Modulation of enrofloxacin binding in OmpF by Mg2+ as revealed by the analysis of fast flickering single-porin current.

One major determinant of the efficacy of antibiotics on gram-negative bacteria is the passage through the outer membrane. During transport of the fluoroquinolone enrofloxacin through the trimeric outer membrane protein OmpF of Escherichia coli, the antibiotic interacts with two binding sites within the pore, thus partially blocking the ionic current. The modulation of one affinity site by Mg(2+) reveals further details of binding sites and binding kinetics. At positive membrane potentials, the slow blocking events induced by enrofloxacin in Mg(2+)-free media are converted to flickery sojourns at the highest apparent current level (all three pores flickering). This indicates weaker binding in the presence of Mg(2+). Analysis of the resulting amplitude histograms with ß distributions revealed the rate constants of blocking (k(OB)) and unblocking (k(BO)) in the range of 1,000 to 120,000 s(-1). As expected for a bimolecular reaction, k(OB) was proportional to blocker concentration and k(BO) independent of it. k(OB) was approximately three times lower for enrofloxacin coming from the cis side than from the trans side. The block was not complete, leading to a residual conductivity of the blocked state being ∼25% of that of the open state. Interpretation of the results has led to the following model: fast flickering as caused by interaction of Mg(2+) and enrofloxacin is related to the binding site at the trans side, whereas the cis site mediates slow blocking events which are also found without Mg(2+). The difference in the accessibility of the binding sites also explains the dependency of k(OB) on the side of enrofloxacin addition and yields a means of determining the most plausible orientation of OmpF in the bilayer. The voltage dependence suggests that the dipole of the antibiotic has to be adequately oriented to facilitate binding.

Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides.

Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS.

Morphology, size distribution, and aggregate structure of lipopolysaccharide and lipid A dispersions from enterobacterial origin.

Lipopolysaccharides (LPSs) from Gram-negative bacteria are strong elicitors of the human immune systems. There is strong evidence that aggregates and not monomers of LPS play a decisive role at least in the initial stages of cell activation of immune cells such as mononuclear cells. In previous reports, it was shown that the biologically most active part of enterobacterial LPS, hexa-acyl bisphosphorylated lipid A, adopts a particular supramolecular conformation, a cubic aggregate structure. However, little is known about the size and morphology of these aggregates, regarding the fact that LPS may have strong variations in the length of the saccharide chains (various rough mutant and smooth-form LPS). Thus, in the present paper, several techniques for the determination of details of the aggregate morphology such as freeze-fracture and cryo-electron microscopy, analytical ultracentrifugation, laser backscattering analysis, and small-angle X-ray scattering were applied for various endotoxin (lipid A and different LPS) preparations. The data show a variety of different morphologies not only for different endotoxins but also when comparing different applied techniques. The data are interpreted with respect to the suitability of the single techniques, in particular on the basis of available literature data.

Lipopolysaccharide interaction is decisive for the activity of the antimicrobial peptide NK-2 against Escherichia coli and Proteus mirabilis.

Phosphatidylglycerol is a widely used mimetic to study the effects of AMPs (antimicrobial peptides) on the bacterial cytoplasmic membrane. However, the antibacterial activities of novel NK-2-derived AMPs could not be sufficiently explained by using this simple model system. Since the LPS (lipopolysaccharide)-containing outer membrane is the first barrier of Gram-negative bacteria, in the present study we investigated interactions of NK-2 and a shortened variant with viable Escherichia coli WBB01 and Proteus mirabilis R45, and with model membranes composed of LPS isolated from these two strains. Differences in net charge and charge distribution of the two LPS have been proposed to be responsible for the differential sensitivity of the respective bacteria to other AMPs. As imaged by TEM (transmission electron microscopy) and AFM (atomic force microscopy), NK-2-mediated killing of these bacteria was corroborated by structural alterations of the outer and inner membranes, the release of E. coli cytoplasma, and the formation of unique fibrous structures inside P. mirabilis, suggesting distinct and novel intracellular targets. NK-2 bound to and intercalated into LPS bilayers, and eventually induced the formation of transient heterogeneous lesions in planar lipid bilayers. However, the discriminative activity of NK-2 against the two bacterial strains was independent of membrane intercalation and lesion formation, which both were indistinguishable for the two LPS. Instead, differences in activity originated from the LPS-binding step, which could be demonstrated by NK-2 attachment to intact bacteria, and to solid-supported LPS bilayers on a surface acoustic wave biosensor.