Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins

The non-fimbrial adhesins, YadA of enteropathogenic YERSINIA: species, and UspA1 and UspA2 of Moraxella catarrhalis, are established pathogenicity factors. In electron micrographs, both surface proteins appear as distinct 'lollipop'-shaped structures forming a novel type of surface projection on the outer membranes. These structures, amino acid sequence analysis of these molecules and yadA gene manipulation suggest a tripartite organization: an N-terminal oval head domain is followed by a putative coiled-coil rod and terminated by a C-terminal membrane anchor domain. In YadA, the head domain is involved in autoagglutination and binding to host cells and collagen. Analysis of the coiled-coil segment of YadA revealed unusual pentadecad repeats with a periodicity of 3.75, which differs significantly from the 3.5 periodicity found in the Moraxella UspAs and other canonical coiled coils. These findings predict that the surface projections are formed by oligomers containing right- (Yersinia) or left-handed (Moraxella) coiled coils. Strikingly, sequence comparison revealed that related proteins are found in many proteobacteria, both human pathogenic and environmental species, suggesting a common role in adaptation to specific ecological niches.     

Distinct mechanisms of integrin binding by Yersinia pseudotuberculosis adhesins determine the phagocytic response of host macrophages

The enteropathogenic yersiniae express two outer membrane adhesins, invasin and YadA, that contribute to pathogenesis. While invasin binds directly to beta1 integrin receptors with high affinity, YadA binds indirectly through extracellular matrix (ECM) components. In this study, Yersinia pseudotuberculosis inv and yadA mutants were used to investigate how these distinct binding mechanisms compare and potentially compete in activating signalling pathways and promoting bacterial uptake by host macrophages. The efficiency of adhesin-mediated phagocytic responses was found to be dependent on the relative expression of invasin and YadA on the bacterial surface as well as the expression of ECM proteins in the extracellular milieu. Under conditions of low concentrations of ECM, invasin was found to be the dominant adhesin, promoting high levels of phagocytosis coincident with robust and sustained activation of the protein tyrosine kinases Fak and Pyk2, phosphorylation of the adaptor molecule Cas and activation of the small GTPase Rac1. In the presence of higher concentrations of ECM, YadA became the dominant functional adhesin through its ability to engage integrin receptors via an ECM bridge. We propose a model whereby invasin promotes robust and prolonged activation of phagocytic signalling cascades by inducing a 'high-affinity' integrin conformation as well as integrin clustering. We postulate that YadA-ECM promotes phagocytosis through a more transient activation of signalling cascades that arises from integrin clustering in the context of a cross-linked fibrillar ECM network.     

Regulatory Protein OmpR Influences the Serum Resistance of Yersinia enterocolitica O:9 by Modifying the Structure of the Outer Membrane

The EnvZ/OmpR two-component system constitutes a regulatory pathway involved in bacterial adaptive responses to environmental cues. Our previous findings indicated that the OmpR regulator in Yersinia enterocolitica O:9 positively regulates the expression of FlhDC, the master flagellar activator, which influences adhesion/invasion properties and biofilm formation. Here we show that a strain lacking OmpR grown at 37°C exhibits extremely high resistance to the bactericidal activity of normal human serum (NHS) compared with the wild-type strain. Analysis of OMP expression in the ompR mutant revealed that OmpR reciprocally regulates Ail and OmpX, two homologous OMPs of Y. enterocolitica, without causing significant changes in the level of YadA, the major serum resistance factor. Analysis of mutants in individual genes belonging to the OmpR regulon (ail, ompX, ompC and flhDC) and strains lacking plasmid pYV, expressing YadA, demonstrated the contribution of the respective proteins to serum resistance. We show that Ail and OmpC act in an opposite way to the OmpX protein to confer serum resistance to the wild-type strain, but are not responsible for the high resistance of the ompR mutant. The serum resistance phenotype of ompR seems to be multifactorial and mainly attributable to alterations that potentiate the function of YadA. Our results indicate that a decreased level of FlhDC in the ompR mutant cells is partly responsible for the serum resistance and this effect can be suppressed by overexpression of flhDC in trans. The observation that the loss of FlhDC enhances the survival of wild-type cells in NHS supports the involvement of FlhDC regulator in this phenotype. In addition, the ompR mutant exhibited a lower level of LPS, but this was not correlated with changes in the level of FlhDC. We propose that OmpR might alter the susceptibility of Y. enterocolitica O:9 to complement-mediated killing through remodeling of the outer membrane.     

Functional mapping of the Yersinia enterocolitica adhesin YadA. Identification Of eight NSVAIG - S motifs in the amino-terminal half of the protein involved in collagen binding

The virulence plasmid-encoded YadA of Yersinia enterocolitica serotype O:3 is a 430-amino-acid outer membrane protein, synthesized with a 25-amino-acid signal peptide. YadA forms homotrimeric surface structures that function as adhesin between bacteria and collagen as well as other host proteins. The structure-function relationships of YadA were studied, and the collagen-binding determinants of YadA were located to its amino-terminal half. Collagen did not bind to any of the overlapping 16-mer YadA peptides, indicating that the collagen binding site of YadA is conformational. Epitope mapping of YadA identified 12 linear antigenic epitopes altogether. Seven epitopes were uniquely recognized by an anti-YadA antiserum able to inhibit collagen binding. Four of these epitopes shared a motif NSVAIG-S that is repeated eight times within the N-terminal half of YadA. Site-directed mutagenesis showed that these motifs are absolutely required for YadA-mediated collagen binding, revealing a novel type of collagen-binding mechanism.     

Unusual, virulence plasmid-dependent growth behavior of Yersinia enterocolitica in three-dimensional collagen gels

As a first approach to establishing a three-dimensional culture infection model, we studied the growth behavior of the extracellular pathogen Yersinia enterocolitica in three-dimensional collagen gels (3D-CoG). Surprisingly, we observed that plasmidless Y. enterocolitica was motile in the 3D-CoG in contrast to its growth in traditional motility agar at 37 degrees C. Motility at 37 degrees C was abrogated in the presence of the virulence plasmid pYV or the exclusive expression of the pYV-located Yersinia adhesion gene yadA. YadA-producing yersiniae formed densely packed (dp) microcolonies, whereas pYVDelta yadA-carrying yersiniae formed loosely packed microcolonies at 37 degrees C in 3D-CoG. Furthermore, we demonstrated that the packing density of the microcolonies was dependent on the head domain of YadA. Moreover, dp microcolony formation did not depend on the capacity of YadA to bind to collagen fibers, as demonstrated by the use of yersiniae producing collagen nonbinding YadA. By using a yopE-gfp reporter, we demonstrated Ca(2+)-dependent expression of this pYV-localized virulence gene by yersiniae in 3D-CoG. In conclusion, this study revealed unique plasmid-dependent growth behavior of yersiniae in a three-dimensional matrix environment that resembles the behavior of yersiniae (e.g., formation of microcolonies) in infected mouse tissue. Thus, this 3D-CoG model may be a first step to a more complex level of in vitro infection models that mimic living tissue, enabling us to study the dynamics of pathogen-host cell interactions.     

Contribution of trimeric autotransporter C-terminal domains of oligomeric coiled-coil adhesin (Oca) family members YadA, UspA1, EibA, and Hia to translocation of the YadA passenger domain and virulence of Yersinia enterocolitica

The Oca family is a novel class of autotransporter-adhesins with highest structural similarity in their C-terminal transmembrane region, which supposedly builds a beta-barrel pore in the outer membrane (OM). The prototype of the Oca family is YadA, an adhesin of Yersinia enterocolitica and Yersinia pseudotuberculosis. YadA forms a homotrimeric lollipop-like structure on the bacterial surface. The C-terminal regions of three YadA monomers form a barrel in the OM and translocate the trimeric N-terminal passenger domain, consisting of stalk, neck, and head region to the exterior. To elucidate the structural and functional role of the C-terminal translocator domain (TLD) and to assess its promiscuous capability with respect to transport of related passenger domains, we constructed chimeric YadA proteins, which consist of the N-terminal YadA passenger domain and C-terminal TLDs of Oca family members UspA1 (Moraxella catarrhalis), EibA (Escherichia coli), and Hia (Haemophilus influenzae). These constructs were expressed in Y. enterocolitica and compared for OM localization, surface exposure, oligomerization, adhesion properties, serum resistance, and mouse virulence. We demonstrate that all chimeric YadA proteins translocated the YadA passenger domain across the OM. Y. enterocolitica strains producing YadA chimeras or wild-type YadA showed comparable binding to collagen and epithelial cells. However, strains producing YadA chimeras were attenuated in serum resistance and mouse virulence. These results demonstrate for the first time that TLDs of Oca proteins of different origin are efficient translocators of the YadA passenger domain and that the cognate TLD of YadA is essential for bacterial survival in human serum and mouse virulence.     

Yersinia enterocolitica exploits different pathways to accomplish adhesion and toxin injection into host cells

The current paradigm suggests that Yersinia enterocolitica (Ye) adheres to host cells via the outer membrane proteins Yersinia adhesin A (YadA) or invasin (Inv) to facilitate injection of Yops by the type III secretion system. In this process Inv binds directly to β1 integrins of host cells while YadA may bind indirectly via extracellular matrix proteins to β1 integrins. Here we challenged this paradigm and investigated the requirements for Yop injection. We demonstrate that Inv‐ but not YadA‐mediated adhesion depends on β1 integrin binding and activation, and that tight adhesion is a prerequisite for Yop injection. By means of novel transgenic cell lines, shRNA approaches and RGD peptides, we found that YadA, in contrast to Inv, may use a broad host cell receptor repertoire for host cell adhesion. In the absence of β1 integrins, YadA mediates Yop injection by interaction with αV integrins in cooperation with yet unknown cofactors expressed by epithelial cells, but not fibroblasts. Electron microscopic and flow chamber studies revealed that a defined intimate contact area between Ye and host cells resulting in adhesion forces resisting shear stress is required for Yop injection. Thus, the indirect binding of YadA to a broad extracellular matrix (ECM) binding host cell receptor repertoire of different cell types makes YadA a versatile tool to ensure Yop injection. In conclusion, given the differential expression of the outer membrane proteins Inv and YadA in the course of Ye infection and differential expression of integrins by various host cell populations, the data demonstrate that Ye is flexibly armed to accomplish Yop injection in different host cell types, a central event in its immune evasion strategy.     

Unique cell adhesion and invasion properties of Yersinia enterocolitica O:3, the most frequent cause of human Yersiniosis

Many enteric pathogens are equipped with multiple cell adhesion factors which are important for host tissue colonization and virulence. Y. enterocolitica, a common food-borne pathogen with invasive properties, uses the surface proteins invasin and YadA for host cell binding and entry. In this study, we demonstrate unique cell adhesion and invasion properties of Y. enterocolitica serotype O:3 strains, the most frequent cause of human yersiniosis, and show that these differences are mainly attributable to variations affecting the function and expression of invasin in response to temperature. In contrast to other enteric Yersinia strains, invasin production in O:3 strains is constitutive and largely enhanced compared to other Y. enterocolitica serotypes, in which invA expression is temperature-regulated and significantly reduced at 37°C. Increase of invasin levels is caused by (i) an IS1667 insertion into the invA promoter region, which includes an additional promoter and RovA and H-NS binding sites, and (ii) a P98S substitution in the invA activator protein RovA rendering the regulator less susceptible to proteolysis. Both variations were shown to influence bacterial colonization in a murine infection model. Furthermore, we found that co-expression of YadA and down-regulation of the O-antigen at 37°C is required to allow efficient internalization by the InvA protein. We conclude that even small variations in the expression of virulence factors can provoke a major difference in the virulence properties of closely related pathogens which may confer better survival or a higher pathogenic potential in a certain host or host environment.     

Yersinia enterocolitica serum resistance proteins YadA and ail bind the complement regulator C4b-binding protein

Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core (OC) do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp), an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host.     

Molecular analysis of transport and oligomerization of the Yersinia enterocolitica adhesin YadA

The Yersinia adhesin YadA is the prototype of a novel class of bacterial adhesins which form oligomeric lollipop-like structures and are anchored in the outer membrane by the C terminus. For YadA, six different regions (R) or domains (D) are predicted from the amino acid sequence: the N-terminal leader sequence, head-D, neck-D, stalk-D, linking-R, and a C-terminal transmembrane region consisting of four beta-strands. To identify structural and functional features of these domains, we performed in-frame deletion mutagenesis and constructed N-terminally tagged YadA variants. Diverse YadA variants were analyzed for outer membrane localization, surface exposure, oligomerization adhesion properties, and ability to protect against complement-mediated lysis. We demonstrated that (i) the C-terminal region (amino acids [aa] 353 to 422) is sufficient for outer membrane insertion and formation of trimers in the outer membrane; (ii) the head, neck, and stalk domains (aa 26 to 330) are surface exposed, forming a passenger domain; and (iii) the linking region (aa 331 to 369) is responsible for outer membrane translocation of the passenger domain. Thus, YadA meets all the criteria of an autotransporter. The same may be true for all other members of the YadA family, forming a subfamily of surface-attached oligomeric autotransporters. Moreover, in-frame truncation mutagenesis suggested that the head and neck domains together form the YadA-binding module which is located on the top of the stalk. However, the YadA-binding module did not confer serum resistance. Mutants lacking the head and neck domain were resistant to complement-mediated lysis. In-frame truncation of the stalk domain did not result in significant attenuation of the mutant in an orogastric mouse infection model.     

Model structure of the prototypical non-fimbrial adhesin YadA of Yersinia enterocolitica

Non-fimbrial adhesins, such as Yersinia YadA, Moraxella UspA1 and A2, Haemophilus Hia and Hsf, or Bartonella BadA represent an important class of molecules by which pathogenic proteobacteria adhere to their hosts. They form trimeric surface structures with a head-stalk-anchor architecture. Whereas head and stalk domains are diverse and appear (frequently repetitively) in different combinations, the anchor domains are homologous and display the properties of autotransporters. We have built a molecular model for the prototypical non-fimbrial adhesin, YadA, by combining the crystal structure of the head (PDB:1P9H) with theoretical models for the stalk and the anchor. The head domain is a single-stranded, left-handed beta-helix, connected to the stalk by a conserved trimerization element (the neck). The stalk consists of a right-handed coiled coil, containing ten 15-residue repeats with a C-terminal stutter (insertion of four residues). The stalk continues into the conserved anchor domain, which is formed by four heptads of a left-handed coiled coil, followed by four transmembrane beta-strands. Our model of the YadA coiled coil, generated with the program BeammotifCC, combines these periodicities into a structure that starts with a pronounced right-handed supercoil and ends with a canonical, left-handed conformation. The last two heptads of the coiled coil are located within a 12-stranded beta-barrel, formed by trimerization of the four transmembrane beta-strands in each monomer. We propose that this pore assembles in the outer membrane to form the opening through which the monomer chains exit the cell. After export is completed, the fiber folds and the pore is occluded by the coiled coil. Our model explains how these proteins can act as autotransporters in the absence of any homology to classical, single-chain autotransporters.     

Substitution of two histidine residues in YadA protein of Yersinia enterocolitica abrogates collagen binding, cell adherence and mouse virulence

The plasmid-encoded surface protein YadA of Yersinia enterocolitica mediates binding to diverse extracellular matrix (ECM) proteins, adherence to epithelial cell lines, resistance to complement lysis, autoagglutination, and is required for mouse virulence. Using site-directed mutagenesis we attempted to analyse the relationship between structural domains and functions of YadA. In a first approach we could abrogate collagen binding by chemical modification of histidyl residues of YadA protein. This result prompted us to substitute histidyl residues (His) of conserved regions of YadA protein of Y. enterocolitica 08 by tyrosine residues using site-directed mutagenesis. Substitution of His-156 and His-159 (YadA-2 mutant) resulted in abrogation of binding to ECM proteins, of cell adherence, and in reduction of mouse virulence, whereas autoagglutination, serum complement resistance and oligomer formation remained unaffected. A striking result was obtained from the orogastric mouse-infection model: the YadA-2 mutant retained the ability to colonize the small intestine and to invade and multiply within the Peyer's patches but was impaired in colonizing mesenteric lymph nodes and spleen in comparison to the wild-type strain.