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.
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 tips bearing specific probes (lectins, antibodies) to analyze the major glycopolymers of pathogenic and non-pathogenic yeast cells at molecular resolution. We show that non-ubiquitous β-1,2-mannans are largely exposed on the surface of native cells from pathogenic Candida albicans and C. glabrata, the former species displaying the highest glycopolymer density and extensions. We also find that chitin, a major component of the inner layer of the yeast cell wall, is much more abundant in C. albicans. These differences in molecular properties, further supported by flow cytometry measurements, may play an important role in strengthening cell wall mechanics and immune interactions. This study demonstrates that single-molecule AFM, combined with immunological and fluorescence methods, is a powerful platform in fungal glycobiology for probing the density, distribution and extension of specific cell wall glycoconjugates. In nanomedicine, we anticipate that this new form of AFM-based nanoglycobiology will contribute to the development of sugar-based drugs, immunotherapeutics, vaccines and diagnostics.
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 function of each transporter was characterized by supplementation experiments, and the kinetic parameters of the uptake transport of uracil were measured using radiolabeled substrate. Fur4p specifically transports uracil and 5-fluorouracil. Dal4p is very close to Fur4p and transports allantoin (glyoxyldiureide). Deletion of the FUR4 gene confers resistance to 5-fluorouracil as well as cross-resistance to triazoles and imidazole antifungals when they are used simultaneously with 5-fluorouracil. However, the nucleobase transporters are not involved in azole uptake. Only fluorinated pyrimidines, not pyrimidines themselves, are able to promote cross-resistance to azoles by both the salvage and the de novo pathway of pyrimidine synthesis. A reinterpretation of the data previously obtained led us to show that subinhibitory doses of 5-fluorocytosine, 5-fluorouracil, and 5-fluorouridine also were able to trigger resistance to fluconazole in susceptible wild-type strains of C. lusitaniae and of different Candida species. Our results suggest that intracellular fluorinated nucleotides play a key role in azole resistance, either by preventing azoles from targeting the lanosterol 14-alpha-demethylase or its catalytic site or by acting as a molecular switch for the triggering of efflux transport.
Series of force-distance profiles obtained by stretching adhesins from various microbial species (see text for details): single adhesion peaks reflecting specific recognition (A), sawtooth patterns ...with multiple force peaks corresponding to the force-induced unfolding of protein secondary structures (B), constant force plateaus originating from the mechanical unzipping of amyloid interactions formed between multiple adhesins (C), and single large adhesion force peaks with linear shapes obtained by pulling on Gram-positive bacterial pili (D). AFM imaging enables microbiologists to decipher the nanoscale architecture of cell surfaces and its remodelling upon growth or interaction with drugs. ...force spectroscopy allows us to understand how cell surface receptors are spatially organized (e.g. clustering) and respond to force (e.g. single specific bonds, sequential unfolding, zipper-like adhesion, and spring-like properties).
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 peptidoglycans remain largely unknown, mainly owing to the paucity of appropriate probing techniques. Here, we use atomic force microscopy to explore the binding mechanism of the major autolysin Acm2 from the probiotic bacterium Lactobacillus plantarum. Atomic force microscopy imaging shows that incubation of bacterial cells with Acm2 leads to major alterations of the cell-surface nanostructure, leading eventually to cell lysis. Single-molecule force spectroscopy demonstrates that the enzyme binds with low affinity to structurally different peptidoglycans and to chitin, and that glucosamine in the glycan chains is the minimal binding motif. We also find that Acm2 recognizes mucin, the main extracellular component of the intestinal mucosal layer, thereby suggesting that this enzyme may also function as a cell adhesion molecule. The binding mechanism (low affinity and broad specificity) of Acm2 may represent a generic mechanism among cell-wall hydrolases for guiding cell division and cell adhesion.
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