Professor Bart Hoogenboom, University College London, London Centre for Nanotechnology, 3C1 17-19 Gordon Street, London, WC1H 0AH, United Kingdom

Atomic force microscopy to view living bacteria, and their disruption by antibiotics, at molecular resolution

Gram-negative bacteria such as E. coli are surrounded by an outer membrane, which represents a major barrier to antibiotics. While the components of this outer membrane are known, it is far from clear how they are organised and how such organisation defines integrity and resistance against antibiotics. We have developed atomic force microscopy methods to resolve the outer membrane of living E. coli at molecular resolution, and to interpret topography of these living cells with biochemical identification. This has enabled us to discover that the outer membrane consists of remarkably tight and near-static networks of outer membrane proteins, that can be interspersed by phase-separated, glycolipid-enriched patches. Using similar methods, we now determine how last-resort polymyxin antibiotics disrupt the bacterial cell envelope and kill the cell.

References:

Benn et al., PNAS 118 (44), e2112237118 (2021)
Webby et al., Sci Adv 8 (44), eadc9566 (2022)
Benn et al., Nat Commun 14 (1), 4772 (2023)
Borrelli et al., bioRxiv, 2025.04. 16.649083 (2025)

Professor Peter Hinterdorfer, Department of Experimental Applied Biophysics, Johannes Kepler University Linz, Altenbergerstr. 69, 4040 Linz, Austria

Dynamic Multimeric Binding Strategies of Pathogens and Therapeutics: Lessons learnt from SARS-CoV-2 Variants of Concern

Recent waves of COVID-19 correlate with the emergence of the Delta and the Omicron variant. We combined high-speed atomic force microscopy with single molecule recognition force spectroscopy to investigate, at single molecule resolution, the interaction dynamics of trimeric Spike with its essential entry receptor ACE2 [1]. Spike trimer undergoes rapid conformational changes on surfaces, resulting in arc-like movements of the three receptor binding domains (RBDs) that collectively screen a circular range of almost 360° degrees. Acting as a highly dynamic molecular caliper, it thereby forms up to three tight bonds through its RBDs with ACE2 expressed on the cell surface. The Spike of both Delta and Omicron (B.1.1.529) variant enhance and markedly prolong viral attachment to the host cell receptor ACE2, which likely not only increases the rate of viral uptake, but also enhances the resistance of the variants against host-cell detachment by shear forces such as airflow, mucus or blood flow. We uncovered distinct binding mechanisms and strategies employed by circulating SARS-CoV-2 variants to enhance infectivity and viral transmission.

The new SARS-CoV-2 variants are continuously emerging with critical implications for therapies or vaccinations. The 22 N-glycan sites of the Spike protein remain highly conserved among SARS-CoV-2 variants, opening an avenue for robust therapeutic intervention. Out of a lectin library, two lectins, Clec4g and CD209c, were identified to strongly bind to the Spike protein of SARS-CoV-2 [2]. We quantified unbinding forces and analyzed the number of bond ruptures between Clec4g/CD209c and the trimeric Spike protein. Multiple bond formations lead to stable complex formation, in which the number of formed bonds enhanced the overall interaction strength and dynamic stability of the lectin/Spike complexes. Equipped with suchlike molecular modalities, Clec4g/CD209c are multivalent efficient competitors in SARS-CoV-2 Spike binding to cellular ACE2.

We also determined the binding capacity of a molecularly engineered lectin cloned from banana, BanLec H84T, which was shown to display broad-spectrum antiviral activity against several RNA viruses. Our studies revealed that H84T-BanLec interacts with the Spike protein of the original viral strain, Wuhan-1 and several variants of concern (Delta, Omicron) [3]. Based on our force probing technique, dynamic molecular interaction patterns with accurate rupture force and length distributions were depicted. The complex multiple binding features between the dimeric H84T and trimeric Spike protein were analyzed with respect to the distribution of the glycosylation sites on the Spike. The capacity of lectins to block SARS-CoV-2 viral entry holds promise for pan-variant therapeutic interventions.

[1] R. Zhu et al., Nature Communications 13, 7926 (2022)
[2] D. Hoffmann, S. Mereiter, Y.J. Oh et al., The EMBO Journal e108375 (2021)
[3] J.F.-W. Chan, Y.J. Oh et al., Cell Reports Medicine 3, 100774 (2022).