Adhesins Research Articles

Architectural dissection of adhesive bacterial cell surface appendages from a "molecular machines" viewpoint.

The ability of bacteria to interact with and respond to their environment is crucial to their lifestyle and survival. Bacterial cells routinely need to engage with extracellular target molecules, in locations spatially separated from their cell surface. Engagement with distant targets allows bacteria to adhere to abiotic surfaces and host cells, sense harmful or friendly molecules in their vicinity, as well as establish symbiotic interactions with neighboring cells in multicellular communities such as biofilms. Binding to extracellular molecules also facilitates transmission of information back to the originating cell, allowing the cell to respond appropriately to external stimuli, which is critical throughout the bacterial life cycle. This requirement of bacteria to bind to spatially separated targets is fulfilled by a myriad of specialized cell surface molecules, which often have an extended, filamentous arrangement. In this review, we compare and contrast such molecules from diverse bacteria, which fulfil a range of binding functions critical for the cell. Our comparison shows that even though these extended molecules have vastly different sequence, biochemical and functional characteristics, they share common architectural principles that underpin bacterial adhesion in a variety of contexts. In this light, we can consider different bacterial adhesins under one umbrella, specifically from the point of view of a modular molecular machine, with each part fulfilling a distinct architectural role. Such a treatise provides an opportunity to discover fundamental molecular principles governing surface sensing, bacterial adhesion, and biofilm formation.

48 Dietary functional fiber properties for improved health and disease outcomes in pigs

Abstract In swine production, using feed antibiotics as antimicrobial growth promotants has been reduced; thus, feed alternatives to manage gut health are required. Dietary fiber and similar carbohydrate structures such as resistant starch, oligosaccharides, and exopolysaccharides are nutritional tools that may enhance gut health in pigs. Dietary fiber may have a role to alleviate diarrheal diseases including in pigs post-weaning but can also reduce constipation in sows, but specific functionality depends on fiber properties. Antibiotics are hypothesized to influence gut health via modulation of intestinal microbial profiles; fermentation and intestinal inflammation are considered important mechanisms. Dietary fiber sources differ in a key functional property: fermentability. Rapid fermentation of fiber and oligosaccharides is associated with changes in microbial profiles and increased metabolite production. Recently, microbial composition was hypothesized to be less important and combined output of metabolites should be the focus. Fiber properties may also manipulate retention time and physico-chemical properties of the undigested residue; hence, non-fermentable fiber may prevent constipation in sows. The fiber-like structure resistant starch is not digested and acts as fermentable fiber but has unique properties, because it specifically increases intestinal abundance of bifidobacteria, which are associated with improved gut health. Using crop breeding, feed processing, and feed additives, the structure of fiber and starch can be altered thereby changing its degradation kinetics and affecting its prebiotic activity. Finally, fiber-like structures such as exopolysaccharides from Limosilactobacillus reuteri and unique oligosaccharides may serve as receptor analogues for pathogenic bacteria, e.g., enterotoxigenic Escherichia coli (ETEC). These receptor analogues block lectin domains of bacterial adhesins and thus prevent adherence of pathogens to the gut wall, thereby avoiding initiation of post-weaning diarrhea. In conclusion, dietary fiber and similar structures possess important functional properties that may also provide important solutions to maintain gut health in pigs.

Molecular basis of bacterial lectin recognition of eukaryotic glycans: The case of Mycoplasma pneumoniae and Mycoplasma genitalium cytoadhesins

Mycoplasma pneumoniae and Mycoplasma genitalium are two emerging bacterial pathogens that colonize the human respiratory and urogenital epithelia, respectively. Both pathogens express cell surface cytoadhesins that play a crucial role in the interaction with the host, mediating the attachment to sialylated glycan receptors and triggering infection. The design of competitive binding inhibitors of Mycoplasma cytoadhesins has potential to disrupt these interactions and lessen bacterial pathogenesis. To this end, we report here molecular insights into the adhesion mechanisms of M. pneumoniae and M. genitalium, which are largely mediated by sialylated glycans on the host cell surface. In detail, a combination of Nuclear Magnetic Resonance (NMR) spectroscopy, fluorescence analysis and computational studies allowed us to explore the recognition by the cytoadhesins P40/P90 in M. pneumoniae and P110 in M. genitalium of sialylated N- and O-glycans. We reveal that, unlike other bacterial adhesins, which are characterized by a wide binding pocket, Mycoplasma cytoadhesins principally accommodate the sialic acid residue, in a similar manner to mammalian Siglecs. These findings represent crucial insight into the future development of novel compounds to counteract Mycoplasma infections by inhibiting bacterial adherence to host tissues.

Acute nitrite exposure causes gut microbiota dysbacteriosis and proliferation of pathogenic Photobacterium in shrimp

Nitrite exposure has become a significant concern in the aquaculture industry, posing a severe threat to aquatic animals such as shrimp. While studies have reported the adverse effects of nitrite on shrimp growth, the part played by the gut microbiota in shrimp mortality resulting from nitrite exposure is poorly understood. Here, the effects of nitrite on shrimp gut bacterial community were investigated using 16S rRNA amplicon sequencing, bacterial isolation, genomic analysis, and infection experiments. Compared to the control_healthy group, changes in the bacterial composition of the nitrite_dead group were associated with reduced abundance of specific beneficial bacteria and increased abundance of certain pathogenic bacteria. Notably, members of the Photobacterium genus were found to be significantly enriched in the nitrite_dead group. Genomic analysis of a representative Photobacterium strain (LvS-8n3) revealed a variety of genes encoding bacterial toxins, including hemolysin, adhesin, and phospholipase. Furthermore, it was also found that LvS-8n3 exhibits strong pathogenicity, probably due to its high production of pathogenic factors and the ability to utilize nitrite for proliferation. Therefore, the proliferation of pathogenic Photobacterium species appears pivotal for driving shrimp mortality caused by nitrite exposure. These findings provide novel insights into the disease mechanism in shrimp under conditions of environmental change.

The serine-rich repeat glycoprotein Srr2 mediates Streptococcus agalactiae interaction with host fibronectin

BackgroundGroup B Streptococcus (GBS) is a commensal of healthy adults and an important pathogen in newborns, the elderly and immunocompromised individuals. GBS displays several virulence factors that promote colonisation and host infection, including the ST-17 strain-specific adhesin Srr2, previously characterised for its binding to fibrinogen. Another common target for bacterial adhesins and for host colonization is fibronectin, a multi-domain glycoprotein found ubiquitously in body fluids, in the extracellular matrix and on the surface of cells.ResultsIn this study, fibronectin was identified as a novel ligand for the Srr2 adhesin of GBS. A derivative of the ST-17 strain BM110 overexpressing the srr2 gene showed an increased ability to bind fibrinogen and fibronectin, compared to the isogenic wild-type strain. Conversely, the deletion of srr2 impaired bacterial adhesion to both ligands. ELISA assays and surface plasmon resonance studies using the recombinant binding region (BR) form of Srr2 confirmed a direct interaction with fibronectin with an estimated Kd of 92 nM. Srr2-BR variants defective in fibrinogen binding also exhibited no interaction with fibronectin, suggesting that Srr2 binds this ligand through the dock-lock-latch mechanism, previously described for fibrinogen binding. The fibronectin site responsible for recombinant Srr2-BR binding was identified and localised in the central cell-binding domain of the protein. Finally, in the presence of fibronectin, the ability of a Δsrr2 mutant to adhere to human cervico-vaginal epithelial cells was significantly lower than that of the wild-type strain.ConclusionBy combining genetic and biochemical approaches, we demonstrate a new role for Srr2, namely interacting with fibronectin. We characterised the molecular mechanism of this interaction and demonstrated that it plays a role in promoting the adhesion of GBS to human cervico-vaginal epithelial cells, further substantiating the role of Srr2 as a factor responsible for the hypervirulence of GBS ST-17 strains. The discovery of the previously undescribed interaction between Srr2 and fibronectin establishes this adhesin as a key factor for GBS colonisation of host tissues.

BPP0974 is a Bordetella parapertussis adhesin expressed in the avirulent phase, implicated in biofilm formation and intracellular survival

B. parapertussis is a bacterium that causes whooping cough, a severe respiratory infection disease, that has shown an increased incidence in the population. Upon transmission through aerosol droplets, the initial steps of host colonization critically depend on the bacterial adhesins. We here described BPP0974, a B. parapertussis protein that exhibits the typical domain architecture of the large repetitive RTX adhesin family. BPP0974 was found to be retained in the bacterial membrane and secreted into the culture medium. This protein was found overexpressed in the avirulent phase of B. parapertussis, the phenotype proposed for initial host colonization. Interestingly, BPP0974 was found relevant for the biofilm formation as well as involved in the bacterial attachment to and survival within the respiratory epithelial cells. Taken together, our results suggest a role for BPP0974 in the early host colonization and pathogenesis of B. parapertussis.

The strongest protein binder is surprisingly labile.

Bacterial adhesins are cell-surface proteins that anchor to the cell wall of the host. The first stage of infection involves the specific attachment to fibrinogen (Fg), a protein found in human blood. This attachment allows bacteria to colonize tissues causing diseases such as endocarditis. The study of this family of proteins is hence essential to develop new strategies to fight bacterial infections. In the case of the Gram-positive bacterium Staphylococcus aureus, there exists a class of adhesins known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Here, we focus on one of them, the clumping factor A (ClfA), which has been found to bind Fg through the dock-lock-latch mechanism. Interestingly, it has recently been discovered that MSCRAMM proteins employ a catch-bond to withstand forces exceeding 2 nN, making this type of interaction as mechanically strong as a covalent bond. However, it is not known whether this strength is an evolved feature characteristic of the bacterial protein or is typical only of the interaction with its partner. Here, we combine single-molecule force spectroscopy, biophysical binding assays, and molecular simulations to study the intrinsic mechanical strength of ClfA. We find that despite the extremely high forces required to break its interactions with Fg, ClfA is not by itself particularly strong. Integrating the results from both theory and experiments we dissect contributions to the mechanical stability of this protein.

A Review of the Mechanisms of Bacterial Colonization of the Mammal Gut.

A healthy animal intestine hosts a diverse population of bacteria in a symbiotic relationship. These bacteria utilize nutrients in the host's intestinal environment for growth and reproduction. In return, they assist the host in digesting and metabolizing nutrients, fortifying the intestinal barrier, defending against potential pathogens, and maintaining gut health. Bacterial colonization is a crucial aspect of this interaction between bacteria and the intestine and involves the attachment of bacteria to intestinal mucus or epithelial cells through nonspecific or specific interactions. This process primarily relies on adhesins. The binding of bacterial adhesins to host receptors is a prerequisite for the long-term colonization of bacteria and serves as the foundation for the pathogenicity of pathogenic bacteria. Intervening in the adhesion and colonization of bacteria in animal intestines may offer an effective approach to treating gastrointestinal diseases and preventing pathogenic infections. Therefore, this paper reviews the situation and mechanisms of bacterial colonization, the colonization characteristics of various bacteria, and the factors influencing bacterial colonization. The aim of this study was to serve as a reference for further research on bacteria-gut interactions and improving animal gut health.

Crystal structure of the long Rib domain of the LPXTG-anchored surface protein from Limosilactobacillus reuteri.

The Rib domain, which is often found as tandem-repeat structural modules in surface proteins of Gram-positive bacteria, plays important roles in mediating interactions of bacteria with their environments and hosts. A comprehensive structural analysis of various Rib domains is essential to fully understand their impact on the structure and functionality of these bacterial adhesins. To date, structural information has been limited for this expansive group of domains. In this study, the high-resolution crystal structure of the second member of the long Rib domain, a unique subclass within the Rib-domain family, derived from Limosilactobacillus reuteri is presented. The data not only demonstrate a highly conserved structure within the long Rib domain, but also highlight an evolutionary convergence in structural architecture with other modular domains found in cell-adhesion molecules.

Modulation of Campylobacter jejuni adhesion to biotic model surfaces by fungal lectins and protease inhibitors.

Campylobacter jejuni, a Gram-negative bacterium, is one of the most common causes of foodborne illness worldwide. Its adhesion mechanism is mediated by several bacterial factors, including flagellum, protein adhesins, lipooligosaccharides, proteases, and host factors, such as surface glycans on epithelial cells and mucins. Fungal lectins, specialized carbohydrate-binding proteins, can bind to specific glycans on host and bacterial cells and thus influence pathogenesis. In this study, we investigated the effects of fungal lectins and protease inhibitors on the adhesion of C. jejuni to model biotic surfaces (mucin, fibronectin, and collagen) and Caco-2 cells as well as the invasion of Caco-2 cells. The lectins Marasmius oreades agglutinin (MOA) and Laccaria bicolor tectonin 2 (Tec2) showed remarkable efficacy in all experiments. In addition, different pre-incubations of lectins with C. jejuni or Caco-2 cells significantly inhibited the ability of C. jejuni to adhere to and invade Caco-2 cells, but to varying degrees. Pre-incubation of Caco-2 cells with selected lectins reduced the number of invasive C. jejuni cells the most, while simultaneous incubation showed the greatest reduction in adherent C. jejuni cells. These results suggest that fungal lectins are a promising tool for the prevention and treatment of C. jejuni infections. Furthermore, this study highlights the potential of fungi as a rich reservoir for novel anti-adhesive agents.

Adhesin domains responsible for binding bacteria to surfaces they colonize project outwards from companion split domains.

Bacterial adhesins attach their hosts to surfaces that the bacteria will colonize. This surface adhesion occurs through specific ligand-binding domains located towards the distal end of the long adhesin molecules. However, recognizing which of the many adhesin domains are structural and which are ligand binding has been difficult up to now. Here we have used the protein structure modeling program AlphaFold2 to predict structures for these giant 0.2- to 1.5-megadalton proteins. Crystal structures previously solved for several adhesin regions are in good agreement with the models. Whereas most adhesin domains are linked in a linear fashion through their N- and C-terminal ends, ligand-binding domains can be recognized by budding out from a companion core domain so that their ligand-binding sites are projected away from the axis of the adhesin for maximal exposure to their targets. These companion domains are "split" in their continuity by projecting the ligand-binding domain outwards. The "split domains" are mostly β-sandwich extender modules, but other domains like a β-solenoid can serve the same function. Bioinformatic analyses of Gram-negative bacterial sequences revealed wide variety ligand-binding domains are used in their Repeats-in-Toxin adhesins. The ligands for many of these domains have yet to be identified but known ligands include various cell-surface glycans, proteins, and even ice. Recognizing the ligands to which the adhesins bind could lead to ways of blocking colonization by bacterial pathogens. Engineering different ligand-binding domains into an adhesin has the potential to change the surfaces to which bacteria bind.

Discovery of Orally Bioavailable FmlH Lectin Antagonists as Treatment for Urinary Tract Infections.

FmlH, a bacterial adhesin of uropathogenic Escherichia coli (UPEC), has been shown to provide a fitness advantage in colonizing the bladder during chronic urinary tract infections (UTIs). Previously reported ortho-biphenyl glycosides based on βGal and βGalNAc have excellent binding affinity to FmlH and potently block binding to its natural carbohydrate receptor, but they lack oral bioavailability. In this paper, we outline studies where we have optimized compounds for improved pharmacokinetics, leading to the discovery of novel analogues with good oral bioavailability. We synthesized galactosides with the anomeric O-linker replaced with more stable S- and C-linked linkers. We also investigated modifications to the GalNAc sugar and modifications to the biphenyl aglycone. We identified GalNAc 69 with an IC50 of 0.19 μM against FmlH and 53% oral bioavailability in mice. We also obtained a FimlH-bound X-ray structure of lead compound 69 (AM4085) which has potential as a new antivirulence therapeutic for UTIs.

Controling the cytoskeleton during CEACAM3-mediated phagocytosis

Phagocytosis, an innate defense mechanism of multicellular animals, is initiated by specialized surface receptors. A phagocytic receptor expressed by human polymorphonuclear granulocytes, the major professional phagocytes in our body, is one of the fastest evolving human proteins implying a special role in human biology. This receptor, CEACAM3, is a member of the CarcinoEmbryonic Antigen-related Cell Adhesion Molecule (CEACAM) family and dedicated to the immediate recognition and rapid internalization of human-restricted pathogens. In this focused contribution, we will review the special adaptations of this protein, which co-evolves with different species of mucosa-colonizing bacteria. While the extracellular Immunoglobulin-variable (IgV)-like domain recognizes various bacterial adhesins, an Immunoreceptor Tyrosine-based Activation Motif (ITAM)-like sequence in the cytoplasmic tail of CEACAM3 constitutes the central signaling hub to trigger actin rearrangements needed for efficient phagocytosis. A major emphasis of this review will be placed on recent findings, which have revealed the multi-level control of this powerful phagocytic device. As tyrosine phosphorylation and small GTPase activity are central for CEACAM3-mediated phagocytosis, the counterregulation of CEACAM3 activity involves the receptor-type protein tyrosine phosphatase J (PTPRJ) as well as the Rac-GTP scavenging protein Cyri-B. Interference with such negative regulatory circuits has revealed that CEACAM3-mediated phagocytosis can be strongly enhanced. In principle, the knowledge gained by studying CEACAM3 can be applied to other phagocytic systems and opens the door to treatments, which boost the phagocytic capacity of professional phagocytes.

Exploring the mechanisms of host-specificity of a hyperparasitic bacterium (Pasteuria spp.) with potential to control tropical root-knot nematodes (Meloidogyne spp.): insights from Caenorhabditis elegans.

Plant-parasitic nematodes are important economic pests of a range of tropical crops. Strategies for managing these pests have relied on a range of approaches, including crop rotation, the utilization of genetic resistance, cultural techniques, and since the 1950's the use of nematicides. Although nematicides have been hugely successful in controlling nematodes, their toxicity to humans, domestic animals, beneficial organisms, and the environment has raised concerns regarding their use. Alternatives are therefore being sought. The Pasteuria group of bacteria that form endospores has generated much interest among companies wanting to develop microbial biocontrol products. A major challenge in developing these bacteria as biocontrol agents is their host-specificity; one population of the bacterium can attach to and infect one population of plant-parasitic nematode but not another of the same species. Here we will review the mechanism by which infection is initiated with the adhesion of endospores to the nematode cuticle. To understand the genetics of the molecular processes between Pasteuria endospores and the nematode cuticle, the review focuses on the nature of the bacterial adhesins and how they interact with the nematode cuticle receptors by exploiting new insights gained from studies of bacterial infections of Carnorhabditis elegans. A new Velcro-like multiple adhesin model is proposed in which the cuticle surface coat, which has an important role in endospore adhesion, is a complex extracellular matrix containing glycans originating in seam cells. The genes associated with these seam cells appear to have a dual role by retaining some characteristics of stem cells.

LppA is a novel plasminogen receptor of Mycoplasma bovis that contributes to adhesion by binding the host extracellular matrix and Annexin A2

Mycoplasma bovis is responsible for various inflammatory diseases in cattle. The prevention and control of M. bovis are complicated by the absence of effective vaccines and the emergence of multidrug-resistant strains, resulting in substantial economic losses worldwide in the cattle industry. Lipoproteins, vital components of the Mycoplasmas cell membrane, are deemed potent antigens for eliciting immune responses in the host upon infection. However, the functions of lipoproteins in M. bovis remain underexplored due to their low sequence similarity with those of other bacteria and the scarcity of genetic manipulation tools for M. bovis. In this study, the lipoprotein LppA was identified in all examined M. bovis strains. Utilizing immunoelectron microscopy and Western blotting, it was observed that LppA localizes to the surface membrane. Recombinant LppA demonstrated dose-dependent adherence to the membrane of embryonic bovine lung (EBL) cells, and this adhesion was inhibited by anti-LppA serum. In vitro binding assays confirmed LppA’s ability to associate with fibronectin, collagen IV, laminin, vitronectin, plasminogen, and tPA, thereby facilitating the conversion of plasminogen to plasmin. Moreover, LppA was found to bind and enhance the accumulation of Annexin A2 (ANXA2) on the cell membrane. Disrupting LppA in M. bovis significantly diminished the bacterium’s capacity to adhere to EBL cells, underscoring LppA’s function as a bacterial adhesin. In conclusion, LppA emerges as a novel adhesion protein that interacts with multiple host extracellular matrix proteins and ANXA2, playing a crucial role in M. bovis’s adherence to host cells and dissemination. These insights substantially deepen our comprehension of the molecular pathogenesis of M. bovis.

Serine-rich repeat proteins: well-known yet little-understood bacterial adhesins.

Serine-rich-repeat proteins (SRRPs) are large mucin-like glycoprotein adhesins expressed by a plethora of pathogenic and symbiotic Gram-positive bacteria. SRRPs play major functional roles in bacterial-host interactions, like adhesion, aggregation, biofilm formation, virulence, and pathogenesis. Through their functional roles, SRRPs aid in the development of host microbiomes but also diseases like infective endocarditis, otitis media, meningitis, and pneumonia. SRRPs comprise shared domains across different species, including two or more heavily O-glycosylated long stretches of serine-rich repeat regions. With loci that can be as large as ~40 kb and can encode up to 10 distinct glycosyltransferases that specifically facilitate SRRP glycosylation, the SRRP loci makes up a significant portion of the bacterial genome. The significance of SRRPs and their glycans in host-microbe communications is becoming increasingly evident. Studies are beginning to reveal the glycosylation pathways and mature O-glycans presented by SRRPs. Here we review the glycosylation machinery of SRRPs across species and discuss the functional roles and clinical manifestations of SRRP glycosylation.

Mechanistic Insights into the Folding Mechanism of Region V in Ice-Binding Protein Secreted by Marinomonas primoryensis Revealed by Single-Molecule Force Spectroscopy.

The Gram-negative bacteria Marinomonas primoryensis secrete an ice-binding protein (MpIBP), which is a vital bacterial adhesin facilitating the adaptation and survival of the bacteria in the harsh Antarctic environment. The C-terminal region of MpIBP, known as region V (RV), is the first domain to be exported into the Ca2+-rich extracellular environment and acts as a folding nucleus for the entire adhesin. However, the mechanisms underlying the secretion and folding of RV remain poorly understood. Here, we used optical tweezers (OT) to investigate the secretion and folding mechanisms of RV at the single-molecule level. In the absence of Ca2+, apo-RV remains unstructured, while Ca2+-bound RV folds into a mechanically stable structure. The folding of RV could occur via the formation of an intermediate state. Even though this folding intermediate is "hidden" during the folding process of wild type RV in vitro, it likely forms in vivo and plays an important role in facilitating protein secretion. Additionally, our results revealed that the N-terminal part of the RV can significantly stabilize its C-terminal structure. Our study paves the way for further investigations into the structure and functions of MpIBP that help bacteria survive in challenging environments.

Modular Bioorthogonal Lipid Nanoparticle Modification Platforms for Cardiac Homing.

Lipid nanoparticles (LNPs) are becoming widely adopted as vectors for the delivery of therapeutic payloads but generally lack intrinsic tissue-homing properties. These extracellular vesicle (EV) mimetics can be targeted toward the liver, lung, or spleen via charge modification of their lipid headgroups. Homing to other tissues has only been achieved via covalent surface modification strategies using small-molecule ligands, peptides, or monoclonal antibodies─methods that are challenging to couple with large-scale manufacturing. Herein, we design a novel modular artificial membrane-binding protein (AMBP) platform for the modification of LNPs postformation. The system is composed of two protein modules that can be readily coupled using bioorthogonal chemistry to yield the AMBP. The first is a membrane anchor module comprising a supercharged green fluorescent protein (scGFP) electrostatically conjugated to a dynamic polymer surfactant corona. The second is a functional module containing a cardiac tissue fibronectin homing sequence from the bacterial adhesin CshA. We demonstrate that LNPs modified using the AMBP exhibit a 20-fold increase in uptake by fibronectin-rich C2C12 cells under static conditions and a 10-fold increase under physiologically relevant shear stresses, with no loss of cell viability. Moreover, we show targeted localization of the AMBP-modified LNPs in zebrafish hearts, highlighting their therapeutic potential as a vector for the treatment of cardiac disease and, more generally, as a smart vector.

Rate limiting step of the allosteric activation of the bacterial adhesin FimH investigated by molecular dynamics simulations.

The bacterial adhesin FimH is a model for the study of protein allostery because its structure has been resolved in multiple configurations, including the active and the inactive state. FimH consists of a pilin domain (PD) that anchors it to the rest of the fimbria and an allosterically regulated lectin domain (LD) that binds mannose on the surface of infected cells. Under normal conditions, the two domains are docked to each other and LD binds mannose weakly. However, in the presence of tensile force generated by shear the domains separate and conformational changes propagate across LD resulting in a stronger bond to mannose. Recently, the crystallographic structure of a variant of FimH has been resolved, called , where PD contains 10 mutations near the inter-domain interface. Although the X-ray structures of FimH and are almost identical, experimental evidence shows that is activated even in the absence of shear. Here, molecular dynamics simulations combined with the Jarzynski equality were used to investigate the discrepancy between the crystallographic structures and the functional assays. The results indicate that the free energy barrier of the unbinding process between LD and PD is drastically reduced in . Rupture of inter-domain hydrogen bonds involving R166 constitutes a rate limiting step of the domain separation process and occurs more readily in than FimH. In conclusion, the mutations in shift the equilibrium toward an equal occupancy of bound and unbound states for LD and PD by reducing a rate limiting step.

Glycosylated extracellular mucin domains protect against SARS-CoV-2 infection at the respiratory surface.

Mucins play an essential role in protecting the respiratory tract against microbial infections while also acting as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance. Transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on epithelial entry of SARS-CoV-2. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of sialic acid and fucose from the epithelial surface did not impact viral entry. In Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are located to the apical surface in close proximity and StcE treatment results in enhanced binding of purified spike protein. Both MUC1 and MUC16 are expressed on the surface of human organoid-derived air-liquid interface (ALI) differentiated airway cultures and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 replication. In these cultures, MUC1 was highly expressed in non-ciliated cells while MUC16 was enriched in goblet cells. In conclusion, the glycosylated extracellular domains of different transmembrane mucins might have similar protective functions in different respiratory cell types by restricting SARS-CoV-2 binding and entry.