In Situ Monitoring of Bacteria under Antimicrobial Stress Using 31P Solid-State NMR

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.

Mechanisms of Action for Antimicrobial Peptides With Antibacterial and Antibiofilm Functions

“…In this review, a number of examples that make use of biophysical methods to determine MOAs that involve DNA/RNA targets (Park et al, 1998;Hsu et al, 2005;Nam et al, 2014), protein targets (Mardirossian et al, 2014(Mardirossian et al, , 2018Krizsan et al, 2015), stringent response inhibition (de la Fuente-Núñez et al, 2014;Pletzer et al, 2017b), and degradation of biofilms (Segev-Zarko et al, 2015;Ansari et al, 2017) were presented. A number of examples of how these biophysical approaches can be applied to bacteria (i.e., termed "biological" assays here) (de la Fuente-Núñez et al, 2014;Brannan et al, 2015;Miyoshi et al, 2017;Overall et al, 2019) serve to illustrate not only how the lines between these methods may become increasingly blurred in the future, but also how all the methods presented in this review, as well as direct visualization approaches [e.g., scanning electron microscopy (SEM) (Chileveru et al, 2015)] and other new approaches [e.g., small angle X-ray scattering (Von Gundlach et al, 2016, von Gundlach et al, 2019], will continue to be relevant.…”

“…Recently, DSC has been used to probe the interaction of the AMP MSI-78 in whole bacteria (Brannan et al, 2015). Likewise, 31 P NMR studies on whole bacterial cells have also been reported (Overall et al, 2019).…”

The mitochondria-targeted peptide SS-31 binds lipid bilayers and modulates surface electrostatics as a key component of its mechanism of action

“…Finally, we measured the effects of SS peptides on mitochondrial calcium stress that may occur independently of Ca 2+ uptake into the matrix. To this end, we used a 31 P ssNMR-based strategy to monitor mitochondrial membrane structural integrity, based on previous studies using NMR analysis of membranes from prokaryotic cells (51) or isolated mitochondria (52)(53)(54). We first characterized 31 P ssNMR spectra of our isolated mitochondria in comparison to model membranes (SI Results H and Figure S19), showing that mitochondria spectra contained a complex superposition of lineshapes that could be deconvoluted for the direct analysis of phospholipid bilayers.…”

Molecular Mechanism of Action of Mitochondrial Therapeutic SS-31 (Elamipretide): Membrane Interactions and Effects on Surface Electrostatics

“…7H-J). To this end, we used a 31 P ssNMR-based strategy to monitor mitochondrial membrane structural integrity, based on previous studies using NMR analysis of membranes from prokaryotic cells (46) or isolated mitochondria (47)(48)(49). We first characterized 31 P ssNMR spectra of our isolated mitochondria in comparison to model membranes (SI Results H and Fig.…”