Detection of Alpha-Toxin and Other Virulence Factors in Biofilms of Staphylococcus aureus on Polystyrene and a Human Epidermal Model.

BACKGROUND & AIM: The ability of Staphylococcus aureus to successfully colonize (a)biotic surfaces may be explained by biofilm formation and the actions of virulence factors. The aim of the present study was to establish the presence of 52 proteins, including virulence factors such as alpha-toxin, during biofilm formation of five different (methicillin resistant) S. aureus strains on Leiden human epidermal models (LEMs) and polystyrene surfaces (PS) using a competitive Luminex-based assay. RESULTS: All five S. aureus strains formed biofilms on PS, whereas only three out of five strains formed biofilms on LEMs. Out of the 52 tested proteins, six functionally diverse proteins (ClfB, glucosaminidase, IsdA, IsaA, SACOL0688 and nuclease) were detected in biofilms of all strains on both PS and LEMs. At the same time, four toxins (alpha-toxin, gamma-hemolysin B and leukocidins D and E), two immune modulators (formyl peptide receptor-like inhibitory protein and Staphylococcal superantigen-like protein 1), and two other proteins (lipase and LytM) were detectable in biofilms by all five S. aureus strains on LEMs, but not on PS. In contrast, fibronectin-binding protein B (FnbpB) was detectable in biofilms by all S. aureus biofilms on PS, but not on LEMs. These data were largely confirmed by the results from proteomic and transcriptomic analyses and in case of alpha-toxin additionally by GFP-reporter technology. CONCLUSION: Functionally diverse virulence factors of (methicillin-resistant) S. aureus are present during biofilm formation on LEMs and PS. These results could aid in identifying novel targets for future treatment strategies against biofilm-associated infections.

Conical Fourier shell correlation applied to electron tomograms.

The resolution of electron tomograms is anisotropic due to geometrical constraints during data collection, such as the limited tilt range and single axis tilt series acquisition. Acquisition of dual axis tilt series can decrease these effects. However, in cryo-electron tomography, to limit the electron radiation damage that occurs during imaging, the total dose should not increase and must be fractionated over the two tilt series. Here we set out to determine whether it is beneficial fractionate electron dose for recording dual axis cryo electron tilt series or whether it is better to perform single axis acquisition. To assess the quality of tomographic reconstructions in different directions here we introduce conical Fourier shell correlation (cFSCe/o). Employing cFSCe/o, we compared the resolution isotropy of single-axis and dual-axis (cryo-)electron tomograms using even/odd split data sets. We show that the resolution of dual-axis simulated and cryo-electron tomograms in the plane orthogonal to the electron beam becomes more isotropic compared to single-axis tomograms and high resolution peaks along the tilt axis disappear. cFSCe/o also allowed us to compare different methods for the alignment of dual-axis tomograms. We show that different tomographic reconstruction programs produce different anisotropic resolution in dual axis tomograms. We anticipate that cFSCe/o can also be useful for comparisons of acquisition and reconstruction parameters, and different hardware implementations.

Electron diffraction study of lipids in non-lesional stratum corneum of atopic eczema patients.

Skin barrier impairment is thought to be an important factor in the pathogenesis of atopic eczema (AE). The skin barrier is located in the stratum corneum (SC), consisting of corneocytes embedded in lipids. Ceramides, cholesterol and free fatty acids are the major lipid classes and are crucial for the skin barrier function, but their role in relation to AE is indistinct. Filaggrin is an epidermal barrier protein and common mutations in the filaggrin gene strongly predispose for AE. However, there is no strong evidence that filaggrin mutations are related to the reduced skin barrier in AE. In this study, electron diffraction is used in order to study the lipid organization of control SC and non-lesional SC of AE patients in vivo. An increased presence of the hexagonal lipid organization was observed in non-lesional SC of AE patients, indicating a less dense lipid organization. These changes correlate with a reduced skin barrier function as measured with transepidermal water loss but do not correlate with the presence of filaggrin mutations. These results are indicative for the importance of the lipid organization for a proper skin barrier function.

Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy.

Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain morphological and structural information from a selected field of view at nm-scale resolution. Previously, an integrated approach to room temperature correlative microscopy was described. Combined use of light and electron microscopy within one instrument greatly simplifies sample handling, avoids cumbersome experimental overheads, simplifies navigation between the two modalities, and improves the success rate of image correlation. Here, an integrated approach for correlative microscopy under cryogenic conditions is presented. Its advantages over the room temperature approach include safeguarding the native hydrated state of the biological specimen, preservation of the fluorescence signal without risk of quenching due to heavy atom stains, and reduced photo bleaching. The potential of cryo integrated light and electron microscopy is demonstrated for the detection of viable bacteria, the study of in vitro polymerized microtubules, the localization of mitochondria in mouse embryonic fibroblasts, and for a search into virus-induced intracellular membrane modifications within mammalian cells.

Monitoring Alzheimer Amyloid Peptide Aggregation by EPR.

Plaques containing the aggregated beta-Amyloid (Abeta) peptide in the brain are the main indicators of Alzheimer's disease. Fibrils, the building blocks of plaques, can also be produced in vitro and consist of a regular arrangement of the peptide. The initial steps of fibril formation are not well understood and could involve smaller aggregates (oligomers) of Abeta. Such oligomers have even been implicated as the toxic agents. Here, a method to study oligomers on the time scale of aggregation is suggested. We have labeled the 40 residue Abeta peptide variant containing an N-terminal cysteine (cys-Abeta) with the MTSL [1-oxyl-2,2,5,5-tetramethyl-Delta-pyrroline-3-methyl] methanethiosulfonate spin label (SL-Abeta). Fibril formation in solutions of pure SL-Abeta and of SL-Abeta mixed with Abeta was shown by Congo-red binding and electron microscopy. Continuous-wave 9 GHz electron paramagnetic resonance reveals three fractions of different spin-label mobility: one attributed to monomeric Abeta, one to a multimer (8-15 monomers), and the last one to larger aggregates or fibrils. The approach, in principle, allows detection of oligomers on the time scale of aggregation.

TYSON: robust searching, sorting, and selecting of single particles in electron micrographs.

We here present TYSON, a new program for automatic and semi-automatic particle selection from electron micrographs. TYSON employs a three-step strategy of searching, sorting and selecting single particles. In the first step, TYSON finds the positions of potential particles by one of three different methods: local averaging, template matching or local variance. The practical merits and drawbacks of these methods are discussed. In the second step, these potential particles are automatically sorted according to their probability of being true positives. Many criteria are provided for this sort. In the final -interactive- step, whole categories of poorly fitting false positives can be removed with a single mouse-click. We present results obtained using cryo-EM micrographs of both spherical virus particles and asymmetric particles. The procedures are fast and use of TYSON allowed, for example, some 20,000 particles to be selected in a single working day.

The 5 A projection structure of the transmembrane domain of the mannitol transporter enzyme II.

The uptake of mannitol in Escherichia coli is controlled by the phosphoenolpyruvate dependent phosphotransferase system. Enzyme II mannitol (EIIMtl) is part of the phosphotransferase system and consists of three covalently bound domains. IICMtl, the integral membrane domain of EIIMtl, is responsible for mannitol transport across the cytoplasmic membrane. In order to understand this molecular process, two-dimensional crystals of IICMtl were grown by reconstitution into lipid bilayers and their structure was investigated by cryo-electron crystallography. The IICMtl crystals obey p22121 symmetry and have a unit cell of 125 Ax65 A, gamma=90 degrees. A projection structure was determined at 5 A resolution using both electron images and electron diffractograms. The unit cell contains two IICMtl dimers with a size of about 40 Ax90 A, which are oriented up and down in the crystal. Each monomer exhibits six domains of high density which most likely correspond to transmembrane alpha-helices and cytoplasmic loops.

A 7.4-A projection structure of outer membrane phospholipase A from Escherichia coli by electron crystallography.

Outer membrane phospholipase A (OMPLA) is one of the few enzymes present in the outer membrane of Escherichia coli. Two-dimensional crystals of OMPLA were grown by reconstitution of purified protein into lipid bilayers via detergent dialysis and were studied by electron crystallography. A 7.4-A projection map reveals OMPLA molecules exhibiting an oval-shaped domain of 30 x 20 A resembling the beta-barrel structure characteristic of porins, which is associated with a 25-A elongated domain of lower density.