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

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.

Goldilocks Dilemma: LPS Works Both as the Initial Target and a Barrier for the Antimicrobial Action of Cationic AMPs on E. coli.

Antimicrobial peptides (AMPs) are generally membrane-active compounds that physically disrupt bacterial membranes. Despite extensive research, the precise mode of action of AMPs is still a topic of great debate. This work demonstrates that the initial interaction between the Gram-negative E. coli and AMPs is driven by lipopolysaccharides (LPS) that act as kinetic barriers for the binding of AMPs to the bacterial membrane. A combination of SPR and NMR experiments provide evidence suggesting that cationic AMPs first bind to the negatively charged LPS before reaching a binding place in the lipid bilayer. In the event that the initial LPS-binding is too strong (corresponding to a low dissociation rate), the cationic AMPs cannot effectively get from the LPS to the membrane, and their antimicrobial potency will thus be diminished. On the other hand, the AMPs must also be able to effectively interact with the membrane to exert its activity. The ability of the studied cyclic hexapeptides to bind LPS and to translocate into a lipid membrane is related to the nature of the cationic charge (arginine vs. lysine) and to the distribution of hydrophobicity along the molecule (alternating vs. clumped tryptophan).

Can the absolute configuration of cyclic peptides be determined with vibrational circular dichroism?

Cyclic peptides show a wide range of biological activities, among others as antibacterial agents. These peptides are often large and flexible with multiple chiral centers. The determination of the stereochemistry of molecules with multiple chiral centers is a challenging and important task in drug development. Chiroptical spectroscopies such as vibrational circular dichroism (VCD) can distinguish between different stereoisomers. The absolute configuration (AC) of a stereoisomer can be determined by comparing its experimental spectra to computed spectra of stereoisomers with known AC. In this way, the AC of rigid molecules with up to seven chiral centers has been assigned (Bogaerts et al., Phys. Chem. Chem. Phys., 2020, 22, 18014). The question arises whether this is possible with more conformationally flexible molecules such as cyclic peptides. We here investigate to what extent the AC of cyclic peptides can be determined with VCD. More specifically, we investigate the maximum number of chiral centers a cyclic peptide can have in order to be able to unambiguously assign the AC with VCD. We present experimental and computed IR and VCD spectra for a series of eight tetrapeptides and hexapeptides with two, three and four chiral centers. We use our recently developed computational protocol with a conformational search based on sampling with meta-dynamics. We use visual inspection to compare the computed spectra of different stereoisomers with an experimental spectrum of the corresponding cyclic peptide with known AC. We find that the AC of the investigated cyclic peptides with two chiral centers can be unambiguously assigned with VCD. This is however not possible for all of the cyclic peptides with three chiral centers and for none of those with four chiral centers. At best, one can limit the number of possible stereoisomers in those cases. Our work shows that other techniques are needed to assign the AC of cyclic peptides with three or more chiral centers. Our study also constitutes a warning that the spectra of all stereoisomers should be computed before attempting to match to an experimental spectrum, to avoid an accidental erroneous match.