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.

Mycobacterial Adhesion: From Hydrophobic to Receptor-Ligand Interactions.

Adhesion is crucial for the infective lifestyles of bacterial pathogens. Adhesion to non-living surfaces, other microbial cells, and components of the biofilm extracellular matrix are crucial for biofilm formation and integrity, plus adherence to host factors constitutes a first step leading to an infection. Adhesion is, therefore, at the core of pathogens' ability to contaminate, transmit, establish residency within a host, and cause an infection. Several mycobacterial species cause diseases in humans and animals with diverse clinical manifestations. Mycobacterium tuberculosis, which enters through the respiratory tract, first adheres to alveolar macrophages and epithelial cells leading up to transmigration across the alveolar epithelium and containment within granulomas. Later, when dissemination occurs, the bacilli need to adhere to extracellular matrix components to infect extrapulmonary sites. Mycobacteria causing zoonotic infections and emerging nontuberculous mycobacterial pathogens follow divergent routes of infection that probably require adapted adhesion mechanisms. New evidence also points to the occurrence of mycobacterial biofilms during infection, emphasizing a need to better understand the adhesive factors required for their formation. Herein, we review the literature on tuberculous and nontuberculous mycobacterial adhesion to living and non-living surfaces, to themselves, to host cells, and to components of the extracellular matrix.

AFM force-clamp spectroscopy captures the nanomechanics of the Tad pilus retraction.

Motorization of bacterial pili is key to generate traction forces to achieve cellular function. The Tad (or Type IVc) pilus from Caulobacter crescentus is a widespread motorized nanomachine crucial for bacterial survival, evolution and virulence. An unusual bifunctional ATPase motor drives Tad pilus retraction, which helps the bacteria to land on target surfaces. Here, we use a novel platform combining a fluorescence-based screening of piliated bacteria and atomic force microscopy (AFM) force-clamp spectroscopy, to monitor over time (30 s) the nanomechanics and dynamics of the Tad nanofilament retraction under a high constant tension (300 pN). We observe striking transient variations of force and height originating from two phenomena: active pilus retraction and passive hydrophobic interactions between the pilus and the hydrophobic substrate. That the Tad pilus is able to retract under high tensile loading - at a velocity of ∼150 nm s-1 - indicates that this nanomachine is stronger than previously anticipated. Our findings show that pilus retraction and hydrophobic interactions work together to mediate bacterial cell landing and surface adhesion. The motorized pilus retraction actively triggers the cell to approach the substrate. At short distances, passive hydrophobic interactions accelerate the approach phenomenon and promote strong cell-substrate adhesion. This mechanism could provide a strategy to save ATP-based energy by the retraction ATPase. Our force-clamp AFM methodology offers promise to decipher the physics of bacterial nanomotors with high sensitivity and temporal resolution.

Force-Induced Changes of PilY1 Drive Surface Sensing by Pseudomonas aeruginosa.

During biofilm formation, the opportunistic pathogen Pseudomonas aeruginosa uses its type IV pili (TFP) to sense a surface, eliciting increased second-messenger production and regulating target pathways required to adapt to a surface lifestyle. The mechanisms whereby TFP detect surface contact are still poorly understood, although mechanosensing is often invoked, with few data supporting this claim. Using a combination of molecular genetics and single-cell analysis, with biophysical, biochemical, and genomics techniques, we show that force-induced changes mediated by the von Willebrand A (vWA) domain-containing, TFP tip-associated protein PilY1 are required for surface sensing. Atomic force microscopy shows that TFP/PilY1 can undergo force-induced, sustained conformational changes akin to those observed for mechanosensitive proteins like titin. We show that mutation of a single cysteine residue in the vWA domain of PilY1 results in modestly lower surface adhesion forces, reduced sustained conformational changes, and increased nanospring-like properties, as well as reduced c-di-GMP signaling and biofilm formation. Mutating this cysteine has allowed us to genetically separate a role for TFP in twitching motility from surface-sensing signaling. The conservation of this Cys residue in all P. aeruginosa PA14 strains and its absence in the ∼720 sequenced strains of P. aeruginosa PAO1 may contribute to explaining the observed differences in surface colonization strategies observed for PA14 versus PAO1. IMPORTANCE Most bacteria live on abiotic and biotic surfaces in surface-attached communities known as biofilms. Surface sensing and increased levels of the second-messenger molecule c-di-GMP are crucial to the transition from planktonic to biofilm growth. The mechanism(s) underlying TFP-mediated surface detection that triggers this c-di-GMP signaling cascade is unclear. Here, we provide key insight into this question; we show that the eukaryote-like vWA domain of the TFP tip-associated protein PilY1 responds to mechanical force, which in turn drives the production of a key second messenger needed to regulate surface behaviors. Our studies highlight a potential mechanism that may account for differing surface colonization strategies.

Staphylococcus aureus binds to the N-terminal region of corneodesmosin to adhere to the stratum corneum in atopic dermatitis.

Staphylococcus aureus colonizes the skin of the majority of patients with atopic dermatitis (AD), and its presence increases disease severity. Adhesion of S. aureus to corneocytes in the stratum corneum is a key initial event in colonization, but the bacterial and host factors contributing to this process have not been defined. Here, we show that S. aureus interacts with the host protein corneodesmosin. Corneodesmosin is aberrantly displayed on the tips of villus-like projections that occur on the surface of AD corneocytes as a result of low levels of skin humectants known as natural moisturizing factor (NMF). An S. aureus mutant deficient in fibronectin binding protein B (FnBPB) and clumping factor B (ClfB) did not bind to corneodesmosin in vitro. Using surface plasmon resonance, we found that FnBPB and ClfB proteins bound with similar affinities. The S. aureus binding site was localized to the N-terminal glycine-serine-rich region of corneodesmosin. Atomic force microscopy showed that the N-terminal region was present on corneocytes containing low levels of NMF and that blocking it with an antibody inhibited binding of individual S. aureus cells to corneocytes. Finally, we found that S. aureus mutants deficient in FnBPB or ClfB have a reduced ability to adhere to low-NMF corneocytes from patients. In summary, we show that FnBPB and ClfB interact with the accessible N-terminal region of corneodesmosin on AD corneocytes, allowing S. aureus to take advantage of the aberrant display of corneodesmosin that accompanies low NMF in AD. This interaction facilitates the characteristic strong binding of S. aureus to AD corneocytes.

O-Methylation of the Glycopeptidolipid Acyl Chain Defines Surface Hydrophobicity of Mycobacterium abscessus and Macrophage Invasion.

Mycobacterium abscessus, an emerging pathogen responsible for severe lung infections in cystic fibrosis patients, displays either smooth (S) or rough (R) morphotypes. The S-to-R transition is associated with reduced levels of glycopeptidolipid (GPL) production and is correlated with increased pathogenicity in animal and human hosts. While the structure of GPL is well established, its biosynthetic pathway is incomplete. In addition, the biological functions of the distinct structural parts of this complex lipid remain elusive. Herein, the fmt gene encoding a putative O-methyltransferase was deleted in the M. abscessus S variant. Subsequent biochemical and structural analyses demonstrated that methoxylation of the fatty acyl chain of GPL was abrogated in the Δfmt mutant, and this defect was rescued upon complementation with a functional fmt gene. In contrast, the introduction of fmt derivatives mutated at residues essential for methyltransferase activity failed to complement GPL defects, indicating that fmt encodes an O-methyltransferase. Unexpectedly, phenotypic analyses showed that Δfmt was more hydrophilic than its parental progenitor, as demonstrated by hexadecane-aqueous buffer partitioning and atomic force microscopy experiments with hydrophobic probes. Importantly, the invasion rate of THP-1 macrophages by Δfmt was reduced by 50% when compared to the wild-type strain. Together, these results indicate that Fmt O-methylates the lipid moiety of GPL and plays a substantial role in conditioning the surface hydrophobicity of M. abscessus as well as in the early steps of the interaction between the bacilli and macrophages.

Fast chemical force microscopy demonstrates that glycopeptidolipids define nanodomains of varying hydrophobicity on mycobacteria.

Mycobacterium abscessus is an emerging multidrug-resistant bacterial pathogen causing severe lung infections in cystic fibrosis patients. A remarkable trait of this mycobacterial species is its ability to form morphologically smooth (S) and rough (R) colonies. The S-to-R transition is caused by the loss of glycopeptidolipids (GPLs) in the outer layer of the cell envelope and correlates with an increase in cording and virulence. Despite the physiological and medical importance of this morphological transition, whether it involves changes in cell surface properties remains unknown. Herein, we combine recently developed quantitative imaging (QI) atomic force microscopy (AFM) with hydrophobic tips to quantitatively map the surface structure and hydrophobicity of M. abscessus at high spatiotemporal resolution, and to assess how these properties are modulated by the S-to-R transition and by treatment with an inhibitor of the mycolic acid transporter MmpL3. We discover that loss of GPLs leads to major modifications in surface hydrophobicity, without any apparent change in cell surface ultrastructure. While R bacilli are homogeneously hydrophobic, S bacilli feature unusual variations of nanoscale hydrophobic properties. These previously undescribed cell surface nanodomains are likely to play critical roles in bacterial adhesion, aggregation, phenotypic heterogeneity and transmission, and in turn in virulence and pathogenicity. Our study also suggests that MmpL3 inhibitors show promise in nanomedicine as chemotherapeutic agents to interfere with the highly hydrophobic nature of the mycobacterial cell wall. The advantages of QI-AFM with hydrophobic tips are the ability to map chemical and structural properties simultaneously and at high resolution, applicable to a wide range of biosystems.

Assembly and disassembly of Aspergillus fumigatus conidial rodlets.

The rodlet structure present on the Aspergillus fumigatus conidial surface hides conidia from immune recognition. In spite of the essential biological role of the rodlets, the molecular basis for their self-assembly and disaggregation is not known. Analysis of the soluble forms of conidia-extracted and recombinant RodA by NMR spectroscopy has indicated the importance of disulfide bonds and identified two dynamic regions as likely candidates for conformational change and intermolecular interactions during conversion of RodA into the amyloid rodlet structure. Point mutations introduced into the RODA sequence confirmed that (1) mutation of a single cysteine was sufficient to block rodlet formation on the conidial surface and (2) both presumed amyloidogenic regions were needed for proper rodlet assembly. Mutations in the two putative amyloidogenic regions retarded and disturbed, but did not completely inhibit, the formation of the rodlets in vitro and on the conidial surface. Even in a disturbed form, the presence of rodlets on the surface of the conidia was sufficient to immunosilence the conidium. However, in contrast to the parental conidia, long exposure of mutant conidia lacking disulfide bridges within RodA or expressing RodA carrying the double (I115S/I146G) mutation activated dendritic cells with the subsequent secretion of proinflammatory cytokines. The immune reactivity of the RodA mutant conidia was not due to a modification in the RodA structure, but to the exposure of different pathogen-associated molecular patterns on the surface as a result of the modification of the rodlet surface layer. The full degradation of the rodlet layer, which occurs during early germination, is due to a complex array of cell wall bound proteases. As reported earlier, this loss of the rodlet layer lead to a strong anti-fumigatus host immune response in mouse lungs.

Force-Induced Strengthening of the Interaction between Staphylococcus aureus Clumping Factor B and Loricrin.

Bacterial pathogens that colonize host surfaces are subjected to physical stresses such as fluid flow and cell surface contacts. How bacteria respond to such mechanical cues is an important yet poorly understood issue. Staphylococcus aureus uses a repertoire of surface proteins to resist shear stress during the colonization of host tissues, but whether their adhesive functions can be modulated by physical forces is not known. Here, we show that the interaction of S. aureus clumping factor B (ClfB) with the squamous epithelial cell envelope protein loricrin is enhanced by mechanical force. We find that ClfB mediates S. aureus adhesion to loricrin through weak and strong molecular interactions both in a laboratory strain and in a clinical isolate. Strong forces (~1,500 pN), among the strongest measured for a receptor-ligand bond, are consistent with a high-affinity "dock, lock, and latch" binding mechanism involving dynamic conformational changes in the adhesin. Notably, we demonstrate that the strength of the ClfB-loricrin bond increases as mechanical force is applied. These findings favor a two-state model whereby bacterial adhesion to loricrin is enhanced through force-induced conformational changes in the ClfB molecule, from a weakly binding folded state to a strongly binding extended state. This force-sensitive mechanism may provide S. aureus with a means to finely tune its adhesive properties during the colonization of host surfaces, helping cells to attach firmly under high shear stress and to detach and spread under low shear stress.IMPORTANCEStaphylococcus aureus colonizes the human skin and the nose and can cause various disorders, including superficial skin lesions and invasive infections. During nasal colonization, the S. aureus surface protein clumping factor B (ClfB) binds to the squamous epithelial cell envelope protein loricrin, but the molecular interactions involved are poorly understood. Here, we unravel the molecular mechanism guiding the ClfB-loricrin interaction. We show that the ClfB-loricrin bond is remarkably strong, consistent with a high-affinity "dock, lock, and latch" binding mechanism. We discover that the ClfB-loricrin interaction is enhanced under tensile loading, thus providing evidence that the function of an S. aureus surface protein can be activated by physical stress.

Live-Cell Nanoscopy in Antiadhesion Therapy.

Live-cell nanoscopy has contributed significantly to assessing the inhibition of adhesion of uropathogenic Escherichia coli and Staphylococcus aureus by glycoconjugates and monoclonal antibodies, respectively, and of S. aureus surface attachment and cell-cell association by a synthetic peptide. This new technology shows promise for the development of antiadhesion therapies against bacterial pathogens.

The Surface Protein SdrF Mediates Staphylococcus epidermidis Adherence to Keratin.

Background: Staphylococcus epidermidis, a major component of skin flora, is an opportunist, often causing prosthetic device infections. A family of structurally related proteins mediates staphylococcal attachment to host tissues, contributing to the success of S. epidermidis as a pathogen. We examined the ability of the surface protein SdrF to adhere to keratin, a major molecule expressed on the skin surface. Methods: A heterologous Lactococcus lactis expression system was used to express SdrF and its ligand-binding domains. Adherence to keratin types 1 and 10, human foreskin keratinocytes, and nasal epithelial cells was examined. Results: SdrF bound human keratins 1 and 10 and adhered to keratinocytes and epithelial cells. Binding involved both the A and B domains. Anti-SdrF antibodies reduced adherence of S. epidermidis to keratin and keratinocytes. RNA interference reduced keratin synthesis in keratinocytes and, as a result, SdrF adherence. Direct force measurements using atomic force microscopy showed that SdrF mediates bacterial adhesion to keratin 10 through strong and weak bonds involving the A and B regions; strong adhesion was primarily mediated by the A region. Conclusions: These studies demonstrate that SdrF mediates adherence to human keratin and suggest that SdrF may facilitate S. epidermidis colonization of the skin.

Clumping Factor B Promotes Adherence of Staphylococcus aureus to Corneocytes in Atopic Dermatitis.

Staphylococcus aureus skin infection is a frequent and recurrent problem in children with the common inflammatory skin disease atopic dermatitis (AD). S. aureus colonizes the skin of the majority of children with AD and exacerbates the disease. The first step during colonization and infection is bacterial adhesion to the cornified envelope of corneocytes in the outer layer, the stratum corneum. Corneocytes from AD skin are structurally different from corneocytes from normal healthy skin. The objective of this study was to identify bacterial proteins that promote the adherence of S. aureus to AD corneocytes. S. aureus strains from clonal complexes 1 and 8 were more frequently isolated from infected AD skin than from the nasal cavity of healthy children. AD strains had increased ClfB ligand binding activity compared to normal nasal carriage strains. Adherence of single S. aureus bacteria to corneocytes from AD patients ex vivo was studied using atomic force microscopy. Bacteria expressing ClfB recognized ligands distributed over the entire corneocyte surface. The ability of an isogenic ClfB-deficient mutant to adhere to AD corneocytes compared to that of its parent clonal complex 1 clinical strain was greatly reduced. ClfB from clonal complex 1 strains had a slightly higher binding affinity for its ligand than ClfB from strains from other clonal complexes. Our results provide new insights into the first step in the establishment of S. aureus colonization in AD patients. ClfB is a key adhesion molecule for the interaction of S. aureus with AD corneocytes and represents a target for intervention.

Molecular interactions and inhibition of the staphylococcal biofilm-forming protein SdrC.

Staphylococcus aureus forms biofilms on indwelling medical devices using a variety of cell-surface proteins. There is growing evidence that specific homophilic interactions between these proteins represent an important mechanism of cell accumulation during biofilm formation, but the underlying molecular mechanisms are still not well-understood. Here we report the direct measurement of homophilic binding forces by the serine-aspartate repeat protein SdrC and their inhibition by a peptide. Using single-cell and single-molecule force measurements, we find that SdrC is engaged in low-affinity homophilic bonds that promote cell-cell adhesion. Low-affinity intercellular adhesion may play a role in favoring biofilm dynamics. We show that SdrC also mediates strong cellular interactions with hydrophobic surfaces, which are likely to be involved in the initial attachment to biomaterials, the first stage of biofilm formation. Furthermore, we demonstrate that a peptide derived from ß-neurexin is a powerful competitive inhibitor capable of efficiently blocking surface attachment, homophilic adhesion, and biofilm accumulation. Molecular modeling suggests that this blocking activity may originate from binding of the peptide to a sequence of SdrC involved in homophilic interactions. Our study opens up avenues for understanding the role of homophilic interactions in staphylococcal adhesion, and for the design of new molecules to prevent biofilm formation during infection.

Blastomyces Virulence Adhesin-1 Protein Binding to Glycosaminoglycans Is Enhanced by Protein Disulfide Isomerase.

UNLABELLED: Blastomyces adhesin-1 (BAD-1) protein mediates the virulence of the yeast Blastomyces dermatitidis, in part by binding host lung tissue, the extracellular matrix, and cellular receptors via glycosaminoglycans (GAGs), such as heparan sulfate. The tandem repeats that make up over 90% of BAD-1 appear in their native state to be tightly folded into an inactive conformation, but recent work has shown that they become activated and adhesive upon reduction of a disulfide linkage. Here, atomic force microscopy (AFM) of a single BAD-1 molecule interacting with immobilized heparin revealed that binding is enhanced upon treatment with protein disulfide isomerase and dithiothreitol (PDI/DTT). PDI/DTT treatment of BAD-1 induced a plateau effect in atomic force signatures that was consistent with sequential rupture of tandem binding domains. Inhibition of PDI in murine macrophages blunted BAD-1 binding to heparin in vitro. Based on AFM, we found that a short Cardin-Weintraub sequence paired with a WxxWxxW sequence in the first, degenerate repeat at the N terminus of BAD-1 was sufficient to initiate heparin binding. Removal of half of the 41 BAD-1 tandem repeats led to weaker adhesion, illustrating their role in enhanced binding. Mass spectroscopy of the tandem repeat revealed that the PDI-induced interaction with heparin is characterized by ruptured disulfide bonds and that cysteine thiols remain reduced. Further binding studies showed direct involvement of thiols in heparin ligation. Thus, we propose that the N-terminal domain of BAD-1 governs the initial association with host GAGs and that proximity to GAG-associated host PDI catalyzes activation of additional binding motifs conserved within the tandem repeats, leading to enhanced avidity and availability of reduced thiols. IMPORTANCE: Pathogenic fungi and other microbes must adhere to host tissue to initiate infection. Surface adhesins promote this event and may be required for disease pathogenesis. We studied a fungal adhesin essential for virulence (BAD-1; Blastomyces adhesin-1) and found that host products induce its structural reconfiguration and foster its optimal binding to tissue structures.

Identification of a supramolecular functional architecture of Streptococcus mutans adhesin P1 on the bacterial cell surface.

P1 (antigen I/II) is a sucrose-independent adhesin of Streptococcus mutans whose functional architecture on the cell surface is not fully understood. S. mutans cells subjected to mechanical extraction were significantly diminished in adherence to immobilized salivary agglutinin but remained immunoreactive and were readily aggregated by fluid-phase salivary agglutinin. Bacterial adherence was restored by incubation of postextracted cells with P1 fragments that contain each of the two known adhesive domains. In contrast to untreated cells, glutaraldehyde-treated bacteria gained reactivity with anti-C-terminal monoclonal antibodies (mAbs), whereas epitopes recognized by mAbs against other portions of the molecule were masked. Surface plasmon resonance experiments demonstrated the ability of apical and C-terminal fragments of P1 to interact. Binding of several different anti-P1 mAbs to unfixed cells triggered release of a C-terminal fragment from the bacterial surface, suggesting a novel mechanism of action of certain adherence-inhibiting antibodies. We also used atomic force microscopy-based single molecule force spectroscopy with tips bearing various mAbs to elucidate the spatial organization and orientation of P1 on living bacteria. The similar rupture lengths detected using mAbs against the head and C-terminal regions, which are widely separated in the tertiary structure, suggest a higher order architecture in which these domains are in close proximity on the cell surface. Taken together, our results suggest a supramolecular organization in which additional P1 polypeptides, including the C-terminal segment originally identified as antigen II, associate with covalently attached P1 to form the functional adhesive layer.

Structural features of the Pseudomonas fluorescens biofilm adhesin LapA required for LapG-dependent cleavage, biofilm formation, and cell surface localization.

The localization of the LapA protein to the cell surface is a key step required by Pseudomonas fluorescens Pf0-1 to irreversibly attach to a surface and form a biofilm. LapA is a member of a diverse family of predicted bacterial adhesins, and although lacking a high degree of sequence similarity, family members do share common predicted domains. Here, using mutational analysis, we determine the significance of each domain feature of LapA in relation to its export and localization to the cell surface and function in biofilm formation. Our previous work showed that the N terminus of LapA is required for cleavage by the periplasmic cysteine protease LapG and release of the adhesin from the cell surface under conditions unfavorable for biofilm formation. We define an additional critical region of the N terminus of LapA required for LapG proteolysis. Furthermore, our results suggest that the domains within the C terminus of LapA are not absolutely required for biofilm formation, export, or localization to the cell surface, with the exception of the type I secretion signal, which is required for LapA export and cell surface localization. In contrast, deletion of the central repetitive region of LapA, consisting of 37 repeats of 100 amino acids, results in an inability to form a biofilm. We also used single-molecule atomic force microscopy to further characterize the role of these domains in biofilm formation on hydrophobic and hydrophilic surfaces. These studies represent the first detailed analysis of the domains of the LapA family of biofilm adhesin proteins.

Surface proteome analysis of a natural isolate of Lactococcus lactis reveals the presence of pili able to bind human intestinal epithelial cells.

Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells.

Single-molecule atomic force microscopy unravels the binding mechanism of a Burkholderia cenocepacia trimeric autotransporter adhesin.

Trimeric autotransporter adhesins (TAAs) are bacterial surface proteins that fulfil important functions in pathogenic Gram-negative bacteria. Prominent examples of TAAs are found in Burkholderia 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 BCAM0224, a prototype TAA from B. cenocepacia K56-2. 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 BCAM0224 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 BCAM0224 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 BCAM0224 is a multifunctional adhesin endowed with remarkable binding properties, which may represent a general mechanism among TAAs for strengthening bacterial adhesion.

Nanoscale imaging of the Candida-macrophage interaction using correlated fluorescence-atomic force microscopy.

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.

Chitin synthases with a myosin motor-like domain control the resistance of Aspergillus fumigatus to echinocandins.

Aspergillus fumigatus has two chitin synthases (CSMA and CSMB) with a myosin motor-like domain (MMD) arranged in a head-to-head configuration. To understand the function of these chitin synthases, single and double csm mutant strains were constructed and analyzed. Although there was a slight reduction in mycelial growth of the mutants, the total chitin synthase activity and the cell wall chitin content were similar in the mycelium of all of the mutants and the parental strain. In the conidia, chitin content in the ΔcsmA strain cell wall was less than half the amount found in the parental strain. In contrast, the ΔcsmB mutant strain and, unexpectedly, the ΔcsmA/ΔcsmB mutant strain did not show any modification of chitin content in their conidial cell walls. In contrast to the hydrophobic conidia of the parental strain, conidia of all of the csm mutants were hydrophilic due to the presence of an amorphous material covering the hydrophobic surface-rodlet layer. The deletion of CSM genes also resulted in an increased susceptibility of resting and germinating conidia to echinocandins. These results show that the deletion of the CSMA and CSMB genes induced a significant disorganization of the cell wall structure, even though they contribute only weakly to the overall cell wall chitin synthesis.