(256g) Protegrin Has Distinct Mechanisms of Action On Prokaryotes and Eukaryotes As Defined by Unique Morphological Changes

Each year in the US, tens of millions of unnecessary antibiotic prescriptions are written (1), livestock feeds are supplemented with over 29 million pounds of antibiotics (2) and at least 50,000 pounds of antibiotics are applied to crops (3). In combination with additional factors, this overuse of antibiotics has caused antibiotic resistance to become a significant concern in the fields of medicine, public health, agriculture and ecology. As the problem grows, alternative antimicrobial treatments are becoming increasingly important areas of study. Antimicrobial peptides (AMPs) are one alternative antimicrobial route currently being pursued. They have a much different activity on bacteria when compared to traditional antibiotics and it is substantially more difficult for bacteria to develope resistance to this kind of activity.

Our work presented here focuses on the transmembrane, pore forming, AMP protegrin (PG-1). PG-1 is highly active across dozens of bacterial strains; unfortunately, it is also toxic to eukaryotic cells. Throughout the last decade, significant efforts have been concentrated on first identifying the killing mechanisms of PG-1 and then engineering mutant peptides that not only remain active against bacteria but also have decreased toxicity relative to eukaryotic cells. To our knowledge, neither of these goals has been decisively realized so far.

Guided by molecular models and experimental observations, we have explicitly identified PG-1’s unique mechanism of action on both prokayotes and eukaryotes. Specifically, the flux of intracellular K+ ions out of the cell via the PG-1 pores is the key to bacterial cell death onset. In contrast, water flux across the cell membrane appears to be critical in eukaryotic cell death onset by PG-1. Notably, we have found no evidence that PG-1 elicits cell death through a means besides its biophysical effects on the cell membrane integrity and membrane potential.  Intracellular domains do not appear to initiate cell death through participation in signal cascades as was previously suggested (4). Based on this characterization, we can now systematically engineer variant PG-1 peptides that maintain very high antibacterial activity in parallel with low toxicity to eukaryotic systems. By doing so, we can custom design peptides for maximum effectiveness against specific microbes of interest while minimizing the toxicity to other cell types.

1. American College of Physicians. Antibiotic Resistance.   http://www.acponline.org/patients_families/diseases_conditions/antibiotic_resistance/ 29 April 2011.
2. Food and Drug Administration. 2009 Summary Report on Antimicrobials Sold or Disbributed for Use in Food Producing Animals.  http://www.fda.gov/downloads/ForIndustry/UserFees/AnimalDrugUserFeeActADUFA/UCM231851.pdf 29 April 2011.
3. National Agricultural Statistics Service. Agricultural Chemical Usage, 1995 Fruits Summary. No. 96172. U.S. Dept. Agriculture.
4. Mangoni M et al. Change in membrane permeability induced by protegrin 1: implication of disulphide bridges and pore formation. FEBS Letters. 383 (1996) 93-98.