Escherichia coli response to subinhibitory concentration of Colistin: Insights from study of membrane dynamics and morphology.

Prevalence of wide spread bacterial infections bring forth a critical need in understanding the molecular mechanisms of the antibiotics as well as the bacterial response to those antibiotics. Improper usage of antibiotics, which can be in sub-lethal concentrations is one among the multiple reasons for acquiring antibiotic resistance which makes it vital to understand the bacterial response towards sub-lethal concentrations of antibiotics. In this work, we have used colistin, a well-known membrane active antibiotic used to treat severe bacterial infections and explored the impact of its subminimum inhibitory concentration (MIC) on the lipid membrane dynamics and morphological changes of E. coli. Upon investigation of live cell membrane properties such as lipid dynamics using fluorescence correlation spectroscopy, we observed that colistin disrupts the lipid membrane at sub-MIC by altering the lipid diffusivity. Interestingly, filamentationlike cell elongation was observed upon colistin treatment which led to further exploration of surface morphology with the help of atomic force spectroscopy. The changes in the surface roughness upon colistin treatment provides additional insight on the colistin-membrane interaction corroborating with the altered lipid diffusion. Although altered lipid dynamics could be attributed to an outcome of lipid rearrangement due to direct disruption by antibiotic molecules on the membrane or an indirect consequence of disruptions in lipid biosynthetic pathways, we were able to ascertain that altered bacterial membrane dynamics is due to direct disruptions. Our results provide a broad overview on the consequence of the cyclic polypeptide, colistin on membrane specific lipid dynamics and morphology of a live Gram-negative bacterial cell.

The dynamical Matryoshka model: 1. Incoherent neutron scattering functions for lipid dynamics in bilayers

Fluid lipid bilayers are the building blocks of biological membranes. Although there is a large amount of experimental data using inconsistent quasi-elastic neutron scattering (QENS) techniques to study membranes, very little theoretical works have been developed to study the local dynamics of membranes. The main objective of this work is to build a theoretical framework to study and describe the local dynamics of lipids and derive analytical expressions of inconsistent diffusion functions (ISF) for QENS. As results, we developed the dynamical Matryoshka model which describes the local dynamics of lipid molecules in membrane layers as a nested hierarchical convolution of three motional processes: (i) individual motions described by the vibrational motions of H-atoms; (ii) internal motions including movements of the lipid backbone, head groups and tails, and (iii) molecule movements of the lipid molecule as a whole. The analytical expressions of the ISF associated with these movements are all derived. For use in analyzing the QENS experimental data, we also derived an analytical expression for the aggregate ISF of the Matryoshka model which involves an elastic term plus three inelastic terms of well-separated time scales and whose amplitudes and rates are functions of the lipid motions. And as an illustrative application, we used the aggregated ISF to analyze the experimental QENS data on a lipid sample of multilamellar bilayers of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine). It is clear from this analysis that the dynamical Matryoshka model describes very well the experimental data and allow extracting the dynamical parameters of the studied system.

The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding

The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.

Structure and lipid dynamics in the maintenance of lipid asymmetry inner membrane complex of A. baumannii

Multi-resistant bacteria are a major threat in modern medicine. The gram-negative coccobacillus Acinetobacter baumannii currently leads the WHO list of pathogens in critical need for new therapeutic development. The maintenance of lipid asymmetry (MLA) protein complex is one of the core machineries that transport lipids from/to the outer membrane in gram-negative bacteria. It also contributes to broad-range antibiotic resistance in several pathogens, most prominently in A. baumannii. Nonetheless, the molecular details of its role in lipid transport has remained largely elusive. Here, we report the cryo-EM maps of the core MLA complex, MlaBDEF, from the pathogen A. baumannii, in the apo-, ATP- and ADP-bound states, revealing multiple lipid binding sites in the cytosolic and periplasmic side of the complex. Molecular dynamics simulations suggest their potential trajectory across the membrane. Collectively with the recently-reported structures of the E. coli orthologue, this data also allows us to propose a molecular mechanism of lipid transport by the MLA system.

Visualizing the Role of Lipid Dynamics during Infrared Neural Stimulation with Hyperspectral Stimulated Raman Scattering Microscopy

Infrared neural stimulation, or INS, is a method of using pulsed infrared light to yield label-free neuronal stimulation with broad experimental and translational utility. Despite its robust demonstration, the mechanistic and biophysical underpinnings of INS have been the subject of debate for more than a decade. The role of lipid membrane thermodynamics appears to play an important role in how fast IR-mediated heating nonspecifically drives action potential generation. Direct observation of lipid membrane dynamics during INS remains to be shown in a live neural model system. To directly test the involvement of lipid dynamics in INS, we used hyperspectral stimulated Raman scattering (hsSRS) microscopy to study biochemical signatures of high speed vibrational dynamics underlying INS in a live neural cell culture model. Findings suggest that lipid bilayer structural changes are occurring during INS in vitro in NG108-15 neuroglioma cells. Lipid-specific signatures of cell SRS spectra were found to vary with stimulation energy and radiant exposure. Spectroscopic observations were verified against high-speed ratiometric fluorescence imaging of a conventional lipophilic membrane structure reporter, di-4-ANNEPS. Overall, the presented data supports the hypothesis that INS causes changes in the lipid membrane of neural cells by changing lipid membrane packing order - which coincides with likelihood of cell stimulation. Furthermore, this work highlights the potential of hsSRS as a method to study biophysical and biochemical dynamics safely in live cells.

Elucidating the structural features of ABCA1 in its heterogeneous membrane environment.

ABCA1 plays an integral part in Reverse Cholesterol Transport (RCT) and is critical for maintaining lipid homeostasis. One theory of lipid efflux by the transporter (alternating access) proposes that ABCA1 harbors two different conformations that provide alternate access for lipid binding and release, leading to sequestration via a direct interaction between ABCA1 and its partner, ApoA1. The alternative theory (lateral access) proposes that ABCA1 obtains lipids laterally from the membrane to form a temporary extracellular reservoir containing an isolated pressurized lipid monolayer caused by the net accumulation of lipids in the exofacial leaflet. Recently, a full-length Cryo-EM structure of this 2,261-residue transmembrane protein showed its discreetly folded domains and conformations, as well as detected the presence of a tunnel enclosed within ECDs. While the tunnel was wide enough at the proximal end for accommodating passage of lipids, the distal end displayed substantial narrowing, making it inaccessible for ApoA1. Therefore, this structure was hypothesized to substantiate the lateral access theory, whereby ApoA1 obtained lipids from the proximal end. Utilizing long time-scale multiple replica atomistic molecular dynamics simulations (MDS), we simulated the structure in a heterogeneous lipid environment and found that along with several large conformational changes, the protein widens enough at the distal end of its ECD tunnel to now enable lipid accommodation. In this study we have characterized ABCA1 and the lipid dynamics along with the protein-lipid interactions in the heterogeneous environment, providing novel insights into understanding ABCA1 conformation at an atomistic level.