Sofiane El-Kirat-Chatel

Laboratoire de Chimie Physique et Microbiologie pour les Matériaux et l’Environnement CNRS

Over the past two decades, atomic force microscopy (AFM) has emerged as one the most popular tools for in situ study of physical, chemical, and biological processes that take place at the cellular to molecular level. Besides, it allows nanoscale characterization in an easy/fast way without the need of any complex procedures or sample preparation. T...

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 phag...

Micro- and Nano-Fibrillated Cellulose (MNFC) have gained increasing attention due to their remarkable properties, but their production usually requires an intensive multi-step process. This study proposes to find a novel approach involving steam explosion for the production of lignin-containing micro- and nano-fibrillated cellulose (L-MNFC) using E...

Squalamine is a natural aminosterol that has been discovered in the tissues of the dogfish shark (Squalus acanthias). Studies have previously demonstrated that this promoter compound and its derivatives exhibit potent bactericidal activity against Gram-negative, Gram-positive bacteria, and multidrug-resistant bacteria. The antibacterial activity of...

Whey protein concentrate powders can be strongly affected by storage conditions inducing both chemical and structural protein modifications. In the present work, a combination of different storage conditions were evaluated, leading to the evaluation of 6 different batches that were chosen in order to evaluate the impact of the following parameters:...

We fabricated an electrochemical MIP chemosensor for rapid identification and quantification of E. coli strain using 2-aminophenyl boronic acid as the functional monomer. This strain is a modified gram-negative strain of Escherichia coli bacterium, an ordinary human gut component. The E. coli strongly interacts with a boronic acid because of porous...

Dairy powders are usually subjected to environmental variations during storage and/or shipment that strongly impact their chemical, nutritional and structural features. Nevertheless, these modifications are rarely investigated at the particle surface level, which represents the interface in contact with air, water, materials or other powders and di...

Microbes employ a variety of strategies to adhere to abiotic and biotic surfaces, as well as host cells. In addition to their surface physicochemical properties (e.g. charge, hydrophobic balance), microbes produce appendages (e.g. pili, fimbriae, flagella) and express adhesion proteins embedded in the cell wall or cell membrane, with adhesive domai...

Pili are polymeric proteins located at the cell surface of bacteria. These filamentous proteins play a pivotal role in bacterial adhesion with the surrounding environment. They are found both in Gram-negative and Gram-positive bacteria but differ in their structural organization. Purifying these high molecular weight proteins is challenging and has...

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 functionali...

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 ta...

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 extracellula...

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 ta...

In the last decade, there has been an increasing interest in the potential health effects associated with the consumption of lactic acid bacteria (LAB) in foods. Some of these bacteria such as Lactobacillus rhamnosus GG (LGG) are known to adhere to milk components, which may impact their distribution and protection within dairy matrices and therefo...

Microbes have evolved sophisticated strategies to colonize biotic and abiotic surfaces. Forces play a central role in microbial cell adhesion processes, yet until recently these were not accessible to study at the molecular scale. Unlike traditional assays, atomic force microscopy (AFM) is capable to study forces in single cell surface molecules an...

Functionalization of AFM probes with biomolecules or microorganisms allows for a better understanding of the interaction mechanisms driving microbial adhesion. Here we describe the most commonly used protocols to graft molecules and bacteria to AFM cantilevers. The bioprobes obtained that way enable to measure forces down to the single-cell and sin...

Lactic Acid Bacteria (LAB) are not homogeneously located in the dairy matrix and their spatial distribution seems to be controlled by the establishment of adhesive interactions between matrix components and bacterial surface biomolecules. However the mechanisms of interaction remain unknown although they constitute an interesting way of study to ap...

Milk is the most popular matrix for the delivery of lactic acid bacteria, but little is known about how milk impacts bacterial functionality. Here, the adhesion mechanisms of Lactobacillus rhamnosus GG (LGG) surface mutants to a milk component, the milk fat globule membrane (MFGM), were compared using atomic force microscopy (AFM). AFM results reve...

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 of the β-1,3-glucan-synthase encoded by FKS genes. Despite its efficiency, the number of gen...

Up to recent years, bacterial adhesion has mostly been evaluated at the population level. Single cell level has improved in the past few years allowing a better comprehension of the implication of individual behaviors as compared to the one of a whole community. A new approach using atomic force microscopy (AFM) to measure adhesion forces between a...

Many fungal adhesins have short, β-aggregation-prone sequences that play important functional roles, and in the Candida albicans adhesin Als5p, these sequences cluster the adhesins after exposure to shear force. Here, we report that Saccharomyces cerevisiae flocculins Flo11p and Flo1p have similar β-aggregation-prone sequences and are similarly sti...

Low-density lipoprotein receptor-related protein 1 (LRP-1) is an endocytic receptor that mediates the clearance of proteases involved in cancer progression and is thus considered a promising therapeutic target. However, it has been demonstrated that LRP-1 is also able to regulate the endocytosis of membrane-anchored proteins. Thus, strategies that...

The development of bacterial strains that are resistant to multiple antibiotics has urged the need for new antibacterial therapies. An exciting approach to fight bacterial diseases is the use of antiadhesive agents capable to block the adhesion of the pathogens to host tissues, the first step of infection. We report the use of a novel atomic force...

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 spectro...

Many fungal pathogens produce cell surface polysaccharides that play essential roles in host-pathogen interactions. In Aspergillus fumigatus, the newly discovered polysaccharide galactosaminogalactan (GAG) mediates adherence to a variety of substrates through molecular mechanisms that are poorly understood. Here we use atomic force microscopy to un...

Unlabelled: 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 un...

P1 (AgI/II) is a sucrose-independent adhesin of Streptococcus mutans whose functional architecture on the cell surface is not fully understood. S. mutans cells subjected to mechanical extraction were significantly diminished in adherence to immobilized salivary agglutinin (SAG), yet remained immunoreactive and readily aggregated by fluid-phase SAG....

Candida glabrata is an opportunistic human fungal pathogen which binds to surfaces mainly through the Epa family of cell adhesion proteins. While some Epa proteins mediate specific lectin-like interactions with human epithelial cells, others promote adhesion and biofilm formation on plastic surfaces via nonspecific interactions that are not yet elu...

Microbial pathogens are highly complex and heterogeneous systems. Cell populations generally contain subgroups of cells which exhibit differences in growth rate, as well as resistance to stress and drug treatment. In addition, individual cells are spatially organized and heterogeneous; this cellular heterogeneity is used to perform key functions. T...

In the baker's yeast Saccharomyces cerevisiae, cell-cell adhesion ("flocculation") is conferred by a family of lectin-like proteins known as the flocculin (Flo) proteins. Knowledge of the adhesive and mechanical properties of flocculins is important for understanding the mechanisms of yeast adhesion, and may help controlling yeast behaviour in biot...

Candida albicans is one of the most medically important pathogens, mainly due to its high frequency and strong ability to form biofilm causing severe human infections. Therefore, a current challenge in public health remains to further elucidate C. albicans virulence factors to help developing new targeted therapeutic strategies. Understanding patho...

A variety of bacterial pathogens use nanoscale protein fibers called type IV pili to mediate cell adhesion, a primary step leading to infection. Currently, how these nanofibers respond to mechanical stimuli and how this response is used to control adhesion is poorly understood. Here, we use atomic force microscopy techniques to quantify the forces...

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 interactio...

We characterized two additional membrane transporters (Fur4p and Dal4p) of the nucleobase cation symporter 1 (NCS1) family involved in the uptake transport of pyrimidines and related molecules in the opportunistic pathogenic yeast Candida lusitaniae. Simple and multiple null mutants were constructed by gene deletion and genetic crosses. The functio...

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. He...

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 tradition...

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...

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...

Although bacterial pili are known to mediate cell adhesion to a variety of substrates, the molecular interactions behind this process are poorly understood. We report the direct measurement of the forces guiding pili-mediated adhesion, focusing on the medically important probiotic bacterium Lactobacillus rhamnosus GG (LGG). Using non-invasive singl...

Cell surface proteins of bacteria play essential roles in mediating the attachment of pathogens to host tissues and, therefore, represent key targets for anti-adhesion therapy. In the opportunistic pathogen Staphylococcus epidermidis , the adhesion protein SdrG mediates attachment of bacteria to the blood plasma protein fibrinogen (Fg) through a bi...

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 anti...

Macroscopic assays that are traditionally used to investigate the adhesion behaviour of microbial cells provide averaged information obtained on large populations of cells and do not measure the fundamental forces driving single-cell adhesion. Here, we use single-cell force spectroscopy (SCFS) to quantify the specific and non-specific forces engage...

Peptidoglycan hydrolases are bacterial secreted enzymes that cleave covalent bonds in the cell-wall peptidoglycan, thereby fulfilling major physiological functions during cell growth and division. Although the molecular structure and functional roles of these enzymes have been widely studied, the molecular details underlying their interaction with...

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 B. cenocepaci...

Single-cell force spectroscopy is a powerful atomic force microscopy modality in which a single living cell is attached to the atomic force microscopy cantilever to quantify the forces that drive cell-cell and cell-substrate interactions. Although various single-cell force spectroscopy protocols are well established for animal cells, application of...

Most microbes are coated with carbohydrates that show remarkable structural variability and play a crucial role in mediating microbial-host interactions. Understanding the functions of cell wall glycoconjugates requires detailed knowledge of their molecular organization, diversity and heterogeneity. Here we use atomic force microscopy (AFM) with ti...

The advent of fungal pathogens that are resistant to the classic repertoire of antifungal drugs has increased the need for new therapeutic agents. A prominent example of such a novel compound is caspofungin, known to alter cell wall biogenesis by inhibiting β-1,3-d-glucan synthesis. Although much progress has been made in understanding the mechanis...

Cellular morphogenesis in the fungal pathogen Candida albicans is associated with changes in cell wall composition that play important roles in biofilm formation and immune responses. Yet, how fungal morphogenesis modulates the biophysical properties and interactions of the cell surface molecules is poorly understood, mainly owing to the paucity of...

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...

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 i...

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 quan...

Representative pictures of the J774 macrophages after 5 hours of infection with the three Candida species in culture flasks at 1M:1Y MOI. Note that the totality of C. glabrata cells were engulfed, whereas C. albicans (mostly in filamentous form) and C. lusitaniae cells were still observed outside the macrophages. The scale bars represent 30 µm. See...

Video-microscopy observation of J774 macrophages infected with C. albicans at 1M:1Y MOI. (AVI)

Macrophage cell death is not due to a nutritional depletion of the medium during infection. The quantity of glucose available was measured in the supernatant of the J774 macrophages that were infected with live or UV-killed yeasts of the three Candida species for 5 and 24 hours at a MOI of 1M:1Y (Method S1), and compared to uninfected macrophages,...

Macrophages viability in cRPMI medium with and without CFW (5 µg/ml) by calcein fluorescence measurements. Each bar is the average of two experiments ± standard error. (TIF)

Calcein fluorescence varies proportionaly with the number of macrophages. Each bar is the average of three experiments ± standard error. (TIF)

Yeast cells muliplication over time by OD600nm and CFW fluorescence measurements in cRPMI medium with 5 µg/ml of CFW. Dashed lines show linear regression lines, and their slopes are indicated. (TIF)

Analysis of the interactions involving the J774 macrophages and stationary-phase living yeast cells at 1M:1Y MOI over 24-hour time course experiments. Figure S5 details how the diagram of Figure 3A was built. Figure S5A shows the flow cytometry analysis of the macrophages and corresponds to the left part of the diagram of Figure 3A. Each bar repres...

Macrophage fungicidal activity toward the different Candida species. To investigate the capacity of the macrophages to kill the different species of Candida, the survival of the yeast cells was determined following 5 and 24 hours of incubation with phagocytic cells at a 1M:1Y MOI (Method S2). 100% of the C. albicans cells survived, while 20% and 40...

Method involved in glucose assay. See Table S1. (DOC)

Method documenting the survival of ingested yeasts. See Figure S7. (DOC)

Video-microscopy observation of J774 macrophages infected with C. lusitaniae at 1M:2Y MOI. (AVI)

Video-microscopy observation of J774 macrophages infected with C. glabrata at 1M:5Y MOI. (AVI)

Yeast cells multiplication in cRPMI medium with and without CFW (5 µg/ml) by OD600nm measurements. C.a: C.albicans, C.g: C.glabrata, C.l: C.lusitaniae. (TIF)

We describe a new cloning-free strategy to delete genes in the opportunistic pathogenic yeast Candida lusitaniae. We first constructed two ura3 Δ strains in C. lusitaniae for their use in transformation experiments. One was deleted for the entire URA3 coding sequence; the other possessed a partial deletion within the coding region, which was used t...

The Agrocybe aegerita mitochondrial genome possesses two polB genes with linear plasmid origin. The cloning and sequencing of the regions flanking Aa-polB P1 revealed two large inverted repeats (higher than 2421 nt) separated by a single copy region of 5834 nt. Both repeats contain identical copies of the nad4 gene. The single copy region contains...