· Force Nanoscopy as a Versatile Platform for Quantifying the Activity of Antiadhesion Compounds Targeting Bacterial Pathogens
A. Beaussart, M. Abellan-Flos, S.
El-Kirat-Chatel, S. P. Vincent, Y.F. Dufrêne, Nano Letetrs, 2016, 16, 1299
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 microscopy (AFM) platform for quantifying the activity of antiadhesion compounds directly on living bacteria, thus without labeling or purification. Novel fullerene-based mannoconjugates bearing 10 carbohydrate ligands and a thiol bond were efficiently prepared. The thiol functionality could be exploited as a convenient handle to graft the multimeric species onto AFM tips. Using a combination of single-molecule and single-cell AFM assays, we demonstrate that, unlike mannosidic monomers, multivalent glycofullerenes strongly block the adhesion of uropathogenic Escherichia coli bacteria to their carbohydrate receptors. We expect that the nanoscopy technique developed here will help designing new antiadhesion drugs to treat microbial infections, including those caused by multidrug resistant organisms.
· Quantifying the forces guiding microbial cell adhesion using single-cell force spectroscopy
A. Beaussart, S. El-Kirat-Chatel, R. M. A. Sullan, D. Alsteens, P. Herman, S. Derclaye, Y. F. Dufrêne, Nature Protocols, 2014, 9, 1049
Highlighted as 'featured protocol' by the journal
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-cell force spectroscopy of probiotic bacteria
A. Beaussart, S. El-Kirat-Chatel, P. Herman, D. Alsteens, J. Mahillon, P. Hols, Y. F. Dufrêne, Biophysical Journal, 2013, 104, 1886Highlighted as 'featured protocol' by the journal
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 the method to individual bacterial cells remains challenging, mainly owing to the lack of appropriate methods for the controlled attachment of single live cells on cantilevers. We present a nondestructive protocol for single-bacterial cell force spectroscopy, which combines the use of colloidal probe cantilevers and of a bioinspired polydopamine wet adhesive. Living cells from the probiotic species Lactobacillus plantarum are picked up with a polydopamine-coated colloidal probe, enabling us to quantify the adhesion forces between single bacteria and biotic (lectin monolayer) or abiotic (hydrophobic monolayer) surfaces. These minimally invasive single-cell experiments provide novel, to our knowledge, insight into the specific and nonspecific forces driving the adhesion of L. plantarum, and represent a generic platform for studying the molecular mechanisms of cell adhesion in probiotic and pathogenic bacteria.
· Surface structure characterization of Aspergillus fumigatus conidia mutated in the melanin synthesis pathway and their human cellular immune response
J. Bayry, A. Beaussart, Y. F. Dufrêne, M. Shrama, K. bansal, O. Kniemeyer, V. Aimanianda, A. A. Brakkage, S. V. Kaveri, K.J. Kwong-Chung, J-P. latgé, A. Beauvais, Infection and Immunity, 2014, 82, 3141Highlighted in the 'Spotlight report' of the journal
In Aspergillus fumigatus, the conidial surface contains dihydroxynaphthalene (DHN)-melanin. Six-clustered gene products have been identified that mediate sequential catalysis of DHN-melanin biosynthesis. Melanin thus produced is known to be a virulence factor, protecting the fungus from the host defense mechanisms. In the present study, individual deletion of the genes involved in the initial three steps of melanin biosynthesis resulted in an altered conidial surface with masked surface rodlet layer, leaky cell wall allowing the deposition of proteins on the cell surface and exposing the otherwise-masked cell wall polysaccharides at the surface. Melanin as such was immunologically inert; however, deletion mutant conidia with modified surfaces could activate human dendritic cells and the subsequent cytokine production in contrast to the wild-type conidia. Cell surface defects were rectified in the conidia mutated in downstream melanin biosynthetic pathway, and maximum immune inertness was observed upon synthesis of vermelone onward. These observations suggest that although melanin as such is an immunologically inert material, it confers virulence by facilitating proper formation of the A. fumigatus conidial surface.
· Single-molecule imaging and functional analysis of Als adhesins and mannan during Candida albicans morphogenesis
A. Beaussart, D. Alsteens, S.
El-Kirat-Chatel, P.N. Lipke, S. Kucharikova, P. Van Dijck, Y.F. Dufrêne, ACS nano, 2012, 6, 10950
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 high-resolution imaging techniques. Here, we use single-molecule atomic force microscopy to localize and analyze the key components of the surface of living C. albicans cells during morphogenesis. We show that the yeast-to-hypha transition leads to a major increase in the distribution, adhesion, unfolding, and extension of Als adhesins and their associated mannans on the cell surface. We also find that morphogenesis dramatically increases cell surface hydrophobicity. These molecular changes are critical for microbe-host interactions, including adhesion, colonization, and biofilm formation. The single-molecule experiments presented here offer promising prospects for understanding how microbial pathogens use cell surface molecules to modulate biofilm and immune interactions
· Single-cell force spectroscopy of the medically-important Staphylococcus epidermis - Candida albicans interaction
A. Beaussart, P. Herman, S. El-Kirat-Chatel, P.N. Lipke, S. Kucharikova, P. Van Dijck, Y.F. Dufrêne, Nanoscale, 2013, 5:22, 10894-10900
Single-cell force spectroscopy of Als mediated fungal adhesion