Walter Richter

A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages

Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3–4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-γ at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in α-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular α-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.

Comparison of Alzheimer Aβ(1–40) and Aβ(1–42) amyloid fibrils reveals similar protofilament structures

We performed mass-per-length (MPL) measurements and electron cryomicroscopy (cryo-EM) with 3D reconstruction on an Aβ(1–42) amyloid fibril morphology formed under physiological pH conditions. The data show that the examined Aβ(1–42) fibril morphology has only one protofilament, although two protofilaments were observed with a previously studied Aβ(1–40) fibril. The latter fibril was resolved at 8 Å resolution showing pairs of β-sheets at the cores of the two protofilaments making up a fibril. Detailed comparison of the Aβ(1–42) and Aβ(1–40) fibril structures reveals that they share an axial twofold symmetry and a similar protofilament structure. Furthermore, the MPL data indicate that the protofilaments of the examined Aβ(1–40) and Aβ(1–42) fibrils have the same number of Aβ molecules per cross-β repeat. Based on this data and the previously studied Aβ(1–40) fibril structure, we describe a model for the arrangement of peptides within the Aβ(1–42) fibril.

Interaction of quorum signals with outer membrane lipids: insights into prokaryotic membrane vesicle formation

Bacteria have evolved elaborate communication strategies to co‐ordinate their group activities, a process termed quorum sensing (QS). Pseudomonas aeruginosa is an opportunistic pathogen that utilizes QS for diverse activities, including disease pathogenesis. P. aeruginosa has evolved a novel communication system in which the signal molecule 2‐heptyl‐3‐hydroxy‐4‐quinolone (Pseudomonas Quinolone Signal, PQS) is trafficked between cells via membrane vesicles (MVs). Not only is PQS packaged into MVs, it is required for MV formation. Although MVs are involved in important biological processes aside from signalling, the molecular mechanism of MV formation is unknown. To provide insight into the molecular mechanism of MV formation, we examined the interaction of PQS with bacterial lipids. Here, we show that PQS interacts strongly with the acyl chains and 4′‐phosphate of bacterial lipopolysaccharide (LPS). Using PQS derivatives, we demonstrate that the alkyl side‐chain and third position hydroxyl of PQS are critical for these interactions. Finally, we show that PQS stimulated purified LPS to form liposome‐like structures. These studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non‐signalling functions.

Hemolysis of human erythrocytes with saponin affects the membrane structure

Incubation of cells and tissues with saponin makes the lipid bilayer permeable to macromolecules. Ghosts (membrane preparations) of saponin-lysed erythrocytes do not reseal, thus indicating an irreversible damage of the lipid bilayer. We investigated the influence of disturbance of the lipid bilayer on membrane proteins by comparing ghosts of saponin-lysed erythrocytes with ghosts of cells lysed in hypotonic buffer. Transmission electron microscopy revealed destruction of the lipid bilayer and emergence of multilamellar buds in saponin-lysed ghosts. Freeze-fracture electron microscopy showed regions with crystalline lipids and an increase in particle-free areas on fracture faces. The number of protein sulfhydryl groups and the binding of hemoglobin were diminished in saponin-lysed ghosts. A Scatchard plot of hemoglobin binding revealed the decrease of high affinity binding sites. All these results indicate an aggregation of band 3 protein also demonstrated by laser scanning microscopy after incubation of cells labelled with eosin-5-maleimide with sublytic concentration of saponin. Hemolysis with saponin also affected the interaction between transmembrane proteins and the cytoskeleton. Dissociation of peripheral membrane proteins by incubation of ghosts in low salt buffer or by blocking sulfhydryl groups was increased and the association of spectrin with spectrin-depleted vesicles was decreased. The increased incorporation of the fluorescent probe Merocyanine 540 into saponin-lysed ghosts and the increased relative fluorescence quantum yield confirmed the perturbation of the lipid bilayer and the changed interaction between membrane lipids and intrinsic membrane proteins. Our results suggest that permeabilization of the lipid bilayer with saponin to admit the access of antibodies to the cytoplasmic surface of cells can aggregate transmembrane proteins and affect the immunocytochemical localization of associated proteins of the cytoskeleton.

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.

Belt-like localisation of caveolin in deep caveolae and its re-distribution after cholesterol depletion

Caveolae are specialised vesicular microdomains of the plasma membrane. Using freeze-fracture immunogold labelling and stereoscopic imaging, the distribution of labelled caveolin 1 in caveolae of 3T3-L1 mouse fibroblast cells was shown. Immunogold-labelled caveolin structures surrounded the basolateral region of deeply invaginated caveolae like a belt whereas in the apical region distal to the plasma membrane, the caveolin labelling was nearly absent. Shallow caveolar membranes showed a dispersed caveolin labelling. After membrane cholesterol reduction by methyl-ß-cyclodextrin treatment, a dynamic re-distribution of labelled caveolin 1 and a flattening of caveolar structures was found. The highly curved caveolar membrane got totally flat, and the initial belt-like caveolin labelling disintegrated to a ring-like structure and later to a dispersed order. Intramembrane particle-free domains were still observable after cholesterol depletion and caveolin re-distribution. These results indicate that cholesterol interacting with caveolin structures at the basolateral part of caveolae is necessary for the maintenance of the deeply invaginated caveolar membranes.

Interaction of LL-37 with Model Membrane Systems of Different Complexity: Influence of the Lipid Matrix ☆

As the main difference between bacterial and mammalian cell membranes is their net charge, the focal point of consideration in many model membrane experiments with antimicrobial peptides is lipid headgroup charge. We studied the interaction of the human multifunctional peptide LL-37 with single phospholipid monolayers, bilayers, and bilayers composed of binary mixtures of the four phospholipid species predominantly used in model membrane experiments (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylserine). We found that 1), the effects on single lipid monolayers are not comparable to those on the corresponding bilayers; 2), there are four different effects of LL-37 on bilayers of the four lipids; 3), the preference of LL-37 for the specific lipids is roughly inversely related to chain packing density; and 4), in the binary lipid mixtures, one lipid-and not necessarily the charged one--generally governs the mode of lipid/peptide interaction. Thus, our results show that lipid net charge is not the decisive factor determining the membrane-perturbing mechanism of LL-37, but only one of several parameters, among them packing density, the ability to form intermolecular H-bonds, and lipid molecular shape, which emphasizes how profoundly the choice of the model system can influence the outcome of a study of lipid/peptide interaction.

Mechanism of interaction of optimized Limulus-derived cyclic peptides with endotoxins: thermodynamic, biophysical and microbiological analysis

On the basis of formerly investigated peptides corresponding to the endotoxin-binding domain from LALF [Limulus anti-LPS (lipopolysaccharide) factor], a protein from Limulus polyphemus, we have designed and synthesized peptides of different lengths with the aim of obtaining potential therapeutic agents against septic shock syndrome. For an understanding of the mechanisms of action, we performed a detailed physicochemical and biophysical analysis of the interaction of rough mutant LPS with these peptides by applying FTIR (Fourier-transform infrared) spectroscopy, SAXS (small-angle X-ray scattering), calorimetric techniques [DSC (differential scanning calorimetry) and ITC (isothermal titration calorimetry)] and FFTEM (freeze-fracture transmission electron microscopy). Also, the action of the peptides on bacteria of different origin in microbial assays was investigated. Using FTIR and DSC, our results indicated a strong fluidization of the lipid A acyl chains due to peptide binding, with a decrease in the endothermic melting enthalpy change of the acyl chains down to a complete disappearance in the 1:0.5 to 1:2 [LPS]:[peptide] molar ratio range. Via ITC, it was deduced that the binding is a clearly exothermic process which becomes saturated at a 1:0.5 to 1:2 [LPS]:[peptide] molar ratio range. The results obtained with SAXS indicated a drastic change of the aggregate structures of LPS into a multilamellar stack, which was visualized in electron micrographs as hundreds of lamellar layers. This can be directly correlated with the inhibition of the LPS-induced production of tumour necrosis factor alpha in human mononuclear cells, but not with the action of the peptides on bacteria.

Growth substrate dependent localization of tetrachloroethene reductive dehalogenase in Sulfurospirillum multivorans

Sulfurospirillum multivorans is a dehalorespiring organism, which is able to utilize tetrachloroethene as terminal electron acceptor in an anaerobic respiratory chain. The localization of the tetrachloroethene reductive dehalogenase in dependence on different growth substrates was studied using the freeze-fracture replica immunogold labeling technique. When the cells were grown with pyruvate plus fumarate, a major part of the enzyme was either localized in the cytoplasm or membrane associated facing the cytoplasm. In cells grown on pyruvate or formate as electron donors and tetrachloroethene as electron acceptor, most of the enzyme was detected at the periplasmic side of the cytoplasmic membrane. These results were confirmed by immunoblots of the enzyme with and without the twin arginine leader peptide. Trichloroethene exhibited the same effect on the enzyme localization as tetrachloroethene. The data indicated that the localization of the enzyme was dependent on the electron acceptor utilized.

The shape of caveolae is omega-like after glutaraldehyde fixation and cup-like after cryofixation

Caveolae were defined as flask- or omega-shaped plasma membrane invaginations, abundant in adipocytes, fibroblasts, endothelial and smooth muscle cells. The major protein component of caveolar membranes is an integral membrane protein named caveolin. We compared the freeze-fracture behavior of caveolae in glutaraldehyde-fixed and cryofixed mouse fibroblast cells and found distinct differences. In glutaraldehyde-fixed cells almost all caveolae were cross-fractured through their pore and only very few caveolar membranes were membrane-fractured. We found the reverse situation in rapid frozen cells without any chemical fixation where most of the caveolae were membrane-fractured, showing different degrees of invagination from nearly flat to deeply invaginated. In ultrathin sections of glutaraldehyde-fixed heart endothelial cells, caveolae exhibit the well known omega-like shape. In high-pressure frozen, freeze-substituted and low temperature embedded heart endothelial cells, the caveolae frequently exhibit a cup-like shape without any constriction or pore. The cup-like caveolar shape could also be shown by tilt series analysis of freeze-fracture replicas obtained from cryofixed cells. Freeze-fracture immunolabeling of caveolin-1 revealed a lateral belt-like caveolin alignment. These findings point out that the constricted “neck” region of caveolae in most cases is an effect that is caused and intensified by the glutaraldehyde fixation. Our data indicate that caveolae in vivo show all degrees of invagination from nearly flat via cup-like depressed to in a few cases omega-like.