Publications by authors named "Yves F Dufrêne"

215 Publications

AFM force-clamp spectroscopy captures the nanomechanics of the Tad pilus retraction.

Nanoscale Horiz 2021 06;6(6):489-496

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte, L7.07.07, Louvain-la-Neuve B-1348, Belgium.

Motorization of bacterial pili is key to generate traction forces to achieve cellular function. The Tad (or Type IVc) pilus from Caulobacter crescentus is a widespread motorized nanomachine crucial for bacterial survival, evolution and virulence. An unusual bifunctional ATPase motor drives Tad pilus retraction, which helps the bacteria to land on target surfaces. Here, we use a novel platform combining a fluorescence-based screening of piliated bacteria and atomic force microscopy (AFM) force-clamp spectroscopy, to monitor over time (30 s) the nanomechanics and dynamics of the Tad nanofilament retraction under a high constant tension (300 pN). We observe striking transient variations of force and height originating from two phenomena: active pilus retraction and passive hydrophobic interactions between the pilus and the hydrophobic substrate. That the Tad pilus is able to retract under high tensile loading - at a velocity of ∼150 nm s-1 - indicates that this nanomachine is stronger than previously anticipated. Our findings show that pilus retraction and hydrophobic interactions work together to mediate bacterial cell landing and surface adhesion. The motorized pilus retraction actively triggers the cell to approach the substrate. At short distances, passive hydrophobic interactions accelerate the approach phenomenon and promote strong cell-substrate adhesion. This mechanism could provide a strategy to save ATP-based energy by the retraction ATPase. Our force-clamp AFM methodology offers promise to decipher the physics of bacterial nanomotors with high sensitivity and temporal resolution.
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http://dx.doi.org/10.1039/d1nh00158bDOI Listing
June 2021

Staphylococcus aureus vWF-binding protein triggers a strong interaction between clumping factor A and host vWF.

Commun Biol 2021 Apr 12;4(1):453. Epub 2021 Apr 12.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium.

The Staphylococcus aureus cell wall-anchored adhesin ClfA binds to the very large blood circulating protein, von Willebrand factor (vWF) via vWF-binding protein (vWbp), a secreted protein that does not bind the cell wall covalently. Here we perform force spectroscopy studies on living bacteria to unravel the molecular mechanism of this interaction. We discover that the presence of all three binding partners leads to very high binding forces (2000 pN), largely outperforming other known ternary complexes involving adhesins. Strikingly, our experiments indicate that a direct interaction involving features of the dock, lock and latch mechanism must occur between ClfA and vWF to sustain the extreme tensile strength of the ternary complex. Our results support a previously undescribed mechanism whereby vWbp activates a direct, ultra-strong interaction between ClfA and vWF. This intriguing interaction represents a potential target for therapeutic interventions, including synthetic peptides inhibiting the ultra-strong interactions between ClfA and its ligands.
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http://dx.doi.org/10.1038/s42003-021-01986-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041789PMC
April 2021

AFM Unravels the Unique Adhesion Properties of the Type IVc Pilus Nanomachine.

Nano Lett 2021 04 23;21(7):3075-3082. Epub 2021 Mar 23.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte, L7.07.07., B-1348 Louvain-la-Neuve, Belgium.

Bacterial pili are proteinaceous motorized nanomachines that play various functional roles including surface adherence, bacterial motion, and virulence. The surface-contact sensor type IVc (or Tad) pilus is widely distributed in both Gram-positive and Gram-negative bacteria. In , this nanofilament, though crucial for surface colonization, has never been thoroughly investigated at the molecular level. As assembles several surface appendages at specific stages of the cell cycle, we designed a fluorescence-based screen to selectively study single piliated cells and combined it with atomic force microscopy and genetic manipulation to quantify the nanoscale adhesion of the type IVc pilus to hydrophobic substrates. We demonstrate that this nanofilament exhibits high stickiness compared to the canonical type IVa/b pili, resulting mostly from multiple hydrophobic interactions along the fiber length, and that it features nanospring mechanical properties. Our findings may be helpful to better understand the structure-function relationship of bacterial pilus nanomachines.
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http://dx.doi.org/10.1021/acs.nanolett.1c00215DOI Listing
April 2021

AFM in cellular and molecular microbiology.

Cell Microbiol 2021 Mar 12:e13324. Epub 2021 Mar 12.

Louvain Institute of Biomolecular Science and Technology, Université Catholique de Louvain, Louvain-la-Neuve, Belgium.

The unique capabilities of the atomic force microscope (AFM), including super-resolution imaging, piconewton force-sensitivity, nanomanipulation and ability to work under physiological conditions, have offered exciting avenues for cellular and molecular biology research. AFM imaging has helped unravel the fine architectures of microbial cell envelopes at the nanoscale, and how these are altered by antimicrobial treatment. Nanomechanical measurements have shed new light on the elasticity, tensile strength and turgor pressure of single cells. Single-molecule and single-cell force spectroscopy experiments have revealed the forces and dynamics of receptor-ligand interactions, the nanoscale distribution of receptors on the cell surface and the elasticity and adhesiveness of bacterial pili. Importantly, recent force spectroscopy studies have demonstrated that extremely stable bonds are formed between bacterial adhesins and their cognate ligands, originating from a catch bond behaviour allowing the pathogen to reinforce adhesion under shear or tensile stress. Here, we survey how the versatility of AFM has enabled addressing crucial questions in microbiology, with emphasis on bacterial pathogens.
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http://dx.doi.org/10.1111/cmi.13324DOI Listing
March 2021

Adhesion of to During Co-Infection Promotes Bacterial Dissemination Through the Host Immune Response.

Front Cell Infect Microbiol 2020 2;10:624839. Epub 2021 Feb 2.

Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Department of Biology, KU Leuven, Leuven, Belgium.

Interspecies interactions greatly influence the virulence, drug tolerance and ultimately the outcome of polymicrobial biofilm infections. A synergistic interaction is observed between the fungus and the bacterium . These species are both normal commensals of most healthy humans and co-exist in several niches of the host. However, under certain circumstances, they can cause hospital-acquired infections with high morbidity and mortality rates. Using a mouse model of oral co-infection, we previously showed that an oral infection with predisposes to a secondary systemic infection with . Here, we unraveled this intriguing mechanism of bacterial dissemination. Using static and dynamic adhesion assays in combination with single-cell force spectroscopy, we identified Als1 and Als3 adhesins as the molecular players involved in the interaction with and in subsequent bacterial dissemination. Remarkably, we identified the host immune response as a key element required for bacterial dissemination. We found that the level of immunosuppression of the host plays a critical yet paradoxical role in this process. In addition, secretion of candidalysin, the peptide responsible for immune activation and cell damage, is required for colonization and subsequent bacterial dissemination. The physical interaction with enhances bacterial uptake by phagocytic immune cells, thereby enabling an opportunity to disseminate.
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http://dx.doi.org/10.3389/fcimb.2020.624839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884861PMC
June 2021

Seeing and Touching the Mycomembrane at the Nanoscale.

J Bacteriol 2021 Apr 21;203(10). Epub 2021 Apr 21.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium

Mycobacteria have unique cell envelopes, surface properties, and growth dynamics, which all play a part in the ability of these important pathogens to infect, evade host immunity, disseminate, and resist antibiotic challenges. Recent atomic force microscopy (AFM) studies have brought new insights into the nanometer-scale ultrastructural, adhesive, and mechanical properties of mycobacteria. The molecular forces with which mycobacterial adhesins bind to host factors, like heparin and fibronectin, and the hydrophobic properties of the mycomembrane have been unraveled by AFM force spectroscopy studies. Real-time correlative AFM and fluorescence imaging have delineated a complex interplay between surface ultrastructure, tensile stresses within the cell envelope, and cellular processes leading to division. The unique capabilities of AFM, which include subdiffraction-limit topographic imaging and piconewton force sensitivity, have great potential to resolve important questions that remain unanswered on the molecular interactions, surface properties, and growth dynamics of this important class of pathogens.
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http://dx.doi.org/10.1128/JB.00547-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8088606PMC
April 2021

Single-cell fluidic force microscopy reveals stress-dependent molecular interactions in yeast mating.

Commun Biol 2021 01 4;4(1):33. Epub 2021 Jan 4.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte L7.07.07, 1348, Louvain-la-Neuve, Belgium.

Sexual agglutinins of the budding yeast Saccharomyces cerevisiae are proteins mediating cell aggregation during mating. Complementary agglutinins expressed by cells of opposite mating types "a" and "α" bind together to promote agglutination and facilitate fusion of haploid cells. By means of an innovative single-cell manipulation assay combining fluidic force microscopy with force spectroscopy, we unravel the strength of single specific bonds between a- and α-agglutinins (~100 pN) which require pheromone induction. Prolonged cell-cell contact strongly increases adhesion between mating cells, likely resulting from an increased expression of agglutinins. In addition, we highlight the critical role of disulfide bonds of the a-agglutinin and of histidine residue H of α-agglutinin. Most interestingly, we find that mechanical tension enhances the interaction strength, pointing to a model where physical stress induces conformational changes in the agglutinins, from a weak-binding folded state, to a strong-binding extended state. Our single-cell technology shows promises for understanding and controlling the complex mechanism of yeast sexuality.
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http://dx.doi.org/10.1038/s42003-020-01498-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782832PMC
January 2021

binds to the N-terminal region of corneodesmosin to adhere to the stratum corneum in atopic dermatitis.

Proc Natl Acad Sci U S A 2021 01;118(1)

Department of Microbiology, Moyne Institute of Preventive Medicine, School of Genetics and Microbiology, Trinity College Dublin, Dublin 2, Ireland;

colonizes the skin of the majority of patients with atopic dermatitis (AD), and its presence increases disease severity. Adhesion of to corneocytes in the stratum corneum is a key initial event in colonization, but the bacterial and host factors contributing to this process have not been defined. Here, we show that interacts with the host protein corneodesmosin. Corneodesmosin is aberrantly displayed on the tips of villus-like projections that occur on the surface of AD corneocytes as a result of low levels of skin humectants known as natural moisturizing factor (NMF). An mutant deficient in fibronectin binding protein B (FnBPB) and clumping factor B (ClfB) did not bind to corneodesmosin in vitro. Using surface plasmon resonance, we found that FnBPB and ClfB proteins bound with similar affinities. The binding site was localized to the N-terminal glycine-serine-rich region of corneodesmosin. Atomic force microscopy showed that the N-terminal region was present on corneocytes containing low levels of NMF and that blocking it with an antibody inhibited binding of individual cells to corneocytes. Finally, we found that mutants deficient in FnBPB or ClfB have a reduced ability to adhere to low-NMF corneocytes from patients. In summary, we show that FnBPB and ClfB interact with the accessible N-terminal region of corneodesmosin on AD corneocytes, allowing to take advantage of the aberrant display of corneodesmosin that accompanies low NMF in AD. This interaction facilitates the characteristic strong binding of to AD corneocytes.
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http://dx.doi.org/10.1073/pnas.2014444118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817190PMC
January 2021

Stress-Induced Catch-Bonds to Enhance Bacterial Adhesion.

Trends Microbiol 2021 Apr 19;29(4):286-288. Epub 2020 Dec 19.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte L7.07.07, B-1348 Louvain-la-Neuve, Belgium. Electronic address:

Physical forces have a profound influence on bacterial cell physiology and disease. A striking example is the formation of catch-bonds that reinforce under mechanical stress. While mannose-binding by the Escherichia coli FimH adhesin has long been the only thoroughly studied microbial catch-bond, it has recently become clear that proteins from other species, such as staphylococci, are also engaged in such stress-dependent interactions.
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http://dx.doi.org/10.1016/j.tim.2020.11.009DOI Listing
April 2021

Single-Molecule Analysis Demonstrates Stress-Enhanced Binding between Surface Protein IsdB and Host Cell Integrins.

Nano Lett 2020 12 25;20(12):8919-8925. Epub 2020 Nov 25.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte L7.07.07, B-1348 Louvain-la-Neuve, Belgium.

Binding of surface proteins to endothelial cell integrins plays essential roles in host cell adhesion and invasion, eventually leading to life-threatening diseases. The staphylococcal protein IsdB binds to β3-containing integrins through a mechanism that has never been thoroughly investigated. Here, we identify and characterize at the nanoscale a previously undescribed stress-dependent adhesion between IsdB and integrin αβ. The strength of single IsdB-αβ interactions is moderate (∼100 pN) under low stress, but it increases dramatically under high stress (∼1000-2000 pN) to exceed the forces traditionally reported for the binding between integrins and Arg-Gly-Asp (RGD) sequences. We suggest a mechanism where high mechanical stress induces conformational changes in the integrin from a low-affinity, weak binding state to a high-affinity, strong binding state. This single-molecule study highlights that direct adhesin-integrin interactions represent potential targets to fight staphylococcal infections.
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http://dx.doi.org/10.1021/acs.nanolett.0c04015DOI Listing
December 2020

Atomic Force Microscopy-Based Force Spectroscopy and Multiparametric Imaging of Biomolecular and Cellular Systems.

Chem Rev 2020 Nov 9. Epub 2020 Nov 9.

Louvain Institute of Biomolecular Science and Technology, Université Catholique de Louvain (UCLouvain), Croix du Sud, 4-5, bte L7.07.07, B-1348 Louvain-la-Neuve, Belgium.

During the last three decades, a series of key technological improvements turned atomic force microscopy (AFM) into a nanoscopic laboratory to directly observe and chemically characterize molecular and cell biological systems under physiological conditions. Here, we review key technological improvements that have established AFM as an analytical tool to observe and quantify native biological systems from the micro- to the nanoscale. Native biological systems include living tissues, cells, and cellular components such as single or complexed proteins, nucleic acids, lipids, or sugars. We showcase the procedures to customize nanoscopic chemical laboratories by functionalizing AFM tips and outline the advantages and limitations in applying different AFM modes to chemically image, sense, and manipulate biosystems at (sub)nanometer spatial and millisecond temporal resolution. We further discuss theoretical approaches to extract the kinetic and thermodynamic parameters of specific biomolecular interactions detected by AFM for single bonds and extend the discussion to multiple bonds. Finally, we highlight the potential of combining AFM with optical microscopy and spectroscopy to address the full complexity of biological systems and to tackle fundamental challenges in life sciences.
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http://dx.doi.org/10.1021/acs.chemrev.0c00617DOI Listing
November 2020

Force-clamp spectroscopy identifies a catch bond mechanism in a Gram-positive pathogen.

Nat Commun 2020 10 27;11(1):5431. Epub 2020 Oct 27.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte L7.07.07, B-1348, Louvain-la-Neuve, Belgium.

Physical forces have profound effects on cellular behavior, physiology, and disease. Perhaps the most intruiguing and fascinating example is the formation of catch-bonds that strengthen cellular adhesion under shear stresses. Today mannose-binding by the Escherichia coli FimH adhesin remains one of the rare microbial catch-bond thoroughly characterized at the molecular level. Here we provide a quantitative demonstration of a catch-bond in living Gram-positive pathogens using force-clamp spectroscopy. We show that the dock, lock, and latch interaction between staphylococcal surface protein SpsD and fibrinogen is strong, and exhibits an unusual catch-slip transition. The bond lifetime first grows with force, but ultimately decreases to behave as a slip bond beyond a critical force (~1 nN) that is orders of magnitude higher than for previously investigated complexes. This catch-bond, never reported for a staphylococcal adhesin, provides the pathogen with a mechanism to tightly control its adhesive function during colonization and infection.
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http://dx.doi.org/10.1038/s41467-020-19216-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591895PMC
October 2020

Multiparametric Atomic Force Microscopy Identifies Multiple Structural and Physical Heterogeneities on the Surface of .

ACS Omega 2020 Aug 13;5(33):20953-20959. Epub 2020 Aug 13.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte L7.07.07, B-1348 Louvain-la-Neuve, Belgium.

A unique feature of the African trypanosome is the presence of an outer layer made of densely packed variable surface glycoproteins (VSGs), which enables the cells to survive in the bloodstream. Although the VSG coat is critical to pathogenesis, how exactly the glycoproteins are organized at the nanoscale is poorly understood. Here, we show that multiparametric atomic force microscopy is a powerful nanoimaging tool for the structural and mechanical characterization of trypanosomes, in a label-free manner and in buffer solution. Directly correlated images of the structure and elasticity of trypanosomes enable us to identify multiple nanoscale mechanical heterogeneities on the cell surface. On a ∼250 nm scale, regions of softer (Young's modulus ∼50 kPa) and stiffer (∼100 kPa) elasticity alternate, revealing variations of the VSG coat and underlying structures. Our nanoimaging experiments show that the cell surface is more heterogeneous than previously anticipated and offer promising prospects for the design of trypanocidal drugs targeting cell surface components.
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http://dx.doi.org/10.1021/acsomega.0c02416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450619PMC
August 2020

-Methylation of the Glycopeptidolipid Acyl Chain Defines Surface Hydrophobicity of and Macrophage Invasion.

ACS Infect Dis 2020 10 14;6(10):2756-2770. Epub 2020 Sep 14.

Centre National de la Recherche Scientifique UMR 9004, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, 1919 route de Mende, 34293 Montpellier, France.

, an emerging pathogen responsible for severe lung infections in cystic fibrosis patients, displays either smooth (S) or rough (R) morphotypes. The S-to-R transition is associated with reduced levels of glycopeptidolipid (GPL) production and is correlated with increased pathogenicity in animal and human hosts. While the structure of GPL is well established, its biosynthetic pathway is incomplete. In addition, the biological functions of the distinct structural parts of this complex lipid remain elusive. Herein, the gene encoding a putative -methyltransferase was deleted in the S variant. Subsequent biochemical and structural analyses demonstrated that methoxylation of the fatty acyl chain of GPL was abrogated in the mutant, and this defect was rescued upon complementation with a functional gene. In contrast, the introduction of derivatives mutated at residues essential for methyltransferase activity failed to complement GPL defects, indicating that encodes an -methyltransferase. Unexpectedly, phenotypic analyses showed that was more hydrophilic than its parental progenitor, as demonstrated by hexadecane-aqueous buffer partitioning and atomic force microscopy experiments with hydrophobic probes. Importantly, the invasion rate of THP-1 macrophages by was reduced by 50% when compared to the wild-type strain. Together, these results indicate that Fmt -methylates the lipid moiety of GPL and plays a substantial role in conditioning the surface hydrophobicity of as well as in the early steps of the interaction between the bacilli and macrophages.
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http://dx.doi.org/10.1021/acsinfecdis.0c00490DOI Listing
October 2020

Binding Strength of Gram-Positive Bacterial Adhesins.

Front Microbiol 2020 25;11:1457. Epub 2020 Jun 25.

Louvain Institute of Biomolecular Science and Technology, Catholic University of Louvain, Louvain-la-Neuve, Belgium.

Bacterial pathogens are equipped with specialized surface-exposed proteins that bind strongly to ligands on host tissues and biomaterials. These adhesins play critical roles during infection, especially during the early step of adhesion where the cells are exposed to physical stress. Recent single-molecule experiments have shown that staphylococci interact with their ligands through a wide diversity of mechanosensitive molecular mechanisms. Adhesin-ligand interactions are activated by tensile force and can be ten times stronger than classical non-covalent biological bonds. Overall these studies demonstrate that Gram-positive adhesins feature unusual stress-dependent molecular interactions, which play essential roles during bacterial colonization and dissemination. With an increasing prevalence of multidrug resistant infections caused by and , chemotherapeutic targeting of adhesins offers an innovative alternative to antibiotics.
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http://dx.doi.org/10.3389/fmicb.2020.01457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7330015PMC
June 2020

The Molecular Complex between Staphylococcal Adhesin SpsD and Fibronectin Sustains Mechanical Forces in the Nanonewton Range.

mBio 2020 07 7;11(4). Epub 2020 Jul 7.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium

The bacterial pathogen is involved in canine otitis externa and pyoderma as well as in surgical wound and urinary tract infections. Invasion of canine epithelial cells is promoted by fibronectin (Fn)-binding proteins SpsD and SpsL through molecular interactions that are currently unknown. By means of single-molecule experiments, we discover that both adhesins have distinct molecular mechanisms for binding to Fn. We show that the SpsD-Fn interaction has a strength equivalent to that of a covalent bond (∼1.5 to 1.8 nN), which is an order of magnitude stronger than the binding force of classical receptor-ligand complexes. We suggest that this extreme mechanostability originates from the β-sheet organization of a tandem β-zipper. Upon binding to FnI modules, the intrinsically disordered binding sequences of SpsD would shift into an ordered structure by forming additional β-strands along triple peptide β-sheets in the Fn molecule. Dynamic force measurements reveal an unexpected behavior, i.e., that strong bonds are activated by mechanical tension as observed with catch bonds. By contrast, the SpsL-Fn interaction involves multiple weak bonds (∼0.2 nN) that rupture sequentially under force. Together with the recently described dock, lock, and latch complex, the ultrastrong interaction unraveled here is among the strongest noncovalent biological interaction measured to date. Our findings may find applications for the identification of inhibitory compounds to treat infections triggered by pathogens engaged in tandem β-zipper interactions. Binding of surface proteins SpsD and SpsL to fibronectin (Fn) plays a critical role in the invasion of canine epithelial cells. Here, we discover that both adhesins have different mechanisms for binding to Fn. The force required to separate SpsD from Fn is extremely strong, consistent with the unusual β-sheet organization of a high-affinity tandem β-zipper. By contrast, unbinding of the SpsL-Fn complex involves the sequential rupture of single weak bonds. Our findings may be of biological relevance as SpsD and SpsL are likely to play complementary roles during invasion. While the SpsD β-zipper supports strong bacterial adhesion and triggers invasion, the weak SpsL interaction would favor fast detachment, enabling the pathogen to colonize new sites.
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http://dx.doi.org/10.1128/mBio.00371-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343985PMC
July 2020

Nanomechanics of the molecular complex between staphylococcal adhesin SpsD and elastin.

Nanoscale 2020 Jul 24;12(26):13996-14003. Epub 2020 Jun 24.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte L7.07.07, B-1348 Louvain-la-Neuve, Belgium.

Staphylococcus pseudintermedius surface protein SpsD binds to extracellular matrix proteins to invade canine epithelial cells. Using single-molecule experiments, we show that SpsD engages in two modes of interaction with elastin that are tightly controlled by physical stress. Binding is weak (∼100 pN) at low tensile force (i.e. loading rate), but is dramatically enhanced (up to ∼1500 pN) by mechanical tension. Consistent with a "dock, lock, and latch" (DLL) mechanism, this force represents among the highest mechanical strengths known for a non-covalent biological interaction. The transition from weak to strong binding correlates with an increase in molecular stiffness but, surprisingly, with a decrease in molecular extension. This unanticipated mechanical behavior indicates that the adhesin is engaged in two distinct interaction mechanisms. Our results emphasize the crucial role of protein nanomechanics in the adhesion of staphylococci, and illustrate their wide diversity of force-dependent ligand-binding activities. These single-molecule mechanical experiments may contribute to the development of antiadhesion approaches to treat infections caused by S. pseudintermedius and other bacterial pathogens engaged in DLL interactions.
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http://dx.doi.org/10.1039/d0nr02745fDOI Listing
July 2020

Bacterial Cell Mechanics Beyond Peptidoglycan.

Trends Microbiol 2020 09 25;28(9):706-708. Epub 2020 May 25.

Institute of Life Sciences, UCLouvain, Croix du Sud, 4-5, bte L7.07.07, B-1348 Louvain-la-Neuve, Belgium; Walloon Excellence in Life sciences and Biotechnology (WELBIO), Wavre, Belgium. Electronic address:

The bacterial cell envelope plays essential roles in controlling cell shape, division, pathogenicity, and resistance against external stresses. In Escherichia coli, peptidoglycan (PG) has long been thought to be the primary component that conveys mechanical strength to the envelope. But a recent publication demonstrates the key contribution of the lipoprotein Lpp in defining the stiffness of the cell envelope and its sensitivity to drugs.
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http://dx.doi.org/10.1016/j.tim.2020.04.013DOI Listing
September 2020

Fast chemical force microscopy demonstrates that glycopeptidolipids define nanodomains of varying hydrophobicity on mycobacteria.

Nanoscale Horiz 2020 06 21;5(6):944-953. Epub 2020 Apr 21.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte L7.07.07, B-1348 Louvain-la-Neuve, Belgium.

Mycobacterium abscessus is an emerging multidrug-resistant bacterial pathogen causing severe lung infections in cystic fibrosis patients. A remarkable trait of this mycobacterial species is its ability to form morphologically smooth (S) and rough (R) colonies. The S-to-R transition is caused by the loss of glycopeptidolipids (GPLs) in the outer layer of the cell envelope and correlates with an increase in cording and virulence. Despite the physiological and medical importance of this morphological transition, whether it involves changes in cell surface properties remains unknown. Herein, we combine recently developed quantitative imaging (QI) atomic force microscopy (AFM) with hydrophobic tips to quantitatively map the surface structure and hydrophobicity of M. abscessus at high spatiotemporal resolution, and to assess how these properties are modulated by the S-to-R transition and by treatment with an inhibitor of the mycolic acid transporter MmpL3. We discover that loss of GPLs leads to major modifications in surface hydrophobicity, without any apparent change in cell surface ultrastructure. While R bacilli are homogeneously hydrophobic, S bacilli feature unusual variations of nanoscale hydrophobic properties. These previously undescribed cell surface nanodomains are likely to play critical roles in bacterial adhesion, aggregation, phenotypic heterogeneity and transmission, and in turn in virulence and pathogenicity. Our study also suggests that MmpL3 inhibitors show promise in nanomedicine as chemotherapeutic agents to interfere with the highly hydrophobic nature of the mycobacterial cell wall. The advantages of QI-AFM with hydrophobic tips are the ability to map chemical and structural properties simultaneously and at high resolution, applicable to a wide range of biosystems.
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http://dx.doi.org/10.1039/c9nh00736aDOI Listing
June 2020

Lipoprotein Lpp regulates the mechanical properties of the E. coli cell envelope.

Nat Commun 2020 04 14;11(1):1789. Epub 2020 Apr 14.

Institute of Life Sciences, UCLouvain, Croix du Sud, 4-5, bte L7.07.06, B-1348, Louvain-la-Neuve, Belgium.

The mechanical properties of the cell envelope in Gram-negative bacteria are controlled by the peptidoglycan, the outer membrane, and the proteins interacting with both layers. In Escherichia coli, the lipoprotein Lpp provides the only covalent crosslink between the outer membrane and the peptidoglycan. Here, we use single-cell atomic force microscopy and genetically engineered strains to study the contribution of Lpp to cell envelope mechanics. We show that Lpp contributes to cell envelope stiffness in two ways: by covalently connecting the outer membrane to the peptidoglycan, and by controlling the width of the periplasmic space. Furthermore, mutations affecting Lpp function substantially increase bacterial susceptibility to the antibiotic vancomycin, indicating that Lpp-dependent effects can affect antibacterial drug efficacy.
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http://dx.doi.org/10.1038/s41467-020-15489-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156740PMC
April 2020

How Microbes Use Force To Control Adhesion.

J Bacteriol 2020 05 27;202(12). Epub 2020 May 27.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium

Microbial adhesion and biofilm formation are usually studied using molecular and cellular biology assays, optical and electron microscopy, or laminar flow chamber experiments. Today, atomic force microscopy (AFM) represents a valuable addition to these approaches, enabling the measurement of forces involved in microbial adhesion at the single-molecule level. In this minireview, we discuss recent discoveries made applying state-of-the-art AFM techniques to microbial specimens in order to understand the strength and dynamics of adhesive interactions. These studies shed new light on the molecular mechanisms of adhesion and demonstrate an intimate relationship between force and function in microbial adhesins.
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http://dx.doi.org/10.1128/JB.00125-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253613PMC
May 2020

Together We Are Stronger: Protein Clustering at the Nanoscale.

Authors:
Yves F Dufrêne

ACS Nano 2020 03 4;14(3):2561-2564. Epub 2020 Mar 4.

Louvain Institute of Biomolecular Science and Technology, Catholic University of Louvain (UCLouvain), Croix du Sud, 4-5, bte L7.07.06, B-1348 Louvain-la-Neuve, Belgium.

Cell surface proteins are known to assemble into nano- and microscale domains in order to govern biological processes, including cell adhesion, endocytosis, and immune responses. The small size and ephemerality of these structures have made their direct observation and functional analysis challenging. In this Perspective, I discuss recent progress made in applying nanotechniques to study protein clustering, emphasizing the use of state-of-the-art single-molecule atomic force microscopy, as reported by Strasser . in this issue of .
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http://dx.doi.org/10.1021/acsnano.0c01451DOI Listing
March 2020

Scratching the Surface: Bacterial Cell Envelopes at the Nanoscale.

mBio 2020 02 25;11(1). Epub 2020 Feb 25.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium

The bacterial cell envelope is essential for viability, the environmental gatekeeper and first line of defense against external stresses. For most bacteria, the envelope biosynthesis is also the site of action of some of the most important groups of antibiotics. It is a complex, often multicomponent structure, able to withstand the internally generated turgor pressure. Thus, elucidating the architecture and dynamics of the cell envelope is important, to unravel not only the complexities of cell morphology and maintenance of integrity but also how interventions such as antibiotics lead to death. To address these questions requires the capacity to visualize the cell envelope via high-spatial resolution approaches. In recent years, atomic force microscopy (AFM) has brought novel molecular insights into the assembly, dynamics, and functions of bacterial cell envelopes. The ultrafine resolution and physical sensitivity of the technique have revealed a wealth of ultrastructural features that are invisible to traditional optical microscopy techniques or imperceptible in their true physiological state by electron microscopy. Here, we discuss recent progress in our use of AFM imaging for understanding the architecture and dynamics of the bacterial envelope. We survey recent studies that demonstrate the power of the technique to observe isolated membranes and live cells at (sub)nanometer resolution and under physiological conditions and to track structural dynamics in response to growth or to drugs.
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http://dx.doi.org/10.1128/mBio.03020-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042696PMC
February 2020

Mechanomicrobiology: how bacteria sense and respond to forces.

Nat Rev Microbiol 2020 04 20;18(4):227-240. Epub 2020 Jan 20.

Institute of Bioengineering and Global Health Institute, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

Microorganisms have evolved to thrive in virtually any terrestrial and marine environment, exposing them to various mechanical cues mainly generated by fluid flow and pressure as well as surface contact. Cellular components enable bacteria to sense and respond to physical cues to optimize their function, ultimately improving bacterial fitness. Owing to newly developed biophysical techniques, we are now starting to appreciate the breadth of bacterial phenotypes influenced by mechanical inputs: adhesion, motility, biofilm formation and pathogenicity. In this Review, we discuss how microbiology and biophysics are converging to advance our understanding of the mechanobiology of microorganisms. We first review the various physical forces that bacteria experience in their natural environments and describe the structures that transmit these forces to a cell. We then discuss how forces can provide feedback to enhance adhesion and motility and how they can be transduced by dedicated cellular machinery to regulate diverse phenotypes. Finally, we provide a perspective on how mechanics influence biofilm spatial organization and homeostasis.
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http://dx.doi.org/10.1038/s41579-019-0314-2DOI Listing
April 2020

What makes bacterial pathogens so sticky?

Mol Microbiol 2020 04 16;113(4):683-690. Epub 2020 Jan 16.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium.

Pathogenic bacteria use a variety of cell surface adhesins to promote binding to host tissues and protein-coated biomaterials, as well as cell-cell aggregation. These cellular interactions represent the first essential step that leads to host colonization and infection. Atomic force microscopy (AFM) has greatly contributed to increase our understanding of the specific interactions at play during microbial adhesion, down to the single-molecule level. A key asset of AFM is that adhesive interactions are studied under mechanical force, which is highly relevant as surface-attached pathogens are often exposed to physical stresses in the human body. These studies have identified sophisticated binding mechanisms in adhesins, which represent promising new targets for antiadhesion therapy.
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http://dx.doi.org/10.1111/mmi.14448DOI Listing
April 2020

An Amyloid Core Sequence in the Major Candida albicans Adhesin Als1p Mediates Cell-Cell Adhesion.

mBio 2019 10 8;10(5). Epub 2019 Oct 8.

Department of Sciences, John Jay College of the City University of New York, New York, New York, USA

The human fungal commensal can become a serious opportunistic pathogen in immunocompromised hosts. The cell adhesion protein Als1p is a highly expressed member of a large family of paralogous adhesins. Als1p can mediate binding to epithelial and endothelial cells, is upregulated in infections, and is important for biofilm formation. Als1p includes an amyloid-forming sequence at amino acids 325 to 331, identical to the sequence in the paralogs Als5p and Als3p. Therefore, we mutated Val326 to test whether this sequence is important for activity. Wild-type Als1p (Als1p) and Als1p with the V326N mutation (Als1p) were expressed at similar levels in a surface display model. Als1p cells adhered to bovine serum albumin (BSA)-coated beads similarly to Als1p cells. However, cells displaying Als1p showed visibly smaller aggregates and did not fluoresce in the presence of the amyloid-binding dye Thioflavin-T. A new analysis tool for single-molecule force spectroscopy-derived surface mapping showed that statistically significant force-dependent Als1p clustering occurred in Als1p cells but was absent in Als1p cells. In single-cell force spectroscopy experiments, strong cell-cell adhesion was dependent on an intact amyloid core sequence on both interacting cells. Thus, the major adhesin Als1p interacts through amyloid-like β-aggregation to cluster adhesin molecules in on the cell surface as well as in to form cell-cell bonds. Microbial cell surface adhesins control essential processes such as adhesion, colonization, and biofilm formation. In the opportunistic fungal pathogen , the gglutinin-ike equence () gene family encodes eight cell surface glycoproteins that mediate adherence to biotic and abiotic surfaces and cell-cell aggregation. Als proteins are critical for commensalism and virulence. Their activities include attachment and invasion of endothelial and epithelial cells, morphogenesis, and formation of biofilms on host tissue and indwelling medical catheters. At the molecular level, Als5p-mediated cell-cell aggregation is dependent on the formation of amyloid-like nanodomains between Als5p-expressing cells. A single-site mutation to valine 326 abolishes cellular aggregation and amyloid formation. Our results show that the binding characteristics of Als1p follow a mechanistic model similar to Als5p, despite its differential expression and biological roles.
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http://dx.doi.org/10.1128/mBio.01766-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786869PMC
October 2019

Mechanostability of the Fibrinogen Bridge between Staphylococcal Surface Protein ClfA and Endothelial Cell Integrin αβ.

Nano Lett 2019 10 18;19(10):7400-7410. Epub 2019 Sep 18.

Louvain Institute of Biomolecular Science and Technology, UCLouvain , Croix du Sud, 4-5, bte L7.07.06, B-1348 Louvain-la-Neuve , Belgium.

Binding of the surface protein clumping factor A (ClfA) to endothelial cell integrin αβ plays a crucial role during sepsis, by causing endothelial cell apoptosis and loss of barrier integrity. ClfA uses the blood plasma protein fibrinogen (Fg) to bind to αβ but how this is achieved at the molecular level is not known. Here we investigate the mechanical strength of the three-component ClfA-Fg-αβ interaction on living bacteria, by means of single-molecule experiments. We find that the ClfA-Fg-αβ ternary complex is extremely stable, being able to sustain forces (∼800 pN) that are much stronger than those of classical bonds between integrins and the Arg-Gly-Asp (RGD) tripeptide sequence (∼100 pN). Adhesion forces between single bacteria and αβ are strongly inhibited by an anti-αβ antibody, the RGD peptide, and the cyclic RGD peptide cilengitide, showing that formation of the complex involves RGD-dependent binding sites and can be efficiently inhibited by αβ blockers. Collectively, our experiments favor a binding mechanism involving the extraordinary elasticity of Fg. In the absence of mechanical stress, RGD sequences in the Aα chains mediate weak binding to αβ, whereas under high mechanical stress exposure of cryptic Aα chain RGD sequences leads to extremely strong binding to the integrin. Our results identify an unexpected and previously undescribed force-dependent binding mechanism between ClfA and αβ on endothelial cells, which could represent a potential target to fight staphylococcal bloodstream infections.
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http://dx.doi.org/10.1021/acs.nanolett.9b03080DOI Listing
October 2019

Bacterial pathogens under high-tension: adhesion to von Willebrand factor is activated by force.

Microb Cell 2019 Jun 11;6(7):321-323. Epub 2019 Jun 11.

Institute of Life Sciences, Université catholique de Louvain, Croix du Sud, 4-5, bte L7.07.06, B-1348 Louvain-la-Neuve, Belgium.

Attachment of to platelets and endothelial cells involves binding of bacterial cell surface protein A (SpA) to the large plasma glycoprotein von Willebrand factor (vWF). SpA-mediated bacterial adhesion to vWF is controlled by fluid shear stress, yet little is currently known about the underlying molecular mechanism. In a recent publication, we showed that the SpA-vWF interaction is tightly regulated by mechanical force. By means of single-molecule pulling experiments, we found that the SpA-vWF bond is extremely strong, being able to resist forces which largely outperform the strength of typical receptor-ligand bonds. In line with flow experiments, strong adhesion is activated by mechanical tension. These results suggest that force induces conformational changes in the vWF molecule, from a globular to an extended state, leading to the exposure of cryptic binding sites to which SpA strongly binds. This force-sensitive mechanism may largely contribute to help bacteria to resist shear stress of flowing blood during infection.
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http://dx.doi.org/10.15698/mic2019.07.684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600117PMC
June 2019

Fluidic Force Microscopy Captures Amyloid Bonds between Microbial Cells.

Trends Microbiol 2019 09 1;27(9):728-730. Epub 2019 Jul 1.

Institute of Life Sciences, Université catholique de Louvain, Croix du Sud, 4-5, bte L7.07.06, B-1348 Louvain-la-Neuve, Belgium; Walloon Excellence in Life sciences and Biotechnology (WELBIO), Brussels, Belgium. Electronic address:

Fluidic force microscopy (FluidFM) is a recent force-controlled pipette technology that enables manipulation of single cells. FluidFM can be used for quantification of forces between single cells, and a novel mode of cell-cell adhesion was uncovered: amyloid-like interactions that mediate homophilic adhesion in the fungal pathogen Candida albicans.
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http://dx.doi.org/10.1016/j.tim.2019.06.001DOI Listing
September 2019

Host-specialized fibrinogen-binding by a bacterial surface protein promotes biofilm formation and innate immune evasion.

PLoS Pathog 2019 06 19;15(6):e1007816. Epub 2019 Jun 19.

The Roslin Institute and Edinburgh Infectious Diseases, University of Edinburgh, Easter Bush Campus, Edinburgh, Scotland, United Kingdom.

Fibrinogen is an essential part of the blood coagulation cascade and a major component of the extracellular matrix in mammals. The interface between fibrinogen and bacterial pathogens is an important determinant of the outcome of infection. Here, we demonstrate that a canine host-restricted skin pathogen, Staphylococcus pseudintermedius, produces a cell wall-associated protein (SpsL) that has evolved the capacity for high strength binding to canine fibrinogen, with reduced binding to fibrinogen of other mammalian species including humans. Binding occurs via the surface-expressed N2N3 subdomains, of the SpsL A-domain, to multiple sites in the fibrinogen α-chain C-domain by a mechanism analogous to the classical dock, lock, and latch binding model. Host-specific binding is dependent on a tandem repeat region of the fibrinogen α-chain, a region highly divergent between mammals. Of note, we discovered that the tandem repeat region is also polymorphic in different canine breeds suggesting a potential influence on canine host susceptibility to S. pseudintermedius infection. Importantly, the strong host-specific fibrinogen-binding interaction of SpsL to canine fibrinogen is essential for bacterial aggregation and biofilm formation, and promotes resistance to neutrophil phagocytosis, suggesting a key role for the interaction during pathogenesis. Taken together, we have dissected a bacterial surface protein-ligand interaction resulting from the co-evolution of host and pathogen that promotes host-specific innate immune evasion and may contribute to its host-restricted ecology.
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http://dx.doi.org/10.1371/journal.ppat.1007816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6602291PMC
June 2019