Characterizing the role of the cell wall in bacterial death caused by treatment with antimicrobial peptides.

Abstract

The rise in pathogenic bacteria becoming resistant to all commercially available antibiotics has compelled scientists to develop new antimicrobial compounds while also identifying effective bacterial targets. Antimicrobial peptides (AMPs), which are part of many different species’ nonspecific immune responses, have shown promising results in eliminating invading bacteria. Several classes of AMPs selectively target bacterial membranes, which have been identified prior as a good target for new antibiotics. The cationic AMP magainin 2 (MAG2) folds into an alpha helix upon contact with the negatively charged bacterial cell wall. Several folded subunits come together to form pores in the bacterial membranes, which leads to the uncontrolled release of cytosol and, eventually, cell death. Using minimum inhibitory concentration (MIC) assays, we found that Staphylococcus epidermidis (Gram positive) was more susceptible to MAG2 than E. coli (Gram negative). We used propidium iodide uptake assays and autofluorescence to evaluate the role of the cell wall in MAG2 activity and bacterial death. We found that despite the increased sensitivity of E. coli to MAG2, pore formation takes longer to reach equilibrium in E. coli than in the S. epidermidis, which is likely due to the difference in the cell walls. We also used fluorescence microscopy and growth assays in order to determine when cell death truly occurred. We found that S. epidermidis was able to regrow at sub-MIC concentrations which shows us that cell membrane integrity is not an adequate way to define cell death.