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Force nanoscopy of staphylococcal biofilms

Periodic Reporting for period 4 - NanoStaph (Force nanoscopy of staphylococcal biofilms)

Reporting period: 2021-04-01 to 2021-09-30

Staphylococcus aureus is a leading cause of hospital-acquired infections, which are often complicated by the ability of this pathogen to grow as biofilms on indwelling medical devices. Because biofilms protect the bacteria from host defenses and are resistant to many antibiotics, biofilm-related infections are difficult to fight and represent a tremendous burden on our healthcare system. Today, a true molecular understanding of the fundamental interactions driving staphylococcal adhesion and biofilm formation is lacking owing to the lack of high-resolution probing techniques. This knowledge would greatly contribute to the development of novel anti-adhesion therapies for combating biofilm infections. We recently established advanced atomic force microscopy (AFM) techniques for analyzing the nanoscale surface architecture and interactions of microbial cells, allowing us to elucidate key cellular functions. This multidisciplinary project aims at developing an innovative AFM-based force nanoscopy platform in biofilm research, enabling us to understand the molecular mechanisms of S. aureus adhesion in a way that was not possible before, and to optimize the use of anti-adhesion compounds capable to inhibit biofilm formation by this pathogen. So the general ambition is to use new tools from physics to make key breakthroughs in biology (biofilms) and medicine (antimicrobials). To this end, the project is composed of three complementary objectives (work packages WPs): Objective 1 aiming at unravelling single-cell adhesion forces in S. aureus, Objective 2 which goal is to understand the binding mechanisms of S. aureus adhesins at the single-molecule level, and Objective 3 which will screen and optimise the use of anti-adhesion compounds targeted against S. aureus. Achieving these targets will involve an unconventional approach combining novel AFM techniques with biological methods. A set of advanced AFM assays will be used for characterizing the nanoscale forces driving bacterial adhesion to host matrix-covered surfaces and bacterial-bacterial cell adhesion, and for assessing the inhibitory effect of anti-adhesion compounds capable to inhibit bacterial adhesion. These analyses will be performed on genetically-defined laboratory mutant strains and on MRSA clinical isolates that are notoriously difficult to fight.
We have been interested in a family of staphylococcal adhesins forming specific “dock, lock, and latch” (DLL) interactions. In a pioneering paper, we demonstrated that the prototypical DLL adhesin SdrG from S. epidermidis binds its ligand fibrinogen (Fg) with an extremely strong force of ~2 nN, similar to that of a covalent bond. This was the strongest non-covalent bond ever reported in biology, despite an ordinary affinity constant. Inspired by our work, the Gaub group used AFM and all-atom steered molecular dynamics to show that this high mechanostability originates from the formation of an elaborate hydrogen bond network between subdomains of SdrG and the Fg peptide backbone. Following this, I was invited to write a perspective article in Science titled "Force matters in hospital-acquired infections", illustrating the medical importance of this emerging field of ultrastrong forces in pathogen adhesion. We next showed that other adhesins engaged in DLL binding. S. aureus clumping factors ClfA and ClfB, bind their targets with similar ultrastrong forces. We made progress in understanding the "beta zipper" interaction of fibronectin (Fn)-binding adhesins (FnBPs), revealing that they mediate bacterial adhesion to soluble Fn via strong forces (∼1,5 nN), consistent with a tandem β-zipper.11 We also identified forces of ~2 nN between a S. aureus protein A and von Willebrand factor, a plasma glycoprotein mediating the pathogen’s binding to endothelial cell surfaces.So ultrastrong adhesion is not restricted to DLL bond-forming proteins.

Surface-attached bacteria are exposed to mechanical stresses, including hydrodynamic flow and cell-surface contacts.We discovered an intriguing mechanism in which mechanical stress dramatically enhances S. aureus adhesion, providing the pathogen with a means to withstand high shear stress during colonization. ClfA and ClfB adhesins were shown to behave as force-sensitive molecular switches, forming very strong bonds only under high stress. This was explained by a mechanism whereby bacterial adhesion to ligands is enhanced through force-induced conformational changes in the Clf molecules, from a weakly binding folded state to a strongly binding extended state. The newly described stress-enhanced adhesion behaviors point to the formation of unusual "catch-bonds" that reinforce under stress (see below).

Perhaps the most fascinating example of the influence of physical force on bacterial behavior is the formation of catch-bonds that strengthen cellular adhesion under shear stress. Using AFM in the force-clamp spectroscopy mode, we recently provided the first evidence of a catch-bond in a Gram-positive bacterium.14 We showed that the DLL interaction between the staphylococcal adhesin SpsD and Fg is strong and exhibits an unusual catch-slip transition. The bond lifetime first grows with force, but ultimately decreases to behave like a slip bond beyond a critical force that is orders of magnitude higher than for previously investigated complexes. Catch-bonds represent a competitive advantage to help the pathogens tune their adhesion to flow conditions, that is, by binding loosely and spreading under low flow while resisting shear and remaining firmly attached under high flow. Uncovering new catch-bond behaviors could help in the design of strategies as alternatives to antibiotics.

Innovative anti-adhesion compounds represent a promising alternative to antibiotics to fight pathogens, especially nosocomial drug-resistant strains. We have identified inhibitors capable of preventing adhesion, aggregation and biofilm formation. In two studies, we unraveled the molecular interactions by which S. aureus SasG and SdrC mediate cell-cell aggregation leading to biofilm formation. Following that, we discovered that a peptide derived from the neuronal cell adhesion molecule β-neurexin inhibits SdrC-dependent attachment to inert surfaces, cell-cell adhesion, and biofilm formation. The peptide could be used as a platform for designing peptidomimetics with potential to prevent biofilm infections. We also showed that monoclonal antibodies can strongly block the adhesion of the S. aureus collagen-binding protein Cna.
Results obtainedin the frame of NanoStaph contribute to radically change our understanding of the mechanisms by which bacterial pathogens form biofilms, and will help in the development of a new generation of anti-adhesion antimicrobials. In the end our ambition is to establish a novel paradigm in microbiology, i.e. the use of an innovative force nanoscopy platform for understanding biofilm formation and for figthing biofilm infections. As testified by a number of top publications, the project has a high-risk/high-gain profile and represents new thinking beyond state-of-the-art in cell nanoscopy and microbiology. See above for more details.
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