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Force nanoscopy of staphylococcal biofilms

Periodic Reporting for period 3 - NanoStaph (Force nanoscopy of staphylococcal biofilms)

Reporting period: 2019-10-01 to 2021-03-31

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.

NanoStaph will have strong scientific, societal and economical impacts. From the technical perspective, force nanoscopy will represent an unconventional methodology for the high throughput and high resolution characterization of adhesion forces in living cells, especially in bacterial pathogens. In microbiology, the results will radically transform our perception of the molecular bases of biofilm formation by S. aureus. In medicine, the project will provide a new screening method for the fast, label-free analysis of anti-adhesion compounds targeting S. aureus strains, including antibiotic-resistant clinical isolates that are notoriously difficult to treat, thus paving the way to the development of anti-adhesion therapies.
Results obtained during the first half of NanoStaph have covered (fully or in part) our three complementary objectives (work packages, WPs): (WP1): unravelling adhesion forces in single S. aureus bacteria; (WP2): understanding the binding mechanisms of single S. aureus adhesins; (WP3): optimising the use of anti-adhesion compounds targeting S. aureus. The methodology involved using atomic force microscopy (AFM) techniques for studying three classes of surface proteins (adhesins): FnBPs, Clfs and Cna. Owing to fruitful collaborations we also studied an additional adhesin, SdrC, fully in line with NanoStaph. These achievements have led to several key publications summarized below.

1. Multifunctionality of Cna (WP1, WP2, WP3). While it is established that the collagen-binding protein Cna binds to collagen via the high-affinity collagen hug mechanism, whether this protein is engaged in other ligand-binding mechanisms is poorly understood. In an ACS Nano (2017) paper, we used AFM to demonstrate that Cna mediates attachment to two structurally and functionally different host proteins, i.e. the complement system protein C1q and the extracellular matrix protein laminin, through binding mechanisms that differ from the collagen hug. Both C1q and laminin interactions can be efficiently blocked by monoclonal antibodies directed against the minimal binding domain of Cna.

2. Discovery of a peptide capable to prevent biofilm formation (WP3). S. aureus biofilm-related infections are difficult to eradicate because bacterial cells are protected from host defenses and are resistant to many antibiotics. An attractive alternative to antibiotics is the use of antiadhesion compounds to block bacterial adhesion and biofilm development. We identified a peptide capable of preventing the formation of S. aureus biofilms. We first unravelled the molecular interactions by which the surface adhesin SdrC mediates biofilm accumulation. SdrC was found to mediate cell-cell adhesion through weak homophilic bonds, and to also promote strong hydrophobic interaction with inert surfaces. We discovered that a peptide derived from the neuronal cell adhesion molecule β-neurexin is able to inhibit SdrC-dependent attachment to inert surfaces, cell-cell adhesion, and biofilm formation. These findings, published in Proc Natl Acad Sci U S A. (2017), raise the possibility that the peptide could be used as a platform for designing a peptidomimetic with potential to prevent biofilm infections.

3. How fibrinogen activates the capture of human plasminogen by staphylococcal surface proteins (WP1, WP2). Invasive bacterial pathogens can capture host plasminogen and allow it to form plasmin, a process of medical importance as surface-bound plasmin promotes bacterial spreading by cleaving tissue components and favours immune evasion by degrading opsonins. We discovered that FnBP proteins bind plasminogen by a sophisticated activation mechanism involving fibrinogen, another ligand found in the blood. The work has been published in mBio (2017).

4. Physical stress activates the adhesive function of surface protein clumping factor B (WP1, WP2). S. aureus colonizes the skin and the nose of humans and can cause various disorders, including superficial skin lesions and invasive infections. During nasal colonization, the S. aureus surface protein ClfB binds to the squamous epithelial cell envelope protein loricrin, but the molecular interactions involved are poorly understood. In a paper published in mBio (2017), we unravelled the molecular mechanism guiding the ClfB-loricrin interaction. We showed that the ClfB-loricrin bond is remarkably strong, consistent with a high affinity "dock, lock and latch" binding mechanism. We discovered that the ClfB-loricrin interaction is enhanced under tensile loading, thus providing evidence that the function of a S. aureus surface protein can be activated by physical stress.

5. Force matters in hospital-acquired infections (WP1, WP2, WP3). In a perspective article in Science (2018) we discuss how extremely strong forces help staphylococci to colonize biomaterials and infect humans, building up on an outstanding paper by Gaub et al.

6. Live-cell nanoscopy in antiadhesion therapy (WP3). In a forum article in Trends Microbiol. (2017), we explain how live-cell nanoscopy has contributed significantly to assessing the inhibition of adhesion of uropathogenic Escherichia coli and Staphylococcus aureus by glycoconjugates and monoclonal antibodies, respectively, and of S. aureus surface attachment and cell–cell association by a synthetic peptide. This new technology, central to NanoStaph, shows promise for the development of antiadhesion therapies against bacterial pathogens.

7. Mechanobiology: Staphylococcus aureus under tension (WP1, WP2, WP3). In a study published in PNAS (2018), our team elucidated the mechanism by which S. aureus responds to mechanical tension. We focused on the bacterial surface protein ClfA, and on its interaction with fibrinogen (Fg), a blood protein that rapidly covers implanted medical devices. We showed that ClfA behaves as a force-sensitive molecular switch that potentiates staphylococcal adhesion under mechanical stress. Strong bonds are inhibited by a peptide mimicking Fg, which offers prospects for the development of antiadhesion therapeutics.
Results obtained so far in the frame of NanoStaph will 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.

Expected results until the end of the project are to progress further on our main objectives (3 WPs). Unique to NanoStaph is the implementation and optimisation of the new FluidFM single-cell (WP1) and multiparametric single-molecule (WP2) techniques for biofilm research, enabling us to address the forces in bacterial biofilms, at a throughput and spatiotemporal resolution never achieved before. Application of these methods, together with more established ones, for analyzing S. aureus, including drug-resistant strains that are difficult to fight, is completely new and challenging. Another unique feature is the application of these tools in the context of anti-adhesion therapy. Unlike existing bioassays, force nanoscopy will enable the high-throughput, label-free analysis of anti-adhesion drugs capable to inhibit biofilms, directly on live cells without the need for labelling or purification. We are not aware of any research groups having addressed – or planning to address - this ambitious, medically-important challenge. More broadly, the project will push the emerging field of live-cell nanophysics beyond state-of-the-art, complementing fluorescence nanoscopy approaches. NanoStaph will also have broad societal and economical impacts: since biofilms are involved in more than 80% of all microbial infections, and multiresistant strains are developing exponentially, there is an urgent need for new screening methods and for alternative drugs capable to fight these infections.