Simple optical nanomotion method for single-bacterium viability and antibiotic response testing.

Antibiotic resistance is nowadays a major public health issue. Rapid antimicrobial susceptibility tests (AST) are one of the options to fight this deadly threat. Performing AST with single-cell sensitivity that is rapid, cheap, and widely accessible, is challenging. Recent studies demonstrated that monitoring bacterial nanomotion by using atomic force microscopy (AFM) upon exposure to antibiotics constitutes a rapid and highly efficient AST. Here, we present a nanomotion detection method based on optical microscopy for testing bacterial viability. This novel technique only requires a very basic microfluidic analysis chamber, and an optical microscope equipped with a camera or a mobile phone. No attachment of the microorganisms is needed, nor are specific bacterial stains or markers. This single-cell technique was successfully tested to obtain AST for motile, nonmotile, gram-positive, and gram-negative bacteria. The simplicity and efficiency of the method make it a game-changer in the field of rapid AST.

Fast Self-Assembly Dynamics of a ß-Sheet Peptide Soft Material.

Peptide-based hydrogels are promising biocompatible materials for wound healing, drug delivery, and tissue engineering applications. The physical properties of these nanostructured materials depend strongly on the morphology of the gel network. However, the self-assembly mechanism of the peptides that leads to a distinct network morphology is still a subject of ongoing debate, since complete assembly pathways have not yet been resolved. To unravel the dynamics of the hierarchical self-assembly process of the model ß-sheet forming peptide KFE8 (Ac-FKFEFKFE-NH2 ), high-speed atomic force microscopy (HS-AFM) in liquid is used. It is demonstrated that a fast-growing network, based on small fibrillar aggregates, is formed at a solid-liquid interface, while in bulk solution, a distinct, more prolonged nanotube network emerges from intermediate helical ribbons. Moreover, the transformation between these morphologies has been visualized. It is expected that this new in situ and in real-time methodology will set the path for the in-depth unravelling of the dynamics of other peptide-based self-assembled soft materials, as well as gaining advanced insights into the formation of fibers involved in protein misfolding diseases.

Dip-pen nanolithography-assisted protein crystallization.

We demonstrate the use of dip-pen nanolithography (DPN) to crystallize proteins on surface-localized functionalized lipid layer arrays. DOPC lipid layers, containing small amounts of biotin-DOPE lipid molecules, were printed on glass substrates and evaluated in vapor diffusion and batch crystallization screening setups, where streptavidin was used as a model protein for crystallization. Independently of the crystallization system used and the geometry of the lipid layers, nucleation of streptavidin crystals occurred specifically on the DPN-printed biotinylated structures. Protein crystallization on lipid array patches is also demonstrated in a microfluidic chip, which opens the way toward high-throughput screening to find suitable nucleation and crystal growth conditions. The results demonstrate the use of DPN in directing and inducing protein crystallization on specific surface locations.

Engineering the carbohydrate-binding site of Epa1p from Candida glabrata: generation of adhesin mutants with different carbohydrate specificity.

The N-terminal domain of the Epa1p adhesin from Candida glabrata (N-Epa1p) is a calcium-dependent lectin, which confers the opportunistic yeast the ability to adhere to human epithelial cells. This lectin domain is able to interact with galactosides and, more precisely, with glycan molecules containing the Galß-1,3-GalNAc disaccharide, also known as the T-antigen. Based on the crystallographic structure of the N-Epa1p domain and the role of the variable loop CBL2 in glycan binding, saturation mutagenesis on some residues of the CBL2 loop was used to increase the binding affinity of N-Epa1p for fibronectin, which was selected as a model of a human glycoprotein. Two adhesin mutants, E227A and Y228W, with improved binding features were obtained. More importantly, a glycan array screening revealed that single-point mutations in the CBL2 could produce significant changes in the carbohydrate specificity of the protein. In particular, lectin molecules were generated with a high affinity for sulfated glycans, which may find an application as molecular probes for the identification of 6-sulfogalactose containing glycans and glycoconjugates.

A simplified version of rapid susceptibility testing of bacteria and yeasts using optical nanomotion detection.

We present a novel optical nanomotion-based rapid antibiotic and antifungal susceptibility test. The technique consisted of studying the effects of antibiotics or antifungals on the nanometric scale displacements of bacteria or yeasts to assess their sensitivity or resistance to drugs. The technique relies on a traditional optical microscope, a video camera, and custom-made image analysis software. It provides reliable results in a time frame of 2-4 h and can be applied to motile, non-motile, fast, and slowly growing microorganisms. Due to its extreme simplicity and low cost, the technique can be easily implemented in laboratories and medical centers in developing countries.

Machine learning method for the classification of the state of living organisms' oscillations.

The World Health Organization highlights the urgent need to address the global threat posed by antibiotic-resistant bacteria. Efficient and rapid detection of bacterial response to antibiotics and their virulence state is crucial for the effective treatment of bacterial infections. However, current methods for investigating bacterial antibiotic response and metabolic state are time-consuming and lack accuracy. To address these limitations, we propose a novel method for classifying bacterial virulence based on statistical analysis of nanomotion recordings. We demonstrated the method by classifying living Bordetella pertussis bacteria in the virulent or avirulence phase, and dead bacteria, based on their cellular nanomotion signal. Our method offers significant advantages over current approaches, as it is faster and more accurate. Additionally, its versatility allows for the analysis of cellular nanomotion in various applications beyond bacterial virulence classification.

Cloning, expression, and purification of the N-terminal domain of the Flo1 flocculation protein from Saccharomyces cerevisiae in Pichia pastoris.

Saccharomyces cerevisiae flocculation is governed by FLO genes, encoding Flo proteins (flocculins). Flo proteins are cell wall proteins consisting of three domains, sticking out of the cell wall and interacting with other yeast cells using their N-terminal mannose-binding domain. Until recently, flocculation research was focused on the genetic and cellular level. To extend the knowledge about flocculation to the protein level, we isolated the N-terminal domain of the Flo1p (N-Flo1p) that contains the mannose-binding domain, which is responsible for the strong interaction (flocculation) of S. cerevisiae cells. To obtain a high production yield and a more uniform and lower glycosylation of N-Flo1p, it was cloned in Pichia pastoris. The expression and the purification of N-Flo1p were optimised towards a one-step purification protocol. The activity of the protein, i.e. the binding of the purified protein to mannose using fluorescence spectroscopy, was demonstrated.

The mannose-specific lectin domains of Flo1p from Saccharomyces cerevisiae and Lg-Flo1p from S. pastorianus: crystallization and preliminary X-ray diffraction analysis of the adhesin-carbohydrate complexes.

Flo1p and Lg-Flo1p are two cell-wall adhesins belonging to the Flo (flocculation) protein family from the yeasts Saccharomyces cerevisiae and S. pastorianus. The main function of these modular proteins endowed with calcium-dependent lectin activity is to mediate cell-cell adhesion events during yeast flocculation, a process which is well known at the cellular level but still not fully characterized from a molecular perspective. Recently, structural features of the N-terminal Flo lectin domains, including the N-terminal domain of Lg-Flo1p (N-Lg-Flo1p), and their interactions with carbohydrate molecules have been investigated. However, structural data concerning the N-terminal domain of Flo1p (N-Flo1p), which is the most specific among the Flo proteins, are missing and information about the N-Lg-Flo1p-carbohydrate interaction still lacks detailed structural insight. Here, the crystallization and preliminary X-ray characterization of the apo form and the mannose complex of N-Flo1p and X-ray analysis of N-Lg-Flo1p crystals soaked in α-1,2-mannobiose are reported. The N-Flo1p crystals diffracted to a resolution of 1.43 Å in the case of the apo form and to 2.12 Å resolution for the mannose complex. Both crystals were orthorhombic and belonged to space group P212121, with one molecule in the asymmetric unit. The N-Lg-Flo1p-α-1,2-mannobiose complex crystal diffracted to 1.73 Å resolution and belonged to the monoclinic space group P1211 with two molecules in the asymmetric unit.

A universal fixation method based on quaternary ammonium salts (RNAlater) for omics-technologies: Saccharomyces cerevisiae as a case study.

Genomics, transcriptomics, proteomics and fluxomics are powerful omics-technologies that play a major role in today's research. For each of these techniques good sample quality is crucial. Major factors contributing to the quality of a sample is the actual sampling procedure itself and the way the sample is stored directly after sampling. It has already been described that RNAlater can be used to store tissues and cells in a way that the RNA quality and quantity are preserved. In this paper, we demonstrate that quaternary ammonium salts (RNAlater) are also suitable to preserve and store samples from Saccharomyces cerevisiae for later use with the four major omics-technologies. Moreover, it is shown that RNAlater also preserves the cell morphology and the potential to recover growth, permitting microscopic analysis and yeast cell culturing at a later stage.

Modulation of the nanoscale motion rate of Candida albicans by X-rays.

Introduction: Patients undergoing cancer treatment by radiation therapy commonly develop Candida albicans infections (candidiasis). Such infections are generally treated by antifungals that unfortunately also induce numerous secondary effects in the patient. Additional to the effect on the immune system, ionizing radiation influences the vital activity of C. albicans cells themselves; however, the reaction of C. albicans to ionizing radiation acting simultaneously with antifungals is much less well documented. In this study, we explored the effects of ionizing radiation and an antifungal drug and their combined effect on C. albicans. Methods: The study essentially relied on a novel technique, referred to as optical nanomotion detection (ONMD) that monitors the viability and metabolic activity of the yeast cells in a label and attachment-free manner. Results and discussion: Our findings demonstrate that after exposure to X-ray radiation alone or in combination with fluconazole, low-frequency nanoscale oscillations of whole cells are suppressed and the nanomotion rate depends on the phase of the cell cycle, absorbed dose, fluconazole concentration, and post-irradiation period. In a further development, the ONMD method can help in rapidly determining the sensitivity of C. albicans to antifungals and the individual concentration of antifungals in cancer patients undergoing radiation therapy.

The epithelial adhesin 1 (Epa1p) from the human-pathogenic yeast Candida glabrata: structural and functional study of the carbohydrate-binding domain.

The yeast Candida glabrata represents the second major cause of clinical candidiasis cases in the world. The ability of this opportunistic pathogen to adhere to human epithelial and endothelial cells relies on the Epa adhesins, a large set of cell-wall proteins whose N-terminal domains are endowed with a calcium-dependent lectin activity. This feature allows the yeast cells to adhere to host cells by establishing multiple interactions with the glycans expressed on their cell membrane. The ligand-binding domain of the Epa1p adhesin, which is one of the best characterized in the Epa family, was expressed in Escherichia coli, purified and crystallized in complex with lactose. Sequence identity with the domain of another yeast adhesin, the Flo5p flocculin from Saccharomyces cerevisiae, was exploited for molecular replacement and the structure of the domain was solved at a resolution of 1.65 Å. The protein is a member of the PA14 superfamily. It has a ß-sandwich core and a DcisD calcium-binding motif, which is also present in the binding site of Flo5p. However, Epa1p differs from this homologue by the lack of a Flo5-like subdomain and by a significantly decreased accessibility of the solvent to the binding site, in which a calcium ion still plays an active role in the interactions with carbohydrates. This structural insight, together with fluorescence-assay data, confirms and explains the higher specificity of Epa1p adhesin for glycan molecules compared with the S. cerevisiae flocculins.

The N-terminal part of Als1 protein from Candida albicans specifically binds fucose-containing glycans.

The opportunistic pathogen Candida albicans expresses on its surface Als (Agglutinin like sequence) proteins, which play an important role in the adhesion to host cells and in the development of candidiasis. The binding specificity of these proteins is broad, as they can bind to various mammalian proteins, such as extracellular matrix proteins, and N- and E-cadherins. The N-terminal part of Als proteins constitutes the substrate-specific binding domain and is responsible for attachment to epithelial and endothelial cells. We have used glycan array screening to identify possible glycan receptors for the binding domain of Als1p-N. Under those conditions, Als1p-N binds specifically to fucose-containing glycans, which adds a lectin function to the functional diversity of the Als1 protein. The binding between Als1p-N and BSA-fucose glycoconjugate was quantitatively characterized using surface plasmon resonance, which demonstrated a weak millimolar affinity between Als1p-N and fucose. Furthermore, we have also quantified the affinity of Als1p-N to the extracellular matrix proteins proteins fibronectin and laminin, which is situated in the micromolar range. Surface plasmon resonance characterization of Als1p-N-Als1p-N interaction was in the micromolar affinity range.

The N-terminal domain of the Flo11 protein from Saccharomyces cerevisiae is an adhesin without mannose-binding activity.

The expression of the Flo11 flocculin in Saccharomyces cerevisiae offers the cell a wide range of phenotypes, depending on the strain and the environmental conditions. The most important are pseudohyphae development, invasive growth and flocculation. The mechanism of cellular adhesion mediated by Flo11p is not well understood. Therefore, the N-terminal domain of Flo11p was purified and studied. Although its amino acid sequence shows less similarity with the other flocculins, Flo11p belongs to the flocculin family. However, the N-terminal domain contains the 'Flo11-domain' (PF10181), but not the mannose-binding PA14 domain, which is present in the other flocculins (Flo1p, Flo5p, Flo9p and Flo10p). Structural and binding properties of the N-terminal domain of Flo11p were studied. It is shown that this domain is O-glycosylated and is structurally composed mainly of ß-sheets, which is typical for the members of the flocculin family. Furthermore, fluorescence spectroscopy binding studies revealed that N-Flo11p does not bind mannose, which is in contrast to the other Flo proteins. However, surface plasmon resonance analysis showed that N-Flo11p self-interacts and explains the cell-cell interaction capacity of FLO11-expressing cells.

The N-terminal domain of the Flo1 flocculation protein from Saccharomyces cerevisiae binds specifically to mannose carbohydrates.

Saccharomyces cerevisiae cells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of ß-sheets. The binding of N-Flo1p to D-mannose, α-methyl-D-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level.

Development of bone cell microarrays in microfluidic chips for studying osteocyte-osteoblast communication under fluid flow mechanical loading.

Bone tissue remodels throughout life in response to mechanical loads. Impaired activities of bone cells (osteocytes, osteoblasts and osteoclasts) result in a disruption of the bone remodelling cycle, which eventually leads to bone disorders such as osteoporosis. To develop efficient therapeutic strategies against bone disorders, new tools are needed to unravel the bone remodelling cycle at the molecular level. Here, we developed a microfluidic platform, which should allow understanding the bone remodelling cycle in much more detail and ultimately be used to discover new therapeutic compounds. We focused specifically on studying cell-cell communication between osteocytes and osteoblasts cells via connexin 43-gap junctions. Therefore, a new cell printing method was developed to create living cellular bone cell arrays in a microfluidic channel. Several cell printing designs where osteocytes and osteoblasts heterotypically interacted at localized interfaces were evaluated. Physical contacts between the bone cells were characterized at high resolution by correlative atomic force microscopy (AFM)-fluorescence microscopy. We demonstrated that the platform is compatible with single-cell mechanostimulation by AFM nanoindentation and subsequent fluorescent analysis of the mechanoresponse. As a proof of concept, we showed the functionality of the platform by analysing the inducedin vivo-like Ca++wave in the printed osteocyte-osteoblast network upon mechanical stimulation by fluid flow shear stress.

High-Speed Atomic Force Microscopy Visualization of Protein-DNA Interactions Using DNA Origami Frames.

Direct, live imaging of protein-DNA interactions under physiological conditions is invaluable for understanding the mechanism and kinetics of binding and understanding the topological changes of the DNA strand. The DNA origami technology allows for precise placement of target molecules in a designed nanostructure. Here, we describe a protocol for the self-assembly of DNA origami frames with 2 stretched DNA sequences containing the binding site of a transcription factor, i.e., the Protein FadR, which is a TetR-family tanscription factor regulator for fatty acid metabolism in the archaeal organism Sulfolobus acidocaldarius. These frames can be used to study the dynamics of transcription factor binding using high-speed AFM and obtain mechanistic insights into the mechanism of action of transcription factors.

The Flo Adhesin Family.

The first step in the infection of fungal pathogens in humans is the adhesion of the pathogen to host tissue cells or abiotic surfaces such as catheters and implants. One of the main players involved in this are the expressed cell wall adhesins. Here, we review the Flo adhesin family and their involvement in the adhesion of these yeasts during human infections. Firstly, we redefined the Flo adhesin family based on the domain architectures that are present in the Flo adhesins and their functions, and set up a new classification of Flo adhesins. Next, the structure, function, and adhesion mechanisms of the Flo adhesins whose structure has been solved are discussed in detail. Finally, we identified from Pfam database datamining yeasts that could express Flo adhesins and are encountered in human infections and their adhesin architectures. These yeasts are discussed in relation to their adhesion characteristics and involvement in infections.

Nanomotion Detection-Based Rapid Antibiotic Susceptibility Testing.

Rapid antibiotic susceptibility testing (AST) could play a major role in fighting multidrug-resistant bacteria. Recently, it was discovered that all living organisms oscillate in the range of nanometers and that these oscillations, referred to as nanomotion, stop as soon the organism dies. This finding led to the development of rapid AST techniques based on the monitoring of these oscillations upon exposure to antibiotics. In this review, we explain the working principle of this novel technique, compare the method with current ASTs, explore its application and give some advice about its implementation. As an illustrative example, we present the application of the technique to the slowly growing and pathogenic Bordetella pertussis bacteria.

Single yeast cell nanomotions correlate with cellular activity.

Living single yeast cells show a specific cellular motion at the nanometer scale with a magnitude that is proportional to the cellular activity of the cell. We characterized this cellular nanomotion pattern of nonattached single yeast cells using classical optical microscopy. The distribution of the cellular displacements over a short time period is distinct from random motion. The range and shape of such nanomotion displacement distributions change substantially according to the metabolic state of the cell. The analysis of the nanomotion frequency pattern demonstrated that single living yeast cells oscillate at relatively low frequencies of around 2 hertz. The simplicity of the technique should open the way to numerous applications among which antifungal susceptibility tests seem the most straightforward.