Summary
SdrG is a cell surface adhesin from Staphylococcus epidermidis which binds to the blood plasma protein fibrinogen (Fg). Ligand binding follows a ‘dock, lock and latch’ model involving dynamic ...conformational changes of the adhesin that result in a greatly stabilized adhesin–ligand complex. To date, the force and dynamics of this multistep interaction are poorly understood. Here we use atomic force microscopy (AFM) to unravel the binding strength and cell surface localization of SdrG at molecular resolution. Single‐cell force spectroscopy shows that SdrG mediates time‐dependent attachment to Fg‐coated surfaces. Single‐molecule force spectroscopy with Fg‐coated AFM tips demonstrates that the adhesin forms nanoscale domains on the cell surface, which we believe contribute to strengthen cell adhesion. Notably, we find that the rupture force of single SdrG–Fg bonds is very large, ∼ 2 nN, equivalent to the strength of a covalent bond, and shows a low dissociation rate, suggesting that the bond is very stable. The strong binding force, slow dissociation and clustering of SdrG provide a molecular foundation for the ability of S. epidermidis to colonize implanted biomaterials and to withstand physiological shear forces.
Living cells use cell surface proteins, such as mechanosensors, to constantly sense and respond to their environment. However, the way in which these proteins respond to mechanical stimuli and ...assemble into large complexes remains poorly understood at the molecular level. In the past years, atomic force microscopy (AFM) has revolutionized the way in which biologists analyze cell surface proteins to molecular resolution. In this Commentary, we discuss how the powerful set of advanced AFM techniques (e.g. live-cell imaging and single-molecule manipulation) can be integrated with the modern tools of molecular genetics (i.e. protein design) to study the localization and molecular elasticity of individual mechanosensors on the surface of living cells. Although we emphasize recent studies on cell surface proteins from yeasts, the techniques described are applicable to surface proteins from virtually all organisms, from bacteria to human cells.
During the past decades, several methods (e.g., electron microscopy, flow chamber experiments, surface chemical analysis, surface charge and surface hydrophobicity measurements) have been developed ...to investigate the mechanisms controlling the adhesion of microbial cells to other cells and to various other substrates. However, none of the traditional approaches are capable of looking at adhesion forces at the single-cell level. In recent years, atomic force microscopy (AFM) has been instrumental in measuring the forces driving microbial adhesion on a single-cell basis. The method, known as single-cell force spectroscopy (SCFS), consists of immobilizing a single living cell on an AFM cantilever and measuring the interaction forces between the cellular probe and a solid substrate or another cell. Here we present SCFS protocols that we have developed for quantifying the cell adhesion forces of medically important microbes. Although we focus mainly on the probiotic bacterium Lactobacillus plantarum, we also show that our procedures are applicable to pathogens, such as the bacterium Staphylococcus epidermidis and the yeast Candida albicans. For well-trained microscopists, the entire protocol can be mastered in 1 week.
Summary
Trimeric autotransporter adhesins (TAAs) are bacterial surface proteins that fulfil important functions in pathogenic Gram‐negative bacteria. Prominent examples of TAAs are found in ...Burkholderia cepacia complex, a group of bacterial species causing severe infections in patients with cystic fibrosis. While there is strong evidence that Burkholderia cenocepacia TAAs mediate adhesion, aggregation and colonization of the respiratory epithelium, we still know very little about the molecular mechanisms behind these interactions. Here, we use single‐molecule atomic force microscopy to unravel the binding mechanism of BCAM0224, a prototype TAA from B. cenocepacia K56‐2. We show that the adhesin forms homophilic trans‐interactions engaged in bacterial aggregation, and that it behaves as a spring capable to withstand high forces. We also find that BCAM0224 binds collagen, a major extracellular component of host epithelia. Both homophilic and heterophilic interactions display low binding affinity, which could be important for epithelium colonization. We then demonstrate that BCAM0224 recognizes receptors on living pneumocytes, and leads to the formation of membrane tethers that may play a role in promoting adhesion. Collectively, our results show that BCAM0224 is a multifunctional adhesin endowed with remarkable binding properties, which may represent a general mechanism among TAAs for strengthening bacterial adhesion.
We developed a new in vitro model for a multi-parameter characterization of the time course interaction of Candida fungal cells with J774 murine macrophages and human neutrophils, based on the use of ...combined microscopy, fluorometry, flow cytometry and viability assays. Using fluorochromes specific to phagocytes and yeasts, we could accurately quantify various parameters simultaneously in a single infection experiment: at the individual cell level, we measured the association of phagocytes to fungal cells and phagocyte survival, and monitored in parallel the overall phagocytosis process by measuring the part of ingested fungal cells among the total fungal biomass that changed over time. Candida albicans, C. glabrata, and C. lusitaniae were used as a proof of concept: they exhibited species-specific differences in their association rate with phagocytes. The fungal biomass uptaken by the phagocytes differed significantly according to the Candida species. The measure of the survival of fungal and immune cells during the interaction showed that C. albicans was the more aggressive yeast in vitro, destroying the vast majority of the phagocytes within five hours. All three species of Candida were able to survive and to escape macrophage phagocytosis either by the intraphagocytic yeast-to-hyphae transition (C. albicans) and the fungal cell multiplication until phagocytes burst (C. glabrata, C. lusitaniae), or by the avoidance of phagocytosis (C. lusitaniae). We demonstrated that our model was sensitive enough to quantify small variations of the parameters of the interaction. The method has been conceived to be amenable to the high-throughput screening of mutants in order to unravel the molecular mechanisms involved in the interaction between yeasts and host phagocytes.
The localization of the LapA protein to the cell surface is a key step required by Pseudomonas fluorescens Pf0-1 to irreversibly attach to a surface and form a biofilm. LapA is a member of a diverse ...family of predicted bacterial adhesins, and although lacking a high degree of sequence similarity, family members do share common predicted domains. Here, using mutational analysis, we determine the significance of each domain feature of LapA in relation to its export and localization to the cell surface and function in biofilm formation. Our previous work showed that the N terminus of LapA is required for cleavage by the periplasmic cysteine protease LapG and release of the adhesin from the cell surface under conditions unfavorable for biofilm formation. We define an additional critical region of the N terminus of LapA required for LapG proteolysis. Furthermore, our results suggest that the domains within the C terminus of LapA are not absolutely required for biofilm formation, export, or localization to the cell surface, with the exception of the type I secretion signal, which is required for LapA export and cell surface localization. In contrast, deletion of the central repetitive region of LapA, consisting of 37 repeats of 100 amino acids, results in an inability to form a biofilm. We also used single-molecule atomic force microscopy to further characterize the role of these domains in biofilm formation on hydrophobic and hydrophilic surfaces. These studies represent the first detailed analysis of the domains of the LapA family of biofilm adhesin proteins.
Staphylococcus aureus is an important opportunistic pathogen which is a leading cause of biofilm-associated infections on indwelling medical devices. The cell surface-located fibronectin-binding ...protein A (FnBPA) plays an important role in the accumulation phase of biofilm formation by methicillin-resistant S. aureus (MRSA), but the underlying molecular interactions are not yet established. Here, we use single-cell and single-molecule atomic force microscopy to unravel the mechanism by which FnBPA mediates intercellular adhesion. We show that FnBPA is responsible for specific cell-cell interactions that involve the FnBPA A domain and cause microscale cell aggregation. We demonstrate that the strength of FnBPA-mediated adhesion originates from multiple low-affinity homophilic interactions between FnBPA A domains on neighboring cells. Low-affinity binding by means of FnBPA may be important for biofilm dynamics. These results provide a molecular basis for the ability of FnBPA to promote cell accumulation during S. aureus biofilm formation. We speculate that homophilic interactions may represent a generic strategy among staphylococcal cell surface proteins for guiding intercellular adhesion. As biofilm formation by MRSA strains depends on proteins rather than polysaccharides, our approach offers exciting prospects for the design of drugs or vaccines to inhibit protein-dependent intercellular interactions in MRSA biofilms.
Staphylococcus aureus is a human pathogen that forms biofilms on indwelling medical devices, such as central venous catheters and prosthetic joints. This leads to biofilm infections that are difficult to treat with antibiotics because many cells within the biofilm matrix are dormant. The fibronectin-binding proteins (FnBPs) FnBPA and FnBPB promote biofilm formation by clinically relevant methicillin-resistant S. aureus (MRSA) strains, but the molecular mechanisms involved remain poorly understood. We used atomic force microscopy techniques to demonstrate that FnBPA mediates cell-cell adhesion via multiple, low-affinity homophilic bonds between FnBPA A domains on adjacent cells. Therefore, FnBP-mediated homophilic interactions represent an interesting target to prevent MRSA biofilms. We propose that such homophilic mechanisms may be widespread among staphylococcal cell surface proteins, providing a means to guide intercellular adhesion and biofilm accumulation.
In the last decades, atomic force microscopy (AFM) has evolved towards an accurate and lasting tool to study the surface of living cells in physiological conditions. Through imaging, single-molecule ...force spectroscopy and single-cell force spectroscopy modes, AFM allows to decipher at multiple scales the morphology and the molecular interactions taking place at the cell surface. Applied to microbiology, these approaches have been used to elucidate biophysical properties of biomolecules and to directly link the molecular structures to their function. In this review, we describe the main methods developed for AFM-based microbial surface analysis that we illustrate with examples of molecular mechanisms unravelled with unprecedented resolution.
Despite the clinical importance of bacterial-fungal interactions, their molecular details are poorly understood. A hallmark of such medically important interspecies associations is the interaction ...between the two nosocomial pathogens Staphylococcus aureus and Candida albicans, which can lead to mixed biofilm-associated infections with enhanced antibiotic resistance. Here, we use single-cell force spectroscopy (SCFS) to quantify the forces engaged in bacterial-fungal co-adhesion, focusing on the poorly investigated S. epidermidis-C. albicans interaction. Force curves recorded between single bacterial and fungal germ tubes showed large adhesion forces (~5 nN) with extended rupture lengths (up to 500 nm). By contrast, bacteria poorly adhered to yeast cells, emphasizing the important role of the yeast-to-hyphae transition in mediating adhesion to bacterial cells. Analysis of mutant strains altered in cell wall composition allowed us to distinguish the main fungal components involved in adhesion, i.e. Als proteins and O-mannosylations. We suggest that the measured co-adhesion forces are involved in the formation of mixed biofilms, thus possibly as well in promoting polymicrobial infections. In the future, we anticipate that this SCFS platform will be used in nanomedicine to decipher the molecular mechanisms of a wide variety of pathogen-pathogen interactions and may help in designing novel anti-adhesion agents.
Infections by pathogenic bacteria have been threatening several fields as food industries, agriculture, textile industries and healthcare products. Layered double hydroxides materials (LDHs), also ...called anionic clays, could be utilized as efficient antibacterial materials due to their several interesting properties such as ease of synthesis, tunable chemical composition, biocompatibility and anion exchange capacity. Pristine LDHs as well as LDH-composites including antibacterial molecules and nanoparticles loaded-LDHs were proven to serve as efficient antibacterial agents against various Gram-positive and Gram-negative bacterial strains. The achieved antibacterial effect was explained by the following mechanisms: (1) Direct contact between the materials and bacterial cells driven by electrostatic interactions between positively charged layers and negatively charged cell membranes, (2) Dissolution and gradual release over time of metallic ions or antibacterial molecules, (3) Generation of reactive oxygen species.
Display omitted
•LDHs antibacterial mechanisms are: direct contact, dissolution and ROS generation.•Direct contact is improved with surface charge, roughness and smaller particle size.•Release of antibacterial molecules depends on their type and intercalation method.•Release of metallic ions depends on molar ratio, stability and intercalated anion.•ROS generation needs a suitable irradiation source and more experimental evidences.
PDF
