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 domains targeting specific ligands or chemical properties. Atomic force microscopy (AFM) is perfectly suited to deciphering the adhesive properties of microbial cells. Notably, AFM imaging has revealed the cell wall topographical organization of live cells at unprecedented resolution, and AFM has a dual capability to probe adhesion at the single-cell and single-molecule levels. AFM is thus a powerful tool for unravelling the molecular mechanisms of microbial adhesion at scales ranging from individual molecular interactions to the behaviours of entire cells. In this review, we cover some of the major breakthroughs facilitated by AFM in deciphering the microbial adhesive arsenal, including the exciting development of anti-adhesive strategies.
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Electrostatics of soft (ion-permeable) (bio)particles (e.g. microorganisms, core/shell colloids) in aqueous electrolytes is commonly formulated by the mean-field Poisson-Boltzmann ...theory and integration of the charge contributions from electrolyte ions and soft material. However, the effects connected to the size of the electrolyte ions and that of the structural charges carried by the particle, to dielectric decrement and ion-ion correlations on soft interface electrostatics have been so far considered at the margin, despite the limits of the Gouy theory for condensed and/or multivalent electrolytes.
Accordingly, we modify herein the Poisson-Boltzmann theory for core/shell (bio)interfaces to include the aforementioned molecular effects considered separately or concomitantly. The formalism is applicable for poorly to highly charged particles in the thin electric double layer regime and to unsymmetrical multivalent electrolytes.
Computational examples of practical interests are discussed with emphasis on how each considered molecular effect or combination thereof affects the interfacial potential distribution depending on size and valence of cations and anions, size of particle charges, length scale of ionic correlations and shell-to-Debye layer thickness ratio. The origins of here-evidenced pseudo-harmonic potential profile and ion size-dependent screening of core/shell particle charges are detailed. In addition, the existence and magnitude of the Donnan potential when reached in the shell layer are shown to depend on the excluded volumes of the electrolyte ions.
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 (EPSs), making 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 taken in physiological conditions revealed typical ultrastructural features with a few nanometers resolution. Using single-cell force spectroscopy, we showed that N. palea strongly adheres to hydrophobic surfaces as compared to hydrophilic ones. Chemical force spectroscopy with hydrophobic tips further confirmed that the 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.
When a charged layer decorating a particle or a macroscopic surface is equilibrated with an electrolyte solution,a constant Donnan potential is established through that layer due to charge-driven ...accumulation of counterionsand companion exclusion of coions. This situation arises when the thickness of the surface layer well exceeds thescreening Debye length, a condition derived from mean-field Poisson-Boltzmann theory within point-like chargeapproximation. Herein, we revisit this condition underlying the applicability of Donnan electrostatic represen-tation with the account of steric effects mediated by the sizes of the electrolyte ions and structural layer charges.A transcendental equation is derived for the Donnan potential as a function of sizes and valences of anions andcations, electrolyte concentration and size of the layer charges, and a closed-form expression is provided forsymmetrical electrolytes. Therefrom we evidence that the existence of a Donnan potential is conditioned not onlyto large values of the layer thickness compared to a here-defined Debye length operative within the shell, but toadditional verification of a criterion that involves space charge density of the layer, solution ionic strength andelectrolyte nondiluteness parameter. Illustrative computational examples show how the existence and magnitudeof the Donnan potential depend on the key molecular descriptors of the electrolyte and soft interface, and theyfurther quantify the deviations from predictions based on classical Donnan potential expression valid for diluteelectrolytes.
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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.
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 guiding the adhesion of Pseudomonas aeruginosa type IV pili to surfaces. Using chemical force microscopy and single-cell force spectroscopy, we show that pili strongly bind to hydrophobic surfaces in a time-dependent manner, while they weakly bind to hydrophilic surfaces. Individual nanofibers are capable of withstanding forces up to 250 pN, thereby explaining how they can resist mechanical stress. Pulling on individual pili yields constant force plateaus, presumably reflecting conformational changes, as well as nanospring properties that may help bacteria to withstand physiological shear forces. Analysis of mutant strains demonstrates that these mechanical responses originate solely from type IV pili, while flagella and the cell surface localized and proposed pili-associated adhesin PilY1 play no direct role. We also demonstrate that bacterial–host interactions involve constant force plateaus, the extension of bacterial pili, and the formation of membrane tethers from host cells. We postulate that the unique mechanical responses of type IV pili unravelled here enable the bacteria to firmly attach to biotic and abiotic surfaces and thus maintain attachment when subjected to high shear forces under physiological conditions, helping to explain why pili play a critical role in colonization of the host.
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.
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.
The Gram-positive bacterium Staphylococcus epidermidis is responsible for important nosocomial infections. With the continuous emergence of antibiotic-resistant strains, the search for new treatments ...has been amplified in the last decades. A potential candidate against multidrug-resistant bacteria is squalamine, a natural aminosterol discovered in dogfish sharks. Despite its broad-spectrum efficiency, little is known about squalamine mode of action. Here, we used atomic force microscopy (AFM) imaging to decipher the effect of squalamine on S. epidermidis morphology, revealing the peptidoglycan structure at the bacterial surface after the drug action. Single-molecule force spectroscopy with squalamine-decorated tips shows that squalamine binds to the cell surface via the spermidine motif, most likely through electrostatic interactions between the amine groups of the molecule and the negatively-charged bacterial cell wall. We demonstrated that - although spermidine is sufficient for the initial attachment of squalamine to S. epidermidis – the integrity of the molecule needs to be conserved for its antimicrobial action. A deeper analysis of the AFM force-distance signatures suggests the implication of the accumulation-associated protein (Aap), one of the main adhesins of S. epidermidis, in the initial binding of squalamine to the bacterial cell wall. This work highlights that AFM -combined with microbiological assays at the bacterial suspension scale- is a valuable approach to better understand the molecular mechanisms behind the efficiency of squalamine antibacterial activity.
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•Squalamine has an antimicrobial action against Staphylococcus epidermidis.•The minimal inhibitory concentration (MIC) against S. epidermidis is 2 µM.•Squalamine binds to the bacterial cell wall via the spermidine motif.•Unfolding signatures suggest that adhesins Aap are involved in the initial binding.
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