The influence of rough lipopolysaccharide structure on molecular interactions with mammalian antimicrobial peptides

Abstract

The influence of Escherichia coli rough lipopolysaccharide chemotype on the membrane activity of the mammalian antimicrobial peptides (AMPs) human cathelicidin (LL37) and bovine lactoferricin (LFb) was studied on bilayers using solid state (2)H NMR (ssNMR) and on monolayers using the subphase injection technique, Brewster angle microscopy (BAM) and neutron reflectivity (NR). The two AMPs were selected because of their differing biological activities. Chain-deuterated dipalmitoylphosphatidylcholine (d62-DPPC) was added to the LPS samples, to highlight alterations in the system properties caused by the presence of the different LPS chemotypes and upon AMP challenge. Both LPS chemotypes showed a temperature dependent influence on the packing of the DPPC molecules, with a fluidizing effect exerted below the DPPC phase transition temperature (Tm), and an ordering effect observed above the Tm. The magnitude of these effects was influenced by LPS structure; the shorter Rc LPS promoted more ordered lipid packing compared to the longer Ra LPS. These differential ordering effects in turn influenced the penetrative activity of the two peptides, as the perturbation induced by both AMPs to Ra LPS-containing models was greater than that observed in those containing Rc LPS. The NR data suggests that in addition to penetrating into the monolayers, both LL37 and LFb formed a non-interacting layer below the LPS/DPPC monolayer. The overall activity of LL37, which showed a deeper penetration into the model membranes, was more marked than that of LFb, which appeared to localise at the interfacial region, thus providing evidence for the molecular origins of their different biological activities.

Mechanism of Action of a Membrane-Active Quinoline-Based Antimicrobial on Natural and Model Bacterial Membranes

“…Nonlinear regression was applied to estimate the lag and slope of the Pressure-Time isotherm curves [27] generated after subphase injection of HT61. A general observation of the data is that the change in surface pressure (ΔΠ) follows a sigmoid increase over time.…”

“…The surface pressure was continually monitored whilst the monolayer was allowed to achieve a stable surface pressure, before the injection into the subphase of 100 µl of HT61, from a syringe fixed prior to monolayer deposition, to give a final concentration of 9.1 µg/ml. Surface pressure changes were continually recorded until a stable plateau was achieved.Nonlinear regression was applied to estimate the lag and slope of the Pressure-Time isotherm curves [27] generated after subphase injection of HT61. A general observation of the data is that the change in surface pressure (ΔΠ) follows a sigmoid increase over time.…”

Neutron Reflectivity as a Tool for Physics-Based Studies of Model Bacterial Membranes

“…16.3, can be sensitive to the precise orientation of the substrate as it is pushed through the monolayer in the Langmuir-Schaeffer step. To address this, the new Level'O'Matic (or LOM), was developed-a laser scanning mechanism removing the human alignment of the sample in the Langmuir-Schaeffer step, during which most deposited membranes are lost (Barker 2011). With these improvements, the fundamental difficulties in building these model systems have been significantly reduced, broadening their usage beyond the specialist and enabling more complex biological problems to be addressed.…”

“…A better approach is provided by the floating bilayer method that was developed by Hughes et al (2008), from the approach of Fragneto (Charitat et al 1999;Fragneto et al 2012), and refined by Barker (2011). In the floating bilayer approach, the lipid bilayer that is to be the subject of the investigation sits above a water layer of 2-5 nm, which sits between the bilayer and a functionalized (typically) silicon substrate.…”

“…There are two approaches which have been followed for the construction of lipid bilayers as models for membranes: supported lipid bilayers which are simpler (Wacklin 2010) and floating lipid bilayers which are more realistic (Fragneto et al 2012;Hughes et al 2008Hughes et al , 2014Barker 2011).…”