Abstract
In-cell NMR offers great insight into the characterization of the effect of toxins and antimicrobial peptides on intact cells. However, the complexity of intact live cells remains a significant challenge for the analysis of the effect these agents have on different cellular components. Here we show that 31P solid-state NMR can be used to quantitatively characterize the dynamic behaviour of DNA within intact live bacteria. Lipids were also identified and monitored, although 31P dynamic filtering methods indicated a range of dynamic states for phospholipid headgroups. We demonstrate the usefulness of this methodology for monitoring the activity of the antibiotic ampicillin and the antimicrobial peptide (AMP) maculatin 1.1 (Mac1.1) against Gram-negative bacteria. Perturbations in the dynamic behaviour of DNA were observed in treated cells, which indicated additional mechanisms of action for the AMP Mac1.1 not previously reported. This work highlights the value of 31P in-cell solid-state NMR as a tool for assessing the antimicrobial activity of antibiotics and AMPs in bacterial cells.
Highlights
The emergence of bacterial strains resistant to many important front line antibiotics has fuelled the urgency for which new efficacious antimicrobial agents are being developed
After 12 h the number of colony forming units (CFUs) decreased by 70% at 30 ◦C while cell survival was greatly improved by maintaining the sample at 10 ◦C, in which CFU declined by only 33% over the same period (Figure 1A). 31P static spectra collected at 10 and 30 ◦C showed a superposition of lineshapes, indicative of a wide range of 31P environments (Figure 1B,C)
In-cell NMR provides a means for understanding the molecular effects of antimicrobial agents in a cellular context
Summary
The emergence of bacterial strains resistant to many important front line antibiotics has fuelled the urgency for which new efficacious antimicrobial agents are being developed. The targeted disruption of bacterial membranes by AMPs can occur by several mechanisms from pore formation to detergent like solubilization of membranes [6,7,8]. It is unclear what the contribution of membrane disruption is to the bactericidal properties of AMPs. The complex architecture of the bacterial membrane could modulate the mode of action of AMPs in situ, altering the threshold of inhibitory concentrations. There are still unknowns in understanding how AMPs interact with bacteria and, in particular, how bacteria respond to AMP activity in situ
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