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A model of the outer membrane of Gram-negative bacteria was created by the deposition of a monolayer of purified rough mutant lipopolysaccharides at an air/water interface. The density profiles of monovalent (K þ ) and divalent (Ca 2þ ) cations normal to the lipopolysaccharides (LPS) monolayers were investigated using grazingincidence X-ray fluorescence. In the absence of Ca 2þ , a K þ concentration peak was found in the negatively charged LPS headgroup region. With the addition of CaCl 2 , Ca 2þ ions almost completely displaced K þ ions from the headgroup region. By integrating the experimentally reconstructed excess ion density profiles, we obtained an accurate measurement of the effective charge density of LPS monolayers. The experimental findings were compared to the results of Monte Carlo simulations based on a coarse-grained minimal model of LPS molecules and showed excellent agreement.monolayer | Monte Carlo simulation | electrostatics | biological interface B iological surfaces expose a variety of charged macromolecules that interact with various sorts of ions under physiological conditions. However, despite the crucial role of charged macromolecules in modulating the interaction between cells and their surrounding environments, the quantitative understanding of electrostatics at such soft, complex interfaces still remains a general scientific challenge. For example, the outer membrane surface of Gram-negative bacteria is mainly composed of lipopolysaccharides (LPSs) (1), whose negatively charged saccharide head groups stabilize the structural integrity of bacteria and protect bacteria against their environment. Several in vivo studies (2, 3) demonstrated that bacteria increase their resistance against cationic antimicrobial peptides (e.g., protamine) in the presence of divalent cations (Ca 2þ , Mg 2þ ). Therefore, for the development of peptide-based antibiotics (4), it is important to understand the molecular mode of action of antimicrobial peptides.A number of theoretical models for the interactions of LPS molecules with divalent cations (5-7), suggested that the ions would bind to the charged 2-keto-3-deoxyoctonoic acid (KDO) groups (the "inner core") thereby stabilizing the membrane. Recently, we measured grazing-incidence X-ray scattering from a monolayer of rough mutant LPS from Salmonella enterica sv. Minnesota at an air/water interface and demonstrated the Ca 2þ -induced increase in the electron density near the inner core (8). These observations were supported by the results of Monte Carlo (MC) simulations of a coarse-grained model (8). A further challenge would be to extend such a strategy to wild-type LPSs that possess polydisperse, specific O-polysaccharide chains (O-side chains). Pink et al. (9) carried out MC simulations of a minimal model of the more complex, wild-type LPSs from Pseudomonas aeruginosa (PAO1) and concluded that divalent cations would induce a collapse of the negatively charged O-sidechains toward the membrane surface. Because it is difficult in practice to deposit LPSs wit...

Lipopolysaccharide (LPS) monolayers deposited on planar, hydrophobic substrates were used as a defined model of outer membranes of Pseudomonas aeruginosa strain dps 89. To investigate the influence of ions on the (out-of-plane) monolayer structure, we measured specular X-ray reflectivity at high energy (22 keV) to ensure transmission through water. Electron density profiles were reconstructed from the reflectivity curves, and they indicate that the presence of Ca 2þ ions induces a significant change in the conformation of the charged polysaccharide head groups (O-side chains). Monte Carlo simulations based on a minimal computer model of LPS molecules allow for the modelling of 100 or more molecules over 10 23 s and theoretically explained the tendency found by experiments.

Nanoscale materials can have cytotoxic effects. Here we present the first combined empirical and theoretical investigation of the influence of electrostatic attraction on nanoparticle cytotoxicity. Modeling electrostatic interactions between cells and 13 nm spheres of zinc oxide nanoparticles provided insight into empirically determined variations of the minimum inhibitory concentrations between four differently charged isogenic strains of Pseudomonas aeruginosa PAO1. We conclude that controlling the electrostatic attraction between nanoparticles and their cellular targets may permit the modulation of nanoparticle cytotoxicity.

Protamine is a cationic antimicrobial peptide, which inhibits or kills a number of Gram-negative bacteria, including Pseudomonas aeruginosa and Escherichia coli. Electrostatic interactions between the outer leaflet of the membrane and protamine are thought to be important for the antimicrobial effect. We hypothesized that divalent ions would compete with protamine for binding to the charged O-sidechain of the liposaccharide and expel protamine from the O-sidechains. Experimentally it was shown that increasing concentrations of divalent cations (Ca2+ and Mg2+) reduced the antimicrobial effect of protamine on P. aeruginosa PA01 and E. coli. We also modeled the electrostatic interactions between five protamine Y1 molecules from Atlantic herring and the surface of a Gram-negative bacterium possessing charged O-sidechains of the B-band lipopolysaccharides of P. aeruginosa PA01 in the presence/absence of calcium ions in an aqueous solution described by linearized Poisson−Boltzmann theory with Debye screening lengths κ-1 of 1.0 nm (∼100 mM) and 3.33 nm (∼10 mM). Our conclusions are as follows. [1] A high concentration of calcium ions brought about a slight polysaccharide chain collapse. The calcium ions formed dynamic bridges between the negatively charged O-sidechains on time scales comparable to that of polymer motion. [2] Without the presence of added calcium, all five protamine molecules were trapped in the charged polysaccharide O-sidechain. The probability of finding segments of protamine molecules closer than ∼0.5 nm to the membrane plane (the x−y plane at z = 0) was effectively zero. [3] Both the calcium distribution and the protamine distribution, when present separately, were essentially independent of monovalent ionic concentration for both values of κ. [4] When calcium and protamine were present simultaneously, the effects depended strongly upon the monovalent ion concentration. Added calcium effectively prevented protamine from entering the O-sidechain brush. Conclusions 3 and 4 show that the minimum inhibitory concentration should depend on monovalent ion concentration since complex growth media contain multivalent ions. [5] Our experiments confirmed our theoretical prediction that the addition of Ca2+ significantly reduced the inhibitory effect of protamine. This also confirmed the importance of electrostatic interactions during the first step in protamine's antibacterial mode of action.