The Expandables: Cracking the Staphylococcal Cell Wall for Expansion Microscopy.
Ontology highlight
ABSTRACT:
SUBMITTER: Kunz TC
PROVIDER: S-EPMC8008081 | biostudies-literature | 2021
REPOSITORIES: biostudies-literature
Publications
The Expandables: Cracking the Staphylococcal Cell Wall for Expansion Microscopy.
Kunz Tobias C TC Rühling Marcel M Moldovan Adriana A Paprotka Kerstin K Kozjak-Pavlovic Vera V Rudel Thomas T Fraunholz Martin M
Frontiers in cellular and infection microbiology 20210316
Expansion Microscopy (ExM) is a novel tool improving the resolution of fluorescence microscopy by linking the sample into a hydrogel that gets physically expanded in water. Previously, we have used ExM to visualize the intracellular Gram-negative pathogens <i>Chlamydia trachomatis</i>, <i>Simkania negevensis</i>, and <i>Neisseria gonorrhoeae</i>. Gram-positive bacteria have a rigid and thick cell wall that impedes classic expansion strategies. Here we developed an approach, which included a seri ...[more]
Similar Datasets
Project description:The major autolysins (Atl) of Staphylococcus epidermidis and S. aureus play an important role in cell separation, and their mutants are also attenuated in virulence. Therefore, autolysins represent a promising target for the development of new types of antibiotics. Here, we report the high-resolution structure of the catalytically active amidase domain AmiE (amidase S. epidermidis) from the major autolysin of S. epidermidis. This is the first protein structure with an amidase-like fold from a bacterium with a gram-positive cell wall architecture. AmiE adopts a globular fold, with several alpha-helices surrounding a central beta-sheet. Sequence comparison reveals a cluster of conserved amino acids that define a putative binding site with a buried zinc ion. Mutations of key residues in the putative active site result in loss of activity, enabling us to propose a catalytic mechanism. We also identified and synthesized muramyltripeptide, the minimal peptidoglycan fragment that can be used as a substrate by the enzyme. Molecular docking and digestion assays with muramyltripeptide derivatives allow us to identify key determinants of ligand binding. This results in a plausible model of interaction of this ligand not only for AmiE, but also for other PGN-hydrolases that share the same fold. As AmiE active-site mutations also show a severe growth defect, our findings provide an excellent platform for the design of specific inhibitors that target staphylococcal cell separation and can thereby prevent growth of this pathogen.
| S-EPMC2837410 | biostudies-literature
Project description:Spurious blinking fluorescent spots are often seen in bacteria during single-molecule localization microscopy experiments. Although this 'autoblinking' phenomenon is widespread, its origin remains unclear. In Deinococcus strains, we observed particularly strong autoblinking at the periphery of the bacteria, facilitating its comprehensive characterization. A systematic evaluation of the contributions of different components of the sample environment to autoblinking levels and the in-depth analysis of the photophysical properties of autoblinking molecules indicate that the phenomenon results from transient binding of fluorophores originating mostly from the growth medium to the bacterial cell wall, which produces single-molecule fluorescence through a Point Accumulation for Imaging in Nanoscale Topography (PAINT) mechanism. Our data suggest that the autoblinking molecules preferentially bind to the plasma membrane of bacterial cells. Autoblinking microscopy was used to acquire nanoscale images of live, unlabeled D. radiodurans and could be combined with PALM imaging of PAmCherry-labeled bacteria in two-color experiments. Autoblinking-based super-resolved images provided insight into the formation of septa in dividing bacteria and revealed heterogeneities in the distribution and dynamics of autoblinking molecules within the cell wall.
| S-EPMC6145920 | biostudies-literature
Project description:The shape of walled cells such as fungi, bacteria, and plants are determined by the cell wall. Models for cell morphogenesis postulate that the effects of turgor pressure and mechanical properties of the cell wall can explain the shapes of these diverse cell types. However, in general, these models await validation through quantitative experiments. Fission yeast Schizosaccharomyces pombe are rod-shaped cells that grow by tip extension and then divide medially through formation of a cell wall septum. Upon cell separation after cytokinesis, the new cell ends adopt a rounded morphology. Here, we show that this shape is generated by a very simple mechanical-based mechanism in which turgor pressure inflates the elastic cell wall in the absence of cell growth. This process is independent of actin and new cell wall synthesis. To model this morphological change, we first estimate the mechanical properties of the cell wall using several approaches. The lateral cell wall behaves as an isotropic elastic material with a Young's modulus of 50 ± 10 MPa inflated by a turgor pressure estimated to be 1.5 ± 0.2 MPa. Based upon these parameters, we develop a quantitative mechanical-based model for new end formation that reveals that the cell wall at the new end expands into its characteristic rounded shape in part because it is softer than the mature lateral wall. These studies provide a simple example of how turgor pressure expands the elastic cell wall to generate a particular cell shape.
| S-EPMC4627367 | biostudies-literature
Project description:Staphylococcus aureus and Staphylococcus epidermidis possess a 42-kDa cell wall transferrin-binding protein (Tpn) which is involved in the acquisition of transferrin-bound iron. To characterize this protein further, cell wall fractions were subjected to two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis blotted, and the N-terminus of Tpn was sequenced. Comparison of the first 20 amino acid residues of Tpn with the protein databases revealed a high degree of homology to the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Analysis of staphylococcal cell wall fractions for GAPDH activity confirmed the presence of a functional enzyme which, like Tpn, is regulated by the availability of iron in the growth medium. To determine whether Tpn is responsible for this GAPDH activity, it was affinity purified with NAD+ agarose. Both S. epidermidis and S. aureus Tpn catalyzed the conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. In contrast, Staphylococcus saprophyticus, which lacks a Tpn, has no cell wall-associated GAPDH activity. Native polyacrylamide gel electrophoresis of the affinity-purified Tpn revealed that it was present in the cell wall as a tetramer, consistent with the structures of all known cytoplasmic GAPDHs. Furthermore, the affinity-purified Tpn retained its ability to bind human transferrin both in its native tetrameric and SDS-denatured monomeric forms. Apart from interacting with human transferrin, Tpn, in common with the group A streptococcal cell wall GAPDH, binds human plasmin. Tpn-bound plasmin is enzymatically active and therefore may contribute to the ability of staphylococci to penetrate tissues during infections. These studies demonstrate that the staphylococcal transferrin receptor protein, Tpn, is a multifunctional cell wall GAPDH.
| S-EPMC96433 | biostudies-literature
Project description:Surface topography on the scale of tens of nanometers to several micrometers substantially affects cell adhesion, migration, and differentiation. Recent studies using electron microscopy and super-resolution microscopy provide insight into how cells interact with surface nanotopography; however, the complex sample preparation and expensive imaging equipment required for these methods makes them not easily accessible. Expansion microscopy (ExM) is an affordable approach to image beyond the diffraction limit, but ExM cannot be readily applied to image the cell-material interface as most materials do not expand. Here, we develop a protocol that allows the use of ExM to resolve the cell-material interface with high resolution. We apply the technique to image the interface between U2OS cells and nanostructured substrates as well as the interface between primary osteoblasts with titanium dental implants. The high spatial resolution enabled by ExM reveals that although AP2 and F-actin both accumulate at curved membranes induced by vertical nanostructures, they are spatially segregated. Using ExM, we also reliably image how osteoblasts interact with roughened titanium implant surfaces below the diffraction limit; this is of great interest to understand osseointegration of the implants but has up to now been a significant technical challenge due to the irregular shape, the large volume, and the opacity of the titanium implants that have rendered them incompatible with other super-resolution techniques. We believe that our protocol will enable the use of ExM as a powerful tool for cell-material interface studies.
| S-EPMC9879138 | biostudies-literature
Project description:Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.
| S-EPMC7147297 | biostudies-literature
Project description:BackgroundFruit cracking occurs easily under unsuitable environmental conditions and is one of the main types of damage that occurs in fruit production. It is widely accepted that plants have developed defence mechanisms and regulatory networks that respond to abiotic stress, which involves perceiving, integrating and responding to stress signals by modulating the expression of related genes. Fruit cracking is also a physiological disease caused by abiotic stress. It has been reported that a single or several genes may regulate fruit cracking. However, almost none of these reports have involved cracking regulatory networks.ResultsHere, RNA expression in 0 h, 8 h and 30 h saturated irrigation-treated fruits from two contrasting tomato genotypes, 'LA1698' (cracking-resistant, CR) and 'LA2683' (cracking-susceptible, CS), was analysed by mRNA and lncRNA sequencing. The GO pathways of the differentially expressed mRNAs were mainly enriched in the 'hormone metabolic process', 'cell wall organization', 'oxidoreductase activity' and 'catalytic activity' categories. According to the gene expression analysis, significantly differentially expressed genes included Solyc02g080530.3 (Peroxide, POD), Solyc01g008710.3 (Mannan endo-1,4-beta-mannosidase, MAN), Solyc08g077910.3 (Expanded, EXP), Solyc09g075330.3 (Pectinesterase, PE), Solyc07g055990.3 (Xyloglucan endotransglucosylase-hydrolase 7, XTH7), Solyc12g011030.2 (Xyloglucan endotransglucosylase-hydrolase 9, XTH9), Solyc10g080210.2 (Polygalacturonase-2, PG2), Solyc08g081010.2 (Gamma-glutamylcysteine synthetase, gamma-GCS), Solyc09g008720.2 (Ethylene receptor, ER), Solyc11g042560.2 (Ethylene-responsive transcription factor 4, ERF4) etc. In addition, the lncRNAs (XLOC_16662 and XLOC_033910, etc) regulated the expression of their neighbouring genes, and genes related to tomato cracking were selected to construct a lncRNA-mRNA network influencing tomato cracking.ConclusionsThis study provides insight into the responsive network for water-induced cracking in tomato fruit. Specifically, lncRNAs regulate the hormone-redox-cell wall network, including plant hormone (auxin, ethylene) and ROS (H2O2) signal transduction and many cell wall-related mRNAs (EXP, PG, XTH), as well as some lncRNAs (XLOC_16662 and XLOC_033910, etc.).
| S-EPMC7161180 | biostudies-literature
Project description:This is the first study of cellulose carbonization in the interior of cell walls. Cotton cellulose was pyrolyzed under nitrogen or in aromatic solvents (benzophenone, diphenyl sulfide, and 1,3-diphenoxybenzene) at 280 °C, and cross sections of the cell walls were examined using ultraviolet (UV) microscopy. After pyrolysis under nitrogen, UV absorption caused by carbonization appeared inside the cell walls. The absorptivity of the cell interiors was homogeneous and slightly lower than that of the cell surfaces. The UV spectra had maximal absorption at ca. 250 nm. The spectra of model compounds and Py-GC/MS analysis data suggested that furanic and polycyclic aromatic structures were present in the carbonized products. The use of aromatic solvents decreased the yields of solid carbonized products and the UV absorptivity, which remained homogeneous throughout the cross sections. The mechanism of cellulose carbonization in cell walls is discussed along with the influence of aromatic solvents.
| S-EPMC9049861 | biostudies-literature
Project description:To reduce staphylococcal food poisoning (SFP), a phage-encoded cell wall hydrolase called endolysin has emerged as an attractive antibacterial agent. In this study, the Staphylococcus aureus infecting phage vB_SauS-SAP27 (ϕSAP27) was isolated from sewage and characterized morphologically and genetically. ϕSAP27 was identified as Siphoviridae temperate phage, with a genome of 43 kbp. A ϕSAP27 endolysin named LysSAP27 was produced recombinantly in Escherichia coli. LysSAP27 exhibited the highest activity at neutral pH and a temperature of 30 °C, and its lytic activity was upregulated by calcium ions. Following optimization of the enzymatic conditions, LysSAP27 was applied to S. aureus-contaminated milk. Treatment with 2 μM LysSAP27 led to a significant bactericidal effect, corresponding to a reduction in bacterial titer by 2.8 log CFU/mL within 1 h and 3.4 log CFU/mL within 2 h. Therefore, LysSAP27 could be used as an effective antimicrobial agent to prevent SFP in food.Supplementary informationThe online version contains supplementary material available at 10.1007/s10068-021-00910-2.
| S-EPMC8144258 | biostudies-literature
Project description:We recently developed a method called expansion microscopy, in which preserved biological specimens are physically magnified by embedding them in a densely crosslinked polyelectrolyte gel, anchoring key labels or biomolecules to the gel, mechanically homogenizing the specimen, and then swelling the gel-specimen composite by ∼4.5× in linear dimension. Here we describe iterative expansion microscopy (iExM), in which a sample is expanded ∼20×. After preliminary expansion a second swellable polymer mesh is formed in the space newly opened up by the first expansion, and the sample is expanded again. iExM expands biological specimens ∼4.5 × 4.5, or ∼20×, and enables ∼25-nm-resolution imaging of cells and tissues on conventional microscopes. We used iExM to visualize synaptic proteins, as well as the detailed architecture of dendritic spines, in mouse brain circuitry.
| S-EPMC5560071 | biostudies-literature