by Cationic Peptides. ACS Chem Biol 13:2161-2169
Yang, Zhilin; Choi, Heejun; Weisshaar, James C (2018) Melittin-Induced Permeabilization, Re-sealing, and Re-permeabilization of E. coli Membranes. Biophys J 114:368-379
Yang, Zhilin; Choi, Heejun (2018) Single-Cell, Time-Lapse Reactive Oxygen Species Detection in E. coli. Curr Protoc Cell Biol 80:e60
Mustafi, Mainak; Weisshaar, James C (2018) Simultaneous Binding of Multiple EF-Tu Copies to Translating Ribosomes in Live Escherichia coli. MBio 9:
Choi, Heejun; Yang, Zhilin; Weisshaar, James C (2017) Oxidative stress induced in E. coli by the human antimicrobial peptide LL-37. PLoS Pathog 13:e1006481
Mohapatra, Sonisilpa; Choi, Heejun; Ge, Xueliang et al. (2017) Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live Escherichia coli. MBio 8:
Choi, Heejun; Chakraborty, Saswata; Liu, Runhui et al. (2016) Single-Cell, Time-Resolved Antimicrobial Effects of a Highly Cationic, Random Nylon-3 Copolymer on Live Escherichia coli. ACS Chem Biol 11:113-20
Li, Wenting; Bouveret, Emmanuelle; Zhang, Yan et al. (2016) Effects of amino acid starvation on RelA diffusive behavior in live Escherichia coli. Mol Microbiol 99:571-85
Choi, Heejun; Rangarajan, Nambirajan; Weisshaar, James C (2016) Lights, Camera, Action! Antimicrobial Peptide Mechanisms Imaged in Space and Time. Trends Microbiol 24:111-122

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