Identification of a novel streptococcal adhesin P (SadP) protein recognizing galactosyl-α1-4-galactose-containing glycoconjugates: convergent evolution of bacterial pathogens to binding of the same host receptor

Bacterial adhesion is often a prerequisite for infection, and host cell surface carbohydrates play a major role as adhesion receptors. Streptococci are a leading cause of infectious diseases. However, only few carbohydrate-specific streptococcal adhesins are known. Streptococcus suis is an important pig pathogen and a zoonotic agent causing meningitis in pigs and humans. In this study, we have identified an adhesin that mediates the binding of S. suis to galactosyl-α1-4-galactose (Galα1-4Gal)-containing host receptors. A functionally unknown S. suis cell wall protein (SSU0253), designated here as SadP (streptococcal adhesin P), was identified using a Galα1-4Gal-containing affinity matrix and LC-ESI mass spectrometry. Although the function of the protein was not previously known, it was recently identified as an immunogenic cell wall protein in a proteomic study. Insertional inactivation of the sadP gene abolished S. suis Galα1-4Gal-dependent binding. The adhesin gene sadP was cloned and expressed in Escherichia coli. Characterization of its binding specificity showed that SadP recognizes Galα1-4Gal-oligosaccharides and binds its natural glycolipid receptor, GbO(3) (CD77). The N terminus of SadP was shown to contain a Galα1-Gal-binding site and not to have apparent sequence similarity to other bacterial adhesins, including the E. coli P fimbrial adhesins, or to E. coli verotoxin or Pseudomonas aeruginosa lectin I also recognizing the same Galα1-4Gal disaccharide. The SadP and E. coli P adhesins represent a unique example of convergent evolution toward binding to the same host receptor structure.     

Cellular interactions by LPxTG-anchored pneumococcal adhesins and their streptococcal homologues

In this review we focus on three important families of LPxTG-anchored adhesins in the human pathogen Streptococcus pneumoniae, but also their homologues in related streptococci. We discuss the contribution of these streptococcal adhesins to host tropism, pathogenesis and their interactions with different host cell types. The first surface structures discussed are the heteropolymeric pili that have been found in important streptococcal pathogens such as S. pneumoniae, S. pyogenes, S. agalactiae and E. faecalis/faecium. Major and minor pilus subunit proteins are covalently joined and finally attached to the cell wall through the action of specific sortases. The role of pili and individual pilin subunits in adhesion and pathogenesis and their structure and assembly in different streptococcal species are being covered. Furthermore, we address recent findings regarding a family of large glycosylated serine-rich repeat (SRR) proteins that act as fibrillar adhesins for which homologues have been found in several streptococcal species including pneumococci. In the pneumococcal genome both pili and its giant SRR protein are encoded by accessory genes present in particular clonal lineages for which epidemiological information is available. Finally, we briefly discuss the role played by the pneumococcal neuraminidase NanA in adhesion and pathogenesis.     

Streptococcus Adherence and Colonization

Summary: Streptococci readily colonize mucosal tissues in the nasopharynx; the respiratory, gastrointestinal, and genitourinary tracts; and the skin. Each ecological niche presents a series of challenges to successful colonization with which streptococci have to contend. Some species exist in equilibrium with their host, neither stimulating nor submitting to immune defenses mounted against them. Most are either opportunistic or true pathogens responsible for diseases such as pharyngitis, tooth decay, necrotizing fasciitis, infective endocarditis, and meningitis. Part of the success of streptococci as colonizers is attributable to the spectrum of proteins expressed on their surfaces. Adhesins enable interactions with salivary, serum, and extracellular matrix components; host cells; and other microbes. This is the essential first step to colonization, the development of complex communities, and possible invasion of host tissues. The majority of streptococcal adhesins are anchored to the cell wall via a C-terminal LPxTz motif. Other proteins may be surface anchored through N-terminal lipid modifications, while the mechanism of cell wall associations for others remains unclear. Collectively, these surface-bound proteins provide Streptococcus species with a “coat of many colors,” enabling multiple intimate contacts and interplays between the bacterial cell and the host. In vitro and in vivo studies have demonstrated direct roles for many streptococcal adhesins as colonization or virulence factors, making them attractive targets for therapeutic and preventive strategies against streptococcal infections. There is, therefore, much focus on applying increasingly advanced molecular techniques to determine the precise structures and functions of these proteins, and their regulatory pathways, so that more targeted approaches can be developed.     

Binding of Dr adhesins of Escherichia coli to carcinoembryonic antigen triggers receptor dissociation

Carcinoembryonic antigen (CEA)-related cell adhesion molecules (CEACAMs) are host receptors for the Dr family of adhesins of Escherichia coli. To define the mechanism for binding of Dr adhesins to CEACAM receptors, we carried out structural studies on the N-terminal domain of CEA and its complex with the Dr adhesin. The crystal structure of CEA reveals a dimer similar to other dimers formed by receptors with IgV-like domains. The structure of the CEA/Dr adhesin complex is proposed based on NMR spectroscopy and mutagenesis data in combination with biochemical characterization. The Dr adhesin/CEA interface overlaps appreciably with the region responsible for CEA dimerization. Binding kinetics, mutational analysis and spectroscopic examination of CEA dimers suggest that Dr adhesins can dissociate CEA dimers prior to the binding of monomeric forms. Our conclusions include a plausible mechanism for how E. coli, and perhaps other bacterial and viral pathogens, exploit CEACAMs. The present structure of the complex provides a powerful tool for the design of novel inhibitory strategies to treat E. coli infections.     

Adaptation of the group A Streptococcus adhesin Scl1 to bind fibronectin type III repeats within wound‐associated extracellular matrix: implications for cancer therapy

The human‐adapted pathogen group A Streptococcus (GAS) utilizes wounds as portals of entry into host tissue, wherein surface adhesins interact with the extracellular matrix, enabling bacterial colonization. The streptococcal collagen‐like protein 1 (Scl1) is a major adhesin of GAS that selectively binds to two fibronectin type III (FnIII) repeats within cellular fibronectin, specifically the alternatively spliced extra domains A and B, and the FnIII repeats within tenascin‐C. Binding to FnIII repeats was mediated through conserved structural determinants present within the Scl1 globular domain and facilitated GAS adherence and biofilm formation. Isoforms of cellular fibronectin that contain extra domains A and B, as well as tenascin‐C, are present for several days in the wound extracellular matrix. Scl1‐FnIII binding is therefore an example of GAS adaptation to the host's wound environment. Similarly, cellular fibronectin isoforms and tenascin‐C are present in the tumor microenvironment. Consistent with this, FnIII repeats mediate GAS attachment to and enhancement of biofilm formation on matrices deposited by cancer‐associated fibroblasts and osteosarcoma cells. These data collectively support the premise for utilization of the Scl1‐FnIII interaction as a novel method of anti‐neoplastic targeting in the tumor microenvironment.     

Molecular phylogenetics of ascomycotal adhesins--a novel family of putative cell-surface adhesive proteins in fission yeasts.

In this work, we identify a family of putative adhesins in the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus. The members of this family share a conserved tandem repeat related to those found in the Candida albicans Als family of adhesins. Unlike previously characterised adhesins that possess conserved ligand-binding domains at the N-terminus, this group of proteins carry ligand-binding domains at their C-termini. We demonstrate that one such domain--the uncharacterised GLEYA domain, is related to the lectin-like ligand-binding domain found in the Saccharomyces cerevisiae Flo proteins. Unlike the Flo and Als proteins, the fission yeast adhesins do not contain detectable glycosyl phosphatidyl inositol (GPI) membrane anchor signals to mediate their attachment to the cell wall, which may suggest a novel cell wall attachment mechanism. Further sequence analysis identified several putative adhesins in the sub-phylum of Pezizomycotina, where only a few adhesins have been described to date.     

Molecular phylogenetics of ascomycotal adhesins--a novel family of putative cell-surface adhesive proteins in fission yeasts

In this work, we identify a family of putative adhesins in the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus. The members of this family share a conserved tandem repeat related to those found in the Candida albicans Als family of adhesins. Unlike previously characterised adhesins that possess conserved ligand-binding domains at the N-terminus, this group of proteins carry ligand-binding domains at their C-termini. We demonstrate that one such domain--the uncharacterised GLEYA domain, is related to the lectin-like ligand-binding domain found in the Saccharomyces cerevisiae Flo proteins. Unlike the Flo and Als proteins, the fission yeast adhesins do not contain detectable glycosyl phosphatidyl inositol (GPI) membrane anchor signals to mediate their attachment to the cell wall, which may suggest a novel cell wall attachment mechanism. Further sequence analysis identified several putative adhesins in the sub-phylum of Pezizomycotina, where only a few adhesins have been described to date.     

Bacterial protein domains with a novel Ig‐like fold target human CEACAM receptors

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is the major cause of neonatal sepsis in humans. A critical step to infection is adhesion of bacteria to epithelial surfaces. GBS adhesins have been identified to bind extracellular matrix components and cellular receptors. However, several putative adhesins have no host binding partner characterised. We report here that surface‐expressed β protein of GBS binds to human CEACAM1 and CEACAM5 receptors. A crystal structure of the complex showed that an IgSF domain in β represents a novel Ig‐fold subtype called IgI3, in which unique features allow binding to CEACAM1. Bioinformatic assessment revealed that this newly identified IgI3 fold is not exclusively present in GBS but is predicted to be present in adhesins from other clinically important human pathogens. In agreement with this prediction, we found that CEACAM1 binds to an IgI3 domain found in an adhesin from a different streptococcal species. Overall, our results indicate that the IgI3 fold could provide a broadly applied mechanism for bacteria to target CEACAMs.     

Crystal structure and mutational analysis of the DaaE adhesin of Escherichia coli

DaaE is a member of the Dr adhesin family of Escherichia coli, members of which are associated with diarrhea and urinary tract infections. A receptor for Dr adhesins is the cell surface protein, decay-accelerating factor (DAF). We have carried out a functional analysis of Dr adhesins, as well as mutagenesis and crystallographic studies of DaaE, to obtain detailed molecular information about interactions of Dr adhesins with their receptors. The crystal structure of DaaE has been solved at 1.48 A resolution. Trimers of the protein are found in the crystal, as has been the case for other Dr adhesins. Naturally occurring variants and directed mutations in DaaE have been generated and analyzed for their ability to bind DAF. Mapping of the mutation sites onto the DaaE molecular structure shows that several of them contribute to a contiguous surface that is likely the primary DAF-binding site. The DAF-binding properties of purified fimbriae and adhesin proteins from mutants and variants correlated with the ability of bacteria expressing these proteins to bind to human epithelial cells in culture. DaaE, DraE, AfaE-III, and AfaE-V interact with complement control protein (CCP) domains 2-4 of DAF, and analysis of the ionic strength dependence of their binding indicates that the intermolecular interactions are highly electrostatic in nature. The adhesins AfaE-I and NfaE-2 bind to CCP-3 and CCP-4 of DAF, and electrostatic interactions contribute significantly less to these interactions. These observations are consistent with structural predictions for these Dr variants and also suggest a role for the positively charged region linking CCP-2 and CCP-3 of DAF in electrostatic Dr adhesin-DAF interactions.     

The conserved PA14 domain of cell wall-associated fungal adhesins governs their glycan-binding specificity

Yeast cell wall-associated, lectin-like adhesins form large families that mediate flocculation and host cell recognition. The glycan specificity of individual adhesins is largely unknown. Zupancic et al . (this issue of Molecular Microbiology ) used glycan microarrays to compare the glycan-binding characteristics of individual adhesins (Epa proteins) of the pathogenic yeast Candida glabrata produced in the non-adherent yeast Saccharomyces cerevisiae . By sequence swapping between the conserved PA14 domains of two related Epa proteins, they identified a pentapeptide that determines binding specificity and cell adherence and is located on a surface loop of the known crystal structure of the anthrax toxin PA14 domain.     

Posttranslational modifications required for cell surface localization and function of the fungal adhesin Aga1p

Adherence of fungal cells to host substrates and each other affects their access to nutrients, sexual conjugation, and survival in hosts. Adhesins are cell surface proteins that mediate these different cell adhesion interactions. In this study, we examine the in vivo functional requirements for specific posttranslational modifications to these proteins, including glycophosphatidylinositol (GPI) anchor addition and O-linked glycosylation. The processing of some fungal GPI anchors, creating links to cell wall β-1,6 glucans, is postulated to facilitate postsecretory traffic of proteins to cell wall domains conducive to their functions. By studying the yeast sexual adhesin subunit Aga1p, we found that deletion of its signal sequence for GPI addition eliminated its activity, while deletions of different internal domains had various effects on function. Substitution of the Aga1p GPI signal domain with those of other GPI-anchored proteins, a single transmembrane domain, or a cysteine capable of forming a disulfide all produced functional adhesins. A portion of the cellular pool of Aga1p was determined to be cell wall resident. Aga1p and the α-agglutinin Agα1p were shown to be under glycosylated in cells lacking the protein mannosyltransferase genes PMT1 and PMT2, with phenotypes manifested only in MATα cells for single mutants but in both cell types when both genes are absent. We conclude that posttranslational modifications to Aga1p are necessary for its biogenesis and activity. Our studies also suggest that in addition to GPI-glucan linkages, other cell surface anchorage mechanisms, such as transmembrane domains or disulfides, may be employed by fungal species to localize adhesins.     

Candida-host cell receptor-ligand interactions

The interaction of Candida species with their cognate host receptors is a key factor in the pathogenesis of different types of candidiasis. The recognition of different forms of Candida albicans by Toll-like receptors 2 and 4 on mononuclear leukocytes has recently been discovered to determine the function and activity of regulatory T-cells, determine the balance of Type 1 and Type 2 cytokines and, thereby, influence the antifungal activity of both the innate and adaptive immune response. Different forms of C. albicans are also recognized by different lectins that are expressed on the surface macrophages. C. albicans and Candida glabrata express the ALS (agglutinin-like sequence) and EPA (epithelial adhesin) families of adhesins, respectively. A key difference between C. glabrata and C. albicans is that EPA expression in C. glabrata is governed by sub-telomeric silencing, whereas ALS expression in C. albicans is regulated by other mechanisms.     

细菌黏附素序列模体分析

目的：认识细菌黏附素序列保守的特征,更好地理解细菌黏附的机理。方法：利用InterProScan、MEME等分析工具对实验确认的155条细菌黏附素序列进行模体搜索。结果：用InterProScan在155个黏附素序列中搜索到119个模体,通过MEME分析发现了50个模体。结论：发现了一些与黏附功能相关的模体,为在细菌基因组内搜索黏附素序列奠定了基础。     

The fimbrial adhesin F17-G of enterotoxigenic Escherichia coli has an immunoglobulin-like lectin domain that binds N-acetylglucosamine

The F17-G adhesin at the tip of flexible F17 fimbriae of enterotoxigenic Escherichia coli mediates binding to N-acetyl-beta-D-glucosamine-presenting receptors on the microvilli of the intestinal epithelium of ruminants. We report the 1.7 A resolution crystal structure of the lectin domain of F17-G, both free and in complex with N-acetylglucosamine. The monosaccharide is bound on the side of the ellipsoid-shaped protein in a conserved site around which all natural variations of F17-G are clustered. A model is proposed for the interaction between F17-fimbriated E. coli and microvilli with enhanced affinity compared with the binding constant we determined for F17-G binding to N-acetylglucosamine (0.85 mM-1). Unexpectedly, the F17-G structure reveals that the lectin domains of the F17-G, PapGII and FimH fimbrial adhesins all share the immunoglobulin-like fold of the structural components (pilins) of their fimbriae, despite lack of any sequence identity. Fold comparisons with pilin and chaperone structures of the chaperone/usher pathway highlight the central role of the C-terminal beta-strand G of the immunoglobulin-like fold and provides new insights into pilus assembly, function and adhesion.     

Use of synthetic peptides to confirm that the Pseudomonas aeruginosa PAK pilus adhesin and the Candida albicans fimbrial adhesin possess a homologous receptor-binding domain

Pseudomonas aeruginosa PAK pili and Candida albicans fimbriae are adhesins present on the microbial cell surfaces which mediate binding to epithelial cell-surface receptors. The receptor-binding domain (adhesintope) of the PAK pilus adhesin has been shown previously to reside in the carboxy-terminal disulphide-bonded region of P. aeruginosa pilin (PAK128-144). The delineation of the C. albicans fimbrial adhesintope was investigated in these studies using synthetic peptides which correspond to the whole (PAK128-144) or part of (PAK134-140) adhesintope of the PAK pilus and their respective anti-peptide antisera and biotinylated PAK pili (Bt-PAK pili), fimbriae (Bt-fimbriae), P. aeruginosa whole cells (Bt- P. aeruginosa ) and C. albicans whole cells (Bt- C. albicans ). The results from these studies confirmed that a structurally conserved motif akin to the PAK(128-144) peptide sequence is present in C. albicans fimbrial adhesin and that the seven-amino-acid residue PAK(134-140) sequence plays an important role in forming the adhesintope for both P. aeruginosa PAK pilus and C. albicans fimbrial adhesins.     

The Extracellular Protein Factor Epf from Streptococcus pyogenes Is a Cell Surface Adhesin That Binds to Cells through an N-terminal Domain Containing a Carbohydrate-binding Module

Streptococcus pyogenes is an exclusively human pathogen. Streptococcal attachment to and entry into epithelial cells is a prerequisite for a successful infection of the human host and requires adhesins. Here, we demonstrate that the multidomain protein Epf from S. pyogenes serotype M49 is a streptococcal adhesin. An epf-deficient mutant showed significantly decreased adhesion to and internalization into human keratinocytes. Cell adhesion is mediated by the N-terminal domain of Epf (EpfN) and increased by the human plasma protein plasminogen. The crystal structure of EpfN, solved at 1.6 Å resolution, shows that it consists of two subdomains: a carbohydrate-binding module and a fibronectin type III domain. Both fold types commonly participate in ligand receptor and protein-protein interactions. EpfN is followed by 18 repeats of a domain classified as DUF1542 (domain of unknown function 1542) and a C-terminal cell wall sorting signal. The DUF1542 repeats are not involved in adhesion, but biophysical studies show they are predominantly α-helical and form a fiber-like stalk of tandem DUF1542 domains. Epf thus conforms with the widespread family of adhesins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), in which a cell wall-attached stalk enables long range interactions via its adhesive N-terminal domain.     

Allosteric catch bond properties of the FimH adhesin from Salmonella enterica serovar Typhimurium

Despite sharing the name and the ability to mediate mannose-sensitive adhesion, the type 1 fimbrial FimH adhesins of Salmonella Typhimurium and Escherichia coli share only 15% sequence identity. In the present study, we demonstrate that even with this limited identity in primary sequence, these two proteins share remarkable similarity of complex receptor binding and structural properties. In silico simulations suggest that, like E. coli FimH, Salmonella FimH has a two-domain tertiary structure topology, with a mannose-binding pocket located on the apex of a lectin domain. Structural analysis of mutations that enhance S. Typhimurium FimH binding to eukaryotic cells and mannose-BSA demonstrated that they are not located proximal to the predicted mannose-binding pocket but rather occur in the vicinity of the predicted interface between the lectin and pilin domains of the adhesin. This implies that the functional effect of such mutations is indirect and probably allosteric in nature. By analogy with E. coli FimH, we suggest that Salmonella FimH functions as an allosteric catch bond adhesin, where shear-induced separation of the lectin and pilin domains results in a shift from a low affinity to a high affinity binding conformation of the lectin domain. Indeed, we observed shear-enhanced binding of whole bacteria expressing S. Typhimurium type 1 fimbriae. In addition, we observed that anti-FimH antibodies activate rather than inhibit S. Typhimurium FimH mannose binding, consistent with the allosteric catch bond properties of this adhesin.     

Nanoscale mapping and functional analysis of individual adhesins on living bacteria

Although much progress has been made in the identification and characterization of adhesins borne by pathogenic bacteria, the molecular details underlying their interaction with host receptors remain largely unknown owing to the lack of appropriate probing techniques. Here we report a method, based on atomic force microscopy (AFM) with tips bearing biologically active molecules, for measuring the specific binding forces of individual adhesins and for mapping their distribution on the surface of living bacteria. First, we determined the adhesion forces between the heparin-binding haemagglutinin adhesin (HBHA) produced by Mycobacterium tuberculosis and heparin, used as a model sulphated glycoconjugate receptor. Both the adhesion frequency and adhesion force increased with contact time, indicating that the HBHA-heparin complex is formed via multiple intermolecular bridges. We then mapped the distribution of single HBHA molecules on the surface of living mycobacteria and found that the adhesin is not randomly distributed over the mycobacterial surface, but concentrated into nanodomains.     

Strengthening relationships: amyloids create adhesion nanodomains in yeasts

Budding yeasts adhere to biotic or abiotic surfaces and aggregate to form biofilms, using wall-anchored glycoprotein adhesins. The process is paradoxical: adhesins often show weak binding to specific ligands, yet mediate remarkably strong adherence. Single-molecule atomic force microscopy (AFM), genomics, biochemistry and cell biology have recently explained the puzzle, with Candida albicans Als adhesins as the paradigm. The strength of adhesion results partly from force-activated amyloid-like clustering of hundreds of adhesin molecules to form arrays of ordered multimeric binding sites. The various protein domains of eukaryotic adhesins cooperate to facilitate this fascinating new mechanism of activation.     

Identification and Differential Gene Expression of Adhesin-Like Wall Proteins in <Emphasis Type="Italic">Candida glabrata</Emphasis> Biofilms

An important initial step in biofilm development and subsequent establishment of fungal infections by the human pathogen Candida glabrata is adherence to a surface. Adherence is mediated through a large number of differentially regulated cell wall-bound adhesins. The fungus can modify the incorporation of adhesins in the cell wall allowing crucial adaptations to new environments. In this study, expression and cell wall incorporation of C. glabrata adhesins were evaluated in biofilms cultured in two different media: YPD and a semi-defined medium SdmYg. Tandem mass spectrometry of isolated C. glabrata cell walls identified 22 proteins including six adhesins: the novel adhesins Awp5 and Awp6, Epa3 and the previously identified adhesins Epa6, Awp2 and Awp4. Regulation of expression of these and other relevant adhesin genes was investigated using real-time qPCR analysis. For most adhesin genes, significant up-regulation was observed in biofilms in at least one of the culturing media. However, this was not the case for EPA6 and AWP2, which is consistent with their gene products already being abundantly present in planktonic cultures grown in YPD medium. Furthermore, most of the adhesin genes tested also show medium-dependent differential regulation. These results underline the idea that many adhesins in C. glabrata are involved in biofilm formation and that their expression is tightly regulated and dependent on environmental conditions and growth phase. This may contribute to its potential to form resilient biofilms and cause infection in various host tissues.