Highlights

> Understanding the role of surface interactions in the antibacterial activity of layered double hydroxide nanoparticles by atomic force microscopy.

Jazia Awassa, Samantha Soulé, Damien Cornu, Christian Ruby and Sofiane El-Kirat-Chatel.

Nanoscale, 2022. https://doi.org/10.1039/D2NR02395D

Understanding the mechanisms of the interactions between zinc-based layered double hydroxides (LDHs) and bacterial surfaces is of great importance to improve the efficiency of these antibiotic-free antibacterial agents. In fact, the role of surface interactions in the antibacterial activity of zinc-based LDH nanoparticles compared to that of dissolution and generation of reactive oxygen species (ROS) is still not well documented. In this study, we show that ZnAl LDH nanoparticles exhibit a strong antibacterial effect against Staphylococcus aureus by inducing serious cell wall damages as revealed by the antibacterial activity tests and atomic force microscopy (AFM) imaging, respectively. The comparison of the antibacterial properties of ZnAl LDH nanoparticles and micron-sized ZnAl LDHs also demonstrated that the antibacterial activity of Zn-based LDHs goes beyond the simple dissolution into Zn2+ antibacterial ions. Furthermore, we developed an original approach to functionalize AFM tips with LDH films in order to probe their interactions with living S. aureus cells by means of AFM-based force spectroscopy (FS). The force spectroscopy analysis revealed that antibacterial ZnAl LDH nanoparticles show specific recognition of S. aureus cells with high adhesion frequency and remarkable force magnitudes. This finding provides a first insight into the antibacterial mechanism of Zn-based LDHs through direct surface interactions by which they are able to recognize and adhere to bacterial surfaces, thus damaging them and leading to subsequent growth inhibition.

> Deciphering the role of monosaccharides during phage infection of Staphylococcus aureus.

Baptiste Arbez, Marion Gardette, Christophe Gantzer, Neus Vilà, Isabelle Bertrand, and Sofiane El-Kirat-Chatel.

Nano Research, 2022. https://doi.org/10.1007/s12274-022-4600-3

As phages are extensively investigated as novel therapy tools but also as transfer agents for antibiotic resistance genes, thorough understanding of phage-host interactions becomes crucial. Prerequisite for phage infection is its adhesion to the host surface. Herein, we used atomic force microscopy-based single-particle force spectroscopy with phage-decorated tips to decipher the adhesion of phage 187 on living Staphylococcus aureus cells. We found that addition of free N-acetyl-Dglucosamine was able to decrease phage adhesion, suggesting that this monosaccharide plays major role in phage 187 infection of S. aureus. Moreover, phage 187 adhesion on monosaccharide-coated model surfaces combined with plaque forming unit counts suggested that a direct link can be established between the propensity to bind to a saccharide and the capability of the latter to inhibit phage infection. On a nanoscale level, single-particle force spectroscopy was successfully used to identify a major receptor required for phage 187 infection of S. aureus but also evidenced that this receptor was responsible for phage adhesion on host-cells. Our work demonstrates that single-particle force spectroscopy is a powerful platform to screen and predict the molecular target of phages on their host surfaces.

> Supported lysozyme for improved antimicrobial surface protection.

Audrey Beaussart, Chloé Retourney, Fabienne Quilès, Raphael Dos Santos Morais, Claire Gaiani, Henri-Pierre Fiérobe and Sofiane El-Kirat-Chatel.

Journal of Colloid and Interface Science, 2021. https://doi.org/10.1016/j.jcis.2020.08.107

Surface protection against biofilms is still an open challenge. Current strategies rely on coatings that are meant to guarantee antiadhesive or antimicrobial effects. While it seems difficult to ensure antiadhesion in complex media and against all the adhesive arsenal of microbes, strategies based on antimicrobials lack from sustainable functionalization methodologies to allow the perfect efficiency of the grafted molecules. Here we used the high affinity ligand-receptor interaction between biotin and streptavidin to functionalize surfaces with lysozyme, an enzyme that degrades the bacterial peptidoglycan cell wall. Biotinylated lysozyme was grafted on surfaces coated with streptavidin receptors. Using atomic force microscopy (AFM)-based single molecule force spectroscopy, we showed that grafting through ligand-receptor interaction allows the correct orientation of the enzyme on the substrate for enhanced activity towards the microbial target. The antibacterial efficiency was tested against Micrococcus luteus and revealed that surface protection was improved when lysozyme was grafted through the ligand-receptor interaction. These results suggest that bio-molecular interactions are promising for a sustainable grafting of antimicrobial agents on surfaces.

> Probing the adhesion of the common freshwater diatom Nitzschia palea at the nanoscale.

Freshwater biofilms play an essential ecological role but they also adversely affect human activities through undesirable biofouling of artificial submerged structures. They form complex aggregates of microorganisms that colonize any type of substratum. In phototrophic biofilms, diatoms dominate in biomass and produce copious amount of extracellular polymeric substances (EPS) that make them efficient early colonizers. Therefore, a better understanding of diatoms adhesive properties is essential to develop new anti-biofouling strategies. In this context, we used atomic force microscopy (AFM) to decipher the topography and adhesive mechanisms of the common freshwater diatom Nitzschia palea. Images in physiological conditions revealed typical ultrastructural features with a few nanometers resolution. Using single-cell force spectroscopy, we showed that N. palea strongly adhere to hydrophobic surfaces as compared to hydrophilic ones. Chemical force spectroscopy with hydrophobic tips further confirmed that adhesion is governed by surface-associated hydrophobic EPS distributed in clusters at the frustule surface, and mostly composed of (glyco)-lipids as revealed by Raman spectroscopy. Collectively, our results demonstrate that AFM-based nanoscopy, combined with Raman spectroscopy, is a powerful tool to provide new insights into the adhesion mechanisms of diatoms.

> AFM combined to ATR-FTIR reveals Candida cell wall changes under caspofungin treatment.

Fungal pathogens from Candida genus are responsible for severe life-threatening infections and the antifungal arsenal is still limited. Caspofungin, an antifungal drug used for human therapy, acts as a blocking agent of the cell wall synthesis by inhibiting the β-1,3-glucan-synthase encoded by FKS genes. Despite its efficiency, the number of genetic mutants that are resistant to caspofungin is increasing. An important challenge to improve antifungal therapy is to understand cellular phenomenon that are associated with drug resistance. Here we used atomic force microscopy (AFM) combined to Fourier transform infrared spectroscopy in attenuated total reflection mode (ATR-FTIR) to decipher the effect of low and high drug concentration on the morphology, mechanics and cell wall composition of two Candida strains, one susceptible and one resistant to caspofungin. Our results confirm that caspofungin induces a dramatic cell wall remodelling via activation of stress responses, even at high drug concentration. Additionally, we highlighted unexpected changes related to drug resistance, suggesting that caspofungin resistance associated with FKS gene mutations comes from a combination of effects: (i) an overall remodelling of yeast cell wall composition; and (ii) cell wall stiffening through chitin synthesis. This work demonstrates that AFM combined to ATR-FTIR is a valuable approach to understand at the molecular scale the biological mechanisms associated with drug resistance.

> Nanoscale adhesion forces between the fungal pathogen Candida albicans and macrophage.

The development of fungal infections is tightly controlled by the interaction of fungal pathogens with host immune cells. While the recognition of specific fungal cell wall components by immune receptors has been widely investigated, the molecular forces involved are not known. In this Communication, we show the ability of single-cell force spectroscopy to quantify the specific adhesion forces between the fungal pathogen Candida albicans and macrophages. The Candida-macrophage adhesion force is strong, up to ∼3000 pN, and corresponds to multiple cumulative bonds between lectin receptors expressed on the macrophage membrane and mannan carbohydrates on the fungal cell surface. Adhesion force signatures show constant force plateaus, up to >100 μm long, reflecting the extraction of elongated tethers from the macrophage membrane, a phenomenon which may increase the duration of intercellular adhesion. Adhesion strengthens with time, suggesting that the macrophage membrane engulfs the pathogen quickly after initial contact, leading to its internalization. The force nanoscopy method developed here holds great promise for understanding and controlling the early stages of microbe-immune interactions.

> Staphyloccocus aureus fibronectin-binding protein A mediates cell-cell adhesion through low affinity homophilic bonds.

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.

> Quantifying the forces guiding microbial cell adhesion using single-cell force spectroscopy.

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.

> Single-molecule analysis of Pseudomonas fluorescens footprints.

El-Kirat-Chatel S, Boyd CD, O'Toole GA and Dufrêne YF.

ACS Nano, 2014, 8:2, 1690-1698. https://doi.org/10.1021/nn4060489

Understanding the molecular mechanisms of bacterial adhesion and biofilm formation is an important topic in current microbiology and a key in nanomedicine for developing new antibacterial strategies. There is growing evidence that the production of extracellular polymeric substances at the cell-substrate interface plays a key role in strengthening bacterial adhesion. Yet, because these adhesive polymers are available in small amounts and are localized at interfaces, they are difficult to study using traditional techniques. Here, we use single-molecule atomic force microscopy (AFM) to functionally analyze the biophysical properties (distribution, adhesion, and extension) of bacterial footprints, that is, adhesive macromolecules left on substrate surfaces after removal of the attached cells. We focus on the large adhesin protein LapA from Pseudomonas fluorescens, which mediates cell attachment to a wide diversity of surfaces. Using AFM tips functionalized with specific antibodies, we demonstrate that adhesion of bacteria to hydrophobic substrates leads to the active accumulation of the LapA protein at the cell-substrate interface. We show that single LapA proteins left on the substrate after cell detachment localize into microscale domains corresponding to the bacterial size and exhibit multiple adhesion peaks reflecting the adhesion and extension of adsorbed LapA proteins. The mechanical behavior of LapA-based footprints makes them ideally suited to function as multipurpose bridging polymers, enabling P. fluorescens to attach to various surfaces. Our experiments show that single-molecule AFM offers promising prospects for characterizing the biophysics and dynamics of the cell-substrate interface in the context of bacterial adhesion, on a scale that was not accessible before.

> Single-cell and single-molecule analysis deciphers the localization, adhesion and mechanics of the biofilm adhesin LapA. 

El-Kirat-Chatel S, Beaussart A, Boyd CD, O'Toole GA and Dufrêne YF.

ACS Chemical Biology,2014, 9:2, 485-494. https://doi.org/10.1021/cb400794

The large adhesin protein LapA mediates adhesion and biofilm formation by Pseudomonas fluorescens. Although adhesion is thought to involve the long multiple repeats of LapA, very little is known about the molecular mechanism by which this protein mediates attachment. Here we use atomic force microscopy to unravel the biophysical properties driving LapA-mediated adhesion. Single-cell force spectroscopy shows that expression of LapA on the cell surface via biofilm-inducing conditions (i.e., phosphate-rich medium) or deletion of the gene encoding the LapG protease (LapA+ mutant) increases the adhesion strength of P. fluorescens toward hydrophobic and hydrophilic substrates, consistent with the adherent phenotypes observed in these conditions. Substrate chemistry plays an unexpected role in modulating the mechanical response of LapA, with sequential unfolding of the multiple repeats occurring only on hydrophilic substrates. Biofilm induction also leads to shortening of the protein extensions, reflecting stiffening of their conformational properties. Using single-molecule force spectroscopy, we next demonstrate that the adhesin is randomly distributed on the surface of wild-type cells and can be released into the solution. For LapA+ mutant cells, we found that the adhesin massively accumulates on the cell surface without being released and that individual LapA repeats unfold when subjected to force. The remarkable adhesive and mechanical properties of LapA provide a molecular basis for the "multi-purpose" adhesion function of LapA, thereby making P. fluorescens capable of colonizing diverse environments.

> Nanoscale imaging of the Candida-macrophage interaction using correlated fluorescence-atomic force microscopy.

El-Kirat-Chatel S and Dufrêne YF. 

ACS Nano, 2012, 6:12, 10792-10799. https://doi.org/10.1021/nn304116f

Knowledge of the molecular bases underlying the interaction of fungal pathogens with immune cells is critical to our understanding of fungal infections and offers exciting perspectives for controlling immune responses for therapy. Although fluorescence microscopy is a valuable tool to visualize pathogen-host interactions, the spatial resolution is low, meaning the fine structural details of the interacting cells cannot be observed. Here, we demonstrate the ability of correlated fluorescence-atomic force microscopy (AFM) to image the various steps of the interaction between fungal pathogens and macrophages with nanoscale resolution. We focus on Candida albicans, known to grow as two morphological forms (yeast cells, filamentous hyphae) that play important roles in modulating the interaction with macrophages. We observe the main steps of macrophage infection, including initial intercellular contact, phagocytosis by internalization of yeast cells, intracellular hyphal growth leading to mechanical stretching, and piercing of the macrophage membrane resulting in pathogen escape. While fluorescence imaging clearly distinguishes fungal cells from macrophages during the various steps of the process, AFM captures nanoscale structural features of the macrophage surface that are of high biological relevance, including ruffles, lamellipodia, filopodia, membrane remnants, and phagocytic cups. As fungal pathogenesis is mainly controlled by the ability of fungi to escape from immune cells, the nanoimaging platform established here has great potential in nanomedicine for understanding and controlling fungal infections.