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Bacterial extracellular vesicles: towards realistic models for bacterial membranes in molecular interaction studies by surface plasmon resonance.

ABSTRACT: One way to mitigate the ongoing antimicrobial resistance crisis is to discover and develop new classes of antibiotics. As all antibiotics at some point need to either cross or just interact with the bacterial membrane, there is a need for representative models of bacterial membranes and efficient methods to characterize the interactions with novel molecules -both to generate new knowledge and to screen compound libraries. Since the bacterial cell envelope is a complex assembly of lipids, lipopolysaccharides, membrane proteins and other components, constructing relevant synthetic liposome-based models of the membrane is both difficult and expensive. We here propose to let the bacteria do the hard work for us. Bacterial extracellular vesicles (bEVs) are naturally secreted by Gram-negative and Gram-positive bacteria, playing a role in communication between bacteria, as virulence factors, molecular transport or being a part of the antimicrobial resistance mechanism. bEVs consist of the bacterial outer membrane and thus inherit many components and properties of the native outer cell envelope. In this work, we have isolated and characterized bEVs from one Escherichia coli mutant and three clinical strains of the ESKAPE pathogens Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. The bEVs were shown to be representative models for the bacterial membrane in terms of lipid composition with speciesstrain specific variations. The bEVs were further used to probe the interactions between bEV and antimicrobial peptides (AMPs) as model compounds by Surface Plasmon Resonance (SPR) and provide proof-of-principle that bEVs can be used as an easily accessible and highly realistic model for the bacterial surface in interaction studies. This further enables direct monitoring of the effect induced by antibiotics, or the response to host-pathogen interactions.

SUBMITTER: Bril'kov MS

PROVIDER: S-EPMC10751319 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Bacterial extracellular vesicles: towards realistic models for bacterial membranes in molecular interaction studies by surface plasmon resonance.

Bril'kov Maxim S MS Stenbakk Victoria V Jakubec Martin M Vasskog Terje T Kristoffersen Tone T Cavanagh Jorunn Pauline JP Ericson Johanna U JU Isaksson Johan J Flaten Gøril Eide GE

Frontiers in molecular biosciences 20231213

One way to mitigate the ongoing antimicrobial resistance crisis is to discover and develop new classes of antibiotics. As all antibiotics at some point need to either cross or just interact with the bacterial membrane, there is a need for representative models of bacterial membranes and efficient methods to characterize the interactions with novel molecules -both to generate new knowledge and to screen compound libraries. Since the bacterial cell envelope is a complex assembly of lipids, lipopol ...[more]

PMID: 38152113

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Project description:Ras GTPases play a crucial role in signal transduction cascades involved in cell differentiation and proliferation, and membrane binding is essential for their proper function. To determine the influence of the nature of the lipid anchor motif and the difference between the active (GTP) and inactive (GDP) forms of N-Ras on partitioning and localization in the lipid membrane, five different N-Ras constructs with different lipid anchors and nucleotide loading (Far/Far (GDP), HD/Far (GDP), HD/HD (GDP), Far (GDP), and HD/Far (GppNHp)) were synthesized. Using the surface plasmon resonance technique, we were able to follow the insertion and dissociation process of the lipidated proteins into and out of model membranes consisting of pure liquid-ordered (l(o)) or liquid-disordered (l(d)) phase and a heterogeneous two-phase mixture, i.e., a raft mixture with l(o) + l(d) phase coexistence. In addition, we examined the influence of negatively charged headgroups and stored curvature elastic stress on the binding properties of the lipidated N-Ras proteins. In most cases, significant differences were found for the various anchor motifs. In general, N-Ras proteins insert preferentially into a fluidlike, rather than a rigid, ordered lipid bilayer environment. Electrostatic interactions with lipid headgroups or stored curvature elastic stress of the membrane seem to have no drastic effect on the binding and dissociation processes of the lipidated proteins. The monofarnesylated N-Ras exhibits generally the highest association rate and fastest dissociation process in fluidlike membranes. Double lipidation, especially including farnesylation, of the protein leads to drastically reduced initial binding rates but strong final association. The change in the nucleotide loading of the natural N-Ras HD/Far induces a slightly different binding and dissociation kinetics, as well as stability of association, and seems to influence the tendency to segregate laterally in the membrane plane. The GDP-bound inactive form of N-Ras with an HD/Far anchor shows stronger membrane association, which might be due to a more pronounced tendency to self-assemble in the membrane matrix than is seen with the active GTP-bound form.

Dataset Information

Bacterial extracellular vesicles: towards realistic models for bacterial membranes in molecular interaction studies by surface plasmon resonance.

Publications

Bacterial extracellular vesicles: towards realistic models for bacterial membranes in molecular interaction studies by surface plasmon resonance.

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