Date Published: May 1, 2014
Publisher: Public Library of Science
Author(s): Justyna Kozlowska, Louic S. Vermeer, Geraint B. Rogers, Nabila Rehnnuma, Sarah-Beth T. A. Amos, Garrit Koller, Michael McArthur, Kenneth D. Bruce, A. James Mason, Jeff H. Chang.
Obtaining an in-depth understanding of the arms races between peptides comprising the innate immune response and bacterial pathogens is of fundamental interest and will inform the development of new antibacterial therapeutics. We investigated whether a whole organism view of antimicrobial peptide (AMP) challenge on Escherichia coli would provide a suitably sophisticated bacterial perspective on AMP mechanism of action. Selecting structurally and physically related AMPs but with expected differences in bactericidal strategy, we monitored changes in bacterial metabolomes, morphological features and gene expression following AMP challenge at sub-lethal concentrations. For each technique, the vast majority of changes were specific to each AMP, with such a plastic response indicating E. coli is highly capable of discriminating between specific antibiotic challenges. Analysis of the ontological profiles generated from the transcriptomic analyses suggests this approach can accurately predict the antibacterial mode of action, providing a fresh, novel perspective for previous functional and biophysical studies.
The isolation of cecropins , magainins  and defensins  from insects, amphibians and mammals in the late 1980’s and early 1990’s, highlighted the potential of host defence peptides as sources of novel antibiotics . This novel antibiotic potential encouraged researchers to develop structure activity relationships for cationic antimicrobial peptides (AMPs), with the anionic bacterial plasma membrane the presumed site of action for bactericidal activity . There is increasing evidence however that each AMP may indeed have multiple effects on a bacterial cell and hence may have multiple ways of killing microbial targets. AMPs may therefore function as “dirty drugs” with different bactericidal strategies possible for distinct bacterial species –. Indeed, the innate immune system may have selected AMPs that can exert their antimicrobial activity in multiple ways since this is less likely to lead to resistance developing as seen with classical antibiotics that have a single, high affinity target . Our understanding of how AMPs function is therefore far from complete. Attempts to optimize AMP potency in the laboratory, that focus on only one possible bactericidal mechanism, ignore the possibilities offered by taking a holistic approach that can reveal the true source(s) of bactericidal potency along with a better understanding of bacterial counter-measures.
1H HR-MAS NMR metabolomics reveals threshold AMP concentration inducing a bacterial response –The four cationic amphipathic AMPS selected to test the response of stationary phase E. coli (Table 1) were of similar length and were all C-terminally amidated with nominal charge ranging from +4 to +9. For the AMP challenge experiments presented here, higher bacterial cell densities (8×108 CFU/ml) were required than is common in the broth microdilution assays  used to generate the MIC data (Table 1), in particular for HR-MAS analysis. At the higher bacterial titre, although the relative potency is similar, the effect of the four AMPs determined using such methods was somewhat different from the minimum inhibitory concentrations (MICs), with D-LAK120-AP13 having a substantially greater effect on bacterial numbers as detected in the challenge and recovery assay (Fig. 1A). Neither magainin 2 nor buforin II had a sufficiently inhibitory effect for a MIC to be determined at the higher titre. Nevertheless the amount of peptide causing a significant reduction in bacterial re-growth can be compared with D-LAK120AP13 effective at 15.6 µg/ml with substantially more pleurocidin (62.5 µg/ml) and magainin 2 (125 µg/ml) required for a significant effect. No effect on bacterial re-growth was observed for buforin II at any of the peptide concentrations tested (Fig. 1A). A multi-parameter assay was taken to assess the effect of peptide challenge on membrane potential (Fig. 1B), esterase activity (Fig. 1C) and membrane integrity in the challenged stationary phase bacteria and suggested that only the higher concentrations of pleurocidin and D-LAK120-AP13 were lethal. A dose dependent response to each of the four AMPs was observed but the membrane potential was not completely lost while the esterase activity was mostly higher than that observed in untreated cells; a hallmark of exposure to sublethal stress in E. coli.