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Review
. 2020 Oct 30;48(5):2139-2149.
doi: 10.1042/BST20200320.

Studying the surfaces of bacteria using neutron scattering: finding new openings for antibiotics

Nicolò Paracini  1 , Luke A Clifton  2 , Jeremy H Lakey  3
Affiliations
Review

Studying the surfaces of bacteria using neutron scattering: finding new openings for antibiotics

Nicolò Paracini et al. Biochem Soc Trans. .

Abstract

The use of neutrons as a scattering probe to investigate biological membranes has steadily grown in the past three decades, shedding light on the structure and behaviour of this ubiquitous and fundamental biological barrier. Meanwhile, the rise of antibiotic resistance has catalysed a renewed interest in understanding the mechanisms underlying the dynamics of antibiotics interaction with the bacterial cell envelope. It is widely recognised that the key reason behind the remarkable success of Gram-negative pathogens in developing antibiotic resistance lies in the effectiveness of their outer membrane (OM) in defending the cell from antibacterial compounds. Critical to its function, the highly asymmetric lipid distribution between the inner and outer bilayer leaflets of the OM, adds an extra level of complexity to the study of this crucial defence barrier. Here we review the opportunities offered by neutron scattering techniques, in particular reflectometry, to provide structural information on the interactions of antimicrobials with in vitro models of the OM. The differential sensitivity of neutrons towards hydrogen and deuterium makes them a unique probe to study the structure and behaviour of asymmetric membranes. Molecular-level understanding of the interactions between antimicrobials and the Gram-negative OM provides valuable insights that can aid drug development and broaden our knowledge of this critically important biological barrier.

Keywords: antibiotics; biological models; gram-negative bacteria; lipopolysaccharides; neutron scattering; outer membrane.

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Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1.
Figure 1.. The Gram-negative cell envelope and its unique defense barrier.
(a) Schematic representation of the three layers of the Gram-negative cell envelope. Above the plasma membrane the periplasm is 10–20 nm wide and contains the rigid peptidoglycan cell wall, which is surrounded by the asymmetric OM made of an inner PL leaflet and an outer LPS layer. LPS molecules are a mixture of short ‘rough' and longer O-antigen bearing ‘smooth' LPS and are cross-linked by divalent cations, shown as green circles. Abundant porin proteins in the OM form a molecular sieve that allows only small hydrophilic molecules across the membrane. (b) Basic structure of E. coli LPS. The structure of rough LPS is from the K-12 strain, shown without non-stoichiometric substitutions [5]. The multiple negative charges of the inner core and lipid A moieties stabilised by divalent cations, together with the six acyl chains of the lipid A, confer on the OM its unique barrier properties. Hep = heptose, Hex = hexose, Kdo = 3-deoxy-D-manno-oct-2-ulosonic acid (c) Physicochemical properties of antibacterial compounds. Commercial drugs are shown in a plot of polarity versus size. Antibiotics active against Gram-negative targets (blue) are not only smaller than ∼600 Da but also more polar (water soluble) than antibiotics against Gram-positive bacteria which lack the OM. The only clear outlier is PmB (circled) which avoids the porin-dictated size limitations by targeting LPS. Reprinted with permission from [11] copyright 2008 American Chemical Society.
Figure 2.
Figure 2.. Neutron reflectometry analysis of asymmetric OM model.
(a) Sample holder for neutron reflectometry experiments. The neutron beam passes through a thick silicon crystal and is reflected at the solid/liquid interface. Here, the supported asymmetric OM model, containing deuterated PL and hydrogenous rough LPS is surrounded by a buffer solution that can be easily exchanged through the cell inlets and outlets. (b) Data obtained from a typical NR experiment. The intensity of the specularly reflected neutron beam is measured as a function of Q, a combination of neutron wavelength and incident angle [53]. The asymmetric PL/LPS OM model is measured at three solution isotopic conditions (data points) which are simultaneously fitted (lines) to a common model that describes the interfacial bilayer structure shown in c. Curves are offset for clarity and the intensity is multiplied by Q4 to correct for the inherent decline in intensity with increasing angle. (c) structure of the deuterated PL (purple)/hydrogenous LPS (black) asymmetric bilayer described by the scattering length density (SLD) distribution of nuclei across the membrane profile. Note how SLD (in effect the neutron refractive index) of the acyl chains differs between natural LPS, deuterated lipids and different concentrations of D2O. The corresponding structure of the interface is shown above the profile. b and c are reproduced from [26].
Figure 3.
Figure 3.. OM models for structural studies of antibiotic interactions.
(a) LPS Langmuir monolayer at the air/water interface enclosed by the movable barriers of a Langmuir trough (b) solid supported asymmetric PL/LPS bilayer (c) floating PL/LPS bilayer. The gold surface is coated with a monolayer of thiolated lipids (purple) which creates a ∼20 Å water cushion between the substrate and the asymmetric model membrane. Dashed lines represent the path of the beam in a NR experiment.
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