Real-time examination of aminoglycoside activity towards bacterial mimetic membranes using Quartz Crystal Microbalance with Dissipation monitoring (QCM-D)

Abstract

The rapid increase in multi-drug resistant bacteria has resulted in previously discontinued treatments being revisited. Aminoglycosides are effective "old" antibacterial agents that fall within this category. Despite extensive usage and understanding of their intracellular targets, there is limited mechanistic knowledge regarding how aminoglycosides penetrate bacterial membranes. Thus, the activity of two well-known aminoglycosides, kanamycin A and neomycin B, towards a bacterial mimetic membrane (DMPC:DMPG (4:1)) was examined using a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D). The macroscopic effect of increasing the aminoglycoside concentration showed that kanamycin A exerts a threshold response, switching from binding to the membrane to disruption of the surface. Neomycin B, however, disrupted the membrane at all concentrations examined. At concentrations above the threshold value observed for kanamycin A, both aminoglycosides revealed similar mechanistic details. That is, they both inserted into the bacterial mimetic lipid bilayer, prior to disruption via loss of materials, presumably aminoglycoside-membrane composites. Depth profile analysis of this membrane interaction was achieved using the overtones of the quartz crystal sensor. The measured data is consistent with a two-stage process in which insertion of the aminoglycoside precedes the 'detergent-like' removal of membranes from the sensor. The results of this study contribute to the insight required for aminoglycosides to be reconsidered as active antimicrobial agents/co-agents by providing details of activity at the bacterial membrane. Kanamycin and neomycin still offer potential as antimicrobial therapeutics for the future and the QCM-D method illustrates great promise for screening new antibacterial or antiviral drug candidates.

A Molecularly Complete Planar Bacterial Outer Membrane Platform

“…QCM-D has been used extensively to study antimicrobial peptide interaction with model membranes due to unique insights it provides into structural/conformational information of membrane-peptide complexes425758. However, these studies were performed on simple artificial bilayers composed of phospholipids like POPC/DOPC, or biomimetic bilayers mimicking bacterial inner membrane (IM).…”

Differential dependencies on [Ca2+] and temperature of the monolayer spontaneous curvatures of DOPE, DOPA and cardiolipin: effects of modulating the strength of the inter-headgroup repulsion

“…In eukaryotes, cardiolipin is nearly exclusively found in the inner membranes of mitochondria, 60 where many vital events, such as intracellular calcium storage, oxidative phosphorylation and apoptosis, take place. 63 To the mitochondrial function of calcium storage, the weak [Ca 2+ ] dependence of the phase preference of cardiolipin can be beneficial: it attenuates the usually strong nonlamellar-forming propensities of anionic phospholipids at high local [Ca 2+ ] (retaining high local [Ca 2+ ] is important to mitochondrial functioning, as reviewed in ref. 63 To the mitochondrial function of calcium storage, the weak [Ca 2+ ] dependence of the phase preference of cardiolipin can be beneficial: it attenuates the usually strong nonlamellar-forming propensities of anionic phospholipids at high local [Ca 2+ ] (retaining high local [Ca 2+ ] is important to mitochondrial functioning, as reviewed in ref.…”

Effects of guanidino modified aminoglycosides on mammalian membranes studied using a quartz crystal microbalance

“…50 This approach is widely used by numerous groups to study membrane interactions 33,47,48,61 and complements our previously studied effects of aminoglycosides on bacterial membranes. 28 The QCM-D technique measures changes in frequency (Δf) and dissipation (ΔD) of a quartz crystal sensor. A decrease in frequency (Δf ) is correlated with an increase in mass (Δm) while an increase in dissipation (ΔD) refers to a softer and less rigid surface structure.…”

“…26,27 A disruptive effect of aminoglycosides towards bacterial mimetic membranes has already been demonstrated leading to its antibiotic activity. 28 Several multidrug-resistant bacteria, such as methicillinresistant Staphylococcus aureus (MRSA), invade mammalian cells. 29 Hence, potential drugs need to be able to penetrate mammalian membranes to target intracellular bacterial infections.…”