A structural overview of mycobacterial adhesins: Key biomarkers for diagnostics and therapeutics

Adherence, colonization, and survival of mycobacteria in host cells require surface adhesins, which are attractive pharmacotherapeutic targets. A large arsenal of pilus and non‐pilus adhesins have been identified in mycobacteria. These adhesins are capable of interacting with host cells, including macrophages and epithelial cells and are essential to microbial pathogenesis. In the last decade, several structures of mycobacterial adhesins responsible for adhesion to either macrophages or extra cellular matrix proteins have been elucidated. In addition, key structural and functional information have emerged for the process of mycobacterial adhesion to epithelial cells, mediated by the Heparin‐binding hemagglutinin (HBHA). In this review, we provide an overview of the structural and functional features of mycobacterial adhesins and discuss their role as important biomarkers for diagnostics and therapeutics. Based on the reported data, it appears clear that adhesins are endowed with a variety of different structures and functions. Most adhesins play important roles in the cell life of mycobacteria and are key virulence factors. However, they have adapted to an extracellular life to exert a role in host‐pathogen interaction. The type of interactions they form with the host and the adhesin regions involved in binding is partly known and is described in this review.     

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.     

A subfamily of Dr adhesins of Escherichia coli bind independently to decay-accelerating factor and the N-domain of carcinoembryonic antigen

Escherichia coli expressing the Dr family of adhesins adheres to epithelial cells by binding to decay-accelerating factor (DAF) and carcinoembryonic antigen (CEA)-related cell surface proteins. The attachment of bacteria expressing Dr adhesins to DAF induces clustering of DAF around bacterial cells and also recruitment of CEA-related cell adhesion molecules. CEA, CEACAM1, and CEACAM6 have been shown to serve as receptors for some Dr adhesins (AfaE-I, AfaE-III, DraE, and DaaE). We demonstrate that AfaE-I, AfaE-V, DraE, and DaaE adhesins bind to the N-domain of CEA. To identify the residues involved in the N-CEA/DraE interaction, we performed SPR binding analyses of naturally occurring variants and a number of randomly generated mutants in DraE and N-CEA. Additionally, we used chemical shift mapping by NMR to determine the surface of DraE involved in N-CEA binding. These results show a distinct CEA binding site located primarily in the A, B, E, and D strands of the Dr adhesin. Interestingly, this site is located opposite to the beta-sheet encompassing the previously determined binding site for DAF, which implies that the adhesin can bind simultaneously to both receptors on the epithelial cell surface. The recognition of CEACAMs from a highly diverse DrCEA subfamily of Dr adhesins indicates that interaction with these receptors plays an important role in niche adaptation of E. coli strains expressing Dr adhesins.     

Selection for functional diversity drives accumulation of point mutations in Dr adhesins of Escherichia coli

Immune escape is considered to be the driving force behind structural variability of major antigens on the surface of bacterial pathogens, such as fimbriae. In the Dr family of Escherichia coli adhesins, structural and adhesive functions are carried out by the same subunit. Dr adhesins have been shown to bind decay-accelerating factor (DAF), collagen IV, and carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). We show that genes encoding Dr adhesins from 100 E. coli strains form eight structural groups with a high level of amino acid sequence diversity between them. However, genes comprising each group differ from each other by only a small number of point mutations. Out of 66 polymorphisms identified within the groups, only three were synonymous mutations, indicating strong positive selection for amino acid replacements. Functional analysis of intragroup variants comprising the Dr haemagglutinin (DraE) group revealed that the point mutations result in distinctly different binding phenotypes, with a tendency of increased affinity to DAF, decreased sensitivity of DAF binding to inhibition by chloramphenicol, and loss of binding capability to collagen, CEACAM3 and CEACAM6. Thus, variability by point mutation of major antigenic proteins on the bacterial surface can be a signature of selection for functional modification.     

Mutational analysis of receptor binding mediated by the Dr family of Escherichia coli adhesins

The fimbrial and afimbrial adhesins of the Dr family mediate the adherence of uropathogenic and diarrhoea-associated Escherichia coli to decay-accelerating factor (DAF) present on erythrocytes and other cell types. The Dr haemagglutinin binds type IV collagen and, unlike other members of the Dr family, mediates an adherence inhibited in the presence of chloramphenicol. We examined the ability of other members of the Dr family—AFAI, AFAIII, and F1845—to bind to type IV collagen, and demonstrated that the collagen-binding phenotype was unique to the Dr haemagglutinin. We employed site-directed mutagenesis to demonstrate the requirement of a negatively charged amino-acid at position 54 of the Dr haemagglutinin subunit for chloramphenicol sensitivity of binding. Mutations at position 32, 40, 54, 90, and 113 differently affected type IV collagen binding and chloramphenicol sensitivity of binding, while retaining DAF-binding capability. These results suggest the existence of a conformational receptor-binding domain in the major structural subunit of Dr family adhesins and demonstrate that chloramphenicol sensitivity of binding and adherence to type IV collagen were independent and separable phenotypes. Finally, we showed that the two conserved cysteine residues of Dr family structural subunits form a disulphide bond and that mutations of these residues abolish haemagglutination and binding to type IV collagen.     

Host-specificity of uropathogenic Escherichia coli depends on differences in binding specificity to Gal alpha 1-4Gal-containing isoreceptors.

Four G adhesins, cloned from uropathogenic Escherichia coli strains, were examined for binding to glycolipids and various eukaryotic cells. PapGAD110 and PapGIA2 showed virtually identical binding patterns to Gal alpha 1-4Gal-containing glycolipids, while PapGJ96 differed slightly and PrsGJ96 markedly with respect to the effect of neighbouring groups on the binding. Their hemagglutination patterns confirmed the existence of three receptor-binding specificities. While the PapG adhesins bound to uroepithelial cells from man (T24) but not to those from the dog (MDCK II), the reverse was true of PrsG. These binding patterns were largely explained by the absence or presence of appropriate glycolipid isoreceptors, although the inability of the PapG adhesins to bind MDCK II cells was attributed to an inappropriate presentation of their receptor epitopes. The high prevalence of PrsG-like specificities observed among wild-type dog uropathogenic E. coli isolates, together with the determined isoreceptor composition of human and dog kidney target tissues, suggest variation in receptor specificity as a mechanism for shifting host specificity, and that this variation has evolved in response to the topography of the host cellular receptors. The receptor-binding half proposed for the predicted amino acid sequences of the four G adhesins and the corresponding adhesin of one of the dog E. coli isolates varied considerably among the three receptor-binding groups of adhesins, but only little within each group.     

Identification of Novel Adhesins of M. tuberculosis H37Rv Using Integrated Approach of Multiple Computational Algorithms and Experimental Analysis

Pathogenic bacteria interacting with eukaryotic host express adhesins on their surface. These adhesins aid in bacterial attachment to the host cell receptors during colonization. A few adhesins such as Heparin binding hemagglutinin adhesin (HBHA), Apa, Malate Synthase of M. tuberculosis have been identified using specific experimental interaction models based on the biological knowledge of the pathogen. In the present work, we carried out computational screening for adhesins of M. tuberculosis. We used an integrated computational approach using SPAAN for predicting adhesins, PSORTb, SubLoc and LocTree for extracellular localization, and BLAST for verifying non-similarity to human proteins. These steps are among the first of reverse vaccinology. Multiple claims and attacks from different algorithms were processed through argumentative approach. Additional filtration criteria included selection for proteins with low molecular weights and absence of literature reports. We examined binding potential of the selected proteins using an image based ELISA. The protein Rv2599 (membrane protein) binds to human fibronectin, laminin and collagen. Rv3717 (N-acetylmuramoyl-L-alanine amidase) and Rv0309 (L,D-transpeptidase) bind to fibronectin and laminin. We report Rv2599 (membrane protein), Rv0309 and Rv3717 as novel adhesins of M. tuberculosis H37Rv. Our results expand the number of known adhesins of M. tuberculosis and suggest their regulated expression in different stages.     

Interaction of Pseudomonas aeruginosa galactophilic lectin PA-IL with pigeon egg white glycoproteins

Pseudomonas aeruginosa produces a galactophilic lectin, PA-IL, that resembles P-fimbrial adhesins of uropathogenic Escherichia coli strains in binding to human P blood group antigens. We examined, in the present study, its interaction with pigeon egg white glycoproteins carrying N-glycans with terminal Galalpha1-4Gal which inhibit the adhesion of P-fimbriae. For comparison, the lectin concanavalin A (Con A) and additional avian egg whites (of hen and quail) were also examined. The results obtained in both hemagglutination inhibition and Western blot analyses showed that PA-IL, unlike Con A, preferentially reacted with the pigeon egg white glycoproteins. These results, which confirmed PA-IL similarity in sugar specificity to E. coli P-fimbriae, demonstrated the advantage of this purified lectin for representing P-type and additional galactophilic microbial adhesins unavailable in purified stable form, in Western blot analyses.     

Staphylococcus aureus adhesion to bone matrix and bone-associated biomaterials

Staphylococcus aureus is a frequent cause of orthopedic infections in humans. The bacterium expresses several adhesins that facilitate bacterial binding to the bone matrix and to bone implant biomaterials coated with host plasma constituents. The relevant S. aureus adhesins are termed microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) and specific MSCRAMMs are involved in bone and joint infections.     

A biochemical guide to yeast adhesins: glycoproteins for social and antisocial occasions.

Fungi are nonmotile eukaryotes that rely on their adhesins for selective interaction with the environment and with other fungal cells. Glycosylphosphatidylinositol (GPI)-cross-linked adhesins have essential roles in mating, colony morphology, host-pathogen interactions, and biofilm formation. We review the structure and binding properties of cell wall-bound adhesins of ascomycetous yeasts and relate them to their effects on cellular interactions, with particular emphasis on the agglutinins and flocculins of Saccharomyces and the Als proteins of Candida. These glycoproteins share common structural motifs tailored to surface activity and biological function. After being secreted to the outer face of the plasma membrane, they are covalently anchored in the wall through modified GPI anchors, with their binding domains elevated beyond the wall surface on highly glycosylated extended stalks. N-terminal globular domains bind peptide or sugar ligands, with between millimolar and nanomolar affinities. These affinities and the high density of adhesins and ligands at the cell surface determine microscopic and macroscopic characteristics of cell-cell associations. Central domains often include Thr-rich tandemly repeated sequences that are highly glycosylated. These domains potentiate cell-to-cell binding, but the molecular mechanism of such an association is not yet clear. These repeats also mediate recombination between repeats and between genes. The high levels of recombination and epigenetic regulation are sources of variation which enable the population to continually exploit new niches and resources.     

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.     

Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier

Members of the Dr family of adhesins of Escherichia coli recognize as a receptor the Dr(a) blood-group antigen present on the complement regulatory and signalling molecule, decay-accelerating factor (DAF). One member of this family, the Dr haemagglutinin, also binds to a second receptor, type IV collagen. Structure/function information regarding these adhesins has been limited and domains directly involved in the interaction with DAF have not been determined. We devised a strategy to identify amino acids in the Dr haemagglutinin that are specifically involved in the interaction with DAF. The gene encoding the adhesive subunit, draE, was subjected to random mutagenesis and used to complement a strain defective for its expression. The resulting mutants were enriched and screened to obtain those that do not bind to DAF, but retain binding to type IV collagen. Individual amino acid changes at positions 10, 63, 65, 75, 77, 79 and 131 of the mature DraE sequence significantly reduced the ability of the DraE adhesin to bind DAF, but not collagen. Over half of the mutants obtained had substitutions within amino acids 63-81. Analysis of predicted structures of DraE suggest that these proximal residues may cluster to form a binding domain for DAF.     

Oral Streptococci Utilize a Siglec-Like Domain of Serine-Rich Repeat Adhesins to Preferentially Target Platelet Sialoglycans in Human Blood

Damaged cardiac valves attract blood-borne bacteria, and infective endocarditis is often caused by viridans group streptococci. While such bacteria use multiple adhesins to maintain their normal oral commensal state, recognition of platelet sialoglycans provides an intermediary for binding to damaged valvular endocardium. We use a customized sialoglycan microarray to explore the varied binding properties of phylogenetically related serine-rich repeat adhesins, the GspB, Hsa, and SrpA homologs from Streptococcus gordonii and Streptococcus sanguinis species, which belong to a highly conserved family of glycoproteins that contribute to virulence for a broad range of Gram-positive pathogens. Binding profiles of recombinant soluble homologs containing novel sialic acid-recognizing Siglec-like domains correlate well with binding of corresponding whole bacteria to arrays. These bacteria show multiple modes of glycan, protein, or divalent cation-dependent binding to synthetic glycoconjugates and isolated glycoproteins in vitro. However, endogenous asialoglycan-recognizing clearance receptors are known to ensure that only fully sialylated glycans dominate in the endovascular system, wherein we find these particular streptococci become primarily dependent on their Siglec-like adhesins for glycan-mediated recognition events. Remarkably, despite an excess of alternate sialoglycan ligands in cellular and soluble blood components, these adhesins selectively target intact bacteria to sialylated ligands on platelets, within human whole blood. These preferred interactions are inhibited by corresponding recombinant soluble adhesins, which also preferentially recognize platelets. Our data indicate that circulating platelets may act as inadvertent Trojan horse carriers of oral streptococci to the site of damaged endocardium, and provide an explanation why it is that among innumerable microbes that gain occasional access to the bloodstream, certain viridans group streptococci have a selective advantage in colonizing damaged cardiac valves and cause infective endocarditis.     

A Biochemical Guide to Yeast Adhesins: Glycoproteins for Social and Antisocial Occasions

Fungi are nonmotile eukaryotes that rely on their adhesins for selective interaction with the environment and with other fungal cells. Glycosylphosphatidylinositol (GPI)-cross-linked adhesins have essential roles in mating, colony morphology, host-pathogen interactions, and biofilm formation. We review the structure and binding properties of cell wall-bound adhesins of ascomycetous yeasts and relate them to their effects on cellular interactions, with particular emphasis on the agglutinins and flocculins of Saccharomyces and the Als proteins of Candida. These glycoproteins share common structural motifs tailored to surface activity and biological function. After being secreted to the outer face of the plasma membrane, they are covalently anchored in the wall through modified GPI anchors, with their binding domains elevated beyond the wall surface on highly glycosylated extended stalks. N-terminal globular domains bind peptide or sugar ligands, with between millimolar and nanomolar affinities. These affinities and the high density of adhesins and ligands at the cell surface determine microscopic and macroscopic characteristics of cell-cell associations. Central domains often include Thr-rich tandemly repeated sequences that are highly glycosylated. These domains potentiate cell-to-cell binding, but the molecular mechanism of such an association is not yet clear. These repeats also mediate recombination between repeats and between genes. The high levels of recombination and epigenetic regulation are sources of variation which enable the population to continually exploit new niches and resources.     

The use of sortase-mediated ligation for the immobilisation of bacterial adhesins onto fluorescence-labelled microspheres: a novel approach to analyse bacterial adhesion to host cells

Commensal and pathogenic bacteria express adhesive proteins on their cell surface, which are important for colonisation of the host. In Gram-positive bacteria, these adhesins are often covalently anchored to the cell wall by a sortase enzyme. A recent bioinformatic study has revealed a total of 860 predicted cell wall-anchored proteins in 94 completely sequenced genomes. The interaction of adhesins with host cells can be analysed with the use of adhesin-coated microbeads. Here we show that sortase-mediated ligation can be used for the site-specific immobilisation of adhesins to red-fluorescence microspheres. This coupling method allows for the native orientation of the adhesins on the beads. Furthermore, the high substrate specificity of the sortase enzyme allows the use of only partially purified recombinant proteins, which reduces preparation time and costs, and also prevents coupling of any contaminants that might interfere with cell binding.     

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.     

Aldolase forms a bridge between cell surface adhesins and the actin cytoskeleton in apicomplexan parasites

Host cell invasion by apicomplexan parasites requires coordinated interactions between cell surface adhesins and the parasite cytoskeleton. We have identified a complex of parasite proteins, including the actin binding protein aldolase, which specifically interacts with the C-terminal domains of several parasite adhesins belonging to the thrombospondin-related anonymous protein (TRAP) family. Binding of aldolase to the adhesin was disrupted by mutation of a critical tryptophan in the C domain, a residue that was previously shown to be essential for parasite motility. Our findings reveal a potential role for aldolase in connecting TRAP family adhesins with the cytoskeleton, and provide a model linking adhesion with motility in apicomplexan parasites.     

Capsule shields the function of short bacterial adhesins

Bacterial surface structures such as capsules and adhesins are generally regarded as important virulence factors. Here we demonstrate that capsules block the function of the self-recognizing protein antigen 43 through physical shielding. The phenomenon is not restricted to Escherichia coli but can occur in other gram-negative bacteria. Likewise, we show that other short adhesins exemplified by the AIDA-I protein are blocked by the presence of a capsule. The results support the notion that capsule polysaccharides sterically prevent receptor-target recognition of short bacterial adhesins. This negative interference has important biological consequences, such as affecting the ability of bacteria to form biofilms.     

Endocytosis-inducer adhesins produced by enteropathogenic serogroups of Escherichia coli participate on bacterial attachment to infant enterocytes

Enteropathogenic E. coli (EPEC) infection of Hep-2 cells proceeds through bacterial attachment to cell surface and internalization of adhered bacteria. EPEC attachment is a prerequisite for cell infection and is mediated by adhesins that recognize carbohydrate-containing receptors on cell membrane. Such endocytosis-inducer adhesins (EIA) also promote EPEC binding to infant enterocytes, suggesting that EIA may have an important role on EPEC gastroenteritis.     

Combining sites of bacterial fimbriae

The few known crystal structures of receptor-binding domains of fimbrial tip adhesins, FimH, PapGII, and F17G, tell us that each of these structures is unique and surprising. Despite little to no sequence identity, common to them all is their variable immunoglobulin (Ig)-fold. Nevertheless, their glycan-binding sites have evolved in different locations onto this similar scaffold, and with distinct, highly specific binding properties. Difficult to capture is the often dominant role played by the fimbrial shaft in host cell recognition and biofilm formation. The major pilin FaeG, building up the shaft of F4 fimbriae, also harbors the carbohydrate receptor-binding property and has thereto an enlarged Ig-domain, with the insertion of two beta-strands and two alpha-helices. Bordetella and CFA/I fimbriae combine a tip adhesin with major subunit adhesins. Still other fimbriae incorporate a specialized invasin at the very tip of polyadhesive fibers for uptake of bacteria in cells of the immune system and host epithelia. Finally, glycan recognition by fimbrial adhesins has often been found to coincide with the binding of cell-surface integrins and components of the extracellular matrix, such as collagen IV and laminin.