The role of polymers in cross-kingdom bioadhesion

Abstract

The secretion of extracellular polymeric substances provides an evolutionary advantage found in many organisms that can adhere to surfaces and cover themselves in a protective matrix. This ability is found in prokaryotes, archaea and eukaryotes, all of which use functionally similar polysaccharides, proteins and nucleic acids to form extracellular matrices, mucus and bioadhesive substances. These macromolecules have been investigated from the perspective of polymer biophysics, and theories to help understand adhesion, viscosity and gelling have been developed. These properties can be measured experimentally using straightforward methods such as cell counting as well as more advanced techniques such as atomic force microscopy and rheometry. An integrated understanding of the properties and uses of adhesive macromolecules across kingdoms is also important and can provide the basis for a range of biotechnological and medical applications.

This article is part of the theme issue ‘Transdisciplinary approaches to the study of adhesion and adhesives in biological systems’.

1. The importance of polymers for microscopic organisms

Polymers are the building blocks of life. A plethora of functions can be achieved by joining many monomers together into polymers, with complex structures and specialized functions. The identity of the monomers defines the nature of the polymer; if the repeating unit is a single monomer, the resulting chain is a homopolymer, and if various monomers are involved, the chain is a heteropolymer. Biological polymers are most often heteropolymers. For instance, DNA is a double-chained polymer with a deoxyribose and phosphate backbone with chains joined together by nucleic acids; this assembly is an exquisite design from the perspective of polymer physics as it is one of the stiffest and most charged polymers known [1]. The nature of a polymer backbone confers its architecture and shape, such as linear, ring, star-branched, H-branched and comb. This architecture in turn dictates its properties, for instance, mucin, one of the main glycoproteins in mucus, folds around foreign objects to entrap them owing to a combination of bare hydrophobic regions and heavily branched hydrophilic regions [2]. Another characteristic of polymers is that they can cross-link, by forming new covalent and ionic bonds with other polymers, and thus they can further modify their physical characteristics (e.g. sol–gel transition) and form complex structures capable of retaining water and suspended particles. The chemical versatility of biopolymers makes them the basis of all biological slime-like substances and thus they define the physics of gel-like materials, which include extracellular polymeric matrices, marine snow, biological adhesives, slimes and mucus.

All living organisms produce some form of slime-like substance, generally composed of similar polymers, which include extracellular DNA (eDNA), polysaccharides, proteins and lipids. These polymers control the adhesion and the mechanical properties of the slime, which are then tuned to the specific needs of the producing organism. A comprehensive study of biological slimes across kingdoms is rarely carried out but is needed to provide a deeper understanding of the general principles and concepts governing this class of substances.

Microscopic organisms such as bacteria, fungi and archaea are prime examples of slime producers, as they normally form biofilms. A biofilm is a self-organized and dynamic assembly of surface-bound cells, forming resilient, flexible and heterogeneous communities. Biofilms are composed of extracellular polymeric substances (EPSs). These form a matrix which first and foremost provides adhesion, whereby cells are kept attached to a surface and to each other, and within which nutrients are concentrated, allowing the community to thrive [3]. Cell proximity increases intercellular interactions in which horizontal gene transfer and quorum sensing enable the rapid transmission of information [4]. The EPSs also surround the microorganisms, protecting them against desiccation, UV damage, predation, host immune defences, and extremes of temperature, pH, salinity and pollutants, allowing them to thrive in a wide variety of habitats [3]. The EPSs also provide a scaffold within which cells can respond as a multi-cellular unit to changing environmental conditions [3].

Biofilms are highly heterogeneous and as such the adhesive properties of their components varies considerably. Firstly, a biofilm is influenced by the fluid mechanics of its environment. Weakly adhered microorganisms either can succeed because they are able to relocate to richer habitats in times of nutrient scarcity, or are outcompeted by strongly attached cells when nutrients are non-limiting [5]. Heterogeneity is thus the key to the continued survival of cells within biofilms. Secondly, heterogeneity can also be influenced by contact with a host: for example, when bacteria colonize the human epithelium, this modulates the secretion of adhesive compounds (e.g. glycans and IgA), which either select for the propagation of a genotype or promote the diversity of commensal microbes [5]. Bacterial adhesins have been found to rewire host signalling pathways and this can prevent detachment, promote invasion and fine-tune inflammatory responses of the immune system by targeting complex receptor systems. Lastly, cells themselves are heterogeneous. They possess patchy adhesive contact points in their membranes owing to an asymmetric production of adhesive polymers [6]. The asymmetry of adhesive contacts around the cell membranes of Pseudomonas aeruginosa and Escherichia coli, for example, balances the membrane tension, which in turn, modulates the cellular orientation during cell division and controls the morphogenesis patterns of the colony [7]. Thus, matrix polymers play a role in biofilm heterogeneity, enabling bacterial proliferation in many complex situations, which underpins the evolution of microbial diversity. All of this is done using a blend of polymers that act in synergy to make microbial life the most successful and widespread on Earth.

Bacteria are generally the most studied microorganisms since their biofilms have the most deleterious effects in medicine and industry. They possess a superb extracellular matrix composed mainly of eDNA, polysaccharides and proteins, which act in synergy to produce a recalcitrant matrix with adhesive, protective and architectural roles [3].

The adhesive polymers create a firm attachment to surfaces and allow colony formation, ensuring the effective transmission of information. Among the adhesive polymers, we can count eDNA, as it is a key component in the establishment of the biofilm of many species. Long strands of eDNA can bridge the gap between cells and can cross electrostatic barriers [8]. Aggregative polysaccharides, such as PIA/PNAG (polysaccharide intercellular adhesin/poly-N-acetyl-glucosamine) in staphylococci and Psl (polysaccharide synthesis locus) and Pel (pellicle polysaccharide) in P. aeruginosa [9] are also involved in the initial establishment of the biofilm [9]. Likewise, biofilm matrix proteins play a key role in initial biofilm formation, as mutants deficient in these proteins have impaired attachment to surfaces. The polysaccharide-binding proteins of P. aeruginosa are well studied: LecA binds to N-acetyl-d-galactosamine and glucose and is essential for biofilm formation on hydrophobic surfaces, LecB binds primarily to fucose and is essential in biofilm formation on hydrophilic surfaces, and CdrA tethers the Psl polysaccharide to strengthen the biofilm matrix [10].

A second major role for EPSs is protection. Protective polymers prevent phagocytosis, desiccation and UV damage and reduce the effects of antibiotics and biocides. Examples of these polymers include alginate in P. aeruginosa, Vibrio cholerae biofilm exopolysaccharide, and cellulose in E. coli [11]. They form hydrogels that retain a large amount of water, which is the main component (up to 97%) of the EPS matrix. eDNA also contributes to cation chelation, thus sequestering antibiotics [3]. In biofilms of the Gram-positive Bacillus subtilis, a surface layer protein BslA, in conjunction with polysaccharides, forms a protective hydrophobic layer [10].

Lastly, biopolymers also play important roles in the architecture of biofilms. Polysaccharides, eDNA and proteins act in synergy to form the scaffold of the biofilm, and the cooperativity of EPSs has been evidenced by recent publications (figure 1). Synergy between proteins and polysaccharides is seen in the interactions between fimbrial or curli proteins and colanic acid or cellulose in enterobacteria [12,13]. Polysaccharides also interact with eDNA by forming fibrils that join the internal structure of the biofilm, as observed in P. aeruginosa with Psl [14], in Vibrio fischeri with cellulose [15] and in Staphylococcus epidermidis with PNAG [16]. The interaction between proteins and eDNA is another clear synergy that contributes to the architecture of the biofilms; these form a mesh in which proteins (e.g. histone-like proteins, integrated host factor, pili proteins and β-toxin [17,18]) are critically positioned at junctions, binding eDNA in a lattice form, maintaining the structural integrity and strength of the biofilm [19]. The tandem contribution to the biofilm scaffold between proteins and eDNA has been observed for Haemophilus influenzae [20], Staphylococcus aureus, S. epidermidis, uropathogenic E. coli [21], P. aeruginosa, Moraxella catarrhalis, Streptococcus pneumoniae and Streptococcus mutans [22]. The interaction between cell-surface proteins and eDNA has also been observed in Neisseria meningitidis [23], where the interaction of the proteins NhbA and IgA protease with eDNA is regulated by the expression of the protein NalP; when NalP is expressed, positively charged polypeptides are produced, blocking the interactions of eDNA, collapsing the biofilm. Taking into account the reported synergistic roles between eDNA and polysaccharides and proteins, it is becoming apparent that eDNA may well be a centrally important component that maintains the architecture of biofilms [8].

Figure 1.

Synergy between the three most important polymer types in bacterial EPS [10,12–23]. (Online version in colour.)

The use of specific biopolymers for bioadhesion appears to have evolved early in the microbial world and is common to bacteria, archaea and fungi. Archaea also form biofilms, often in extreme habitats (pH, temperature, toxic chemicals). They attach to surfaces using structures such as hami (three-pronged pilus like fibres), cannulae (a network of extracellular tubules) and archaella (archaeal flagella) that allow them to attach irreversibly to surfaces. Archaea also produce EPS, rich in extracellular glycans, glycoproteins and amyloid proteins [24]. Archaea inhabit the human body, on the skin and in the intestinal, oral and vaginal mucosae, and have syntrophic relations with bacteria, forming a complex community. Although they do not cause disease directly, their presence affects bacterial populations and they have been associated with periodontitis and irritable bowel syndrome [24].

Microscopic fungi form biofilms and are common sources of nosocomial infections. Fungi adhere strongly to host tissues before infection and to other surfaces. They produce hyphal filaments during growth to absorb nutrients and often possess melanized cell walls that increase hydrophobicity and protect them against environmental stresses (e.g. UV light, oxidizing agents). The EPS of fungi is heavily reliant on glucans such as glycosaminoglycan, galactomannan and 1,3-glucan. EPS-associated proteins in fungi are very diverse, with 458 being identified, for example, in the medically important Candida albicans. Hydrophobins are proteins that allow fungi to attach to the surfaces of hydrophobic leaves and subsequently infect plants [25]. Fungal EPS is thus central to the success of this kingdom; it underpins their ability to attach to almost any surface and establish symbiotic relationships with algae, cyanobacteria and bacteria, including nitrogen-fixing rhizobia [26]. Fungal EPS also allows symbiosis with plants (mycorrhiza), and has important effects on soil, contributing to clumping of soil particles, which is essential to soil health and food production [26].

2. Similarities between bacterial EPS and other colloidal systems

Chemical similarities due to functional convergence can be observed when comparing microbial biofilms and other colloidal and adhesive systems in biology (table 1). In the ocean, colloids aggregate and form transparent exopolymeric particles. These are mucoidal particles made of polymers from phytoplankton, zooplankton, bacteria and archaea and they can further aggregate to form marine snow which falls to the sea floor [27]. Larger organisms such as giant mesopelagic larvaceans also contribute to this process by forming outstanding mucoidal filter-feeding structures that eventually sink to the bottom [28]. Marine microorganisms produce EPS with high levels of uronic acids and other polyanions and amphiphilic glycoproteins [29]. Other systems that are chemically similar to bacterial EPS are biological adhesives. Marine bioadhesives include extracellular polymers produced by echinoderms, molluscs, barnacles and other animals. They are characterized by their high adhesive strength and their capacity to sustain large deformation. They form remarkable glues that function on a variety of often wet substrates, at different salinities and temperatures. Bioadhesives are generally protein-rich and rely on chemical cross-linking to attach to surfaces. Algae also produce bioadhesives, often referred to as mucilage. Microalgae form assemblies in the form of flocs, sludge, mats and biofilms and are major contributors to underwater fouling. Microalgal adhesion also relies on β-1,3-glucan or galactan, polysaccharides with mannose–sugar complexes that bind heavy metals, proteins, eDNA, lipids, glycoproteins and uronic acids in ways that can be chemically similar to the adhesion of bacteria and fungi [29].

Table 1.

Convergent chemical evolution of bioadhesion strategies in nature. Observing these similarities is confounded by similar substances being referred to by a range of different terminologies.

terminology	organism	functions		composition
extracellular polymeric substances	bacteria [12]	protection against desiccation, UV damage, predation, host defences, antibiotics, pollutants and extreme pH, temperature or salinity	adhesion to surface and three-dimensional architecture; colony formation; transmission of information; genetic exchange	eDNA, polysaccharides, proteins
	archaea [24]	protection against extreme pH, temperature or salinity	establish symbiotic relations with bacteria	eDNA, glycans, glycoproteins, amyloid proteins
	e.g. Haloferax spp. [24]	protection in highly saline environments	attachment in the form of mats and dense pillar structures	eDNA, EPS nanofibres, sulfated polysaccharide with mannose and N-acetyl-d-glucosaminuronic acid units
	fungi [26]	symbiosis and pathogenesis with bacteria, nitrogen-fixing rhizobia and plants	weathering rocks; clumping soil particles	eDNA, melanins to increase cell hydrophobicity, hydrophobins to attach to leaves, glucans
	e.g. C. albicans [25]	interaction with bacteria in polymicrobial diseases	multi-drug resistance	eDNA, mannan–glucan complex, β-1,3-glucan glycoproteins, polysaccharides, 458 different proteins, lipids
marine gels (marine snow, transparent exopolymeric particles)	gel-forming organisms [27]	carbon flux contribution; mediate fate of heavy metals and trace nutrients	emulsification of hydrocarbons	polysaccharides with high levels of uronic acids and polyanions, amphiphilic glycoproteins
	e.g. giant mesopelagic larvaceans [28]	filter feeding	vertical nutrient flux to the seabed	glycoproteins
mucilage	algae [29]	fighting drag and lift; algal mat formation	binding heavy metals	β-1,3-glucan or galactan, polysaccharides with mannose–sugar complexes, eDNA, proteins, lipids, glycolipids
	e.g. diatom Craspedostaouros australis [29]	colonization of underwater surfaces	protection against variable and challenging conditions in the sea; excellent adhesives for high-energy marine environments [30]	anionic polysaccharides with heterogeneous monosaccharides with sulfated esters and uronic acids (similar to marine bacteria) and proteins
bioadhesives	adhesive-producing organisms [29]	high adhesive strength	resistance to deformation	proteins and carbohydrates, proteins with small side chains and polar amino acids
	echinoderms (sea stars) [29]	quick attachment and detachment; building tubes or burrows	handling of food; attachment for extended periods	non-permanent adhesion made by a mixture of proteins and polysaccharides, with acids, sulfated sugars and glycoproteins
	molluscs (mussel) [29]	cross-linking with surfaces	permanent adhesion to withstand large currents	20 byssal proteins with varying degrees of DOPA
	barnacles [29]	cement bioadhesive	permanent adhesion to withstand large currents	90% proteins
mucus	mucus-producing organisms [31]	barrier against microorganisms	lubrication	glycoproteins
	molluscs (snails) [32]	trail and adhesive mucus; locomotion; feeding; reproduction	desiccation protection, antibacterial protection, defensive substances versus predators	alternate between proteins that trigger gelling and stiffening, cross-linking with gel proteins
	cnidarians [31]	allows exchange of oxygen, CO2, nutrients, metabolites; nutrient capture	immobilization of metabolites and enzymes; pollutant capture	glycoproteins, O-linked glycans covalently linked to the polypeptide backbone, phospholipids and glycolipids
	humans [31]	offensive and defensive mechanisms versus invading microorganisms: particle traps: lubrication	desiccation protection; size exclusion barrier to molecules; particle transport; self-cleaning	glycoproteins, O-linked glycans covalently linked to the polypeptide backbone, phospholipids and glycolipids

Bacterial EPS is also chemically like mucus. Mucus is a hydrated matrix of polymers, which can often be hundreds of micrometres in length. It is made primarily of heavily glycosylated glycoproteins in an extended conformation that allows them to retain large amounts of water and imparts viscoelastic characteristics. It is an interface that covers the tissues of metazoans. In the Ctenophora (comb jellies) and cnidarians (corals, jellyfish, anemones), mucus is primarily used for trapping food and cleaning, by ciliary-mucus-driven entrainment. The use of mucus to effectively exclude bacteria from their own tissues was a major evolutionary event first seen in the Cnidaria [31]. Mammals also have mucin glycoproteins with sequences shared with the cnidarians, and mammalian mucus-producing systems are similar. Other species such as gastropods can also tailor the viscoelastic properties of their mucus depending on their needs. Snails and slugs produce trail mucus to aid in their locomotion and protect their underbellies from microbial infection. Snails can alter their mucus composition by secreting small glue-like proteins that mix with larger proteins in their mucus to stiffen the resulting gel. This allows snails to stick to surfaces and protects against dehydration [32]. Mucus preserves tissue integrity, is a key part of our immune system and acts as a barrier to pollutants and microorganisms, preventing disease. Several species of bacteria can penetrate mucus and colonize tissues. Understanding how they do this, and how mucus successfully prevents penetration of most bacteria, might lead to better anti-infective therapies. Many of these unique properties of biofilms and mucus are due to their biophysical properties.

3. Physical characterization of polymeric systems used in biological adhesion

Early investigations into bacterial adhesion were purely qualitative, as there was no established technique that could quantitatively measure the force of adhesion. These techniques involved counting the number of cells attached to a given area of a surface and inferring that a larger number of attached cells indicated stronger adhesion. From this assumption, the relative adhesive capacities of different surfaces could be implied. The simplest technique used to count cells on a surface is optical microscopy. Owing to the resolution limit of this being the wavelength of light (400–700 nm for the visible spectrum), there are limited possibilities for observing any details at the cellular level. As researchers began to require greater resolution, new techniques were developed. These include super-resolution microscopy, a group of techniques that probe beyond the resolution limit of light and view much smaller objects, for example, structured illumination microscopy (SIM) [33] and stimulated emission depletion (STED) [34] microscopy. Moving away from the use of light, cell count studies have also been performed using scanning electron microscopy (SEM) [35]. This technique uses a beam of electrons instead of a beam of light to image the surface. As the wavelength of electrons is of the order of a few nanometres, rather than a few hundred nanometres, it is possible to achieve much greater resolution. The complications with SEM are that the measurements must be performed in vacuum and the surface must be conductive, and to achieve this with biomaterials the surface is coated with a thin layer of a conductive material. This is clearly not ideal for any study in which the adhesion of a cell to a surface may be affected by this sample preparation. A high-resolution device that allows cells to be counted on a surface while in buffer, without any coating, is the atomic force microscope (AFM) [35]. In this case, a sharp probe is raster scanned over the surface, building up an image of the sample with resolution determined by the dimensions of the probe, which can be as small as a few nanometres. A key issue identified with these techniques is that they count all adhered cells—regardless of viability. There are many reasons that only viable cells should be counted in studies on bacterial adhesion, so new techniques were required to distinguish between live and dead cells. The key method for this is using fluorescence. For example, BacLight Live–Dead stain will colour viable cells bright green and dead cells red [36]. A fluorescence microscope will then allow the user to count only live cells, giving a much better representation of the number of cells on the surface than the previous counting techniques. A further improvement on these techniques is to use a flow chamber to remove loosely adhered cells from the surface in question, leaving behind only the well-adhered cells. Several types of flow chamber are used, such as parallel plate, radial and rotating flow chambers. Counting the number of cells using one of the aforementioned techniques after the loosely adhered cells have been removed provides a much more accurate representation of adhesion onto the surface under investigation.

All of the techniques discussed so far have been qualitative, where adhesion between cells and surfaces can be ranked and compared but the specific adhesion between one cell and one surface cannot be calculated. The most common way of quantifying the adhesion of slime-producing organisms is to use force spectroscopy with the AFM [30,37]. With the AFM, a probe is brought into contact with a surface and then separated, and the deflection of the probe is monitored with sub-nanometre resolution throughout the whole probe–sample interaction cycle. By knowing the properties of the probe, it is then possible to measure the interaction forces on both approach and retraction, and therefore accurately calculate the adhesion between the probe and the surface during separation (figure 2a,b). As AFM hardware and software have developed, new techniques have been brought in to improve on force spectroscopy. As well as being able to accurately measure the interaction force between the probe and a specific point on the sample, it is possible to move the probe accurately in the x and y directions at relatively high speed so that a large area of the sample can be covered easily, measuring the probe–sample adhesion at each point on the surface. This creates a quantifiable two-dimensional image, over the course of several minutes, of the probe–surface adhesion. This technique is known as force mapping. In the last few years, this has been further improved by two leading AFM manufacturers to bring the time for a complete force image down to a couple of seconds, using techniques known as PeakForce Tapping (developed by Bruker) and quantitative imaging (QI; developed by JPK) [38]. Furthermore, different aspects of adhesion can be measured by using the AFM. The AFM probe can have a point of contact of just a few nanometres and can be coated with a variety of materials of interest. By securing a bacterium to the surface via mechanical or chemical immobilization, and attaching a molecule of interest to the AFM probe, a map of the bacterial surface adhesion is easily created. Examples of AFM probes modified in this way include hydrophobic/hydrophilic coatings and coating with proteins corresponding to bacterial surface proteins [39]. This allows high-resolution maps of the bacterial surface to be created, with bacterial surface properties not always as homogeneous as might have been expected. In addition, several studies have been performed where bacterial cells themselves have been attached to the probe, allowing the study of the interaction of the whole bacterial cell either to a surface of choice, or to a second bacterial cell immobilized onto the surface as mentioned previously, or even to larger-scale biofilms and tissues.

Figure 2.

(a) Schematic of tip of a cantilever mapping a surface. (b) Plot of tip deflection versus the cantilever position. (c) Schematic of adhesive strength testing device. (d) Plot of forces experienced during adhesive strength testing. (e) Schematic of a rheometer and the different geometries. (f) Plots from various tests that can be performed on a rheometer. (g) Schematic showing rheometer output during a small/large amplitude oscillatory shear test. (Online version in colour.)

Adhesion tests can also be performed at a macroscopic level using tack adhesion measurements [40]. These involve bringing a rigid probe into contact with the polymer gel and then holding the probe in contact for a defined period. The probe is then separated and the normal force to pull the adhesive apart is measured as shown in figure 2c. The plot in 2d shows the peak adhesive force, the strain required before failure (or strain to failure), and the area under the curve, which represents the total amount of work done during the process. These parameters depend on the roughness of the surface, the rate of pulling and the viscoelastic and molecular properties of the gel, as well as any debonding mechanisms in play. The peak force seen in the plots is a result of the formation of cavitation bubbles and this causes the formation of thin slender structures called fibrils that strain-harden with applied force. The fibrils finally fracture, which results in debonding of the material from the bottom plate. These techniques and approaches to characterize adhesion forces are essential to a better understanding of the mechanisms of action of these biological substances.

4. Rheology

Certain EPSs, in their capacity as bioadhesives and structural elements of biofilms, are known to withstand elevated mechanical stresses and operate under extreme temperatures. These polymers can stretch many times their original length to fill cracks and voids and hold structures together. Owing to these outstanding characteristics, these polymers have inspired the creation of adhesives with tuneable characteristics and thus it is vital to measure their mechanical performance. The study of flow and deformation of materials is known as rheology and it provides insights into the relationship between structure and functionality of materials.

The rheological characteristics of materials are commonly studied using a rheometer. A rheometer applies oscillatory shear strain on the sample and records the response (shear stress) of the sample (figure 2e). By recording the resulting strain and stress waveforms, a wide array of parameters of the material, like the elastic modulus (measure of elasticity) and viscous modulus (measure of fluidity), can be obtained [41]. Four measurement approaches that are commonly used for biological materials are: sol–gel transition, amplitude sweep, frequency sweep and multiple creep tests with recovery. Firstly, the sol–gel transition is crucial for the study of self-healing systems such as gastric mucins, which line and protect stomach epithelium. This phenomenon refers to the transition from a liquid-like system to a gel-like network or vice versa and can be triggered by a change of pH, for example, in the case of gastric mucins. The transition phenomena can be studied in a rheometer with a concentric cylinder geometry (figure 2e), and recording the temporal variation in elastic and viscous moduli of the polymer system. The magnitude of the elastic and viscous moduli, which are representative of the rigidity and fluidity of polymeric systems like biofilms, can be probed using amplitude and frequency sweeps in a rheometer. Figure 2f shows an amplitude sweep with a typical plot for biofilms grown on agar plates where the magnitude of the elastic modulus is greater than the viscous modulus. In the linear viscoelastic regime (LVER), the magnitudes of the moduli do not exhibit significant variations; beyond the LVER, the elastic modulus decreases and the viscous modulus shows a hump that is characteristic of many colloidal systems [42]. The frequency sweep (figure 2f) is useful for characterizing the relaxation time (w_r) of the polymer, which denotes the transition from a fluid-like behaviour to solid-like behaviour. Both amplitude and frequency sweeps have been used to characterize the role of polysaccharides like Pel, Psl and alginate in modulating viscoelastic behaviour of P. aeruginosa biofilms in cystic fibrosis patients [43]. Finally, creep tests (figure 2f) study the ability of a material to recover after periodic loading and unloading cycles and can also be used to determine the yield point of the material, at which it stops behaving like a solid.

Commonly, studies in rheology involve experiments with a small applied strain; however, biological materials in nature can be subjected to very large strains and can behave unexpectedly by stiffening or softening. For example, during slug motility, there is a large deformation of the underlying mucus layer which causes stiffening of the polymer network. Large amplitude oscillatory shear (LAOS) experiments apply a large strain on the materials and allow a greater understanding of nonlinear responses which can be more representative of biological systems in nature (figure 2g) [42]. Using LAOS experiments, the architecture of polymers (i.e. branched or unbranched) as well as yielding, reformation and cross-linking of biopolymers can be investigated. This allows one to gain a much broader perspective of the behaviours exhibited by the natural polymeric systems, which are usually a complex mixture of multiple polymers with varying degrees of cross-linking.

In summary, the remarkable properties of biopolymers used across kingdoms in biological adhesion are beginning to be more fully understood, but a greater use of quantitative biophysical techniques is required.

Data accessibility

This article has no additional data.

Authors' contributions

A.L.M.-G., R.G.B. and S.J. contributed equally to the writing of the manuscript under the supervision of J.G.B. All authors contributed to the conception, design and structure of the manuscript, as well as its critical revision.

Competing interests

We declare we have no competing interests.

Funding

J.G.B. thanks the NERC and the BBSRC for funding. R.G.B. acknowledges the Wellcome Trust and the Engineering and Physical Sciences Research Council (UK) (EPSRC) for funding. S.J. acknowledges funding from the EPSRC through award number EP/K039083/1 to Newcastle University.