Candida glabrata is an opportunistic human fungal pathogen which binds to surfaces mainly through the Epa family of cell adhesion proteins. While some Epa proteins mediate specific lectin-like ...interactions with human epithelial cells, others promote adhesion and biofilm formation on plastic surfaces via nonspecific interactions that are not yet elucidated. We report the measurement of hydrophobic forces engaged in Epa6-mediated cell adhesion by means of atomic force microscopy (AFM). Using single-cell force spectroscopy, we found that C. glabrata wild-type (WT) cells attach to hydrophobic surfaces via strongly adhesive macromolecular bonds, while mutant cells impaired in Epa6 expression are weakly adhesive. Nanoscale mapping of yeast cells using AFM tips functionalized with hydrophobic groups shows that Epa6 is massively exposed on WT cells and conveys strong hydrophobic properties to the cell surface. Our results demonstrate that Epa6 mediates strong hydrophobic interactions, thereby providing a molecular basis for the ability of this adhesin to drive biofilm formation on abiotic surfaces.
Knowledge of the molecular bases underlying the interaction of fungal pathogens with immune cells is critical to our understanding of fungal infections and offers exciting perspectives for ...controlling immune responses for therapy. Although fluorescence microscopy is a valuable tool to visualize pathogen–host interactions, the spatial resolution is low, meaning the fine structural details of the interacting cells cannot be observed. Here, we demonstrate the ability of correlated fluorescence-atomic force microscopy (AFM) to image the various steps of the interaction between fungal pathogens and macrophages with nanoscale resolution. We focus on Candida albicans, known to grow as two morphological forms (yeast cells, filamentous hyphae) that play important roles in modulating the interaction with macrophages. We observe the main steps of macrophage infection, including initial intercellular contact, phagocytosis by internalization of yeast cells, intracellular hyphal growth leading to mechanical stretching, and piercing of the macrophage membrane resulting in pathogen escape. While fluorescence imaging clearly distinguishes fungal cells from macrophages during the various steps of the process, AFM captures nanoscale structural features of the macrophage surface that are of high biological relevance, including ruffles, lamellipodia, filopodia, membrane remnants, and phagocytic cups. As fungal pathogenesis is mainly controlled by the ability of fungi to escape from immune cells, the nanoimaging platform established here has great potential in nanomedicine for understanding and controlling fungal infections.
Summary
Trimeric autotransporter adhesins (
TAAs
) are bacterial surface proteins that fulfil important functions in pathogenic
G
ram‐negative bacteria. Prominent examples of
TAAs
are found in
B
...urkholderia cepacia
complex, a group of bacterial species causing severe infections in patients with cystic fibrosis. While there is strong evidence that
Burkholderia cenocepacia
TAAs
mediate adhesion, aggregation and colonization of the respiratory epithelium, we still know very little about the molecular mechanisms behind these interactions. Here, we use single‐molecule atomic force microscopy to unravel the binding mechanism of
BCAM
0224, a prototype
TAA
from
B
. cenocepacia
K
56‐2. We show that the adhesin forms homophilic trans‐interactions engaged in bacterial aggregation, and that it behaves as a spring capable to withstand high forces. We also find that
BCAM
0224 binds collagen, a major extracellular component of host epithelia. Both homophilic and heterophilic interactions display low binding affinity, which could be important for epithelium colonization. We then demonstrate that
BCAM
0224 recognizes receptors on living pneumocytes, and leads to the formation of membrane tethers that may play a role in promoting adhesion. Collectively, our results show that
BCAM
0224 is a multifunctional adhesin endowed with remarkable binding properties, which may represent a general mechanism among
TAAs
for strengthening bacterial adhesion.
In the last decade, there has been an increasing interest in the potential health effects associated with the consumption of lactic acid bacteria (LAB) in foods. Some of these bacteria such as
GG ...(LGG) are known to adhere to milk components, which may impact their distribution and protection within dairy matrices and therefore is likely to modulate the efficiency of their delivery. However, the adhesive behavior of most LAB, as well as its effect on food structuration and on the final bacterial distribution within the food matrix remain very poorly studied. Using a recently developed high-throughput approach, we have screened a collection of 73 LAB strains for their adhesive behavior toward the major whey protein β-lactoglobulin. Adhesion was then studied by genomics in relation to common bacterial surface characteristics such as pili and adhesion-related domain containing proteins. Representative adhesive and non-adhesive strains have been studied in further depth through biophysical measurement using atomic force microscopy (AFM) and a relation with bacterial distribution in whey protein isolate (WPI) solution has been established. AFM measurements have revealed that bacterial adhesion to β-lactoglobulin is highly specific and cannot be predicted accurately using only genomic information. Non-adhesive strains were found to remain homogeneously distributed in solution whereas adhesive strains gathered in flocs. These findings show that several LAB strains are able to adhere to β-lactoglobulin, whereas this had only been previously observed on LGG. We also show that these adhesive interactions present similar characteristics and are likely to impact bacterial location and distribution in dairy matrices containing β-lactoglobulin. This may help with designing more efficient dairy food matrices for optimized LAB delivery.
Microbes employ a variety of strategies to adhere to abiotic and biotic surfaces, as well as host cells. In addition to their surface physicochemical properties (e.g. charge, hydrophobic balance), ...microbes produce appendages (e.g. pili, fimbriae, flagella) and express adhesion proteins embedded in the cell wall or cell membrane, with adhesive domains targeting specific ligands or chemical properties. Atomic force microscopy (AFM) is perfectly suited to deciphering the adhesive properties of microbial cells. Notably, AFM imaging has revealed the cell wall topographical organization of live cells at unprecedented resolution, and AFM has a dual capability to probe adhesion at the single-cell and single-molecule levels. AFM is thus a powerful tool for unravelling the molecular mechanisms of microbial adhesion at scales ranging from individual molecular interactions to the behaviours of entire cells. In this review, we cover some of the major breakthroughs facilitated by AFM in deciphering the microbial adhesive arsenal, including the exciting development of anti-adhesive strategies.
Squalamine is a natural aminosterol that has been discovered in the tissues of the dogfish shark (Squalus acanthias). Studies have previously demonstrated that this promoter compound and its ...derivatives exhibit potent bactericidal activity against Gram-negative, Gram-positive bacteria, and multidrug-resistant bacteria. The antibacterial activity of squalamine was found to correlate with that of other antibiotics, such as colistin and polymyxins. Still, in the field of microbiology, evidence has shown that squalamine and its derivatives have antifungal activity, antiprotozoa effect against a limited list of protozoa, and could exhibit antiviral activity against both RNA- and DNA-enveloped viruses. Furthermore, squalamine and its derivatives have been identified as being antiangiogenic compounds in the case of several types of cancers and induce a potential positive effect in the case of other diseases such as experimental retinopathy and Parkinson’s disease. Given the diverse effects of the squalamine and its derivatives, in this review we provide the different advances in our understanding of the various effects of these promising molecules and try to draw up a non-exhaustive list of the different mechanisms of actions of squalamine and its derivatives on the human organism and on different pathogens.
Many fungal adhesins have short, β-aggregation-prone sequences that play important functional roles, and in the Candida albicans adhesin Als5p, these sequences cluster the adhesins after exposure to ...shear force. Here, we report that Saccharomyces cerevisiae flocculins Flo11p and Flo1p have similar β-aggregation-prone sequences and are similarly stimulated by shear force, despite being nonhomologous. Shear from vortex mixing induced the formation of small flocs in cells expressing either adhesin. After the addition of Ca(2+), yeast cells from vortex-sheared populations showed greatly enhanced flocculation and displayed more pronounced thioflavin-bright surface nanodomains. At high concentrations, amyloidophilic dyes inhibited Flo1p- and Flo11p-mediated agar invasion and the shear-induced increase in flocculation. Consistent with these results, atomic force microscopy of Flo11p showed successive force-distance peaks characteristic of sequentially unfolding tandem repeat domains, like Flo1p and Als5p. Flo11p-expressing cells bound together through homophilic interactions with adhesion forces of up to 700 pN and rupture lengths of up to 600 nm. These results are consistent with the potentiation of yeast flocculation by shear-induced formation of high-avidity domains of clustered adhesins at the cell surface, similar to the activation of Candida albicans adhesin Als5p. Thus, yeast adhesins from three independent gene families use similar force-dependent interactions to drive cell adhesion. IMPORTANCE The Saccharomyces cerevisiae flocculins mediate the formation of cellular aggregates and biofilm-like mats, useful in clearing yeast from fermentations. An important property of fungal adhesion proteins, including flocculins, is the ability to form catch bonds, i.e., bonds that strengthen under tension. This strengthening is based, at least in part, on increased avidity of binding due to clustering of adhesins in cell surface nanodomains. This clustering depends on amyloid-like β-aggregation of short amino acid sequences in the adhesins. In Candida albicans adhesin Als5, shear stress from vortex mixing can unfold part of the protein to expose aggregation-prone sequences, and then adhesins aggregate into nanodomains. We therefore tested whether shear stress from mixing can increase flocculation activity by potentiating similar protein remodeling and aggregation in the flocculins. The results demonstrate the applicability of the Als adhesin model and provide a rational framework for the enhancement or inhibition of flocculation in industrial applications.
The large adhesin protein LapA mediates adhesion and biofilm formation by Pseudomonas fluorescens. Although adhesion is thought to involve the long multiple repeats of LapA, very little is known ...about the molecular mechanism by which this protein mediates attachment. Here we use atomic force microscopy to unravel the biophysical properties driving LapA-mediated adhesion. Single-cell force spectroscopy shows that expression of LapA on the cell surface via biofilm-inducing conditions (i.e., phosphate-rich medium) or deletion of the gene encoding the LapG protease (LapA+ mutant) increases the adhesion strength of P. fluorescens toward hydrophobic and hydrophilic substrates, consistent with the adherent phenotypes observed in these conditions. Substrate chemistry plays an unexpected role in modulating the mechanical response of LapA, with sequential unfolding of the multiple repeats occurring only on hydrophilic substrates. Biofilm induction also leads to shortening of the protein extensions, reflecting stiffening of their conformational properties. Using single-molecule force spectroscopy, we next demonstrate that the adhesin is randomly distributed on the surface of wild-type cells and can be released into the solution. For LapA+ mutant cells, we found that the adhesin massively accumulates on the cell surface without being released and that individual LapA repeats unfold when subjected to force. The remarkable adhesive and mechanical properties of LapA provide a molecular basis for the “multi-purpose” adhesion function of LapA, thereby making P. fluorescens capable of colonizing diverse environments.
Pili are polymeric proteins located at the cell surface of bacteria. These filamentous proteins play a pivotal role in bacterial adhesion with the surrounding environment. They are found both in ...Gram-negative and Gram-positive bacteria but differ in their structural organization. Purifying these high molecular weight proteins is challenging and has certainly slowed down their characterization. Here, we propose a chromatography-based protocol, mainly relying on multimodal chromatography (core bead technology using Capto Core 700 resin), to purify sortase-dependent SpaCBA pili from the probiotic strain
GG (LGG). Contrary to previously published methods, this purification protocol does not require specific antibodies nor complex laboratory equipment, including for the multimodal chromatography step, and provides high degree of protein purity. No other proteins were detectable by SDS-PAGE and the 260/280 nm ratio (∼0.6) of the UV spectrum confirmed the absence of any other co-purified macromolecules. One can obtain ∼50 μg of purified pili, starting from 1 L culture at OD
≈ 1, in 2-3 working days. This simple protocol could be useful to numerous laboratories to purify pili from LGG easily. Therefore, the present work should boost specific studies dedicated to LGG SpaCBA pili and the characterization of the interactions occurring with their protein partners at the molecular level. Moreover, this straightforward purification process might be extended to the purification of sortase-dependant pili from other Gram-positive bacteria.
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