Pathogenic infections represent a persistent threat to human health. The rapid development of resistance to drug therapies creates a continuing need for new anti-infective agents. The proposed research focuses on the development of sequence-random copolymers having a nylon-3 backbone as general, inexpensive antibacterial agents. We will synthesize and characterize a variety of these copolymers designed to mimic the activity of natural host-defense peptides (HDPs), which have been discovered in plants, animals, and humans. HDPs are remarkable for their general ability to halt growth of both Gram negative and Gram positive bacteria while leaving animal cells largely unaffected. The relative inability of bacteria to resist HDPs is usually attributed to a general mode of action involving degradation of bacterial cell membranes. Cationic HDPs are known to form globally amphipathic helices (hydrophobic side chains on one side, charged and other hydrophilic side chains on the opposite side) when they bind to anionic cell membranes. In contrast, the nylon-3 copolymers of interest contain a random sequence of cationic and hydrophobic side chains;they also vary in length. Preliminary work suggests that the random copolymers attack membranes by forming irregular amphipathic surface structures enabled by the flexibility of the nylon-3 backbone. Several of the random nylon-3 copolymers have bacteriostatic properties rivaling those of natural HDPs;they hemolytically attack red blood cells only at high concentration. This work will obtain a better fundamental understanding of how these copolymers degrade membranes in order to inform design improvements. The approach includes both chemical synthesis and detailed analysis of function by single-cell fluorescence imaging. The synthetic methodology enables control of mean copolymer length and the type and percentage of hydrophobic, cationic, and polar side chains. For each individual cell, the analytical methods enable direct correlation in real time of the development of bacterial "symptoms" with the amount of antimicrobial polymer absorbed and the halting of cell growth. The initial work will focus on E. coli and B. subtilis as representative Gram negative and Gram positive species. Observable symptoms include translocation across the outer membrane (OM), lysing of the OM, translocation across the cytoplasmic membrane (CM) and lysing of the CM. The same techniques will determine the special properties of "survivor cells" that are unusually resistant to attack. Time lapse observations after restoration of normal growth medium will reveal which short-term symptoms are sufficient to kill cells, i.e. to prevent subsequent recovery and growth. Detailed mechanistic data from the experiments will feed back into the effort to design cheap and effective antimicrobial polymers. The novel techniques and concepts developed in this work will find wide application in all efforts to design antimicrobial agents involving membrane-based functions. Random copolymers may find applications in the context of antifungal activity, antiplasmodial activity, and lung-surfactant activity as well.

Public Health Relevance

Bacterial infections are increasingly resistant to antibiotic therapies. This work explores random copolymers based on the nylon-3 backbone as a new class of antibacterial agents. Fundamental insights from detailed experimental work will inform the design of more effective antibacterial polymers. These can be synthesized in large quantities at low cost.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM093265-02
Application #
8301533
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
2011-08-01
Project End
2015-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
2
Fiscal Year
2012
Total Cost
$316,879
Indirect Cost
$96,879
Name
University of Wisconsin Madison
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Lee, Michelle W; Chakraborty, Saswata; Schmidt, Nathan W et al. (2014) Two interdependent mechanisms of antimicrobial activity allow for efficient killing in nylon-3-based polymeric mimics of innate immunity peptides. Biochim Biophys Acta 1838:2269-79
Liu, Runhui; Chen, Xinyu; Falk, Shaun P et al. (2014) Structure-activity relationships among antifungal nylon-3 polymers: identification of materials active against drug-resistant strains of Candida albicans. J Am Chem Soc 136:4333-42
Liu, Runhui; Suárez, Jose M; Weisblum, Bernard et al. (2014) Synthetic polymers active against Clostridium difficile vegetative cell growth and spore outgrowth. J Am Chem Soc 136:14498-504
Chakraborty, Saswata; Liu, Runhui; Hayouka, Zvi et al. (2014) Ternary nylon-3 copolymers as host-defense peptide mimics: beyond hydrophobic and cationic subunits. J Am Chem Soc 136:14530-5
Bakshi, Somenath; Choi, Heejun; Rangarajan, Nambirajan et al. (2014) Nonperturbative imaging of nucleoid morphology in live bacterial cells during an antimicrobial peptide attack. Appl Environ Microbiol 80:4977-86
Liu, Runhui; Chen, Xinyu; Chakraborty, Saswata et al. (2014) Tuning the biological activity profile of antibacterial polymers via subunit substitution pattern. J Am Chem Soc 136:4410-8
Bakshi, Somenath; Choi, Heejun; Mondal, Jagannath et al. (2014) Time-dependent effects of transcription- and translation-halting drugs on the spatial distributions of the Escherichia coli chromosome and ribosomes. Mol Microbiol 94:871-87
Choi, Heejun; Chakraborty, Saswata; Liu, Runhui et al. (2014) Medium effects on minimum inhibitory concentrations of nylon-3 polymers against E. coli. PLoS One 9:e104500
Hayouka, Zvi; Chakraborty, Saswata; Liu, Runhui et al. (2013) Interplay among subunit identity, subunit proportion, chain length, and stereochemistry in the activity profile of sequence-random peptide mixtures. J Am Chem Soc 135:11748-51
Liu, Runhui; Chen, Xinyu; Hayouka, Zvi et al. (2013) Nylon-3 polymers with selective antifungal activity. J Am Chem Soc 135:5270-3

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