Cell-Cell Mating Interactions: Overview and Potential of Single-Cell Force Spectroscopy.

It is an understatement that mating and DNA transfer are key events for living organisms. Among the traits needed to facilitate mating, cell adhesion between gametes is a universal requirement. Thus, there should be specific properties for the adhesion proteins involved in mating. Biochemical and biophysical studies have revealed structural information about mating adhesins, as well as their specificities and affinities, leading to some ideas about these specialized adhesion proteins. Recently, single-cell force spectroscopy (SCFS) has added important findings. In SCFS, mating cells are brought into contact in an atomic force microscope (AFM), and the adhesive forces are monitored through the course of mating. The results have shown some remarkable characteristics of mating adhesins and add knowledge about the design and evolution of mating adhesins.

Fluidic Force Microscopy Demonstrates That Homophilic Adhesion by Candida albicans Als Proteins Is Mediated by Amyloid Bonds between Cells.

The fungal pathogen Candida albicans frequently forms drug-resistant biofilms in hospital settings and in chronic disease patients. Cell adhesion and biofilm formation involve a family of cell surface Als (agglutinin-like sequence) proteins. It is now well documented that amyloid-like clusters of laterally arranged Als proteins activate cell-cell adhesion under mechanical stress, but whether amyloid-like bonds form between aggregating cells is not known. To address this issue, we measure the forces driving Als5-mediated intercellular adhesion using an innovative fluidic force microscopy platform. Strong cell-cell adhesion is dependent on expression of amyloid-forming Als5 at high cell surface density and is inhibited by a short antiamyloid peptide. Furthermore, there is greatly attenuated binding between cells expressing amyloid-forming Als5 and cells with a nonamyloid form of Als5. Thus, homophilic bonding between Als5 proteins on adhering cells is the major mode of fungal aggregation, rather than protein-ligand interactions. These results point to a model whereby amyloid-like ß-sheet interactions play a dual role in cell-cell adhesion, that is, in formation of adhesin nanoclusters ( cis-interactions) and in homophilic bonding between amyloid sequences on opposing cells ( trans-interactions). Because potential amyloid-forming sequences are found in many microbial adhesins, we speculate that this novel mechanism of amyloid-based homophilic adhesion might be widespread and could represent an interesting target for treating biofilm-associated infections.

Garcinia xanthochymus Benzophenones Promote Hyphal Apoptosis and Potentiate Activity of Fluconazole against Candida albicans Biofilms.

Xanthochymol and garcinol, isoprenylated benzophenones purified from Garcinia xanthochymus fruits, showed multiple activities against Candida albicans biofilms. Both compounds effectively prevented emergence of fungal germ tubes and were also cytostatic, with MICs of 1 to 3 µM. The compounds therefore inhibited development of hyphae and subsequent biofilm maturation. Xanthochymol treatment of developing and mature biofilms induced cell death. In early biofilm development, killing had the characteristics of apoptosis, including externalization of phosphatidyl serine and DNA fragmentation, as evidenced by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) fluorescence. These activities resulted in failure of biofilm maturation and hyphal death in mature biofilms. In mature biofilms, xanthochymol and garcinol caused the death of biofilm hyphae, with 50% effective concentrations (EC50s) of 30 to 50 µM. Additionally, xanthochymol-mediated killing was complementary with fluconazole against mature biofilms, reducing the fluconazole EC50 from >1,024 µg/ml to 13 µg/ml. Therefore, xanthochymol has potential as an adjuvant for antifungal treatments as well as in studies of fungal apoptosis.

Role of force-sensitive amyloid-like interactions in fungal catch bonding and biofilms.

The Candida albicans Als adhesin Als5p has an amyloid-forming sequence that is required for aggregation and formation of model biofilms on polystyrene. Because amyloid formation can be triggered by force, we investigated whether laminar flow could activate amyloid formation and increase binding to surfaces. Shearing Saccharomyces cerevisiae cells expressing Als5p or C. albicans at 0.8 dyne/cm(2) increased the quantity and strength of cell-to-surface and cell-to-cell binding compared to that at 0.02 dyne/cm(2). Thioflavin T fluorescence showed that the laminar flow also induced adhesin aggregation into surface amyloid nanodomains in Als5p-expressing cells. Inhibitory concentrations of the amyloid dyes thioflavin S and Congo red or a sequence-specific anti-amyloid peptide decreased binding and biofilm formation under flow. Shear-induced binding also led to formation of robust biofilms. There was less shear-activated increase in adhesion, thioflavin fluorescence, and biofilm formation in cells expressing the amyloid-impaired V326N-substituted Als5p. Similarly, S. cerevisiae cells expressing Flo1p or Flo11p flocculins also showed shear-dependent binding, amyloid formation, biofilm formation, and inhibition by anti-amyloid compounds. Together, these results show that laminar flow activated amyloid formation and led to enhanced adhesion of yeast cells to surfaces and to biofilm formation.

Atomic force microscopy - looking at mechanosensors on the cell surface.

Living cells use cell surface proteins, such as mechanosensors, to constantly sense and respond to their environment. However, the way in which these proteins respond to mechanical stimuli and assemble into large complexes remains poorly understood at the molecular level. In the past years, atomic force microscopy (AFM) has revolutionized the way in which biologists analyze cell surface proteins to molecular resolution. In this Commentary, we discuss how the powerful set of advanced AFM techniques (e.g. live-cell imaging and single-molecule manipulation) can be integrated with the modern tools of molecular genetics (i.e. protein design) to study the localization and molecular elasticity of individual mechanosensors on the surface of living cells. Although we emphasize recent studies on cell surface proteins from yeasts, the techniques described are applicable to surface proteins from virtually all organisms, from bacteria to human cells.

Does Candida albicans Als5p amyloid play a role in commensalism in Caenorhabditis elegans?

Candida albicans, a dimorphic fungus and an opportunistic pathogen, possesses a myriad of adherence factors, including members of the agglutinin-like sequence (Als) family of mannoproteins. The adhesin Als5p mediates adhesion to many substrates and is upregulated during commensal interactions but is downregulated during active C. albicans infections. An amyloid-forming core sequence at residues 325 to 331 is important for Als5p function, because a single-amino-acid substitution at position 326 (V326N) greatly reduces Als5p-mediated adherence. We evaluated the role of Als5p in host-microbe interactions by using Caenorhabditis elegans nematodes as a host model and feeding them Saccharomyces cerevisiae expressing Als5p on the surface. Als5p-expressing yeast had 8.5- and 3.5-fold-increased intestinal accumulation rates compared to Als5p-nonexpressing S. cerevisiae or yeast expressing amyloid-deficient Als5p(V326N), respectively. Surprisingly, this accumulation delayed S. cerevisiae-induced killing of C. elegans. The median survival time was nearly twice as long as that of nematodes fed nonexpressing or non-amyloid-forming Als5p(V326N)-expressing S. cerevisiae. Treatment with the amyloid-inhibiting dye Congo red or repression of Als5p expression abrogated the protective effect of Als5p. Furthermore, Als5p had no effect on oocyte quantity or quality, since nematodes fed either empty vector (EV)- or Als5p(V326N)-expressing S. cerevisiae had similar egg-laying and egg-hatching rates. This study is the first, to our knowledge, to show that expression of an amyloid-forming protein can attenuate pathogenicity in C. elegans.

Quantifying the forces driving cell-cell adhesion in a fungal pathogen.

Owing to its ability to form biofilms on implanted medical devices, the fungal pathogen Candida albicans causes frequent infections in humans. A hallmark of C. albicans biofilms is the presence of two types of cells, budding yeast cells and growing hyphae, which are bound together and embedded in extracellular matrix material. Although cell-cell adhesion is critical to biofilm formation, architecture, and cohesion, we know little about the fundamental forces behind this interaction. Here, we use single-cell force spectroscopy to quantify the forces engaged in yeast-hyphae adhesion, focusing on the role of Als (agglutinin-like sequence) proteins as prototypes of cell adhesion molecules. We show that adhesion between individual yeast and hyphal cells involves strong, short-range cohesive interactions (1.1 ± 0.2 nN; 86 ± 33 nm) and weak, long-range tether interactions (0.4 ± 0.2 nN; 234 ± 81 nm). Control experiments demonstrate that these interactions originate from cell surface proteins that are specific to C. albicans. Using mutant strains deficient for Als expression, we find that Als3 proteins, primarily expressed on the germ tube, play a key role in establishing strong cohesive adhesion. We suggest a model in which cohesive adhesion during biofilm formation originates from tight hydrophobic interactions between Als tandem repeat domains on adjacent cells. When subjected to force, the two interacting cell surfaces detach, but the cell bodies remain tethered through macromolecular extensions. Our results represent the first direct, noninvasive measurement of adhesion forces between interacting fungal cells and provide novel insights into the molecular origin of the cohesive strength of fungal biofilms.

Force-induced formation and propagation of adhesion nanodomains in living fungal cells.

Understanding how cell adhesion proteins form adhesion domains is a key challenge in cell biology. Here, we use single-molecule atomic force microscopy (AFM) to demonstrate the force-induced formation and propagation of adhesion nanodomains in living fungal cells, focusing on the covalently anchored cell-wall protein Als5p from Candida albicans. We show that pulling on single adhesins with AFM tips terminated with specific antibodies triggers the formation of adhesion domains of 100-500 nm and that the force-induced nanodomains propagate over the entire cell surface. Control experiments (with cells lacking Als5p, single-site mutation in the protein, bare tips, and tips modified with irrelevant antibodies) demonstrate that Als5p nanodomains result from protein redistribution triggered by force-induced conformational changes in the initially probed proteins, rather than from nonspecific cell-wall perturbations. Als5p remodeling is independent of cellular metabolic activity because heat-killed cells show the same behavior as live cells. Using AFM and fluorescence microscopy, we also find that nanodomains are formed within ∼30 min and migrate at a speed of ∼20 nm·min(-1), indicating that domain formation and propagation are slow, time-dependent processes. These results demonstrate that mechanical stimuli can trigger adhesion nanodomains in fungal cells and suggest that the force-induced clustering of adhesins may be a mechanism for activating cell adhesion.

New features of invasive candidiasis in humans: amyloid formation by fungi and deposition of serum amyloid P component by the host.

BACKGROUND: Invasive candidiasis occurs in the gastrointestinal tract, especially in neutropenic patients. We were interested in determining whether invasive fungi formed amyloid in humans as they are known to do in vitro. We also sought to characterize the consequence(s) of such amyloid formation. METHODS: Tissue from 25 autopsy patients with invasive candidiasis of the gastrointestinal tract was stained with amyloidophilic dyes and for the presence of serum amyloid P component (SAP). Confirmation of the interaction of SAP and Candida was demonstrated using Candida albicans and mutants for amyloid formation. RESULTS: Amyloid was present on the cellular surface of fungi invading gut tissue. Moreover, SAP bound to the fungal cell walls, confirming the presence of amyloid. In vitro observations showed SAP bound avidly to fungi when amyloid formed in fungal cell walls. An unexpected result was the lack of host neutrophils in response to the invading fungi, not only in neutropenic patients but also in patients with normal or increased white blood counts. CONCLUSIONS: We report the first demonstration of functional fungal amyloid in human tissue and the binding of SAP to invading fungi. It is postulated that fungal amyloid, SAP, or a complex of the proteins may inhibit the neutrophil response.

Sticking to the Subject: Multifunctionality in Microbial Adhesins.

Bacterial and fungal adhesins mediate microbial aggregation, biofilm formation, and adhesion to host. We divide these proteins into two major classes: professional adhesins and moonlighting adhesins that have a non-adhesive activity that is evolutionarily conserved. A fundamental difference between the two classes is the dissociation rate. Whereas moonlighters, including cytoplasmic enzymes and chaperones, can bind with high affinity, they usually dissociate quickly. Professional adhesins often have unusually long dissociation rates: minutes or hours. Each adhesin has at least three activities: cell surface association, binding to a ligand or adhesive partner protein, and as a microbial surface pattern for host recognition. We briefly discuss Bacillus subtilis TasA, pilin adhesins, gram positive MSCRAMMs, and yeast mating adhesins, lectins and flocculins, and Candida Awp and Als families. For these professional adhesins, multiple activities include binding to diverse ligands and binding partners, assembly into molecular complexes, maintenance of cell wall integrity, signaling for cellular differentiation in biofilms and in mating, surface amyloid formation, and anchorage of moonlighting adhesins. We summarize the structural features that lead to these diverse activities. We conclude that adhesins resemble other proteins with multiple activities, but they have unique structural features to facilitate multifunctionality.

Strain and temperature dependent aggregation of Candida auris is attenuated by inhibition of surface amyloid proteins.

Candida auris is a multi-drug resistant human fungal pathogen that has become a global threat to human health due to its drug resistant phenotype, persistence in the hospital environment and propensity for patient to patient spread. Isolates display variable aggregation that may affect the relative virulence of strains. Therefore, dissection of this phenotype has gained substantial interest in recent years. We studied eight clinical isolates from four different clades (I-IV); four of which had a strongly aggregating phenotype and four of which did not. Genome analysis identified polymorphisms associated with loss of cell surface proteins were enriched in weakly-aggregating strains. Additionally, we identified down-regulation of chitin synthase genes involved in the synthesis of the chitinous septum. Characterisation of the cells revealed no ultrastructural defects in cytokinesis or cell separation in aggregating isolates. Strongly and weakly aggregating strains did not differ in net surface charge or in cell surface hydrophobicity. The capacity for aggregation and for adhesion to polystyrene microspheres were also not correlated. However, aggregation and extracellular matrix formation were all increased at higher growth temperatures, and treatment with the amyloid protein inhibitor Thioflavin-T markedly attenuated aggregation. Genome analysis further indicated strain specific differences in the genome content of GPI-anchored proteins including those encoding genes with the potential to form amyloid proteins. Collectively our data suggests that aggregation is a complex strain and temperature dependent phenomenon that may be linked in part to the ability to form extracellular matrix and cell surface amyloids.

Accelerated and adaptive evolution of yeast sexual adhesins.

There is a recent emergence of interest in the genes involved in gametic recognition as drivers of reproductive isolation. The recent population genomic sequencing of two species of sexually primitive yeasts (Liti G, Carter DM, Moses AM, Warringer J, Parts L, James SA, Davey RP, Roberts IN, Burt A, Koufopanou V et al. [23 co-authors]. 2009. Population genomics of domestic and wild yeasts. Nature 458:337-341.) has provided data for systematic study of the roles these genes play in the early evolution of sex and speciation. Here, we discovered that among genes encoding cell surface proteins, the sexual adhesin genes have evolved significantly more rapidly than others, both within and between Saccharomyces cerevisiae and its closest relative S. paradoxus. This result was supported by analyses using the PAML pairwise model, a modified McDonald-Kreitman test, and the PAML branch model. Moreover, using a combination of a new statistic of neutrality, an information theory-based measure of evolutionary variability, and functional characterization of amino acid changes, we found that a higher proportion of amino acid changes are fixed in the sexual adhesins than in other proteins and a greater proportion of the fixed amino acid changes either between the two species or the two subgroups of S. paradoxus are functionally dissimilar or radically different. These results suggest that the accelerated evolution of sexual adhesin genes may facilitate speciation, or incipient speciation, and promote sexual selection in general.

Blocking Serum Amyloid-P Component from Binding to Macrophages and Augmenting Fungal Functional Amyloid Increases Macrophage Phagocytosis of Candida albicans.

Candida-macrophage interactions are important immune defense responses associated with disseminated and deep-seated candidiasis in humans. Cells of Candida spp. express functional amyloids on their surfaces during the pathogenesis of disseminated candidiasis. These amyloids become decorated with serum amyloid P-component (SAP) that binds to Candida cells and macrophages and downregulates the cellular and cytokine response to the fungi. In this report, further characterization of the interactions of SAP and fungal functional amyloid are demonstrated. Blocking the binding of SAP to macrophage FcγR1 receptors increases phagocytosis of yeast cells; seeding a pro-amyloid-forming peptide on the yeast cell surface also increases phagocytosis of yeasts by macrophages; and, lastly, miridesap, a small palindromic molecule, prevents binding of SAP to yeasts and removes SAP that is bound to C. albicans thus, potentially increasing phagocytosis of yeasts by macrophages. Some, or all, of these interventions may be useful in boosting the host immune response to disseminated candidiasis.

The Paradoxical Effects of Serum Amyloid-P Component on Disseminated Candidiasis.

Serum amyloid P component (SAP) may play an important role in human fungal diseases. SAP binds to functional amyloid on the fungal surface and masks fungi from host immune processes, skewing the macrophage population from the pro-inflammatory M1 to the quiescent M2 type. We assessed the role of SAP in a murine model of disseminated candidiasis. Mice were injected with human SAP subcutaneously (SQ) followed by intravenous injection of Candida albicans. Male, BALBcJ mice were administered 2 mg human SAP or the homologous human pro-inflammatory pentraxin CRP, SQ on day −1 followed by 1 mg on days 0 thru 4; yeast cells were administered intravenously on day 0. Mice not receiving a pentraxin were morbid on day 1, surviving 4−7 days. Mice administered SAP survived longer than mice receiving yeast cells alone (p < 0.022), although all mice died. Mice given CRP died faster than mice receiving yeast cells alone (p < 0.017). Miridesap is a molecule that avidly binds SAP, following which the complex is broken down by the liver. Miridesap administered in the drinking water removed SAP from the serum and yeast cells and significantly prolonged the life of mice (p < 0.020). Some were "cured" of candidiasis. SAP administered early in the septic process provided short-lived benefit to mice, probably by blunting cytokine secretion associated with disseminated candidiasis. The most important finding was that removal of SAP with miridesap led to prolonged survival by removing SAP and preventing its dampening effects on the host immune response.

Structure and Conservation of Amyloid Spines From the Candida albicans Als5 Adhesin.

Candida Als family adhesins mediate adhesion to biological and abiotic substrates, as well as fungal cell aggregation, fungal-bacterial co-aggregation and biofilm formation. The activity of at least two family members, Als5 and Als1, is dependent on amyloid-like protein aggregation that is initiated by shear force. Each Als adhesin has a ∼300-residue N-terminal Ig-like/invasin region. The following 108-residue, low complexity, threonine-rich (T) domain unfolds under shear force to expose a critical amyloid-forming segment 322SNGIVIVATTRTV334 at the interface between the Ig-like/invasin domain 2 and the T domain of Candida albicans Als5. Amyloid prediction programs identified six potential amyloidogenic sequences in the Ig-like/invasin region and three others in the T domain of C. albicans Als5. Peptides derived from four of these sequences formed fibrils that bound thioflavin T, the amyloid indicator dye, and three of these revealed atomic-resolution structures of cross-ß spines. These are the first atomic-level structures for fungal adhesins. One of these segments, from the T domain, revealed kinked ß-sheets, similarly to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked segments) found in human functional amyloids. Based on the cross-ß structures in Als proteins, we use evolutionary arguments to identify functional amyloidogenic sequences in other fungal adhesins, including adhesins from Candida auris. Thus, cross-ß structures are often involved in fungal pathogenesis and potentially in antifungal therapy.

A biochemical guide to yeast adhesins: glycoproteins for social and antisocial occasions.

Fungi are nonmotile eukaryotes that rely on their adhesins for selective interaction with the environment and with other fungal cells. Glycosylphosphatidylinositol (GPI)-cross-linked adhesins have essential roles in mating, colony morphology, host-pathogen interactions, and biofilm formation. We review the structure and binding properties of cell wall-bound adhesins of ascomycetous yeasts and relate them to their effects on cellular interactions, with particular emphasis on the agglutinins and flocculins of Saccharomyces and the Als proteins of Candida. These glycoproteins share common structural motifs tailored to surface activity and biological function. After being secreted to the outer face of the plasma membrane, they are covalently anchored in the wall through modified GPI anchors, with their binding domains elevated beyond the wall surface on highly glycosylated extended stalks. N-terminal globular domains bind peptide or sugar ligands, with between millimolar and nanomolar affinities. These affinities and the high density of adhesins and ligands at the cell surface determine microscopic and macroscopic characteristics of cell-cell associations. Central domains often include Thr-rich tandemly repeated sequences that are highly glycosylated. These domains potentiate cell-to-cell binding, but the molecular mechanism of such an association is not yet clear. These repeats also mediate recombination between repeats and between genes. The high levels of recombination and epigenetic regulation are sources of variation which enable the population to continually exploit new niches and resources.

Structure and function of glycosylated tandem repeats from Candida albicans Als adhesins.

Tandem repeat (TR) regions are common in yeast adhesins, but their structures are unknown, and their activities are poorly understood. TR regions in Candida albicans Als proteins are conserved glycosylated 36-residue sequences with cell-cell aggregation activity (J. M. Rauceo, R. De Armond, H. Otoo, P. C. Kahn, S. A. Klotz, N. K. Gaur, and P. N. Lipke, Eukaryot. Cell 5:1664-1673, 2006). Ab initio modeling with either Rosetta or LINUS generated consistent structures of three-stranded antiparallel beta-sheet domains, whereas randomly shuffled sequences with the same composition generated various structures with consistently higher energies. O- and N-glycosylation patterns showed that each TR domain had exposed hydrophobic surfaces surrounded by glycosylation sites. These structures are consistent with domain dimensions and stability measurements by atomic force microscopy (D. Alsteen, V. Dupres, S. A. Klotz, N. K. Gaur, P. N. Lipke, and Y. F. Dufrene, ACS Nano 3:1677-1682, 2009) and with circular dichroism determination of secondary structure and thermal stability. Functional assays showed that the hydrophobic surfaces of TR domains supported binding to polystyrene surfaces and other TR domains, leading to nonsaturable homophilic binding. The domain structures are like "classic" subunit interaction surfaces and can explain previously observed patterns of promiscuous interactions between TR domains in any Als proteins or between TR domains and surfaces of other proteins. Together, the modeling techniques and the supporting data lead to an approach that relates structure and function in many kinds of repeat domains in fungal adhesins.

Yeast cell adhesion molecules have functional amyloid-forming sequences.

The occurrence of highly conserved amyloid-forming sequences in Candida albicans Als proteins (H. N. Otoo et al., Eukaryot. Cell 7:776-782, 2008) led us to search for similar sequences in other adhesins from C. albicans and Saccharomyces cerevisiae. The beta-aggregation predictor TANGO found highly beta-aggregation-prone sequences in almost all yeast adhesins. These sequences had an unusual amino acid composition: 77% of their residues were beta-branched aliphatic amino acids Ile, Thr, and Val, which is more than 4-fold greater than their prevalence in the S. cerevisiae proteome. High beta-aggregation potential peptides from S. cerevisiae Flo1p and C. albicans Eap1p rapidly formed insoluble amyloids, as determined by Congo red absorbance, thioflavin T fluorescence, and fiber morphology. As examples of the amyloid-forming ability of the native proteins, soluble glycosylphosphatidylinositol (GPI)-less fragments of C. albicans Als5p and S. cerevisiae Muc1p also formed amyloids within a few days under native conditions at nM concentrations. There was also evidence of amyloid formation in vivo: the surfaces of cells expressing wall-bound Als1p, Als5p, Muc1p, or Flo1p were birefringent and bound the fluorescent amyloid-reporting dye thioflavin T. Both of these properties increased upon aggregation of the cells. In addition, amyloid binding dyes strongly inhibited aggregation and flocculation. The results imply that amyloid formation is an intrinsic property of yeast cell adhesion proteins from many gene families and that amyloid formation is an important component of cellular aggregation mediated by these proteins.

A New Function for Amyloid-Like Interactions: Cross-Beta Aggregates of Adhesins form Cell-to-Cell Bonds.

Amyloid structures assemble through a repeating type of bonding called "cross-ß", in which identical sequences in many protein molecules form ß-sheets that interdigitate through side chain interactions. We review the structural characteristics of such bonds. Single cell force microscopy (SCFM) shows that yeast expressing Als5 adhesin from Candida albicans demonstrate the empirical characteristics of cross-ß interactions. These properties include affinity for amyloid-binding dyes, birefringence, critical concentration dependence, repeating structure, and inhibition by anti-amyloid agents. We present a model for how cross-ß bonds form in trans between two adhering cells. These characteristics also apply to other fungal adhesins, so the mechanism appears to be an example of a new type of cell-cell adhesion.