The primary structural component of the bacterial cell wall is peptidoglycan which is crucial for survival and division of the cells. Peptidoglycan (PG) is a heterogeneous macromolecule composed of glycan chains (sugars) and small peptides that unify the structure by crosslinking the glycan chains. PG is tens of nanometres thick in Gram-positive bacteria and acts as a constraint to interrupt turgor. This component of the cell is also one of the major targets by cell wall antibiotics such as Methicillin or Vancomycin. 

In this project, we applied several imaging modes of atomic force microscopy (AFM) to interrogate the morphology of this heterogeneous hydrogel. The bacteria of study were Staphylococcus aureus and Bacillus subtilis which are two Gram-positive species with distinct cell shape (sphere vs rod). High resolution tapping was used to image the external surface of live cells and PeakForce Tapping was used to study both the internal and external surface of purified PG. Then, quantitative image analysis methods were developed to obtain robust conclusions when comparing different samples. The results was that contrary to stablished theories, the PG is not an homogeneous impenetrable wall, it is a highly porous heterogeneous hydrogel [1].

Then, once the architecture of PG from healthy cells was well characterised, the same techniques were applied to study the effect of different antibiotics (Methicillin and Vancomycin) to the PG morphology. We used Staphylococcus aureus wild type, together with different mutants to decipher the role of PG modification enzymes during cell death [2]. The results corroborate the model that for the cell to survive there must be an equilibrium between synthesis and hydrolisis of PG, this equilibrium is disrupted when antibiotics are applied, therefore leading to cell death. This brings us one step closer to obtain a complete bigger picture of how the bacterial cell wall evolves during the life and death cycle.

AFM, High-resolution, Bacterial cells, Peptidoglycan, Antibiotics, Image Analysis, Biophysics

The recruitment of viral pathogens to host cells is often mediated by carbohydrates on the cell surface. For example, glycosaminoglycans (GAGs), negatively charged oligosaccharides, play a decisive role in initially recruiting and accumulating viruses at the cell surface. Moreover, the degree and type of GAG sulfation has been suggested to be important in modulating the attachment/detachment behavior of enveloped viruses like herpes simplex virus type I (1-3). However, little is known about the biophysical properties of the interactions between non-enveloped virus and GAGs, specifically when it comes to their dynamics. In this study, we probe how the attachment, detachment and diffusion of a non-enveloped virus, human papillomavirus type 16 (HPV16), is regulated on the cell surface. HPV16 is a double-stranded DNA virus of high medical important, as it is the leading cause of cervical cancer (4).Using two powerful single particle techniques: single particle tracking by total internal reflection fluorescence (TIRF) and atomic force microscopy-based single molecule force spectroscopy (AFM-FS) (3,5) alongside with surface-immobilized GAGs (a cell-surface mimic platform that offers tight control on grafting density and presentation of GAGs), we study how specific sulfation patterns on highly sulphated GAGs like heparin influence the binding kinetics and diffusion behaviors of HPV type 16. Our TIRF data show that HPV16 binds with higher affinity to parental heparin as compare to selectively N-Desulphated heparin with no major difference in their diffusion behavior. Our AFM-FS data in excellent agreement with TIRF shows higher binding probability and lower dissociation rate for the interaction of HPV16 and parental heparin further highlighting the importance of N-sulphation on the binding behavior. Taken together, this implies that the type of sulphation of the GAGs has an important functional implication for the viral attachment which may influence entry. 

Human Papillomavirus, glycosaminoglycan, TIRF, single particle tracking, AFM force spectroscopy

1. Spear et al, J. Virol. 65 1090–1098 (1991) 
2. Tufaro et al., Virology 208 531–539. (1995)
3. Bally et al., Biophys. J113, 1223 (2017) 
4. Kast et al., J. Viorology 87, 6062, (2013) and Schelhaas et al. Cellular microbiol. 15, 1818 (2013)
5. Bally et. al., ACS Chem. Biol. 14, 534−542 (2019)

Methicillin-resistant Staphylococcus aureus (MRSA) is a gram-positive bacteria that is genetically-distinct from the antibiotic-sensitive Staphylococcus aureus. Also, MRSA is part of WHO priority group of Superdrug that could lead to 10 million death in the year 2050¹. So far, the studies performed on resistance in MRSA have focussed on the genetic-mutation and evolution associated with MRSA but little has been done as touching understanding the physics that underpins resistance in MRSA².   Here are some of the questions that we seek to address; 1) what are the imprints of resistance on the cell wall architecture? 2) Can we distinguish the antimicrobial strains based on the material properties of their associated cell wall? 3) What is the link between the architectural differences and the inherent macroscopic resistance expressed by MRSA? In addition, 4) when the native penicillin-binding proteins are turned off via methicillin treatment, what are the architectural changes observed?

Our goal is to address these questions by using high-resolution atomic force microscopy (AFM) to decipher the associated cell wall, treated and not treated with an antibiotic. We utilized Tapping mode and PeakForce Tapping mode to examine the thickness of the purified sacculi and the 3D molecular architecture associated with the internal and the external surface of MRSA sacculi (extracted cell wall), without and with treatment with antibiotic. In a complementary fashion, we used wide-field fluorescence microscopy to characterize the cell size and cell cycle associated with MRSA cells and other derivatives of S.aureus, with the latter consisting different genetic modifications but within the same genetic background. 

We find that MRSA is associated with thicker cell wall (by approximately 35%) and reduced cell size (by approximately 30%) when compared to other S.aureus derivatives with no and low-level resistance. For the external surface, AFM reveals different subdomain of porous-rich mesh network, with changing depth, and an extra layer of mesh matrix in the Z-direction. In addition, the nascent peptidoglycan structure is characterized by a concentric rings-like structure in the absence of methicillin treatment, but this is replaced with dense but random structure when such cells are treated with methicillin. By studying the impact of antibiotics on cells with mutations in different cell wall synthesis proteins, we are able to gain new insights into which enzymes are responsible for which architectural features in the bacterial cell wall. This work brings us closer to methods for determining the molecular phenotype associated with particular genes, as well as to understanding how MRSA evades antibiotic-induced cell death.

High-resolution AFM, peptidoglycan, MRSA, 3D architecture.

References

1. de Kraker MEA, Stewardson AJ, Harbarth S. Will 10 Million People Die a Year due to Antimicrobial Resistance by 2050? PLoS Med. 2016;13(11):1002184. 
2. Panchal V V., Griffiths C, Mosaei H, et al. Evolving MRSA: High-level β-lactam resistance in Staphylococcus aureus is associated with RNA Polymerase alterations and fine-tuning of gene expression. Peschel A, ed. PLOS Pathog. 2020; 16(7):e1008672. 
3. Pasquina-Lemonche L, Burns J, Turner RD, et al. The architecture of the Gram-positive bacterial cell wall. Nature. 2020;582(7811):294-297

Extracellular nanovesicles (EVs) are small (30-150 nm) phospholipid-based vesicles present in most, if not all bodily fluids. They are naturally released by cells into the surrounding medium and are used as vehicles to cargo small molecules, proteins and nucleic acids throughout the body. EVs mediate active communication between cells and can help regulate the growth and the fate of adjacent and distant cells [1]. Recently, EVs have drawn considerable attention for their potential in nanomedicine due to the fact that they carry distinct markers (proteins and nucleotides) which concentration may be correlated with diseases such as cancers, diabetes, and neurodegenerative diseases [1, 2]. In the case of cancer, for example, it has been shown that both cancer cells and Tumour MicroEnvironment stromal cells release EVs that promote tumour-induced immune suppression, angiogenesis and metastasis [3]. Aside from promoting tumour proliferation, multiple studies conducted on EVs extracted from cancer patients suggest the existence of distinct markers specific to most types of cancerous tumours [1], suggesting that EVs could be used as an early diagnostic tool, when cancer is most treatable. This would be further facilitated by the fact that EVs collection from liquid biopsies (mainly blood, saliva and urine) is relatively straightforward and minimally invasive compared to surgery.

However, characterising EVs from biopsies remains currently a significant challenge. Quantifying the proteomic and genetic content of the EVs is lengthy (> hours), costly, and typically requires significant quantities of biopsy liquid. This is because the EV sample are obtained by purification and concentration from the biopsies. There are no accepted standards for these crucial steps, and it is not clear whether the process alters the EVs, let alone the subsequent characterisation. This often results in contradictory or confusing findings, leading to difficulties in comparing studies and building a reliable picture. This confusion is best illustrated by the existence of a review paper for every three original publications in the field, with reviews attempting to bridge studies and build a global picture.

There is hence a strong need to develop techniques able to characterise EVs in-situ, directly in raw bodily fluid samples. Ideally such techniques should also be rapid from sample collection to quantitative results, relatively cheap, have inbuilt references, and be able to function on small volumes of fluid. 

Here we test the suitability of using atomic force microscopy (AFM) microcantilevers to quantify a set model EV sub-population exhibiting a specific marker directly inside saliva. Aside from bypassing extraction and purification-related issues, a microcantilever-based approach offers multiple advantages over existing approaches: (i) it uses using small amount of biopsy (<0.1 mL), (ii) it can be multiplexed easily, (iii) it is relatively flexible for functionalisation, and (iv) it can be integrated into microfluidics chips, potentially using self-actuated cantilever to bypass the expensive AFM detection. 

Microcantilevers have long been used for bio-detection with excellent sensitivity [4,5], but usually with the cantilever operated in air or in vapour, but not directly immersed into the fluid of interest. Here we use as a model system AFM microcantilevers functionalised with streptavidin to detect small quantities of synthetic phospholipid vesicles dissolved in raw saliva. 0.5% of the vesicles’ lipids are biotinylated, allowing for strong, irreversible binding to the cantilever’s streptavidin. The main challenge to overcome is the need for the cantilever to achieve reliable dynamic sensing inside a ‘dirty’ non-Newtonian fluid containing biopolymers in suspension while operating for extended periods of time. We show that it is possible to achieve a detection sensitivity better than 1 microgram/ml of vesicles in raw saliva within minutes, limited mainly by the vesicle’s diffusion timescale within the sample. The detection sensitivity can track model EVs at concentrations two orders of magnitude lower than the typical total EV concentration naturally occurring in blood [6]. This suggests microcantilever-based approaches to targeted EVs as a promising route for in-situ detection. 

[1] S. Fais, et al. ACS Nano 10 (2016) 3886–3899.

[2] L. J. Vella, et al. Int. J. Mol. Sci. 17, 173 (2016)

[3] J. Armstrong, et al. ACS Nano 11, 69 (2017).

[4] H. P. Lang, M. Hegner, & C. Gerber Materials Today 8 (2005) 30–36. 

[5] T. Braun, et al. Nat Nanotechnol 4 (2009) 179–185.

[6] I. Helwa, et al. PLoS ONE 12 (2017) e0170628.

Excessively oily skin can often cause unwanted skin traits in patients, such as excessive shine, enlarged pores, frequent outbreaks or acne. Comedones are small skin-colored papules frequently found on the T-zone (forehead, nose and chin). Sodium salicylate (NaSal) is an ingredient commonly used in anti-inflammatory drugs, anti-bacterial agents, anti-blemish and anti-aging cosmetic products. Although the exact mechanism of NaSal on comedones is not fully understood at present, it appears to exhibit a significant exfoliation effect on the skin after repeated use. Recent advances in metrology have led to novel methods being implemented using atomic force microscopy (AFM) to probe the structure of biological samples and materials. Indeed, characterization of the nanomechanical properties of comedones at sub-cellular levels remains key in understanding the dynamic processes of comodone outbreak. Herein, we investigated the physical properties of comedones pre- and post-treatment using 2% NaSal under ambient temperature and humidity. When treating comedones with 2% NaSal, samples appeared significantly softer when compared to their pre-treated measurements. Furthermore, the force-volume maps generated, showed that after NaSal treatment, areas in the comedone appeared softer suggesting the beneficial impact of the 2% NaSal solution on loosening the inner content of comedones. Our results provide evidence that NaSal is indeed beneficial as an active ingredient in topical creams aimed at targeting eruptive skin conditions.

Atomic force microscopy, force-volume mapping, microcomedones, sodium salicylate and elastic modulus

We introduce an improved platform based on atomic force microscopy (AFM) to quantify the micro-mechanical properties, including both elastic moduli and viscosity, of relatively intact metastatic breast tumour in bone. A unique mechanical distribution of extremely high deformability (Young’s modulus down to a few Pa) and low viscosity (viscosity down to a few Pa×s) was identified, which is significantly more compliant (> 10 times) than the mechanical properties found using in-vitro systems (e.g. cancer cells on a petri-dish) both by ourselves and others. These findings shed light on how mechanics plays a deterministic role in cancer development and metastasis, as well as spotlight the gap between in-vitro models and real tissues in studies of mechanobiology.

Bone metastases of breast cancer (metastatic tumours, MT) were established using GFP expressed MDA-MB-231 cells based on a mouse model [1]. Point force (F) vs. indentation (δ) and creep measurements by colloidal probe AFM [2] were applied at randomly selected positions within the MT area contained by the fresh (un-fixed) murine bones in physiological buffer, aided by the in-situ fluorescent imaging. The weighted Sneddon model and Kelvin-Voigt model were employed to obtain the Young’s moduli and viscosity from the AFM data. Such mechanical characterisations were also applied on the 2D in-vitro model (MDA-MB-231 cells on petri-dish), the explanted subcutaneous tumour (SCT) and the metaphysis of bones (BM) from mice with or without tumours.

The resultant mechanical properties of the 2D models are over an order of magnitude higher than those of tumours, indicating the importance of a proper 3D environment and acellular components in cancer mechanics. The MT is considerably more compliant than the SCT, suggesting other mechanical cues in addition to 3D environment contribute to bone metastases of breast cancer. Meanwhile, the MT was found to be mechanically distinct from its surrounding environment (i.e. the BM) and it did not mechanically alter the environment at meso-scale distance (> 200 µm). These findings could inspire the design of more realistic in-vitro cancer research models or mechanical interventions as anti-cancer drugs/treatments. This improved AFM based system is powerful for further characterisations of bones containing tumour in the presence of anti-cancer therapy or other complex tissues.

AFM, viscoelasticity, cancer models, bone microenvironment

[1] P.D. Ottewell, et al. (2014) Zoledronic acid has differential antitumor activity in the pre- and postmenopausal bone microenvironment in vivo, Clin Cancer Res, 20: 2922-2932. 

[2] X. Chen, et al. (2020) Mechanical Heterogeneity in the Bone Microenvironment as Characterised by Atomic Force Microscopy, Biophys. J., 119(3): 502-513.

Biological membranes perform a staggering array of functions in vivo, with their lipid molecules acting not just as passive structural components, but actively driving processes related to protein function, oncogenesis and disease signalling, and even intrinsic sensing capabilities [1–5]. The versatility of lipid bilayers originates from their fluidity and flexibility – that is, their dynamics – which allows molecular transport along and across them, membrane restructuring, and the ability to robustly sustain large shear forces, all with minimal energy cost. In-plane lipid motion, as well as the slowly-evolving hydration structures at their headgroups contributes to the strongly lubricating regimes observed in synovial joints which outperform artificial lubricants with friction coefficients as low as µ = 10^-5 [6,7]. Further, a bilayer’s fluidity and ability to restructure governs the motion of bound nanostructures and transport of proteins [8,9] as well as modulating mesoscale events such as the generation and detection of exosomes [4].

Despite the clear need for a holistic understanding of lipid dynamics, experimental techniques rarely have access to local membrane viscosity, frictional or diffusive coefficients over a broad range of timescales, relying instead upon either equilibrium fluctuations or experimentally friendly velocities that are typically two orders of magnitude too small for modelling real-life applications [9,10].

Here, we demonstrate the use of an atomic force microscope-based high-frequency shearing device to probe the dissipation and lubrication abilities of supported lipid membranes. We can access velocities ranging from hundreds of nms^-1 to mms^-1, easily capturing quasistatic (v_shear << lipid motion), glassy (v_shear >> lipid motion) and potential non-linear transitional regimes. Crucially, the technique does not require molecular labels and has contact areas of order ~nm², allowing for direct, local energetic measurements on the scale of single proteins. We explore the impact of membrane and buffer composition and unpick the complex interplay between lipid dynamics, headgroup hydration and experimental timescale. The results have important implications for our understanding of biolubrication, as well as dynamic membrane binding events.

Biolubrication, nano-rheology, membrane dynamics, lipid diffusion, AFM

[1]       E. Sezgin, I. Levental, S. Mayor, and C. Eggeling, The Mystery of Membrane Organization: Composition, Regulation and Roles of Lipid Rafts, Nat. Rev. Mol. Cell Biol. 18, 361 (2017).

[2]       P. A. Janmey and P. K. J. Kinnunen, Biophysical Properties of Lipids and Dynamic Membranes, Trends Cell Biol. 16, 538 (2006).

[3]       S. Beloribi-Djefaflia, S. Vasseur, and F. Guillaumond, Lipid Metabolic Reprogramming in Cancer Cells, Oncogenesis 5, e189 (2016).

[4]       N. P. Hessvik and A. Llorente, Current Knowledge on Exosome Biogenesis and Release, Cell. Mol. Life Sci. 75, 193 (2018).

[5]       B. P. Young, J. J. H. Shin, R. Orij, J. T. Chao, S. C. Li, X. L. Guan, A. Khong, E. Jan, M. R. Wenk, W. A. Prinz, G. J. Smits, and C. J. R. Loewen, Phosphatidic Acid Is a PH Biosensor That Links Membrane Biogenesis to Metabolism, Science (80-. ). 329, 1085 (2010).

[6]       W. H. Briscoe, S. Titmuss, F. Tiberg, R. K. Thomas, D. J. McGillivray, and J. Klein, Boundary Lubrication under Water, Nature 444, 191 (2006).

[7]       W. H. Briscoe, Aqueous Boundary Lubrication: Molecular Mechanisms, Design Strategy, and Terra Incognita, Curr. Opin. Colloid Interface Sci. 27, 1 (2017).

[8]       B. Plochberger, C. Röhrl, J. Preiner, C. Rankl, M. Brameshuber, J. Madl, R. Bittman, R. Ros, E. Sezgin, C. Eggeling, P. Hinterdorfer, H. Stangl, and G. J. Schütz, HDL Particles Incorporate into Lipid Bilayers – a Combined AFM and Single Molecule Fluorescence Microscopy Study, Sci. Rep. 7, 15886 (2017).

[9]       T. T. Hormel, S. Q. Kurihara, M. K. Brennan, M. C. Wozniak, and R. Parthasarathy, Measuring Lipid Membrane Viscosity Using Rotational and Translational Probe Diffusion, Phys. Rev. Lett. 112, 188101 (2014).

[10]     L. Ma, A. Gaisinskaya-Kipnis, N. Kampf, and J. Klein, Origins of Hydration Lubrication, Nat. Commun. 6, 6060 (2015).

The basic architecture of a virus consists of the capsid, a shell made up of repeating protein subunits, which packs, shuttles and delivers their genome at the right place and moment. Viral particles are endorsed with specific physicochemical properties which confer to their structures certain meta-stability whose modulation permits fulfilling each task of the viral cycle. These natural designed capabilities have impelled using viral capsids as protein containers of artificial cargoes (drugs, polymers, enzymes, minerals) with applications in biomedical and materials sciences. Both natural and artificial protein cages (1) have to protect their cargo against a variety of physicochemical aggressive environments, including molecular impacts of highly crowded media, thermal and chemical stresses, and osmotic shocks. Viral cages stability under these ambiences depend not only on the ultimate structure of the external capsid, which rely on the interactions between protein subunits, but also on the nature of the cargo. During the last decade our lab has focused on the study of protein cages with Atomic Force Microscopy (AFM). We are interested in stablishing links of their mechanical properties with their structure and function. In particular, mechanics provide information about the cargo storage strategies of both natural and virus-derived protein cages (2,3,4). Mechanical fatigue has revealed as a nanosurgery tool to unveil the strength of the capisd subunit bonds (5).