Nanomechanics of CCN1-Mediated Staphylococcus aureus Phagocytosis.

Phagocytosis is an essential mechanism of the human immune system where pathogens are eliminated by immune cells. The CCN1 protein plays an important role in the phagocytosis of Staphylococcus aureus by favoring the bridging of the αVß3 integrin to the bacterial peptidoglycan (PG), through mechanical forces that remain unknown. Here, we employ single-molecule experiments to unravel the nanomechanics of the PG-CCN1-αVß3 ternary complex. While CCN1 binds αVß3 integrins with moderate force (∼60 pN), much higher binding strengths (up to ∼800 pN) are observed between CCN1 and PG. Notably, the strength of both CCN1-αVß3 and CCN1-PG bonds is dramatically enhanced by tensile loading, favoring a model in which mechanical stress induces the exposure of cryptic integrin binding sites in CCN1 and multivalent binding between CCN1 lectin sites and monosaccharides along the PG glycan chains.

Atomic force microscopy analysis of Pel polysaccharide- and type IV pili-mediated adhesion of Pseudomonas aeruginosa PA14 to an abiotic surface.

Type IV pili (TFP) contribute to the ability of microbes such as Pseudomonas aeruginosa to engage with and move across surfaces. We reported previously that P. aeruginosa TFP generate retractive forces of ∼30 pN and provided indirect evidence that TFP-mediated surface attachment was enhanced in the presence of the Pel polysaccharide. Here, we use different mutants defective in flagellar, Pel production or TFP production - alone or in combination - to decipher the relative contribution of these biofilm-promoting factors for P. aeruginosa adhesion. By means of atomic force microscopy (AFM), we show that mutating the flagellum (ΔflgK mutant) results in an increase in Pel polysaccharide production, but this increase in Pel does not result in an increase in surface adhesive properties compared to those previously described for the WT strain. By blocking Pel production in the ΔflgK mutant (ΔflgKΔpel), we directly show that TFP play a major role in the adhesion of the bacteria to hydrophobic AFM tips, but that the adhesion force is only slightly impaired by the absence of Pel. Inversely, performing single-cell force spectroscopy measurements with the mutant lacking TFP (ΔflgKΔpilA) reveals that the Pel can modulate the attachment of the bacteria to a hydrophobic substrate in a time-dependent manner. Finally, little adhesion was detected for the ΔflgKΔpilAΔpelA triple mutant, suggesting that both TFP and Pel polysaccharide make a substantial contribution to bacteria-substratum interaction events. Altogether, our data allow us to decipher the relative contribution of Pel and TFP in the early attachment by P. aeruginosa.

Mechanobiology: Shaping the future of cellular form and function.

Mechanobiology-the field studying how cells produce, sense, and respond to mechanical forces-is pivotal in the analysis of how cells and tissues take shape in development and disease. As we venture into the future of this field, pioneers share their insights, shaping the trajectory of future research and applications.

Functional Redundancy in Candida auris Cell Surface Adhesins Crucial for Cell-Cell Interaction and Aggregation.

Candida auris is an emerging nosocomial fungal pathogen associated with life-threatening invasive disease due to its persistent colonization, high level of transmissibility and multi-drug resistance. Aggregative and non-aggregative growth phenotypes for C. auris strains with different biofilm forming abilities, drug susceptibilities and virulence characteristics have been described. Using comprehensive transcriptional analysis we identified key cell surface adhesins that were highly upregulated in the aggregative phenotype during in vitro and in vivo grown biofilms using a mouse model of catheter infection. Phenotypic and functional evaluations of generated null mutants demonstrated crucial roles for the adhesins Als5 and Scf1 in mediating cell-cell adherence, coaggregation and biofilm formation. While individual mutants were largely non-aggregative, in combination cells were able to co-adhere and aggregate, as directly demonstrated by measuring cell adhesion forces using single-cell atomic force spectroscopy. This co-adherence indicates their role as complementary adhesins, which despite their limited similarity, may function redundantly to promote cell-cell interaction and biofilm formation. Functional diversity of cell wall proteins may be a form of regulation that provides the aggregative phenotype of C. auris with flexibility and rapid adaptation to the environment, potentially impacting persistence and virulence.

Functional Redundancy in Candida auris Cell Surface Adhesins Crucial for Cell-Cell Interaction and Aggregation.

Candida auris is an emerging nosocomial fungal pathogen associated with life-threatening invasive disease due to its persistent colonization, high level of transmissibility and multi-drug resistance. Aggregative and non-aggregative growth phenotypes for C. auris strains with different biofilm forming abilities, drug susceptibilities and virulence characteristics have been described. Using comprehensive transcriptional analysis we identified key cell surface adhesins that were highly upregulated in the aggregative phenotype during in vitro and in vivo grown biofilms using a mouse model of catheter infection. Phenotypic and functional evaluations of generated null mutants demonstrated crucial roles for the adhesins Als5 and Scf1 in mediating cell-cell adherence, coaggregation and biofilm formation. While individual mutants were largely non-aggregative, in combination cells were able to co-adhere and aggregate, as directly demonstrated by measuring cell adhesion forces using single-cell atomic force spectroscopy. This co-adherence indicates their role as complementary adhesins, which despite their limited similarity, may function redundantly to promote cell-cell interaction and biofilm formation. Functional diversity of cell wall proteins may be a form of regulation that provides the aggregative phenotype of C. auris with flexibility and rapid adaptation to the environment, potentially impacting persistence and virulence.

Tight-packing of large pilin subunits provides distinct structural and mechanical properties for the Myxococcus xanthus type IVa pilus.

Type IVa pili (T4aP) are ubiquitous cell surface filaments important for surface motility, adhesion to surfaces, DNA uptake, biofilm formation, and virulence. T4aP are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While major pilins of structurally characterized T4aP have lengths of <165 residues, the major pilin PilA of Myxococcus xanthus is unusually large with 208 residues. All major pilins have a conserved N-terminal domain and a variable C-terminal domain, and the additional residues of PilA are due to a larger C-terminal domain. We solved the structure of the M. xanthus T4aP (T4aPMx) at a resolution of 3.0 Å using cryo-EM. The T4aPMx follows the structural blueprint of other T4aP with the pilus core comprised of the interacting N-terminal α1-helices, while the globular domains decorate the T4aP surface. The atomic model of PilA built into this map shows that the large C-terminal domain has more extensive intersubunit contacts than major pilins in other T4aP. As expected from these greater contacts, the bending and axial stiffness of the T4aPMx is significantly higher than that of other T4aP and supports T4aP-dependent motility on surfaces of different stiffnesses. Notably, T4aPMx variants with interrupted intersubunit interfaces had decreased bending stiffness, pilus length, and strongly reduced motility. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4aP that expands the environmental conditions in which the T4aP system functions.

The inactivation of tolC sensitizes Escherichia coli to perturbations in lipopolysaccharide transport.

The Escherichia coli outer membrane channel TolC complexes with several inner membrane efflux pumps to export compounds across the cell envelope. All components of these complexes are essential for robust efflux activity, yet E. coli is more sensitive to antimicrobial compounds when tolC is inactivated compared to the inactivation of genes encoding the inner membrane drug efflux pumps. While investigating these susceptibility differences, we identified a distinct class of inhibitors targeting the core-lipopolysaccharide translocase, MsbA. We show that tolC null mutants are sensitized to structurally unrelated MsbA inhibitors and msbA knockdown, highlighting a synthetic-sick interaction. Phenotypic profiling revealed that tolC inactivation induced cell envelope softening and increased outer membrane permeability. Overall, this work identified a chemical probe of MsbA, revealed that tolC is associated with cell envelope mechanics and integrity, and highlighted that these findings should be considered when using tolC null mutants to study efflux deficiency.

Staphylococcus aureus skin colonization is mediated by SasG lectin variation.

Staphylococcus aureus causes the majority of skin and soft tissue infections, but this pathogen only transiently colonizes healthy skin. However, this transient skin exposure enables S. aureus to transition to infection. The initial adhesion of S. aureus to skin corneocytes is mediated by surface protein G (SasG). Here, phylogenetic analyses reveal the presence of two major divergent SasG alleles in S. aureus: SasG-I and SasG-II. Structural analyses of SasG-II identify a nonaromatic arginine in the binding pocket of the lectin subdomain that mediates adhesion to corneocytes. Atomic force microscopy and corneocyte adhesion assays indicate that SasG-II can bind to a broader variety of ligands than SasG-I. Glycosidase treatment results in different binding profiles between SasG-I and SasG-II on skin cells. In addition, SasG-mediated adhesion is recapitulated using differentiated N/TERT keratinocytes. Our findings indicate that SasG-II has evolved to adhere to multiple ligands, conferring a distinct advantage to S. aureus during skin colonization.

Staphylococcus aureus skin colonization is mediated by SasG lectin variation.

Staphylococcus aureus causes the majority of skin and soft tissue infections, but this pathogen only transiently colonizes healthy skin. However, this transient skin exposure enables S. aureus to transition to infection. Initial adhesion of S. aureus to skin corneocytes is mediated by surface protein G (SasG). Here, phylogenetic analyses reveal the presence of two major divergent SasG alleles in S. aureus, SasG-I and SasG-II. Structural analyses of SasG-II identified a unique non-aromatic arginine in the binding pocket of the lectin subdomain that mediates adhesion to corneocytes. Atomic force microscopy and corneocyte adhesion assays indicated SasG-II can bind to a broader variety of ligands than SasG-I. Glycosidase treatment resulted in different binding profiles between SasG-I and SasG-II on skin cells. Additionally, SasG-mediated adhesion was recapitulated using differentiated N/TERT keratinocytes. Our findings indicate that SasG-II has evolved to adhere to multiple ligands, conferring a distinct advantage to S. aureus during skin colonization.

Mechanobiology: How pathogens use mechanics to modulate host interactions.

Mechanobiology explores how cells sense and respond to mechanical cues and how mechanics guide cell function, physiology, and disease. In this issue of Cell, Thacker and colleagues reveal how the tuberculosis-causing pathogen exploits the mechanical behavior of cord-like structures to promote infection, impacting immune response, antibiotic susceptibility, and treatment strategies.

Large pilin subunits provide distinct structural and mechanical properties for the Myxococcus xanthus type IV pilus.

Type IV pili (T4P) are ubiquitous bacterial cell surface filaments important for surface motility, adhesion to biotic and abiotic surfaces, DNA uptake, biofilm formation, and virulence. T4P are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While the major pilins of structurally characterized T4P have lengths of up to 161 residues, the major pilin PilA of Myxococcus xanthus is unusually large with 208 residues. All major pilins have a highly conserved N-terminal domain and a highly variable C-terminal domain, and the additional residues in the M. xanthus PilA are due to a larger C-terminal domain. We solved the structure of the M. xanthus T4P (T4P Mx ) at a resolution of 3.0 Å using cryo-electron microscopy (cryo-EM). The T4P Mx follows the structural blueprint observed in other T4P with the pilus core comprised of the extensively interacting N-terminal α1-helices while the globular domains decorate the T4P surface. The atomic model of PilA built into this map shows that the large C-terminal domain has much more extensive intersubunit contacts than major pilins in other T4P. As expected from these greater contacts, the bending and axial stiffness of the T4P Mx is significantly higher than that of other T4P and supports T4P-dependent motility on surfaces of different stiffnesses. Notably, T4P Mx variants with interrupted intersubunit interfaces had decreased bending stiffness and strongly reduced motility on all surfaces. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4P that expands the environmental conditions in which the T4P system functions.

Catch Bond-Mediated Adhesion Drives Staphylococcus aureus Host Cell Invasion.

Various viruses and pathogenic bacteria interact with annexin A2 to invade mammalian cells. Here, we show that Staphylococcus aureus engages in extremely strong catch bonds for host cell invasion. By means of single-molecule atomic force microscopy, we find that bacterial surface-located clumping factors bind annexin A2 with extraordinary strength, indicating that these bonds are extremely resilient to mechanical tension. By determining the lifetimes of the complexes under increasing mechanical stress, we demonstrate that the adhesins form catch bonds with their ligand that are capable to sustain forces of 1500-1700 pN. The force-dependent adhesion mechanism identified here provides a molecular framework to explain how S. aureus pathogens tightly attach to host cells during invasion and shows promise for the design of new therapeutics against intracellular S. aureus.

Nanoscale clustering of mycobacterial ligands and DC-SIGN host receptors are key determinants for pathogen recognition.

The bacterial pathogen Mycobacterium tuberculosis binds to the C-type lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) on dendritic cells to evade the immune system. While DC-SIGN glycoconjugate ligands are ubiquitous among mycobacterial species, the receptor selectively binds pathogenic species from the M. tuberculosis complex (MTBC). Here, we unravel the molecular mechanism behind this intriguing selective recognition by means of a multidisciplinary approach combining single-molecule atomic force microscopy with Förster resonance energy transfer and bioassays. Molecular recognition imaging of mycobacteria demonstrates that the distribution of DC-SIGN ligands markedly differs between Mycobacterium bovis Bacille Calmette-Guérin (BCG) (model MTBC species) and Mycobacterium smegmatis (non-MTBC species), the ligands being concentrated into dense nanodomains on M. bovis BCG. Upon bacteria-host cell adhesion, ligand nanodomains induce the recruitment and clustering of DC-SIGN. Our study highlights the key role of clustering of both ligands on MTBC species and DC-SIGN host receptors in pathogen recognition, a mechanism that might be widespread in host-pathogen interactions.

High-force catch bonds between the Staphylococcus aureus surface protein SdrE and complement regulator factor H drive immune evasion.

The invasive bacterial pathogen Staphylococcus aureus recruits the complement regulatory protein factor H (fH) to its surface to evade the human immune system. Here, we report the identification of an extremely high-force catch bond used by the S. aureus surface protein SdrE to efficiently capture fH under mechanical stress. We find that increasing the external force applied to the SdrE-fH complex prolongs the lifetime of the bond at an extraordinary high force, 1,400 pN, above which the bond lifetime decreases as an ordinary slip bond. This catch-bond behavior originates from a variation of the dock, lock and latch interaction, where the SdrE ligand binding domains undergo conformational changes under stress, enabling the formation of long-lived hydrogen bonds with fH. The binding mechanism dissected here represents a potential target for new therapeutics against multidrug-resistant S. aureus strains.

Mechanistic basis of staphylococcal interspecies competition for skin colonization.

Staphylococci, whether beneficial commensals or pathogens, often colonize human skin, potentially leading to competition for the same niche. In this multidisciplinary study we investigate the structure, binding specificity, and mechanism of adhesion of the Aap lectin domain required for Staphylococcus epidermidis skin colonization and compare its characteristics to the lectin domain from the orthologous Staphylococcus aureus adhesin SasG. The Aap structure reveals a legume lectin-like fold with atypical architecture, showing specificity for N-acetyllactosamine and sialyllactosamine. Bacterial adhesion assays using human corneocytes confirmed the biological relevance of these Aap-glycan interactions. Single-cell force spectroscopy experiments measured individual binding events between Aap and corneocytes, revealing an extraordinarily tight adhesion force of nearly 900 nN and a high density of receptors at the corneocyte surface. The SasG lectin domain shares similar structural features, glycan specificity, and corneocyte adhesion behavior. We observe cross-inhibition of Aap-and SasG-mediated staphylococcal adhesion to corneocytes. Together, these data provide insights into staphylococcal interspecies competition for skin colonization and suggest potential avenues for inhibition of S. aureus colonization.

A molecular device for the redox quality control of GroEL/ES substrates.

Hsp60 chaperonins and their Hsp10 cofactors assist protein folding in all living cells, constituting the paradigmatic example of molecular chaperones. Despite extensive investigations of their structure and mechanism, crucial questions regarding how these chaperonins promote folding remain unsolved. Here, we report that the bacterial Hsp60 chaperonin GroEL forms a stable, functionally relevant complex with the chaperedoxin CnoX, a protein combining a chaperone and a redox function. Binding of GroES (Hsp10 cofactor) to GroEL induces CnoX release. Cryoelectron microscopy provided crucial structural information on the GroEL-CnoX complex, showing that CnoX binds GroEL outside the substrate-binding site via a highly conserved C-terminal α-helix. Furthermore, we identified complexes in which CnoX, bound to GroEL, forms mixed disulfides with GroEL substrates, indicating that CnoX likely functions as a redox quality-control plugin for GroEL. Proteins sharing structural features with CnoX exist in eukaryotes, suggesting that Hsp60 molecular plugins have been conserved through evolution.

Interaction of the Staphylococcus aureus Surface Protein FnBPB with Corneodesmosin Involves Two Distinct, Extremely Strong Bonds.

Attachment of Staphylococcus aureus to human skin corneocyte cells plays a critical role in exacerbating the severity of atopic dermatitis (AD). Pathogen-skin adhesion is mediated by bacterial cell-surface proteins called adhesins, including fibronectin-binding protein B (FnBPB). FnBPB binds to corneodesmosin (CDSN), a glycoprotein exposed on AD patient corneocytes. Using single-molecule experiments, we demonstrate that CDSN binding by FnBPB relies on a sophisticated two-site mechanism. Both sites form extremely strong bonds with binding forces of ∼1 and ∼2.5 nN albeit with faster dissociation rates than those reported for homologues of the adhesin. This previously unidentified two-binding site interaction in FnBPB illustrates its remarkable variety of adhesive functions and is of biological significance as the high strength and short bond lifetime will favor efficient skin colonization by the pathogen.

Fibronectin binding protein B binds to loricrin and promotes corneocyte adhesion by Staphylococcus aureus.

Colonisation of humans by Staphylococcus aureus is a major risk factor for infection, yet the bacterial and host factors involved are not fully understood. The first step during skin colonisation is adhesion of the bacteria to corneocytes in the stratum corneum where the cornified envelope protein loricrin is the main ligand for S. aureus. Here we report a novel loricrin-binding protein of S. aureus, the cell wall-anchored fibronectin binding protein B (FnBPB). Single-molecule force spectroscopy revealed both weak and ultra-strong (2 nN) binding of FnBPB to loricrin and that mechanical stress enhanced the strength of these bonds. Treatment with a peptide derived from fibrinogen decreased the frequency of strong interactions, suggesting that both ligands bind to overlapping sites within FnBPB. Finally, we show that FnBPB promotes adhesion to human corneocytes by binding strongly to loricrin, highlighting the relevance of this interaction to skin colonisation.

Force-induced unfolding of an antibiotic-bound outer-membrane protein.

In this issue of Structure, Ritzmann et al. characterize the unfolding of the ß-barrel assembly machinery component BamA with single-molecule force spectroscopy and reveal how an antibiotic changes BamA's mechanical properties and inhibits its activity. This work helps us understand the effects antibiotics have on Gram-negative outer membrane proteins.