Outer Membrane Adhesin Research Articles

Outer membrane vesicles shield Moraxella catarrhalis -lactamase from neutralization by serum IgG

The aim of this study was to detect the presence of IgG against Moraxella catarrhalis β-lactamase in healthy adults, and to determine whether outer membrane vesicles (OMVs) could protect the enzyme from inhibition by anti-β-lactamase IgG. Transmission electron microscopy was used to detect the presence of β-lactamase in OMVs. Sera were examined by ELISA for specific IgG directed against recombinant M. catarrhalis β-lactamase in addition to the outer membrane adhesins MID/Hag, UspA1 and A2. Binding of anti-β-lactamase IgG from serum to OMVs was analysed by flow cytometry. The chromogenic substrate nitrocefin was used to quantify β-lactamase enzyme activity. The presence of β-lactamase was determined in OMVs from a 9-year-old child suffering from M. catarrhalis sinusitis. Furthermore, anti-β-lactamase IgG was detected in sera obtained from healthy adults. Out of 40 adult blood donors (aged 18-65 years) tested, 6 (15.0%) carried anti-β-lactamase IgG. No correlation between IgG titres against β-lactamase and the adhesins was found. Flow cytometry analyses revealed that anti-β-lactamase IgG from serum bound to β-lactamase-positive OMVs. By comparing the β-lactamase activity of intact OMV with OMV that were permeabilized with saponin we found that OMVs shielded active β-lactamase from the anti-β-lactamase IgG. Moraxella catarrhalis β-lactamase is found in, or associated with, OMVs, providing clinical relevance for the vesicles in the spread of antibiotic resistance. Furthermore, OMVs protect β-lactamase from specific IgG.

HtrA chaperone activity contributes to host cell binding in Campylobacter jejuni

BackgroundAcute gastroenteritis caused by the food-borne pathogen Campylobacter jejuni is associated with attachment of bacteria to the intestinal epithelium and subsequent invasion of epithelial cells. In C. jejuni, the periplasmic protein HtrA is required for efficient binding to epithelial cells. HtrA has both protease and chaperone activity, and is important for virulence of several bacterial pathogens.ResultsThe aim of this study was to determine the role of the dual activities of HtrA in host cell interaction of C. jejuni by comparing an htrA mutant lacking protease activity, but retaining chaperone activity, with a ΔhtrA mutant and the wild type strain. Binding of C. jejuni to both epithelial cells and macrophages was facilitated mainly by HtrA chaperone activity that may be involved in folding of outer membrane adhesins. In contrast, HtrA protease activity played only a minor role in interaction with host cells.ConclusionWe show that HtrA protease and chaperone activities contribute differently to C. jejuni's interaction with mammalian host cells, with the chaperone activity playing the major role in host cell binding.

Identification and characterisation of a novel adhesin Ifp in Yersinia pseudotuberculosis

BackgroundIn order to identify new virulence determinants in Y. pseudotuberculosis a comparison between its genome and that of Yersinia pestis was undertaken. This reveals dozens of pseudogenes in Y. pestis, which are still putatively functional in Y. pseudotuberculosis and may be important in the enteric lifestyle. One such gene, YPTB1572 in the Y. pseudotuberculosis IP32953 genome sequence, encodes a protein with similarity to invasin, a classic adhesion/invasion protein, and to intimin, the attaching and effacing protein from enteropathogenic (EPEC) and enterohaemorraghic (EHEC) Escherichia coli.ResultsWe termed YPTB1572 Ifp (Intimin family protein) and show that it is able to bind directly to human HEp-2 epithelial cells. Cysteine and tryptophan residues in the C-terminal region of intimin that are essential for function in EPEC and EHEC are conserved in Ifp. Protein binding occurred at distinct foci on the HEp-2 cell surface and can be disrupted by mutation of a single cysteine residue at the C-terminus of the protein. Temporal expression analysis using lux reporter constructs revealed that ifp is expressed at late log phase at 37°C in contrast to invasin, suggesting that Ifp is a late stage adhesin. An ifp defined mutant showed a reduction in adhesion to HEp-2 cells and was attenuated in the Galleria mellonella infection model.ConclusionA new Y. pseudotuberculosis adhesin has been identified and characterised. This Ifp is a new member in the family of invasin/intimin outer membrane adhesins.

Subcutaneous and intranasal immunization with Stx2B–Tir–Stx1B–Zot reduces colonization and shedding of Escherichia coli O157:H7 in mice

The type III secretion system of Escherichia coli O157:H7 is involved in colonization of mammalian hosts by the organism. The translocated intimin receptor (Tir) is inserted into the mammalian host cell plasma membrane in a hairpin loop topology with the central loop of the molecule exposed to the host cell surface and accessible for interaction with an LEE-encoded bacterial outer membrane adhesin called intimin. Shiga toxin type 1 and 2 produced by E. coli O157:H7 are responsible for hemolytic uremic syndrome and able to promote intestinal colonization. Zonula occludens toxin (Zot) is a single polypeptide chain encoded by the filamentous bacteriophage CTXφ of Vibrio cholerae. Zot binds a receptor on intestinal epithelial cells and increases mucosal permeability by affecting the structure of epithelial tight junctions. Because of these properties, Zot is a promising tool for mucosal drug and antigen (Ag) delivery. In the current study, we constructed a novel fusion protein carrying both of the immunogenic B subunits derived from the two toxins, Tir and Zot, designated Stx2B–Tir–Stx1B–Zot, expressed in the E. coli BL21 and harvested the purified protein by a simple GST·Bind Resin chromatography method. We used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs. intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Immune responses against Stx2B–Tir–Stx1B–Zot, Stx2B–Tir–Stx1B and control agent (GST/PBS) were also monitored. Subcutaneous immunization of mice with Stx2B–Tir–Stx1B–Zot induced significant Stx2B–Tir–Stx1B–Zot-specific serum IgG antibodies but did not significantly induce any antigen-specific IgA in feces, whereas intranasal immunization elicited significant Stx2B–Tir–Stx1B–Zot-specific serum IgG antibodies with some animals developing antigen-specific IgA in feces. Mice that were immunized intranasally with Stx2B–Tir–Stx1B–Zot showed dramatically decreased E. coli O157:H7 shedding compared to those of Stx2B–Tir–Stx1B and control agent following experimental infection. Mice immunized subcutaneously with Stx2B–Tir–Stx1B–Zot or Stx2B–Tir–Stx1B both showed reduced shedding in feces, moreover, Stx2B–Tir–Stx1B–Zot did better. These results demonstrate the perspective for the use of Stx2B–Tir–Stx1B–Zot to prevent colonization and shedding of E. coli O157:H7.

A c-di-GMP Effector System Controls Cell Adhesion by Inside-Out Signaling and Surface Protein Cleavage

In Pseudomonas fluorescens Pf0-1 the availability of inorganic phosphate (Pi) is an environmental signal that controls biofilm formation through a cyclic dimeric GMP (c-di-GMP) signaling pathway. In low Pi conditions, a c-di-GMP phosphodiesterase (PDE) RapA is expressed, depleting cellular c-di-GMP and causing the loss of a critical outer-membrane adhesin LapA from the cell surface. This response involves an inner membrane protein LapD, which binds c-di-GMP in the cytoplasm and exerts a periplasmic output promoting LapA maintenance on the cell surface. Here we report how LapD differentially controls maintenance and release of LapA: c-di-GMP binding to LapD promotes interaction with and inhibition of the periplasmic protease LapG, which targets the N-terminus of LapA. We identify conserved amino acids in LapA required for cleavage by LapG. Mutating these residues in chromosomal lapA inhibits LapG activity in vivo, leading to retention of the adhesin on the cell surface. Mutations with defined effects on LapD's ability to control LapA localization in vivo show concomitant effects on c-di-GMP-dependent LapG inhibition in vitro. To establish the physiological importance of the LapD-LapG effector system, we track cell attachment and LapA protein localization during Pi starvation. Under this condition, the LapA adhesin is released from the surface of cells and biofilms detach from the substratum. This response requires c-di-GMP depletion by RapA, signaling through LapD, and proteolytic cleavage of LapA by LapG. These data, in combination with the companion study by Navarro et al. presenting a structural analysis of LapD's signaling mechanism, give a detailed description of a complete c-di-GMP control circuit—from environmental signal to molecular output. They describe a novel paradigm in bacterial signal transduction: regulation of a periplasmic enzyme by an inner membrane signaling protein that binds a cytoplasmic second messenger.

The HP0256 gene product is involved in motility and cell envelope architecture of Helicobacter pylori

BackgroundHelicobacter pylori is the causative agent for gastritis, and peptic and duodenal ulcers. The bacterium displays 5-6 polar sheathed flagella that are essential for colonisation and persistence in the gastric mucosa. The biochemistry and genetics of flagellar biogenesis in H. pylori has not been fully elucidated. Bioinformatics analysis suggested that the gene HP0256, annotated as hypothetical, was a FliJ homologue. In Salmonella, FliJ is a chaperone escort protein for FlgN and FliT, two proteins that themselves display chaperone activity for components of the hook, the rod and the filament.ResultsAblation of the HP0256 gene in H. pylori significantly reduced motility. However, flagellin and hook protein synthesis was not affected in the HP0256 mutant. Transmission electron transmission microscopy revealed that the HP0256 mutant cells displayed a normal flagellum configuration, suggesting that HP0256 was not essential for assembly and polar localisation of the flagella in the cell. Interestingly, whole genome microarrays of an HP0256 mutant revealed transcriptional changes in a number of genes associated with the flagellar regulon and the cell envelope, such as outer membrane proteins and adhesins. Consistent with the array data, lack of the HP0256 gene significantly reduced adhesion and the inflammatory response in host cells.ConclusionsWe conclude that HP0256 is not a functional counterpart of FliJ in H. pylori. However, it is required for full motility and it is involved, possibly indirectly, in expression of outer membrane proteins and adhesins involved in pathogenesis and adhesion.

Immunization of cattle with a combination of purified intimin-531, EspA and Tir significantly reduces shedding of Escherichia coli O157:H7 following oral challenge

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen that can cause gastrointestinal disease with potentially fatal consequences as a result of systemic Shiga toxin activity. Cattle are the main reservoir host of EHEC O157 and interventions need to be developed that prevent cattle colonization or limit shedding of the organism from this host. EHEC O157 predominately colonizes the bovine terminal rectum and requires a type III secretion system (T3SS) for adherence and persistence at this site. A vaccine based on concentrated bacterial supernatant that contains T3S proteins has shown some efficacy. Here we have demonstrated that vaccination with a combination of antigens associated with T3S-mediated adherence; the translocon filament protein, EspA, the extracellular region of the outer membrane adhesin, intimin, and the translocated intimin receptor (Tir) significantly reduced shedding of EHEC O157 from experimentally infected animals. Furthermore, this protection may be augmented by addition of H7 flagellin to the vaccine preparation that has been previously demonstrated to be partially protective in cattle. Protection correlates with systemic and mucosal antibody responses to the defined antigens and validates the targeting of these colonization factors.

Invasiveness as a putative additional virulence mechanism of some atypical Enteropathogenic Escherichia colistrains with different uncommon intimin types

BackgroundEnteropathogenic Escherichia coli (EPEC) produce attaching/effacing (A/E) lesions on eukaryotic cells mediated by the outer membrane adhesin intimin. EPEC are sub-grouped into typical (tEPEC) and atypical (aEPEC). We have recently demonstrated that aEPEC strain 1551-2 (serotype O non-typable, non-motile) invades HeLa cells by a process dependent on the expression of intimin sub-type omicron. In this study, we evaluated whether aEPEC strains expressing other intimin sub-types are also invasive using the quantitative gentamicin protection assay. We also evaluated whether aEPEC invade differentiated intestinal T84 cells.ResultsFive of six strains invaded HeLa and T84 cells in a range of 13.3%–20.9% and 5.8%–17.8%, respectively, of the total cell-associated bacteria. The strains studied were significantly more invasive than prototype tEPEC strain E2348/69 (1.4% and 0.5% in HeLa and T84 cells, respectively). Invasiveness was confirmed by transmission electron microscopy. We also showed that invasion of HeLa cells by aEPEC 1551-2 depended on actin filaments, but not on microtubules. In addition, disruption of tight junctions enhanced its invasion efficiency in T84 cells, suggesting preferential invasion via a non-differentiated surface.ConclusionSome aEPEC strains may invade intestinal cells in vitro with varying efficiencies and independently of the intimin sub-type.

MrkF is a component of type 3 fimbriae in Klebsiella pneumoniae

Klebsiella pneumoniae type 3 fimbriae are encoded by mrkABCDF genes which produce the major pilin subunit MrkA, chaperone MrkB, outer membrane usher MrkC, adhesin MrkD and MrkF of unknown function, respectively. RT-PCR analysis demonstrated that the mrkF gene is contained within the mrk operon. Deletion of mrkF in K. pneumoniae CG43 was found to reduce biofilm formation. A higher level of biofilm formation activity was also observed in recombinant Escherichia coli JM109[p mrkABCDF] compared to that observed for JM109[p mrkABCD]. Immunoelectron microscopy analysis of recombinant type 3 fimbriae using anti-MrkA and anti-MrkF antibody-labeled gold particles revealed that MrkF intermittently inserted into the MrkA filament. An interaction between recombinant MrkA and MrkF was demonstrated by co-immunoprecipitation analysis, further supporting the notion that MrkF is a structural component of the fimbriae. Intriguingly, the incorporation of MrkF appeared to decrease fimbrial length but increased activity of autoaggregation and biofilm formation in the bacteria JM109[p mrkABCDF]. This suggested that MrkF may play a role in assembly of the filament.

CadA negatively regulates Escherichia coli O157:H7 adherence and intestinal colonization.

Adherence of pathogenic Escherichia coli strains to intestinal epithelia is essential for infection. For enterohemorrhagic E. coli (EHEC) serotype O157:H7, we have previously demonstrated that multiple factors govern this pathogen's adherence to HeLa cells (A. G. Torres and J. B. Kaper, Infect. Immun. 71:4985-4995, 2003). One of these factors is CadA, a lysine decarboxylase, and this protein has been proposed to negatively regulate virulence in several enteric pathogens. In the case of EHEC strains, CadA modulates expression of the intimin, an outer membrane adhesin involved in pathogenesis. Here, we inactivated cadA in O157:H7 strain 86-24 to investigate the role of this gene in EHEC adhesion to tissue-cultured monolayers, global gene expression patterns, and colonization of the infant rabbit intestine. The cadA mutant did not possess lysine decarboxylation activity and was hyperadherent to tissue-cultured cells. Adherence of the cadA mutant was nearly twofold greater than that of the wild type, and the adherence phenotype was independent of pH, lysine, or cadaverine in the media. Additionally, complementation of the cadA defect reduced adherence back to wild-type levels, and it was found that the mutation affected the expression of the intimin protein. Disruption of the eae gene (intimin-encoding gene) in the cadA mutant significantly reduced its adherence to tissue-cultured cells. However, adherence of the cadA eae double mutant was greater than that of an 86-24 eae mutant, suggesting that the enhanced adherence of the cadA mutant is not entirely attributable to enhanced expression of intimin in this background. Gene array analysis revealed that the cadA mutation significantly altered EHEC gene expression patterns; expression of 1,332 genes was downregulated and that of 132 genes was upregulated in the mutant compared to the wild-type strain. Interestingly, the gene expression variation shows an EHEC-biased gene alteration including intergenic regions. Two putative adhesins, flagella and F9 fimbria, were upregulated in the cadA mutant, suggestive of their association with adherence in the absence of the Cad regulatory mechanism. In the infant rabbit model, the cadA mutant outcompeted the wild-type strain in the ileum but not in the cecum or mid-colon, raising the possibility that CadA negatively regulates EHEC pathogenicity in a tissue-specific fashion.

In meso crystal structure and docking simulations suggest an alternative proteoglycan binding site in the OpcA outer membrane adhesin

OpcA is an integral outer membrane adhesin protein from Neisseria meningitidis, the causative agent of meningococcal meningitis and septicemia. It binds to sialic acid (SA)-containing polysaccharides on the surface of epithelial cells. The crystal structure of OpcA showed that the protein adopts a 10-stranded beta-barrel structure, with five extensive loop regions on the extracellular side of the membrane. These form a crevice structure, lined with basic residues, which was hypothesized to act as the binding site for polysaccharide ligands. In the current study, a distinctly different OpcA structure has been obtained using crystals grown from a lipidic mesophase. Comparison of the two structures shows that the largest loop (L2), which closes over the end of the beta-barrel in the original crystal form, adopts a much more extended structure by reaching outward and away from the protein. The difference in conformation may be attributable to the absence of zinc ions from the crystallization conditions for the in meso crystal form: in the original structure, two zinc ions were bound to the external loops. Molecular dynamics (MD) simulations performed on the two OpcA models in a lipid bilayer environment demonstrated pronounced loop mobility. These observations support the view that the loop regions of OpcA are capable of a high degree of conformational flexibility. The original binding site for polysaccharide is not present in the in meso crystal form, and is disrupted during MD simulations. Docking analysis suggests a putative alternative location for the SA ligand in the new crystal form of OpcA.

The localized adherence pattern of an atypical enteropathogenic Escherichia coli is mediated by intimin omicron and unexpectedly promotes HeLa cell invasion

Enteropathogenic Escherichia coli (EPEC) forms attaching and effacing lesions in the intestinal mucosa characterized by intimate attachment to the epithelium by means of intimin (an outer membrane adhesin encoded by eae). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC); only tEPEC carries the EAF (EPEC adherence factor) plasmid that encodes the bundle-forming pilus (BFP). Characteristically, after 3 h of incubation, tEPEC produces localized adherence (LA) (with compact microcolonies) in HeLa/HEp-2 cells by means of BFP, whereas most aEPEC form looser microcolonies. We have previously identified nine aEPEC strains displaying LA in extended (6 h) assays (LA6). In this study, we analysed the kinetics of LA6 pattern development and the role of intimin in the process. Transmission electron microscopy and confocal laser microscopy showed that the invasive process of strain 1551-2 displays a LA phenotype. An eae-defective mutant of strain 1551-2 prevented the invasion although preserving intense diffused adherence. Sequencing of eae revealed that strain 1551-2 expresses the omicron subtype of intimin. We propose that the LA phenotype of aEPEC strain 1551-2 is mediated by intimin omicron and hypothesize that this strain expresses an additional novel adhesive structure. The present study is the first to report the association of compact microcolony formation and an intense invasive ability in aEPEC.

TccP2-mediated subversion of actin dynamics by EPEC 2 – a distinct evolutionary lineage of enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhoea in developing countries. While colonizing the gut mucosa, EPEC triggers extensive actin-polymerization activity at the site of intimate bacterial attachment, which is mediated by avid interaction between the outer-membrane adhesin intimin and the type III secretion system (T3SS) effector Tir. The prevailing dogma is that actin polymerization by EPEC is achieved following tyrosine phosphorylation of Tir, recruitment of Nck and activation of neuronal Wiskott–Aldrich syndrome protein (N-WASP). In closely related enterohaemorrhagic E. coli (EHEC) O157 : H7, actin polymerization is triggered following recruitment of the T3SS effector TccP/EspFU (instead of Nck) and local activation of N-WASP. In addition to tccP, typical EHEC O157 : H7 harbour a pseudogene (tccP2). However, it has recently been found that atypical, sorbitol-fermenting EHEC O157 carries functional tccP and tccP2 alleles. Interestingly, intact tccP2 has been identified in the incomplete genome sequence of the prototype EPEC strain B171 (serotype O111 : H−), but it is missing from another prototype EPEC strain E2348/69 (O127 : H7). E2348/69 and B171 belong to two distinct evolutionary lineages of EPEC, termed EPEC 1 and EPEC 2, respectively. Here, it is reported that while both EPEC 1 and EPEC 2 triggered actin polymerization via the Nck pathway, tccP2 was found in 26 of 27 (96.2 %) strains belonging to EPEC 2, and in none of the 34 strains belonging to EPEC 1. It was shown that TccP2 was: (i) translocated by the locus of enterocyte effacement-encoded T3SS; (ii) localized at the tip of the EPEC 2-induced actin-rich pedestals in infected HeLa cells and human intestinal in vitro organ cultures ex vivo; and (iii) essential for actin polymerization in infected Nck−/− cells. Therefore, unlike strains belonging to EPEC 1, strains belonging to EPEC 2 can trigger actin polymerization using both Nck and TccP2 actin-polymerization signalling cascades.

Functional studies of intimin in vivo and ex vivo: implications for host specificity and tissue tropism.

Intimin is an outer-membrane adhesin that is essential for colonization of the host gastrointestinal tract by attaching and effacing pathogens including enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR). The N-terminus of intimin from the different strains is highly conserved while the C-terminus, which harnesses the active receptor-binding site, shows sequence and antigenic polymorphism. This diversity was used to define a number of distinct intimin types, the most common of which are α, β and γ. Intimin binds the type III secretion system effector protein Tir. However, a large body of evidence suggests that intimin also binds a host-cell-encoded receptor(s) (Hir), and interaction of different intimin types with Hir contributes to tissue and host specificity. The aims of this study were to compare the activity of the major intimin types (α, β and γ) in vivo and ex vivo, using the CR mouse model and in vitro organ culture (IVOC), and to determine their exchangeability. The results confirm that intimin γ is not functional in the CR mouse model. In the pig, intimin β can substitute for EPEC intimin α but when placed in an EHEC O157 : H7 background it does not produce an intimin α-like tropism, although some adhesion to the small and large intestine was observed. In contrast, in human IVOC, intimin β in an EHEC background produces small intestinal colonization in a similar manner to intimin α.

Increased adherence and actin pedestal formation by dam‐deficient enterohaemorrhagic Escherichia coli O157:H7

Enterohaemorrhagic Escherichia coli (EHEC) are highly infectious pathogens capable of causing severe diarrhoeal illnesses. As a critical step during their colonization, EHEC adhere intimately to intestinal epithelial cells and generate F-actin 'pedestal' structures that elevate them above surrounding cell surfaces. Intimate adhesion and pedestal formation result from delivery of the EHEC type III secretion system (TTSS) effector proteins Tir and EspF(U) into the host cell and expression of the bacterial outer membrane adhesin, intimin. To investigate a role for DNA methylation during the regulation of adhesion and pedestal formation in EHEC, we deleted the dam (DNA adenine methyltransferase) gene from EHEC O157:H7 and demonstrate that this mutation results in increased interactions with cultured host cells. EHECDeltadam exhibits dramatically elevated levels of adherence and pedestal formation when compared with wild-type EHEC, and expresses significantly higher protein levels of intimin, Tir and EspF(U). Analyses of GFP fusions, Northern blotting, reverse transcription polymerase chain reaction, and microarray experiments indicate that the abundance of Tir in the dam mutant is not due to increased transcription levels, raising the possibility that Dam methylation can indirectly control protein expression by a post-transcriptional mechanism. In contrast to other dam-deficient pathogens, EHECDeltadam is capable of robust intestinal colonization of experimentally infected animals.

Isolation and Characterization of the P5 Adhesin Protein of Haemophilus parasuis Serotype 5

Haemophilus parasuis is a Gram‐negative respiratory pathogen of young pigs that colonizes the upper respiratory tract and produces a number of symptoms collectiviely described as Glässer's disease. Recently, an H. parasuis P5‐like outer membrane adhesin protein homologous to H. influenzae P5 was identified. The P5 adhesin was partially purified by anion exchange and size‐exclusion chromatography. Final purification for functional studies was performed by elution of the protein from a polyacrylamide gel. Identification of the protein as a P5 adhesin homolog of H. influenzae was confirmed by N‐terminal sequencing. The P5 protein had a molecular mass of 32,000 and a pI of 5.5. Unlike the H. influenzae P5 adhesin, the H. parasuis P5 protein did not bind carcinoembryonic antigen.

Co-ordinate action of bacterial adhesins and human carcinoembryonic antigen receptors in enhanced cellular invasion by capsulate serum resistant Neisseria meningitidis

Neisseria meningitidis (Nm) is a human specific opportunistic pathogen that occasionally penetrates mucosal barriers via the action of adhesins and invasins and evades host immune mechanisms during further dissemination via capsule expression. From in vitro studies, the primary adhesion of capsulate bacteria is believed to be mediated by polymeric pili, followed by invasion via outer membrane adhesins such as Opa proteins. As the latter requires the surface capsule to be down-modulated, invading bacteria would be serum sensitive and thus avirulent. However, there is recent evidence that capsulate bacteria may interact via Opa proteins when host cells express high levels of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), their target receptors. Such a situation may arise following increased circulation of inflammatory cytokines that upregulate certain adhesion molecules on host cells. In this study, using a tetracycline controlled expression system, we have developed cell lines with inducible CEACAM expression to mimic post-inflammation state of target tissues and analysed the interplay between the three surface components capsule, pili and Opa proteins in cellular interactions. With two distinct cell lines, not only the level but also the rate of adhesion of capsulate Opa-expressing Nm increased concurrently with CEACAM density. Moreover, when threshold levels of receptor were reached, cellular invasion ensued in an Opa-dependent manner. In studies with cell lines intrinsically expressing pilus receptors, notable synergism in cellular interactions between pili and Opa of several meningococcal strains was observed and was independent of capsule type. A number of internalized bacteria were shown to express capsule and when directly isolated from host cells, these bacteria were as serum resistant as the inoculated phenotype. Furthermore, we observed that agents that block Opa-CEACAM binding substantially reduced cellular invasion, while maintaining a low level of cellular adhesion. These studies highlight some of the factors that may determine increased host susceptibility to infection by serum resistant phenotypes; and demonstrate the potential of selective inhibition of key interactions in preventing target tissue penetration while maintaining a level of colonization.

Identification of a domain in Yersinia virulence factor YadA that is crucial for extracellular matrix-specific cell adhesion and uptake

For many pathogens, cell adhesion factors are critical virulence determinants. Enteropathogenic Yersinia species express the afimbrial adhesin YadA, the prototype of a class of homotrimeric outer membrane adhesins, which mediates adherence to host cells by binding to extracellular matrix components. In this study, we demonstrate that different pathogenic functions are attributable to highly homologous YadA proteins. YadA of Yersinia pseudotuberculosis (YadA(pstb)) and Yersinia enterocolitica (YadA(ent)) exhibit fundamental differences in their specificity of extracellular matrix substrate binding, they cause dissimilar bacterial aggregation behaviors, and YadA(pstb), but not YadA(ent), promotes efficient uptake into human cells. Evidence is presented here that a unique N-terminal amino acid sequence of YadA(pstb), which is absent in YadA(ent), acts as an "uptake domain" by mediating tight binding to fibronectin bound on alpha(5)beta(1) integrin receptors, which are crucial for initiating the entry process. Deleting this motif in YadA(pstb) generated all features of the YadA(ent) protein, i.e., the molecule lost its adhesiveness to fibronectin and its invasiveness, but gained adhesion potential to collagen and laminin. Loss of the "uptake region" also attenuated host tissue colonization by Y. pseudotuberculosis during oral infections of mice, demonstrating that this motif plays a crucial role in defining pathogen-host cell interaction and pathogenesis. We conclude that even small variations in adhesion factors can provoke major differences in the virulence properties of related pathogens.

A novel category of enteropathogenic Escherichia coli simultaneously utilizes the Nck and TccP pathways to induce actin remodelling

Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) induce drastic reorganization of the microfilament cytoskeleton. EHEC and EPEC translocate Tir (translocated intimin receptor) which, once inserted into the host plasma membrane, binds the bacterial outer membrane adhesin intimin. Tir(EPEC) then becomes tyrosine phosphorylated facilitating the recruitment and site-specific binding of the eukaryotic adaptor Nck, which in turn binds and activates the Wiskott-Aldrich syndrome protein (N-WASP), leading to actin-related protein 2/3 (Arp2/3) complex-mediated actin polymerization. In contrast, Tir(EHEC) has no Nck binding site; instead, EHEC utilizes the translocated effector TccP (Tir-cytoskeleton coupling protein) to bind and activate N-WASP. Here we report a novel class of EPEC that translocates both TccP and Tir(EPEC)-like effector molecules. Consistent with these characteristics, we show that both the Tir-Nck and Tir:TccP actin remodelling pathways function simultaneously during infection, making this a novel and versatile EPEC category.

Cold Shock Response of the UspA1 Outer Membrane Adhesin of Moraxella catarrhalis

Colonization of the human nasopharynx exposes Moraxella catarrhalis, a common cause of otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, to sudden downshifts in temperature, occurring when the host breathes cold air. We investigated whether in vitro cold shock influences the expressions of the outer membrane adhesins UspA1 and hemagglutinin, which are considered virulence factors, and of an M. catarrhalis homolog of recA, a housekeeping gene, which in Escherichia coli is induced by cold shock. Quantitative real-time reverse transcriptase PCR was used for measuring mRNA copy number. A screening experiment revealed that a cold shock at 26 degrees C maximally induced the copy number of uspA1. In comparison with 37 degrees C conditions, a 1-hour cold shock at 26 degrees C increased copy numbers of uspA1 and recA by 2.5-fold (11.2 +/- 1.8 versus 4.5 +/- 0.8 copies/CFU) and 2.7-fold (0.30 +/- 0.10 versus 0.11 +/- 0.06), respectively, but did not induce transcription of hag. Exposure to 26 degrees C increased surface expression of UspA1, as assessed by fluorescence-activated cell sorter analysis, and resulted in a significant increase in adherence of strain O35E to Chang human conjunctival cells (97.1% +/- 2.0% versus 48.3% +/- 9.2% at 37 degrees C; P = 0.01). Cold shock induction of uspA1 and recA was detected in strains belonging to either phylogenetic subpopulation of M. catarrhalis (16S rRNA types 1 and 2/3) and was most pronounced in type 2/3 strains (4- to 25-fold for uspA1), which do not express detectable amounts of UspA1 protein at 37 degrees C. These data indicate that cold shock at a physiologically relevant temperature of 26 degrees C induces the expression of at least one virulence factor (UspA1). To our knowledge, no similar data are available for other nasopharyngeal pathogens.