Abstract
A fundamental issue in the design and development of antimicrobials is the lack of understanding of complex modes of action and how this complexity affects potential pathways for resistance evolution. Bac8c (RIWVIWRR-NH2) is an 8 amino acid antimicrobial peptide (AMP) that has been shown to have enhanced activity against a range of pathogenic Gram-positive and Gram-negative bacteria, as well as yeast. We have previously demonstrated that Bac8c appears to interfere with multiple targets, at least in part through the disruption of cytoplasmic membrane related functions, and that resistance to this peptide does not easily develop using standard laboratory methods. Here, we applied a genomics approach, SCalar Analysis of Library Enrichement (SCALEs), to map the effect of gene overexpression onto Bac8c resistance in parallel for all genes and gene combinations (up to ∼ 10 adjacent genes) in the E. coli genome (a total of ∼ 500,000 individual clones were mapped). Our efforts identified an elaborate network of genes for which overexpression leads to low-level resistance to Bac8c (including biofilm formation, multi-drug transporters, etc). This data was analyzed to provide insights into the complex relationships between mechanisms of action and potential routes by which resistance to this synthetic AMP can develop.
Highlights
A major barrier to the development of antimicrobial peptidebased therapies is the lack of a complete understanding of the complex modes of killing by antimicrobial peptides (AMPs) [1]
It is smaller than bactenecin, the smallest known broad spectrum natural antimicrobial peptide, but has enhanced activity against a range of pathogenic Gram-positive and Gram-negative bacteria, as well as yeast [9]
We have previously described efforts to decipher the mode of action of Bac8c [10] and have shown that this AMP appears to interfere with multiple targets, apparently through the disruption of cytoplasmic membrane related functions
Summary
A major barrier to the development of antimicrobial peptidebased therapies is the lack of a complete understanding of the complex modes of killing by antimicrobial peptides (AMPs) [1]. Based on the complexity of the Bac8c mode of action, it is not surprising that we were unable to identify mutants with substantial resistance to Bac8c even when we performed comprehensive screening of knockout, over-expression, and chemical mutant libraries (unpublished data). Since this recalcitrance to resistance is a very attractive property for any antimicrobial compound, we sought to improve our understanding of the complex mechanisms employed by this peptide to avoid resistance using a genome-scale library screening method reported previously by our group [11,12,13,14]
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