A reflection on ‘Nanoscale adhesion forces between the fungal pathogen Candida albicans and macrophages’

Abstract

Quantifying the nanoscale forces between pathogens (yeasts, bacteria and viruses) and human host cells is key to understanding the first stage of microbial infection, and offers promising prospects in nanomedicine for therapy. In 2016, we published an article in Nanoscale Horizons (S. El-Kirat-Chatel and Y. F. Dufrêne, Nanoscale Horiz., 2016, 1, 69–74, https://doi.org/10.1039/C5NH00049A) in which we established atomic force microscopy as a nanoscopy platform for studying the adhesion forces between the fungal pathogen Candida albicans and immune cells. The adhesion between the pathogen and macrophages was strong and primarily involved multiple specific molecular bonds between lectin receptors on the host membrane and mannan carbohydrates on the fungal cell surface. This paper has since then inspired many nanoscopy experiments that have led to a deeper understanding of the adhesion of various pathogens, from fungi to bacteria and viruses, to their target host cells. Collectively, these studies emphasise the importance of protein and glycan mechanobiology in regulating pathogen–host adhesion and infection, and offer promising prospects for the development of therapeutic approaches against intracellular pathogens.

1. Introduction

The adhesion of microbial cells to host cells is the initial step leading to infection, colonization, and symbiosis. This complex process is often governed by microbial surface adhesins and/or appendages that mediate specific ligand–receptor interactions, hydrogen bonding, hydrophobic forces, electrostatic attraction, or van der Waals interactions. Understanding the mechanisms driving these early interactions is a prerequisite for developing innovative infection-blocking strategies in nanomedicine. However, quantifying the molecular forces driving pathogen–host adhesion has long been a technical challenge.

The application of atomic force microscopy (AFM) to microbial cells in the early 2000s opened up a new window into understanding their mechanical and adhesive properties.¹ Functionalization of AFM tips with (i) specific chemical groups for chemical force microscopy (CFM), or (ii) biological ligands for single-molecule force spectroscopy (SMFS), enabled the elucidation of the remarkable diversity of adhesive strategies evolved by microbes for their efficient attachment in their environments. Among the adhesive mechanisms uncovered through CFM and SMFS are: the strong “dock, lock, and latch” (DLL) adhesion of SdrG in Staphylococcus epidermidis; the SpsD β-zipper in Staphylococcus pseudintermedius; the homophilic Zn-dependent interaction of SasG in Staphylococcus aureus; the catch-bond and lectin-mediated adhesion of FimH pili in Escherichia coli; the hydrophobic and hydrophilic contributions of type IV pili in Pseudomonas aeruginosa; and the multifunctional adhesive roles of agglutinin-like sequence (Als) proteins in Candida albicans.

These insights into the binding mechanisms of cell-surface-associated molecules rely on the mechanical detection of individual molecules, made possible by the nanoscale precision of AFM, which probes areas of only a few nm² on the cell surface. However, while single-molecule approaches provide invaluable mechanistic insights, microbial pathogen adhesion in vivo involves the entire cell surface interacting simultaneously with host interfaces. This highlights the need for AFM-based methods capable of measuring adhesion at the level of single pathogenic cells, allowing for a better understanding of the relative and collective contributions of all interacting surface molecules.

Almost two decades ago, the development of single-cell force spectroscopy (SCFS) transformed AFM into a quantitative biophysical probe capable of measuring the forces driving the adhesion of single whole cells under physiological conditions.² The conceptual roots of SCFS can be traced back to the early work of Benoit et al., who used the method to quantify cell–cell adhesion, thereby establishing the methodological framework for single-cell force measurements.³ The authors used tipless cantilevers coated with lectins to capture single cells, allowing repeated approach–retract cycles to record adhesion dynamics.

The next step in nanomicrobiology was therefore to adapt SCFS to explore pathogen–substrate and pathogen–host interactions. To enable more robust cell attachment and broader application to different cell types, lectin-coated cantilevers were replaced with cantilevers coated with polydopamine (DOPA), a mussel-inspired adhesive capable of binding to virtually any biotic or abiotic surface under liquid conditions. Using DOPA-coated tipless cantilevers, Alsteens et al. measured the interactions of single Candida yeast cells with hydrophobic and fibronectin-coated surfaces, demonstrating the major contribution of Als proteins as multipurpose adhesins.⁴

An additional challenge emerged for SCFS when applied to bacteria. Due to their smaller size (∼1 µm vs. 5 µm for yeasts), the precise positioning of bacterial cells on tipless cantilevers was difficult, as the cantilever itself could contribute to the measured adhesion signal. To overcome this limitation, Beaussart et al. developed a modified approach in which tipless cantilevers were first functionalised with silica beads before DOPA coating and bacterial attachment.⁵ This geometry enabled precise positioning of the bacterium, ensuring that only the bacterial probe made contact with the test surface. Importantly, these early SCFS studies on microbial cells also demonstrated that the proposed protocols preserved cell viability. Neither the AFM laser nor the DOPA-based immobilization affected the physiological state of the cells, confirming the non-toxic and bioadhesive nature of DOPA as a versatile cell-immobilization strategy. Moreover, this method does not rely on specific ligand–receptor fixation, which could potentially alter the interactions measured during SCFS experiments.

2. Single-cell force spectroscopy of pathogen–host adhesion: proof of concept

The development of SCFS for analyzing microbial adhesion opened the way to increase our understanding of pathogen–host interactions that lead to infection. In 2016, El-Kirat-Chatel and Dufrêne implemented SCFS to investigate the nanomechanical dialogue between pathogenic C. albicans yeast cells and macrophages (Fig. 1a and b) (https://doi.org/10.1039/C5NH00049A).⁶ There were two key motivations here. From the biological standpoint, the initial contact between a pathogen and host immune cells is of particular importance, as it determines whether internalization occurs and guides subsequent immune responses. Although the main immune receptors—grouped as pathogen recognition receptors (PRRs)—and the key C. albicans cell-wall components—classified as pathogen-associated molecular patterns (PAMPs)—had been identified previously, the nanoscale forces generated by these molecular interactions remained unknown. From the single-cell and single-molecule technology view, we wanted to establish a SCFS nanoplatform to help understand the interaction mechanisms between fungal pathogens and immune cells, which is a key technological challenge, with implications for biology and medicine.

Fig. 1 SCFS of pathogen–host interactions. (a) SCFS setup for fungal–macrophage adhesion: scheme showing a yeast cell (blue) attached to a tipless AFM cantilever, approaching a macrophage (green) spread on a solid substrate. (b) Representative retraction force curve recorded after contact between C. albicans and a macrophage. The largest adhesion peak corresponds to the adhesion force (in piconewtons, pN). Smaller jumps and tethers/force plateaus at longer distances (micrometers, µm) reflect the sequential rupture of individual adhesive bonds until complete detachment. (c) Bio-probes obtained by immobilizing: (i) a single yeast on a tipless cantilever, (ii) and (iii) single bacteria on colloidal probes, and (iv) a single phage or (v) a single virus on AFM tips.

To tackle this problem, single C. albicans cells were attached to DOPA-coated tipless cantilevers. Macrophages were cultured in glass-bottom Petri dishes, and a few tens of yeast cells were deposited in the center of the dish before the experiment. After allowing the yeasts to settle, a DOPA-coated cantilever was gently brought into contact with a single yeast cell to form a living “bio-probe.” The attached yeast was then positioned over an isolated macrophage, and adhesion forces were measured at different locations on the macrophage surface (Fig. 1a). The precise alignment of the yeast on the cantilever and, subsequently, the positioning of the living probe over the macrophage were achieved using an integrated AFM–optical microscopy setup, which allowed simultaneous mechanical measurements and visual control. This configuration ensured accurate targeting and reproducibility while maintaining the physiological integrity of both the fungal and host cells. Macrophages are highly deformable and can produce long membrane extensions, such as membrane tethers. Because most conventional AFMs are equipped with short Z-range scanners (5–15 µm), an extended Z-range scanner capable of achieving retraction distances up to 100 µm was used. Repeated force–distance (FD) curves captured the entire adhesion and detachment process at the single-cell level (Fig. 1b). High forces were observed at short separation distances, corresponding to the initial detachment of the two cells. These events were followed by multiple force jumps occurring between 5 and 25 µm, which correspond to the sequential rupture of individual or clustered molecular bonds. These unbinding events likely represent a combination of nonspecific interactions and specific PAMP–PRR-mediated binding. At longer separation distances, extending up to 100 µm, force plateaus characteristic of membrane tethers being pulled from the macrophage surface were observed. Such long-range elongation highlights the ability of phagocytes to extensively deform their membrane in order to retain or internalise their targets.

Quantitative analysis showed strong force peaks ranging between 500 pN and 3 nN, which corresponds to multiple molecules interacting simultaneously, since single lectin bonds typically rupture in the 50–100 pN range. The work of adhesion, extracted from the area under the curves, ranged from 2 to 50 fJ. Surprisingly, these values are comparable to those reported for mammalian cell–cell detachment, despite C. albicans being smaller and mechanically stiffer. Increasing the interaction time was found to enhance the maximum adhesion force, work of adhesion, and rupture distance, suggesting that longer contact allows the engagement of more specific PAMP–PRR interactions, resulting in stronger adhesion. Because macrophages are highly dynamic, longer contact durations may promote the early formation of a phagocytic cup, which would increase the effective contact area with the yeast cell. Addition of soluble mannoside or concanavalin A was found to block the C. albicans-macrophage adhesion, indicating that mannose–lectin bonds represent the primary driving force of this interaction.

In summary, this study underscores the key role of membrane adhesion and deformation in phagocyte–pathogen interactions, owing to an advanced nanoplatform. What were the important challenges that were encountered? Technically, this work wouldn’t have been possible without: (i) a reliable methodology to build up single-pathogen probes, without altering the fungal cell surface or inducing cell death; that is where DOPA turned out to be the best option; (ii) having a non-conventional Z-piezo scanner that allows for long rupture distance measurements (100 µm), with a low signal/noise ratio, which is not available on conventional set-ups.

Would the authors have done anything differently with the technical and biological insights of today? Not really, as at that time this was a pioneering study; but for sure, today, using super-resolution microscopy (see STED, Section 4), in parallel with AFM, to specifically locate macrophage receptors and follow their relocalisation and dynamics upon contact with the pathogen, is an exciting challenge! Furthermore, in future research, genetic manipulation of the receptors and ligands involved will certainly contribute to a more detailed understanding of the adhesion mechanism.

3. SCFS of other pathogen–host interactions

Following this early work, several groups then applied SCFS to other pathogens, from fungi to bacteria and viruses. While several studies have focused on pathogen–solid-substrate interactions, these will not be covered here.

Fungi (Fig. 1c(i)). In the fungal area, the contribution of PRRs in immune recognition of C. albicans was dissected by te Riet et al., who applied SCFS to immature dendritic cells expressing both macrophage mannose receptors and DC-SIGN.⁷ Their results demonstrated that both lectin receptors contribute comparably to C. albicans binding, and that DC-SIGN-mediated mannose recognition is strengthened when the receptor is present in a tetrameric state and when the host cell glycocalyx is intact. These findings illustrate how the receptor organization and membrane microenvironment modulate recognition forces. On the pathogen side, SCFS has helped identify fungal determinants controlling host adhesion. For instance, it was shown that the transcription factor CgEfg1 in Candida glabrata is required to initiate adhesion to vaginal epithelial cells.⁸ This demonstrates that transcriptional regulation of surface adhesins plays a direct role in determining early host colonization. In addition to biochemical interactions, host cell mechanics also contribute critically to Candida adhesion.

In a biological study, SCFS enabled the quantification of adhesion forces between Candida auris cells, but also the observation of adhesion profiles with both low and high intensities. Based on the different force peak profiles, the distinction between specific and non-specific interactions could be made.⁹ In biotechnology, the ability of Saccharomyces cerevisiae to form aggregates influences its behavior, for instance by enhancing tolerance to alcohol stress. This phenotype is mainly controlled by the expression of flocculin lectins of the Flo surface protein family. SCFS studies probing the role of Flo1 revealed that this adhesin (i) initiates cell–cell aggregation through calcium-dependent lectin binding to mannose residues on neighboring cells, and (ii) unfolds under mechanical load, exposing hydrophobic tandem repeat domains that further stabilise cohesion.¹⁰

Bacteria (Fig. 1c(ii) and (iii)). SCFS has also been instrumental in deciphering the forces guiding the adhesion of bacterial pathogens. S. aureus can invade various types of mammalian cells, thereby enabling it to evade host immune defenses and antibiotics. Prystopiuk et al. unravelled the molecular forces guiding S. aureus cellular invasion, focusing on the prototypical three-component FnBPA–fibronectin (Fn)–integrin interaction.¹¹ The bacterial protein FnBPA mediates bacterial adhesion to soluble Fn via strong forces (∼1500 pN), consistent with a high-affinity tandem β-zipper. Furthermore, the FnBPA-Fn complex binds to immobilised α5β1 integrins with a much higher strength than the classical Fn-integrin bond (∼100 pN). These data suggest an invasion model in which Fn-binding by FnBPA leads to the exposure of cryptic integrin binding sites via allosteric activation, which in turn engage into a strong interaction with integrins. Fn-dependent adhesion between S. aureus and endothelial cells strengthens with time, suggesting that internalization occurs within a few minutes.

S. aureus colonises the human skin, thereby causing various disorders, including atopic eczema, also known as atopic dermatitis (AD). Attachment of the pathogens to the outermost surface of the epidermis, the stratum corneum, involves specific cell-surface proteins on the bacterial cell surface that bind to target ligands on corneocytes, that is, keratinized cells found on the outermost surface of the stratum corneum. Identifying the various adhesins involved in skin adhesion and understanding their molecular binding mechanisms would greatly contribute to the development of new therapies to prevent or treat skin infections. AFM showed that the adhesin clumping factor B (ClfB) mediates the adhesion of S. aureus to corneocytes from AD patients, via binding to ligand proteins loricrin and cytokeratin through the well-known DLL mechanism. A unique relationship was found between the NMF level in the skin and the strength of ClfB-mediated bacterium–corneocyte adhesion, and it was discovered that FnBPB and ClfB interact with the N-terminal region of the ligand corneodesmosin on AD corneocytes, allowing S. aureus to take advantage of the aberrant display of corneodesmosin that accompanies low natural moisturizing factor (NMF) in AD.^12–14

Today, a very exciting challenge is to combine AFM experiments with modelling approaches. A recent example of this is the use of in vitro and in silico SMFS to demonstrate that the staphylococcal serine-aspartate repeat D (SdrD) protein forms ultrastrong bonds with the skin protein desmoglein-1 (DSG-1).¹⁵ This is the strongest non-covalent protein–protein interaction ever reported, explaining why the pathogen remains attached to the skin even after scratching or washing and helping us understand why these infections are so difficult to get rid of. Remarkably, the teams discovered that calcium, an element better known for strengthening bones, plays a key role in fortifying this bacterial grip. When calcium levels are reduced, the bond between SdrD and DSG-1 weakens significantly. When calcium is added back, the bond becomes even stronger. This finding is particularly relevant for patients with eczema, where disrupted calcium gradients amplify SdrD interactions, which could potentially intensify S. aureus virulence. These findings provide crucial insights into the calcium-dependent regulation of bacterial adhesion and open the door to new strategies for combating antibiotic-resistant infections. Instead of trying to kill bacteria directly, which often drives the evolution of resistance, scientists could design therapies that block or weaken bacterial adhesion.

Advances have also been made in understanding bacterial–bacterial adhesion mechanisms. The SasG surface protein from S. aureus displayed remarkable zinc-dependent mechanical properties that are critical for its adhesive function during biofilm formation. Zinc activates SasG-mediated adhesion through a mechanism whereby adsorption of zinc ions to the bacterial cell surface increases cell-wall cohesion and favors zinc-dependent homophilic bonds between SasG proteins protruding from opposing cell surfaces. The zinc-dependent adhesive function of SasG represents a promising target for the design of antibacterial compounds.¹⁶ Similar adhesive behavior has been reported for the SasG homolog in S. epidermidis, suggesting a conserved evolutionary mechanism adapted to the ecological niches of these pathogens.¹⁷

Finally, SCFS has been applied to understand interactions between plant growth-promoting rhizobacteria and plant roots. These studies revealed distinct preferential colonization zones along Arabidopsis thaliana roots, with Bacillus velezensis predominantly adhering to elongation zones, while Pseudomonas defensor preferentially interacts with cell division zones.¹⁸ This nanoscale insight into plant–microbe adhesion mechanisms may inform the design of next-generation biofertilization strategies.

Viruses (Fig. 1c(iv) and (v)). Finally, understanding the mechanisms by which viruses recognise and infect their host cells has also been an area of rapid progress. AFM has opened new avenues for studying virus interactions, which is quite challenging given their small size and their inherent orientation, possessing distinct head and body regions. Several groups have developed methodologies for single-virus force spectroscopy (SVFS), in which AFM tips are functionalised with virus particles to probe host receptors directly. Using this approach, comparison of the adhesion forces of wild-type and mutant herpes virus particles revealed that the viral surface glycoprotein gH/gL is the major effector responsible for binding to glycosaminoglycans on host cells.¹⁹ In the context of bacteriophages—viruses that infect bacteria—Arbez et al. used a similar strategy to investigate interactions between the head–tail phage 187 and the surface of S. aureus.²⁰ Their results demonstrated that initial phage attachment is governed by the recognition of N-acetylglucosamine residues on wall teichoic acids by the phage tail receptor-binding proteins. In both viral systems, adhesion involves a combination of specific and nonspecific interactions. The specificity conferred by viral proteins was demonstrated not only on host cells but also validated using model surfaces functionalised with purified receptors, confirming that the measured adhesion forces originate from bona fide receptor–ligand pairing.^19,20

A study of MDCK cell surfaces revealed a heterogeneous topography caused by microvilli, as shown by a high-resolution AFM image. The AFM tip was functionalised with a single EnvA–RABV(ΔG::eGFP) virus using a flexible PEG linker, allowing controlled and physiologically relevant interactions with the cell membrane.²¹ Microscopy confirmed that only one virus was attached to the apex of the tip, ensuring highly precise measurements. By combining AFM imaging with fluorescence microscopy, the researchers were able to simultaneously map cell topography and detect specific virus–receptor adhesion events on TVA-expressing cells. This approach enables direct quantification of viral binding forces and provides a powerful tool for studying viral specificity and infection mechanisms at the nanoscale. In another study, AFM tips functionalised with T3 reovirus were used to image a confluent monolayer of co-cultured cells expressing (CHO) or lacking (Lec2) α-linked sialic acid (α-SA) on the cell surface.²² These cells were engineered to express distinct fluorescent markers so they could be distinguished during the AFM experiments. Corresponding AFM height and adhesion maps revealed the spatial localisation of specific adhesion events. The technique was unable to differentiate CHO cells because of their high density of adhesion sites, in contrast to the sparse distribution observed in Lec2 cells. This finding supports the formation of specific T3SA+–α-SA interactions on living cells. Together, these studies show that combining single-particle force spectroscopy (SPFS) with fluorescently labelled host cells allows researchers to investigate how the same virus interacts with different cell types under identical experimental conditions.

4. What is next? Towards new nanoplatforms to decipher pathogen–host adhesion

In future research, the community will greatly benefit from increasing the throughput of SCFS analyses, for which the FluidFM technology is ideally suited.²³ Here, microchanneled cantilevers with nano-sized apertures enable the fast manipulation of single living cells under physiological conditions. In classical SCFS, immobilization of the cell on the probe is labor-intensive, meaning performing statistically significant numbers of measurements is time-consuming. To circumvent this problem, FluidFM allows us to quickly and reversibly immobilise cells on the pyramidal tip of the microchanneled cantilever. The hollow cantilever is connected to a pressure controller, enabling its operation in liquid as a force-controlled nanopipette under optical control. Unlike other assays, many cells can be probed in a short time frame, meaning statistically relevant data sets will be obtained within a few hours (up to 200 cells in a day vs. 10 cells in classical assays), which is a major asset for probing the numerous interactions planned in the project (various adhesins and adhesin targets, multiple strains), but also for the fast screening of anti-adhesion compounds.

Combining AFM with optical nanoscopy is another exciting direction. As highlighted by the Nobel Prize in Chemistry 2014, super-resolution microscopy (optical nanoscopy) has revolutionised the way biologists explore the living cell at a molecular resolution, enabling probing of the localization and motion of single molecules in (or on) living cells with a resolution in the range of a few tens of nanometers. Current optical nanoscopies all aim at overcoming the diffraction limit of light, which imposes a finite magnification limit in conventional microscopy, by switching on and off the fluorescence of a subpopulation of fluorophores within a diffraction-limited area, enabling molecules that are spatially indistinguishable from each other to be separated in time. In stimulated emission depletion (STED) microscopy, the point spread function (PSF) is optically confined to a spot smaller than a diffraction-limited area, achieved by overlaying two beams, an excitation beam to induce fluorescence, and a donut-shaped STED beam, for stimulated-emission-depletion, to inhibit fluorescence at the outer rim of the excitation beam,²⁴ thus allowing only the fluorescent molecules at the center of the excitation beam to provide a signal for image construction.

AFM has been combined with STED to produce high-resolution fluorescence imaging, topographical imaging and nano-mechanical imaging of biological samples.²⁵ The choice of STED is motivated by the unique capabilities of the method, e.g., fast image acquisition. STED-AFM-based biomanipulation demonstrated the capability to monitor real-time responses of filament movements to mechanical force inside the cell, opening new perspectives for correlative STED-AFM experiments.²⁶ Hence, combining AFM and super-resolution optical microscopy (STED) appears to be a powerful platform to address the following questions: how do adhesins and their ligands localise across the bacterial and corneocyte surfaces? Are these randomly distributed, or do they form clusters (nanodomains) which could favour multivalency and strong binding? STED microscopy is the ideal option here given its resolution (<50 nm) and its versatility. For many years, AFM and STED have been used separately, but very few groups have combined them for biology.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

S. E. K. C. and O. K. thank the University of Bordeaux for the RIE grant 2024 ‘Canlys’. S. E. K. C. and G. R. thank CNRS Chimie for the Emergence grant 2025 ‘Matolys’. Work at the Université Catholique de Louvain was supported by the National Fund for Scientific Research (FNRS). Y. F. D. is a Research Director at the FNRS. Z. Z. is a postdoctoral researcher at the FNRS.

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