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https://doi.org/10.1016/j.bpj.2016.11.2075
Copy DOIJournal: Biophysical Journal | Publication Date: Feb 1, 2017 |
Citations: 1 | License type: publisher-specific-oa |
Antimicrobial peptides (AMPs) have long been considered excellent candidates for development as antibiotic agents to combat the threat of drug resistant bacterial pathogens. Yet, the promise AMPs have displayed in the laboratory has not yielded correlative clinical success. We hypothesized that a major reason for the disparity between outcomes at the bench and in the clinic is AMP interaction with and toxicity towards host cells. Here, we validate this theory using human red blood cells as model eukaryotic cells. Indeed, the potency of several well-studied antimicrobial peptides decreases from minimum sterilizing concentrations (MSC) of 1-5 μM to >30 μM in the presence of RBCs at 1x109 cells/ml; equivalent to 20% of the human physiological concentration. To address this issue, we have developed a high-throughput method to screen a combinatorial peptide library for antimicrobial activity in the presence of concentrated human cells. The screen consists of assays that assess the effect of RBCs on antimicrobial activity as well as toxicity toward eukaryotic cells. We present data on peptides identified and characterized from the high-throughput library screen and show that these new peptides were not as effective as they initially appeared during the screen. We demonstrate that the difference between observed and expected results is due to unforeseen proteolytic activity sequestered within the cytosol of the red blood cells. Finally, we show that a library consensus sequence synthesized using all d-amino acids outperforms all library isolates as well as the template sequence in terms of microbicidal activity in the presence of RBCs. Ultimately, our results suggest that combining the power of combinatorial library screening with the nuance of rational sequence engineering is an effective approach to engineering novel antimicrobial peptides that retain activity under physiological conditions.
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