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Sample records for amyloid-like adhesins produced

  1. Subtractive hybridization and identification of putative adhesins in a Shiga toxin-producing eae-negative Escherichia coli.

    PubMed

    Vidal, Maricel; Prado, Valeria; Whitlock, Gregory C; Solari, Aldo; Torres, Alfredo G; Vidal, Roberto M

    2008-12-01

    Adherence to epithelial cells by specific adhesins is a characteristic of Shiga toxin-producing Escherichia coli (STEC) strains. The eae-encoded protein intimin is the main adhesin implicated in intestinal colonization in vivo. We recently showed that STEC strains isolated in Chile displayed a wide variety of adhesins; here we demonstrate that some of these STEC strains are eae-negative and still adhere to epithelial cells at a level 100-fold higher than enterohaemorrhagic E. coli (EHEC) O157 : H7 prototype strain EDL933. This phenotype is associated with the presence of adherence factors different from the intimin protein. Subtractive hybridization between EHEC EDL933 and STEC eae-negative strain 472-1 was used to identify regions implicated in adhesion. In addition to the saa gene, we identified 18 specific genes in STEC 472-1, 16 of which had nucleotide identity to Salmonella ST46 phage genes; the two remaining ones shared identity to a gene encoding a hypothetical protein of uropathogenic E. coli. The DNA sequence of the STEC 472-1 psu-int region identified five open reading frames with homology to phage genes. We constructed mutant strains in the saa gene and the psu-int region to study the participation of these genes in the adherence to epithelial cells and our results demonstrated that STECDeltasaa and STECDeltapsu-int mutants displayed a 10-fold decrease in adherence as compared to the STEC 472-1 wild-type strain. Overall, our results suggest that STEC strain 472-1 adheres to epithelial cells in an eae-independent matter and that saa and psu-int participate in this adhesion process.

  2. Amyloid-Like Fibril Elongation Follows Michaelis-Menten Kinetics

    PubMed Central

    Milto, Katazyna; Botyriute, Akvile; Smirnovas, Vytautas

    2013-01-01

    A number of proteins can aggregate into amyloid-like fibrils. It was noted that fibril elongation has similarities to an enzymatic reaction, where monomers or oligomers would play a role of substrate and nuclei/fibrils would play a role of enzyme. The question is how similar these processes really are. We obtained experimental data on insulin amyloid-like fibril elongation at the conditions where other processes which may impact kinetics of fibril formation are minor and fitted it using Michaelis-Menten equation. The correlation of the fit is very good and repeatable. It speaks in favour of enzyme-like model of fibril elongation. In addition, obtained and values at different conditions may help in better understanding influence of environmental factors on the process of fibril elongation. PMID:23874721

  3. EHEC Adhesins

    PubMed Central

    McWilliams, Brian D.; Torres, Alfredo G.

    2014-01-01

    Adhesins are a group of proteins in enterohemorrhagic Escherichia coli (EHEC) that are involved in the attachment or colonization of this pathogen to abiotic (plastic or steel) and biological surfaces, such as those found in bovine and human intestines. This review provides the most up-to-date information on these essential adhesion factors, summarizing important historical discoveries and analyzing the current and future state of this research. In doing so, the proteins intimin and Tir are discussed in depth, especially regarding their role in the development of attaching and effacing lesions and in EHEC virulence. Further, a series of fimbrial proteins (Lpf1, Lpf2, curli, ECP, F9, ELF, Sfp, HCP, and type 1 fimbriae) are also described, emphasizing their various contributions to adherence and colonization of different surfaces and their potential use as genetic markers in detection and classification of different EHEC serotypes. This review also discusses the role of several autotransporter proteins (EhaA-D, EspP, Saa and Sab, and Cah), as well as other proteins associated with adherence, such as flagella, EibG, Iha, and OmpA. While these proteins have all been studied to varying degrees, all of the adhesins summarized in this chapter have been linked to different stages of the EHEC life cycle, making them good targets for the development of more effective diagnostics and therapeutics. PMID:25635238

  4. Amyloid-like ribbons of amelogenins in enamel mineralization.

    PubMed

    Carneiro, Karina M M; Zhai, Halei; Zhu, Li; Horst, Jeremy A; Sitlin, Melody; Nguyen, Mychi; Wagner, Martin; Simpliciano, Cheryl; Milder, Melissa; Chen, Chun-Long; Ashby, Paul; Bonde, Johan; Li, Wu; Habelitz, Stefan

    2016-01-01

    Enamel, the outermost layer of teeth, is an acellular mineralized tissue that cannot regenerate; the mature tissue is composed of high aspect ratio apatite nanocrystals organized into rods and inter-rod regions. Amelogenin constitutes 90% of the protein matrix in developing enamel and plays a central role in guiding the hierarchical organization of apatite crystals observed in mature enamel. To date, a convincing link between amelogenin supramolecular structures and mature enamel has yet to be described, in part because the protein matrix is degraded during tissue maturation. Here we show compelling evidence that amelogenin self-assembles into an amyloid-like structure in vitro and in vivo. We show that enamel matrices stain positive for amyloids and we identify a specific region within amelogenin that self-assembles into β-sheets. We propose that amelogenin nanoribbons template the growth of apatite mineral in human enamel. This is a paradigm shift from the current model of enamel development.

  5. Amyloid-like ribbons of amelogenins in enamel mineralization

    PubMed Central

    Carneiro, Karina M. M.; Zhai, Halei; Zhu, Li; Horst, Jeremy A.; Sitlin, Melody; Nguyen, Mychi; Wagner, Martin; Simpliciano, Cheryl; Milder, Melissa; Chen, Chun-Long; Ashby, Paul; Bonde, Johan; Li, Wu; Habelitz, Stefan

    2016-01-01

    Enamel, the outermost layer of teeth, is an acellular mineralized tissue that cannot regenerate; the mature tissue is composed of high aspect ratio apatite nanocrystals organized into rods and inter-rod regions. Amelogenin constitutes 90% of the protein matrix in developing enamel and plays a central role in guiding the hierarchical organization of apatite crystals observed in mature enamel. To date, a convincing link between amelogenin supramolecular structures and mature enamel has yet to be described, in part because the protein matrix is degraded during tissue maturation. Here we show compelling evidence that amelogenin self-assembles into an amyloid-like structure in vitro and in vivo. We show that enamel matrices stain positive for amyloids and we identify a specific region within amelogenin that self-assembles into β-sheets. We propose that amelogenin nanoribbons template the growth of apatite mineral in human enamel. This is a paradigm shift from the current model of enamel development. PMID:27009419

  6. Haemagglutination induced by Bordetella pertussis filamentous haemagglutinin adhesin (FHA) is inhibited by antibodies produced against FHA(430-873) fragment expressed in Lactobacillus casei.

    PubMed

    Colombi, Débora; Oliveira, Maria L S; Campos, Ivana B; Monedero, Vicente; Pérez-Martinez, Gaspar; Ho, Paulo L

    2006-12-01

    Filamentous haemagglutinin adhesin (FHA) is an important virulence factor from Bordetella pertussis related to the adhesion and spread of the bacteria through the respiratory tract. Three distinct domains have been characterized in mature FHA, and among them, the FHA(442-863) fragment was suggested to be responsible for the heparin-binding activity. In this study, we cloned the gene encoding the HEP fragment (FHA(430-873)) in a Lactobacillus casei-inducible expression vector based on the lactose operon. The recombinant bacteria, transformed with the resulting construct (L. casei-HEP), were able to express the heterologous protein depending on the sugar added to the culture. Subcutaneous inoculation of L. casei-HEP in Balb/C mice, using the cholera toxin B subunit as adjuvant, induced systemic anti-HEP antibodies that were able to inhibit in vitro erythrocyte haemagglutination induced by FHA. This is the first example of a B. pertussis antigen produced in lactic acid bacteria and opens new perspectives for alternative vaccine strategies against whooping cough. PMID:17106803

  7. Amyloid-like Fibril Formation by Trypsin in Aqueous Ethanol. Inhibition of Fibrillation by PEG.

    PubMed

    Kotormán, Márta; Simon, Mária L; Borics, Attila; Szabó, Márton Richárd; Szabó, Kitti; Szögi, Titanilla; Fülöp, Lívia

    2015-01-01

    The formation of amyloid-like fibrils was studied by using the well-known serine protease trypsin as a model protein in the presence of ethanol as organic solvent. Trypsin forms amyloid-like fibrils in aqueous ethanol at pH = 7.0. The dye Congo red (CR) was used to detect the presence of amyloid-like fibrils in the samples. The binding of CR to fibrils led to an increase in absorption intensity and a red shift in the absorption band of CR. Thioflavin T (ThT) and 8-anilino-1- naphthalenesulfonic acid (ANS) binding assays were employed to characterize amyloid-like fibril formation. The ThT binding assay revealed that the protein exhibited maximum aggregation in 60% (v/v) ethanol after incubation for 24 h at 24 (o)C. The ANS binding results indicated that the hydrophobic residues were more exposed to the solvent in the aggregated form of the protein. The effects of polyethylene glycol (PEG) on the formation of amyloid-like fibrils was studied in vitro. The aggregation of trypsin was followed via the kinetics of aggregation, the far-UV circular dichroism (CD) and transmission electron microscopy (TEM) in the presence and absence of PEG. The CD measurements indicated that the protein aggregates have a cross-beta structure in 60% ethanol. TEM revealed that trypsin forms fibrils with a thread-like structure. The inhibitory effect of PEG on the aggregation of trypsin increased with rising PEG concentration. PEG therefore inhibits the formation of amyloid-like fibrils of trypsin in aqueous ethanol. PMID:26428300

  8. An adhesin from hydrogen-utilizing rumen methanogen Methanobrevibacter ruminantium M1 binds a broad range of hydrogen-producing microorganisms.

    PubMed

    Ng, Filomena; Kittelmann, Sandra; Patchett, Mark L; Attwood, Graeme T; Janssen, Peter H; Rakonjac, Jasna; Gagic, Dragana

    2016-09-01

    Symbiotic associations are ubiquitous in the microbial world and have a major role in shaping the evolution of both partners. One of the most interesting mutualistic relationships exists between protozoa and methanogenic archaea in the fermentative forestomach (rumen) of ruminant animals. Methanogens reside within and on the surface of protozoa as symbionts, and interspecies hydrogen transfer is speculated to be the main driver for physical associations observed between the two groups. In silico analyses of several rumen methanogen genomes have previously shown that up to 5% of genes encode adhesin-like proteins, which may be central to rumen interspecies attachment. We hypothesized that adhesin-like proteins on methanogen cell surfaces facilitate attachment to protozoal hosts. Using phage display technology, we have identified a protein (Mru_1499) from Methanobrevibacter ruminantium M1 as an adhesin that binds to a broad range of rumen protozoa (including the genera Epidinium and Entodinium). This unique adhesin also binds the cell surface of the bacterium Butyrivibrio proteoclasticus, suggesting a broad adhesion spectrum for this protein.

  9. An adhesin from hydrogen-utilizing rumen methanogen Methanobrevibacter ruminantium M1 binds a broad range of hydrogen-producing microorganisms.

    PubMed

    Ng, Filomena; Kittelmann, Sandra; Patchett, Mark L; Attwood, Graeme T; Janssen, Peter H; Rakonjac, Jasna; Gagic, Dragana

    2016-09-01

    Symbiotic associations are ubiquitous in the microbial world and have a major role in shaping the evolution of both partners. One of the most interesting mutualistic relationships exists between protozoa and methanogenic archaea in the fermentative forestomach (rumen) of ruminant animals. Methanogens reside within and on the surface of protozoa as symbionts, and interspecies hydrogen transfer is speculated to be the main driver for physical associations observed between the two groups. In silico analyses of several rumen methanogen genomes have previously shown that up to 5% of genes encode adhesin-like proteins, which may be central to rumen interspecies attachment. We hypothesized that adhesin-like proteins on methanogen cell surfaces facilitate attachment to protozoal hosts. Using phage display technology, we have identified a protein (Mru_1499) from Methanobrevibacter ruminantium M1 as an adhesin that binds to a broad range of rumen protozoa (including the genera Epidinium and Entodinium). This unique adhesin also binds the cell surface of the bacterium Butyrivibrio proteoclasticus, suggesting a broad adhesion spectrum for this protein. PMID:26643468

  10. Regulated Formation of an Amyloid-like Translational Repressor Governs Gametogenesis.

    PubMed

    Berchowitz, Luke E; Kabachinski, Greg; Walker, Margaret R; Carlile, Thomas M; Gilbert, Wendy V; Schwartz, Thomas U; Amon, Angelika

    2015-10-01

    Message-specific translational control is required for gametogenesis. In yeast, the RNA-binding protein Rim4 mediates translational repression of numerous mRNAs, including the B-type cyclin CLB3, which is essential for establishing the meiotic chromosome segregation pattern. Here, we show that Rim4 forms amyloid-like aggregates and that it is the amyloid-like form of Rim4 that is the active, translationally repressive form of the protein. Our data further show that Rim4 aggregation is a developmentally regulated process. Starvation induces the conversion of monomeric Rim4 into amyloid-like aggregates, thereby activating the protein to bring about repression of translation. At the onset of meiosis II, Rim4 aggregates are abruptly degraded allowing translation to commence. Although amyloids are best known for their role in the etiology of diseases such as Alzheimer's, Parkinson's, and diabetes by forming toxic protein aggregates, our findings show that cells can utilize amyloid-like protein aggregates to function as central regulators of gametogenesis. PMID:26411291

  11. Adhesins of Bartonella spp.

    PubMed

    O'Rourke, Fiona; Schmidgen, Thomas; Kaiser, Patrick O; Linke, Dirk; Kempf, Volkhard A J

    2011-01-01

    Adhesion to host cells represents the first step in the infection process and one of the decisive features in the pathogenicity of Bartonella spp. B. henselae and B. quintana are considered to be the most important human pathogenic species, responsible for cat scratch disease, bacillary angiomatosis, trench fever and other diseases. The ability to cause vasculoproliferative disorders and intraerythrocytic bacteraemia are unique features of the genus Bartonella. Consequently, the interaction with endothelial cells and erythrocytes is a focus in Bartonella research. The genus harbours a variety of trimeric autotransporter adhesins (TAAs) such as the Bartonella adhesin A (BadA) of B. henselae and the variably expressed outer-membrane proteins (Vomps) of B. quintana, which display remarkable variations in length and modular construction. These adhesins mediate many of the biologically-important properties of Bartonella spp. such as adherence to endothelial cells and extracellular matrix proteins and induction of angiogenic gene programming. There is also significant evidence that the laterally acquired Trw-conjugation systems of Bartonella spp. mediate host-specific adherence to erythrocytes. Other potential adhesins are the filamentous haemagglutinins and several outer membrane proteins. The exact molecular functions of these adhesins and their interplay with other pathogenicity factors (e.g., the VirB/D4 type 4 secretion system) need to be analysed in detail to understand how these pathogens adapt to their mammalian hosts.

  12. [Adhesins of oral streptococci].

    PubMed

    Takahashi, Yukihiro; Urano-Tashiro, Yumiko; Konishi, Kiyoshi

    2013-01-01

    Oral streptococci comprise a numerically prominent group of oral bacteria that occur primarily on the human tooth surface as members of the biofilm community, commonly referred to as dental plaque. These streptococci are not only causative of dental caries and are primers for colonization of periodontopathic bacteria, but also well known for their ability to colonize damaged heart valves, identified most frequently as primary etiological agents of infective endocarditis. A number of streptococcal cell surface components are known to contribute to colonization of the tooth surface including putative adhesins recognizing host sialic acid (sialic acid-binding adhesins). Interactions mediated by these adhesins include the attachment of these bacteria to saliva-coated hydroxyapatite and their adhesion to erythrocytes, both of which are abolished or reduced by sialidase pretreatment of the corresponding host sialoglycoconjugate receptors. The sialic acid-binding adhesin on Streptococcus gordonii, an early colonizer on the tooth surface, has been molecularly analyzed. The adhesin, Hsa (203-kDa protein), consists of an N-terminal non repetitive region (NR1) including a signal sequence, a relatively short serine-rich region (SR1), a second non repetitive region (NR2), a long serine-rich region (SR2) containing 113 dodecapeptide repeats accounting for 75% of the whole protein, and a C-terminal cell wall anchoring domain. Therefore, it has been suggested that NR2, the putative sialic acid-binding domain of Hsa, is presented on the bacterial surface at the end of a long molecular stalk formed by SR2. The present review deals with the function and pathogenicity of oral streptococcal adhesins. PMID:23727707

  13. Atomic models of de novo designed cc beta-Met amyloid-like fibrils.

    PubMed

    Steinmetz, Michel O; Gattin, Zrinka; Verel, Rene; Ciani, Barbara; Stromer, Thusnelda; Green, Janelle M; Tittmann, Peter; Schulze-Briese, Clemens; Gross, Heinz; van Gunsteren, Wilfred F; Meier, Beat H; Serpell, Louise C; Müller, Shirley A; Kammerer, Richard A

    2008-02-22

    The common characteristics of amyloid and amyloid-like fibrils from disease- and non-disease-associated proteins offer the prospect that well-defined model systems can be used to systematically dissect the driving forces of amyloid formation. We recently reported the de novo designed cc beta peptide model system that forms a native-like coiled-coil structure at low temperatures and which can be switched to amyloid-like fibrils by increasing the temperature. Here, we report a detailed molecular description of the system in its fibrillar state by characterizing the cc beta-Met variant using several microscopic techniques, circular dichroism spectroscopy, X-ray fiber diffraction, solid-state nuclear magnetic resonance, and molecular dynamics calculations. We show that cc beta-Met forms amyloid-like fibrils of different morphologies on both the macroscopic and atomic levels, which can be controlled by variations of assembly conditions. Interestingly, heterogeneity is also observed along single fibrils. We propose atomic models of the cc beta-Met amyloid-like fibril, which are in good agreement with all experimental data. The models provide a rational explanation why oxidation of methionine residues completely abolishes cc beta-Met amyloid fibril formation, indicating that a small number of site-specific hydrophobic interactions can play a major role in the packing of polypeptide-chain segments within amyloid fibrils. The detailed structural information available for the cc beta model system provides a strong molecular basis for understanding the influence and relative contribution of hydrophobic interactions on native-state stability, kinetics of fibril formation, fibril packing, and polymorphism.

  14. Looking for a generic inhibitor of amyloid-like fibril formation among flavone derivatives

    PubMed Central

    Šneideris, Tomas; Baranauskienė, Lina; Cannon, Jonathan G.; Rutkienė, Rasa; Meškys, Rolandas

    2015-01-01

    A range of diseases is associated with amyloid fibril formation. Despite different proteins being responsible for each disease, all of them share similar features including beta-sheet-rich secondary structure and fibril-like protein aggregates. A number of proteins can form amyloid-like fibrils in vitro, resembling structural features of disease-related amyloids. Given these generic structural properties of amyloid and amyloid-like fibrils, generic inhibitors of fibril formation would be of interest for treatment of amyloid diseases. Recently, we identified five outstanding inhibitors of insulin amyloid-like fibril formation among the pool of 265 commercially available flavone derivatives. Here we report testing of these five compounds and of epi-gallocatechine-3-gallate (EGCG) on aggregation of alpha-synuclein and beta-amyloid. We used a Thioflavin T (ThT) fluorescence assay, relying on halftimes of aggregation as the measure of inhibition. This method avoids large numbers of false positive results. Our data indicate that four of the five flavones and EGCG inhibit alpha-synuclein aggregation in a concentration-dependent manner. However none of these derivatives were able to increase halftimes of aggregation of beta-amyloid. PMID:26421240

  15. Desmin modifications associate with amyloid-like oligomers deposition in heart failure

    PubMed Central

    Agnetti, Giulio; Halperin, Victoria L.; Kirk, Jonathan A.; Chakir, Khalid; Guo, Yurong; Lund, Linda; Nicolini, Francesco; Gherli, Tiziano; Guarnieri, Carlo; Caldarera, Claudio M.; Tomaselli, Gordon F.; Kass, David A.; Van Eyk, Jennifer E.

    2014-01-01

    Aims The ultimate cause of heart failure (HF) is not known to date. The cytoskeletal protein desmin is differentially modified and forms amyloid-like oligomers in HF. We postulated that desmin post-translational modifications (PTMs) could drive aberrant desmin aggregation in HF. Therefore, we identified these PTMs and investigated their impact on desmin amyloidogenicity in human and experimental HF. Methods and results We detected increased levels of selectively phosphorylated and cleaved desmin in a canine pacing model of dyssynchronous HF (DHF) compared with either controls or animals treated with cardiac resynchronization therapy (CRT). This unique animal model combines clinically relevant features with the possibility of a partly rescued phenotype. We confirmed analogous changes in desmin modifications in human HF and identified two phosphorylation sites within a glycogen synthase kinase 3 (GSK3) consensus sequence. Desmin-positive oligomers were also increased in DHF hearts compared with controls. Their amyloid properties were decreased by treatment with CRT or an anti-amyloid small molecule. Finally, we confirmed GSK3's involvement with desmin phosphorylation using an in vitro model. Conclusions Based on these findings, we postulate a new mechanism of cardiac toxicity based on the PTM-driven accumulation of desmin amyloid-like oligomers. Phosphorylation and cleavage as well as oligomers formation are reduced by treatment (CRT) indicating a relationship between the three. Finally, the decrease of desmin amyloid-like oligomers with CRT or small molecules points both to a general mechanism of HF based on desmin toxicity that is independent of protein mutations and to novel potential therapies. PMID:24413773

  16. pH-Driven Polymorphism of Insulin Amyloid-Like Fibrils

    PubMed Central

    Sneideris, Tomas; Darguzis, Domantas; Botyriute, Akvile; Grigaliunas, Martynas; Winter, Roland; Smirnovas, Vytautas

    2015-01-01

    Prions are infective proteins, which can self-assemble into different strain conformations, leading to different disease phenotypes. An increasing number of studies suggest that prion-like self-propagation may be a common feature of amyloid-like structures. Thus it is important to unravel every possible factor leading to the formation of different amyloid strains. Here we report on the formation of two types of insulin amyloid-like fibrils with distinct infrared spectroscopic features grown under slightly different pH conditions. Similar to prion strains, both insulin fibril types are able to self-propagate their conformational template under conditions, favoring spontaneous formation of different type fibrils. The low-pH-induced insulin amyloid strain is structurally very similar to previously reported strains formed either in the presence of 20% ethanol, or by modification of the amino acid sequence of insulin. A deeper analysis of literature data in the context of our current findings suggests a shift of the monomer-dimer equilibrium of insulin as a possible factor controlling the formation of different strains. PMID:26313643

  17. pH-Driven Polymorphism of Insulin Amyloid-Like Fibrils.

    PubMed

    Sneideris, Tomas; Darguzis, Domantas; Botyriute, Akvile; Grigaliunas, Martynas; Winter, Roland; Smirnovas, Vytautas

    2015-01-01

    Prions are infective proteins, which can self-assemble into different strain conformations, leading to different disease phenotypes. An increasing number of studies suggest that prion-like self-propagation may be a common feature of amyloid-like structures. Thus it is important to unravel every possible factor leading to the formation of different amyloid strains. Here we report on the formation of two types of insulin amyloid-like fibrils with distinct infrared spectroscopic features grown under slightly different pH conditions. Similar to prion strains, both insulin fibril types are able to self-propagate their conformational template under conditions, favoring spontaneous formation of different type fibrils. The low-pH-induced insulin amyloid strain is structurally very similar to previously reported strains formed either in the presence of 20% ethanol, or by modification of the amino acid sequence of insulin. A deeper analysis of literature data in the context of our current findings suggests a shift of the monomer-dimer equilibrium of insulin as a possible factor controlling the formation of different strains. PMID:26313643

  18. Mechanical properties of amyloid-like fibrils defined by secondary structures.

    PubMed

    Bortolini, C; Jones, N C; Hoffmann, S V; Wang, C; Besenbacher, F; Dong, M

    2015-05-01

    Amyloid and amyloid-like fibrils represent a generic class of highly ordered nanostructures that are implicated in some of the most fatal neurodegenerative diseases. On the other hand, amyloids, by possessing outstanding mechanical robustness, have also been successfully employed as functional biomaterials. For these reasons, physical and chemical factors driving fibril self-assembly and morphology are extensively studied - among these parameters, the secondary structures and the pH have been revealed to be crucial, since a variation in pH changes the fibril morphology and net chirality during protein aggregation. It is important to quantify the mechanical properties of these fibrils in order to help the design of effective strategies for treating diseases related to the presence of amyloid fibrils. In this work, we show that by changing pH the mechanical properties of amyloid-like fibrils vary as well. In particular, we reveal that these mechanical properties are strongly related to the content of secondary structures. We analysed and estimated the Young's modulus (E) by comparing the persistence length (Lp) - measured from the observation of TEM images by using statistical mechanics arguments - with the mechanical information provided by peak force quantitative nanomechanical property mapping (PF-QNM). The secondary structure content and the chirality are investigated by means of synchrotron radiation circular dichroism (SR-CD). Results arising from this study could be fruitfully used as a protocol to investigate other medical or engineering relevant peptide fibrils. PMID:25839069

  19. Amyloid-like IgM deposition neuropathy: a distinct clinico-pathologic and proteomic profiled disorder

    PubMed Central

    Figueroa, Juan J.; Bosch, E. Peter; Dyck, P. James B.; Singer, Wolfgang; Vrana, Julie A.; Theis, Jason D.; Dogan, Ahmet; Klein, Christopher J.

    2014-01-01

    Some patients with immunoglobulin paraproteinemic neuropathy have intra-nerve deposits that morphologically mimick amyloid, but do no stain with Congo red. Patients with amyloid-like deposits were identified. The nerve amyloid-like aggregates were studied by laser microdissection and dual mass spectrometry. Three male patients, all with IgM gammopathy, and neuropathy were identified. Follow-up, disease duration was 5, 19, and 7 years, respectively. All had progressive asymmetric sensory-onset distal axonal polyneuropathy with late motor involvement. Autonomic symptoms occurred in only one after 13 years of symptoms. None had clinical cardio-renal involvement. One had skin papules with dermal amyloid-like deposits. Endoneurial amyloid-like deposits had granulo-fibrillar ultrastructure. Mass spectrometry of laser-dissected deposits identified IgM pentameric macroglobulin (heavy, light, and joining chains) without amyloid-associated proteins including absent apolipoprotein E and serum amyloid P-component. Amyloid-like neuropathy has distinct clinical, pathologic, and proteomic features which expand the spectrum of IgM neuropathies. Patients have favorable survival, relative absence of autonomic features, and distinct proteomic profiles of the infiltrative protein in nerve. PMID:22734903

  20. Amyloid-like properties of Saccharomyces cerevisiae cell wall glucantransferase Bgl2p

    PubMed Central

    Plotnikova, Tatyana A; Gorkovskii, Anton A; Selyakh, Irina O; Galzitskaya, Oxana V; Bezsonov, Evgeniy E; Gellissen, Gerd; Kulaev, Igor S

    2008-01-01

    Glucantransferase Bgl2p is a major conserved cell wall constituent described for a wide range of yeast species. In the baker's yeast Saccharomyces cerevisiae it is the only non-covalently bound cell wall protein that cannot be released from cell walls by sequential SDS and trypsin treatment. It contains seven amyloidogenic determinants. Circular dichroism analysis and fluorescence spectroscopy with thioflavin T indicate the presence of β-sheet structures in Bgl2p isolates. Bgl2p forms fibrils, a process that is enforced in the presence of other cell wall components. Thus the data obtained is the first evidence for amyloid-like properties of yeast cell wall protein—glucantransferase Bgl2p. PMID:19098439

  1. Mechanical properties of amyloid-like fibrils defined by secondary structures

    NASA Astrophysics Data System (ADS)

    Bortolini, C.; Jones, N. C.; Hoffmann, S. V.; Wang, C.; Besenbacher, F.; Dong, M.

    2015-04-01

    Amyloid and amyloid-like fibrils represent a generic class of highly ordered nanostructures that are implicated in some of the most fatal neurodegenerative diseases. On the other hand, amyloids, by possessing outstanding mechanical robustness, have also been successfully employed as functional biomaterials. For these reasons, physical and chemical factors driving fibril self-assembly and morphology are extensively studied - among these parameters, the secondary structures and the pH have been revealed to be crucial, since a variation in pH changes the fibril morphology and net chirality during protein aggregation. It is important to quantify the mechanical properties of these fibrils in order to help the design of effective strategies for treating diseases related to the presence of amyloid fibrils. In this work, we show that by changing pH the mechanical properties of amyloid-like fibrils vary as well. In particular, we reveal that these mechanical properties are strongly related to the content of secondary structures. We analysed and estimated the Young's modulus (E) by comparing the persistence length (Lp) - measured from the observation of TEM images by using statistical mechanics arguments - with the mechanical information provided by peak force quantitative nanomechanical property mapping (PF-QNM). The secondary structure content and the chirality are investigated by means of synchrotron radiation circular dichroism (SR-CD). Results arising from this study could be fruitfully used as a protocol to investigate other medical or engineering relevant peptide fibrils.Amyloid and amyloid-like fibrils represent a generic class of highly ordered nanostructures that are implicated in some of the most fatal neurodegenerative diseases. On the other hand, amyloids, by possessing outstanding mechanical robustness, have also been successfully employed as functional biomaterials. For these reasons, physical and chemical factors driving fibril self-assembly and morphology

  2. Preparation of amyloid-like fibrils containing magnetic iron oxide nanoparticles: Effect of protein aggregation on proton relaxivity

    SciTech Connect

    Viktor Andersson, B.; Skoglund, Caroline; Uvdal, Kajsa; Solin, Niclas

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer Preparation of amyloid materials labeled with magnetic iron oxide nanoparticles. Black-Right-Pointing-Pointer Characterization of amyloid materials by electron tomography. Black-Right-Pointing-Pointer Influence of protein aggregation on the magnetic nanoparticle properties. -- Abstract: A method to prepare amyloid-like fibrils functionalized with magnetic nanoparticles has been developed. The amyloid-like fibrils are prepared in a two step procedure, where insulin and magnetic nanoparticles are mixed simply by grinding in the solid state, resulting in a water soluble hybrid material. When the hybrid material is heated in aqueous acid, the insulin/nanoparticle hybrid material self assembles to form amyloid-like fibrils incorporating the magnetic nanoparticles. This results in magnetically labeled amyloid-like fibrils which has been characterized by Transmission Electron Microscopy (TEM) and electron tomography. The influence of the aggregation process on proton relaxivity is investigated. The prepared materials have potential uses in a range of bio-imaging applications.

  3. Self-Assembled Amyloid-Like Oligomeric-Cohesin Scaffoldin for Augmented Protein Display on the Saccharomyces cerevisiae Cell Surface

    PubMed Central

    Han, Zhenlin; Zhang, Bei; Wang, Yi E.; Zuo, Yi Y.

    2012-01-01

    In this study, a molecular self-assembly strategy to develop a novel protein scaffold for amplifying the extent and variety of proteins displayed on the surface of Saccharomyces cerevisiae is presented. The cellulosomal scaffolding protein cohesin and its upstream hydrophilic domain (HD) were genetically fused with the yeast Ure2p N-terminal fibrillogenic domain consisting of residues 1 to 80 (Ure2p1-80). The resulting Ure2p1-80-HD-cohesin fusion protein was successfully expressed in Escherichia coli to produce self-assembled supramolecular nanofibrils that serve as a novel protein scaffold displaying multiple copies of functional cohesin domains. The amyloid-like property of the nanofibrils was confirmed via thioflavin T staining and atomic force microscopy. These cohesin nanofibrils attached themselves, via a green fluorescent protein (GFP)-dockerin fusion protein, to the cell surface of S. cerevisiae engineered to display a GFP-nanobody. The excess cohesin units on the nanofibrils provide ample sites for binding to dockerin fusion proteins, as exemplified using an mCherry-dockerin fusion protein as well as the Clostridium cellulolyticum CelA endoglucanase. More than a 24-fold increase in mCherry fluorescence and an 8-fold increase in CelA activity were noted when the cohesin nanofibril scaffold-mediated yeast display was used, compared to using yeast display with GFP-cohesin that contains only a single copy of cohesin. Self-assembled supramolecular cohesin nanofibrils created by fusion with the yeast Ure2p fibrillogenic domain provide a versatile protein scaffold that expands the utility of yeast cell surface display. PMID:22344635

  4. Tensile deformation and failure of amyloid and amyloid-like protein fibrils

    NASA Astrophysics Data System (ADS)

    Solar, Max; Buehler, Markus J.

    2014-03-01

    Here we report a series of full atomistic molecular dynamics simulations of six amyloid or amyloid-like protein fibrils in order to systematically understand the effect of different secondary structure motifs on the mechanical tensile and failure response of cross-\\beta protein fibrils. We find a similar failure behavior across the six structures; an initial failure event occurs at small strains involving cooperative rupture of a group of hydrogen bonds, followed by a slow one-by-one hydrogen bond rupture process as the remaining \\beta -sheets peel off with very low applied stress. We also find that the ultimate tensile strength of the protein fibrils investigated scales directly with the number of hydrogen bonds per unit area which break in the initial rupture event. Our results provide insights into structure-property relationships in protein fibrils important for disease and engineering applications and lay the groundwork for the development of materials selection criteria for the design of de novo amyloid-based functional biomaterials.

  5. Salts drive controllable multilayered upright assembly of amyloid-like peptides at mica/water interface

    PubMed Central

    Dai, Bin; Kang, Seung-gu; Huynh, Tien; Lei, Haozhi; Castelli, Matteo; Hu, Jun; Zhang, Yi; Zhou, Ruhong

    2013-01-01

    Surface-assisted self-assembly of amyloid-like peptides has received considerable interest in both amyloidosis research and nanotechnology in recent years. Despite extensive studies, some controlling factors, such as salts, are still not well understood, even though it is known that some salts can promote peptide self-assemblies through the so-called “salting-out” effect. However, they are usually noncontrollable, disordered, amorphous aggregates. Here, we show via a combined experimental and theoretical approach that a conserved consensus peptide NH2-VGGAVVAGV-CONH2 (GAV-9) (from representative amyloidogenic proteins) can self-assemble into highly ordered, multilayered nanofilaments, with surprising all-upright conformations, under high-salt concentrations. Our atomic force microscopy images also demonstrate that the vertical stacking of multiple layers is highly controllable by tuning the ionic strength, such as from 0 mM (monolayer) to 100 mM (mainly double layer), and to 250 mM MgCl2 (double, triple, quadruple, and quintuple layers). Our atomistic molecular dynamics simulations then reveal that these individual layers have very different internal nanostructures, with parallel β-sheets in the first monolayer but antiparallel β-sheets in the subsequent upper layers due to their different microenvironment. Further studies show that the growth of multilayered, all-upright nanostructures is a common phenomenon for GAV-9 at the mica/water interface, under a variety of salt types and a wide range of salt concentrations. PMID:23650355

  6. Salts drive controllable multilayered upright assembly of amyloid-like peptides at mica/water interface.

    PubMed

    Dai, Bin; Kang, Seung-gu; Huynh, Tien; Lei, Haozhi; Castelli, Matteo; Hu, Jun; Zhang, Yi; Zhou, Ruhong

    2013-05-21

    Surface-assisted self-assembly of amyloid-like peptides has received considerable interest in both amyloidosis research and nanotechnology in recent years. Despite extensive studies, some controlling factors, such as salts, are still not well understood, even though it is known that some salts can promote peptide self-assemblies through the so-called "salting-out" effect. However, they are usually noncontrollable, disordered, amorphous aggregates. Here, we show via a combined experimental and theoretical approach that a conserved consensus peptide NH2-VGGAVVAGV-CONH2 (GAV-9) (from representative amyloidogenic proteins) can self-assemble into highly ordered, multilayered nanofilaments, with surprising all-upright conformations, under high-salt concentrations. Our atomic force microscopy images also demonstrate that the vertical stacking of multiple layers is highly controllable by tuning the ionic strength, such as from 0 mM (monolayer) to 100 mM (mainly double layer), and to 250 mM MgCl2 (double, triple, quadruple, and quintuple layers). Our atomistic molecular dynamics simulations then reveal that these individual layers have very different internal nanostructures, with parallel β-sheets in the first monolayer but antiparallel β-sheets in the subsequent upper layers due to their different microenvironment. Further studies show that the growth of multilayered, all-upright nanostructures is a common phenomenon for GAV-9 at the mica/water interface, under a variety of salt types and a wide range of salt concentrations. PMID:23650355

  7. Quantum dots induce charge-specific amyloid-like fibrillation of insulin at physiological conditions

    NASA Astrophysics Data System (ADS)

    Sukhanova, Alyona; Poly, Simon; Shemetov, Anton; Nabiev, Igor R.

    2012-10-01

    Agglomeration of some proteins may give rise to aggregates that have been identified as the main cause of amyloid diseases. For example, fibrillation of insulin is related to diabetes mellitus. Quantum dots (QDs) are of special interest as tagging agents for diagnostic and therapeutic studies due to their broad absorption spectra, narrow emission spectra, and high photostability. In this study, PEGylated CdSe/ZnS QDs have been shown to induce the formation of amyloid-like fibrils of human insulin under physiological conditions, this process being dependent on the variation of the surface charge of the nanoparticles (NPs) used. Circular dichroism (CD), protein secondary structure analysis, thioflavin T (ThT) fluorescence assay, and the dynamic light scattering (DLS) technique have been used for comparative analysis of different stages of the fibrillation process. In particular, insulin secondary structure remodelling accompanied by a considerable increase in the rate of amyloid fiber formation have been observed after insulin was mixed with PEGylated QDs. Nanoparticles may significantly influence the rate of protein fibrillation and induce new mechanisms of amyloid diseases, as well as offer opportunities for their treatment.

  8. MAAP: malarial adhesins and adhesin-like proteins predictor.

    PubMed

    Ansari, Faraz Alam; Kumar, Naveen; Bala Subramanyam, Mekapati; Gnanamani, Muthiah; Ramachandran, Srinivasan

    2008-02-15

    Malaria caused by protozoan parasites belonging to the genus Plasmodium is a dreaded disease, second only to tuberculosis. The emergence of parasites resistant to commonly used drugs and the lack of availability of vaccines aggravates the problem. One of the preventive approaches targets adhesion of parasites to host cells and tissues. Adhesion of parasites is mediated by proteins called adhesins. Abrogation of adhesion by either immunizing the host with adhesins or inhibiting the interaction using structural analogs of host cell receptors holds the potential to develop novel preventive strategies. The availability of complete genome sequence offers new opportunities for identifying adhesin and adhesin-like proteins. Development of computational algorithms can simplify this task and accelerate experimental characterization of the predicted adhesins from complete genomes. A curated positive dataset of experimentally known adhesins from Plasmodium species was prepared by careful examination of literature reports. "Controversial" or "hypothetical" adhesins were excluded. The negative dataset consisted of proteins representing various intracellular functions including information processing, metabolism, and interface (transporters). We did not include proteins likely to be on the surface with unknown adhesin properties or which are linked even indirectly to the adhesion process in either of the training sets. A nonhomology-based approach using 420 compositional properties of amino acid dipeptide and multiplet frequencies was used to develop MAAP Web server with Support Vector Machine (SVM) model classifier as its engine for the prediction of malarial adhesins and adhesin-like proteins. The MAAP engine has six SVM classifier models identified through an exhaustive search from 728 kernel parameters set. These models displayed an efficiency (Mathews correlation coefficient) of 0.860-0.967. The final prediction P(maap) score is the maximum score attained by a given

  9. Amyloid-like conformation and interaction for the self-assembly in barnacle underwater cement.

    PubMed

    Nakano, Masahiro; Kamino, Kei

    2015-01-27

    Barnacles are unique marine sessile crustaceans and permanently attach to various foreign surfaces during most of their lifespan. The protein complex secreted from their body and used to attach their calcareous shell to almost all surfaces in water has long fascinated us because we have limited technology with which to attach materials in water. Unraveling the mechanism of underwater attachment by barnacles is thus important for interface science, for the understanding of the biology and physiology of barnacles, and for the development of technology to prevent fouling. Previous studies have indicated that the intermolecular interactions optimized by conformations of the adhesive proteins are crucial in the self-assembly and/or curing of the adhesive. This study aimed to identify the possible structural determinants responsible for the self-assembly. Thioflavin T binding screening of peptides designed on the basis of the primary structure of a bulk 52 kDa cement protein indicated the presence of some amyloidogenic motifs in the protein. The conformation of the peptide was transformed to a β-sheet by an increase in either pH or ionic strength, resulting in its self-assembly. Thioflavin T binding was inhibited by small polyphenolic molecules, suggesting the contribution of aromatic interactions during self-assembly. The occurrence of amyloid-like units in the protein implies that the protein conformation is an important factor contributing to the self-assembly of the cement, the first event of the curing, as the adhesive material is secreted into the seawater out of the animal's body.

  10. Double-stranded DNA stereoselectively promotes aggregation of amyloid-like fibrils and generates peptide/DNA matrices.

    PubMed

    Yamada, Masanori; Hara, Sachiko; Yamada, Tetsuya; Katagiri, Fumihiko; Hozumi, Kentaro; Nomizu, Motoyoshi

    2014-11-01

    An amyloidogenic LAM-L peptide (AASIKVAVSADR, all-L configuration) derived from laminin promoted cell adhesion, neurite outgrowth, and angiogenesis. Here, we prepared novel matrices using double-stranded DNA and the LAM-L peptide. Double-stranded DNA promoted aggregation of amyloid-like fibrils and generated a LAM-L/DNA matrix through electrostatic interactions between the phosphate groups of DNA and the amino groups of LAM-L. This formation of peptide/DNA matrix depends on the Ile-Lys-Val-Ala-Val (IKVAV) sequence in the peptide, since LAM-RM peptide (AASVVIAKSADR), which is scrambled peptide of LAM-L, did not form a matrix with DNA. Further, LAM-D (all-D configuration of LAM-L), which forms amyloid-like fibrils and promotes similar biological activities as LAM-L, did not form amyloid-like fibrils with DNA, suggesting that DNA selectively interacts with the L-configured peptide. Moreover, the LAM-L/DNA matrices showed stronger cell attachment activity compared with LAM-L alone, suggesting the LAM-L/DNA matrices have potential for use as a novel biomaterial in tissue engineering.

  11. The Biology of Neisseria Adhesins

    PubMed Central

    Hung, Miao-Chiu; Christodoulides, Myron

    2013-01-01

    Members of the genus Neisseria include pathogens causing important human diseases such as meningitis, septicaemia, gonorrhoea and pelvic inflammatory disease syndrome. Neisseriae are found on the exposed epithelia of the upper respiratory tract and the urogenital tract. Colonisation of these exposed epithelia is dependent on a repertoire of diverse bacterial molecules, extending not only from the surface of the bacteria but also found within the outer membrane. During invasive disease, pathogenic Neisseriae also interact with immune effector cells, vascular endothelia and the meninges. Neisseria adhesion involves the interplay of these multiple surface factors and in this review we discuss the structure and function of these important molecules and the nature of the host cell receptors and mechanisms involved in their recognition. We also describe the current status for recently identified Neisseria adhesins. Understanding the biology of Neisseria adhesins has an impact not only on the development of new vaccines but also in revealing fundamental knowledge about human biology. PMID:24833056

  12. Tuning calcium carbonate growth through physical confinement and templating with amyloid-like polypeptide aggregates

    NASA Astrophysics Data System (ADS)

    Colaco, Martin Francis

    that this methodology does not extend to three-dimensional confined systems, as the water has no method of escape. Through the addition of an insoluble hydroscopic polymer to our microreactors, amorphous calcium carbonate of controllable sizes can be grown. However, crystalline calcium carbonate cannot be grown without some type of templating. Studies of calcium carbonate templating have predominantly been performed on SAMs or in poorly characterized gels or protein films. The use of ordered protein or polypeptide aggregates for templating permits both geometry and charge surface density to be varied. We have studied the kinetics and final morphology of ordered aggregates of poly-L-glutamic acid and a copolymer of glutamic acid and alanine through experiments and simulations. Electrostatics, not structure, of the monomer appeared to be the dominating factor in the aggregation, as pH and salt concentration changes led to dramatic changes in the kinetics. Examining our experimental with existing models provided inconsistent results, so we developed a new model that yielded physically realistic rate constants, while generating better fits with longer lag phases and faster growths. However, despite the similarity of aggregation conditions, the two polypeptides yielded vastly different morphologies, with the PEA forming typical amyloid-like fibrils and PE forming larger, twisted lamellar aggregates. Templating with these aggregates also yielded dramatically different patterns. Polycrystalline rhombohedral calcite with smooth faces and edges grew on PEA fibrils, with minimal templating in evidence. However, on PE, numerous calcite crystals with triangular projections tracked the surface of the aggregate. The PE lamellae are characterized by extensive beta-sheet structure. In this conformation, the glutamic acid spacings on the surface of the aggregates can mimic the spacings of the carboxylates in the calcite lattice. In addition, the high negative charge density on the

  13. A domain dictionary of trimeric autotransporter adhesins.

    PubMed

    Bassler, Jens; Hernandez Alvarez, Birte; Hartmann, Marcus D; Lupas, Andrei N

    2015-02-01

    Trimeric autotransporter adhesins (TAAs) are modular, highly repetitive outer membrane proteins that mediate adhesion to external surfaces in many Gram-negative bacteria. In recent years, several TAAs have been investigated in considerable detail, also at the structural level. However, in their vast majority, putative TAAs in prokaryotic genomes remain poorly annotated, due to their sequence diversity and changeable domain architecture. In order to achieve an automated annotation of these proteins that is both detailed and accurate we have taken a domain dictionary approach, in which we identify recurrent domains by sequence comparisons, produce bioinformatic descriptors for each domain type, and connect these to structural information where available. We implemented this approach in a web-based platform, daTAA, in 2008 and demonstrated its applicability by reconstructing the complete fiber structure of a TAA conserved in enterobacteria. Here we review current knowledge on the domain structure of TAAs.

  14. Antibodies derived from an enterotoxigenic Escherichia coli (ETEC) adhesin tip MEFA (multiepitope fusion antigen) against adherence of nine ETEC adhesins: CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21 and EtpA.

    PubMed

    Nandre, Rahul M; Ruan, Xiaosai; Duan, Qiangde; Sack, David A; Zhang, Weiping

    2016-06-30

    Diarrhea continues to be a leading cause of death in children younger than 5 years in developing countries. Enterotoxigenic Escherichia coli (ETEC) is a leading bacterial cause of children's diarrhea and travelers' diarrhea. ETEC bacteria initiate diarrheal disease by attaching to host receptors at epithelial cells and colonizing in small intestine. Therefore, preventing ETEC attachment has been considered the first line of defense against ETEC diarrhea. However, developing vaccines effectively against ETEC bacterial attachment encounters challenge because ETEC strains produce over 23 immunologically heterogeneous adhesins. In this study, we applied MEFA (multiepitope fusion antigen) approach to integrate epitopes from adhesin tips or adhesive subunits of CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21 and EtpA adhesins and to construct an adhesin tip MEFA peptide. We then examined immunogenicity of this tip MEFA in mouse immunization, and assessed potential application of this tip MEFA for ETEC vaccine development. Data showed that mice intraperitoneally immunized with this adhesin tip MEFA developed IgG antibody responses to all nine ETEC adhesins. Moreover, ETEC and E. coli bacteria expressing these nine adhesins, after incubation with serum of the immunized mice, exhibited significant reduction in attachment to Caco-2 cells. These results indicated that anti-adhesin antibodies induced by this adhesin tip MEFA blocked adherence of the most important ETEC adhesins, suggesting this multivalent tip MEFA may be useful for developing a broadly protective anti-adhesin vaccine against ETEC diarrhea. PMID:27228947

  15. Time-lapse atomic force microscopy in the characterization of amyloid-like fibril assembly and oligomeric intermediates.

    PubMed

    Goldsbury, Claire; Green, Janelle

    2005-01-01

    The atomic force microscope (AFM) images the topography of biological structures adsorbed to surfaces with nanometer to angstrom scale resolution. Amyloid-like fibrils and oligomers can be imaged in buffer solutions, allowing the samples to retain physiological-like properties while temporal changes in structure are monitored, e.g., the elongation of fibrils or the growth of single oligomers. These qualities distinguish AFM from conventional imaging techniques of comparable resolution, i.e., electron microscopy (EM). However, AFM is limited in that the specimen must be firmly attached to a solid support for measurement and that time-lapse imaging of individual assemblies can thus only be achieved for fibrils and oligomers growing on this support. Nevertheless, AFM has provided several insights into the in vitro assembly mechanism and structures of amyloid-like fibrils. The first section of this chapter provides a methodological introduction to AFM, whilst the second details the application of this technique to the investigation of amyloidogenic proteins, specifically amylin and amyloid-beta (Abeta) peptides. PMID:15980598

  16. A genomic region involved in the formation of adhesin fibers in Bacillus cereus biofilms

    PubMed Central

    Caro-Astorga, Joaquín; Pérez-García, Alejandro; de Vicente, Antonio; Romero, Diego

    2015-01-01

    Bacillus cereus is a bacterial pathogen that is responsible for many recurrent disease outbreaks due to food contamination. Spores and biofilms are considered the most important reservoirs of B. cereus in contaminated fresh vegetables and fruits. Biofilms are bacterial communities that are difficult to eradicate from biotic and abiotic surfaces because of their stable and extremely strong extracellular matrix. These extracellular matrixes contain exopolysaccharides, proteins, extracellular DNA, and other minor components. Although B. cereus can form biofilms, the bacterial features governing assembly of the protective extracellular matrix are not known. Using the well-studied bacterium B. subtilis as a model, we identified two genomic loci in B. cereus, which encodes two orthologs of the amyloid-like protein TasA of B. subtilis and a SipW signal peptidase. Deletion of this genomic region in B. cereus inhibited biofilm assembly; notably, mutation of the putative signal peptidase SipW caused the same phenotype. However, mutations in tasA or calY did not completely prevent biofilm formation; strains that were mutated for either of these genes formed phenotypically different surface attached biofilms. Electron microscopy studies revealed that TasA polymerizes to form long and abundant fibers on cell surfaces, whereas CalY does not aggregate similarly. Heterologous expression of this amyloid-like cassette in a B. subtilis strain lacking the factors required for the assembly of TasA amyloid-like fibers revealed (i) the involvement of this B. cereus genomic region in formation of the air-liquid interphase pellicles and (ii) the intrinsic ability of TasA to form fibers similar to the amyloid-like fibers produced by its B. subtilis ortholog. PMID:25628606

  17. Assessment of Adhesins as an Indicator of Pathovar-Associated Virulence Factors in Bovine Escherichia coli.

    PubMed

    Valat, Charlotte; Forest, Karine; Auvray, Frédéric; Métayer, Véronique; Méheut, Thomas; Polizzi, Charlène; Gay, Emilie; Haenni, Marisa; Oswald, Eric; Madec, Jean-Yves

    2014-12-01

    The CS31A, F17, and F5 adhesins are usually targeted by serology-based methods to detect pathogenic Escherichia coli associated with calf enteritis. However, the virulence traits of the selected isolates are still poorly described. Here, from a set of 349 diarrheagenic E. coli isolates from cattle, we demonstrated a 70.8% concordance rate (Cohen's kappa, 0.599) between serology- and PCR-based approaches for the detection of adhesins under field conditions. A 79% to 82.4% correspondence between the two methods was found for fimbrial adhesins, whereas major discrepancies (33%) were observed for CS31A-type antigens. Various F17A variants were found, such as F17Ac (20K) (50%), F17Aa (FY) (18.9%), F17Ab (8.1%), and F17Ad (111K) (5.4%), including a high proportion (17.6%) of new F17A internal combinations (F17Aab, F17Aac, and F17Abc) or untypeable variants. In addition, the highest proportion of pathovar-associated virulence factor (VF) genes was observed among E. coli isolates that produced F5/F41 adhesins. A specific link between the heat-stable toxins related to the enterotoxigenic E. coli (ETEC) pathovar and adhesins was identified. STa was significantly linked to F5/F41 and EAST1 to CS31A adhesins (P < 0.001), respectively, whereas NTEC was associated with F17 adhesin (P = 0.001). Clustering between phylogroups according to the adhesin types was also observed. Also, few Shiga toxin-producing E. coli (STEC) or enteropathogenic E. coli (EPEC) pathovars were identified. Finally, no statistically significant difference was observed in the occurrence of extended-spectrum beta lactamase (ESBL) production according to the adhesins expressed by the isolates (P = 0.09). Altogether, this study gives new insights into the relationship between adhesins, VF, and antimicrobial resistance in calf enteritis and supports the need for further standardization of methodologies for such approaches. PMID:25217019

  18. HecA, a member of a class of adhesins produced by diverse pathogenic bacteria, contributes to the attachment, aggregation, epidermal cell killing, and virulence phenotypes of Erwinia chrysanthemi EC16 on Nicotiana clevelandii seedlings

    PubMed Central

    Rojas, Clemencia M.; Ham, Jong Hyun; Deng, Wen-Ling; Doyle, Jeff J.; Collmer, Alan

    2002-01-01

    Erwinia chrysanthemi is representative of a broad class of bacterial pathogens that are capable of inducing necrosis in plants. The E. chrysanthemi EC16 hecA gene predicts a 3,850-aa member of the Bordetella pertussis filamentous hemagglutinin family of adhesins. A hecA∷Tn7 mutant was reduced in virulence on Nicotiana clevelandii seedlings after inoculation without wounding. Epifluorescence and confocal laser-scanning microscopy observations of hecA and wild-type cells expressing the green fluorescent protein revealed that the mutant is reduced in its ability to attach and then form aggregates on leaves and to cause an aggregate-associated killing of epidermal cells. Cell killing also depended on production of the major pectate lyase isozymes and the type II, but not the type III, secretion pathway in E. chrysanthemi. HecA homologs were found in bacterial pathogens of plants and animals and appear to be unique to pathogens and universal in necrogenic plant pathogens. Phylogenetic comparison of the conserved two-partner secretion domains in the proteins and the 16S rRNA sequences in respective bacteria revealed the two datasets to be fundamentally incongruent, suggesting horizontal acquisition of these genes. Furthermore, hecA and its two homologs in Yersinia pestis had a G+C content that was 10% higher than that of their genomes and similar to that of plant pathogenic Ralstonia, Xylella, and Pseudomonas spp. Our data suggest that filamentous hemagglutinin-like adhesins are broadly important virulence factors in both plant and animal pathogens. PMID:12271135

  19. FaaPred: A SVM-Based Prediction Method for Fungal Adhesins and Adhesin-Like Proteins

    PubMed Central

    Ramana, Jayashree; Gupta, Dinesh

    2010-01-01

    Adhesion constitutes one of the initial stages of infection in microbial diseases and is mediated by adhesins. Hence, identification and comprehensive knowledge of adhesins and adhesin-like proteins is essential to understand adhesin mediated pathogenesis and how to exploit its therapeutic potential. However, the knowledge about fungal adhesins is rudimentary compared to that of bacterial adhesins. In addition to host cell attachment and mating, the fungal adhesins play a significant role in homotypic and xenotypic aggregation, foraging and biofilm formation. Experimental identification of fungal adhesins is labor- as well as time-intensive. In this work, we present a Support Vector Machine (SVM) based method for the prediction of fungal adhesins and adhesin-like proteins. The SVM models were trained with different compositional features, namely, amino acid, dipeptide, multiplet fractions, charge and hydrophobic compositions, as well as PSI-BLAST derived PSSM matrices. The best classifiers are based on compositional properties as well as PSSM and yield an overall accuracy of 86%. The prediction method based on best classifiers is freely accessible as a world wide web based server at http://bioinfo.icgeb.res.in/faap. This work will aid rapid and rational identification of fungal adhesins, expedite the pace of experimental characterization of novel fungal adhesins and enhance our knowledge about role of adhesins in fungal infections. PMID:20300572

  20. Recombinant Human Semenogelin-1 (Sg1) and Sg1 (1-159) form Detergent Stable Amyloid like Aggregates in vitro.

    PubMed

    Sharma, Neetu; Vishwanath, S; Patel, Basant K

    2016-01-01

    Senile seminal vesicle amyloidosis (SSVA) is associated with deposition of semenogelin-1 (Sg1) protein aggregates in seminal vesicles that may manifest as hematospermia. Sg1 is the predominant protein that entraps spermatozoa which are freed upon fragmentation of Sg1 by the protease prostate specific antigen (PSA), post semen release. Certain small peptide fragments of Sg1 have been reported to form amyloid aggregates in vitro that can enhance HIV infectivity to cell cultures. However, the amyloid deposits in the seminal vesicles are expected to be that of the full length Sg1, as PSA is encountered downstream. So far, amyloid forming ability of full length Sg1 has not been established in vitro. Here, we examined the amyloidogenicity of full length Sg1 and a large fragment Sg1 (1-159), using recombinant proteins and tested if Zinc has any effect on their aggregation. Levels of Zinc, which is essential for health of male reproductive system, gradually decline with age. We succeeded in forming amyloid-like aggregates of Sg1 full length and Sg1 (1-159) fragment showing detergent stability and found that presence of Zn2+ substantially inhibits their amyloid aggregation in vitro. Possibly, high Zn2+ found in seminal plasma of young individuals may have preventive role against aggregation of Sg1 in seminal vesicles.

  1. Hevea brasiliensis prohevein possesses a conserved C-terminal domain with amyloid-like properties in vitro.

    PubMed

    Berthelot, Karine; Lecomte, Sophie; Coulary-Salin, Bénédicte; Bentaleb, Ahmed; Peruch, Frédéric

    2016-04-01

    Prohevein is a wound-induced protein and a main allergen from latex of Hevea brasiliensis (rubber tree). This 187 amino-acid protein is cleaved in two fragments: a N-terminal 43 amino-acids called hevein, a lectin bearing a chitin-binding motif with antifungal properties and a C-terminal domain (C-ter) far less characterized. We provide here new insights on the characteristics of prohevein, hevein and C-terminal domain. Using complementary biochemical (ThT/CR/chitin binding, agglutination) and structural (modeling, ATR-FTIR, TEM, WAXS) approaches, we show that this domain clearly displays all the characteristics of an amyloid-like proteins in vitro, that could confer agglutination activity in synergy with its chitin-binding activity. Additionally, this C-ter domain is highly conserved and present in numerous plant prohevein-like proteins or pathogenesis-related (PR and WIN) proteins. This could be the hallmark of the eventual presence of proteins with amyloid properties in plants, that could potentially play a role in defense through aggregation properties. PMID:26805576

  2. An alternative structural isoform in amyloid-like aggregates formed from thermally denatured human γD-crystallin.

    PubMed

    Moran, Sean D; Zhang, Tianqi O; Zanni, Martin T

    2014-03-01

    The eye lens protein γD-crystallin contributes to cataract formation in the lens. In vitro experiments show that γD-crystallin has a high propensity to form amyloid fibers when denatured, and that denaturation by acid or UV-B photodamage results in its C-terminal domain forming the β-sheet core of amyloid fibers. Here, we show that thermal denaturation results in sheet-like aggregates that contain cross-linked oligomers of the protein, according to transmission electron microscopy and SDS-PAGE. We use two-dimensional infrared spectroscopy to show that these aggregates have an amyloid-like secondary structure with extended β-sheets, and use isotope dilution experiments to show that each protein contributes approximately one β-strand to each β-sheet in the aggregates. Using segmental (13) C labeling, we show that the organization of the protein's two domains in thermally induced aggregates results in a previously unobserved structure in which both the N-terminal and C-terminal domains contribute to β-sheets. We propose a model for the structural organization of the aggregates and attribute the recruitment of the N-terminal domain into the fiber structure to intermolecular cross linking. PMID:24415662

  3. Evaluation of protease resistance and toxicity of amyloid-like food fibrils from whey, soy, kidney bean, and egg white.

    PubMed

    Lassé, Moritz; Ulluwishewa, Dulantha; Healy, Jackie; Thompson, Dion; Miller, Antonia; Roy, Nicole; Chitcholtan, Kenny; Gerrard, Juliet A

    2016-02-01

    The structural properties of amyloid fibrils combined with their highly functional surface chemistry make them an attractive new food ingredient, for example as highly effective gelling agents. However, the toxic role of amyloid fibrils in disease may cause some concern about their food safety because it has not been established unequivocally if consumption of food fibrils poses a health risk to consumers. Here we present a study of amyloid-like fibrils from whey, kidney bean, soy bean, and egg white to partially address this concern. Fibrils showed varied resistance to proteolytic digestion in vitro by either Proteinase K, pepsin or pancreatin. The toxicity of mature fibrils was measured in vitro and compared to native protein, early-stage-fibrillar protein, and sonicated fibrils in two immortalised human cancer cell lines, Caco-2 and Hec-1a. There was no reduction in the viability of either Caco-2 or Hec-1a cells after treatment with a fibril concentration of up to 0.25 mg/mL.

  4. Distribution and degree of heterogeneity of the afimbrial-adhesin-encoding operon (afa) among uropathogenic Escherichia coli isolates.

    PubMed

    Labigne-Roussel, A; Falkow, S

    1988-03-01

    The afimbrial adhesin (AFA-I) from a pyelonephritic Escherichia coli isolate (KS52) is a mannose-resistant, P-independent, X-binding adhesin, expressed by the afa-1 operon. It is distinct from the E. coli X-binding adhesins with M and S specificity. A total of 138 E. coli isolates belonging to various serotypes, mostly from urinary tract infections, were screened for the presence of DNA sequences related to the afa operon and for the expression of an X-adhesin able to mediate mannose-resistant hemagglutination (MRHA) and adhesion to uroepithelial cells. Fifteen strains were shown to harbor DNA sequences related to the AFA-I-encoding operon, and 13 of them expressed an X-adhesin. Using as probes different DNA segments of the AFA-I-encoding operon in Southern experiments, we demonstrated that only three of these clinical isolates contained genetic determinants closely related to those identified in the original afa prototype strain (KS52): presence of the afaA, afaB, afaC, afaD, and afaE genes associated with the expression of a 16,000-dalton hemagglutinin-adhesin which strongly cross-reacted with AFA-I-specific antibodies. The other E. coli isolates harbored DNA sequences homologous to the afaA, afaB, afaC, and afaD genes, but lacked the sequence corresponding to the adhesin-producing gene afaE; Western blots allowed the detection of polypeptides (15,000, 15,500, or 16,000 daltons) in these strains which cross-reacted with variable intensity with antibodies raised against the denatured AFA-I protein, but did not cross-react with native AFA-I-specific antibodies. Following DNA cloning experiments from chromosomal DNA of two of those strains (A22 and A30), we demonstrated that although the AFA-related operon in A22 and A30 strains lacked the AFA-I adhesin-encoding gene, they synthesized a functional X-adhesin. Thus, strains A22 and A30 encode adhesins designated AFA-II and AFA-III, which were cloned on recombinant plasmids pILL72 and pILL61, respectively. Southern

  5. FimH adhesin of Escherichia coli K1 type 1 fimbriae activates BV-2 microglia

    SciTech Connect

    Lee, Jongseok; Shin, Sooan; Teng, C.-H.; Hong, Suk Jin; Kim, Kwang Sik . E-mail: kwangkim@jhmi.edu

    2005-09-02

    The generation of intense inflammation in the subarachnoid space in response to meningitis-causing bacteria contributes to brain dysfunction and neuronal injury in bacterial meningitis. Microglia, the major immune effector cells in the central nervous system (CNS), become activated by bacterial components to produce proinflammatory immune mediators. In this study, we showed that FimH adhesin, a tip component of type 1 fimbriae of meningitis-causing Escherichia coli K1, activated the murine microglial cell line, BV-2, which resulted in the production of nitric oxide and the release of tumor necrosis factor-{alpha}. Mitogen-activated protein kinases, ERK and p-38, and nuclear factor-{kappa}B were involved in FimH adhesin-mediated microglial activation. These findings suggest that FimH adhesin contributes to the CNS inflammatory response by virtue of activating microglia in E. coli meningitis.

  6. Mid-infrared free-electron laser tuned to the amide I band for converting insoluble amyloid-like protein fibrils into the soluble monomeric form.

    PubMed

    Kawasaki, Takayasu; Fujioka, Jun; Imai, Takayuki; Torigoe, Kanjiro; Tsukiyama, Koichi

    2014-09-01

    A mid-infrared free-electron laser (FEL) is operated as a pulsed and linearly polarized laser with tunable wavelengths within infrared region. Although the FEL can ablate soft tissues with minimum collateral damage in surgery, the potential of FEL for dissecting protein aggregates is not fully understood. Protein aggregates such as amyloid fibrils are in some cases involved in serious diseases. In our previous study, we showed that amyloid-like lysozyme fibrils could be disaggregated into the native form with FEL irradiation specifically tuned to the amide I band (1,620 cm(-1)). Here, we show further evidence for the FEL-mediated disaggregation of amyloid-like fibrils using insulin fibrils. Insulin fibrils were prepared in acidic solution and irradiated by the FEL, which was tuned to either 1,620 or 2,000 cm(-1) prior to the experiment. The Fourier transform infrared spectroscopy (FT-IR) spectrum after irradiation with the FEL at 1,620 cm(-1) indicated that the broad peak (1,630-1,660 cm(-1)) became almost a single peak (1,652 cm(-1)), and the β-sheet content was reduced to 25 from 40% in the fibrils, while that following the irradiation at 2,000 cm(-1) remained at 38%. The Congo Red assay as well as transmission electron microscopy observation confirmed that the number of fibrils was reduced by FEL irradiation at the amide I band. Size-exclusion chromatography analysis indicated that the disaggregated form of fibrils was the monomeric form. These results confirm that FEL irradiation at the amide I band can dissect amyloid-like protein fibrils into the monomeric form in vitro.

  7. Neisseria meningitidis adhesin NadA targets beta1 integrins: functional similarity to Yersinia invasin.

    PubMed

    Nägele, Virginie; Heesemann, Jürgen; Schielke, Stephanie; Jiménez-Soto, Luisa F; Kurzai, Oliver; Ackermann, Nikolaus

    2011-06-10

    Meningococci are facultative-pathogenic bacteria endowed with a set of adhesins allowing colonization of the human upper respiratory tract, leading to fulminant meningitis and septicemia. The Neisseria adhesin NadA was identified in about 50% of N. meningitidis isolates and is closely related to the Yersinia adhesin YadA, the prototype of the oligomeric coiled-coil adhesin (Oca) family. NadA is known to be involved in cell adhesion, invasion, and induction of proinflammatory cytokines. Because of the enormous diversity of neisserial cell adhesins the analysis of the specific contribution of NadA in meningococcal host interactions is limited. Therefore, we used a non-invasive Y. enterocolitica mutant as carrier to study the role of NadA in host cell interaction. NadA was shown to be efficiently produced and localized in its oligomeric form on the bacterial surface of Y. enterocolitica. Additionally, NadA mediated a β1 integrin-dependent adherence with subsequent internalization of yersiniae by a β1 integrin-positive cell line. Using recombinant NadA(24-210) protein and human and murine β1 integrin-expressing cell lines we could demonstrate the role of the β1 integrin subunit as putative receptor for NadA. Subsequent inhibition assays revealed specific interaction of NadA(24-210) with the human β1 integrin subunit. Cumulatively, these results indicate that Y. enterocolitica is a suitable toolbox system for analysis of the adhesive properties of NadA, revealing strong evidence that β1 integrins are important receptors for NadA. Thus, this study demonstrated for the first time a direct interaction between the Oca-family member NadA and human β1 integrins.

  8. [The influence of dipole modifiers on the channel-forming activity of amyloid and amyloid-like peptides in lipid bilayers].

    PubMed

    Efimova, S S; Zakharov, V V; Ostroumova, O S

    2015-01-01

    We have studied the steady-state transmembrane current induced by amyloid and amyloid-like peptides in lipid bilayers in the presence of dipole modifiers. It has been shown that the addition of dipole modifier, phloretin, to the membrane bathing solutions leads to an increase in the multichannel activity of amyloid beta-peptide fragment 25-35, [Gly35]-amyloid beta-peptide fragment 25--35, prion protein fragment 106-126 and amyloid-like peptides myr-BASP1 (1--13), myr-BASP1(1--19) and GAP-43(1--40). We have found that the effect of phloretin is not the result of dipole potential changes due to adsorption of this modifier on the membrane. Using the various fragments of amyloid beta-peptide, presenilin, prion protein and neuronal proteins BASP1 and GAP-43 allowes to conclude that the steady-state peptide-induced transmembrane current in the case of addition of phloretin is due to the electrostatic interaction between the positively charged channel-forming agents and negatively charged dipole modifier. The results obtained by electron microscopy have demonstrated that this interaction increases degree of peptide oligomerization. PMID:26035972

  9. Comparison of adhesin genes and antimicrobial susceptibilities between uropathogenic and intestinal commensal Escherichia coli strains.

    PubMed

    Qin, Xiaohua; Hu, Fupin; Wu, Shi; Ye, Xinyu; Zhu, Demei; Zhang, Ying; Wang, Minggui

    2013-01-01

    The presence of adhesins is arguably an important determinant of pathogenicity for Uropathogenic Escherichia coli (UPEC). Antimicrobial susceptibilities were tested by agar dilution method, fifteen adhesin genes were detected by polymerase chain reaction, and multilocus sequence typing (MLST) was analyzed in 70 UPEC isolates and 41 commensal E. coli strains. Extended-spectrum β-lactamase (ESBL) was determined with confirmatory test. The prevalence of ESBL-producers in UPEC (53%, 37/70) was higher than the commensal intestinal isolates (7%, 3/41), and 97% (36/37) of the ESBL-producing UPEC harbored bla CTX-M genes. afa was present in 36% (10/28) UPEC isolates from recurrent lower urinary tract infection (UTI), and none in the acute pyelonephritis, acute uncomplicated cystitis or commensal strains (P<0.0001). papG was detected in 28% (20/70) of UPEC isolates, while 5% (2/41) of the commensal strains were papG positive (P = 0.0025), and the prevalence of papG was significantly higher in acute pyelonephritis group (71%) than the other two UTI groups (P<0.0001). The prevalence of flu, yqi, yadN and ygiL was significantly higher in UPEC isolates than in the commensal strains. ESBL-producing UPEC showed a lower prevalence of adhesin genes compared with non-ESBL-producing strains. The MLST profiles were different between UPEC and commensal strains, with ST131 (19%, 13/70) and ST10 (20%, 8/41) being the most common MLSTs, respectively. This study demonstrated that several adhesin genes were more prevalent in UPEC isolates than in commensal E. coli, and afa may be associated with recurrent lower UTI whereas papG is more frequently associated with acute pyelonephritis.

  10. Amyloid-like fibrils from an 18-residue peptide analogue of a part of the central domain of the B-family of silkmoth chorion proteins.

    PubMed

    Iconomidou, V A; Chryssikos, G D; Gionis, V; Vriend, G; Hoenger, A; Hamodrakas, S J

    2001-06-22

    Chorion is the major component of silkmoth eggshell. More than 95% of its dry mass consists of the A and B families of low molecular weight structural proteins, which have remarkable mechanical and chemical properties, and protect the oocyte and the developing embryo from the environment. We present data from negative staining, Congo red binding, X-ray diffraction, Fourier transform-Raman, attenuated total reflectance infrared spectroscopy and modelling studies of a synthetic peptide analogue of a part of the central domain of the B family of silkmoth chorion proteins, indicating that this peptide folds and self-assembles, forming amyloid-like fibrils. These results support further our proposal, based on experimental data from a synthetic peptide analogue of the central domain of the A family of chorion proteins, that silkmoth chorion is a natural, protective amyloid [Iconomidou et al., FEBS Lett. 479 (2000) 141-145]. PMID:11423129

  11. Adhesin contribution to nanomechanical properties of the virulent Bordetella pertussis envelope.

    PubMed

    Arnal, L; Serra, D O; Cattelan, N; Castez, M F; Vázquez, L; Salvarezza, R C; Yantorno, O M; Vela, M E

    2012-05-15

    Adherence to a biological surface allows bacteria to colonize and persist within the host and represents an essential first step in the pathogenesis of most bacterial diseases. Consequently, the physicochemical properties of the outer membrane in bacteria play a key role for attachment to surfaces and therefore for biofilm formation. Bordetella pertussis is a Gram-negative bacterium that colonizes the respiratory tract of humans, producing whooping cough or pertussis, a highly infectious disease. B. pertussis uses various adhesins exposed on its surface to promote cell-surface and cell-cell interactions. The most dominant adhesin function is displayed by filamentous hemagglutinin (FHA). B. pertussis Tohama I wild-type (Vir+) strain and two defective mutants, an avirulent (Vir-) and a FHA-deficient (FHA-) B. pertussis strains were studied by AFM under physiological conditions to evaluate how the presence or absence of adhesins affects the mechanical properties of the B. pertussis cell surface. Quantitative information on the nanomechanical properties of the bacterial envelope was obtained by AFM force-volume analysis. These studies suggested that the presence of virulence factors is correlated with an increase in the average membrane rigidity, which is largely influenced by the presence of FHA. Moreover, for this system we built a nanoscale stiffness map that reveals an inhomogeneous spatial distribution of Young modulus as well as the presence of rigid nanodomains on the cell surface.

  12. Importance of adhesins in virulence of Paracoccidioides spp.

    PubMed Central

    de Oliveira, Haroldo C.; da Silva, Julhiany de Fátima; Scorzoni, Liliana; Marcos, Caroline M.; Rossi, Suelen A.; de Paula e Silva, Ana C. A.; Assato, Patrícia A.; da Silva, Rosângela A. M.; Fusco-Almeida, Ana M.; Mendes-Giannini, Maria J. S.

    2015-01-01

    Members of the Paracoccidioides genus are the etiologic agents of paracoccidioidomycosis (PCM). This genus is composed of two species: Paracoccidioides brasiliensis and Paracoccidioides lutzii. The correct molecular taxonomic classification of these fungi has created new opportunities for studying and understanding their relationships with their hosts. Paracoccidioides spp. have features that permit their growth under adverse conditions, enable them to adhere to and invade host tissues and may contribute to disease development. Cell wall proteins called adhesins facilitate adhesion and are capable of mediating fungi-host interactions during infection. This study aimed to evaluate the adhesion profile of two species of the genus Paracoccidioides, to analyze the expression of adhesin-encoding genes by real-time PCR and to relate these results to the virulence of the species, as assessed using a survival curve in mice and in Galleria mellonella after blocking the adhesins. A high level of heterogeneity was observed in adhesion and adhesin expression, showing that the 14-3-3 and enolase molecules are the most highly expressed adhesins during pathogen-host interaction. Additionally, a survival curve revealed a correlation between the adhesion rate and survival, with P. brasiliensis showing higher adhesion and adhesin expression levels and greater virulence when compared with P. lutzii. After blocking 14-3-3 and enolase adhesins, we observed modifications in the virulence of these two species, revealing the importance of these molecules during the pathogenesis of members of the Paracoccidioides genus. These results revealed new insights into the host-pathogen interaction of this genus and may enhance our understanding of different isolates that could be useful for the treatment of this mycosis. PMID:25914695

  13. Evaluation of Cell Binding Activities of Leptospira ECM Adhesins

    PubMed Central

    Robbins, Gregory T.; Hahn, Beth L.; Evangelista, Karen V.; Padmore, Lavinia; Aranda, Patrick S.; Coburn, Jenifer

    2015-01-01

    Pathogenic spirochetes of the genus Leptospira are the causative agents of leptospirosis, a zoonotic infection that occurs globally. The bacteria colonize the renal proximal tubules of many animals and are shed in the urine. Contact with the urine, or with water contaminated with the urine of infected animals can cause infection of new host animals, including humans. Mechanisms of colonization of the proximal tubule and other tissues are not known, but specific interactions between bacterial adhesins and host substrates are likely to be critical in this process. Several extracellular matrix (ECM) adhesins have been previously identified, but more recently, it has been shown that Leptospira bind more efficiently to cells than ECM. In this work, recombinant forms of five putative Leptospira ECM adhesins, namely LipL32, Loa22, OmpL1, p31/LipL45, and LenA were evaluated for binding to cells as well as an expanded variety of ECM components. Reproducible and significant adhesin activity was demonstrated only for OmpL1, which bound to both mammalian cell lines tested and to glycosaminoglycans (GAGs). While determination of biologically significant bacterial adhesion activity will require generation of site-directed mutant strains, our results suggest that OmpL1 is a strong candidate for future evaluation regarding the roles of the adhesin activity of the protein during L. interrogans infection. PMID:25875373

  14. Adhesins in Human Fungal Pathogens: Glue with Plenty of Stick

    PubMed Central

    de Groot, Piet W. J.; Bader, Oliver; de Boer, Albert D.; Weig, Michael

    2013-01-01

    Understanding the pathogenesis of an infectious disease is critical for developing new methods to prevent infection and diagnose or cure disease. Adherence of microorganisms to host tissue is a prerequisite for tissue invasion and infection. Fungal cell wall adhesins involved in adherence to host tissue or abiotic medical devices are critical for colonization leading to invasion and damage of host tissue. Here, with a main focus on pathogenic Candida species, we summarize recent progress made in the field of adhesins in human fungal pathogens and underscore the importance of these proteins in establishment of fungal diseases. PMID:23397570

  15. Transformation of amyloid-like fibers, formed from an elastin-based biopolymer, into a hydrogel: an X-ray photoelectron spectroscopy and atomic force microscopy study.

    PubMed

    Flamia, R; Salvi, A M; D'Alessio, L; Castle, J E; Tamburro, A M

    2007-01-01

    Previous studies have revealed the propensity of elastin-based biopolymers to form amyloid-like fibers when dissolved in water. These are of interest when considered as "ancestral units" of elastin in which they represent the simplest sequences in the hydrophobic regions of the general type XxxGlyGlyZzzGly (Xxx, Zzz = Val, Leu). We normally refer to these biopolymers based on elastin or related to elastin units as "elastin-like polypeptides". The requirement of water for the formation of amyloids seems quite interesting and deserves investigation, the water representing the natural transport medium in human cells. As a matter of fact, the "natural" supramolecular organization of elastin is in the form of beaded-string-like filaments and not in the form of amyloids whose "in vivo" deposition is associated with some important human diseases. Our work is directed, therefore, to understanding the mechanism by which such hydrophobic sequences form amyloids and any conditions by which they might regress to a non-amyloid filament. The elastin-like sequence here under investigation is the ValGlyGlyValGly pentapeptide that has been previously analyzed both in its monomer and polymer form. In particular, we have focused our investigation on the apparent stability of amyloids formed from poly(ValGlyGlyValGly), and we have observed these fibers evolving to a hydrogel after prolonged aging in water. We will show how atomic force microscopy can be combined with X-ray photoelectron spectroscopy to gain an insight into the spontaneous organization of an elastin-like polypeptide driven by interfacial interactions. The results are discussed also in light of fractal-like assembly and their implications from a biomedical point of view. PMID:17206798

  16. Polysaccharide intercellular adhesin in biofilm: structural and regulatory aspects

    PubMed Central

    Arciola, Carla Renata; Campoccia, Davide; Ravaioli, Stefano; Montanaro, Lucio

    2015-01-01

    Staphylococcus aureus and Staphylococcus epidermidis are the leading etiologic agents of implant-related infections. Biofilm formation is the main pathogenetic mechanism leading to the chronicity and irreducibility of infections. The extracellular polymeric substances of staphylococcal biofilms are the polysaccharide intercellular adhesin (PIA), extracellular-DNA, proteins, and amyloid fibrils. PIA is a poly-β(1-6)-N-acetylglucosamine (PNAG), partially deacetylated, positively charged, whose synthesis is mediated by the icaADBC locus. DNA sequences homologous to ica locus are present in many coagulase-negative staphylococcal species, among which S. lugdunensis, however, produces a biofilm prevalently consisting of proteins. The product of icaA is an N-acetylglucosaminyltransferase that synthetizes PIA oligomers from UDP-N-acetylglucosamine. The product of icaD gives optimal efficiency to IcaA. The product of icaC is involved in the externalization of the nascent polysaccharide. The product of icaB is an N-deacetylase responsible for the partial deacetylation of PIA. The expression of ica locus is affected by environmental conditions. In S. aureus and S. epidermidis ica-independent alternative mechanisms of biofilm production have been described. S. epidermidis and S. aureus undergo to a phase variation for the biofilm production that has been ascribed, in turn, to the transposition of an insertion sequence in the icaC gene or to the expansion/contraction of a tandem repeat naturally harbored within icaC. A role is played by the quorum sensing system, which negatively regulates biofilm formation, favoring the dispersal phase that disseminates bacteria to new infection sites. Interfering with the QS system is a much debated strategy to combat biofilm-related infections. In the search of vaccines against staphylococcal infections deacetylated PNAG retained on the surface of S. aureus favors opsonophagocytosis and is a potential candidate for immune-protection. PMID

  17. Polysaccharide intercellular adhesin in biofilm: structural and regulatory aspects.

    PubMed

    Arciola, Carla Renata; Campoccia, Davide; Ravaioli, Stefano; Montanaro, Lucio

    2015-01-01

    Staphylococcus aureus and Staphylococcus epidermidis are the leading etiologic agents of implant-related infections. Biofilm formation is the main pathogenetic mechanism leading to the chronicity and irreducibility of infections. The extracellular polymeric substances of staphylococcal biofilms are the polysaccharide intercellular adhesin (PIA), extracellular-DNA, proteins, and amyloid fibrils. PIA is a poly-β(1-6)-N-acetylglucosamine (PNAG), partially deacetylated, positively charged, whose synthesis is mediated by the icaADBC locus. DNA sequences homologous to ica locus are present in many coagulase-negative staphylococcal species, among which S. lugdunensis, however, produces a biofilm prevalently consisting of proteins. The product of icaA is an N-acetylglucosaminyltransferase that synthetizes PIA oligomers from UDP-N-acetylglucosamine. The product of icaD gives optimal efficiency to IcaA. The product of icaC is involved in the externalization of the nascent polysaccharide. The product of icaB is an N-deacetylase responsible for the partial deacetylation of PIA. The expression of ica locus is affected by environmental conditions. In S. aureus and S. epidermidis ica-independent alternative mechanisms of biofilm production have been described. S. epidermidis and S. aureus undergo to a phase variation for the biofilm production that has been ascribed, in turn, to the transposition of an insertion sequence in the icaC gene or to the expansion/contraction of a tandem repeat naturally harbored within icaC. A role is played by the quorum sensing system, which negatively regulates biofilm formation, favoring the dispersal phase that disseminates bacteria to new infection sites. Interfering with the QS system is a much debated strategy to combat biofilm-related infections. In the search of vaccines against staphylococcal infections deacetylated PNAG retained on the surface of S. aureus favors opsonophagocytosis and is a potential candidate for immune-protection. PMID

  18. The apicomplexan glideosome and adhesins -- structures and function

    PubMed Central

    Boucher, Lauren E.; Bosch, Jürgen

    2015-01-01

    The apicomplexan family of pathogens, which includes Plasmodium spp. and Toxoplasma gondii, are primarily obligate intracellular parasites and invade multiple cell types. These parasites express extracellular membrane protein receptors, adhesins, to form specific pathogen-host cell interaction complexes. Various adhesins are used to invade a variety of cell types. The receptors are linked to an actomyosin motor, which is part of a complex comprised of many proteins known as the invasion machinery or glideosome. To date, reviews on invasion have focused primarily on the molecular pathways and signals of invasion, with little or no structural information presented. Over 75 structures of parasite receptors and glideosome proteins have been deposited with the Protein Data Bank. These structures include adhesins, motor proteins, bridging proteins, inner membrane complex and cytoskeletal proteins, as well as co-crystal structures with peptides and antibodies. These structures provide information regarding key interactions necessary for target receptor engagement, machinery complex formation, how force is transmitted, and the basis of inhibitory antibodies. Additionally, these structures can provide starting points for the development of antibodies and inhibitory molecules targeting protein-protein interactions, with the aim to inhibit invasion. This review provides an overview of the parasite adhesin protein families, the glideosome components, glideosome architecture, and discuss recent work regarding alternative models. PMID:25764948

  19. The apicomplexan glideosome and adhesins - Structures and function.

    PubMed

    Boucher, Lauren E; Bosch, Jürgen

    2015-05-01

    The apicomplexan family of pathogens, which includes Plasmodium spp. and Toxoplasma gondii, are primarily obligate intracellular parasites and invade multiple cell types. These parasites express extracellular membrane protein receptors, adhesins, to form specific pathogen-host cell interaction complexes. Various adhesins are used to invade a variety of cell types. The receptors are linked to an actomyosin motor, which is part of a complex comprised of many proteins known as the invasion machinery or glideosome. To date, reviews on invasion have focused primarily on the molecular pathways and signals of invasion, with little or no structural information presented. Over 75 structures of parasite receptors and glideosome proteins have been deposited with the Protein Data Bank. These structures include adhesins, motor proteins, bridging proteins, inner membrane complex and cytoskeletal proteins, as well as co-crystal structures with peptides and antibodies. These structures provide information regarding key interactions necessary for target receptor engagement, machinery complex formation, how force is transmitted, and the basis of inhibitory antibodies. Additionally, these structures can provide starting points for the development of antibodies and inhibitory molecules targeting protein-protein interactions, with the aim to inhibit invasion. This review provides an overview of the parasite adhesin protein families, the glideosome components, glideosome architecture, and discuss recent work regarding alternative models.

  20. T4 Phage Tail Adhesin Gp12 Counteracts LPS-Induced Inflammation In Vivo

    PubMed Central

    Miernikiewicz, Paulina; Kłopot, Anna; Soluch, Ryszard; Szkuta, Piotr; Kęska, Weronika; Hodyra-Stefaniak, Katarzyna; Konopka, Agnieszka; Nowak, Marcin; Lecion, Dorota; Kaźmierczak, Zuzanna; Majewska, Joanna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

    2016-01-01

    Bacteriophages that infect Gram-negative bacteria often bind to the bacterial surface by interaction of specific proteins with lipopolysaccharide (LPS). Short tail fiber proteins (tail adhesin, gp12) mediate adsorption of T4-like bacteriophages to Escherichia coli, binding surface proteins or LPS. Produced as a recombinant protein, gp12 retains its ability to bind LPS. Since LPS is able to exert a major impact on the immune response in animals and in humans, we have tested LPS-binding phage protein gp12 as a potential modulator of the LPS-induced immune response. We have produced tail adhesin gp12 in a bacterial expression system and confirmed its ability to form trimers and to bind LPS in vitro by dynamic light scattering. This product had no negative effect on mammalian cell proliferation in vitro. Further, no harmful effects of this protein were observed in mice. Thus, gp12 was used in combination with LPS in a murine model, and it decreased the inflammatory response to LPS in vivo, as assessed by serum levels of cytokines IL-1 alpha and IL-6 and by histopathological analysis of spleen, liver, kidney and lungs. Thus, in future studies gp12 may be considered as a potential tool for modulating and specifically for counteracting LPS-related physiological effects in vivo. PMID:27471503

  1. T4 Phage Tail Adhesin Gp12 Counteracts LPS-Induced Inflammation In Vivo.

    PubMed

    Miernikiewicz, Paulina; Kłopot, Anna; Soluch, Ryszard; Szkuta, Piotr; Kęska, Weronika; Hodyra-Stefaniak, Katarzyna; Konopka, Agnieszka; Nowak, Marcin; Lecion, Dorota; Kaźmierczak, Zuzanna; Majewska, Joanna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

    2016-01-01

    Bacteriophages that infect Gram-negative bacteria often bind to the bacterial surface by interaction of specific proteins with lipopolysaccharide (LPS). Short tail fiber proteins (tail adhesin, gp12) mediate adsorption of T4-like bacteriophages to Escherichia coli, binding surface proteins or LPS. Produced as a recombinant protein, gp12 retains its ability to bind LPS. Since LPS is able to exert a major impact on the immune response in animals and in humans, we have tested LPS-binding phage protein gp12 as a potential modulator of the LPS-induced immune response. We have produced tail adhesin gp12 in a bacterial expression system and confirmed its ability to form trimers and to bind LPS in vitro by dynamic light scattering. This product had no negative effect on mammalian cell proliferation in vitro. Further, no harmful effects of this protein were observed in mice. Thus, gp12 was used in combination with LPS in a murine model, and it decreased the inflammatory response to LPS in vivo, as assessed by serum levels of cytokines IL-1 alpha and IL-6 and by histopathological analysis of spleen, liver, kidney and lungs. Thus, in future studies gp12 may be considered as a potential tool for modulating and specifically for counteracting LPS-related physiological effects in vivo. PMID:27471503

  2. Lectin-Glycan Interaction Network-Based Identification of Host Receptors of Microbial Pathogenic Adhesins

    PubMed Central

    Ielasi, Francesco S.; Alioscha-Perez, Mitchel; Donohue, Dagmara; Claes, Sandra; Sahli, Hichem; Schols, Dominique

    2016-01-01

    ABSTRACT The first step in the infection of humans by microbial pathogens is their adherence to host tissue cells, which is frequently based on the binding of carbohydrate-binding proteins (lectin-like adhesins) to human cell receptors that expose glycans. In only a few cases have the human receptors of pathogenic adhesins been described. A novel strategy—based on the construction of a lectin-glycan interaction (LGI) network—to identify the potential human binding receptors for pathogenic adhesins with lectin activity was developed. The new approach is based on linking glycan array screening results of these adhesins to a human glycoprotein database via the construction of an LGI network. This strategy was used to detect human receptors for virulent Escherichia coli (FimH adhesin), and the fungal pathogens Candida albicans (Als1p and Als3p adhesins) and C. glabrata (Epa1, Epa6, and Epa7 adhesins), which cause candidiasis. This LGI network strategy allows the profiling of potential adhesin binding receptors in the host with prioritization, based on experimental binding data, of the most relevant interactions. New potential targets for the selected adhesins were predicted and experimentally confirmed. This methodology was also used to predict lectin interactions with envelope glycoproteins of human-pathogenic viruses. It was shown that this strategy was successful in revealing that the FimH adhesin has anti-HIV activity. PMID:27406561

  3. K88 Fimbrial Adhesin Targeting of Microspheres Containing Gentamicin Made with Albumin Glycated with Lactose

    PubMed Central

    Sarabia-Sainz, Andre-i; Sarabia-Sainz, Hector Manuel; Ramos-Clamont Montfort, Gabriela; Mata-Haro, Veronica; Guzman-Partida, Ana María; Guzman, Roberto; Garcia-Soto, Mariano; Vazquez-Moreno, Luz

    2015-01-01

    The formulation and characterization of gentamicin-loaded microspheres as a delivery system targeting enterotoxigenic Escherichia coli K88 (E. coli K88) was investigated. Glycated albumin with lactose (BSA-glucose-β (4-1) galactose) was used as the microsphere matrix (MS-Lac) and gentamicin included as the transported antibiotic. The proposed target strategy was that exposed galactoses of MS-Lac could be specifically recognized by E. coli K88 adhesins, and the delivery of gentamicin would inhibit bacterial growth. Lactosylated microspheres (MS-Lac1, MS-Lac2 and MS-Lac3) were obtained using a water-in-oil emulsion, containing gentamicin, followed by crosslinking with different concentrations of glutaraldehyde. Electron microscopy displayed spherical particles with a mean size of 10–17 µm. In vitro release of gentamicin from MS-Lac was best fitted to a first order model, and the antibacterial activity of encapsulated and free gentamicin was comparable. MS-Lac treatments were recognized by plant galactose-specific lectins from Ricinus communis and Sophora japonica and by E. coli K88 adhesins. Results indicate MS-Lac1, produced with 4.2 mg/mL of crosslinker, as the best treatment and that lactosylated microsphere are promising platforms to obtain an active, targeted system against E. coli K88 infections. PMID:26389896

  4. K88 Fimbrial Adhesin Targeting of Microspheres Containing Gentamicin Made with Albumin Glycated with Lactose.

    PubMed

    Sarabia-Sainz, Andre-I; Sarabia-Sainz, Hector Manuel; Montfort, Gabriela Ramos-Clamont; Mata-Haro, Veronica; Guzman-Partida, Ana María; Guzman, Roberto; Garcia-Soto, Mariano; Vazquez-Moreno, Luz

    2015-09-16

    The formulation and characterization of gentamicin-loaded microspheres as a delivery system targeting enterotoxigenic Escherichia coli K88 (E. coli K88) was investigated. Glycated albumin with lactose (BSA-glucose-β (4-1) galactose) was used as the microsphere matrix (MS-Lac) and gentamicin included as the transported antibiotic. The proposed target strategy was that exposed galactoses of MS-Lac could be specifically recognized by E. coli K88 adhesins, and the delivery of gentamicin would inhibit bacterial growth. Lactosylated microspheres (MS-Lac1, MS-Lac2 and MS-Lac3) were obtained using a water-in-oil emulsion, containing gentamicin, followed by crosslinking with different concentrations of glutaraldehyde. Electron microscopy displayed spherical particles with a mean size of 10-17 µm. In vitro release of gentamicin from MS-Lac was best fitted to a first order model, and the antibacterial activity of encapsulated and free gentamicin was comparable. MS-Lac treatments were recognized by plant galactose-specific lectins from Ricinus communis and Sophora japonica and by E. coli K88 adhesins. Results indicate MS-Lac1, produced with 4.2 mg/mL of crosslinker, as the best treatment and that lactosylated microsphere are promising platforms to obtain an active, targeted system against E. coli K88 infections.

  5. K88 Fimbrial Adhesin Targeting of Microspheres Containing Gentamicin Made with Albumin Glycated with Lactose.

    PubMed

    Sarabia-Sainz, Andre-I; Sarabia-Sainz, Hector Manuel; Montfort, Gabriela Ramos-Clamont; Mata-Haro, Veronica; Guzman-Partida, Ana María; Guzman, Roberto; Garcia-Soto, Mariano; Vazquez-Moreno, Luz

    2015-01-01

    The formulation and characterization of gentamicin-loaded microspheres as a delivery system targeting enterotoxigenic Escherichia coli K88 (E. coli K88) was investigated. Glycated albumin with lactose (BSA-glucose-β (4-1) galactose) was used as the microsphere matrix (MS-Lac) and gentamicin included as the transported antibiotic. The proposed target strategy was that exposed galactoses of MS-Lac could be specifically recognized by E. coli K88 adhesins, and the delivery of gentamicin would inhibit bacterial growth. Lactosylated microspheres (MS-Lac1, MS-Lac2 and MS-Lac3) were obtained using a water-in-oil emulsion, containing gentamicin, followed by crosslinking with different concentrations of glutaraldehyde. Electron microscopy displayed spherical particles with a mean size of 10-17 µm. In vitro release of gentamicin from MS-Lac was best fitted to a first order model, and the antibacterial activity of encapsulated and free gentamicin was comparable. MS-Lac treatments were recognized by plant galactose-specific lectins from Ricinus communis and Sophora japonica and by E. coli K88 adhesins. Results indicate MS-Lac1, produced with 4.2 mg/mL of crosslinker, as the best treatment and that lactosylated microsphere are promising platforms to obtain an active, targeted system against E. coli K88 infections. PMID:26389896

  6. A novel Plasmodium falciparum rhoptry associated adhesin mediates erythrocyte invasion through the sialic-acid dependent pathway

    PubMed Central

    Anand, Gaurav; Reddy, K. Sony; Pandey, Alok Kumar; Mian, Syed Yusuf; Singh, Hina; Mittal, Shivani Arora; Amlabu, Emmanuel; Bassat, Quique; Mayor, Alfredo; Chauhan, Virander Singh; Gaur, Deepak

    2016-01-01

    Erythrocyte invasion by Plasmodium falciparum merozoites is central to blood-stage infection and malaria pathogenesis. This intricate process is coordinated by multiple parasite adhesins that bind erythrocyte receptors and mediate invasion through several alternate pathways. P. falciparum expresses 2700 genes during the blood-stages, of which the identity and function of many remains unknown. Here, we have identified and characterized a novel P. falciparum rhoptry associated adhesin (PfRA) that mediates erythrocyte invasion through the sialic-acid dependent pathway. PfRA appears to play a significant functional role as it is conserved across different Plasmodium species. It is localized in the rhoptries and further translocated to the merozoite surface. Both native and recombinant PfRA specifically bound erythrocytes in a sialic-acid dependent, chymotrypsin and trypsin resistant manner, which was abrogated by PfRA antibodies confirming a role in erythrocyte invasion. PfRA antibodies inhibited erythrocyte invasion and in combination with antibodies against other parasite ligands produced an additive inhibitory effect, thus validating its important role in erythrocyte invasion. We have thus identified a novel P. falciparum adhesin that binds with a sialic acid containing erythrocyte receptor. Our observations substantiate the strategy to block P. falciparum erythrocyte invasion by simultaneously targeting multiple conserved merozoite antigens involved in alternate invasion pathways. PMID:27383149

  7. The giant adhesin SiiE of Salmonella enterica.

    PubMed

    Barlag, Britta; Hensel, Michael

    2015-01-01

    Salmonella enterica is a Gram-negative, food-borne pathogen, which colonizes the intestinal tract and invades enterocytes. Invasion of polarized cells depends on the SPI1-encoded type III secretion system (T3SS) and the SPI4-encoded type I secretion system (T1SS). The substrate of this T1SS is the non-fimbrial giant adhesin SiiE. With a size of 595 kDa, SiiE is the largest protein of the Salmonella proteome and consists of 53 repetitive bacterial immunoglobulin (BIg) domains, each containing several conserved residues. As known for other T1SS substrates, such as E. coli HlyA, Ca2+ ions bound by conserved D residues within the BIg domains stabilize the protein and facilitate secretion. The adhesin SiiE mediates the first contact to the host cell and thereby positions the SPI1-T3SS to initiate the translocation of a cocktail of effector proteins. This leads to actin remodeling, membrane ruffle formation and bacterial internalization. SiiE binds to host cell apical membranes in a lectin-like manner. GlcNAc and α2-3 linked sialic acid-containing structures are ligands of SiiE. Since SiiE shows repetitive domain architecture, we propose a zipper-like binding mediated by each individual BIg domain. In this review, we discuss the characteristics of the SPI4-T1SS and the giant adhesin SiiE. PMID:25587788

  8. The giant adhesin SiiE of Salmonella enterica.

    PubMed

    Barlag, Britta; Hensel, Michael

    2015-01-12

    Salmonella enterica is a Gram-negative, food-borne pathogen, which colonizes the intestinal tract and invades enterocytes. Invasion of polarized cells depends on the SPI1-encoded type III secretion system (T3SS) and the SPI4-encoded type I secretion system (T1SS). The substrate of this T1SS is the non-fimbrial giant adhesin SiiE. With a size of 595 kDa, SiiE is the largest protein of the Salmonella proteome and consists of 53 repetitive bacterial immunoglobulin (BIg) domains, each containing several conserved residues. As known for other T1SS substrates, such as E. coli HlyA, Ca2+ ions bound by conserved D residues within the BIg domains stabilize the protein and facilitate secretion. The adhesin SiiE mediates the first contact to the host cell and thereby positions the SPI1-T3SS to initiate the translocation of a cocktail of effector proteins. This leads to actin remodeling, membrane ruffle formation and bacterial internalization. SiiE binds to host cell apical membranes in a lectin-like manner. GlcNAc and α2-3 linked sialic acid-containing structures are ligands of SiiE. Since SiiE shows repetitive domain architecture, we propose a zipper-like binding mediated by each individual BIg domain. In this review, we discuss the characteristics of the SPI4-T1SS and the giant adhesin SiiE.

  9. Essential Functional Role of the Polysaccharide Intercellular Adhesin of Staphylococcus epidermidis in Hemagglutination

    PubMed Central

    Mack, Dietrich; Riedewald, Joachim; Rohde, Holger; Magnus, Tim; Feucht, Hubert H.; Elsner, Holger-A.; Laufs, Rainer; Rupp, Mark E.

    1999-01-01

    Hemagglutination of erythrocytes is a common property of Staphylococcus epidermidis strains, which is related to adherence and biofilm formation and may be essential for the pathogenesis of biomaterial-associated infections caused by S. epidermidis. In three independent biofilm-producing, hemagglutination-positive S. epidermidis isolates, interruption of the icaADBC operon essential for polysaccharide intercellular adhesin (PIA) synthesis by Tn917 insertions led to a hemagglutination-negative phenotype. An immunoglobulin G fraction of antiserum to PIA greatly reduced hemagglutination. Purified PIA led to a 64-fold decrease of hemagglutination titers of these strains; however, it did not mediate hemagglutination by itself. These observations define PIA as the hemagglutinin of S. epidermidis or at least as its major functional component. PMID:9916125

  10. Adhesin degradation accelerates delivery of heat-labile toxin by enterotoxigenic Escherichia coli.

    PubMed

    Roy, Koushik; Kansal, Rita; Bartels, Scott R; Hamilton, David J; Shaaban, Salwa; Fleckenstein, James M

    2011-08-26

    Many enteric pathogens, including enterotoxigenic Escherichia coli (ETEC), produce one or more serine proteases that are secreted via the autotransporter (or type V) bacterial secretion pathway. These molecules have collectively been referred to as SPATE proteins (serine protease autotransporter of the Enterobacteriaceae). EatA, an autotransporter previously identified in ETEC, possesses a functional serine protease motif within its secreted amino-terminal passenger domain. Although this protein is expressed by many ETEC strains and is highly immunogenic, its precise function is unknown. Here, we demonstrate that EatA degrades a recently characterized adhesin, EtpA, resulting in modulation of bacterial adhesion and accelerated delivery of the heat-labile toxin, a principal ETEC virulence determinant. Antibodies raised against the passenger domain of EatA impair ETEC delivery of labile toxin to epithelial cells suggesting that EatA may be an effective target for vaccine development. PMID:21757737

  11. Binding Forces of Streptococcus mutans P1 Adhesin

    PubMed Central

    Sullan, Ruby May A.; Li, James K.; Crowley, Paula J.; Brady, L. Jeannine; Dufrêne, Yves F.

    2015-01-01

    Streptococcus mutans is a Gram-positive oral bacterium that is a primary etiological agent associated with human dental caries. In the oral cavity, S. mutans adheres to immobilized salivary agglutinin (SAG) contained within the salivary pellicle on the tooth surface. Binding to SAG is mediated by cell surface P1, a multifunctional adhesin that is also capable of interacting with extracellular matrix proteins. This may be of particular importance outside of the oral cavity as S. mutans has been associated with infective endocarditis and detected in atherosclerotic plaque. Despite the biomedical importance of P1, its binding mechanisms are not completely understood. In this work, we use atomic force microscopy-based single-molecule and single-cell force spectroscopy to quantify the nanoscale forces driving P1-mediated adhesion. Single-molecule experiments show that full-length P1, as well as fragments containing only the P1 globular head or C-terminal region, binds to SAG with relatively weak forces (~50 pN). In contrast, single-cell analyses reveal that adhesion of a single S. mutans cell to SAG is mediated by strong (~500 pN) and long-range (up to 6000 nm) forces. This is likely due to the binding of multiple P1 adhesins to self-associated gp340 glycoproteins. Such a cooperative, long-range character of the S. mutans–SAG interaction would therefore dramatically increase the strength and duration of cell adhesion. We also demonstrate, at single-molecule and single-cell levels, the interaction of P1 with fibronectin and collagen, as well as with hydrophobic, but not hydrophilic, substrates. The binding mechanism (strong forces, cooperativity, broad specificity) of P1 provides a molecular basis for its multifunctional adhesion properties. Our methodology represents a valuable approach to probe the binding forces of bacterial adhesins and offers a tractable methodology to assess anti-adhesion therapy. PMID:25671413

  12. Adhesins and host serum factors drive Yop translocation by yersinia into professional phagocytes during animal infection.

    PubMed

    Maldonado-Arocho, Francisco J; Green, Carlos; Fisher, Michael L; Paczosa, Michelle K; Mecsas, Joan

    2013-01-01

    Yersinia delivers Yops into numerous types of cultured cells, but predominantly into professional phagocytes and B cells during animal infection. The basis for this cellular tropism during animal infection is not understood. This work demonstrates that efficient and specific Yop translocation into phagocytes by Yersinia pseudotuberculosis (Yptb) is a multi-factorial process requiring several adhesins and host complement. When WT Yptb or a multiple adhesin mutant strain, ΔailΔinvΔyadA, colonized tissues to comparable levels, ΔailΔinvΔyadA translocated Yops into significantly fewer cells, demonstrating that these adhesins are critical for translocation into high numbers of cells. However, phagocytes were still selectively targeted for translocation, indicating that other bacterial and/or host factors contribute to this function. Complement depletion showed that complement-restricted infection by ΔailΔinvΔyadA but not WT, indicating that adhesins disarm complement in mice either by prevention of opsonophagocytosis or by suppressing production of pro-inflammatory cytokines. Furthermore, in the absence of the three adhesins and complement, the spectrum of cells targeted for translocation was significantly altered, indicating that Yersinia adhesins and complement direct Yop translocation into neutrophils during animal infection. In summary, these findings demonstrate that in infected tissues, Yersinia uses adhesins both to disarm complement-dependent killing and to efficiently translocate Yops into phagocytes.

  13. Adhesins involved in attachment to abiotic surfaces by Gram-negative bacteria

    PubMed Central

    Berne, Cécile; Ducret, Adrien; Hardy, Gail G; Brun, Yves V.

    2015-01-01

    During the first step of biofilm formation, initial attachment is dictated by physicochemical and electrostatic interactions between the surface and the bacterial envelope. Depending upon the nature of these interactions, attachment can be transient or permanent. To achieve irreversible attachment, bacterial cells have developed a series of surface adhesins promoting specific or non-specific adhesion under various environmental conditions. This chapter will review the recent advances in our understanding of the secretion, assembly and regulation of the bacterial adhesins during biofilm formation with a particular emphasis on the fimbrial, non-fimbrial and discrete polysaccharide adhesins in Gram-negative bacteria. PMID:26350310

  14. Iron and contact with host cells induce expression of adhesins on surface of Trichomonas vaginalis

    PubMed Central

    Garcia, Ana F.; Chang, Te-Hung; Benchimol, Marlene; Klumpp, David Jichael; Lehker, Michael W.; Alderete, John F.

    2007-01-01

    Summary The proteins AP65, AP51, AP33 and AP23 synthesized by Trichomonas vaginalis organisms in high iron play a role in adherence. Multigene families encode enzymes of the hydrogenosome organelles, which have identity to adhesins. This fact raises questions regarding the compartmentalization of the proteins outside the organelle and about the interactions of adhesins with host cells. Data here demonstrate the presence of the proteins outside the organelle under high-iron conditions. Fluorescence and immunocytochemical experiments show that high-iron-grown organisms coexpressed adhesins on the surface and intracellularly in contrast with low-iron parasites. Furthermore, the AP65 epitopes seen by rabbit anti-AP65 serum that blocks adherence and detects surface proteins were identified, and a mAb reacting to those epitopes recognized the trichomonal surface. Two-dimensional electrophoresis and immunoblot of adhesins from surface-labelled parasites provided evidence that all members of the multigene family were co-ordinately expressed and placed on the trichomonal surface. Similar two-dimensional analysis of proteins from purified hydrogenosomes obtained from iodinated trichomonads confirmed the specific surface labelling of proteins. Contact of trichomonads with vaginal epithelial cells increased the amount of surface-expressed adhesins. Moreover, we found a direct relationship between the levels of adherence and amount of adhesins bound to immortalized vaginal and ureter epithelial cells, further reinforcing specific associations. Finally, trichomonads of MR100, a drug-resistant isolate absent in hydrogenosome proteins and adhesins, were non-adherent. Overall, the results confirm an important role for iron and contact in the surface expression of adhesins of T. vaginalis organisms. PMID:12603729

  15. The Staphylococcus aureus collagen adhesin is a virulence determinant in experimental septic arthritis.

    PubMed Central

    Patti, J M; Bremell, T; Krajewska-Pietrasik, D; Abdelnour, A; Tarkowski, A; Rydén, C; Höök, M

    1994-01-01

    The importance of a collagen-binding adhesin in the pathogenesis of septic arthritis has been examined by comparing the virulence of two sets of Staphylococcus aureus mutants in an animal model. Collagen adhesin-negative mutant PH100 was constructed by replacing the chromosomal collagen adhesin gene (cna) in a clinical strain, Phillips, with an inactivated copy of the gene. Collagen adhesin-positive mutant S. aureus CYL574 was generated by introducing the cna gene into CYL316, a strain that normally lacks the cna gene. Biochemical, immunological, and functional analyses of the generated mutants and their respective parent strains showed that binding of 125I-labeled collagen, expression of an immunoreactive collagen adhesin, and bacterial adherence to cartilage were directly correlated with the presence of a functional cna gene. Greater than 70% of the mice injected with the Cna+ strains developed clinical signs of arthritis, whereas less than 27% of the animals injected with Cna- strains showed symptoms of disease. Furthermore, mice injected with the Cna+ strain Phillips had remarkably elevated levels of immunoglobulin G1 and interleukin-6 compared with mice injected with the Cna- mutant PH100. Taken together, these results demonstrate that collagen adhesin plays an important role in the pathogenesis of septic arthritis induced by S. aureus. Images PMID:8262622

  16. Localization of adhesins on the surface of a pathogenic bacterial envelope through atomic force microscopy.

    PubMed

    Arnal, L; Longo, G; Stupar, P; Castez, M F; Cattelan, N; Salvarezza, R C; Yantorno, O M; Kasas, S; Vela, M E

    2015-11-01

    Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract infection, an important protein participating in the adhesion process is a 220 kDa adhesin named filamentous haemagglutinin (FHA), an outer membrane and also secreted protein that contains recognition domains to adhere to ciliated respiratory epithelial cells and macrophages. In this work, we obtained information on the cell-surface localization and distribution of the B. pertussis adhesin FHA using an antibody-functionalized AFM tip. Through the analysis of specific molecular recognition events we built a map of the spatial distribution of the adhesin which revealed a non-homogeneous pattern. Moreover, our experiments showed a force induced reorganization of the adhesin on the surface of the cells, which could explain a reinforced adhesive response under external forces. This single-molecule information contributes to the understanding of basic molecular mechanisms used by bacterial pathogens to cause infectious disease and to gain insights into the structural features by which adhesins can act as force sensors under mechanical shear conditions. PMID:26446736

  17. Glycosaminoglycan binding by Borrelia burgdorferi adhesin BBK32 specifically and uniquely promotes joint colonization

    PubMed Central

    Lin, Yi-Pin; Chen, Qiang; Ritchie, Jennifer A.; Dufour, Nicholas P.; Fischer, Joshua R.; Coburn, Jenifer; Leong, John M.

    2014-01-01

    SUMMARY Microbial pathogens that colonize multiple tissues commonly produce adhesive surface proteins that mediate attachment to cells and/or extracellular matrix in target organs. Many of these ‘adhesins’ bind to multiple ligands, complicating efforts to understand the role of each ligand-binding activity. Borrelia burgdorferi, the causative agent of Lyme disease, produces BBK32, first identified as a fibronectin-binding adhesin that promotes skin and joint colonization. BBK32 also binds to glycosaminoglycan (GAG), which, like fibronectin is ubiquitously present on cell surfaces. To determine which binding activity is relevant for BBK32-promoted infectivity, we generated a panel of BBK32 truncation and internal deletion mutants, and identified variants specifically defective for binding to either fibronectin or GAG. These variants promoted bacterial attachment to different mammalian cell types in vitro, suggesting that fibronectin and GAG binding may play distinct roles during infection. Intravenous inoculation of mice with a high-passage non-infectious B. burgdorferi strain that produced wild type BBK32 or BBK32 mutants defective for GAG or fibronectin binding, revealed that only GAG-binding activity was required for significant localization to joints at 60 minutes post-infection. An otherwise infectious B. burgdorferi strain producing BBK32 specifically deficient in fibronectin binding was fully capable of both skin and joint colonization in the murine model, whereas a strain producing BBK32 selectively attenuated for GAG binding colonized the inoculation site but not knee or tibiotarsus joints. Thus, the BBK32 fibronectin- and GAG-binding activities are separable in vivo, and BBK32-mediated GAG binding, but not fibronectin binding, contributes to joint colonization. PMID:25486989

  18. Molecular epidemiology of adhesin and hemolysin virulence factors among uropathogenic Escherichia coli.

    PubMed

    Arthur, M; Johnson, C E; Rubin, R H; Arbeit, R D; Campanelli, C; Kim, C; Steinbach, S; Agarwal, M; Wilkinson, R; Goldstein, R

    1989-02-01

    The pap, prs, pil, and hly operons of the pyelonephritic Escherichia coli isolate J96 code for the expression of P, F, and type 1 adhesins and the production of hemolysin, respectively; the afaI operon of the pyelonephritic E. coli KS52 encodes an X adhesin. Using different segments of these operons as probes, colony hybridizations were performed on 97 E. coli urinary tract and 40 fecal clinical isolates to determine (i) the presence in the infecting bacteria of nucleotide sequences related to virulence operons, and (ii) the phenotypic properties associated with such sequences. Coexpression of P and F adhesins encoded by pap-related sequences was detected more frequently among isolates from patients with pyelonephritis (32 of 49, 65%) than among those with cystitis (11 of 48, 23%; P less than 0.0001) or from fecal specimens (6 of 40, 15%; P less than 0.0001). Therefore, the expression of both adhesins appears to be critical in the colonization of the upper urinary tract. In contrast, afaI-related sequences were detected significantly more frequently among isolates from patients with cystitis, suggesting that this class of X adhesin may have a role in lower urinary tract infections. Urinary tract isolates differed from fecal isolates by a low incidence of type 1 adhesin expression among pil probe-positive isolates. hly-related sequences were only detected in pap probe-positive isolates. The frequency of hemolysin production among pap probe-positive isolates was not associated with a particular pattern of infection. The distribution of these virulence factors was similar in the presence or absence of reflux, indicating that structural abnormalities of the urinary tract did not facilitate colonization by adhesin-negative isolates.

  19. Essential roles and regulation of the Legionella pneumophila collagen-like adhesin during biofilm formation.

    PubMed

    Mallegol, Julia; Duncan, Carla; Prashar, Akriti; So, Jannice; Low, Donald E; Terebeznik, Mauricio; Guyard, Cyril

    2012-01-01

    Legionellosis is mostly caused by Legionella pneumophila (Lp) and is defined by a severe respiratory illness with a case fatality rate ranging from 5 to 80%. In a previous study, we showed that a glycosaminoglycan (GAG)-binding adhesin of Lp, named Lcl, is produced during legionellosis and is unique to the L. pneumophila species. Importantly, a mutant depleted in Lcl (Δlpg2644) is impaired in adhesion to GAGs and epithelial cells and in biofilm formation. Here, we examine the molecular function(s) of Lcl and the transcriptional regulation of its encoding gene during different stages of the biofilm development. We show that the collagen repeats and the C-terminal domains of Lcl are crucial for the production of biofilm. We present evidence that Lcl is involved in the early step of surface attachment but also in intercellular interactions. Furthermore, we address the relationship between Lcl gene regulation during biofilm formation and quorum sensing (QS). In a static biofilm assay, we show that Lcl is differentially regulated during growth phases and biofilm formation. Moreover, we show that the transcriptional regulation of lpg2644, mediated by a prototype of QS signaling homoserine lactone (3OC12-HSL), may play a role during the biofilm development. Thus, transcriptional down-regulation of lpg2644 may facilitate the dispersion of Lp to reinitiate biofilm colonization on a distal surface. PMID:23029523

  20. Description of a Novel Adhesin of Mycobacterium avium Subsp. paratuberculosis

    PubMed Central

    Viale, Mariana Noelia; Echeverria-Valencia, Gabriela; Romasanta, Pablo; Mon, María Laura; Fernandez, Marisa; Malchiodi, Emilio; Romano, María Isabel; Gioffré, Andrea Karina; Santangelo, María de la Paz

    2014-01-01

    The binding and ingestion of Mycobacterium avium subsp. paratuberculosis (MAP) by host cells are fibronectin (FN) dependent. In several species of mycobacteria, a specific family of proteins allows the attachment and internalization of these bacteria by epithelial cells through interaction with FN. Thus, the identification of adhesion molecules is essential to understand the pathogenesis of MAP. The aim of this study was to identify and characterize FN binding cell wall proteins of MAP. We searched for conserved adhesins within a large panel of surface immunogenic proteins of MAP and investigated a possible interaction with FN. For this purpose, a cell wall protein fraction was obtained and resolved by 2D electrophoresis. The immunoreactive spots were identified by MALDI-TOF MS and a homology search was performed. We selected elongation factor Tu (EF-Tu) as candidate for further studies. We demonstrated the FN-binding capability of EF-Tu using a ligand blot assay and also confirmed the interaction with FN in a dose-dependent manner by ELISA. The dissociation constant of EF-Tu was determined by surface plasmon resonance and displayed values within the μM range. These data support the hypothesis that this protein could be involved in the interaction of MAP with epithelial cells through FN binding. PMID:25136616

  1. The Staphylococcal Biofilm: Adhesins, regulation, and host response

    PubMed Central

    Paharik, Alexandra E.; Horswill, Alexander R.

    2015-01-01

    The Staphylococci comprise a diverse genus of Gram-positive, non-motile commensal organisms that inhabit the skin and mucous membranes of humans and other mammals. In general, Staphylococci are benign members of the natural flora, but many species have the capacity to be opportunistic pathogens, mainly infecting individuals who have medical device implants or are otherwise immunocompromised. S. aureus and S. epidermidis are a major source of hospital-acquired infections and are the most common causes of surgical site infections and central line-associated bloodstream infections. The ability of Staphylococci to form biofilms in vivo makes them highly resistant to chemotherapeutics and leads to chronic diseases. These biofilm infections include osteomyelitis, endocarditis, medical device implants, and persistence in the cystic fibrosis lung. Here, we provide a comprehensive analysis of our current understanding of Staphylococcal biofilm formation, with an emphasis on adhesins and regulation, while also addressing how Staphylococcal biofilms interact with the immune system. On the whole, this review will provide a thorough picture of biofilm formation of the Staphylococcus genus and how this mode of growth impacts the host. PMID:27227309

  2. The Staphylococcal Biofilm: Adhesins, Regulation, and Host Response.

    PubMed

    Paharik, Alexandra E; Horswill, Alexander R

    2016-04-01

    The staphylococci comprise a diverse genus of Gram-positive, nonmotile commensal organisms that inhabit the skin and mucous membranes of humans and other mammals. In general, staphylococci are benign members of the natural flora, but many species have the capacity to be opportunistic pathogens, mainly infecting individuals who have medical device implants or are otherwise immunocompromised. Staphylococcus aureus and Staphylococcus epidermidis are major sources of hospital-acquired infections and are the most common causes of surgical site infections and medical device-associated bloodstream infections. The ability of staphylococci to form biofilms in vivo makes them highly resistant to chemotherapeutics and leads to chronic diseases. These biofilm infections include osteomyelitis, endocarditis, medical device infections, and persistence in the cystic fibrosis lung. Here, we provide a comprehensive analysis of our current understanding of staphylococcal biofilm formation, with an emphasis on adhesins and regulation, while also addressing how staphylococcal biofilms interact with the immune system. On the whole, this review will provide a thorough picture of biofilm formation of the staphylococcus genus and how this mode of growth impacts the host. PMID:27227309

  3. Description of a novel adhesin of Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Viale, Mariana Noelia; Echeverria-Valencia, Gabriela; Romasanta, Pablo; Mon, María Laura; Fernandez, Marisa; Malchiodi, Emilio; Romano, María Isabel; Gioffré, Andrea Karina; Santangelo, María de la Paz

    2014-01-01

    The binding and ingestion of Mycobacterium avium subsp. paratuberculosis (MAP) by host cells are fibronectin (FN) dependent. In several species of mycobacteria, a specific family of proteins allows the attachment and internalization of these bacteria by epithelial cells through interaction with FN. Thus, the identification of adhesion molecules is essential to understand the pathogenesis of MAP. The aim of this study was to identify and characterize FN binding cell wall proteins of MAP. We searched for conserved adhesins within a large panel of surface immunogenic proteins of MAP and investigated a possible interaction with FN. For this purpose, a cell wall protein fraction was obtained and resolved by 2D electrophoresis. The immunoreactive spots were identified by MALDI-TOF MS and a homology search was performed. We selected elongation factor Tu (EF-Tu) as candidate for further studies. We demonstrated the FN-binding capability of EF-Tu using a ligand blot assay and also confirmed the interaction with FN in a dose-dependent manner by ELISA. The dissociation constant of EF-Tu was determined by surface plasmon resonance and displayed values within the μM range. These data support the hypothesis that this protein could be involved in the interaction of MAP with epithelial cells through FN binding. PMID:25136616

  4. Identification of Coli Surface Antigen 23, a Novel Adhesin of Enterotoxigenic Escherichia coli

    PubMed Central

    Del Canto, Felipe; Botkin, Douglas J.; Valenzuela, Patricio; Popov, Vsevolod; Ruiz-Perez, Fernando; Nataro, James P.; Levine, Myron M.; Stine, O. Colin; Pop, Mihai

    2012-01-01

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea, mainly in developing countries. Although there are 25 different ETEC adhesins described in strains affecting humans, between 15% and 50% of the clinical isolates from different geographical regions are negative for these adhesins, suggesting that additional unidentified adhesion determinants might be present. Here, we report the discovery of Coli Surface Antigen 23 (CS23), a novel adhesin expressed by an ETEC serogroup O4 strain (ETEC 1766a), which was negative for the previously known ETEC adhesins, albeit it has the ability to adhere to Caco-2 cells. CS23 is encoded by an 8.8-kb locus which contains 9 open reading frames (ORFs), 7 of them sharing significant identity with genes required for assembly of K88-related fimbriae. This gene locus, named aal (adhesion-associated locus), is required for the adhesion ability of ETEC 1766a and was able to confer this adhesive phenotype to a nonadherent E. coli HB101 strain. The CS23 major structural subunit, AalE, shares limited identity with known pilin proteins, and it is more closely related to the CS13 pilin protein CshE, carried by human ETEC strains. Our data indicate that CS23 is a new member of the diverse adhesin repertoire used by ETEC strains. PMID:22645287

  5. Identification of Coli Surface Antigen 23, a novel adhesin of enterotoxigenic Escherichia coli.

    PubMed

    Del Canto, Felipe; Botkin, Douglas J; Valenzuela, Patricio; Popov, Vsevolod; Ruiz-Perez, Fernando; Nataro, James P; Levine, Myron M; Stine, O Colin; Pop, Mihai; Torres, Alfredo G; Vidal, Roberto

    2012-08-01

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea, mainly in developing countries. Although there are 25 different ETEC adhesins described in strains affecting humans, between 15% and 50% of the clinical isolates from different geographical regions are negative for these adhesins, suggesting that additional unidentified adhesion determinants might be present. Here, we report the discovery of Coli Surface Antigen 23 (CS23), a novel adhesin expressed by an ETEC serogroup O4 strain (ETEC 1766a), which was negative for the previously known ETEC adhesins, albeit it has the ability to adhere to Caco-2 cells. CS23 is encoded by an 8.8-kb locus which contains 9 open reading frames (ORFs), 7 of them sharing significant identity with genes required for assembly of K88-related fimbriae. This gene locus, named aal (adhesion-associated locus), is required for the adhesion ability of ETEC 1766a and was able to confer this adhesive phenotype to a nonadherent E. coli HB101 strain. The CS23 major structural subunit, AalE, shares limited identity with known pilin proteins, and it is more closely related to the CS13 pilin protein CshE, carried by human ETEC strains. Our data indicate that CS23 is a new member of the diverse adhesin repertoire used by ETEC strains. PMID:22645287

  6. Identification of Coli Surface Antigen 23, a novel adhesin of enterotoxigenic Escherichia coli.

    PubMed

    Del Canto, Felipe; Botkin, Douglas J; Valenzuela, Patricio; Popov, Vsevolod; Ruiz-Perez, Fernando; Nataro, James P; Levine, Myron M; Stine, O Colin; Pop, Mihai; Torres, Alfredo G; Vidal, Roberto

    2012-08-01

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea, mainly in developing countries. Although there are 25 different ETEC adhesins described in strains affecting humans, between 15% and 50% of the clinical isolates from different geographical regions are negative for these adhesins, suggesting that additional unidentified adhesion determinants might be present. Here, we report the discovery of Coli Surface Antigen 23 (CS23), a novel adhesin expressed by an ETEC serogroup O4 strain (ETEC 1766a), which was negative for the previously known ETEC adhesins, albeit it has the ability to adhere to Caco-2 cells. CS23 is encoded by an 8.8-kb locus which contains 9 open reading frames (ORFs), 7 of them sharing significant identity with genes required for assembly of K88-related fimbriae. This gene locus, named aal (adhesion-associated locus), is required for the adhesion ability of ETEC 1766a and was able to confer this adhesive phenotype to a nonadherent E. coli HB101 strain. The CS23 major structural subunit, AalE, shares limited identity with known pilin proteins, and it is more closely related to the CS13 pilin protein CshE, carried by human ETEC strains. Our data indicate that CS23 is a new member of the diverse adhesin repertoire used by ETEC strains.

  7. Identification of novel adhesins of M. tuberculosis H37Rv using integrated approach of multiple computational algorithms and experimental analysis.

    PubMed

    Kumar, Sanjiv; Puniya, Bhanwar Lal; Parween, Shahila; Nahar, Pradip; Ramachandran, Srinivasan

    2013-01-01

    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.

  8. A Structural Model for Binding of the Serine-Rich Repeat Adhesin GspB to Host Carbohydrate Receptors

    SciTech Connect

    Pyburn, Tasia M.; Bensing, Barbara A.; Xiong, Yan Q.; Melancon, Bruce J.; Tomasiak, Thomas M.; Ward, Nicholas J.; Yankovskaya, Victoria; Oliver, Kevin M.; Cecchini, Gary; Sulikowski, Gary A.; Tyska, Matthew J.; Sullam, Paul M.; Iverson, T.M.

    2014-10-02

    GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIb{alpha}. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspB{sub BR}), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspB{sub BR} structure revealed that it is comprised of three independently folded subdomains or modules: (1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; (2) a second Ig-fold resembling the binding region of mammalian Siglecs; (3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIb{alpha}. Further examination of purified GspB{sub BR}-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspBBR. This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.

  9. Neisseria adhesin A variation and revised nomenclature scheme.

    PubMed

    Bambini, Stefania; De Chiara, Matteo; Muzzi, Alessandro; Mora, Marirosa; Lucidarme, Jay; Brehony, Carina; Borrow, Ray; Masignani, Vega; Comanducci, Maurizio; Maiden, Martin C J; Rappuoli, Rino; Pizza, Mariagrazia; Jolley, Keith A

    2014-07-01

    Neisseria adhesin A (NadA), involved in the adhesion and invasion of Neisseria meningitidis into host tissues, is one of the major components of Bexsero, a novel multicomponent vaccine licensed for protection against meningococcal serogroup B in Europe, Australia, and Canada. NadA has been identified in approximately 30% of clinical isolates and in a much lower proportion of carrier isolates. Three protein variants were originally identified in invasive meningococci and named NadA-1, NadA-2, and NadA-3, whereas most carrier isolates either lacked the gene or harbored a different variant, NadA-4. Further analysis of isolates belonging to the sequence type 213 (ST-213) clonal complex identified NadA-5, which was structurally similar to NadA-4, but more distantly related to NadA-1, -2, and -3. At the time of this writing, more than 89 distinct nadA allele sequences and 43 distinct peptides have been described. Here, we present a revised nomenclature system, taking into account the complete data set, which is compatible with previous classification schemes and is expandable. The main features of this new scheme include (i) the grouping of the previously named NadA-2 and NadA-3 variants into a single NadA-2/3 variant, (ii) the grouping of the previously assigned NadA-4 and NadA-5 variants into a single NadA-4/5 variant, (iii) the introduction of an additional variant (NadA-6), and (iv) the classification of the variants into two main groups, named groups I and II. To facilitate querying of the sequences and submission of new allele sequences, the nucleotide and amino acid sequences are available at http://pubmlst.org/neisseria/NadA/.

  10. Localization of adhesins on the surface of a pathogenic bacterial envelope through atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Arnal, L.; Longo, G.; Stupar, P.; Castez, M. F.; Cattelan, N.; Salvarezza, R. C.; Yantorno, O. M.; Kasas, S.; Vela, M. E.

    2015-10-01

    Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract infection, an important protein participating in the adhesion process is a 220 kDa adhesin named filamentous haemagglutinin (FHA), an outer membrane and also secreted protein that contains recognition domains to adhere to ciliated respiratory epithelial cells and macrophages. In this work, we obtained information on the cell-surface localization and distribution of the B. pertussis adhesin FHA using an antibody-functionalized AFM tip. Through the analysis of specific molecular recognition events we built a map of the spatial distribution of the adhesin which revealed a non-homogeneous pattern. Moreover, our experiments showed a force induced reorganization of the adhesin on the surface of the cells, which could explain a reinforced adhesive response under external forces. This single-molecule information contributes to the understanding of basic molecular mechanisms used by bacterial pathogens to cause infectious disease and to gain insights into the structural features by which adhesins can act as force sensors under mechanical shear conditions.Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract

  11. Immunogenicity of a prototype enterotoxigenic Escherichia coli adhesin vaccine in mice and nonhuman primates.

    PubMed

    Sincock, Stephanie A; Hall, Eric R; Woods, Colleen M; O'Dowd, Aisling; Poole, Steven T; McVeigh, Annette L; Nunez, Gladys; Espinoza, Nereyda; Miller, Milagros; Savarino, Stephen J

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC) are the most common cause of bacterial diarrhea in young children in developing countries and in travelers. Efforts to develop an ETEC vaccine have intensified in the past decade, and intestinal colonization factors (CFs) are somatic components of most investigational vaccines. CFA/I and related Class 5 fimbrial CFs feature a major stalk-forming subunit and a minor, antigenically conserved tip adhesin. We hypothesized that the tip adhesin is critical for stimulating antibodies that specifically inhibit ETEC attachment to the small intestine. To address this, we compared the capacity of donor strand complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, and CFA/I fimbriae to elicit anti-adhesive antibodies in mice, using hemagglutination inhibition (HAI) as proxy for neutralization of intestinal adhesion. When given with genetically attenuated heat-labile enterotoxin LTR192G as adjuvant by intranasal (IN) or orogastric (OG) vaccination, dscCfaE exceeded CFA/I fimbriae in eliciting serum HAI titers and anti-CfaE antibody titers. Based on these findings, we vaccinated Aotus nancymaae nonhuman primates (NHP) with dscCfaE alone or admixed with one of two adjuvants, LTR192G and cholera toxin B-subunit, by IN and OG administration. Only IN vaccination with dscCfaE with either adjuvant elicited substantial serum HAI titers and IgA and IgG anti-adhesin responses, with the latter detectable a year after vaccination. In conclusion, we have shown that dscCfaE elicits robust HAI and anti-adhesin antibody responses in both mice and NHPs when given with adjuvant by IN vaccination, encouraging further evaluation of an ETEC adhesin-based vaccine approach. PMID:26597148

  12. Immunogenicity of a prototype enterotoxigenic Escherichia coli adhesin vaccine in mice and nonhuman primates.

    PubMed

    Sincock, Stephanie A; Hall, Eric R; Woods, Colleen M; O'Dowd, Aisling; Poole, Steven T; McVeigh, Annette L; Nunez, Gladys; Espinoza, Nereyda; Miller, Milagros; Savarino, Stephen J

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC) are the most common cause of bacterial diarrhea in young children in developing countries and in travelers. Efforts to develop an ETEC vaccine have intensified in the past decade, and intestinal colonization factors (CFs) are somatic components of most investigational vaccines. CFA/I and related Class 5 fimbrial CFs feature a major stalk-forming subunit and a minor, antigenically conserved tip adhesin. We hypothesized that the tip adhesin is critical for stimulating antibodies that specifically inhibit ETEC attachment to the small intestine. To address this, we compared the capacity of donor strand complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, and CFA/I fimbriae to elicit anti-adhesive antibodies in mice, using hemagglutination inhibition (HAI) as proxy for neutralization of intestinal adhesion. When given with genetically attenuated heat-labile enterotoxin LTR192G as adjuvant by intranasal (IN) or orogastric (OG) vaccination, dscCfaE exceeded CFA/I fimbriae in eliciting serum HAI titers and anti-CfaE antibody titers. Based on these findings, we vaccinated Aotus nancymaae nonhuman primates (NHP) with dscCfaE alone or admixed with one of two adjuvants, LTR192G and cholera toxin B-subunit, by IN and OG administration. Only IN vaccination with dscCfaE with either adjuvant elicited substantial serum HAI titers and IgA and IgG anti-adhesin responses, with the latter detectable a year after vaccination. In conclusion, we have shown that dscCfaE elicits robust HAI and anti-adhesin antibody responses in both mice and NHPs when given with adjuvant by IN vaccination, encouraging further evaluation of an ETEC adhesin-based vaccine approach.

  13. FungalRV: adhesin prediction and immunoinformatics portal for human fungal pathogens

    PubMed Central

    2011-01-01

    Background The availability of sequence data of human pathogenic fungi generates opportunities to develop Bioinformatics tools and resources for vaccine development towards benefitting at-risk patients. Description We have developed a fungal adhesin predictor and an immunoinformatics database with predicted adhesins. Based on literature search and domain analysis, we prepared a positive dataset comprising adhesin protein sequences from human fungal pathogens Candida albicans, Candida glabrata, Aspergillus fumigatus, Coccidioides immitis, Coccidioides posadasii, Histoplasma capsulatum, Blastomyces dermatitidis, Pneumocystis carinii, Pneumocystis jirovecii and Paracoccidioides brasiliensis. The negative dataset consisted of proteins with high probability to function intracellularly. We have used 3945 compositional properties including frequencies of mono, doublet, triplet, and multiplets of amino acids and hydrophobic properties as input features of protein sequences to Support Vector Machine. Best classifiers were identified through an exhaustive search of 588 parameters and meeting the criteria of best Mathews Correlation Coefficient and lowest coefficient of variation among the 3 fold cross validation datasets. The "FungalRV adhesin predictor" was built on three models whose average Mathews Correlation Coefficient was in the range 0.89-0.90 and its coefficient of variation across three fold cross validation datasets in the range 1.2% - 2.74% at threshold score of 0. We obtained an overall MCC value of 0.8702 considering all 8 pathogens, namely, C. albicans, C. glabrata, A. fumigatus, B. dermatitidis, C. immitis, C. posadasii, H. capsulatum and P. brasiliensis thus showing high sensitivity and specificity at a threshold of 0.511. In case of P. brasiliensis the algorithm achieved a sensitivity of 66.67%. A total of 307 fungal adhesins and adhesin like proteins were predicted from the entire proteomes of eight human pathogenic fungal species. The immunoinformatics

  14. The heat-resistant agglutinin family includes a novel adhesin from enteroaggregative Escherichia coli strain 60A.

    PubMed

    Mancini, Justin; Weckselblatt, Brooke; Chung, Yoonjie K; Durante, Julia C; Andelman, Steven; Glaubman, Jessica; Dorff, Justin D; Bhargava, Samhita; Lijek, Rebeccah S; Unger, Katherine P; Okeke, Iruka N

    2011-09-01

    Heat-resistant agglutinin 1 (Hra1) is an accessory colonization factor of enteroaggregative Escherichia coli (EAEC) strain 042. Tia, a close homolog of Hra1, is an invasin and adhesin that has been described in enterotoxigenic E. coli. We devised a PCR-restriction fragment length polymorphism screen for the associated genes and found that they occur among 55 (36.7%) of the enteroaggregative E. coli isolates screened, as well as lower proportions of enterotoxigenic, enteropathogenic, enterohemorrhagic, and commensal E. coli isolates. Overall, 25%, 8%, and 3% of 150 EAEC strains harbored hra1 alone, tia alone, or both genes, respectively. One EAEC isolate, 60A, produced an amplicon with a unique restriction profile, distinct from those of hra1 and tia. We cloned and sequenced the full-length agglutinin gene from strain 60A and have designated it hra2. The hra2 gene was not detected in any of 257 diarrheagenic E. coli isolates in our collection but is present in the genome of Salmonella enterica serovar Heidelberg strain SL476. The cloned hra2 gene from strain 60A, which encodes a predicted amino acid sequence that is 64% identical to that of Hra1 and 68% identical to that of Tia, was sufficient to confer adherence on E. coli K-12. We constructed an hra2 deletion mutant of EAEC strain 60A. The mutant was deficient in adherence but not autoaggregation or invasion, pointing to a functional distinction from the autoagglutinin Hra1 and the Tia invasin. Hra1, Tia, and the novel accessory adhesin Hra2 are members of a family of integral outer membrane proteins that confer different colonization-associated phenotypes. PMID:21764925

  15. Recognition of bacterial lipopolysaccharide using bacteriophage-adhesin-coated long-period gratings.

    PubMed

    Brzozowska, Ewa; Śmietana, Mateusz; Koba, Marcin; Górska, Sabina; Pawlik, Krzysztof; Gamian, Andrzej; Bock, Wojtek J

    2015-05-15

    In this paper we present a new type of highly sensitive label-free sensor based on long-period gratings (LPG) coated with T4 bacteriophage (phage) adhesin. The adhesin (gp37) binds Escherichia coli B (E. coli B) by recognizing its bacterial lipopolysaccharide (LPS). The LPG biofunctionalization methodology is based on coating LPG surface with nickel ions capable of gp37 histidine tag reversible binding. For the first time recombinant adhesive phage protein has been used as a receptor molecule in biosensing scheme. The specificity of LPS binding by adhesin has been tested with LPG-based device and confirmed using Western blot, Enzyme-Linked Immunosorbent Assay (ELISA) and BIACORE methods. The LPG-based sensor can measure bacterial contamination in real time and with a high accuracy. We show that T4 phage adhesin binds E. coli B LPS in its native or denatured form. The binding is highly specific and irreversible. The applied procedure allows for obtaining reusable biosensors. PMID:25067838

  16. Analysis of the Mycoplasma genitalium MgpB Adhesin to Predict Membrane Topology, Investigate Antibody Accessibility, Characterize Amino Acid Diversity, and Identify Functional and Immunogenic Epitopes

    PubMed Central

    Iverson-Cabral, Stefanie L.; Wood, Gwendolyn E.; Totten, Patricia A.

    2015-01-01

    Mycoplasma genitalium is a sexually transmitted pathogen and is associated with reproductive tract disease that can be chronic in nature despite the induction of a strong antibody response. Persistent infection exacerbates the likelihood of transmission, increases the risk of ascension to the upper tract, and suggests that M. genitalium may possess immune evasion mechanism(s). Antibodies from infected patients predominantly target the MgpB adhesin, which is encoded by a gene that recombines with homologous donor sequences, thereby generating sequence variation within and among strains. We have previously characterized mgpB heterogeneity over the course of persistent infection and have correlated the induction of variant-specific antibodies with the loss of that particular variant from the infected host. In the current study, we examined the membrane topology, antibody accessibility, distribution of amino acid diversity, and the location of functional and antigenic epitopes within the MgpB adhesin. Our results indicate that MgpB contains a single transmembrane domain, that the majority of the protein is surface exposed and antibody accessible, and that the attachment domain is located within the extracellular C-terminus. Not unexpectedly, amino acid diversity was concentrated within and around the three previously defined variable regions (B, EF, and G) of MgpB; while nonsynonymous mutations were twice as frequent as synonymous mutations in regions B and G, region EF had equal numbers of nonsynonymous and synonymous mutations. Interestingly, antibodies produced during persistent infection reacted predominantly with the conserved C-terminus and variable region B. In contrast, infection-induced antibodies reacted poorly with the N-terminus, variable regions EF and G, and intervening conserved regions despite the presence of predicted B cell epitopes. Overall, this study provides an important foundation to define how different segments of the MgpB adhesin contribute to

  17. Characterization of the binding activities of proteinase-adhesin complexes from Porphyromonas gingivalis.

    PubMed Central

    Pike, R N; Potempa, J; McGraw, W; Coetzer, T H; Travis, J

    1996-01-01

    Adhesins from oral bacteria perform an important function in colonizing target tissues within the dentogingival cavity. In Porphyromonas gingivalis certain of these adhesion proteins exist as a complex with either of two major proteinases referred to as gingipain R (arginine-specific gingipain) and gingipain K (lysine-specific gingipain) (R. N. Pike, W. T. McGraw, J. Potempa, and J. Travis, J. Biol. Chem. 269:406-411, 1994). With specific proteinase inhibitors, it was shown that hemagglutination by either proteinase-adhesin complex could occur independently of proteinase activity. Significantly, low concentrations of fibrinogen, fibronectin, and laminin inhibited hemagglutination, indicating that adherence to these proteins and not the hemagglutination activity was a primary property of the adhesin activity component of complexes. Binding studies with gingipain K and gingipain R suggest that interaction with fibrinogen is a major function of the adhesin domain, with dissociation constants for binding to fibrinogen being 4 and 8.5 nM, respectively. Specific association with fibronectin and laminin was also found. All bound proteins were degraded by the functional proteinase domain, with gingipain R being more active on laminin and fibronectin and gingipain K being more effective in the digestion of fibrinogen. Cumulatively, these data suggest that gingipain R and gingipain K, acting as proteinase-adhesin complexes, progressively attach to, degrade, and detach from target proteins. Since such complexes appear to be present on the surfaces of both vesicles and membranes of P. gingivalis, they may play an important role in the attachment of this bacterium to host cell surfaces. PMID:8631676

  18. A two-plasmid Escherichia coli system for expression of Dr adhesins.

    PubMed

    Kur, Marta; Piatek, Rafał; Kur, Józef

    2007-10-01

    This paper presents a very efficient expression system for production of Dr adhesins. The system consists of two plasmids. One is the pACYCpBAD-DraC-C-His, which contains the draC gene under the control of the arabinose promoter (pBAD), encoding the DraC usher. The second is the pET30b-syg-DraBE, which contains the draB and draE genes under the control of the T7lac promoter, encoding the DraB chaperone and the DraE adhesin, respectively. Those plasmids have different origin of replication and can therefore coexist in one cell. Since different promoters are present, the protein expression can be controlled. The Dr adhesion expression system constructed opens up a lot of possibilities, and could be very useful in experiments focusing on understanding the biogenesis of Gram-negative bacteria adhesins. For this purpose we showed that the AfaE-III adhesin (98.1% identity between the DraE and the AfaE-III adhesins, with three divergent amino acids within the sequences) was able to pass through the DraC channel in the Escherichia coli BL21(DE3) strain. Immunoblotting analysis and immunofluorescence microscopy showed the presence of AfaE-III on the bacterial cell surface. In addition, the system described can be useful for displaying the immune-relevant sectors of foreign proteins on the bacterial cell. The heterologous epitope sequence of the HSV1 glycoprotein D was inserted into the draE gene in place of the N-terminal region of surface exposed domain 2. Chimeric proteins were exposed on the bacterial surface as evidenced by immunoblotting and immunofluorescence microscopy. The effective display of peptide segments on Dr fimbriae expressed at the bacterial cell surface, can be used for the development of a fimbrial vaccine.

  19. Identification of factors in human urine that inhibit the binding of Escherichia coli adhesins.

    PubMed Central

    Parkkinen, J; Virkola, R; Korhonen, T K

    1988-01-01

    Earlier studies on the binding of Escherichia coli adhesins to the human urinary tract have indicated that the ability to recognize binding sites on the urinary tract epithelial cells is not a characteristic for P fimbriae only, but is also shared by some other adhesins that are not associated with pyelonephritis, especially S fimbriae. In the present study we have investigated whether human urine contains inhibitors of the binding of E. coli adhesins. Normal human urine was found to inhibit hemagglutination by S and type 1 fimbriae but not P fimbriae. The major inhibitor of S fimbriae in normal urine was identified as Tamm-Horsfall glycoprotein, and the interaction with S fimbriae is probably mediated by its sialyloligosaccharide chains. No significant variation was observed in the inhibitory effect of T-H glycoprotein preparations originating from different individuals. In contrast to S fimbriae, the major inhibitors of type 1 fimbriae in urine were identified as low-molecular-weight compounds. Gel filtration and ion-exchange chromatography and alpha-mannosidase treatment indicated that they were neutral alpha-mannosides, probably manno-oligosaccharides with three to five saccharides. Studies of urine samples collected from several individuals indicated the common occurrence of these inhibitory alpha-mannosides. Type 1 fimbriae bound to immobilized T-H glycoprotein, but, unlike S fimbriae, their binding was poorly inhibited by soluble T-H glycoprotein. Some urine samples were also found to contain low-molecular-weight inhibitors for the O75X adhesin of E. coli. These results emphasize that to function as a virulence factor in human urinary tract infections, an adhesin must evidently recognize such receptor structures at the infection sites that are not excreted in soluble form in urine. This prerequisite is filled by P fimbriae but not by type 1 or S fimbriae. PMID:2901405

  20. Prokaryotic High-Level Expression System in Producing Adhesin Recombinant Protein E of Nontypeable Haemophilus influenzae

    PubMed Central

    Tavakoli, Minoo; Bouzari, Saeed; Siadat, Seyed Davar; Najar Peerayeh, Shahin; Jafari, Anis

    2015-01-01

    Background: Adhesion protein E (PE) of Haemophilus influenzae is a 16 - 18 kDa protein with 160 amino acids which causes adhesion to epithelial cells and acts as a major factor in pathogenesis. Objectives: In this study, we performed cloning, expression and purification of PE as a candidate antigen for vaccine design upon further study. Materials and Methods: At first, the pe gene of NTHi ATCC 49766 strain (483 bp) was amplified by PCR. Then, to sequence the resulted amplicon, it was cloned into TA vector (pTZ57R/T). In the next step, the sequenced gene was sub-cloned in pBAD/gIII A vector and transformed into competent Escherichia coli TOP10. For overexpression, the recombinant bacteria were grown in broth medium containing arabinose and the recombinant protein was purified using metal affinity chromatography (Ni-nitrilotriacetic acid) (Ni-NTA agarose). Finally, the protein was detected using sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAG) and confirmed by western blotting. Results: The cloned gene was confirmed by PCR, restriction digestion and sequencing. The sequenced gene was searched for homology in GenBank and 99% similarity was found to the already deposited genes in GenBank. Then we obtained PE using Ni-NTA agarose with up to 7 mg/mL concentration. Conclusions: The pe gene was successfully cloned and confirmed by sequencing. Finally, PE was obtained with high concentration. Due to high homology and similarity among the pe gene from NTHi ATCC 49766 and other NTHi strains in GenBank, we believe that the protein is a universal antigen to be used as a vaccine design candidate and further studies to evaluate its immunogenicity is underway. PMID:26034537

  1. Amyloid-producing odontogenic tumour (calcifying epithelial odontogenic tumour) in the mandible of a Bengal tiger (Panthera tigris tigris).

    PubMed

    Kang, M-S; Park, M-S; Kwon, S-W; Ma, S-A; Cho, D-Y; Kim, D-Y; Kim, Y

    2006-01-01

    A 13-year-old male tiger (Panthera tigris tigris) had a marked mandibular swelling noticed 12 months earlier and associated with progressive anorexia and weight loss. Radiological and post-mortem examination revealed a mass (13x15 cm) which was firm and poorly defined, with destruction of the adjacent bone tissue. Histologically, the mass was poorly demarcated, with infiltrative growth, and composed of nests, cords and islands of epithelial cells with characteristic basal cell features. Also observed were extensive squamous metaplasia, ghost cells, stellate reticulum, and fibroblastic connective tissue stroma containing inflammatory cells. A prominent feature of this tumour consisted of abundant nodular deposits of congophilic amyloid-like material with partial mineralization (Liesegang rings). Immunohistochemically, the neoplastic cells and the amyloid-like material were positive for pancytokeratin and negative for vimentin. The findings supported the diagnosis of an amyloid-producing odontogenic tumour (APOT), also known as calcifying epithelial odontogenic tumour in man and animals.

  2. Amyloid-producing odontogenic tumour (calcifying epithelial odontogenic tumour) in the mandible of a Bengal tiger (Panthera tigris tigris).

    PubMed

    Kang, M-S; Park, M-S; Kwon, S-W; Ma, S-A; Cho, D-Y; Kim, D-Y; Kim, Y

    2006-01-01

    A 13-year-old male tiger (Panthera tigris tigris) had a marked mandibular swelling noticed 12 months earlier and associated with progressive anorexia and weight loss. Radiological and post-mortem examination revealed a mass (13x15 cm) which was firm and poorly defined, with destruction of the adjacent bone tissue. Histologically, the mass was poorly demarcated, with infiltrative growth, and composed of nests, cords and islands of epithelial cells with characteristic basal cell features. Also observed were extensive squamous metaplasia, ghost cells, stellate reticulum, and fibroblastic connective tissue stroma containing inflammatory cells. A prominent feature of this tumour consisted of abundant nodular deposits of congophilic amyloid-like material with partial mineralization (Liesegang rings). Immunohistochemically, the neoplastic cells and the amyloid-like material were positive for pancytokeratin and negative for vimentin. The findings supported the diagnosis of an amyloid-producing odontogenic tumour (APOT), also known as calcifying epithelial odontogenic tumour in man and animals. PMID:16540113

  3. Proteomic analysis of hyperadhesive Candida glabrata clinical isolates reveals a core wall proteome and differential incorporation of adhesins.

    PubMed

    Gómez-Molero, Emilia; de Boer, Albert D; Dekker, Henk L; Moreno-Martínez, Ana; Kraneveld, Eef A; Ichsan; Chauhan, Neeraj; Weig, Michael; de Soet, Johannes J; de Koster, Chris G; Bader, Oliver; de Groot, Piet W J

    2015-12-01

    Attachment to human host tissues or abiotic medical devices is a key step in the development of infections by Candida glabrata. The genome of this pathogenic yeast codes for a large number of adhesins, but proteomic work using reference strains has shown incorporation of only few adhesins in the cell wall. By making inventories of the wall proteomes of hyperadhesive clinical isolates and reference strain CBS138 using mass spectrometry, we describe the cell wall proteome of C. glabrata and tested the hypothesis that hyperadhesive isolates display differential incorporation of adhesins. Two clinical strains (PEU382 and PEU427) were selected, which both were hyperadhesive to polystyrene and showed high surface hydrophobicity. Cell wall proteome analysis under biofilm-forming conditions identified a core proteome of about 20 proteins present in all C. glabrata strains. In addition, 12 adhesin-like wall proteins were identified in the hyperadherent strains, including six novel adhesins (Awp8-13) of which only Awp12 was also present in CBS138. We conclude that the hyperadhesive capacity of these two clinical C. glabrata isolates is correlated with increased and differential incorporation of cell wall adhesins. Future studies should elucidate the role of the identified proteins in the establishment of C. glabrata infections. PMID:26546455

  4. The gp38 Adhesins of the T4 Superfamily: A Complex Modular Determinant of the Phage’s Host Specificity

    PubMed Central

    Trojet, Sabrina N.; Caumont-Sarcos, Anne; Perrody, Elsa; Comeau, André M.; Krisch, H. M.

    2011-01-01

    The tail fiber adhesins are the primary determinants of host range in the T4-type bacteriophages. Among the indispensable virion components, the sequences of the long tail fiber genes and their associated adhesins are among the most variable. The predominant form of the adhesin in the T4-type phages is not even the version of the gene encoded by T4, the archetype of the superfamily, but rather a small unrelated protein (gp38) encoded by closely related phages such as T2 and T6. This gp38 adhesin has a modular design: its N-terminal attachment domain binds at the tip of the tail fiber, whereas the C-terminal specificity domain determines its host receptor affinity. This specificity domain has a series of four hypervariable segments (HVSs) that are separated by a set of highly conserved glycine-rich motifs (GRMs) that apparently form the domain’s conserved structural core. The role of gp38’s various components was examined by a comparative analysis of a large series of gp38 adhesins from T-even superfamily phages with differing host specificities. A deletion analysis revealed that the individual HVSs and GRMs are essential to the T6 adhesin’s function and suggests that these different components all act in synergy to mediate adsorption. The evolutionary advantages of the modular design of the adhesin involving both conserved structural elements and multiple independent and easily interchanged specificity determinants are discussed. PMID:21746838

  5. Vaccination with a recombinant fragment of collagen adhesin provides protection against Staphylococcus aureus-mediated septic death.

    PubMed

    Nilsson, I M; Patti, J M; Bremell, T; Höök, M; Tarkowski, A

    1998-06-15

    Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. Morbidity and mortality due to infections such as sepsis, osteomyelitis, septic arthritis, and invasive endocarditis remain high despite the use of antibiotics. The emergence of antibiotic resistant super bugs mandates that alternative strategies for the prevention and treatment of S. aureus infections are developed. We investigated the ability of vaccination with a recombinant fragment of the S. aureus collagen adhesin to protect mice against sepsis-induced death. Actively immunized NMRI mice were intravenously inoculated with the S. aureus clinical isolate strain Phillips. 14 d after inoculation, mortality in the collagen adhesin-vaccinated group was only 13%, compared with 87% in the control antigen immunized group (P < 0.001). To determine if the protective effect was antibody mediated, we passively immunized naive mice with collagen adhesin-specific antibodies. Similar to the active immunization strategy, passive transfer of collagen adhesin-specific antibodies protected mice against sepsis-induced death. In vitro experiments indicated that S. aureus opsonized with sera from collagen adhesin immunized mice promoted phagocytic uptake and enhanced intracellular killing compared with bacteria opsonized with sera from control animals. These results indicate that the collagen adhesin is a viable target in the development of immunotherapeutics against S. aureus.

  6. Structural Basis for Sialoglycan Binding by the Streptococcus sanguinis SrpA Adhesin.

    PubMed

    Bensing, Barbara A; Loukachevitch, Lioudmila V; McCulloch, Kathryn M; Yu, Hai; Vann, Kendra R; Wawrzak, Zdzislaw; Anderson, Spencer; Chen, Xi; Sullam, Paul M; Iverson, T M

    2016-04-01

    Streptococcus sanguinisis a leading cause of infective endocarditis, a life-threatening infection of the cardiovascular system. An important interaction in the pathogenesis of infective endocarditis is attachment of the organisms to host platelets.S. sanguinisexpresses a serine-rich repeat adhesin, SrpA, similar in sequence to platelet-binding adhesins associated with increased virulence in this disease. In this study, we determined the first crystal structure of the putative binding region of SrpA (SrpABR) both unliganded and in complex with a synthetic disaccharide ligand at 1.8 and 2.0 Å resolution, respectively. We identified a conserved Thr-Arg motif that orients the sialic acid moiety and is required for binding to platelet monolayers. Furthermore, we propose that sequence insertions in closely related family members contribute to the modulation of structural and functional properties, including the quaternary structure, the tertiary structure, and the ligand-binding site.

  7. Programming Controlled Adhesion of E. coli to Target Surfaces, Cells, and Tumors with Synthetic Adhesins

    PubMed Central

    2014-01-01

    In this work we report synthetic adhesins (SAs) enabling the rational design of the adhesion properties of E. coli. SAs have a modular structure comprising a stable β-domain for outer membrane anchoring and surface-exposed immunoglobulin domains with high affinity and specificity that can be selected from large repertoires. SAs are constitutively and stably expressed in an E. coli strain lacking a conserved set of natural adhesins, directing a robust, fast, and specific adhesion of bacteria to target antigenic surfaces and cells. We demonstrate the functionality of SAs in vivo, showing that, compared to wild type E. coli, lower doses of engineered E. coli are sufficient to colonize solid tumors expressing an antigen recognized by the SA. In addition, lower levels of engineered bacteria were found in non-target tissues. Therefore, SAs provide stable and specific adhesion capabilities to E. coli against target surfaces of interest for diverse applications using live bacteria. PMID:25045780

  8. Bacteriophage adhesin-coated long-period gratings for bacterial lipopolysaccharide recognition

    NASA Astrophysics Data System (ADS)

    Koba, Marcin; Śmietana, Mateusz; Brzozowska, Ewa; Górska, Sabina; Mikulic, Predrag; Bock, Wojtek J.

    2014-05-01

    In this work we report an application of the optical fiber long-period gratings (LPGs) working near the dispersion turning point of higher order cladding modes for bacterial lipopolysaccharide (LPS) recognition. We show that when the LPG is functionalized with the bacteriophage adhesin, it is capable of very specific LPS detection. Thus, we compare label-free binding effect for specific to the adhesin LPS-positive and non-specific LPS-negative. The resonance wavelength shift induced by the LPS-positive reaches 2.9 nm, while for LPS-negative the shift is negligible. The LPG-based sensing structure allows for monitoring of the binding phenomenon in real time and with good accuracy.

  9. Cloning of an Enterococcus faecalis endocarditis antigen: homology with adhesins from some oral streptococci.

    PubMed Central

    Lowe, A M; Lambert, P A; Smith, A W

    1995-01-01

    Serum from a patient with Enterococcus faecalis endocarditis was used to identify the gene efaA cloned in Lambda ZapII in Escherichia coli. Nucleotide sequence analysis revealed a 924-bp open reading frame encoding a protein with a predicted molecular weight of 34,768. The amino acid sequence of EfaA shows 55 to 60% homology to a group of streptococcal proteins, FimA from Streptococcus parasanguis, SsaB from Streptococcus sanguis, ScaA from Streptococcus gordonii, and PsaA from Streptococcus pneumoniae. Members of this group have been shown to be adhesins, and we hypothesize that EfaA may function as an adhesin in endocarditis. PMID:7822045

  10. Functional characterization of a mucus-specific LPXTG surface adhesin from probiotic Lactobacillus rhamnosus GG.

    PubMed

    von Ossowski, Ingemar; Satokari, Reetta; Reunanen, Justus; Lebeer, Sarah; De Keersmaecker, Sigrid C J; Vanderleyden, Jos; de Vos, Willem M; Palva, Airi

    2011-07-01

    In spite of the wealth of clinical evidence supporting the health benefits of Lactobacillus rhamnosus GG in humans, there is still a lack of understanding of the molecular mechanisms behind its probiosis. Current knowledge suggests that the health-promoting effects of this probiotic strain might be partly dependent on its persistence in the intestine and adhesion to mucosal surfaces. Moreover, L. rhamnosus GG contains mucus-binding pili that might also explain the occupation of its ecological niche as a comparatively less stringent allochthonous intestine-dwelling bacterium. To uncover additional surface proteins involved in mucosal adhesion, we investigated the adherence properties of the only predicted protein (LGG_02337) in L. rhamnosus GG that exhibits homology with a known mucus-binding domain. We cloned a recombinant form of the gene for this putative mucus adhesin and established that the purified protein readily adheres to human intestinal mucus. We also showed that this mucus adhesin is visibly distributed throughout the cell surface and participates in the adhesive interaction between L. rhamnosus GG and mucus, although less prominently than the mucus-binding pili in this strain. Based on primary structural comparisons, we concluded that the current annotation of the LGG_02337 protein likely does not accurately reflect its predicted properties, and we propose that this mucus-specific adhesin be called the mucus-binding factor (MBF). Finally, we interpret our results to mean that L. rhamnosus GG MBF, as an active mucus-specific surface adhesin with a presumed ancillary involvement in pilus-mediated mucosal adhesion, plays a part in the adherent mechanisms during intestinal colonization by this probiotic.

  11. Functional characterization of a mucus-specific LPXTG surface adhesin from probiotic Lactobacillus rhamnosus GG.

    PubMed

    von Ossowski, Ingemar; Satokari, Reetta; Reunanen, Justus; Lebeer, Sarah; De Keersmaecker, Sigrid C J; Vanderleyden, Jos; de Vos, Willem M; Palva, Airi

    2011-07-01

    In spite of the wealth of clinical evidence supporting the health benefits of Lactobacillus rhamnosus GG in humans, there is still a lack of understanding of the molecular mechanisms behind its probiosis. Current knowledge suggests that the health-promoting effects of this probiotic strain might be partly dependent on its persistence in the intestine and adhesion to mucosal surfaces. Moreover, L. rhamnosus GG contains mucus-binding pili that might also explain the occupation of its ecological niche as a comparatively less stringent allochthonous intestine-dwelling bacterium. To uncover additional surface proteins involved in mucosal adhesion, we investigated the adherence properties of the only predicted protein (LGG_02337) in L. rhamnosus GG that exhibits homology with a known mucus-binding domain. We cloned a recombinant form of the gene for this putative mucus adhesin and established that the purified protein readily adheres to human intestinal mucus. We also showed that this mucus adhesin is visibly distributed throughout the cell surface and participates in the adhesive interaction between L. rhamnosus GG and mucus, although less prominently than the mucus-binding pili in this strain. Based on primary structural comparisons, we concluded that the current annotation of the LGG_02337 protein likely does not accurately reflect its predicted properties, and we propose that this mucus-specific adhesin be called the mucus-binding factor (MBF). Finally, we interpret our results to mean that L. rhamnosus GG MBF, as an active mucus-specific surface adhesin with a presumed ancillary involvement in pilus-mediated mucosal adhesion, plays a part in the adherent mechanisms during intestinal colonization by this probiotic. PMID:21602388

  12. Heterologous expression in Tritrichomonas foetus of functional Trichomonas vaginalis AP65 adhesin

    PubMed Central

    Kucknoor, Ashwini S; Mundodi, Vasanthakrishna; Alderete, JF

    2005-01-01

    Background Trichomonosis, caused by Trichomonas vaginalis, is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs), a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus, adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus, as an alternative approach to confirm adhesin function for this virulence factor. Results In this study, we show stable transfection and expression of the T. vaginalis ap65 gene in T. foetus from an episomal pBS-ap65-neo plasmid. Expression of the gene and protein was confirmed by RT-PCR and immunoblots, respectively. AP65 in transformed T. foetus bound to host cells. Specific mAbs revealed episomally-expressed AP65 targeted to the parasite surface and hydrogenosome organelles. Importantly, surface-expression of AP65 in T. foetus paralleled increased levels of adherence of transfected bovine trichomonads to human VECs. Conclusion The T. vaginalis AP65 adhesin was stably expressed in T. foetus, and the data obtained using this heterologous system strongly supports the role of AP65 as a prominent adhesin for T. vaginalis. In addition, the heterologous expression in T. foetus of a T. vaginalis gene offers an important, new approach for confirming and characterizing virulence factors. PMID:15748280

  13. An Acinetobacter trimeric autotransporter adhesin reaped from cells exhibits its nonspecific stickiness via a highly stable 3D structure

    PubMed Central

    Yoshimoto, Shogo; Nakatani, Hajime; Iwasaki, Keita; Hori, Katsutoshi

    2016-01-01

    Trimeric autotransporter adhesins (TAAs), cell surface proteins of Gram-negative bacteria, mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific, high adhesiveness to abiotic material surfaces as well as to biotic surfaces. AtaA is a homotrimer of polypeptides comprising 3,630 amino acids and forms long nanofibers; therefore, it is too large and structurally complex to be produced as a recombinant protein. In this study, we isolated AtaA’s passenger domain (AtaA PSD), which is translocated to the cell surface through the C-terminal transmembrane domain and exhibits biological functions, using a new method. We introduced a protease recognition site and reaped AtaA nanofibers 225 nm in length from the cell surface through proteolytic cleavage with a specific protease. Biochemical and biophysical analyses of the purified native AtaA PSD revealed that it has a stable structure under alkaline and acidic conditions. Temperatures above 80 °C, which disrupted AtaA’s higher-order structure but maintained the full-length AtaA polypeptide, inactivated AtaA’s nonspecific adhesiveness, suggesting that the stickiness of AtaA requires its 3D structure. This finding refutes the widespread but vague speculation that large unfolded polypeptides readily stick to various surfaces. PMID:27305955

  14. Subcutaneous or oral immunization of mice with Lactococcus lactis expressing F4 fimbrial adhesin FaeG.

    PubMed

    Liu, Shujie; Li, Yongming; Xu, Ziwei; Wang, Yicheng

    2013-01-01

    Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrhea in neonatal and postweaning piglets. Fimbrial adhesion of ETEC has been considered an important colonization factor with antigenicity. To safely and effectively deliver the F4 (K88) fimbrial adhesin FaeG to the immune system, we have previously constructed the secretory expression vector pNZ8112-faeG, and FaeG was produced in cytoplasmic form in Lactococcus lactis. In this work, BALB/c mice were immunized with recombinant L. lactis to further determine the immunogenicity of recombinant FaeG (rFaeG) via the subcutaneous or oral route. Subcutaneous immunization in mice with recombinant L. lactis induced a significant increase in the F4-specific serum IgG titer and the number of antibody-secreting cells (ASCs) in the spleen. Oral immunization of mice with recombinant L. lactis induced mucosal and systemic F4-specific immune responses and increased the number of ASCs in the spleen, mesenteric lymph nodes and Peyer's patches. High-dose (2.8 × 10(11) CFU) recombinant strains and adjuvant cholera toxin B subunit enhanced specific mucosal immune responses. The results suggest the feasibility of delivering rFaeG expressed in L. lactis to the immune system in order to induce an F4-specific immune response. PMID:23386358

  15. Oligosaccharide-receptor interaction of the Gal alpha 1-4Gal binding adhesin of Streptococcus suis. Combining site architecture and characterization of two variant adhesin specificities.

    PubMed

    Haataja, S; Tikkanen, K; Nilsson, U; Magnusson, G; Karlsson, K A; Finne, J

    1994-11-01

    The sugar binding specificities of two groups of Streptococcus suis, a pig pathogen that causes meningitis also in man, were determined. Both the group represented by a recently characterized strain inhibitable by galactose and N-acetylgalactosamine (type PN) and the group inhibitable by galactose (type PO) were found by hemagglutination and solid-phase binding inhibition experiments to recognize the disaccharide Gal alpha 1-4Gal of the P1 and Pk blood group antigens. Both types preferred the disaccharide in terminal position. PN showed some, whereas PO showed almost no, binding to the globoside oligosaccharide containing an additional GalNAc beta 1-3 residue. The complete hydrogen bonding patterns were determined by using deoxy and other synthetic derivatives of the receptor disaccharide, and the constructed models of the interactions were compared with that of Escherichia coli PapG396 adhesin. The essential hydroxyls for binding were the HO-4', HO-6', HO-2, and HO-3 hydroxyls on the beta' alpha-side of the Gal alpha 1-4Gal molecule. Type PO adhesin also formed weak interactions with the hydroxyls HO-6 and HO-3'. The mechanism differed from that of E. coli, which binds to a cluster of five hydroxyls (HO-6, HO-2', HO-3', HO-4', and HO-6') and thus to a different part of the receptor disaccharide. These results represent the first example of the comparison of the saccharide receptor hydrogen bonding patterns of two bacterial organisms of different origin and show that the same saccharide may be recognized by two different binding mechanisms.

  16. Structural Sampling of Glycan Interaction Profiles Reveals Mucosal Receptors for Fimbrial Adhesins of Enterotoxigenic Escherichia coli

    PubMed Central

    Lonardi, Emanuela; Moonens, Kristof; Buts, Lieven; de Boer, Arjen R.; Olsson, Johan D. M.; Weiss, Manfred S.; Fabre, Emeline; Guérardel, Yann; Deelder, André M.; Oscarson, Stefan; Wuhrer, Manfred; Bouckaert, Julie

    2013-01-01

    Fimbriae are long, proteinaceous adhesion organelles expressed on the bacterial envelope, evolutionarily adapted by Escherichia coli strains for the colonization of epithelial linings. Using glycan arrays of the Consortium for Functional Glycomics (CFG), the lectin domains were screened of the fimbrial adhesins F17G and FedF from enterotoxigenic E. coli (ETEC) and of the FimH adhesin from uropathogenic E. coli. This has led to the discovery of a more specific receptor for F17G, GlcNAcβ1,3Gal. No significant differences emerged from the glycan binding profiles of the F17G lectin domains from five different E. coli strains. However, strain-dependent amino acid variations, predominantly towards the positively charged arginine, were indicated by sulfate binding in FedF and F17G crystal structures. For FedF, no significant binders could be observed on the CFG glycan array. Hence, a shotgun array was generated from microvilli scrapings of the distal jejunum of a 3-week old piglet about to be weaned. On this array, the blood group A type 1 hexasaccharide emerged as a receptor for the FedF lectin domain and remarkably also for F18-fimbriated E. coli. F17G was found to selectively recognize glycan species with a terminal GlcNAc, typifying intestinal mucins. In conclusion, F17G and FedF recognize long glycan sequences that could only be identified using the shotgun approach. Interestingly, ETEC strains display a large capacity to adapt their fimbrial adhesins to ecological niches via charge-driven interactions, congruent with binding to thick mucosal surfaces displaying an acidic gradient along the intestinal tract. PMID:24833052

  17. Crystallization and preliminary X-ray data of the FadA adhesin from Fusobacterium nucleatum

    SciTech Connect

    Nithianantham, Stanley; Xu, Minghua; Wu, Nan; Han, Yiping W.; Shoham, Menachem

    2006-12-01

    The FadA adhesin from F. nucleatum, which is involved in bacterial attachment and invasion of human oral epithelial cells, has been crystallized in space group P6{sub 1} or P6{sub 5}, and X-ray data have been collected to 1.9 Å resolution. Fusobacterium nucleatum is a Gram-negative anaerobe prevalent in the oral cavity that is associated with periodontal disease, preterm birth and infections in other parts of the human body. The bacteria attach to and invade epithelial and endothelial cells in the gum tissue and elsewhere via a 13.7 kDa adhesin protein FadA (Fusobacterium adhesin A). FadA exists in two forms: the intact form (pre-FadA), consisting of 129 amino acids, and the mature form (mFadA), which lacks an 18-residue signal sequence. Both forms have been expressed in Escherichia coli and purified. mFadA has been crystallized. The crystals belong to the hexagonal space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 59.3, c = 125.7 Å and one molecule per asymmetric unit. The crystals exhibit an unusually high solvent content of 74%. Synchrotron X-ray data have been collected to 1.9 Å. The crystals are suitable for X-ray structure determination. The crystal structure of FadA may provide a basis for the development of therapeutic agents to combat periodontal disease and other infections associated with F. nucleatum.

  18. Structural Sampling of Glycan Interaction Profiles Reveals Mucosal Receptors for Fimbrial Adhesins of Enterotoxigenic Escherichia coli.

    PubMed

    Lonardi, Emanuela; Moonens, Kristof; Buts, Lieven; de Boer, Arjen R; Olsson, Johan D M; Weiss, Manfred S; Fabre, Emeline; Guérardel, Yann; Deelder, André M; Oscarson, Stefan; Wuhrer, Manfred; Bouckaert, Julie

    2013-01-01

    Fimbriae are long, proteinaceous adhesion organelles expressed on the bacterial envelope, evolutionarily adapted by Escherichia coli strains for the colonization of epithelial linings. Using glycan arrays of the Consortium for Functional Glycomics (CFG), the lectin domains were screened of the fimbrial adhesins F17G and FedF from enterotoxigenic E. coli (ETEC) and of the FimH adhesin from uropathogenic E. coli. This has led to the discovery of a more specific receptor for F17G, GlcNAcb1,3Gal. No significant differences emerged from the glycan binding profiles of the F17G lectin domains from five different E. coli strains. However, strain-dependent amino acid variations, predominantly towards the positively charged arginine, were indicated by sulfate binding in FedF and F17G crystal structures. For FedF, no significant binders could be observed on the CFG glycan array. Hence, a shotgun array was generated from microvilli scrapings of the distal jejunum of a 3-week old piglet about to be weaned. On this array, the blood group A type 1 hexasaccharide emerged as a receptor for the FedF lectin domain and remarkably also for F18-fimbriated E. coli. F17G was found to selectively recognize glycan species with a terminal GlcNAc, typifying intestinal mucins. In conclusion, F17G and FedF recognize long glycan sequences that could only be identified using the shotgun approach. Interestingly, ETEC strains display a large capacity to adapt their fimbrial adhesins to ecological niches via charge-driven interactions, congruent with binding to thick mucosal surfaces displaying an acidic gradient along the intestinal tract. PMID:24833052

  19. Minimum chemical requirements for adhesin activity of the acid-stable part of Candida albicans cell wall phosphomannoprotein complex.

    PubMed

    Kanbe, T; Cutler, J E

    1998-12-01

    This study was conducted to define adhesive characteristics of the acid-stable moiety of the Candida albicans phosphomannoprotein complex (PMPC) on adherence of this fungus to marginal zone macrophages of the mouse spleen. Complete digestion of the acid-stable moiety (Fr.IIS) of the C. albicans PMPC with an alpha-mannosidase or hydrolysis with 0.6 N sulfuric acid destroyed adhesin activity, as determined by the inability of the soluble digests to inhibit yeast cell adherence to the splenic marginal zone. Fr.IIS adhesin activity was decreased following digestion with an alpha-1,2-specific mannosidase. Oligomannosyls consisting of one to six mannose units, which were isolated from the acid-stable part of the PMPC, did not inhibit yeast cell binding and thus do not function alone as adhesin sites in the PMPC. To gain more insight into the minimum requirements for adhesin activity, PMPCs were isolated from a Saccharomyces cerevisiae wild-type strain and from mutant strains mnn1, mnn2, and mnn4; the PMPCs were designated scwt/Fr.II, scmn1/Fr.II, scmn2/Fr.II, and scmn4/Fr.II, respectively. S. cerevisiae scmn2/Fr.II lacks oligomannosyl side chain branches from the outer core mannan, and scmn2/Fr.II was the only PMPC without adhesin activity. S. cerevisiae scwt/Fr.II, scmn1/Fr.II, and scmn4/Fr.II showed adhesin activities less than that of C. albicans Fr.II. These three S. cerevisiae PMPCs are generally similar to Fr. IIS, except that the S. cerevisiae structure has fewer and shorter side chains. Immunofluorescence microscopy show that the acid-stable part of the PMPC is displayed homogeneously on the C. albicans yeast cell surface, which would be expected for a surface adhesin. Our results indicate that both the mannan core and the oligomannosyl side chains are responsible for the adhesin activity of the acid-stable part of the PMPC. PMID:9826359

  20. Surface contact stimulates the just-in-time deployment of bacterial adhesins.

    PubMed

    Li, Guanglai; Brown, Pamela J B; Tang, Jay X; Xu, Jing; Quardokus, Ellen M; Fuqua, Clay; Brun, Yves V

    2012-01-01

    The attachment of bacteria to surfaces provides advantages such as increasing nutrient access and resistance to environmental stress. Attachment begins with a reversible phase, often mediated by surface structures such as flagella and pili, followed by a transition to irreversible attachment, typically mediated by polysaccharides. Here we show that the interplay between pili and flagellum rotation stimulates the rapid transition between reversible and polysaccharide-mediated irreversible attachment. We found that reversible attachment of Caulobacter crescentus cells is mediated by motile cells bearing pili and that their contact with a surface results in the rapid pili-dependent arrest of flagellum rotation and concurrent stimulation of polar holdfast adhesive polysaccharide. Similar stimulation of polar adhesin production by surface contact occurs in Asticcacaulis biprosthecum and Agrobacterium tumefaciens. Therefore, single bacterial cells respond to their initial contact with surfaces by triggering just-in-time adhesin production. This mechanism restricts stable attachment to intimate surface interactions, thereby maximizing surface attachment, discouraging non-productive self-adherence, and preventing curing of the adhesive.

  1. In vitro Paracoccidioides brasiliensis biofilm and gene expression of adhesins and hydrolytic enzymes

    PubMed Central

    Sardi, Janaina de Cássia Orlandi; Pitangui, Nayla de Souza; Voltan, Aline Raquel; Braz, Jaqueline Derissi; Machado, Marcelo Pelajo; Fusco Almeida, Ana Marisa; Mendes Giannini, Maria Jose Soares

    2015-01-01

    Paracoccidioides species are dimorphic fungi that initially infect the lungs but can also spread throughout the body. The spreading infection is most likely due to the formation of a biofilm that makes it difficult for the host to eliminate the infection. Biofilm formation is crucial for the development of infections and confines the pathogen to an extracellular matrix. Its presence is associated with antimicrobial resistance and avoidance of host defenses. This current study provides the first description of biofilm formation by Paracoccidioides brasiliensis (Pb18) and an analysis of gene expression, using real-time PCR, associated with 3 adhesins and 2 hydrolytic enzymes that could be associated with the virulence profile. Biofilm formation was analyzed using fluorescence microscopy, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Metabolic activity was determined using the XTT reduction assay. P. brasiliensis was able to form mature biofilm in 144 h with a thickness of 100 μm. The presence of a biofilm was found to be associated with an increase in the expression of adhesins and enzymes. GP43, enolase, GAPDH and aspartyl proteinase genes were over-expressed, whereas phospholipase was down-regulated in biofilm. The characterization of biofilm formed by P. brasiliensis may contribute to a better understanding of the pathogenesis of paracoccidioidomycosis as well as the search for new therapeutic alternatives; while improving the effectiveness of treatment. PMID:26055497

  2. Human monocytes/macrophages are a target of Neisseria meningitidis Adhesin A (NadA).

    PubMed

    Franzoso, Susanna; Mazzon, Cristina; Sztukowska, Maryta; Cecchini, Paola; Kasic, Tihana; Capecchi, Barbara; Tavano, Regina; Papini, Emanuele

    2008-05-01

    Specific surface proteins of Neisseria meningitidis have been proposed to stimulate leukocytes during tissue invasion and septic shock. In this study, we demonstrate that the adhesin N. meningitidis Adhesin A (NadA) involved in the colonization of the respiratory epithelium by hypervirulent N. meningitidis B strains also binds to and activates human monocytes/macrophages. Expression of NadA on the surface on Escherichia coli does not increase bacterial-monocyte association, but a NadA-positive strain induced a significantly higher amount of TNF-alpha and IL-8 compared with the parental NadA-negative strain, suggesting that NadA has an intrinsic stimulatory action on these cells. Consistently, highly pure, soluble NadA(Delta351-405), a proposed component of an antimeningococcal vaccine, efficiently stimulates monocytes/macrophages to secrete a selected pattern of cytokines and chemotactic factors characterized by high levels of IL-8, IL-6, MCP-1, and MIP-1alpha and low levels of the main vasoactive mediators TNF-alpha and IL-1. NadA(Delta351-405) also inhibited monocyte apoptosis and determined its differentiation into a macrophage-like phenotype.

  3. Structure of the Head of the Bartonella Adhesin BadA

    PubMed Central

    Szczesny, Pawel; Linke, Dirk; Ursinus, Astrid; Bär, Kerstin; Schwarz, Heinz; Riess, Tanja M.; Kempf, Volkhard A. J.; Lupas, Andrei N.; Martin, Jörg; Zeth, Kornelius

    2008-01-01

    Trimeric autotransporter adhesins (TAAs) are a major class of proteins by which pathogenic proteobacteria adhere to their hosts. Prominent examples include Yersinia YadA, Haemophilus Hia and Hsf, Moraxella UspA1 and A2, and Neisseria NadA. TAAs also occur in symbiotic and environmental species and presumably represent a general solution to the problem of adhesion in proteobacteria. The general structure of TAAs follows a head-stalk-anchor architecture, where the heads are the primary mediators of attachment and autoagglutination. In the major adhesin of Bartonella henselae, BadA, the head consists of three domains, the N-terminal of which shows strong sequence similarity to the head of Yersinia YadA. The two other domains were not recognizably similar to any protein of known structure. We therefore determined their crystal structure to a resolution of 1.1 Å. Both domains are β-prisms, the N-terminal one formed by interleaved, five-stranded β-meanders parallel to the trimer axis and the C-terminal one by five-stranded β-meanders orthogonal to the axis. Despite the absence of statistically significant sequence similarity, the two domains are structurally similar to domains from Haemophilus Hia, albeit in permuted order. Thus, the BadA head appears to be a chimera of domains seen in two other TAAs, YadA and Hia, highlighting the combinatorial evolutionary strategy taken by pathogens. PMID:18688279

  4. Identification of Mycobacterium tuberculosis adherence-mediating components: a review of key methods to confirm adhesin function

    PubMed Central

    Ramsugit, Saiyur; Pillay, Manormoney

    2016-01-01

    Anti-adhesion therapy represents a potentially promising avenue for the treatment and prevention of tuberculosis in a post-antibiotic era. Adhesins are surface-exposed microbial structures or molecules that enable pathogenic organisms to adhere to host surfaces, a fundamental step towards host infection. Although several Mycobacterium tuberculosis adhesins have been identified, it is predicted that numerous additional adherence-mediating components contribute to the virulence and success of this pathogen. Significant further research to discern and characterize novel M. tuberculosis adhesins is, therefore, required to gain a holistic account of M. tuberculosis adhesion to the host. This would enable the identification of potential drug and vaccine targets for attenuating M. tuberculosis adherence and infectivity. Several methods have been successfully applied to the study and identification of M. tuberculosis adhesins. In this manuscript, we review these methods, which include adherence assays that utilize wild-type and gene knockout mutant strains, epitope masking and competitive inhibition analyses, extracellular matrix protein binding assays, microsphere adhesion assays, M. tuberculosis auto-aggregation assays, and in silico analyses. PMID:27482337

  5. Flavobacterium johnsoniae GldK, GldL, GldM, and SprA Are Required for Secretion of the Cell Surface Gliding Motility Adhesins SprB and RemA

    PubMed Central

    Shrivastava, Abhishek; Johnston, Joseph J.; van Baaren, Jessica M.

    2013-01-01

    Flavobacterium johnsoniae cells move rapidly over surfaces by gliding motility. Gliding results from the movement of adhesins such as SprB and RemA along the cell surface. These adhesins are delivered to the cell surface by a Bacteroidetes-specific secretion system referred to as the type IX secretion system (T9SS). GldN, SprE, SprF, and SprT are involved in secretion by this system. Here we demonstrate that GldK, GldL, GldM, and SprA are each also involved in secretion. Nonpolar deletions of gldK, gldL, or gldM resulted in the absence of gliding motility and in T9SS defects. The mutant cells produced SprB and RemA proteins but failed to secrete them to the cell surface. The mutants were resistant to phages that use SprB or RemA as a receptor, and they failed to attach to glass, presumably because of the absence of cell surface adhesins. Deletion of sprA resulted in similar but slightly less dramatic phenotypes. sprA mutant cells failed to secrete SprB and RemA, but cells remained susceptible to some phages and retained some limited ability to glide. The phenotype of the sprA mutant was similar to those previously described for sprE and sprT mutants. SprA, SprE, and SprT are needed for secretion of SprB and RemA but may not be needed for secretion of other proteins targeted to the T9SS. Genetic and molecular experiments demonstrate that gldK, gldL, gldM, and gldN form an operon and suggest that the proteins encoded by these genes may interact to form part of the F. johnsoniae T9SS. PMID:23667240

  6. Phase variation and host immunity against high molecular weight (HMW) adhesins shape population dynamics of nontypeable Haemophilus influenzae within human hosts.

    PubMed

    Davis, Gregg S; Marino, Simeone; Marrs, Carl F; Gilsdorf, Janet R; Dawid, Suzanne; Kirschner, Denise E

    2014-08-21

    Nontypeable Haemophilus influenzae (NTHi) is a bacterium that resides within the human pharynx. Because NTHi is human-restricted, its long-term survival is dependent upon its ability to successfully colonize new hosts. Adherence to host epithelium, mediated by bacterial adhesins, is one of the first steps in NTHi colonization. NTHi express several adhesins, including the high molecular weight (HMW) adhesins that mediate attachment to the respiratory epithelium where they interact with the host immune system to elicit a strong humoral response. hmwA, which encodes the HMW adhesin, undergoes phase variation mediated by 7-base pair tandem repeats located within its promoter region. Repeat number affects both hmwA transcription and HMW-adhesin production such that as the number of repeats increases, adhesin production decreases. Cells expressing large amounts of HMW adhesins may be critical for the establishment and maintenance of NTHi colonization, but they might also incur greater fitness costs when faced with an adhesin-specific antibody-mediated immune response. We hypothesized that the occurrence of large deletion events within the hmwA repeat region allows NTHi cells to maintain adherence in the presence of antibody-mediated immunity. To study this, we developed a mathematical model, incorporating hmwA phase variation and antibody-mediated immunity, to explore the trade-off between bacterial adherence and immune evasion. The model predicts that antibody levels and avidity, catastrophic loss rates, and population carrying capacity all significantly affected numbers of adherent NTHi cells within a host. These results suggest that the occurrence of large, yet rare, deletion events allows for stable maintenance of a small population of adherent cells in spite of HMW adhesin specific antibody-mediated immunity. These adherent subpopulations may be important for sustaining colonization and/or maintaining transmission. PMID:24747580

  7. The knockdown of each component of the cysteine proteinase-adhesin complex of Entamoeba histolytica (EhCPADH) affects the expression of the other complex element as well as the in vitro and in vivo virulence.

    PubMed

    Ocádiz-Ruiz, Ramón; Fonseca, Wendy; Linford, Alicia S; Yoshino, Timothy P; Orozco, Esther; Rodríguez, Mario A

    2016-01-01

    Entamoeba histolytica is the protozoan parasite causative of human amoebiasis, disease responsible for 40 000-100 000 deaths annually. The cysteine proteinase-adhesin complex of this parasite (EhCPADH) is a heterodimeric protein formed by a cysteine protease (EhCP112) and an adhesin (EhADH) that plays an important role in the cytopathic mechanism of this parasite. The coding genes for EhCP112 and EhADH are adjacent in the E. histolytica genome, suggesting that their expression may be co-regulated, but this hypothesis has not yet been confirmed. Here, we performed the knockdown of EhCP112 and EhADH using gene-specific short-hairpin RNAs (shRNA), and the effect of these knockdowns on the expression of both complex components as well as on the in vitro and in vivo virulence was analysed. Results showed that the knockdown of one of the EhCPADH components produced a simultaneous downregulation of the other protein. Accordingly, a concomitant reduction in the overall expression of the complex was observed. The downregulation of each component also produced a significant decrease in the in vitro and in vivo virulence of trophozoites. These results demonstrated that the expression of EhCP112 and EhADH is co-regulated and confirmed that the EhCPADH complex plays an important role in E. histolytica virulence.

  8. Structure and copy number of gene clusters related to the pap P-adhesin operon of uropathogenic Escherichia coli.

    PubMed

    Arthur, M; Campanelli, C; Arbeit, R D; Kim, C; Steinbach, S; Johnson, C E; Rubin, R H; Goldstein, R

    1989-02-01

    The structurally related pap and prs operons of the uropathogenic Escherichia coli isolate J96 encode a P and an F adhesin that mediate bacterial attachment to the human P blood group antigen and the Forssman antigen, respectively. Using probes prepared from different segments of the pap operon, Southern blot hybridizations were performed to characterize pap-related sequences of 30 E. coli clinical isolates expressing different adhesin phenotypes. Gene clusters encoding P and F adhesins displayed no restriction site polymorphism in sequences homologous to the papH, papC, and papD genes that encode proteins essential to the transport and polymerization of the subunits of the P-pilus adhesin. In contrast, pap-related genetic elements associated with a null phenotype either lacked homology to the papH, papC, and papD genes or displayed a restriction site polymorphism in this region. Sequences within and surrounding the J96 papG and prsG adhesin genes that determine the binding specificities to the P and F antigens, respectively, were not conserved. However, gene clusters encoding different binding specificities could not be distinguished based on such restriction site polymorphisms. The majority of clinical isolates had more than one copy of pap-related sequences that involved gene clusters similar to the J96 pap operon, as well as genetic elements that were related only to a part of this operon. The implications of this unexpected copy number polymorphism with respect to possible recombination events involving pap-related sequences are discussed.

  9. Staphylococcus aureus adherence to Candida albicans hyphae is mediated by the hyphal adhesin Als3p

    PubMed Central

    Peters, Brian M.; Ovchinnikova, Ekaterina S.; Krom, Bastiaan P.; Schlecht, Lisa Marie; Zhou, Han; Hoyer, Lois L.; Busscher, Henk J.; van der Mei, Henny C.; Jabra-Rizk, Mary Ann

    2012-01-01

    The bacterium Staphylococcus (St.) aureus and the opportunistic fungus Candida albicans are currently among the leading nosocomial pathogens, often co-infecting critically ill patients, with high morbidity and mortality. Previous investigations have demonstrated preferential adherence of St. aureus to C. albicans hyphae during mixed biofilm growth. In this study, we aimed to characterize the mechanism behind this observed interaction. C. albicans adhesin-deficient mutant strains were screened by microscopy to identify the specific receptor on C. albicans hyphae recognized by St. aureus. Furthermore, an immunoassay was developed to validate and quantify staphylococcal binding to fungal biofilms. The findings from these experiments implicated the C. albicans adhesin agglutinin-like sequence 3 (Als3p) in playing a major role in the adherence process. This association was quantitatively established using atomic force microscopy, in which the adhesion force between single cells of the two species was significantly reduced for a C. albicans mutant strain lacking als3. Confocal microscopy further confirmed these observations, as St. aureus overlaid with a purified recombinant Als3 N-terminal domain fragment (rAls3p) exhibited robust binding. Importantly, a strain of Saccharomyces cerevisiae heterologously expressing Als3p was utilized to further confirm this adhesin as a receptor for St. aureus. Although the parental strain does not bind bacteria, expression of Als3p on the cell surface conferred upon the yeast the ability to strongly bind St. aureus. To elucidate the implications of these in vitro findings in a clinically relevant setting, an ex vivo murine model of co-infection was designed using murine tongue explants. Fluorescent microscopic images revealed extensive hyphal penetration of the epithelium typical of C. albicans mucosal infection. Interestingly, St. aureus bacterial cells were only seen within the epithelial tissue when associated with the invasive

  10. Detection specificity studies of bacteriophage adhesin-coated long-period grating-based biosensor

    NASA Astrophysics Data System (ADS)

    Koba, Marcin; Śmietana, Mateusz; Brzozowska, Ewa; Górska, Sabina; Mikulic, Predrag; Cusano, Andrea; Bock, Wojtek J.

    2015-09-01

    In this work, we present a label-free detection specificity study of an optical fiber long-period grating (LPG) biosensor working near the dispersion turning point of higher order cladding modes. The LPG sensor functionalized with bacteriophage adhesin is tested with specific and non-specific bacteria dry weight. We show that such biosensor is able to selectively bind, thus recognize different bacteria. We use bacteria dry weights of E. coli B as positive test and E. coli K12 and Salmonella enterica as negative tests. The resonance wavelength shift induced by E. coli B reaches over 90 nm, while for E. coli K12 and Salmonella enterica approximately 40 and 20 nm, respectively.

  11. The Binding of Plasmodium falciparum Adhesins and Erythrocyte Invasion Proteins to Aldolase Is Enhanced by Phosphorylation.

    PubMed

    Diaz, Suraya A; Martin, Stephen R; Howell, Steven A; Grainger, Munira; Moon, Robert W; Green, Judith L; Holder, Anthony A

    2016-01-01

    Aldolase has been implicated as a protein coupling the actomyosin motor and cell surface adhesins involved in motility and host cell invasion in the human malaria parasite Plasmodium falciparum. It binds to the cytoplasmic domain (CTD) of type 1 membrane proteins of the thrombospondin-related anonymous protein (TRAP) family. Other type 1 membrane proteins located in the apical organelles of merozoites, the form of the parasite that invades red blood cells, including apical membrane antigen 1 (AMA1) and members of the erythrocyte binding ligand (EBL) and reticulocyte binding homologue (RH) protein families have been implicated in host cell binding and invasion. Using a direct binding method we confirm that TRAP and merozoite TRAP (MTRAP) bind aldolase and show that the interaction is mediated by more than just the C-terminal six amino acid residues identified previously. Single amino acid substitutions in the MTRAP CTD abolished binding to aldolase. The CTDs of AMA1 and members of the EBL and RH protein families also bound to aldolase. MTRAP competed with AMA1 and RH4 for binding to aldolase, indicating overlapping binding sites. MTRAP CTD was phosphorylated in vitro by both calcium dependent kinase 1 (CDPK1) and protein kinase A, and this modification increased the affinity of binding to aldolase by ten-fold. Phosphorylation of the CTD of members of the EBL and RH protein families also increased their affinity for aldolase in some cases. To examine whether or not MTRAP expressed in asexual blood stage parasites is phosphorylated, it was tagged with GFP, purified and analysed, however no phosphorylation was detected. We propose that CTD binding to aldolase may be dynamically modulated by phosphorylation, and there may be competition for aldolase binding between different CTDs. The use and efficiency of alternate invasion pathways may be determined by the affinity of adhesins and cell invasion proteins for aldolase, in addition to their host ligand specificity. PMID

  12. Re-Evaluation of a Bacterial Antifreeze Protein as an Adhesin with Ice-Binding Activity

    PubMed Central

    Guo, Shuaiqi; Garnham, Christopher P.; Whitney, John C.; Graham, Laurie A.; Davies, Peter L.

    2012-01-01

    A novel role for antifreeze proteins (AFPs) may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII) and region IV (RIV), divide MpAFP into five distinct regions, all of which require mM Ca2+ levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca2+-bound beta-helix containing thirteen Repeats-In-Toxin (RTX)-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2) server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice. PMID:23144980

  13. The novel chlamydial adhesin CPn0473 mediates the lipid raft‐dependent uptake of Chlamydia pneumoniae

    PubMed Central

    Fechtner, Tim; Galle, Jan N.

    2016-01-01

    Summary Chlamydiae are Gram‐negative, obligate intracellular pathogens that pose a serious threat to public health worldwide. Chlamydial surface molecules are essential for host cell invasion. The first interaction with the host cell is thereby accomplished by the Outer membrane complex protein B (OmcB) binding to heparan sulfate moieties on the host cell surface, followed by the interaction of the chlamydial polymorphic membrane proteins (Pmps) with host cell receptors. Specifically, the interaction of the Pmp21 adhesin and invasin with its human interaction partner, the epidermal growth factor receptor, results in receptor activation, down‐stream signalling and finally internalization of the bacteria. Blocking both, the OmcB and Pmp21 adhesion pathways, did not completely abolish infection, suggesting the presence of additional factors relevant for host cell invasion. Here, we show that the novel surface protein CPn0473 of Chlamydia pneumoniae contributes to the binding and invasion of infectious chlamydial particles. CPn0473 is expressed late in the infection cycle and located on the infectious chlamydial cell surface. Soluble recombinant CPn0473 as well as rCPn0473‐coupled fluorescent latex beads adhere to human epithelial HEp‐2 cells. Interestingly, in classical infection blocking experiments pretreatment of HEp‐2 cells with rCPn0473 does not attenuate adhesion but promotes dose‐dependently internalization by C. pneumoniae suggesting an unusual mode of action for this adhesin. This CPn0473‐dependent promotion of infection by C. pneumoniae depends on two different domains within the protein and requires intact lipid rafts. Thus, inhibition of the interaction of CPn0473 with the host cell could provide a way to reduce the virulence of C. pneumoniae. PMID:26780295

  14. Genetic analysis of the gene cluster encoding nonfimbrial adhesin I from an Escherichia coli uropathogen.

    PubMed Central

    Ahrens, R; Ott, M; Ritter, A; Hoschützky, H; Bühler, T; Lottspeich, F; Boulnois, G J; Jann, K; Hacker, J

    1993-01-01

    The chromosomally encoded nonfimbrial adhesion I (NFA-I) from Escherichia coli urinary tract isolate 827 (O83:K1:H4) mediates agglutination of human erythrocytes. Subclones were constructed from an NFA-I-expressing recombinant E. coli K-12 clone, derived from a genomic library of E. coli 827. Minicell analysis and nucleotide sequencing revealed that proteins of 30.5, 9, 80, 15, and 19 kDa encoded on a stretch of approximately 6 kb are involved in the expression of NFA-I. NFA-I exhibits a polymeric structure, which disintegrates with elevated temperature into a 19-kDa monomer but with some relatively stable dimers. By using gold-conjugated monoclonal antibodies directed against NFA-I in electron microscopy, the adhesin could be localized on the outer surface of the recombinant E. coli K-12 bacteria. The nucleotide sequence of the nfaA gene encoding the monomeric structural subunit of the adhesin was determined. An open reading frame of 184 amino acids encoding the NfaA precursor, which is processed to the mature protein, was found; it consisted of 156 amino acids with a calculated molecular weight of 16,000. Peptide sequencing of the NFA-I subunit protein confirmed that this open reading frame corresponds to the NfaA coding locus. Furthermore, the nucleotide sequence of the open reading frame termed NfaE, located at the proximal part of the DNA stretch responsible for NFA-I expression, was elaborated. NfaE consists of 247 amino acids, including a presumptive 29-amino-acid signal peptide, leading to a molecular weight of 24,000 for the mature protein. The nfaE sequence shares homology with the 27-kDa CS3 protein, which is involved in the assembly of CS3 fibrillae, and might encode the 30.5-kDa protein, detected in minicells. Images PMID:8099066

  15. The novel chlamydial adhesin CPn0473 mediates the lipid raft-dependent uptake of Chlamydia pneumoniae.

    PubMed

    Fechtner, Tim; Galle, Jan N; Hegemann, Johannes H

    2016-08-01

    Chlamydiae are Gram-negative, obligate intracellular pathogens that pose a serious threat to public health worldwide. Chlamydial surface molecules are essential for host cell invasion. The first interaction with the host cell is thereby accomplished by the Outer membrane complex protein B (OmcB) binding to heparan sulfate moieties on the host cell surface, followed by the interaction of the chlamydial polymorphic membrane proteins (Pmps) with host cell receptors. Specifically, the interaction of the Pmp21 adhesin and invasin with its human interaction partner, the epidermal growth factor receptor, results in receptor activation, down-stream signalling and finally internalization of the bacteria. Blocking both, the OmcB and Pmp21 adhesion pathways, did not completely abolish infection, suggesting the presence of additional factors relevant for host cell invasion. Here, we show that the novel surface protein CPn0473 of Chlamydia pneumoniae contributes to the binding and invasion of infectious chlamydial particles. CPn0473 is expressed late in the infection cycle and located on the infectious chlamydial cell surface. Soluble recombinant CPn0473 as well as rCPn0473-coupled fluorescent latex beads adhere to human epithelial HEp-2 cells. Interestingly, in classical infection blocking experiments pretreatment of HEp-2 cells with rCPn0473 does not attenuate adhesion but promotes dose-dependently internalization by C. pneumoniae suggesting an unusual mode of action for this adhesin. This CPn0473-dependent promotion of infection by C. pneumoniae depends on two different domains within the protein and requires intact lipid rafts. Thus, inhibition of the interaction of CPn0473 with the host cell could provide a way to reduce the virulence of C. pneumoniae.

  16. Ca2+-stabilized adhesin helps an Antarctic bacterium reach out and bind ice

    PubMed Central

    Vance, Tyler D. R.; Olijve, Luuk L. C.; Campbell, Robert L.; Voets, Ilja K.; Davies, Peter L.; Guo, Shuaiqi

    2014-01-01

    The large size of a 1.5-MDa ice-binding adhesin [MpAFP (Marinomonas primoryensis antifreeze protein)] from an Antarctic Gram-negative bacterium, M. primoryensis, is mainly due to its highly repetitive RII (Region II). MpAFP_RII contains roughly 120 tandem copies of an identical 104-residue repeat. We have previously determined that a single RII repeat folds as a Ca2+-dependent immunoglobulin-like domain. Here, we solved the crystal structure of RII tetra-tandemer (four tandem RII repeats) to a resolution of 1.8 Å. The RII tetra-tandemer reveals an extended (~190-Å × ~25-Å), rod-like structure with four RII-repeats aligned in series with each other. The inter-repeat regions of the RII tetra-tandemer are strengthened by Ca2+ bound to acidic residues. SAXS (small-angle X-ray scattering) profiles indicate the RII tetra-tandemer is significantly rigidified upon Ca2+ binding, and that the protein's solution structure is in excellent agreement with its crystal structure. We hypothesize that >600 Ca2+ help rigidify the chain of ~120 104-residue repeats to form a ~0.6 μm rod-like structure in order to project the ice-binding domain of MpAFP away from the bacterial cell surface. The proposed extender role of RII can help the strictly aerobic, motile bacterium bind ice in the upper reaches of the Antarctic lake where oxygen and nutrients are most abundant. Ca2+-induced rigidity of tandem Ig-like repeats in large adhesins might be a general mechanism used by bacteria to bind to their substrates and help colonize specific niches. PMID:24892750

  17. The Binding of Plasmodium falciparum Adhesins and Erythrocyte Invasion Proteins to Aldolase Is Enhanced by Phosphorylation

    PubMed Central

    Diaz, Suraya A.; Martin, Stephen R.; Howell, Steven A.; Grainger, Munira; Moon, Robert W.; Green, Judith L.

    2016-01-01

    Aldolase has been implicated as a protein coupling the actomyosin motor and cell surface adhesins involved in motility and host cell invasion in the human malaria parasite Plasmodium falciparum. It binds to the cytoplasmic domain (CTD) of type 1 membrane proteins of the thrombospondin-related anonymous protein (TRAP) family. Other type 1 membrane proteins located in the apical organelles of merozoites, the form of the parasite that invades red blood cells, including apical membrane antigen 1 (AMA1) and members of the erythrocyte binding ligand (EBL) and reticulocyte binding homologue (RH) protein families have been implicated in host cell binding and invasion. Using a direct binding method we confirm that TRAP and merozoite TRAP (MTRAP) bind aldolase and show that the interaction is mediated by more than just the C-terminal six amino acid residues identified previously. Single amino acid substitutions in the MTRAP CTD abolished binding to aldolase. The CTDs of AMA1 and members of the EBL and RH protein families also bound to aldolase. MTRAP competed with AMA1 and RH4 for binding to aldolase, indicating overlapping binding sites. MTRAP CTD was phosphorylated in vitro by both calcium dependent kinase 1 (CDPK1) and protein kinase A, and this modification increased the affinity of binding to aldolase by ten-fold. Phosphorylation of the CTD of members of the EBL and RH protein families also increased their affinity for aldolase in some cases. To examine whether or not MTRAP expressed in asexual blood stage parasites is phosphorylated, it was tagged with GFP, purified and analysed, however no phosphorylation was detected. We propose that CTD binding to aldolase may be dynamically modulated by phosphorylation, and there may be competition for aldolase binding between different CTDs. The use and efficiency of alternate invasion pathways may be determined by the affinity of adhesins and cell invasion proteins for aldolase, in addition to their host ligand specificity. PMID

  18. FimH adhesin from host unrestricted Salmonella Enteritidis binds to different glycoprotein ligands expressed by enterocytes from sheep, pig and cattle than FimH adhesins from host restricted Salmonella Abortus-ovis, Salmonella Choleraesuis and Salmonella Dublin.

    PubMed

    Grzymajło, Krzysztof; Ugorski, Maciej; Kolenda, Rafał; Kędzierska, Anna; Kuźmińska-Bajor, Marta; Wieliczko, Alina

    2013-10-25

    Adhesion to gut tissues and colonization of the alimentary tract, two important stages in the pathogenesis of Salmonella, are mediated by FimH adhesin of type 1 fimbriae. It was suggested that minor differences in the structure of FimH are most likely associated with differences in adhesion specificities, and may determine the tropism of various Salmonella serovars to different species and tissues. We investigated this hypothesis by comparing the binding properties of FimH proteins from three Salmonella enterica serovars with limited (Choleraesuis, Dublin) or restricted (Abortusovis) host ranges to FimH from broad host range S. Enteritidis and mannose inactive FimH from S. Gallinarum. Although all active variants of FimH protein were able to bind mannose-rich glycoproteins (RNase B, HRP and Man-BSA) with comparable affinity measured by surface plasmon resonance, there were significant differences in the binding profiles of the FimH proteins from host restricted serovars and host unrestricted serovar Enteritidis, to glycoproteins from enterocyte cell lines established in vitro and derived from sheep, pig and cattle. When low-binding FimH adhesin from S. Enteritidis was subjected to Western blot analysis, it bound to surface membrane protein of about 130 kDa, and high-binding FimH adhesins from S. Abortusovis, S. Choleraesuis and S. Dublin bound to surface membrane protein of about 55 kDa present in each cell line. Differential binding of FimH proteins from host-restricted and broad-host-range Salmonella to intestinal receptors was confirmed using mutant FimH adhesins obtained by site-directed mutagenesis. It was found that the low-binding variant of FimH from S. Choleraesuis with mutation Leu57Pro lost the ability to bind protein band of 55 kDa, but instead interacted with glycoprotein of about 130 kDa. On the other hand, the high-binding variant of FimH adhesin from S. Enteritids with mutation Asn101Ser did not bind to its receptor of 130 kDa, but instead it

  19. Versatility of Biofilm Matrix Molecules in Staphylococcus epidermidis Clinical Isolates and Importance of Polysaccharide Intercellular Adhesin Expression during High Shear Stress

    PubMed Central

    Schaeffer, Carolyn R.; Hoang, Tra-My N.; Sudbeck, Craig M.; Alawi, Malik; Tolo, Isaiah E.; Robinson, D. Ashley; Horswill, Alexander R.; Rohde, Holger

    2016-01-01

    ABSTRACT Staphylococcus epidermidis is a leading cause of hospital-associated infections, including those of intravascular catheters, cerebrospinal fluid shunts, and orthopedic implants. Multiple biofilm matrix molecules with heterogeneous characteristics have been identified, including proteinaceous, polysaccharide, and nucleic acid factors. Two of the best-studied components in S. epidermidis include accumulation-associated protein (Aap) and polysaccharide intercellular adhesin (PIA), produced by the enzymatic products of the icaADBC operon. Biofilm composition varies by strain as well as environmental conditions, and strains producing PIA-mediated biofilms are more robust. Clinically, biofilm-mediated infections occur in a variety of anatomical sites with diverse physiological properties. To test the hypothesis that matrix composition exhibits niche specificity, biofilm-related genetic and physical properties were compared between S. epidermidis strains isolated from high-shear and low-shear environments. Among a collection of 105 clinical strains, significantly more isolates from high-shear environments carried the icaADBC operon than did those from low-shear settings (43.9% versus 22.9%, P < 0.05), while there was no significant difference in the presence of aap (77.2% versus 75.0%, P > 0.05). Additionally, a significantly greater number of high-shear isolates were capable of forming biofilm in vitro in a microtiter assay (82.5% versus 45.8%, P < 0.0001). However, even among high-shear clinical isolates, less than half contained the icaADBC locus; therefore, we selected for ica-negative variants with increased attachment to abiotic surfaces to examine PIA-independent biofilm mechanisms. Sequencing of selected variants identified substitutions capable of enhancing biofilm formation in multiple genes, further highlighting the heterogeneity of S. epidermidis biofilm molecules and mechanisms. IMPORTANCE Staphylococcus epidermidis is a leading cause of

  20. Efficiency of Direct Microscopy of Stool Samples Using an Antigen-Specific Adhesin Test for Entamoeba Histolytica

    PubMed Central

    İrvem, Arzu; Özdil, Kamil; Çalışkan, Zuhal; Yücel, Muhterem

    2016-01-01

    Background: E. histolytica is among the common causes of acute gastroenteritis. The pathogenic species E. histolytica and the nonpathogenic species E. dispar cannot be morphologically differentiated, although correct identification of these protozoans is important for treatment and public health. In many laboratories, the screening of leukocytes, erythrocytes, amoebic cysts, trophozoites and parasite eggs is performed using Native-Lugol’s iodine for pre-diagnosis. Aims: In this study, we aimed to investigate the frequency of E. histolytica in stool samples collected from 788 patients residing in the Anatolian region of İstanbul who presented with gastrointestinal complaints. We used the information obtained to evaluate the effectiveness of microscopic examinations when used in combination with the E. histolytica adhesin antigen test. Study Design: Retrospective cross-sectional study Methods: Preparations of stool samples stained with Native-Lugol’s iodine were evaluated using the E. histolytica adhesin test and examined using standard light microscopy at ×40 magnification. Pearson’s Chi-square and Fisher’s exact tests were used for statistical analysis. Logistic regression analysis was used for multivariate analysis. Results: Of 788 samples, 38 (4.8%) were positive for E. histolytica adhesin antigens. When evaluated together with the presences of erythrocytes, leukocytes, cysts, and trophozoites, respectively, using logistic regression analysis, leukocyte positivity was significantly higher. The odds ratio of leukocyte positivity increased adhesin test-positivity by 2,530-fold (95% CI=1.01–6.330). Adhesin test-positivity was significant (p=0.047). Conclusion: In line with these findings, the consistency between the presence of cysts and erythrocytes and adhesin test-positivity was found to be highly significant, but that of higher levels of leukocytes was found to be discordant. It was concluded that leukocytes and trophozoites were easily misjudged

  1. Regulation of Expression of Uropathogenic Escherichia coli Nonfimbrial Adhesin TosA by PapB Homolog TosR in Conjunction with H-NS and Lrp.

    PubMed

    Engstrom, Michael D; Mobley, Harry L T

    2016-01-11

    Urinary tract infections (UTIs) are a major burden to human health. The overwhelming majority of UTIs are caused by uropathogenic Escherichia coli (UPEC) strains. Unlike some pathogens, UPEC strains do not have a fixed core set of virulence and fitness factors but do have a variety of adhesins and regulatory pathways. One such UPEC adhesin is the nonfimbrial adhesin TosA, which mediates adherence to the epithelium of the upper urinary tract. The tos operon is AT rich, resides on pathogenicity island aspV, and is not expressed under laboratory conditions. Because of this, we hypothesized that tosA expression is silenced by H-NS. Lrp, based on its prominent function in the regulation of other adhesins, is also hypothesized to contribute to tos operon regulation. Using a variety of in vitro techniques, we mapped both the tos operon promoter and TosR binding sites. We have now identified TosR as a dual regulator of the tos operon, which could control the tos operon in association with H-NS and Lrp. H-NS is a negative regulator of the tos operon, and Lrp positively regulates the tos operon. Exogenous leucine also inhibits Lrp-mediated tos operon positive regulation. In addition, TosR binds to the pap operon, which encodes another important UPEC adhesin, P fimbria. Induction of TosR synthesis reduces production of P fimbria. These studies advance our knowledge of regulation of adhesin expression associated with uropathogen colonization of a host.

  2. A conserved domain of previously unknown function in Gap1 mediates protein-protein interaction and is required for biogenesis of a serine-rich streptococcal adhesin

    PubMed Central

    Li, Yirong; Chen, Yabing; Huang, Xiang; Zhou, Meixian; Wu, Ren; Dong, Shengli; Pritchard, David G.; Fives-Taylor, Paula; Wu, Hui

    2010-01-01

    Summary Fap1-like serine-rich proteins are a new family of bacterial adhesins found in a variety of streptococci and staphylococci that have been implicated in bacterial pathogenesis. A gene cluster encoding glycosyltransferases and accessory Sec components is required for Fap1 glycosylation and biogenesis in Streptococcus parasanguinis. Here we report that the glycosylation-associated protein, Gap1, contributes to glycosylation and biogenesis of Fap1 by interacting with another glycosylation-associated protein, Gap3. Gap1 shares structural homology with glycosyltransferases. The gap1 mutant, like the gap3 mutant, produced an aberrantly-glycosylated Fap1 precursor and failed to produce mature Fap1, suggesting that Gap1 and Gap3 might function in concert in the Fap1 glycosylation and biogenesis. Indeed, Gap1 interacted with Gap3 in vitro and in vivo. A Gap1 amino-terminal motif, within a highly conserved domain of unknown function (DUF1975) identified in many bacterial glycosyltrasnferases, was required for the Gap1-Gap3 interaction. Deletion of one, four, and nine amino acids within the conserved motif gradually inhibited the Gap1-Gap3 interaction and diminished production of mature Fap1 and concurrently increased production of the Fap1 precursor. Consequently, bacterial adhesion to an in vitro tooth model was also reduced. These data demonstrate that the Gap1-Gap3 interaction is required for Fap1 biogenesis and Fap1-dependent bacterial adhesion. PMID:18826412

  3. The dynamics and pH-dependence of Ag43 adhesins' self-association probed by atomic force spectroscopy

    NASA Astrophysics Data System (ADS)

    Jacquot, Adrien; Sakamoto, Chizuko; Razafitianamarahavo, Angelina; Caillet, Céline; Merlin, Jenny; Fahs, Ahmad; Ghigo, Jean-Marc; Duval, Jérôme F. L.; Beloin, Christophe; Francius, Grégory

    2014-10-01

    Self-associating auto-transporter (SAAT) adhesins are two-domain cell surface proteins involved in bacteria auto-aggregation and biofilm formation. Antigen 43 (Ag43) is a SAAT adhesin commonly found in Escherichia coli whose variant Ag43a has been shown to promote persistence of uropathogenic E. coli within the bladder. The recent resolution of the tri-dimensional structure of the 499 amino-acids' β-domain in Ag43a has shed light on the possible mechanism governing the self-recognition of SAAT adhesins, in particular the importance of trans-interactions between the L shaped β-helical scaffold of two α-domains of neighboring adhesins. In this study, we use single-molecule force spectroscopy (SMFS) and dynamic force spectroscopy (DFS) to unravel the dynamics of Ag43-self association under various pH and molecular elongation rate conditions that mimic the situations encountered by E. coli in its natural environment. Results evidenced an important stretchability of Ag43α with unfolding of sub-domains leading to molecular extension as long as 150 nm. Nanomechanical analysis of molecular stretching data suggested that self-association of Ag43 can lead to the formation of dimers and tetramers driven by rapid and weak cis- as well as slow but strong trans-interaction forces with a magnitude as large as 100-250 pN. The dynamics of cis- and trans-interactions were demonstrated to be strongly influenced by pH and applied shear force, thus suggesting that environmental conditions can modulate Ag43-mediated aggregation of bacteria at the molecular level.Self-associating auto-transporter (SAAT) adhesins are two-domain cell surface proteins involved in bacteria auto-aggregation and biofilm formation. Antigen 43 (Ag43) is a SAAT adhesin commonly found in Escherichia coli whose variant Ag43a has been shown to promote persistence of uropathogenic E. coli within the bladder. The recent resolution of the tri-dimensional structure of the 499 amino-acids' β-domain in Ag43a has shed

  4. VirB2 and VirB5 proteins: specialized adhesins in bacterial type-IV secretion systems?

    PubMed

    Backert, Steffen; Fronzes, Remi; Waksman, Gabriel

    2008-09-01

    Many type-IV secretion systems (T4SSs) of plant and human pathogens assemble a pilus used to inject virulence molecules (effectors) into host target cells. The T4SS of Agrobacterium tumefaciens consists of VirB1-VirB11 and VirD4 proteins. Whether targeting of T4SSs to the host requires a T4SS-adhesin that specifically engages host receptors for delivery of effectors has, until recently, remained unclear. Recent data of Agrobacterium and Helicobacter indicate that two classes of T4SS components, VirB2 and VirB5, might function as adhesins that mediate host-cell targeting through binding to specific host receptors. Here, we discuss this important issue and recent progress in the field. PMID:18706815

  5. Characterization of porcine intestinal receptors for the K88ac fimbrial adhesin of Escherichia coli as mucin-type sialoglycoproteins.

    PubMed Central

    Erickson, A K; Baker, D R; Bosworth, B T; Casey, T A; Benfield, D A; Francis, D H

    1994-01-01

    We have previously identified two K88ac adhesion receptors (210 and 240 kDa) which are present in membrane preparations from adhesive but not nonadhesive porcine intestinal brush border cells; these adhesin receptors are postulated to be important determinants of the susceptibility of pigs to K88ac+ enterotoxigenic Escherichia coli infections (A.K. Erickson, J.A. Willgohs, S.Y. McFarland, D.A. Benfield, and D.F. Francis, Infect. Immun. 60:983-988, 1992). We now describe a procedure for the purification of these two receptors. Receptors were solubilized from adhesive intestinal brush border vesicles using deoxycholate and were purified by gel filtration chromatography on Sepharose CL-4B and then by hydroxyapatite chromatography. Amino acid compositional analyses indicated that the two receptors have similar amino acid compositions. The most distinguishing characteristic of both receptors is a high percentage of threonine and proline residues. Neuraminidase treatment caused the K88ac adhesin receptors to migrate with a slower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, indicating that these receptors are sialoglycoproteins. Results from lectin-binding studies indicated that the receptors contain O-linked oligosaccharides composed of galactosyl (beta-1,3)N-acetylgalactosamine, alpha-linked fucose, galactosyl(beta-1,4)N-acetylglucosamine, sialic acid, galactose, and N-acetylgalactosamine. Collectively, these characteristics indicate that the K88ac adhesin receptors are mucin-type sialoglycoproteins. Images PMID:7960120

  6. Fap2 of Fusobacterium nucleatum is a galactose-inhibitable adhesin involved in coaggregation, cell adhesion, and preterm birth.

    PubMed

    Coppenhagen-Glazer, S; Sol, A; Abed, J; Naor, R; Zhang, X; Han, Y W; Bachrach, G

    2015-03-01

    Fusobacterium nucleatum is a common oral anaerobe involved in periodontitis that is known to translocate and cause intrauterine infections. In the oral environment, F. nucleatum adheres to a large diversity of species, facilitating their colonization and creating biological bridges that stabilize the multispecies dental biofilm. Many of these interactions (called coadherences or coaggregations) are galactose sensitive. Galactose-sensitive interactions are also involved in the binding of F. nucleatum to host cells. Hemagglutination of some F. nucleatum strains is also galactose sensitive, suggesting that a single galactose-sensitive adhesin might mediate the interaction of fusobacteria with many partners and targets. In order to identify the fusobacterial galactose-sensitive adhesin, a system for transposon mutagenesis in fusobacteria was created. The mutant library was screened for hemagglutination deficiency, and three clones were isolated. All three clones were found to harbor the transposon in the gene coding for the Fap2 outer membrane autotransporter. The three fap2 mutants failed to show galactose-inhibitable coaggregation with Porphyromonas gingivalis and were defective in cell binding. A fap2 mutant also showed a 2-log reduction in murine placental colonization compared to that of the wild type. Our results suggest that Fap2 is a galactose-sensitive hemagglutinin and adhesin that is likely to play a role in the virulence of fusobacteria.

  7. Label-free Gram-negative bacteria detection using bacteriophage-adhesin-coated long-period gratings.

    PubMed

    Brzozowska, Ewa; Koba, Marcin; Śmietana, Mateusz; Górska, Sabina; Janik, Monika; Gamian, Andrzej; Bock, Wojtek J

    2016-03-01

    This paper presents a novel application of a highly sensitive sensor based on long-period gratings (LPGs) coated with T4 bacteriophage adhesin for Gram-negative bacteria detection. We show here, that the sensor evidently recognizes Escherichia coli K-12 (PCM2560), whereas in the reference tests - ELISA and BIAcore - the results are questionable. For LPGs sensor the resonant wavelength shift observed for E. coli K-12 was approximately half of that measured for E.coli B (positive control). The BIAcore readings (RU) for E. coli K-12 were at 10% level of the signal obtained for E .coli B. These results confirm the improved sensitivity of the LPGs sensor. Moreover, we also show that application of adhesin may allow for efficient detection of E. coli O111 (PCM418), Klebsiella pneumoniae O1 (PCM1) and Yersinia enterocolitica O1 (PCM1879). The specificity of binding bacteria by the adhesin is discussed and it is determined by a distinct region of lipopolysaccharide receptors and/or by the presence of outer-membrane protein C in an outer membrane of Gram-negative bacteria.

  8. Fap2 of Fusobacterium nucleatum Is a Galactose-Inhibitable Adhesin Involved in Coaggregation, Cell Adhesion, and Preterm Birth

    PubMed Central

    Coppenhagen-Glazer, S.; Sol, A.; Abed, J.; Naor, R.; Zhang, X.

    2015-01-01

    Fusobacterium nucleatum is a common oral anaerobe involved in periodontitis that is known to translocate and cause intrauterine infections. In the oral environment, F. nucleatum adheres to a large diversity of species, facilitating their colonization and creating biological bridges that stabilize the multispecies dental biofilm. Many of these interactions (called coadherences or coaggregations) are galactose sensitive. Galactose-sensitive interactions are also involved in the binding of F. nucleatum to host cells. Hemagglutination of some F. nucleatum strains is also galactose sensitive, suggesting that a single galactose-sensitive adhesin might mediate the interaction of fusobacteria with many partners and targets. In order to identify the fusobacterial galactose-sensitive adhesin, a system for transposon mutagenesis in fusobacteria was created. The mutant library was screened for hemagglutination deficiency, and three clones were isolated. All three clones were found to harbor the transposon in the gene coding for the Fap2 outer membrane autotransporter. The three fap2 mutants failed to show galactose-inhibitable coaggregation with Porphyromonas gingivalis and were defective in cell binding. A fap2 mutant also showed a 2-log reduction in murine placental colonization compared to that of the wild type. Our results suggest that Fap2 is a galactose-sensitive hemagglutinin and adhesin that is likely to play a role in the virulence of fusobacteria. PMID:25561710

  9. Label-free Gram-negative bacteria detection using bacteriophage-adhesin-coated long-period gratings.

    PubMed

    Brzozowska, Ewa; Koba, Marcin; Śmietana, Mateusz; Górska, Sabina; Janik, Monika; Gamian, Andrzej; Bock, Wojtek J

    2016-03-01

    This paper presents a novel application of a highly sensitive sensor based on long-period gratings (LPGs) coated with T4 bacteriophage adhesin for Gram-negative bacteria detection. We show here, that the sensor evidently recognizes Escherichia coli K-12 (PCM2560), whereas in the reference tests - ELISA and BIAcore - the results are questionable. For LPGs sensor the resonant wavelength shift observed for E. coli K-12 was approximately half of that measured for E.coli B (positive control). The BIAcore readings (RU) for E. coli K-12 were at 10% level of the signal obtained for E .coli B. These results confirm the improved sensitivity of the LPGs sensor. Moreover, we also show that application of adhesin may allow for efficient detection of E. coli O111 (PCM418), Klebsiella pneumoniae O1 (PCM1) and Yersinia enterocolitica O1 (PCM1879). The specificity of binding bacteria by the adhesin is discussed and it is determined by a distinct region of lipopolysaccharide receptors and/or by the presence of outer-membrane protein C in an outer membrane of Gram-negative bacteria. PMID:27231592

  10. Label-free Gram-negative bacteria detection using bacteriophage-adhesin-coated long-period gratings

    PubMed Central

    Brzozowska, Ewa; Koba, Marcin; Śmietana, Mateusz; Górska, Sabina; Janik, Monika; Gamian, Andrzej; Bock, Wojtek J.

    2016-01-01

    This paper presents a novel application of a highly sensitive sensor based on long-period gratings (LPGs) coated with T4 bacteriophage adhesin for Gram-negative bacteria detection. We show here, that the sensor evidently recognizes Escherichia coli K-12 (PCM2560), whereas in the reference tests – ELISA and BIAcore – the results are questionable. For LPGs sensor the resonant wavelength shift observed for E. coli K-12 was approximately half of that measured for E.coli B (positive control). The BIAcore readings (RU) for E. coli K-12 were at 10% level of the signal obtained for E .coli B. These results confirm the improved sensitivity of the LPGs sensor. Moreover, we also show that application of adhesin may allow for efficient detection of E. coli O111 (PCM418), Klebsiella pneumoniae O1 (PCM1) and Yersinia enterocolitica O1 (PCM1879). The specificity of binding bacteria by the adhesin is discussed and it is determined by a distinct region of lipopolysaccharide receptors and/or by the presence of outer-membrane protein C in an outer membrane of Gram-negative bacteria. PMID:27231592

  11. Erythrocyte gangliosides act as receptors for Neisseria subflava: identification of the Sia-1 adhesin.

    PubMed Central

    Nyberg, G; Strömberg, N; Jonsson, A; Karlsson, K A; Normark, S

    1990-01-01

    Neisseria gonorrhoeae was recently shown to bind to a subset of lactose-containing glycolipids (N. Strömberg, C. Deal, G. Nyberg, S. Normark, M. So, and K.-A. Karlsson, Proc. Natl. Acad. Sci. USA 85:4902-4906, 1988). A number of commensal Neisseria strains were also shown to be lactose binders. In addition, Neisseria subflava bound to immobilized gangliosides, such as hematoside and sialosyl paragloboside, carrying the NeuAc alpha 2-3Gal beta 1-4Glc sequence. To a lesser extent, N. gonorrhoeae also bound to this receptor in vitro. In N. subflava GN01, this binding property mediated agglutination of human erythrocytes in a neuraminidase-sensitive fashion. Nitrosoguanidine-induced nonhemagglutinative mutants of N. subflava GN01 had lost the ability to bind hematoside and sialosylparagloboside but remained able to bind lactosylceramide and gangliotetraosylceramide. These mutants fell into three classes with respect to their outer membrane protein profiles in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Class 1 mutants were identical to the parent strain save for the loss of a 27-kilodalton (kDa) protein. Class 2 mutants showed an outer membrane protein profile identical to that of the wild type, whereas mutants belonging to class 3 showed a number of changes, including the apparent absence of the 27-kDa protein. The 27-kDa protein from N. subflava GN01 was purified from the supernatant. A polyclonal antiserum to the purified Sia-1 protein as well as a Sia-1-specific monoclonal antibody inhibited hemagglutination by strain GN01. The purified Sia-1 protein in the presence of diluted anti-Sia-1 antiserum mediated a neuraminidase-sensitive hemagglutination. The purified Sia protein from a class 2 mutant was not able to hemagglutinate when cross-linked with antibodies, suggesting that it is a mutant form of Sia-1 affected in the receptor-binding site. Immunoelectron microscopy with a Sia-1-specific monoclonal antibody revealed that the adhesin was

  12. Bile salts induce expression of the afimbrial LDA adhesin of atypical enteropathogenic Escherichia coli.

    PubMed

    Torres, Alfredo G; Tutt, Christopher B; Duval, Lisabeth; Popov, Vsevolod; Nasr, Abdelhakim Ben; Michalski, Jane; Scaletsky, Isabel C A

    2007-04-01

    Atypical enteropathogenic Escherichia coli (aEPEC) strains are frequently implicated in infant diarrhoea in developing countries. Not much is known about the adherence properties of aEPEC; however, it has been shown that these strains can adhere to tissue-cultured cells. A chromosomal region designated the locus for diffuse adherence (LDA) confers aEPEC strain 22 the ability to adhere to culture cells. LDA is an afimbrial adhesin that contains a major subunit, LdaG, whose expression is induced on MacConkey agar at 37 degrees C. We hypothesized that the bile salts found in this culture media induce the expression of LdaG. Strain 22 and the LdaG mutant were grown in Luria-Bertani (LB) media in the presence or absence of bile salts and heat-extracted surface-expressed proteins were separated by SDS-PAGE to determine whether expression of the 25 kDa LdaG protein was induced. Western blot analysis with anti-LdaG confirmed that bile salts enhance LdaG expression at 37 degrees C. Adhesion assays on HeLa cells revealed that adhesion in a diffuse pattern of strain 22 increased in the presence of bile salts. We also confirmed that expression of the localized adherence pattern observed in the ldaG mutant required the presence of a large cryptic plasmid found in strain 22 and that this phenotype was not induced by bile salts. At the transcriptional level, the ldaG-lacZ promoter fusion displayed maximum beta-galactosidase activity when the parent strain was grown in LB supplemented with bile salts. Fluorescence Activated Cell Sorting analysis, immunogold labelling electron microscopy and immunofluorescence using anti-LdaG sera confirmed that LDA is a bile salts-inducible surface-expressed afimbrial adhesin. Finally, LdaG expression was induced in presence of individual bile salts but not by other detergents. We concluded that bile salts increase expression of LDA, conferring a diffuse adherence pattern and having an impact on the adhesion properties of this aEPEC strain.

  13. Structure of the Streptococcus pneumoniae Surface Protein and Adhesin PfbA

    PubMed Central

    Suits, Michael D.; Boraston, Alisdair B.

    2013-01-01

    PfbA (plasmin- and fibronectin-binding protein A) is an extracellular Streptococcus pneumoniae cell-wall attached surface protein that binds to fibronectin, plasmin, and plasminogen. Here we present a structural analysis of the surface exposed domains of PfbA using a combined approach of X-ray crystallography and small-angle X-ray scattering (SAXS). The crystal structure of the PfbA core domain, here called PfbAβ, determined to 2.28 Å resolution revealed an elongated 12-stranded parallel β-helix fold, which structure-based comparisons reveal is most similar to proteins with carbohydrate modifying activity. A notable feature of the PfbAβ is an extensive cleft on one face of the protein with electrochemical and spatial features that are analogous to structurally similar carbohydrate-active enzymes utilizing this feature for substrate accommodation. Though this cleft displays a combination of basic amino acid residues and solvent exposed aromatic amino acids that are distinct features for recognition of carbohydrates, no obvious arrangement of amino acid side chains that would constitute catalytic machinery is evident. The pseudo-atomic SAXS model of a larger fragment of PfbA suggests that it has a relatively well-ordered structure with the N-terminal and core domains of PfbA adopting an extend organization and reveals a novel structural class of surface exposed pneumococcal matrix molecule adhesins. PMID:23894284

  14. The Streptococcus pneumoniae adhesin PsrP binds to Keratin 10 on lung cells

    PubMed Central

    Shivshankar, Pooja; Sanchez, Carlos; Rose, Lloyd F.; Orihuela, Carlos J.

    2009-01-01

    Pneumococcal serine-rich repeat protein (PsrP) is a pathogenicity island encoded adhesin that mediates attachment to lung cells. It is a member of the Serine-rich repeat protein (SRRP) family and the largest bacterial protein known. PsrP production by S. pneumoniae was confirmed by immunoblotting and a truncated version of the protein was determined to be glycosylated. Using isogenic psrP mutants complemented with various PsrP constructs and competitive inhibition assays with recombinant proteins, we determined that PsrP requires an extended SRR2 domain for function and that adhesion is mediated through amino acids 273-341 of its Basic Region (BR) domain. Affinity chromatography, immunoprecipitation, ELISA, FACS, and immunofluorescent co-localization studies determined that PsrP binds to Keratin 10 (K10) on the surface of lung but not nasopharyngeal epithelial cells. Unglycosylated K10 bound to wild type but not psrP deficient pneumococci; suggesting that unlike other SRRPs, PsrP-mediated adhesion was independent of lectin activity. Finally, mice immunized with recombinant (r)PsrPBR had significantly less bacteria in their blood and improved survival versus controls following intranasal challenge. We conclude that the BR domain of PsrP binds to K10 in a lectin-independent manner; that K10 is expressed on lung cells; and that vaccination with rPsrPBR is protective against pneumococcal disease. PMID:19627498

  15. Apa is a trimeric autotransporter adhesin of Actinobacillus pleuropneumoniae responsible for autoagglutination and host cell adherence.

    PubMed

    Xiao, Longwen; Zhou, Liang; Sun, Changjiang; Feng, Xin; Du, ChongTao; Gao, Yu; Ji, Qun; Yang, Shuxin; Wang, Yu; Han, Wenyu; Langford, P R; Lei, Liancheng

    2012-10-01

    Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, and adherence to host cells is a key step in the pathogenic process. Although trimeric autotransporter adhesins (TAAs) were identified in many pathogenic bacteria in recent years, none in A. pleuropneumoniae have been characterized. In this study, we identified a TAA from A. pleuropneumoniae, Apa, and characterized the contribution of its amino acid residues to the adhesion process. Sequence analysis of the C-terminal amino acid residues of Apa revealed the presence of a putative translocator domain and six conserved HsfBD1-like or HsfBD2-like binding domains. Western blot analysis revealed that the 126 C-terminal amino acids of Apa could form trimeric molecules. By confocal laser scanning microscopy, one of these six domains (ApaBD3) was determined to mediate adherence to epithelial cells. Adherence assays and adherence inhibition assays using a recombinant E. coli- ApaBD3 strain which expressed ApaBD3 on the surface of E. coli confirmed that this domain was responsible for the adhesion activity. Moreover, cellular enzyme-linked immunosorbent assays demonstrated that ApaBD3 mediated high-level adherence to epithelial cell lines. Intriguingly, autoagglutination was observed with the E. coli- ApaBD3 strain, and this phenomenon was dependent upon the association of the expressed ApaBD3 with the C-terminal translocator domain.

  16. Targeted Gene Disruption Reveals an Adhesin Indispensable for Pathogenicity of Blastomyces dermatitidis

    PubMed Central

    Tristan Brandhorst, T.; Wüthrich, Marcel; Warner, Thomas; Klein, Bruce

    1999-01-01

    Systemic fungal infections are becoming more common and difficult to treat, yet the pathogenesis of these infectious diseases remains poorly understood. In many cases, pathogenicity can be attributed to the ability of the fungi to adhere to target tissues, but the lack of tractable genetic systems has limited progress in understanding and interfering with the offending fungal products. In Blastomyces dermatitidis, the agent of blastomycosis, a respiratory and disseminated mycosis of people and animals worldwide, expression of the putative adhesin encoded by the WI-1 gene was investigated as a possible virulence factor. DNA-mediated gene transfer was used to disrupt the WI-1 locus by allelic replacement, resulting in impaired binding and entry of yeasts into macrophages, loss of adherence to lung tissue, and abolishment of virulence in mice; each of these properties was fully restored after reconstitution of WI-1 by means of gene transfer. These findings establish the pivotal role of WI-1 in adherence and virulence of B. dermatitidis yeasts. To our knowledge, they offer the first example of a genetically proven virulence determinant among systemic dimorphic fungi, and underscore the value of reverse genetics for studies of pathogenesis in these organisms. PMID:10209038

  17. sae is essential for expression of the staphylococcal adhesins Eap and Emp.

    PubMed

    Harraghy, Niamh; Kormanec, Jan; Wolz, Christiane; Homerova, Dagmar; Goerke, Christiane; Ohlsen, Knut; Qazi, Saara; Hill, Philip; Herrmann, Mathias

    2005-06-01

    Eap and Emp are two Staphylococcus aureus adhesins initially described as extracellular matrix binding proteins. Eap has since emerged as being important in adherence to and invasion of eukaryotic cells, as well as being described as an immunomodulator and virulence factor in chronic infections. This paper describes the mapping of the transcription start point of the eap and emp promoters. Moreover, using reporter-gene assays and real-time PCR in defined regulatory mutants, environmental conditions and global regulators affecting expression of eap and emp were investigated. Marked differences were found in expression of eap and emp between strain Newman and the 8325 derivatives SH1000 and 8325-4. Moreover, both genes were repressed in the presence of glucose. Analysis of expression of both genes in various regulatory mutants revealed that sarA and agr were involved in their regulation, but the data suggested that there were additional regulators of both genes. In a sae mutant, expression of both genes was severely repressed. sae expression was also reduced in the presence of glucose, suggesting that repression of eap and emp in glucose-containing medium may, in part, be a consequence of a decrease in expression of sae.

  18. Staphylococcus epidermidis Polysaccharide Intercellular Adhesin Production Significantly Increases during Tricarboxylic Acid Cycle Stress

    PubMed Central

    Vuong, Cuong; Kidder, Joshua B.; Jacobson, Erik R.; Otto, Michael; Proctor, Richard A.; Somerville, Greg A.

    2005-01-01

    Staphylococcal polysaccharide intercellular adhesin (PIA) is important for the development of a mature biofilm. PIA production is increased during growth in a nutrient-replete or iron-limited medium and under conditions of low oxygen availability. Additionally, stress-inducing stimuli such as heat, ethanol, and high concentrations of salt increase the production of PIA. These same environmental conditions are known to repress tricarboxylic acid (TCA) cycle activity, leading us to hypothesize that altering TCA cycle activity would affect PIA production. Culturing Staphylococcus epidermidis with a low concentration of the TCA cycle inhibitor fluorocitrate dramatically increased PIA production without impairing glucose catabolism, the growth rate, or the growth yields. These data lead us to speculate that one mechanism by which staphylococci perceive external environmental change is through alterations in TCA cycle activity leading to changes in the intracellular levels of biosynthetic intermediates, ATP, or the redox status of the cell. These changes in the metabolic status of the bacteria result in the attenuation or augmentation of PIA production. PMID:15838022

  19. Catch-bond mechanism of the bacterial adhesin FimH

    PubMed Central

    Sauer, Maximilian M.; Jakob, Roman P.; Eras, Jonathan; Baday, Sefer; Eriş, Deniz; Navarra, Giulio; Bernèche, Simon; Ernst, Beat; Maier, Timm; Glockshuber, Rudi

    2016-01-01

    Ligand–receptor interactions that are reinforced by mechanical stress, so-called catch-bonds, play a major role in cell–cell adhesion. They critically contribute to widespread urinary tract infections by pathogenic Escherichia coli strains. These pathogens attach to host epithelia via the adhesin FimH, a two-domain protein at the tip of type I pili recognizing terminal mannoses on epithelial glycoproteins. Here we establish peptide-complemented FimH as a model system for fimbrial FimH function. We reveal a three-state mechanism of FimH catch-bond formation based on crystal structures of all states, kinetic analysis of ligand interaction and molecular dynamics simulations. In the absence of tensile force, the FimH pilin domain allosterically accelerates spontaneous ligand dissociation from the FimH lectin domain by 100,000-fold, resulting in weak affinity. Separation of the FimH domains under stress abolishes allosteric interplay and increases the affinity of the lectin domain. Cell tracking demonstrates that rapid ligand dissociation from FimH supports motility of piliated E. coli on mannosylated surfaces in the absence of shear force. PMID:26948702

  20. Amyloid-like ribbons of amelogenins in enamel mineralization

    DOE PAGES

    Carneiro, Karina M. M.; Zhai, Halei; Zhu, Li; Horst, Jeremy A.; Sitlin, Melody; Nguyen, Mychi; Wagner, Martin; Simpliciano, Cheryl; Milder, Melissa; Chen, Chun-Long; et al

    2016-03-24

    We report that enamel, the outermost layer of teeth, is an acellular mineralized tissue that cannot regenerate; the mature tissue is composed of high aspect ratio apatite nanocrystals organized into rods and inter-rod regions. Amelogenin constitutes 90% of the protein matrix in developing enamel and plays a central role in guiding the hierarchical organization of apatite crystals observed in mature enamel. To date, a convincing link between amelogenin supramolecular structures and mature enamel has yet to be described, in part because the protein matrix is degraded during tissue maturation. Here we show compelling evidence that amelogenin self-assembles into an amyloid-likemore » structure in vitro and in vivo. We show that enamel matrices stain positive for amyloids and we identify a specific region within amelogenin that self-assembles into β-sheets. Lastly, we propose that amelogenin nanoribbons template the growth of apatite mineral in human enamel. This is a paradigm shift from the current model of enamel development.« less

  1. Specificity of Campylobacter jejuni Adhesin PEB3 for Phosphates and Structural Differences among Its Ligand Complexes

    SciTech Connect

    Min, Tongpil; Vedadi, Masoud; Watson, David C.; Wasney, Gregory A.; Munger, Christine; Cygler, Miroslaw; Matte, Allan; Young, N. Martin

    2009-04-22

    PEB3 is a glycoprotein adhesin from Campylobacter jejuni whose structure suggested a role in transport. We have investigated potential ligands for PEB3 and characterized their binding properties using biophysical methods in solution and by X-ray crystallography. A thermal aggregation assay of PEB3 with a library of physiological compounds identified three possible ligands [3-phosphoglycerate (3-PG), phosphoenolpyruvate (PEP), and aconitate], which stabilized wild-type PEB3 but did not stabilize either a PEB3 form containing two mutations at the ligand-binding site, T138A/S139A, or a second PEB3 mutant, K135E, at a site {approx}14 {angstrom} away. Fluorescence titration experiments and cocrystal structures with various ligands were used to characterize the binding of 3-PG, PEP, and phosphate to PEB3. Further, a C. jejuni growth experiment in minimal medium supplemented with 3-PG showed that this molecule enhances the growth of wild-type C. jejuni, but not of the PEB3 mutants. Crystallographic analysis of PEB3 complexes revealed that the Ser171-Gln180 region in the presence of 3-PG or other phosphates is helical and similar to those of other transport proteins, but it is nonhelical when citrate is bound. The K135E mutation resulted in expression of a more highly glycosylated form of PEB3 in vivo, and its crystal structure showed the conformation of the first two residues of the glycan. On the basis of our findings, we suggest that PEB3 is a transport protein that may function in utilization of 3-PG or other phosphate-containing molecules from the host.

  2. Blastomyces Virulence Adhesin-1 Protein Binding to Glycosaminoglycans Is Enhanced by Protein Disulfide Isomerase

    PubMed Central

    Beaussart, Audrey; Brandhorst, Tristan

    2015-01-01

    ABSTRACT Blastomyces adhesin-1 (BAD-1) protein mediates the virulence of the yeast Blastomyces dermatitidis, in part by binding host lung tissue, the extracellular matrix, and cellular receptors via glycosaminoglycans (GAGs), such as heparan sulfate. The tandem repeats that make up over 90% of BAD-1 appear in their native state to be tightly folded into an inactive conformation, but recent work has shown that they become activated and adhesive upon reduction of a disulfide linkage. Here, atomic force microscopy (AFM) of a single BAD-1 molecule interacting with immobilized heparin revealed that binding is enhanced upon treatment with protein disulfide isomerase and dithiothreitol (PDI/DTT). PDI/DTT treatment of BAD-1 induced a plateau effect in atomic force signatures that was consistent with sequential rupture of tandem binding domains. Inhibition of PDI in murine macrophages blunted BAD-1 binding to heparin in vitro. Based on AFM, we found that a short Cardin-Weintraub sequence paired with a WxxWxxW sequence in the first, degenerate repeat at the N terminus of BAD-1 was sufficient to initiate heparin binding. Removal of half of the 41 BAD-1 tandem repeats led to weaker adhesion, illustrating their role in enhanced binding. Mass spectroscopy of the tandem repeat revealed that the PDI-induced interaction with heparin is characterized by ruptured disulfide bonds and that cysteine thiols remain reduced. Further binding studies showed direct involvement of thiols in heparin ligation. Thus, we propose that the N-terminal domain of BAD-1 governs the initial association with host GAGs and that proximity to GAG-associated host PDI catalyzes activation of additional binding motifs conserved within the tandem repeats, leading to enhanced avidity and availability of reduced thiols. PMID:26396244

  3. Atomic force microscopy measurements reveal multiple bonds between Helicobacter pylori blood group antigen binding adhesin and Lewis b ligand.

    PubMed

    Parreira, P; Shi, Q; Magalhaes, A; Reis, C A; Bugaytsova, J; Borén, T; Leckband, D; Martins, M C L

    2014-12-01

    The strength of binding between the Helicobacter pylori blood group antigen-binding adhesin (BabA) and its cognate glycan receptor, the Lewis b blood group antigen (Le(b)), was measured by means of atomic force microscopy. High-resolution measurements of rupture forces between single receptor-ligand pairs were performed between the purified BabA and immobilized Le(b) structures on self-assembled monolayers. Dynamic force spectroscopy revealed two similar but statistically different bond populations. These findings suggest that the BabA may form different adhesive attachments to the gastric mucosa in ways that enhance the efficiency and stability of bacterial adhesion.

  4. That's my story, and I'm sticking to it—an update on B. burgdorferi adhesins

    PubMed Central

    Brissette, Catherine A.; Gaultney, Robert A.

    2014-01-01

    Adhesion is the initial event in the establishment of any infection. Borrelia burgdorferi, the etiological agent of Lyme disease, possesses myriad proteins termed adhesins that facilitate contact with its vertebrate hosts. B. burgdorferi adheres to host tissues through interactions with host cells and extracellular matrix, as well as other molecules present in serum and extracellular fluids. These interactions, both general and specific, are critical in the establishment of infection. Modulation of borrelial adhesion to host tissues affects the microorganisms's ability to colonize, disseminate, and persist. In this review, we update the current knowledge on structure, function, and role in pathogenesis of these “sticky” B. burgdorferi infection-associated proteins. PMID:24772392

  5. Shiga toxin-producing Escherichia coli

    PubMed Central

    Etcheverría, Analía Inés; Padola, Nora Lía

    2013-01-01

    Shiga toxin-producing Escherichia coli (STEC) cause hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in humans. Outbreaks are linked to bovine food sources. STEC O157:H7 has been responsible for the most severe outbreaks worldwide. However, non-O157 serotypes have emerged as important enteric pathogens in several countries. The main virulence factor of STEC is the production of Shiga toxins 1 and 2. Additional virulence markers are a plasmid-encoded enterohemolysin (ehxA), an autoagglutinating adhesin (Saa), a catalase-peroxidase (katP), an extracellular serine protease (espP), a zinc metalloprotease (stcE), a subtilase cytotoxin (subAB), among others. Other virulence factors are intimin and adhesins that had a roll in the adherence of STEC to bovine colon. This review focuses on the virulence traits of STEC and especially on those related to the adhesion to bovine colon. The known of the interaction between STEC and the bovine host is crucial to develop strategies to control cattle colonization. PMID:23624795

  6. Potential use of a recombinant replication-defective adenovirus vector carrying the C-terminal portion of the P97 adhesin protein as a vaccine against Mycoplasma hyopneumoniae in swine.

    PubMed

    Okamba, Faust René; Arella, Maximilien; Music, Nedzad; Jia, Jian Jun; Gottschalk, Marcelo; Gagnon, Carl A

    2010-07-01

    Mycoplasma hyopneumoniae causes severe economic losses to the swine industry worldwide and the prevention of its related disease, enzootic porcine pneumonia, remains a challenge. The P97 adhesin protein of M. hyopneumoniae should be a good candidate for the development of a subunit vaccine because antibodies produced against P97 could prevent the adhesion of the pathogen to the respiratory epithelial cells in vitro. In the present study, a P97 recombinant replication-defective adenovirus (rAdP97c) subunit vaccine efficiency was evaluated in pigs. The rAdP97c vaccine was found to induce both strong P97 specific humoral and cellular immune responses. The rAdP97c vaccinated pigs developed a lower amount of macroscopic lung lesions (18.5 + or - 9.6%) compared to the unvaccinated and challenged animals (45.8 + or - 11.5%). rAdP97c vaccine reduced significantly the severity of inflammatory response and the amount of M. hyopneumoniae in the respiratory tract. Furthermore, the average daily weight gain was slightly improved in the rAdP97c vaccinated pigs (0.672 + or - 0.068 kg/day) compared to the unvaccinated and challenged animals (0.568 + or - 0.104 kg/day). A bacterin-based commercial vaccine (Suvaxyn MH-one) was more efficient to induce a protective immune response than rAdP97c even if it did not evoke a P97 specific immune response. These results suggest that immunodominant antigens other than P97 adhesin are also important in the induction of a protective immune response and should be taken into account in the future development of M. hyopneumoniae subunit vaccines. PMID:20472025

  7. Oral immunization of a live attenuated Escherichia coli strain expressing a holotoxin-structured adhesin-toxoid fusion (1FaeG-FedF-LTA₂:5LTB) protected young pigs against enterotoxigenic E. coli (ETEC) infection.

    PubMed

    Ruan, Xiaosai; Zhang, Weiping

    2013-03-01

    ETEC strains expressing K88 (F4) or F18 fimbriae and enterotoxins are the predominant cause of porcine post-weaning diarrhea (PWD). PWD continues causing significant economic losses to swine producers worldwide. Vaccines effectively protecting against PWD are needed. Our recent study revealed that a tripartite adhesin-toxin monomer (FaeG-FedF-LT(A2-B)) elicited protective antibodies. In this study, we constructed a new adhesin-toxoid fusion, expressed it as a 1A:5B holotoxin-structured antigen (1FaeG-FedF-LT(192A2):5LT(B)) in an avirulent Escherichia coli strain, and evaluated its vaccine potential in pig challenge studies. Piglets orally inoculated with this live strain showed no adverse effects but developed systemic and mucosal antibodies that neutralized cholera toxin and inhibited adherence of K88 and F18 fimbriae in vitro. Moreover, the immunized piglets, when were challenged with ETEC strain 3030-2 (K88ac/LT/STb), had significant fewer bacteria colonized at small intestines and did not develop diarrhea; whereas the control piglets developed severe diarrhea and died. These results indicated the 1FaeG-FedF-LT(192A2):5LT(B) fusion antigen induced protective antiadhesin and antitoxin immunity in pigs, and suggested a live attenuated vaccine can be potentially developed against porcine ETEC diarrhea. Additionally, presenting antigens in a holotoxin structure to target host local mucosal immunity can be used in vaccine development against other enteric diseases. PMID:23375979

  8. [Role of Bacterial Adhesin RAPA1 in Formation of Efficient Symbiosis of Rhizobium leguminosarum with Bean Plants].

    PubMed

    Nigmatullina, L R; Lavina, A M; Vershinina, Z R; Baimiev, Al Kh

    2015-01-01

    Bacterial adhesins, the proteins responsible for attachment of plant growth-promoting rhizobacteria to plant roots, are involved in formation of stable associative symbioses. In the present work enhanced expression of the rapA1 adhesin gene in Rhizobium leguminosarum PVu5 was shown to improve the efficiency of nodulation on bean roots inoculated with the modified strain. The rapA1 gene was cloned into the pJN105Turbo plasmid, this construct was used for transformation of R. leguminosarum PVu5, bean plants were inoculated by this transgenic strain, and efficiency of root nodule formation was determined. In the plants treated with rapA1-transgenic rhizobia, the number of root nodules was on average two times higher than in the plants inoculated with the original strain. Aggregation of R. leguminosarum was achieved when the rapA1 gene expression was enhanced either in rhizobia or in the co-cultured modified strain E. coli pJN105TurboRapA1.

  9. In vitro effect of temperature on the conformational structure and collagen binding of SdrF, a Staphylococcus epidermidis adhesin.

    PubMed

    Di Poto, Antonella; Papi, Massimiliano; Trivedi, Sheetal; Maiorana, Alessandro; Gavazzo, Paola; Vassalli, Massimo; Lowy, Franklin D; De Spirito, Marco; Montanaro, Lucio; Imbriani, Marcello; Arciola, Carla Renata; Visai, Livia

    2015-07-01

    Staphylococcus epidermidis is the leading etiologic agent of device-related infections. S. epidermidis is able to bind, by means of the adhesins of its cell wall, the host matrix proteins filming the artificial surfaces. Thence, bacteria cling to biomaterials and infection develops. The effect of temperature on integrity, structure, and biological activity of the collagen-binding adhesin (SdrF) of S. epidermidis has been here investigated. By cloning in E. coli XL1-Blue, a recombinant of the SdrF binding domain B (rSdrFB), carrying an N-terminal polyhistidine, was obtained. Purification was by HiTrap(TM) Chelating HP columns. Assessment of purity, molecular weight, and integrity was by SDS-PAGE. The rSdrFB-collagen binding was investigated by ELISA. A full three-dimensional reconstruction of rSdrFB was achieved by small-angle X-ray scattering (SAXS). At 25 °C, rSdrFB bound to type I collagen in a dose-dependent, saturable manner, with a Kd of 2.48 × 10(-7) M. When temperature increased from 25 to 37 °C, a strong conformational change occurred, together with the abolition of the rSdrFB-collagen binding. The rSdrFB integrity was not affected by temperature variation. SdrFB-collagen binding is switched on/off depending on the temperature. Implications with the infection pathogenesis are enlightened.

  10. Nanowire Arrays as Cell Force Sensors To Investigate Adhesin-Enhanced Holdfast of Single Cell Bacteria and Biofilm Stability.

    PubMed

    Sahoo, Prasana K; Janissen, Richard; Monteiro, Moniellen P; Cavalli, Alessandro; Murillo, Duber M; Merfa, Marcus V; Cesar, Carlos L; Carvalho, Hernandes F; de Souza, Alessandra A; Bakkers, Erik P A M; Cotta, Monica A

    2016-07-13

    Surface attachment of a planktonic bacteria, mediated by adhesins and extracellular polymeric substances (EPS), is a crucial step for biofilm formation. Some pathogens can modulate cell adhesiveness, impacting host colonization and virulence. A framework able to quantify cell-surface interaction forces and their dependence on chemical surface composition may unveil adhesiveness control mechanisms as new targets for intervention and disease control. Here we employed InP nanowire arrays to dissect factors involved in the early stage biofilm formation of the phytopathogen Xylella fastidiosa. Ex vivo experiments demonstrate single-cell adhesion forces up to 45 nN, depending on the cell orientation with respect to the surface. Larger adhesion forces occur at the cell poles; secreted EPS layers and filaments provide additional mechanical support. Significant adhesion force enhancements were observed for single cells anchoring a biofilm and particularly on XadA1 adhesin-coated surfaces, evidencing molecular mechanisms developed by bacterial pathogens to create a stronger holdfast to specific host tissues. PMID:27336224

  11. Induction of specific immune responses in piglets by intramuscular immunization with fimbrial adhesin FaeG expressed in Lactococcus lactis.

    PubMed

    Liu, Shujie; Li, Yongming; Xu, Ziwei

    2013-08-01

    Fimbrial adhesin plays a critical role in the pathogenesis of enterotoxigenic Escherichia coli (ETEC)-induced piglet diarrhoea. Lactococcus lactis is an attractive food-grade host for the production of heterologous antigens. We previously demonstrated that fimbrial adhesin FaeG was expressed in L. lactis and that oral immunization in mice with recombinant L. lactis expressing FaeG induced F4-specific mucosal and systemic immune responses. In the present study, we explored the immune responses of piglets induced by intramuscular vaccination with recombinant L. lactis expressing rFaeG. Intramuscular vaccination resulted in significantly elevated serum IgG level and modest increases in serum IgA and IgM levels. In addition, IgG, IgA, and IgM antibody secreting cells were induced in the spleen, mesenteric lymph nodes, and jejunum. The growth performance of piglets was not influenced by intramuscular vaccination. The results suggest that L. lactis expressing FaeG is a promising candidate vaccine against ETEC. PMID:23540979

  12. [Role of Bacterial Adhesin RAPA1 in Formation of Efficient Symbiosis of Rhizobium leguminosarum with Bean Plants].

    PubMed

    Nigmatullina, L R; Lavina, A M; Vershinina, Z R; Baimiev, Al Kh

    2015-01-01

    Bacterial adhesins, the proteins responsible for attachment of plant growth-promoting rhizobacteria to plant roots, are involved in formation of stable associative symbioses. In the present work enhanced expression of the rapA1 adhesin gene in Rhizobium leguminosarum PVu5 was shown to improve the efficiency of nodulation on bean roots inoculated with the modified strain. The rapA1 gene was cloned into the pJN105Turbo plasmid, this construct was used for transformation of R. leguminosarum PVu5, bean plants were inoculated by this transgenic strain, and efficiency of root nodule formation was determined. In the plants treated with rapA1-transgenic rhizobia, the number of root nodules was on average two times higher than in the plants inoculated with the original strain. Aggregation of R. leguminosarum was achieved when the rapA1 gene expression was enhanced either in rhizobia or in the co-cultured modified strain E. coli pJN105TurboRapA1. PMID:26964360

  13. BslA, the S-layer adhesin of B. anthracis, is a virulence factor for anthrax pathogenesis

    PubMed Central

    Kern, Justin; Schneewind, Olaf

    2010-01-01

    Summary Microbial pathogens use adhesive surface proteins to bind to and interact with host tissues, events that are universal for the pathogenesis of infectious diseases. A surface adhesin of Bacillus anthracis, the causative agent of anthrax, required to mediate these steps has not been discovered. Previous work identified BslA, an S-layer protein, to be necessary and sufficient for adhesion of the anthrax vaccine strain, Bacillus anthracis Sterne, to host cells. Here we asked whether encapsulated bacilli require BslA for anthrax pathogenesis in guinea pigs. Compared with the highly virulent parent strain B. anthracis Ames, bslA mutants displayed a dramatic increase in the lethal dose and in mean time-to-death. Whereas all tissues of animals infected with B. anthracis Ames contained high numbers of bacilli, only few vegetative forms could be recovered from internal organs of animals infected with the bslA mutant. Surface display of BslA occurred at the poles of encapsulated bacilli and enabled the binding of vegetative forms to host cells. Together these results suggest that BslA functions as the surface adhesin of the anthrax pathogen B. anthracis strain Ames. PMID:19906175

  14. The RNA Chaperone Hfq Is Essential for Virulence and Modulates the Expression of Four Adhesins in Yersinia enterocolitica

    PubMed Central

    Kakoschke, Tamara Katharina; Kakoschke, Sara Carina; Zeuzem, Catharina; Bouabe, Hicham; Adler, Kristin; Heesemann, Jürgen; Rossier, Ombeline

    2016-01-01

    In Enterobacteriaceae, the RNA chaperone Hfq mediates the interaction of small RNAs with target mRNAs, thereby modulating transcript stability and translation. This post-transcriptional control helps bacteria adapt quickly to changing environmental conditions. Our previous mutational analysis showed that Hfq is involved in metabolism and stress survival in the enteropathogen Yersinia enterocolitica. In this study we demonstrate that Hfq is essential for virulence in mice and influences production of surface pathogenicity factors, in particular lipopolysaccharide and adhesins mediating interaction with host tissue. Hfq inhibited the production of Ail, the Ail-like protein OmpX and the MyfA pilin post-transcriptionally. In contrast Hfq promoted production of two major autotransporter adhesins YadA and InvA. While protein secretion in vitro was not affected, hfq mutants exhibited decreased protein translocation by the type III secretion system into host cells, consistent with decreased production of YadA and InvA. The influence of Hfq on YadA resulted from a complex interplay of transcriptional, post-transcriptional and likely post-translational effects. Hfq regulated invA by modulating the expression of the transcriptional regulators rovA, phoP and ompR. Therefore, Hfq is a global coordinator of surface virulence determinants in Y. enterocolitica suggesting that it constitutes an attractive target for developing new antimicrobial strategies. PMID:27387855

  15. The RNA Chaperone Hfq Is Essential for Virulence and Modulates the Expression of Four Adhesins in Yersinia enterocolitica.

    PubMed

    Kakoschke, Tamara Katharina; Kakoschke, Sara Carina; Zeuzem, Catharina; Bouabe, Hicham; Adler, Kristin; Heesemann, Jürgen; Rossier, Ombeline

    2016-07-08

    In Enterobacteriaceae, the RNA chaperone Hfq mediates the interaction of small RNAs with target mRNAs, thereby modulating transcript stability and translation. This post-transcriptional control helps bacteria adapt quickly to changing environmental conditions. Our previous mutational analysis showed that Hfq is involved in metabolism and stress survival in the enteropathogen Yersinia enterocolitica. In this study we demonstrate that Hfq is essential for virulence in mice and influences production of surface pathogenicity factors, in particular lipopolysaccharide and adhesins mediating interaction with host tissue. Hfq inhibited the production of Ail, the Ail-like protein OmpX and the MyfA pilin post-transcriptionally. In contrast Hfq promoted production of two major autotransporter adhesins YadA and InvA. While protein secretion in vitro was not affected, hfq mutants exhibited decreased protein translocation by the type III secretion system into host cells, consistent with decreased production of YadA and InvA. The influence of Hfq on YadA resulted from a complex interplay of transcriptional, post-transcriptional and likely post-translational effects. Hfq regulated invA by modulating the expression of the transcriptional regulators rovA, phoP and ompR. Therefore, Hfq is a global coordinator of surface virulence determinants in Y. enterocolitica suggesting that it constitutes an attractive target for developing new antimicrobial strategies.

  16. Novel adhesin from Pasteurella multocida that binds to the integrin-binding fibronectin FnIII9-10 repeats.

    PubMed

    Mullen, Lisa M; Nair, Sean P; Ward, John M; Rycroft, Andrew N; Williams, Rachel J; Robertson, Giles; Mordan, Nicky J; Henderson, Brian

    2008-03-01

    Phage display screening with fragmented genomic DNA from the animal pathogen Pasteurella multocida has identified a gene encoding a putative fibronectin binding protein (19). Homologues of this gene (PM1665) are found in all other sequenced members of the Pasteurellaceae. Gene PM1665 has been cloned, and the protein has been expressed. Recombinant PM1665 protein binds to both soluble and immobilized fibronectin and is unique in that it interacts with the integrin-binding fibronectin type III (FnIII) repeats FnIII(9-10) and not, as is the case for almost all other fibronectin adhesins, to the N-terminal type I repeats. Surface plasmon resonance analysis revealed a complex binding mechanism with a K(D) (equilibrium dissociation constant) of 150 nM +/- 70 nM. Bioinformatics analysis suggests that the PM1665 protein contains two helix-hairpin-helix (HhH) motifs, and truncation mutation studies have identified the binding site in the protein as a combination of these two HhH motifs in conjunction with a conserved amino acid motif, VNINTA. We have shown that the PM1665 protein is on the cell surface and that binding of P. multocida to fibronectin is almost completely inhibited by anti-PM1665 antiserum. These results support the hypothesis that the PM1665 protein is a member of a new family of fibronectin binding adhesins that are important in the adhesion of P. multocida to fibronectin. PMID:18160478

  17. Exploiting chimeric human antibodies to characterize a protective epitope of Neisseria adhesin A, one of the Bexsero vaccine components.

    PubMed

    Bertoldi, Isabella; Faleri, Agnese; Galli, Barbara; Lo Surdo, Paola; Liguori, Alessia; Norais, Nathalie; Santini, Laura; Masignani, Vega; Pizza, Mariagrazia; Giuliani, Marzia Monica

    2016-01-01

    Neisseria adhesin A (NadA) is one of the antigens of Bexsero, the recently licensed multicomponent vaccine against serogroup B Neisseria meningitidis (MenB). NadA belongs to the class of oligomeric coiled-coil adhesins and is able to mediate adhesion and invasion of human epithelial cells. As a vaccine antigen, NadA has been shown to induce high levels of bactericidal antibodies; however, the domains important for protective response are still unknown. In order to further investigate its immunogenic properties, we have characterized the murine IgG1 mAb (6E3) that was able to recognize the 2 main antigenic variants of NadA on the surface of MenB strains. The epitope targeted by mAb 6E3 was mapped by hydrogen-deuterium exchange mass spectrometry and shown to be located on the coiled-coil stalk region of NadA (aa 206-249). Although no serum bactericidal activity was observed for murine IgG1 mAb 6E3, functional activity was restored when using chimeric antibodies in which the variable regions of the murine mAb 6E3 were fused to human IgG3 constant regions, thus confirming the protective nature of the mAb 6E3 epitope. The use of chimeric antibody molecules will enable future investigations of complement-mediated antibody functionality independently of the Fc-mediated differences in complement activation.

  18. O-Glycosylation of the N-terminal Region of the Serine-rich Adhesin Srr1 of Streptococcus agalactiae Explored by Mass Spectrometry *

    PubMed Central

    Chaze, Thibault; Guillot, Alain; Valot, Benoît; Langella, Olivier; Chamot-Rooke, Julia; Di Guilmi, Anne-Marie; Trieu-Cuot, Patrick; Dramsi, Shaynoor; Mistou, Michel-Yves

    2014-01-01

    Serine-rich (Srr) proteins exposed at the surface of Gram-positive bacteria are a family of adhesins that contribute to the virulence of pathogenic staphylococci and streptococci. Lectin-binding experiments have previously shown that Srr proteins are heavily glycosylated. We report here the first mass-spectrometry analysis of the glycosylation of Streptococcus agalactiae Srr1. After Srr1 enrichment and trypsin digestion, potential glycopeptides were identified in collision induced dissociation spectra using X! Tandem. The approach was then refined using higher energy collisional dissociation fragmentation which led to the simultaneous loss of sugar residues, production of diagnostic oxonium ions and backbone fragmentation for glycopeptides. This feature was exploited in a new open source software tool (SpectrumFinder) developed for this work. By combining these approaches, 27 glycopeptides corresponding to six different segments of the N-terminal region of Srr1 [93–639] were identified. Our data unambiguously indicate that the same protein residue can be modified with different glycan combinations including N-acetylhexosamine, hexose, and a novel modification that was identified as O-acetylated-N-acetylhexosamine. Lectin binding and monosaccharide composition analysis strongly suggested that HexNAc and Hex correspond to N-acetylglucosamine and glucose, respectively. The same protein segment can be modified with a variety of glycans generating a wide structural diversity of Srr1. Electron transfer dissociation was used to assign glycosylation sites leading to the unambiguous identification of six serines and one threonine residues. Analysis of purified Srr1 produced in mutant strains lacking accessory glycosyltransferase encoding genes demonstrates that O-GlcNAcylation is an initial step in Srr1 glycosylation that is likely required for subsequent decoration with Hex. In summary, our data obtained by a combination of fragmentation mass spectrometry techniques

  19. Detection of pap, sfa, afa, foc, and fim Adhesin-Encoding Operons in Uropathogenic Escherichia coli Isolates Collected From Patients With Urinary Tract Infection

    PubMed Central

    Rahdar, Masoud; Rashki, Ahmad; Miri, Hamid Reza; Rashki Ghalehnoo, Mehdi

    2015-01-01

    Background: Uropathogenic Escherichia coli (UPEC) with its virulence factors is the most prevalent cause of urinary tract infection (UTI). Objectives; This study aimed to determine the occurrence of fim, pap, sfa, and afa genes among 100 UPEC isolates collected from patients diagnosed with UTI. Materials and Methods A total of 100 UPEC isolates were obtained from urine samples of patients with UTI. The prevalence of 5 virulence genes encoding type 1 fimbriae (fimH), pili associated with pyelonephritis (pap), S and F1C fimbriae (sfa and foc) and afimbrial adhesins (afa) were determined through PCR method. We also investigated the phylogenetic background of all isolates. In addition, the distribution of adhesin-encoding operons between the phylogroups was assessed. Results: The prevalence of genes encoding for fimbrial adhesive systems was 95% for fim, 57% for pap, 16% for foc, and 81% for sfa. The operons encoding for afa afimbrial adhesins were identified in 12% of isolates. The various combinations of detected genes were designated as virulence patterns. The fim gene, which occurred in strains from all phylogenetic groups (A, B1, B2, and D) was evaluated and no significant differences were found among these groups. Conversely, significant differences were observed in relation to pap, afa, foc, and sfa operons. Conclusions: These results indicate that the PCR method is a powerful genotypic assay for the detection of adhesin-encoding operons. Thus, this assay can be recommended for clinical use to detect virulent urinary E. coli strains, as well as epidemiological studies. PMID:26464770

  20. Mannitol promotes adherence of an outbreak strain of Burkholderia multivorans via an exopolysaccharide-independent mechanism that is associated with upregulation of newly identified fimbrial and afimbrial adhesins.

    PubMed

    Denman, Carmen C; Brown, Alan R

    2013-04-01

    Burkholderia multivorans, a member of the Burkholderia cepacia complex (Bcc), is an important pathogen of the cystic fibrosis (CF) lung. Mannitol, approved as an inhaled osmolyte therapy for use in CF patients, promotes exopolysaccharide (EPS) production by the Bcc. In the present study, we investigated the role of mannitol-induced EPS in the adherence of B. multivorans. We report that mannitol promoted adherence of two representative B. multivorans strains. However, whilst this enhanced adherence was largely EPS-dependent in an environmental isolate, it was EPS-independent within a CF outbreak strain, suggesting strain-to-strain variation in adhesins. Genome sequencing of the outbreak strain enabled the identification of two distinct loci encoding putative fimbrial and afimbrial adhesins. The putative fimbriae-encoding locus was found to be widely distributed amongst clinical and environmental B. multivorans. In contrast, the locus encoding the putative afimbrial adhesin (of the filamentous haemagglutinin family, FHA) was restricted to clinical isolates. Both loci contributed to biofilm formation and mucin adherence. Furthermore, we report that mannitol promoted expression of both loci, and that the locus encoding the putative FHA-family adhesin is a key determinant of the enhanced adherence observed following growth in mannitol. Our studies provide the first characterization, to our knowledge, of B. multivorans adhesins, and in so doing highlight the strain-dependent role of EPS in the Bcc and the difficulties in assigning phenotypic traits to Bcc EPS due to the wider response to mannitol. Our observations also highlight the need to monitor the microbiological effects of inhaled mannitol therapy in Bcc-infected CF patients.

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

    PubMed Central

    Deng, Lingquan; Bensing, Barbara A.; Thamadilok, Supaporn; Yu, Hai; Lau, Kam; Chen, Xi; Ruhl, Stefan; Sullam, Paul M.; Varki, Ajit

    2014-01-01

    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

  2. Oral vaccination of weaned rabbits against enteropathogenic Escherichia coli-like E. coli O103 infection: use of heterologous strains harboring lipopolysaccharide or adhesin of pathogenic strains.

    PubMed Central

    Milon, A; Esslinger, J; Camguilhem, R

    1992-01-01

    To test the importance of lipopolysaccharide (LPS) and adhesin as major antigens in vaccination against rabbit enteropathogenic Escherichia coli (EPEC)-like E. coli O103 infection, we used two nonpathogenic wild-type strains to immunize rabbits at weaning. One of these strains (C127) harbors the O103 LPS but does not express the 32,000-molecular-weight adhesin that characterizes the highly pathogenic O103 strains. The other (C6) belongs to the O128 serogroup, which does not cross-react with the O103 serogroup, but expresses the adhesin. These strains were administered orally, either live or after Formalin inactivation. After vaccination, the animals were challenged with highly pathogenic O103 strain B10. Compared with rabbits vaccinated with the Formalin-killed homologous strain, rabbits vaccinated with killed C127 or C6 showed partial but significant protection. When given live, these strains colonized more or less heavily the digestive tract of the animals and provided nearly complete (C127) or complete (C6) protection against challenge. They induced a quick local immune response, as judged by fecal immunoglobulin A anti-LPS kinetics. Furthermore, strain C6 induced an ecological effect of "resistance to colonization" against challenge strain B10. This effect may have been due to the adhesin that is shared by both strains and to the production of a colicin. Strain C6 could inhibit in vitro the growth of highly pathogenic O103 strains. On the whole, our results show that adhesins and LPS are important, although probably not exclusive, protection-inducing components in rabbit EPEC-like colibacillosis and provide insight into possible protection of rabbits against EPEC-like E. coli infection with live strains. Images PMID:1351880

  3. Molecular mechanisms that mediate colonization of Shiga toxin-producing Escherichia coli strains.

    PubMed

    Farfan, Mauricio J; Torres, Alfredo G

    2012-03-01

    Shiga toxin-producing Escherichia coli (STEC) is a group of pathogens which cause gastrointestinal disease in humans and have been associated with numerous food-borne outbreaks worldwide. The intimin adhesin has been considered for many years to be the only colonization factor in these strains. However, the rapid progress in whole-genome sequencing of different STEC serotypes has accelerated the discovery of other adhesins (fimbrial and afimbrial), which have emerged as important contributors to the intestinal colonization occurring during STEC infection. This review summarizes recent progress to identify and characterize, at the molecular level, novel adhesion and colonization factors in STEC strains, with an emphasis on their contribution to virulence traits, their host-pathogen interactions, the regulatory mechanisms controlling their expression, and their role as targets eliciting immune responses in the host.

  4. Molecular mechanisms that mediate colonization of Shiga toxin-producing Escherichia coli strains.

    PubMed

    Farfan, Mauricio J; Torres, Alfredo G

    2012-03-01

    Shiga toxin-producing Escherichia coli (STEC) is a group of pathogens which cause gastrointestinal disease in humans and have been associated with numerous food-borne outbreaks worldwide. The intimin adhesin has been considered for many years to be the only colonization factor in these strains. However, the rapid progress in whole-genome sequencing of different STEC serotypes has accelerated the discovery of other adhesins (fimbrial and afimbrial), which have emerged as important contributors to the intestinal colonization occurring during STEC infection. This review summarizes recent progress to identify and characterize, at the molecular level, novel adhesion and colonization factors in STEC strains, with an emphasis on their contribution to virulence traits, their host-pathogen interactions, the regulatory mechanisms controlling their expression, and their role as targets eliciting immune responses in the host. PMID:22144484

  5. Molecular Mechanisms That Mediate Colonization of Shiga Toxin-Producing Escherichia coli Strains

    PubMed Central

    Farfan, Mauricio J.

    2012-01-01

    Shiga toxin-producing Escherichia coli (STEC) is a group of pathogens which cause gastrointestinal disease in humans and have been associated with numerous food-borne outbreaks worldwide. The intimin adhesin has been considered for many years to be the only colonization factor in these strains. However, the rapid progress in whole-genome sequencing of different STEC serotypes has accelerated the discovery of other adhesins (fimbrial and afimbrial), which have emerged as important contributors to the intestinal colonization occurring during STEC infection. This review summarizes recent progress to identify and characterize, at the molecular level, novel adhesion and colonization factors in STEC strains, with an emphasis on their contribution to virulence traits, their host-pathogen interactions, the regulatory mechanisms controlling their expression, and their role as targets eliciting immune responses in the host. PMID:22144484

  6. Redefinition of the Carbohydrate Binding Specificity of Helicobacter pylori BabA Adhesin*

    PubMed Central

    Benktander, John; Ångström, Jonas; Breimer, Michael E.; Teneberg, Susann

    2012-01-01

    Certain Helicobacter pylori strains adhere to the human gastric epithelium using the blood group antigen-binding adhesin (BabA). All BabA-expressing H. pylori strains bind to the blood group O determinants on type 1 core chains, i.e. to the Lewis b antigen (Fucα2Galβ3(Fucα4)GlcNAc; Leb) and the H type 1 determinant (Fucα2Galβ3GlcNAc). Recently, BabA strains have been categorized into those recognizing only Leb and H type 1 determinants (designated specialist strains) and those that also bind to A and B type 1 determinants (designated generalist strains). Here, the structural requirements for carbohydrate recognition by generalist and specialist BabA were further explored by binding of these types of strains to a panel of different glycosphingolipids. Three glycosphingolipids recognized by both specialist and generalist BabA were isolated from the small intestine of a blood group O pig and characterized by mass spectrometry and proton NMR as H type 1 pentaglycosylceramide (Fucα2Galβ3GlcNAcβ3Galβ4Glcβ1Cer), Globo H hexaglycosylceramide (Fucα2Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), and a mixture of three complex glycosphingolipids (Fucα2Galβ4GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer, Fucα2Galβ3GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer, and Fucα2Galβ4(Fucα3)GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer). In addition to the binding of both strains to the Globo H hexaglycosylceramide, i.e. a blood group O determinant on a type 4 core chain, the generalist strain bound to the Globo A heptaglycosylceramide (GalNAcα3(Fucα2)Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), i.e. a blood group A determinant on a type 4 core chain. The binding of BabA to the two sets of isoreceptors is due to conformational similarities of the terminal disaccharides of H type 1 and Globo H and of the terminal trisaccharides of A type 1 and Globo A. PMID:22822069

  7. Structural and Functional Analysis of Cell Wall-anchored Polypeptide Adhesin BspA in Streptococcus agalactiae.

    PubMed

    Rego, Sara; Heal, Timothy J; Pidwill, Grace R; Till, Marisa; Robson, Alice; Lamont, Richard J; Sessions, Richard B; Jenkinson, Howard F; Race, Paul R; Nobbs, Angela H

    2016-07-29

    Streptococcus agalactiae (group B Streptococcus, GBS) is the predominant cause of early-onset infectious disease in neonates and is responsible for life-threatening infections in elderly and immunocompromised individuals. Clinical manifestations of GBS infection include sepsis, pneumonia, and meningitis. Here, we describe BspA, a deviant antigen I/II family polypeptide that confers adhesive properties linked to pathogenesis in GBS. Heterologous expression of BspA on the surface of the non-adherent bacterium Lactococcus lactis confers adherence to scavenger receptor gp340, human vaginal epithelium, and to the fungus Candida albicans Complementary crystallographic and biophysical characterization of BspA reveal a novel β-sandwich adhesion domain and unique asparagine-dependent super-helical stalk. Collectively, these findings establish a new bacterial adhesin structure that has in effect been hijacked by a pathogenic Streptococcus species to provide competitive advantage in human mucosal infections. PMID:27311712

  8. The Screw-Like Movement of a Gliding Bacterium Is Powered by Spiral Motion of Cell-Surface Adhesins.

    PubMed

    Shrivastava, Abhishek; Roland, Thibault; Berg, Howard C

    2016-09-01

    Flavobacterium johnsoniae, a rod-shaped bacterium, glides over surfaces at speeds of ∼2 μm/s. The propulsion of a cell-surface adhesin, SprB, is known to enable gliding. We used cephalexin to generate elongated cells with irregular shapes and followed their displacement in three dimensions. These cells rolled about their long axes as they moved forward, following a right-handed trajectory. We coated gold nanoparticles with an SprB antibody and tracked them in three dimensions in an evanescent field where the nanoparticles appeared brighter when they were closer to the glass. The nanoparticles followed a right-handed spiral trajectory on the surface of the cell. Thus, if SprB were to adhere to the glass rather than to a nanoparticle, the cell would move forward along a right-handed trajectory, as observed, but in a direction opposite to that of the nanoparticle. PMID:27602728

  9. Differential expression of insect and plant specific adhesin genes, Mad1 and Mad2, in Metarhizium robertsii.

    PubMed

    Barelli, Larissa; Padilla-Guerrero, Israel Enrique; Bidochka, Michael J

    2011-11-01

    Metarhizium robertsii is an entomopathogenic fungus that is also plant rhizosphere competent. Two adhesin-encoding genes, Metarhizium adhesin-like protein 1 (Mad1) and Mad2, are involved in insect pathogenesis or plant root colonization, respectively. Here we examined the differential expression of the Mad genes when grown on a variety of soluble (carbohydrates and plant root exudate) and insoluble substrates (locust, tobacco hornworm, and cockroach cuticle, chitin, tomato stems, cellulose, and starch) and during insect, Plutella xylostella, infection. On insect cuticles Mad1 was up regulated, whereas bean root exudate and tomato stems resulted in the up regulation of Mad2. During the early stages of insect infection Mad1 was expressed while Mad2 was not expressed until fungal hyphae emerged and conidiated on the insect cadaver. The regulation of Mad2 was compared to that of other stress-related genes (heat shock protein (Hsp)30, Hsp70, and starvation stress gene A (ssgA)). Mad2 was generally up regulated by nutrient starvation (similar to ssgA) but not by pH, temperature, oxidative or osmotic stresses. Whereas Hsp30 and Hsp70 were generally up regulated at 37 °C or by oxidative stress even under nutrient enriched conditions. We fused the promoter of the Mad2 gene to a marker gene (green fluorescent protein (GFP)) and confirmed that Mad2 was up regulated when M. robertsii was grown in the presence of nutrient starvation. Examination of the promoter region of Mad2 revealed that it possessed two copies of a stress-response element (STRE) known to be regulated under the general stress-response pathway. PMID:22036295

  10. Maternal Vaccination with a Fimbrial Tip Adhesin and Passive Protection of Neonatal Mice against Lethal Human Enterotoxigenic Escherichia coli Challenge

    PubMed Central

    Luiz, Wilson B.; Rodrigues, Juliana F.; Crabb, Joseph H.

    2015-01-01

    Globally, enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood and travelers' diarrhea, for which an effective vaccine is needed. Prevalent intestinal colonization factors (CFs) such as CFA/I fimbriae and heat-labile enterotoxin (LT) are important virulence factors and protective antigens. We tested the hypothesis that donor strand-complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, is a protective antigen, using a lethal neonatal mouse ETEC challenge model and passive dam vaccination. For CFA/I-ETEC strain H10407, which has been extensively studied in volunteers, an inoculum of 2 × 107 bacteria resulted in 50% lethal doses (LD50) in neonatal DBA/2 mice. Vaccination of female DBA/2 mice with CFA/I fimbriae or dscCfaE, each given with a genetically attenuated LT adjuvant (LTK63) by intranasal or orogastric delivery, induced high antigen-specific serum IgG and fecal IgA titers and detectable milk IgA responses. Neonates born to and suckled by dams antenatally vaccinated with each of these four regimens showed 78 to 93% survival after a 20× LD50 challenge with H10407, compared to 100% mortality in pups from dams vaccinated with sham vaccine or LTK63 only. Crossover experiments showed that high pup survival rates after ETEC challenge were associated with suckling but not birthing from vaccinated dams, suggesting that vaccine-specific milk antibodies are protective. In corroboration, preincubation of the ETEC inoculum with antiadhesin and antifimbrial bovine colostral antibodies conferred a dose-dependent increase in pup survival after challenge. These findings indicate that the dscCfaE fimbrial tip adhesin serves as a protective passive vaccine antigen in this small animal model and merits further evaluation. PMID:26371126

  11. Combined inactivation and expression strategy to study gene function under physiological conditions: application to identification of new Escherichia coli adhesins.

    PubMed

    Roux, Agnès; Beloin, Christophe; Ghigo, Jean-Marc

    2005-02-01

    In bacteria, whereas disruption methods have been improved recently, the use of plasmid complementation strategies are still subject to limitations, such as cloning difficulties, nonphysiological levels of gene expression, or a requirement for antibiotics as plasmid selection pressure. Moreover, because of the pleiotropic modifications of cell physiology often induced by plasmid-based complementation, these strategies may introduce biases when biological process such as adhesion or biofilm formation are studied. We developed a plasmid-free approach that combines the lambda-red linear DNA recombination method with site-directed insertion of a repression and expression (RExBAD) cassette which places a functional pBAD promoter upstream of a target gene. We showed that this method permits both inactivation and modulation of most Escherichia coli gene expression, including expression of toxin and essential genes. We used this strategy to study adhesion and bacterial biofilms by placing the RExBAD cassette in front of the tra operon, encoding the DNA transfer and pilus genes on the F conjugative plasmid, and in front of flu, the antigen 43 (Ag43) autotransporter adhesin-encoding gene. In silico analysis revealed the existence of 10 genes with homology to the Ag43 gene that were good candidates for genes that encode putative new adhesins in E. coli. We used the RExBAD strategy to study these genes and demonstrated that induction of expression of four of them is associated with adhesion of E. coli to abiotic surfaces. The potential use of the RExBAD approach to study the function of cryptic or uncharacterized genes in large-scale postgenomic functional analyses is discussed.

  12. Number of positive blood cultures, biofilm formation, and adhesin genes in differentiating true coagulase-negative staphylococci bacteremia from contamination.

    PubMed

    Papadimitriou-Olivgeri, I; Giormezis, N; Papadimitriou-Olivgeris, M; Zotou, A; Kolonitsiou, F; Koutsileou, K; Fligou, F; Marangos, M; Anastassiou, E D; Spiliopoulou, I

    2016-01-01

    The significance of the number of coagulase-negative staphylococci (CNS)-positive blood cultures remains obscure in regards to determining true bacteremia versus contamination. The goal of this study was to determine the predictors of real CNS bloodstream infection among intensive care unit (ICU) patients. ICU patients with at least one CNS-positive blood culture were identified from the microbiology database. Biofilm formation was tested by glass tube and microtiter plate assay. mecA gene, ica operon genes (icaA, icaB, icaD), and adhesin genes (aap, bap, atlE, fbe, fnbA) were detected by polymerase chain reaction (PCR). CNS were recovered from 120 septic episodes, 20 of which were true CNS bacteremias, whereas from the remaining 100 episodes, the isolated CNS were characterized as contaminants. The number of positive blood cultures was significantly associated with true CNS bacteremia. Nineteen true bacteremic Staphylococcus epidermidis strains were compared to 38 contaminants. Biofilm synthesis was documented in 37 isolates associated with the presence of the ica operon (p = 0.048). There were 39, 26, 38, 21, and 10 strains positive for the presence of atlE, bap, fbe, aap, and fnbA genes, respectively. Rifampicin resistance, absence of severe sepsis, number of S. epidermidis-positive blood cultures, and absence of the bap gene were independently associated with true S. epidermidis bacteremia as compared to contaminant strains. The number of positive blood cultures is associated with true CNS bacteremia. The presence of adhesin genes may play a role in differentiating true infection from contamination, whereas absence of the bap gene is associated with true S. epidermidis bacteremia. PMID:26490138

  13. Tight conformational coupling between the domains of the enterotoxigenic Escherichia coli fimbrial adhesin CfaE regulates binding state transition.

    PubMed

    Liu, Yang; Esser, Lothar; Interlandi, Gianluca; Kisiela, Dagmara I; Tchesnokova, Veronika; Thomas, Wendy E; Sokurenko, Evgeni; Xia, Di; Savarino, Stephen J

    2013-04-01

    CfaE, the tip adhesin of enterotoxigenic Escherichia coli colonization factor antigen I fimbriae, initiates binding of this enteropathogen to the small intestine. It comprises stacked β-sandwich adhesin (AD) and pilin (PD) domains, with the putative receptor-binding pocket at one pole and an equatorial interdomain interface. CfaE binding to erythrocytes is enhanced by application of moderate shear stress. A G168D replacement along the AD facing the CfaE interdomain region was previously shown to decrease the dependence on shear by increasing binding at lower shear forces. To elucidate the structural basis for this functional change, we studied the properties of CfaE G168D (with a self-complemented donor strand) and solved its crystal structure at 2.6 Å resolution. Compared with native CfaE, CfaE G168D showed a downward shift in peak erythrocyte binding under shear stress and greater binding under static conditions. The thermal melting transition of CfaE G168D occurred 10 °C below that of CfaE. Compared with CfaE, the atomic structure of CfaE G168D revealed a 36% reduction in the buried surface area at the interdomain interface. Despite the location of this single modification in the AD, CfaE G168D exhibited structural derangements only in the adjoining PD compared with CfaE. In molecular dynamics simulations, the G168D mutation was associated with weakened interdomain interactions under tensile force. Taken together, these findings indicate that the AD and PD of CfaE are conformationally tightly coupled and support the hypothesis that opening of the interface plays a critical modulatory role in the allosteric activation of CfaE. PMID:23393133

  14. Maternal vaccination with a fimbrial tip adhesin and passive protection of neonatal mice against lethal human enterotoxigenic Escherichia coli challenge.

    PubMed

    Luiz, Wilson B; Rodrigues, Juliana F; Crabb, Joseph H; Savarino, Stephen J; Ferreira, Luis C S

    2015-12-01

    Globally, enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood and travelers' diarrhea, for which an effective vaccine is needed. Prevalent intestinal colonization factors (CFs) such as CFA/I fimbriae and heat-labile enterotoxin (LT) are important virulence factors and protective antigens. We tested the hypothesis that donor strand-complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, is a protective antigen, using a lethal neonatal mouse ETEC challenge model and passive dam vaccination. For CFA/I-ETEC strain H10407, which has been extensively studied in volunteers, an inoculum of 2 × 10(7) bacteria resulted in 50% lethal doses (LD50) in neonatal DBA/2 mice. Vaccination of female DBA/2 mice with CFA/I fimbriae or dscCfaE, each given with a genetically attenuated LT adjuvant (LTK63) by intranasal or orogastric delivery, induced high antigen-specific serum IgG and fecal IgA titers and detectable milk IgA responses. Neonates born to and suckled by dams antenatally vaccinated with each of these four regimens showed 78 to 93% survival after a 20× LD50 challenge with H10407, compared to 100% mortality in pups from dams vaccinated with sham vaccine or LTK63 only. Crossover experiments showed that high pup survival rates after ETEC challenge were associated with suckling but not birthing from vaccinated dams, suggesting that vaccine-specific milk antibodies are protective. In corroboration, preincubation of the ETEC inoculum with antiadhesin and antifimbrial bovine colostral antibodies conferred a dose-dependent increase in pup survival after challenge. These findings indicate that the dscCfaE fimbrial tip adhesin serves as a protective passive vaccine antigen in this small animal model and merits further evaluation. PMID:26371126

  15. Role of β1 integrins and bacterial adhesins for Yop injection into leukocytes in Yersinia enterocolitica systemic mouse infection.

    PubMed

    Deuschle, Eva; Keller, Birgit; Siegfried, Alexandra; Manncke, Birgit; Spaeth, Tanja; Köberle, Martin; Drechsler-Hake, Doreen; Reber, Julia; Böttcher, Ralph T; Autenrieth, Stella E; Autenrieth, Ingo B; Bohn, Erwin; Schütz, Monika

    2016-02-01

    Injection of Yersinia outer proteins (Yops) into host cells by a type III secretion system is an important immune evasion mechanism of Yersinia enterocolitica (Ye). In this process Ye invasin (Inv) binds directly while Yersinia adhesin A (YadA) binds indirectly via extracellular matrix (ECM) proteins to β1 integrins on host cells. Although leukocytes turned out to be an important target of Yop injection by Ye, it was unclear which Ye adhesins and which leukocyte receptors are required for Yop injection. To explain this, we investigated the role of YadA, Inv and β1 integrins for Yop injection into leukocytes and their impact on the course of systemic Ye infection in mice. Ex vivo infection experiments revealed that adhesion of Ye via Inv or YadA is sufficient to promote Yop injection into leukocytes as revealed by a β-lactamase reporter assay. Serum factors inhibit YadA- but not Inv-mediated Yop injection into B and T cells, shifting YadA-mediated Yop injection in the direction of neutrophils and other myeloid cells. Systemic Ye mouse infection experiments demonstrated that YadA is essential for Ye virulence and Yop injection into leukocytes, while Inv is dispensable for virulence and plays only a transient and minor role for Yop injection in the early phase of infection. Ye infection of mice with β1 integrin-depleted leukocytes demonstrated that β1 integrins are dispensable for YadA-mediated Yop injection into leukocytes, but contribute to Inv-mediated Yop injection. Despite reduced Yop injection into leukocytes, β1 integrin-deficient mice exhibited an increased susceptibility for Ye infection, suggesting an important role of β1 integrins in immune defense against Ye. This study demonstrates that Yop injection into leukocytes by Ye is largely mediated by YadA exploiting, as yet unknown, leukocyte receptors.

  16. Structural and Functional Analysis of a New Subfamily of Glycosyltransferases Required for Glycosylation of Serine-rich Streptococcal Adhesins

    SciTech Connect

    Zhu, Fan; Erlandsen, Heidi; Ding, Lei; Li, Jingzhi; Huang, Ying; Zhou, Meixian; Liang, Xiaobo; Ma, Jinbiao; Wu, Hui

    2011-09-16

    Serine-rich repeat glycoproteins (SRRPs) are a growing family of bacterial adhesins found in many streptococci and staphylococci; they play important roles in bacterial biofilm formation and pathogenesis. Glycosylation of this family of adhesins is essential for their biogenesis. A glucosyltransferase (Gtf3) catalyzes the second step of glycosylation of a SRRP (Fap1) from an oral streptococcus, Streptococcus parasanguinis. Although Gtf3 homologs are highly conserved in SRRP-containing streptococci, they share minimal homology with functionally known glycosyltransferases. We report here the 2.3 {angstrom} crystal structure of Gtf3. The structural analysis indicates that Gtf3 forms a tetramer and shares significant structural homology with glycosyltransferases from GT4, GT5, and GT20 subfamilies. Combining crystal structural analysis with site-directed mutagenesis and in vitro glycosyltransferase assays, we identified residues that are required for UDP- or UDP-glucose binding and for oligomerization of Gtf3 and determined their contribution to the enzymatic activity of Gtf3. Further in vivo studies revealed that the critical amino acid residues identified by the structural analysis are crucial for Fap1 glycosylation in S. parasanguinis in vivo. Moreover, Gtf3 homologs from other streptococci were able to rescue the gtf3 knock-out mutant of S. parasanguinis in vivo and catalyze the sugar transfer to the modified SRRP substrate in vitro, demonstrating the importance and conservation of the Gtf3 homologs in glycosylation of SRRPs. As the Gtf3 homologs only exist in SRRP-containing streptococci, we conclude that the Gtf3 homologs represent a unique subfamily of glycosyltransferases.

  17. Suppression subtractive hybridization identifies an autotransporter adhesin gene of E. coli IMT5155 specifically associated with avian pathogenic Escherichia coli (APEC)

    PubMed Central

    2010-01-01

    Background Extraintestinal pathogenic E. coli (ExPEC) represent a phylogenetically diverse group of bacteria which are implicated in a large range of infections in humans and animals. Although subgroups of different ExPEC pathotypes, including uropathogenic, newborn meningitis causing, and avian pathogenic E. coli (APEC) share a number of virulence features, there still might be factors specifically contributing to the pathogenesis of a certain subset of strains or a distinct pathotype. Thus, we made use of suppression subtractive hybridization and compared APEC strain IMT5155 (O2:K1:H5; sequence type complex 95) with human uropathogenic E. coli strain CFT073 (O6:K2:H5; sequence type complex 73) to identify factors which may complete the currently existing model of APEC pathogenicity and further elucidate the position of this avian pathoype within the whole ExPEC group. Results Twenty-eight different genomic loci were identified, which are present in IMT5155 but not in CFT073. One of these loci contained a gene encoding a putative autotransporter adhesin. The open reading frame of the gene spans a 3,498 bp region leading to a putative 124-kDa adhesive protein. A specific antibody was raised against this protein and expression of the adhesin was shown under laboratory conditions. Adherence and adherence inhibition assays demonstrated a role for the corresponding protein in adhesion to DF-1 chicken fibroblasts. Sequence analyses revealed that the flanking regions of the chromosomally located gene contained sequences of mobile genetic elements, indicating a probable spread among different strains by horizontal gene transfer. In accordance with this hypothesis, the adhesin was found to be present not only in different phylogenetic groups of extraintestinal pathogenic but also of commensal E. coli strains, yielding a significant association with strains of avian origin. Conclusions We identified a chromosomally located autotransporter gene in a highly virulent APEC

  18. Adherence of Pseudomonas aeruginosa and Candida albicans to glycosphingolipid (Asialo-GM1) receptors is achieved by a conserved receptor-binding domain present on their adhesins.

    PubMed Central

    Yu, L; Lee, K K; Hodges, R S; Paranchych, W; Irvin, R T

    1994-01-01

    Pseudomonas aeruginosa, a gram-negative bacterium, and Candida albicans, a dimorphic yeast, are evolutionarily distant microorganisms which can utilize filamentous structures termed pili and fimbriae, respectively, to mediate adherence to glycosphingolipids (asialoganglioside-GM1) receptors. The mechanism of adherence to glycosphingolipid receptors was investigated in these studies. By using monoclonal antibodies (MAbs) against purified pili of P. aeruginosa PAK (PK99H) and monospecific anti-peptide antibodies against the PAK pilin peptides [anti-PAK(128-144) and anti-PAK(134-140)], we demonstrated that these antibodies agglutinated C. albicans whole cells and cross-reacted with C. albicans fimbriae in immunoblots. A control MAb, PKL1, and anti-PAK(75-84) peptide antibodies failed to agglutinate C. albicans whole cells or cross-react with the fimbrial proteins. Conversely, the anti-C. albicans fimbrial MAb Fm16, but not Fm34, agglutinated P. aeruginosa PAK whole cells and Western blots (immunoblots). The interactions between PK99H and Fm16 and their respective homologous antigens were competitively inhibited by heterologous antigens; this demonstrated that the interactions between the antibodies and the heterologous antigens, i.e., PK99H with C. albicans fimbriae and Fm16 with P. aeruginosa pili, were highly specific and suggested that both adhesins share a common antigenic determinant. The immunological cross-reactivity between Fm16 and P. aeruginosa PAK pilin is localized onto the PAK(134-140) region as shown by a competitive enzyme-linked immunosorbent assay. The PAK(134-140) region of PAK pilin contains the epitope recognized by PK99H and also constitutes part of the receptor-binding domain of the pilus adhesin. Thus, the results from these studies suggest that common cell surface receptors are recognized by the P. aeruginosa and C. albicans adhesins because of a conserved receptor-binding domain on the adhesins. Images PMID:7525482

  19. Pathogenesis of Human Diffusely Adhering Escherichia coli Expressing Afa/Dr Adhesins (Afa/Dr DAEC): Current Insights and Future Challenges

    PubMed Central

    2014-01-01

    SUMMARY The pathogenicity and clinical pertinence of diffusely adhering Escherichia coli expressing the Afa/Dr adhesins (Afa/Dr DAEC) in urinary tract infections (UTIs) and pregnancy complications are well established. In contrast, the implication of intestinal Afa/Dr DAEC in diarrhea is still under debate. These strains are age dependently involved in diarrhea in children, are apparently not involved in diarrhea in adults, and can also be asymptomatic intestinal microbiota strains in children and adult. This comprehensive review analyzes the epidemiology and diagnosis and highlights recent progress which has improved the understanding of Afa/Dr DAEC pathogenesis. Here, I summarize the roles of Afa/Dr DAEC virulence factors, including Afa/Dr adhesins, flagella, Sat toxin, and pks island products, in the development of specific mechanisms of pathogenicity. In intestinal epithelial polarized cells, the Afa/Dr adhesins trigger cell membrane receptor clustering and activation of the linked cell signaling pathways, promote structural and functional cell lesions and injuries in intestinal barrier, induce proinflammatory responses, create angiogenesis, instigate epithelial-mesenchymal transition-like events, and lead to pks-dependent DNA damage. UTI-associated Afa/Dr DAEC strains, following adhesin-membrane receptor cell interactions and activation of associated lipid raft-dependent cell signaling pathways, internalize in a microtubule-dependent manner within urinary tract epithelial cells, develop a particular intracellular lifestyle, and trigger a toxin-dependent cell detachment. In response to Afa/Dr DAEC infection, the host epithelial cells generate antibacterial defense responses. Finally, I discuss a hypothetical role of intestinal Afa/Dr DAEC strains that can act as “silent pathogens” with the capacity to emerge as “pathobionts” for the development of inflammatory bowel disease and intestinal carcinogenesis. PMID:25278576

  20. Protection of gerbils from amebic liver abscess by immunization with a recombinant protein derived from the 170-kilodalton surface adhesin of Entamoeba histolytica.

    PubMed Central

    Zhang, T; Stanley, S L

    1994-01-01

    The protozoan parasite Entamoeba histolytica causes extensive morbidity and mortality worldwide through intestinal infection and amebic liver abscess. Here we show that vaccination of gerbils, a standard model for amebic liver abscess, with recombinant proteins derived from the 170-kDa galactose-binding adhesin of E. histolytica and the serine-rich E. histolytica protein or a combination of the two recombinant antigens provides excellent protection against subsequent hepatic challenge with virulent E. histolytica trophozoites. PMID:8188384

  1. Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium

    PubMed Central

    Sillanpää, Jouko; Nallapareddy, Sreedhar R.; Prakash, Vittal P.; Qin, Xiang; Hook, Magnus; Weinstock, George M.; Murray, Barbara E.

    2009-01-01

    SUMMARY Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about pathogenic determinants of this organism. We have previously identified a cell wall anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad spectrum binding to extracellular matrix proteins. Here, we analyzed the draft genome of strain TX0016 for potential MSCRAMMs (microbial surface component recognizing adhesive matrix molecules). Genome-based bioinformatics identified 22 predicted cell wall anchored E. faeciumsurface proteins (Fms) of which 15 (including Acm) have typical characteristics of MSCRAMMs including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one (Fms10, redesignated Scm for second collagen adhesin of E. faeciu m) revealed that recombinant Scm65 (A- and B-domains) and Scm36 (A-domain) bound efficiently to collagen type V in a concentration dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism of recombinant Scm36 and of Acm37 indicated that these proteins are rich in β-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; 9 of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated as EbpCfm), detected a “ladder” pattern of high-molecular weight protein bands in a Western blot

  2. Nucleotide sequence of the afimbrial-adhesin-encoding afa-3 gene cluster and its translocation via flanking IS1 insertion sequences.

    PubMed Central

    Garcia, M I; Labigne, A; Le Bouguenec, C

    1994-01-01

    The afa gene clusters encode afimbrial adhesins (AFAs) that are expressed by uropathogenic and diarrhea-associated Escherichia coli strains. The plasmid-borne afa-3 gene cluster is responsible for the biosynthesis of the AFA-III adhesin that belongs to the Dr family of hemagglutinins. Reported in this work is the nucleotide sequence of the 9.2-kb insert of the recombinant plasmid pILL61, which contains the afa-3 gene cluster cloned from a cystitis-associated E. coli strain (A30). The afa-3 gene cluster was shown to contain six open reading frames, designated afaA to afaF. It was organized in two divergent transcriptional units. Five of the six Afa products showed marked homologies with proteins encoded by previously described adhesion systems that allowed us to attribute to each of them a putative function in the biogenesis of the AFA-III adhesin. AfaE was identified as the structural adhesin product, whereas AfaB and AfaC were recognized as periplasmic chaperone and outer membrane anchor proteins, respectively. The AfaA and AfaF products were shown to be homologous to the PapI-PapB transcriptional regulatory proteins. No function could be attributed to the AfaD product, the gene of which was previously shown to be dispensable for the synthesis of a functional adhesin. Upstream of the afa-3 gene cluster, a 1.2-kb region was found to be 96% identical to the RepFIB sequence of one of the enterotoxigenic E. coli plasmids (P307), suggesting a common ancestor plasmid. This region contains an integrase-like gene (int). Sequence analysis revealed the presence of an IS1 element between the int gene and the afa-3 gene cluster. Two other IS1 elements were detected and located in the vicinity of the afa-3 gene cluster by hybridization experiments. The afa-3 gene cluster was therefore found to be flanked by two IS1 elements in direct orientation and two in opposite orientations. The afa-3 gene cluster, flanked by two directly oriented IS1 elements, was shown to translocate

  3. A distinct sortase SrtB anchors and processes a streptococcal adhesin AbpA with a novel structural property.

    PubMed

    Liang, Xiaobo; Liu, Bing; Zhu, Fan; Scannapieco, Frank A; Haase, Elaine M; Matthews, Steve; Wu, Hui

    2016-01-01

    Surface display of proteins by sortases in Gram-positive bacteria is crucial for bacterial fitness and virulence. We found a unique gene locus encoding an amylase-binding adhesin AbpA and a sortase B in oral streptococci. AbpA possesses a new distinct C-terminal cell wall sorting signal. We demonstrated that this C-terminal motif is required for anchoring AbpA to cell wall. In vitro and in vivo studies revealed that SrtB has dual functions, anchoring AbpA to the cell wall and processing AbpA into a ladder profile. Solution structure of AbpA determined by NMR reveals a novel structure comprising a small globular α/β domain and an extended coiled-coil heliacal domain. Structural and biochemical studies identified key residues that are crucial for amylase binding. Taken together, our studies document a unique sortase/adhesion substrate system in streptococci adapted to the oral environment rich in salivary amylase. PMID:27492581

  4. Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of [alpha]- and PPII-helices

    SciTech Connect

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Patel, Manisha H.; Robinette, Rebekah A.; Crowley, Paula J.; Michalek, Suzanne; Brady, L. Jeannine; Deivanayagam, Champion

    2010-08-18

    Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 {angstrom}) crystal structure of the A{sub 3}VP{sub 1} fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 {angstrom}) through the interaction of two noncontiguous regions in the primary sequence. The A{sub 3} repeat of the alanine-rich domain adopts an extended {alpha}-helix that intertwines with the P{sub 1} repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A{sub 3} and P{sub 1} helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length.

  5. A distinct sortase SrtB anchors and processes a streptococcal adhesin AbpA with a novel structural property

    PubMed Central

    Liang, Xiaobo; Liu, Bing; Zhu, Fan; Scannapieco, Frank A.; Haase, Elaine M.; Matthews, Steve; Wu, Hui

    2016-01-01

    Surface display of proteins by sortases in Gram-positive bacteria is crucial for bacterial fitness and virulence. We found a unique gene locus encoding an amylase-binding adhesin AbpA and a sortase B in oral streptococci. AbpA possesses a new distinct C-terminal cell wall sorting signal. We demonstrated that this C-terminal motif is required for anchoring AbpA to cell wall. In vitro and in vivo studies revealed that SrtB has dual functions, anchoring AbpA to the cell wall and processing AbpA into a ladder profile. Solution structure of AbpA determined by NMR reveals a novel structure comprising a small globular α/β domain and an extended coiled-coil heliacal domain. Structural and biochemical studies identified key residues that are crucial for amylase binding. Taken together, our studies document a unique sortase/adhesion substrate system in streptococci adapted to the oral environment rich in salivary amylase. PMID:27492581

  6. Characterization of an Acidic-pH-Inducible Stress Protein (hsp70), a Putative Sulfatide Binding Adhesin, from Helicobacter pylori

    PubMed Central

    Huesca, Mario; Goodwin, Avery; Bhagwansingh, Arianna; Hoffman, Paul; Lingwood, Clifford A.

    1998-01-01

    The in vitro glycolipid binding specificity of the gastric pathogen Helicobacter pylori is altered to include sulfated glycolipids (sulfatides) following brief exposure of the organism to acid pH typical of the stomach. This change is prevented by anti-hsp70 antibodies, suggesting that hsp70 may be a stress-induced surface adhesin, mediating sulfatide recognition. To facilitate investigation of the role of hsp70 in attachment, we have cloned and sequenced the H. pylori hsp70 gene (dnaK). The hsp70 gene was identified by probing a cosmid DNA library made from H. pylori 439 with a PCR amplicon generated with oligonucleotides synthesized to highly conserved regions of dnaK. The 1.9-kb H. pylori hsp70 gene encodes a product of 616 amino acids. Primer extension analysis revealed a single transcription start site, while Northern blot analysis established that hsp70 was preferentially induced by low pH rather than by heat shock. The ability of H. pylori to alter its glycolipid binding specificity following exposure to low pH by upregulating hsp70 and by expressing hsp70 on the bacterial surface may provide a survival advantage during periods of high acid stress. PMID:9712748

  7. A food-grade fimbrial adhesin FaeG expression system in Lactococcus lactis and Lactobacillus casei.

    PubMed

    Lu, W W; Wang, T; Wang, Y; Xin, M; Kong, J

    2016-03-01

    Enterotoxigenic Escherichia coli (ETEC) infection is the major cause of diarrhea in neonatal piglets. The fimbriae as colonizing factor in the pathogenesis of ETEC constitute a primary target for vaccination against ETEC. Lactic acid bacteria (LAB) are attractive tools to deliver antigens at the mucosal level. With the safety of genetically modified LAB in mind, a food-grade secretion vector (pALRc or pALRb) was constructed with DNA entirely from LAB, including the replicon, promoter, signal peptide, and selection marker alanine racemase gene (alr). To evaluate the feasibility of the system, the nuclease gene (nuc) from Staphylococcus aureus was used as a reporter to be expressed in both Lactococcus lactis and Lactobacillus casei. Subsequently, the extracellular secretion of the fimbrial adhesin FaeG of ETEC was confirmed by Western blot analysis. These results showed that this food-grade expression system has potential as the delivery vehicle for the safe use of genetically modified LAB for the development of vaccines against ETEC infection.

  8. Crystal Structure of FadA Adhesin from Fusobacterium nucleatum Reveals a Novel Oligomerization Motif, the Leucine Chain

    SciTech Connect

    Nithianantham, Stanley; Xu, Minghua; Yamada, Mitsunori; Ikegami, Akihiko; Shoham, Menachem; Han, Yiping W.

    2009-04-07

    Many bacterial appendages have filamentous structures, often composed of repeating monomers assembled in a head-to-tail manner. The mechanisms of such linkages vary. We report here a novel protein oligomerization motif identified in the FadA adhesin from the Gram-negative bacterium Fusobacterium nucleatum. The 2.0 {angstrom} crystal structure of the secreted form of FadA (mFadA) reveals two antiparallel {alpha}-helices connected by an intervening 8-residue hairpin loop. Leucine-leucine contacts play a prominent dual intra- and intermolecular role in the structure and function of FadA. First, they comprise the main association between the two helical arms of the monomer; second, they mediate the head-to-tail association of monomers to form the elongated polymers. This leucine-mediated filamentous assembly of FadA molecules constitutes a novel structural motif termed the 'leucine chain.' The essential role of these residues in FadA is corroborated by mutagenesis of selected leucine residues, which leads to the abrogation of oligomerization, filament formation, and binding to host cells.

  9. A food-grade fimbrial adhesin FaeG expression system in Lactococcus lactis and Lactobacillus casei.

    PubMed

    Lu, W W; Wang, T; Wang, Y; Xin, M; Kong, J

    2016-03-01

    Enterotoxigenic Escherichia coli (ETEC) infection is the major cause of diarrhea in neonatal piglets. The fimbriae as colonizing factor in the pathogenesis of ETEC constitute a primary target for vaccination against ETEC. Lactic acid bacteria (LAB) are attractive tools to deliver antigens at the mucosal level. With the safety of genetically modified LAB in mind, a food-grade secretion vector (pALRc or pALRb) was constructed with DNA entirely from LAB, including the replicon, promoter, signal peptide, and selection marker alanine racemase gene (alr). To evaluate the feasibility of the system, the nuclease gene (nuc) from Staphylococcus aureus was used as a reporter to be expressed in both Lactococcus lactis and Lactobacillus casei. Subsequently, the extracellular secretion of the fimbrial adhesin FaeG of ETEC was confirmed by Western blot analysis. These results showed that this food-grade expression system has potential as the delivery vehicle for the safe use of genetically modified LAB for the development of vaccines against ETEC infection. PMID:26825016

  10. Expression of the meningococcal adhesin NadA is controlled by a transcriptional regulator of the MarR family.

    PubMed

    Schielke, Stephanie; Huebner, Claudia; Spatz, Carolin; Nägele, Virginie; Ackermann, Nikolaus; Frosch, Matthias; Kurzai, Oliver; Schubert-Unkmeir, Alexandra

    2009-05-01

    Two closely related pathogenic species have evolved in the genus Neisseria: N. meningitidis and N. gonorrhoeae, which occupy different host niches and cause different clinical entities. In contrast to the pathogen N. gonorrhoeae, N. meningitidis is a commensal and only rarely becomes invasive. Little is known about the genetic background of the entirely different lifestyles in these closely related species. Meningococcal NMB1843 encodes a transcriptional regulator of the MarR family. The gonococcal homologue FarR regulates expression of farAB, mediating fatty acid resistance. We show that NmFarR also directly interacts with NmfarAB. Yet, by contrast to N. gonorrhoeae, no significant sensitivity to fatty acids was observed in a DeltafarR mutant due to intrinsic resistance of meningococci. Further analyses identified an NmFarR-repressed protein absent from N. gonorrhoeae. This protein is the meningococcus-specific adhesin and vaccine component NadA that has most likely been acquired by horizontal gene transfer. NmFarR binds to a 16 base pair palindromic repeat within the nadA promoter. De-repression of nadA resulted in significantly higher association of a DeltafarR strain with epithelial cells. Hence NmFarR has gained control over a meningococcus-specific gene involved in host colonization and thus contributed to divergent niche adaptation in pathogenic Neisseriae.

  11. Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-cadherin/β-catenin signaling via its FadA adhesin

    PubMed Central

    Liu, Wendy; Hao, Yujun; Cai, Guifang; Han, Yiping W.

    2013-01-01

    SUMMARY Fusobacterium nucleatum (Fn) has been associated with colorectal cancer (CRC), but causality and underlying mechanisms remain to be established. We demonstrate that Fn adheres to, invades and induces oncogenic and inflammatory responses to stimulate growth of CRC cells through its unique FadA adhesin. FadA binds to E-cadherin, activates β-catenin signaling, and differentially regulates the inflammatory and oncogenic responses. The FadA-binding site on E-cadherin is mapped to an 11 amino acid region. A synthetic peptide derived from this region of E-cadherin abolishes FadA-induced CRC cell growth, and oncogenic and inflammatory responses. FadA levels in the colon tissue from patients with adenomas and adenocarcinomas is >10–100 times higher compared to normal individuals. The increased FadA expression in CRC correlates with increased expression of oncogenic and inflammatory genes. This study unveils a mechanism by which Fn can drive CRC and identifies FadA as a potential diagnostic and therapeutic target for CRC. PMID:23954158

  12. MHJ_0125 is an M42 glutamyl aminopeptidase that moonlights as a multifunctional adhesin on the surface of Mycoplasma hyopneumoniae

    PubMed Central

    Robinson, Mark W.; Buchtmann, Kyle A.; Jenkins, Cheryl; Tacchi, Jessica L.; Raymond, Benjamin B. A.; To, Joyce; Roy Chowdhury, Piklu; Woolley, Lauren K.; Labbate, Maurizio; Turnbull, Lynne; Whitchurch, Cynthia B.; Padula, Matthew P.; Djordjevic, Steven P.

    2013-01-01

    Bacterial aminopeptidases play important roles in pathogenesis by providing a source of amino acids from exogenous proteins, destroying host immunological effector peptides and executing posttranslational modification of bacterial and host proteins. We show that MHJ_0125 from the swine respiratory pathogen Mycoplasma hyopneumoniae represents a new member of the M42 class of bacterial aminopeptidases. Despite lacking a recognizable signal sequence, MHJ_0125 is detectable on the cell surface by fluorescence microscopy and LC-MS/MS of (i) biotinylated surface proteins captured by avidin chromatography and (ii) peptides released by mild trypsin shaving. Furthermore, surface-associated glutamyl aminopeptidase activity was detected by incubation of live M. hyopneumoniae cells with the diagnostic substrate H-Glu-AMC. MHJ_0125 moonlights as a multifunctional adhesin, binding to both heparin and plasminogen. Native proteomics and comparative modelling studies suggest MHJ_0125 forms a dodecameric, homopolymeric structure and provide insight into the positions of key residues that are predicted to interact with heparin and plasminogen. MHJ_0125 is the first aminopeptidase shown to both bind plasminogen and facilitate its activation by tissue plasminogen activator. Plasmin cleaves host extracellular matrix proteins and activates matrix metalloproteases, generating peptide substrates for MHJ_0125 and a source of amino acids for growth of M. hyopneumoniae. This unique interaction represents a new paradigm in microbial pathogenesis. PMID:23594879

  13. Inhibition of leukocyte-endothelial cell interactions and inflammation by peptides from a bacterial adhesin which mimic coagulation factor X.

    PubMed Central

    Rozdzinski, E; Sandros, J; van der Flier, M; Young, A; Spellerberg, B; Bhattacharyya, C; Straub, J; Musso, G; Putney, S; Starzyk, R

    1995-01-01

    Factor X (factor ten) of the coagulation cascade binds to the integrin CD11b/CD18 during inflammation, initiating procoagulant activity on the surface of leukocytes (Altieri, D.C., O.R. Etingin, D.S. Fair, T.K. Brunk, J.E. Geltosky, D.P. Hajjar, and T. S. Edgington. 1991. Science [Wash.DC]. 254:1200-1202). Filamentous hemagglutinin (FHA), an adhesin of Bordetella pertussis also binds to the CD11b/CD18 integrin (Relman D., E. Tuomanen, S. Falkow, D.T. Golenbock, K. Saukkonen, and S.D. Wright. 1990. Cell. 61:1375-1382). FHA and the CD11b/CD18 binding loops of Factor X share amino acid sequence similarity. FHA peptides similar to Factor X binding loops inhibited 125I-Factor X binding to human neutrophils and prolonged clotting time. In addition, ETKEVDG and its Factor X analogue prevented transendothelial migration of leukocytes in vitro and reduced leukocytosis and blood brain barrier disruption in vivo. Interference with leukocyte migration by a coagulation-based peptide suggests a novel strategy for antiinflammatory therapy. PMID:7883955

  14. Immune responses elicited in mice with recombinant Lactococcus lactis expressing F4 fimbrial adhesin FaeG by oral immunization.

    PubMed

    Liu, Shujie; Li, Yongming; Xu, Ziwei; Wang, Yicheng

    2010-08-01

    Enterotoxigenic Escherichia coli (ETEC) is a major pathogenic agent causing piglet diarrhea. The major subunit and adhesin FaeG of F4(+) ETEC is an important virulence factor with strong immunogenicity. To determine whether Lactococcus lactis (L. lactis) could effectively deliver FaeG to the mucosal immune system, recombinant L. lactis expressing FaeG was constructed, and immune responses in mice following oral route delivery of recombinant L. lactis were explored. The production of FaeG expressed in L. lactis was up to approximately 10% of soluble whole-cell proteins, and recombinant FaeG (rFaeG) possessed good immunoreactivity by Western blot analysis. Oral immunization with recombinant L. lactis expressing FaeG induced F4-specific mucosal and systemic immune responses in the mice. In addition, high dose recombinant L. lactis or co-administration of high dose recombinant L. lactis with CTB enhanced the immune responses. These results suggested that L. lactis expressing FaeG was a promising candidate vaccine against ETEC. PMID:20532816

  15. Phage display revisited: Epitope mapping of a monoclonal antibody directed against Neisseria meningitidis adhesin A using the PROFILER technology.

    PubMed

    Cariccio, Veronica Lanza; Domina, Maria; Benfatto, Salvatore; Venza, Mario; Venza, Isabella; Faleri, Agnese; Bruttini, Marco; Bartolini, Erika; Giuliani, Marzia Monica; Santini, Laura; Brunelli, Brunella; Norais, Nathalie; Borgogni, Erica; Midiri, Angelina; Galbo, Roberta; Romeo, Letizia; Biondo, Carmelo; Masignani, Vega; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2016-01-01

    There is a strong need for rapid and reliable epitope mapping methods that can keep pace with the isolation of increasingly larger numbers of mAbs. We describe here the identification of a conformational epitope using Phage-based Representation OF ImmunoLigand Epitope Repertoire (PROFILER), a recently developed high-throughput method based on deep sequencing of antigen-specific lambda phage-displayed libraries. A novel bactericidal monoclonal antibody (mAb 9F11) raised against Neisseria meningitidis adhesin A (NadA), an important component of the Bexsero(®) anti-meningococcal vaccine, was used to evaluate the technique in comparison with other epitope mapping methods. The PROFILER technology readily identified NadA fragments that were capable of fully recapitulating the reactivity of the entire antigen against mAb 9F11. Further analysis of these fragments using mutagenesis and hydrogen-deuterium exchange mass-spectrometry allowed us to identify the binding site of mAb 9F11 (A250-D274) and an adjoining sequence (V275-H312) that was also required for the full functional reconstitution of the epitope. These data suggest that, by virtue of its ability to detect a great variety of immunoreactive antigen fragments in phage-displayed libraries, the PROFILER technology can rapidly and reliably identify epitope-containing regions and provide, in addition, useful clues for the functional characterization of conformational mAb epitopes. PMID:26963435

  16. Immunogenicity of recombinant F4 (K88) fimbrial adhesin FaeG expressed in tobacco chloroplast.

    PubMed

    Shen, Huifeng; Qian, Bingjun; Chen, Weiwei; Liu, Zhenhua; Yang, Litao; Zhang, Dabing; Liang, Wanqi

    2010-08-01

    To test the possibility of producing the novel vaccine in plants against diarrhea normally found in neonatal and newly weaned piglets, the faeG gene, encoding a major F4ac fimbrial subunit protein, was introduced into the tobacco chloroplast genome. After two rounds of selection under spectinomycin, we obtained the transgenic plants nearly homoplasmic. RNA gel blot analysis indicated that faeG and the antibiotic selective gene aminoglycoside 3' adenylyltransferase (aadA) were highly transcribed as a dicistron, while the translational level of recombinant FaeG in transplastomic tobacco was about 0.15% of total soluble protein. The immunogenicity of recombinant FaeG produced in tobacco chloroplasts was confirmed by the observation that FaeG-specific antibodies were elicited in mice immunized with total soluble protein of transgenic plants, as well as the result that mouse sera stimulated by chloroplast-derived recombinant FaeG could neutralize F4ac enterotoxigenic Escherichia coli (ETEC) in vivo. This study provides a new alternative for producing the ETEC vaccine using the chloroplast expression system.

  17. Immunogenicity of recombinant F4 (K88) fimbrial adhesin FaeG expressed in tobacco chloroplast.

    PubMed

    Shen, Huifeng; Qian, Bingjun; Chen, Weiwei; Liu, Zhenhua; Yang, Litao; Zhang, Dabing; Liang, Wanqi

    2010-08-01

    To test the possibility of producing the novel vaccine in plants against diarrhea normally found in neonatal and newly weaned piglets, the faeG gene, encoding a major F4ac fimbrial subunit protein, was introduced into the tobacco chloroplast genome. After two rounds of selection under spectinomycin, we obtained the transgenic plants nearly homoplasmic. RNA gel blot analysis indicated that faeG and the antibiotic selective gene aminoglycoside 3' adenylyltransferase (aadA) were highly transcribed as a dicistron, while the translational level of recombinant FaeG in transplastomic tobacco was about 0.15% of total soluble protein. The immunogenicity of recombinant FaeG produced in tobacco chloroplasts was confirmed by the observation that FaeG-specific antibodies were elicited in mice immunized with total soluble protein of transgenic plants, as well as the result that mouse sera stimulated by chloroplast-derived recombinant FaeG could neutralize F4ac enterotoxigenic Escherichia coli (ETEC) in vivo. This study provides a new alternative for producing the ETEC vaccine using the chloroplast expression system. PMID:20705597

  18. Discovery of a novel periplasmic protein that forms a complex with a trimeric autotransporter adhesin and peptidoglycan.

    PubMed

    Ishikawa, Masahito; Yoshimoto, Shogo; Hayashi, Ayumi; Kanie, Junichi; Hori, Katsutoshi

    2016-08-01

    Trimeric autotransporter adhesins (TAAs), fibrous proteins on the cell surface of Gram-negative bacteria, have attracted attention as virulence factors. However, little is known about the mechanism of their biogenesis. AtaA, a TAA of Acinetobacter sp. Tol 5, confers nonspecific, high adhesiveness to bacterial cells. We identified a new gene, tpgA, which forms a single operon with ataA and encodes a protein comprising two conserved protein domains identified by Pfam: an N-terminal SmpA/OmlA domain and a C-terminal OmpA_C-like domain with a peptidoglycan (PGN)-binding motif. Cell fractionation and a pull-down assay showed that TpgA forms a complex with AtaA, anchoring it to the outer membrane (OM). Isolation of total PGN-associated proteins showed TpgA binding to PGN. Disruption of tpgA significantly decreased the adhesiveness of Tol 5 because of a decrease in surface-displayed AtaA, suggesting TpgA involvement in AtaA secretion. This is reminiscent of SadB, which functions as a specific chaperone for SadA, a TAA in Salmonella species; however, SadB anchors to the inner membrane, whereas TpgA anchors to the OM through AtaA. The genetic organization encoding the TAA-TpgA-like protein cassette can be found in diverse Gram-negative bacteria, suggesting a common contribution of TpgA homologues to TAA biogenesis. PMID:27074146

  19. A novel lily anther-specific gene encodes adhesin-like proteins associated with exine formation during anther development

    PubMed Central

    Liu, Ming-Che; Yang, Cheng-Shou; Wang, Co-Shine

    2014-01-01

    The anther-specific gene LLA1271 isolated from lily (Lilium longiflorum Thunb.) anthers is novel and exists in two forms. The protein encoded by LLA1271 may represent an adhesin-like protein first found in higher plants. The protein contains a typical N-terminal signal peptide followed by a highly conserved repeat domain. The LLA1271 gene is temporally expressed at the phase of microspore development. RNA blot and RNA in situ hybridization analyses demonstrated that the gene was expressed both in the tapetum and in the microspore. The gene is endo- and exogenously induced by gibberellin. Studies with the gibberellin biosynthesis inhibitor uniconazole and an inhibitor of ethylene activity, 2,5-norbornadien (NBD), revealed that LLA1271 is negatively regulated by ethylene, and a cross-talk of regulation between gibberellin and ethylene occurs in young anthers. The treatment with NBD caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole arrested tapetal development in a state close to that of a tapetum without treatment. The LLA1271 protein is heat stable and heterogeneous. An immunoblot of separated protein fractions of the anther revealed that the LLA1271 protein was detected in protein fraction of the microspore released from the cell wall by treatment with either 0.5% or 2% Triton X-100. Ectopic expression of LLA1271 resulted in impaired stamen and low pollen germination. Scanning electron microscopy of TAP::LLA1271 pollen showed distorted exine formation and patterning. The LLA1271 protein once synthesized in both the tapetum and microspore is secreted and deposited on the surface of microspores, moderately affecting exine formation and patterning. PMID:24591055

  20. Identification of adhesin-like protein ALP41 from Spiroplasma eriocheiris and induction immune response of Eriocheir sinensis.

    PubMed

    Meng, Qingguo; Li, Wenjie; Liang, Tingming; Jiang, Xuejiao; Gu, Wei; Wang, Wen

    2010-10-01

    Spiroplasma eriocheiris is a causative agent of the tremor disease (TD) of Chinese mitten crab Eriocheir sinensis which is a novel pathogen of aquatic animals found in recent years. A gene, adhesin-like protein (ALP41), of S. eriocheiris from E. sinensis was identified and its characteristics were analyzed in present paper. The role of this pathogen's host-binding protein in promoting immune responses was characterized through analyzing the interaction between S. eriocheiris and E. sinensis. The full-length DNA of ALP41 is 1074 bp and encodes 357 amino acid residues. The theoretical molecular weight and isoelectric point for the ALP41 are 40.94 kDa and 4.79, respectively. Since UGA is read as a tryptophan codon and not as a termination signal in most Mollicute species, the ALP41 gene was site-mutated from TGA to TGG and transcribed in Escherichia coli to full expression; the titer of rabbits anti-ALP41 serum was about 1:6000. A specific immunoreactive band was identified when rabbits anti-S. eriocheiris serum was opposed to the recombinant protein. The ALP41 band was detected using anti-ALP41 serum and the total proteins of S. eriocheiris. Realtime-PCR was used for detection of expression levels of the immune genes in E. sinensis. Among the examined genes, the mRNA expression of anti-lipopolysaccharide factor (ALF), prophenoloxidase (proPO), peroxiredoxin 6 (Prx6) and pacifastin light chain (PLC) in E. sinensis were significantly induced after ALP41 treatment. PMID:20538062

  1. Structural mechanisms underlying sequence-dependent variations in GAG affinities of decorin binding protein A, a Borrelia burgdorferi adhesin.

    PubMed

    Morgan, Ashli M; Wang, Xu

    2015-05-01

    Decorin-binding protein A (DBPA) is an important surface adhesin of the bacterium Borrelia burgdorferi, the causative agent of Lyme disease. DBPA facilitates the bacteria's colonization of human tissue by adhering to glycosaminoglycan (GAG), a sulfated polysaccharide. Interestingly, DBPA sequence variation among different strains of Borrelia spirochetes is high, resulting in significant differences in their GAG affinities. However, the structural mechanisms contributing to these differences are unknown. We determined the solution structures of DBPAs from strain N40 of B. burgdorferi and strain PBr of Borrelia garinii, two DBPA variants whose GAG affinities deviate significantly from strain B31, the best characterized version of DBPA. Our structures revealed that significant differences exist between PBr DBPA and B31/N40 DBPAs. In particular, the C-terminus of PBr DBPA, unlike C-termini from B31 and N40 DBPAs, is positioned away from the GAG-binding pocket and the linker between helices one and two of PBr DBPA is highly structured and retracted from the GAG-binding pocket. The repositioning of the C-terminus allowed the formation of an extra GAG-binding epitope in PBr DBPA and the retracted linker gave GAG ligands more access to the GAG-binding epitopes than other DBPAs. Characterization of GAG ligands' interactions with wild-type (WT) PBr and mutants confirmed the importance of the second major GAG-binding epitope and established the fact that the two epitopes are independent of one another and the new epitope is as important to GAG binding as the traditional epitope.

  2. Immunogenicity and Protective Efficacy against Enterotoxigenic Escherichia coli Colonization following Intradermal, Sublingual, or Oral Vaccination with EtpA Adhesin.

    PubMed

    Luo, Qingwei; Vickers, Tim J; Fleckenstein, James M

    2016-07-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a common cause of diarrhea. Extraordinary antigenic diversity has prompted a search for conserved antigens to complement canonical approaches to ETEC vaccine development. EtpA, an immunogenic extracellular ETEC adhesin relatively conserved in the ETEC pathovar, has previously been shown to be a protective antigen following intranasal immunization. These studies were undertaken to explore alternative routes of EtpA vaccination that would permit use of a double mutant (R192G L211A) heat-labile toxin (dmLT) adjuvant. Here, oral vaccination with EtpA adjuvanted with dmLT afforded significant protection against small intestinal colonization, and the degree of protection correlated with fecal IgG, IgA, or total fecal antibody responses to EtpA. Sublingual vaccination yielded compartmentalized mucosal immune responses with significant increases in anti-EtpA fecal IgG and IgA, and mice vaccinated via this route were also protected against colonization. In contrast, while intradermal (i.d.) vaccination achieved high levels of both serum and fecal antibodies against both EtpA and dmLT, mice vaccinated via the i.d. route were not protected against subsequent colonization and the avidity of serum IgG and IgA EtpA-specific antibodies was significantly lower after i.d. immunization compared to other routes. Finally, we demonstrate that antiserum from vaccinated mice significantly impairs binding of LT to cognate GM1 receptors and shows near complete neutralization of toxin delivery by ETEC in vitro Collectively, these data provide further evidence that EtpA could complement future vaccine strategies but also suggest that additional effort will be required to optimize its use as a protective immunogen. PMID:27226279

  3. Characterization and Functional Analysis of AatB, a Novel Autotransporter Adhesin and Virulence Factor of Avian Pathogenic Escherichia coli

    PubMed Central

    ZhuGe, Xiangkai; Wang, Shaohui; Fan, Hongjie; Pan, Zihao; Ren, Jianluan; Yi, Li; Meng, Qingmei; Yang, Xuqiu; Lu, Chengping

    2013-01-01

    Autotransporter (AT) proteins constitute a large family of extracellular proteins that contribute to bacterial virulence. A novel AT adhesin gene, aatB, was identified in avian pathogenic Escherichia coli (APEC) DE205B via genomic analyses. The open reading frame of aatB was 1,017 bp, encoding a putative 36.3-kDa protein which contained structural motifs characteristic for AT proteins: a signal peptide, a passenger domain, and a translocator domain. The predicted three-dimensional structure of AatB consisted of two distinct domains, the C-terminal β-barrel translocator domain and an N-terminal passenger domain. The prevalence analyses of aatB in APEC indicated that aatB was detected in 26.4% (72/273) of APEC strains and was strongly associated with phylogenetic groups D and B2. Quantitative real-time reverse transcription-PCR analyses revealed that AatB expression was increased during infection in vitro and in vivo. Moreover, AatB could elicit antibodies in infected ducks, suggesting that AatB is involved in APEC pathogenicity. Thus, APEC DE205B strains with a mutated aatB gene and mutated strains complemented with the aatB gene were constructed. Inactivation of aatB resulted in a reduced capacity to adhere to DF-1 cells, defective virulence capacity in vivo, and decreased colonization capacity in lung during systemic infection compared with the capacities of the wild-type strain. Furthermore, these capacities were restored in the complementation strains. These results indicated that AatB makes a significant contribution to APEC virulence through bacterial adherence to host tissues in vivo and in vitro. In addition, biofilm formation assays with strain AAEC189 expressing AatB indicated that AatB mediates biofilm formation. PMID:23630958

  4. BibA: a novel immunogenic bacterial adhesin contributing to group B Streptococcus survival in human blood.

    PubMed

    Santi, Isabella; Scarselli, Maria; Mariani, Massimo; Pezzicoli, Alfredo; Masignani, Vega; Taddei, Annarita; Grandi, Guido; Telford, John L; Soriani, Marco

    2007-02-01

    By the analysis of the recently sequenced genomes of Group B Streptococcus (GBS) we have identified a novel immunogenic adhesin with anti-phagocytic activity, named BibA. The bibA gene is present in 100% of the 24 GBS strains analysed. BibA-specific IgG were found in human sera from normal healthy donors. The putative protein product is a polypeptide of 630 amino acids containing a helix-rich N-terminal domain, a proline-rich region and a canonical LPXTG cell wall-anchoring domain. BibA is expressed on the surface of several GBS strains, but is also recovered in GBS culture supernatants. BibA specifically binds to human C4-binding protein, a regulator of the classic complement pathway. Deletion of the bibA gene severely reduced the capacity of GBS to survive in human blood and to resist opsonophagocytic killing by human neutrophils. In addition, BibA expression increased the virulence of GBS in a mouse infection model. The role of BibA in GBS adhesion was demonstrated by the impaired ability of a bibA knockout mutant strain to adhere to both human cervical and lung epithelial cells. Furthermore, we calculated that recombinant BibA bound to human epithelial cells of distinct origin with an affinity constant of approximately 10(-8) M for cervical epithelial cells. Hence BibA is a novel multifunctional protein involved in both resistance to phagocytic killing and adhesion to host cells. The identification of this potential new virulence factor represents an important step in the development of strategies to combat GBS-associated infections.

  5. Structural Features of the Pseudomonas fluorescens Biofilm Adhesin LapA Required for LapG-Dependent Cleavage, Biofilm Formation, and Cell Surface Localization

    PubMed Central

    Boyd, Chelsea D.; Smith, T. Jarrod; El-Kirat-Chatel, Sofiane; Newell, Peter D.; Dufrêne, Yves F.

    2014-01-01

    The localization of the LapA protein to the cell surface is a key step required by Pseudomonas fluorescens Pf0-1 to irreversibly attach to a surface and form a biofilm. LapA is a member of a diverse family of predicted bacterial adhesins, and although lacking a high degree of sequence similarity, family members do share common predicted domains. Here, using mutational analysis, we determine the significance of each domain feature of LapA in relation to its export and localization to the cell surface and function in biofilm formation. Our previous work showed that the N terminus of LapA is required for cleavage by the periplasmic cysteine protease LapG and release of the adhesin from the cell surface under conditions unfavorable for biofilm formation. We define an additional critical region of the N terminus of LapA required for LapG proteolysis. Furthermore, our results suggest that the domains within the C terminus of LapA are not absolutely required for biofilm formation, export, or localization to the cell surface, with the exception of the type I secretion signal, which is required for LapA export and cell surface localization. In contrast, deletion of the central repetitive region of LapA, consisting of 37 repeats of 100 amino acids, results in an inability to form a biofilm. We also used single-molecule atomic force microscopy to further characterize the role of these domains in biofilm formation on hydrophobic and hydrophilic surfaces. These studies represent the first detailed analysis of the domains of the LapA family of biofilm adhesin proteins. PMID:24837291

  6. The Soluble Recombinant Neisseria meningitidis Adhesin NadAΔ351–405 Stimulates Human Monocytes by Binding to Extracellular Hsp90

    PubMed Central

    Cecchini, Paola; Tavano, Regina; Polverino de Laureto, Patrizia; Franzoso, Susanna; Mazzon, Cristina; Montanari, Paolo; Papini, Emanuele

    2011-01-01

    The adhesin NadA favors cell adhesion/invasion by hypervirulent Neisseria meningitidis B (MenB). Its recombinant form NadAΔ351–405, devoid of the outer membrane domain, is an immunogenic candidate for an anti-MenB vaccine able to stimulate monocytes, macrophages and dendritic cells. In this study we investigated the molecular mechanism of NadAΔ351–405 cellular effects in monocytes. We show that NadAΔ351–405 (against which we obtained polyclonal antibodies in rabbits), binds to hsp90, but not to other extracellular homologous heat shock proteins grp94 and hsp70, in vitro and on the surface of monocytes, in a temperature dependent way. Pre-incubation of monocytes with the MenB soluble adhesin interfered with the binding of anti-hsp90 and anti-hsp70 antibodies to hsp90 and hsp70 at 37°C, a condition in which specific cell-binding occurs, but not at 0°C, a condition in which specific cell-binding is very diminished. Conversely, pre-incubation of monocytes with anti-hsp90 and anti-hsp70 antibodies did not affected NadAΔ351–405 cell binding in any temperature condition, indicating that it associates to another receptor on their plasma membrane and then laterally diffuses to encounter hsp90. Consistently, polymixin B interfered with NadAΔ351–405 /hsp90 association, abrogated the decrease of anti-hsp90 antibodies binding to the cell surface due to NadAΔ351–405 and inhibited adhesin-induced cytokine/chemokine secretion without affecting monocyte-adhesin binding. Co-stimulation of monocytes with anti-hsp90 antibodies and NadAΔ351–405 determined a stronger but polymixin B insensitive cell activation. This indicated that the formation of a recombinant NadA/hsp90/hsp70 complex, although essential for full monocyte stimulation, can be replaced by anti-hsp90 antibody/hsp90 binding. Finally, the activation of monocytes by NadAΔ351–405 alone or in the presence of anti-hsp90 antibodies were both inhibited by neutralizing anti-TLR4 antibodies, but not by

  7. The soluble recombinant Neisseria meningitidis adhesin NadA(Δ351-405) stimulates human monocytes by binding to extracellular Hsp90.

    PubMed

    Cecchini, Paola; Tavano, Regina; Polverino de Laureto, Patrizia; Franzoso, Susanna; Mazzon, Cristina; Montanari, Paolo; Papini, Emanuele

    2011-01-01

    The adhesin NadA favors cell adhesion/invasion by hypervirulent Neisseria meningitidis B (MenB). Its recombinant form NadA(Δ351-405,) devoid of the outer membrane domain, is an immunogenic candidate for an anti-MenB vaccine able to stimulate monocytes, macrophages and dendritic cells. In this study we investigated the molecular mechanism of NadA(Δ351-405) cellular effects in monocytes. We show that NadA(Δ351-405) (against which we obtained polyclonal antibodies in rabbits), binds to hsp90, but not to other extracellular homologous heat shock proteins grp94 and hsp70, in vitro and on the surface of monocytes, in a temperature dependent way. Pre-incubation of monocytes with the MenB soluble adhesin interfered with the binding of anti-hsp90 and anti-hsp70 antibodies to hsp90 and hsp70 at 37°C, a condition in which specific cell-binding occurs, but not at 0°C, a condition in which specific cell-binding is very diminished. Conversely, pre-incubation of monocytes with anti-hsp90 and anti-hsp70 antibodies did not affected NadA(Δ351-405) cell binding in any temperature condition, indicating that it associates to another receptor on their plasma membrane and then laterally diffuses to encounter hsp90. Consistently, polymixin B interfered with NadA(Δ351-405) /hsp90 association, abrogated the decrease of anti-hsp90 antibodies binding to the cell surface due to NadA(Δ351-405) and inhibited adhesin-induced cytokine/chemokine secretion without affecting monocyte-adhesin binding. Co-stimulation of monocytes with anti-hsp90 antibodies and NadA(Δ351-405) determined a stronger but polymixin B insensitive cell activation. This indicated that the formation of a recombinant NadA/hsp90/hsp70 complex, although essential for full monocyte stimulation, can be replaced by anti-hsp90 antibody/hsp90 binding. Finally, the activation of monocytes by NadA(Δ351-405) alone or in the presence of anti-hsp90 antibodies were both inhibited by neutralizing anti-TLR4 antibodies, but not by

  8. Relationship between adhesin genes and biofilm formation in vancomycin-intermediate Staphylococcus aureus clinical isolates.

    PubMed

    Mirzaee, Mohsen; Najar-Peerayeh, Shahin; Behmanesh, Mehrdad; Moghadam, Mahdi Forouzandeh

    2015-05-01

    The adherence ability and biofilm production are the characteristic of enhanced virulence among isolates of vancomycin-intermediate Staphylococcus aureus (VISA) strains. Although biofilm-forming properties have been well demonstrated in S. aureus, they still remain unclear among the recently emerged types of VISA strains. The aim of this study was to investigate correlations between the distribution of genes encoding staphylococcal microbial surface components which recognise adhesive matrix molecules (MSCRAMMs), the surface protein A (Spa) types, MLST types and the ability of VISA strains to biofilm formation. Microtiter plate assay (Mtp) results showed that all eleven biofilm producer isolates were adherent at various levels. PCR experiments showed that nine MSCRAMM genes, clfA, clfB, fnbA and fib were detected in all of the strains, indicating a high prevalence. The prevalences of other MSCRAMMs and icaABCD genes were found to be variable and not equally distributed among the VISA strains. There was no direct correlation between the distribution of adhesion-related genes and biofilm formation, which indicates that the presence or absence of these genes cannot be employed as an indicator of the ability to biofilm formation. Isolates which belong to the same Spa and ST types showed similar adherence capacities in the Mtp assay, but significant differences were observed between different Spa types. The findings of this study, using quantitative methods, have shown that genotypically different strains of VISA have different capabilities to produce biofilms. This may be caused by a difference in the spa types of VISA isolates or due to their differences in the expression of MSCRAMM and icaABCD genes.

  9. Structure determination and analysis of a haemolytic gingipain adhesin domain from Porphyromonas gingivalis

    SciTech Connect

    Li, N.; Yun, P.; Nadkarni, M.A.; Ghadikolaee, N.B.; Nguyen, K.A.; Lee, M.; Hunter, N.; Collyer, C.A.

    2010-08-27

    Porphyromonas gingivalis is an obligately anaerobic bacterium recognized as an aetiological agent of adult periodontitis. P. gingivalis produces cysteine proteinases, the gingipains. The crystal structure of a domain within the haemagglutinin region of the lysine gingipain (Kgp) is reported here. The domain was named K2 as it is the second of three homologous structural modules in Kgp. The K2 domain structure is a 'jelly-roll' fold with two anti-parallel {beta}-sheets. This fold topology is shared with adhesive domains from functionally diverse receptors such as MAM domains, ephrin receptor ligand binding domains and a number of carbohydrate binding modules. Possible functions of K2 were investigated. K2 induced haemolysis of erythrocytes in a dose-dependent manner that was augmented by the blocking of anion transport. Further, cysteine-activated arginine gingipain RgpB, which degrades glycophorin A, sensitized erythrocytes to the haemolytic effect of K2. Cleaved K2, similar to that found in extracted Kgp, lacks the haemolytic activity indicating that autolysis of Kgp may be a staged process which is artificially enhanced by extraction of the protein. The data indicate a functional role for K2 in the integrated capacity conferred by Kgp to enable the porphyrin auxotroph P. gingivalis to capture essential haem from erythrocytes.

  10. Biofilm formation by Psychrobacter arcticus and the role of a large adhesin in attachment to surfaces.

    PubMed

    Hinsa-Leasure, Shannon M; Koid, Cassandra; Tiedje, James M; Schultzhaus, Janna N

    2013-07-01

    Psychrobacter arcticus strain 273-4, an isolate from a Siberian permafrost core, is capable of forming biofilms when grown in minimal medium under laboratory conditions. Biofilms form at 4 to 22°C when acetate is supplied as the lone carbon source and with 1 to 7% sea salt. P. arcticus is also capable of colonizing quartz sand. Transposon mutagenesis identified a gene important for biofilm formation by P. arcticus. Four transposon mutants were mapped to a 20.1-kbp gene, which is predicted to encode a protein of 6,715 amino acids (Psyc_1601). We refer to this open reading frame as cat1, for cold attachment gene 1. The cat1 mutants are unable to form biofilms at levels equivalent to that of the wild type, and there is no impact on the planktonic growth characteristics of the strains, indicating a specific role in biofilm formation. Through time course studies of the static microtiter plate assay, we determined that cat1 mutants are unable to form biofilms equivalent to that of the wild type under all conditions tested. In flow cell experiments, cat1 mutants initially are unable to attach to the surface. Over time, however, they form microcolonies, an architecture very different from that produced by wild-type biofilms. Our results demonstrate that Cat1 is involved in the initial stages of bacterial attachment to surfaces. PMID:23603675

  11. Biofilm Formation by Psychrobacter arcticus and the Role of a Large Adhesin in Attachment to Surfaces

    PubMed Central

    Koid, Cassandra; Tiedje, James M.; Schultzhaus, Janna N.

    2013-01-01

    Psychrobacter arcticus strain 273-4, an isolate from a Siberian permafrost core, is capable of forming biofilms when grown in minimal medium under laboratory conditions. Biofilms form at 4 to 22°C when acetate is supplied as the lone carbon source and with 1 to 7% sea salt. P. arcticus is also capable of colonizing quartz sand. Transposon mutagenesis identified a gene important for biofilm formation by P. arcticus. Four transposon mutants were mapped to a 20.1-kbp gene, which is predicted to encode a protein of 6,715 amino acids (Psyc_1601). We refer to this open reading frame as cat1, for cold attachment gene 1. The cat1 mutants are unable to form biofilms at levels equivalent to that of the wild type, and there is no impact on the planktonic growth characteristics of the strains, indicating a specific role in biofilm formation. Through time course studies of the static microtiter plate assay, we determined that cat1 mutants are unable to form biofilms equivalent to that of the wild type under all conditions tested. In flow cell experiments, cat1 mutants initially are unable to attach to the surface. Over time, however, they form microcolonies, an architecture very different from that produced by wild-type biofilms. Our results demonstrate that Cat1 is involved in the initial stages of bacterial attachment to surfaces. PMID:23603675

  12. Comparison of antibiotic resistance and virulence between biofilm-producing and non-producing clinical isolates of Enterococcus faecium.

    PubMed

    Sieńko, Anna; Wieczorek, Piotr; Majewski, Piotr; Ojdana, Dominika; Wieczorek, Anna; Olszańska, Dorota; Tryniszewska, Elżbieta

    2015-01-01

    An increase in the antibiotic resistance among Enterococcus faecium strains has been observed worldwide. Moreover, this bacteria has the ability to produce several virulence factors and to form biofilm that plays an important role in human infections. This study was designed to compare the antibiotic resistance and the prevalence of genes encoding surface protein (esp), aggregation substance (as), surface adhesin (efaA), collagen adhesin (ace), gelatinase (gelE), and hialuronidase (hyl) between biofilm-producing and non-producing E. faecium strains. Therefore, ninety E. faecium clinical isolates were tested for biofilm-forming ability, and then were assigned to two groups: biofilm-positive (BIO(+), n =70) and biofilm-negative (BIO(-), n = 20). Comparison of these groups showed that BIO(+) isolates were resistant to β-lactams, whereas 10% of BIO(-) strains were susceptible to ampicillin (statistically significant difference, p = 0.007) and 5% to imipenem. Linezolid and tigecycline were the only antibiotics active against all tested isolates. Analysis of the virulence factors revealed that ace, efaA, and gelE genes occurred more frequently in BIO(-) strains (ace in 50% BIO(+) vs. 75% BIO(-); efaA 44.3% vs. 85%; gelE 2.9% vs. 15%, respectively), while hyl gene appeared more frequently in BIO(+) isolates (87.1% BIO(+) vs. 65% BIO(-)). These differences were significant (p < 0.05). We concluded that BIO(+) strains were more resistant to antibiotics than BIO(-) strains, but interestingly, BIO(-) isolates were characterized by possession of higher virulence capabilities.

  13. Sulfated glycoconjugate receptors for the Bordetella pertussis adhesin filamentous hemagglutinin (FHA) and mapping of the heparin-binding domain on FHA.

    PubMed Central

    Hannah, J H; Menozzi, F D; Renauld, G; Locht, C; Brennan, M J

    1994-01-01

    Filamentous hemagglutinin (FHA) is a major adhesin present on the surface of the gram-negative respiratory pathogen Bordetella pertussis. A number of binding mechanisms have been described for the interaction of FHA with eukaryotic cells. We have focused on its function as a sulfated polysaccharide-binding protein and on identifying potential receptors for FHA on the epithelial cell surface. Using a thin-layer overlay technique, we found that FHA binds specifically to sulfated glycolipids but not to gangliosides or other neutral glycolipids. These results suggest that epithelial cell surface sulfated glycolipids function as receptors for FHA. Further studies demonstrated that a Chinese hamster ovary (CHO) cell strain deficient in glycosaminoglycan expression exhibits greatly diminished attachment to FHA. By FHA-Affi-Gel chromatography, a putative receptor for FHA that has characteristics consistent with a heparan sulfate proteoglycan was isolated from epithelial cell extracts. In addition, by using recombinant FHA fusion proteins, a specific glycosaminoglycan-binding domain located near the N terminus of the FHA molecule was identified. Our results indicate that the B. pertussis adhesin FHA may utilize sulfated glycolipids and proteoglycans commonly found on the surface of human cells and tissues to initiate infection. Images PMID:7927782

  14. The HMW1 and HMW2 Adhesins Enhance the Ability of Nontypeable Haemophilus influenzae To Colonize the Upper Respiratory Tract of Rhesus Macaques.

    PubMed

    Rempe, Katherine A; Porsch, Eric A; Wilson, Jolaine M; St Geme, Joseph W

    2016-10-01

    Nontypeable Haemophilus influenzae (NTHi) initiates infection by colonizing the upper respiratory tract and is a common cause of localized respiratory tract disease. Previous work has established that the NTHi HMW1 and HMW2 proteins are potent adhesins that mediate efficient in vitro adherence to cultured human respiratory epithelial cells. In this study, we used a rhesus macaque model to assess the contributions of HMW1 and HMW2 to in vivo colonization. In experiments involving inoculation of individual isogenic derivatives of NTHi strain 12, the parent strain expressing both HMW1 and HMW2 and the mutant strains expressing either HMW1 or HMW2 were able to colonize more frequently than the double mutant strain lacking HMW1 and HMW2. In competition experiments, the parent strain efficiently outcompeted the double mutant lacking HMW1 and HMW2. Colonization with strains expressing HMW2 resulted in development of antibody against HMW2 in a number of the animals, demonstrating that colonization can stimulate an antibody response. In conclusion, we have established that the HMW1 and HMW2 adhesins play a major role in facilitating colonization of the upper respiratory tract of rhesus macaques, in some cases associated with stimulation of an immune response.

  15. Infection of human mucosal tissue by Pseudomonas aeruginosa requires sequential and mutually dependent virulence factors and a novel pilus-associated adhesin

    PubMed Central

    Heiniger, Ryan W.; Winther-Larsen, Hanne C.; Pickles, Raymond J.; Koomey, Michael; Wolfgang, Matthew C.

    2010-01-01

    Summary Tissue damage predisposes humans to life-threatening disseminating infection by the opportunistic pathogen Pseudomonas aeruginosa. Bacterial adherence to host tissue is a critical first step in this infection process. It is well established that P. aeruginosa attachment to host cells involves type IV pili (TFP), which are retractile surface fibers. The molecular details of attachment and the identity of the bacterial adhesin and host receptor remain controversial. Using a mucosal epithelium model system derived from primary human tissue, we show that the pilus-associated protein PilY1 is required for bacterial adherence. We establish that P. aeruginosa preferentially binds to exposed basolateral host cell surfaces, providing a mechanistic explanation for opportunistic infection of damaged tissue. Further, we demonstrate that invasion and fulminant infection of intact host tissue requires the coordinated and mutually dependent action of multiple bacterial factors, including pilus fiber retraction and the host cell intoxication system, termed type III secretion. Our findings offer new and important insights into the complex interactions between a pathogen and its human host and provide compelling evidence that PilY1 serves as the principal P. aeruginosa adhesin for human tissue and that it specifically recognizes a host receptor localized or enriched on basolateral epithelial cell surfaces. PMID:20331639

  16. A Trichomonas vaginalis 120 kDa protein with identity to hydrogenosome pyruvate:ferredoxin oxidoreductase is a surface adhesin induced by iron.

    PubMed

    Moreno-Brito, Verónica; Yáñez-Gómez, Carmina; Meza-Cervantez, Patricia; Avila-González, Leticia; Rodríguez, Mario Alberto; Ortega-López, Jaime; González-Robles, Arturo; Arroyo, Rossana

    2005-02-01

    Trichomonas vaginalis, a human sexually transmitted protozoan, relies on adherence to the vaginal epithelium for colonization and maintenance of infection in the host. Thus, adherence molecules play a fundamental role in the trichomonal infection. Here, we show the identification and characterization of a 120 kDa surface glycoprotein (AP120) induced by iron, which participates in cytoadherence. AP120 is synthesized by the parasite when grown in 250 microM iron medium. Antibodies to AP120 and the electro-eluted AP120 inhibited parasite adherence in a concentration-dependent manner, demonstrating its participation in cytoadherence. In addition, a protein of 130 kDa was detected on the surface of HeLa cells as the putative receptor for AP120. By peptide matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), the AP120 adhesin showed homology with a hydrogenosomal enzyme, the pyruvate:ferredoxin oxidoreductase (PFO) encoded by the pfoa gene. This homology was confirmed by immunoblot and indirect immunofluorescence assays with an antibody to the carboxy-terminus region of the Entamoeba histolytica PFO. Reverse transcription polymerase chain reaction (RT-PCR) assays showed that a pfoa-like gene was better transcribed in trichomonads grown in iron-rich medium. In conclusion, the homology of AP120 to PFO suggests that this novel adhesin induced by iron could be an example of moonlighting protein in T. vaginalis.

  17. Lactobacillus rhamnosus GG and its SpaC pilus adhesin modulate inflammatory responsiveness and TLR-related gene expression in the fetal human gut

    PubMed Central

    Ganguli, Kriston; Collado, Maria Carmen; Rautava, Jaana; Lu, Lei; Satokari, Reetta; von Ossowski, Ingemar; Reunanen, Justus; de Vos, Willem M.; Palva, Airi; Isolauri, Erika; Salminen, Seppo; Walker, W. Allan; Rautava, Samuli

    2015-01-01

    Background Bacterial contact in utero modulates fetal and neonatal immune responses. Maternal probiotic supplementation reduces the risk of immune-mediated disease in the infant. We investigated the immunomodulatory properties of live Lactobacillus rhamnosus GG and its SpaC pilus adhesin in human fetal intestinal models. Methods TNF-α mRNA expression was measured by qPCR in a human fetal intestinal organ culture model exposed to live L. rhamnosus GG and proinflammatory stimuli. Binding of recombinant SpaC pilus protein to intestinal epithelial cells was assessed in human fetal intestinal organ culture and the human fetal intestinal epithelial cell line H4 by immunohistochemistry and immunofluorescence, respectively. TLR-related gene expression in fetal ileal organ culture after exposure to recombinant SpaC was assessed by qPCR. Results Live L. rhamnosus GG significantly attenuates pathogen-induced TNF-α mRNA expression in the human fetal gut. Recombinant SpaC protein was found to adhere to the fetal gut and to modulate varying levels of TLR-related gene expression. Conclusion The human fetal gut is responsive to luminal microbes. L. rhamnosus GG significantly attenuates fetal intestinal inflammatory responses to pathogenic bacteria. The L. rhamnosus GG pilus adhesin SpaC binds to immature human intestinal epithelial cells and directly modulates intestinal epithelial cell innate immune gene expression. PMID:25580735

  18. Comparison of Surface Proteomes of Adherence Variants of Listeria Monocytogenes Using LC-MS/MS for Identification of Potential Surface Adhesins

    PubMed Central

    Tiong, Hung King; Hartson, Steven D.; Muriana, Peter M.

    2016-01-01

    The ability of Listeria monocytogenes to adhere and form biofilms leads to persistence in food processing plants and food-associated listeriosis. The role of specific surface proteins as adhesins to attach Listeria cells to various contact surfaces has not been well characterized to date. In prior research comparing different methods for surface protein extraction, the Ghost urea method revealed cleaner protein content as verified by the least cytoplasmic protein detected in surface extracts using LC-MS/MS. The same technique was utilized to extract and detect surface proteins among two surface-adherent phenotypic strains of L. monocytogenes (i.e., strongly and weakly adherent). Of 640 total proteins detected among planktonic and sessile cells, 21 protein members were exclusively detected in the sessile cells. Relative LC-MS/MS detection and quantification of surface-extracted proteins from the planktonic weakly adherent (CW35) and strongly adherent strains (99-38) were examined by protein mass normalization of proteins. We found that L. monocytogenes 99-38 exhibited a total of 22 surface proteins that were over-expressed: 11 proteins were detected in surface extracts of both sessile and planktonic 99-38 that were ≥5-fold over-expressed while another 11 proteins were detected only in planktonic 99-38 cells that were ≥10-fold over-expressed. Our results suggest that these protein members are worthy of further investigation for their involvement as surface adhesins. PMID:27196934

  19. Cooperative role of antibodies against heat-labile toxin and the EtpA Adhesin in preventing toxin delivery and intestinal colonization by enterotoxigenic Escherichia coli.

    PubMed

    Roy, Koushik; Hamilton, David J; Fleckenstein, James M

    2012-10-01

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease in developing countries, where it is responsible for hundreds of thousands of deaths each year. Vaccine development for ETEC has been hindered by the heterogeneity of known molecular targets and the lack of broad-based sustained protection afforded by existing vaccine strategies. In an effort to explore the potential role of novel antigens in ETEC vaccines, we examined the ability of antibodies directed against the ETEC heat-labile toxin (LT) and the recently described EtpA adhesin to prevent intestinal colonization in vivo and toxin delivery to epithelial cells in vitro. We demonstrate that EtpA is required for the optimal delivery of LT and that antibodies against this adhesin play at least an additive role in preventing delivery of LT to target intestinal cells when combined with antibodies against either the A or B subunits of the toxin. Moreover, vaccination with a combination of LT and EtpA significantly impaired intestinal colonization. Together, these results suggest that the incorporation of recently identified molecules such as EtpA could be used to enhance current approaches to ETEC vaccine development. PMID:22875600

  20. Molecular Basis for Strain Variation in the Saccharomyces cerevisiae Adhesin Flo11p

    PubMed Central

    Li, Li; Lipke, Peter N.; Dranginis, Anne M.

    2016-01-01

    ABSTRACT FLO11 encodes a yeast cell wall flocculin that mediates a variety of adhesive phenotypes in Saccharomyces cerevisiae. Flo11p is implicated in many developmental processes, including flocculation, formation of pseudohyphae, agar invasion, and formation of microbial mats and biofilms. However, Flo11p mediates different processes in different yeast strains. To investigate the mechanisms by which FLO11 determines these differences in colony morphology, flocculation, and invasion, we studied gene structure, function, and expression levels. Nonflocculent Saccharomyces cerevisiae Σ1278b cells exhibited significantly higher FLO11 mRNA expression, especially in the stationary phase, than highly flocculent S. cerevisiae var. diastaticus. The two strains varied in cell surface hydrophobicity, and Flo11p contributed significantly to surface hydrophobicity in S. cerevisiae var. diastaticus but not in strain Σ1278b. Sequencing of the FLO11 gene in S. cerevisiae var. diastaticus revealed strain-specific differences, including a 15-amino-acid insertion in the adhesion domain. Flo11p adhesion domains from strain Σ1278b and S. cerevisiae var. diastaticus were expressed and used to coat magnetic beads. The adhesion domain from each strain bound preferentially to homologous cells, and the preferences were independent of the cells in which the adhesion domains were produced. These results are consistent with the idea that strain-specific variations in the amino acid sequences in the adhesion domains cause different Flo11p flocculation activities. The results also imply that strain-specific differences in expression levels, posttranslational modifications, and allelic differences outside the adhesion domains have little effect on flocculation. IMPORTANCE As a nonmotile organism, Saccharomyces cerevisiae employs the cell surface flocculin Flo11/Muc1 as an important means of adapting to environmental change. However, there is a great deal of strain variation in the

  1. Molecular Basis for Strain Variation in the Saccharomyces cerevisiae Adhesin Flo11p.

    PubMed

    Barua, Subit; Li, Li; Lipke, Peter N; Dranginis, Anne M

    2016-01-01

    FLO11 encodes a yeast cell wall flocculin that mediates a variety of adhesive phenotypes in Saccharomyces cerevisiae. Flo11p is implicated in many developmental processes, including flocculation, formation of pseudohyphae, agar invasion, and formation of microbial mats and biofilms. However, Flo11p mediates different processes in different yeast strains. To investigate the mechanisms by which FLO11 determines these differences in colony morphology, flocculation, and invasion, we studied gene structure, function, and expression levels. Nonflocculent Saccharomyces cerevisiae Σ1278b cells exhibited significantly higher FLO11 mRNA expression, especially in the stationary phase, than highly flocculent S. cerevisiae var. diastaticus. The two strains varied in cell surface hydrophobicity, and Flo11p contributed significantly to surface hydrophobicity in S. cerevisiae var. diastaticus but not in strain Σ1278b. Sequencing of the FLO11 gene in S. cerevisiae var. diastaticus revealed strain-specific differences, including a 15-amino-acid insertion in the adhesion domain. Flo11p adhesion domains from strain Σ1278b and S. cerevisiae var. diastaticus were expressed and used to coat magnetic beads. The adhesion domain from each strain bound preferentially to homologous cells, and the preferences were independent of the cells in which the adhesion domains were produced. These results are consistent with the idea that strain-specific variations in the amino acid sequences in the adhesion domains cause different Flo11p flocculation activities. The results also imply that strain-specific differences in expression levels, posttranslational modifications, and allelic differences outside the adhesion domains have little effect on flocculation. IMPORTANCE As a nonmotile organism, Saccharomyces cerevisiae employs the cell surface flocculin Flo11/Muc1 as an important means of adapting to environmental change. However, there is a great deal of strain variation in the expression of

  2. Structural insight in the inhibition of adherence of F4 fimbriae producing enterotoxigenic Escherichia coli by llama single domain antibodies.

    PubMed

    Moonens, Kristof; Van den Broeck, Imke; Okello, Emmanuel; Pardon, Els; De Kerpel, Maia; Remaut, Han; De Greve, Henri

    2015-01-01

    Enterotoxigenic Escherichia coli that cause neonatal and post-weaning diarrhea in piglets express F4 fimbriae to mediate attachment towards host receptors. Recently we described how llama single domain antibodies (VHHs) fused to IgA, produced in Arabidopsis thaliana seeds and fed to piglets resulted in a progressive decline in shedding of F4 positive ETEC bacteria. Here we present the structures of these inhibiting VHHs in complex with the major adhesive subunit FaeG. A conserved surface, distant from the lactose binding pocket, is targeted by these VHHs, highlighting the possibility of targeting epitopes on single-domain adhesins that are non-involved in receptor binding. PMID:25828907

  3. Project Produce

    ERIC Educational Resources Information Center

    Wolfinger, Donna M.

    2005-01-01

    The grocery store produce section used to be a familiar but rather dull place. There were bananas next to the oranges next to the limes. Broccoli was next to corn and lettuce. Apples and pears, radishes and onions, eggplants and zucchinis all lay in their appropriate bins. Those days are over. Now, broccoli may be next to bok choy, potatoes beside…

  4. Relationship between phylogenetic groups, antibiotic resistance and patient characteristics in terms of adhesin genes in cystitis and pyelonephritis isolates of Escherichia coli.

    PubMed

    Er, Doganhan Kadir; Dundar, Devrim; Uzuner, Huseyin; Osmani, Agim

    2015-12-01

    Extraintestinal pathogenic Escherichia coli (E. coli) is considered as the main causative agent of urinary tract infections worldwide. The relationship between antimicrobial resistance, phylogenetic groups, patient characteristics and adhesin virulence genes are complex and not fully understood. In this study, among 146 urinary isolates of E. coli, phylogenetic groups and various adhesin virulence genes were examined with multiplex Polymerase Chain Reaction methods. Patient characteristics divided into sex, cystitis and pyelonephritis; community-acquired and hospital-acquired; complicated and uncomplicated infection. Antimicrobial resistance was also determined. The papAH gene was seen more often in pyelonephritis than cystitis and female than male patients. iha gene was more frequent in hospital-acquired infections than in community-acquired infections. sfa/focDE was more frequent in ampicillin, amikacin, gentamicin, nalidixic acid, norfloxacin, cefuroxime, ceftriaxone, cefazolin, cefotaxime, ciprofloxacin and trimethoprim/sulfamethoxazole susceptible and extended-spectrum β-lactamase (ESBL) and multi-drug resistance (MDR) negative isolates. focG was seen more often in nalidixic acid, norfloxacin, cefuroxime, ceftriaxone, ciprofloxacin susceptible and MDR negative isolates. fimH and papAH were more commonly observed in amoxicillin/clavulanic acid and cefotaxime susceptible isolates, respectively. iha and afa/draBC genes were more frequent in resistant isolates than the susceptible ones; for iha, in ampicillin, amoxicillin/clavulanic acid, nalidixic acid, cefuroxime, ceftriaxone resistant and ESBL and MDR positive isolates; for afa/draBC, in cefotaxime, cefuroxime, ciprofloxacin, trimethoprim/sulfamethoxazole resistant and ESBL and MDR positive isolates, this trend was observed. ST 131 E. coli virulence gene pattern has a direct effect on resistance profile. Isolates belong to that clonal group has MDR and commonly harbour afa/draBC and iha genes. Our findings may

  5. UafB is a serine-rich repeat adhesin of Staphylococcus saprophyticus that mediates binding to fibronectin, fibrinogen and human uroepithelial cells.

    PubMed

    King, Nathan P; Beatson, Scott A; Totsika, Makrina; Ulett, Glen C; Alm, Richard A; Manning, Paul A; Schembri, Mark A

    2011-04-01

    Staphylococcus saprophyticus is an important cause of urinary tract infection (UTI), particularly among young women, and is second only to uropathogenic Escherichia coli as the most frequent cause of UTI. The molecular mechanisms of urinary tract colonization by S. saprophyticus remain poorly understood. We have identified a novel 6.84 kb plasmid-located adhesin-encoding gene in S. saprophyticus strain MS1146 which we have termed uro-adherence factor B (uafB). UafB is a glycosylated serine-rich repeat protein that is expressed on the surface of S. saprophyticus MS1146. UafB also functions as a major cell surface hydrophobicity factor. To characterize the role of UafB we generated an isogenic uafB mutant in S. saprophyticus MS1146 by interruption with a group II intron. The uafB mutant had a significantly reduced ability to bind to fibronectin and fibrinogen. Furthermore, we show that a recombinant protein containing the putative binding domain of UafB binds specifically to fibronectin and fibrinogen. UafB was not involved in adhesion in a mouse model of UTI; however, we observed a striking UafB-mediated adhesion phenotype to human uroepithelial cells. We have also identified genes homologous to uafB in other staphylococci which, like uafB, appear to be located on transposable elements. Thus, our data indicate that UafB is a novel adhesin of S. saprophyticus that contributes to cell surface hydrophobicity, mediates adhesion to fibronectin and fibrinogen, and exhibits tropism for human uroepithelial cells.

  6. BtaE, an Adhesin That Belongs to the Trimeric Autotransporter Family, Is Required for Full Virulence and Defines a Specific Adhesive Pole of Brucella suis

    PubMed Central

    Ruiz-Ranwez, Verónica; Posadas, Diana M.; Van der Henst, Charles; Estein, Silvia M.; Arocena, Gastón M.; Abdian, Patricia L.; Martín, Fernando A.; Sieira, Rodrigo; De Bolle, Xavier

    2013-01-01

    Brucella is responsible for brucellosis, one of the most common zoonoses worldwide that causes important economic losses in several countries. Increasing evidence indicates that adhesion of Brucella spp. to host cells is an important step to establish infection. We have previously shown that the BmaC unipolar monomeric autotransporter mediates the binding of Brucella suis to host cells through cell-associated fibronectin. Our genome analysis shows that the B. suis genome encodes several additional potential adhesins. In this work, we characterized a predicted trimeric autotransporter that we named BtaE. By expressing btaE in a nonadherent Escherichia coli strain and by phenotypic characterization of a B. suis ΔbtaE mutant, we showed that BtaE is involved in the binding of B. suis to hyaluronic acid. The B. suis ΔbtaE mutant exhibited a reduction in the adhesion to HeLa and A549 epithelial cells compared with the wild-type strain, and it was outcompeted by the wild-type strain in the binding to HeLa cells. The knockout btaE mutant showed an attenuated phenotype in the mouse model, indicating that BtaE is required for full virulence. BtaE was immunodetected on the bacterial surface at one cell pole. Using old and new pole markers, we observed that both the BmaC and BtaE adhesins are consistently associated with the new cell pole, suggesting that, in Brucella, the new pole is functionally differentiated for adhesion. This is consistent with the inherent polarization of this bacterium, and its role in the invasion process. PMID:23319562

  7. The ShdA adhesin binds to the cationic cradle of the fibronectin 13FnIII repeat module: evidence for molecular mimicry of heparin binding.

    PubMed

    Kingsley, Robert A; Keestra, A Marijke; de Zoete, Marcel R; Bäumler, Andreas J

    2004-04-01

    Introduction of Salmonella enterica serotype Typhimurium into food products results from its ability to persist in the intestine of healthy livestock by mechanisms that are poorly understood. The non-fimbrial adhesin ShdA is a fibronectin binding protein required for persistent intestinal carriage of S. Typhimurium. We further investigated the molecular mechanism of ShdA-mediated intestinal persistence by determining the binding-site of this receptor in fibronectin. Analysis of ShdA binding to fibronectin proteolytic fragments and to recombinant fibronectin fusion proteins identified the (13)FnIII repeat module of the Hep-2 domain as the primary binding site for this adhesin. The (13)FnIII repeat module of fibronectin contains a cationic cradle formed by six basic residues (R6, R7, R9, R23, K25 and R54) that is a high affinity heparin-binding site conserved among fibronectin sequences from frogs to man. Binding of ShdA to the (13)FnIII repeat module of fibronectin and to a second extracellular matrix protein, Collagen I, could be inhibited by heparin. Furthermore, binding of ShdA to the Hep-2 domain was sensitive to the ionic buffer strength, suggesting that binding involved ionic interactions. We therefore determined whether amino acid substitutions of basic residues in the cationic cradle of the Hep-2 domain that inhibit heparin binding also abrogate binding of ShdA. Combined substitution of R6S and R7S strongly reduced ShdA binding to (13)FnIII. These data suggest that ShdA binds the Hep-2 domain of fibronectin by a mechanism that may mimic binding of the host polysaccharide heparin. PMID:15066025

  8. Verocytotoxin-producing Escherichia coli (VTEC).

    PubMed

    Karmali, Mohamed A; Gannon, Victor; Sargeant, Jan M

    2010-01-27

    Escherichia coli O157:H7 and other Verocytotoxin-producing E. coli (VTEC) are zoonotic pathogens associated with food and waterborne illness around the world. E. coli O157:H7 has been implicated in large outbreaks as well as in sporadic cases of haemorrhagic colitis and the sometimes fatal haemolytic uremic syndrome. VTs produced by these bacteria are thought to damage host endothelial cells in small vessels of the intestine, kidney and brain resulting in thrombotic microangiopathy. All VTs have the same subunit structure, glycolipid cell receptor and inhibit protein synthesis. During VTEC infection, it is thought one or more bacterial adhesins initiates colonization and establishes intimate attachment and is responsible for the translocation of a variety of effectors which alter the structure and function of host cells. VTEC are widespread in animals but ruminants are thought to be their natural reservoir. E. coli O157:H7 colonizes the terminal colon of cattle and can be shed in very large numbers by specific herdmates known as "supershedders". Faeces containing these organisms act as a source of contamination for a variety of foods and the environment. Many VTEC control efforts have been investigated along the "farm to fork" continuum including, vaccination of cattle with colonization factors, and the use of novel antimicrobials, such as bacteriocins, chloral hydrate, bacteriophage and substances which disrupt quorum sensing. In addition, many barriers have been developed for use in the slaughter and food processing industry such as steam pasteurization and irradiation. Despite these efforts many scientific, technical and regulatory challenges remain in the control and prevention of VTEC-associated human illness.

  9. Emerging ST121/agr4 community-associated methicillin-resistant Staphylococcus aureus (MRSA) with strong adhesin and cytolytic activities: trigger for MRSA pneumonia and fatal aspiration pneumonia in an influenza-infected elderly.

    PubMed

    Wan, T-W; Tomita, Y; Saita, N; Konno, K; Iwao, Y; Hung, W-C; Teng, L-J; Yamamoto, T

    2016-09-01

    The pathogenesis of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) pneumonia in influenza-infected elderly individuals has not yet been elucidated in detail. In the present study, a 92-year-old man infected with influenza developed CA-MRSA pneumonia. His CA-MRSA was an emerging type, originated in ST121/agr4 S. aureus, with diversities of Panton-Valentine leucocidin (PVL)(-)/spat5110/SCCmecV(+) versus PVL(+)/spat159((etc.))/SCCmec (-), but with common virulence potentials of strong adhesin and cytolytic activities. Resistance to erythromycin/clindamycin (inducible-type) and gentamicin was detected. Pneumonia improved with the administration of levofloxacin, but with the subsequent development of fatal aspiration pneumonia. Hence, characteristic CA-MRSA with strong adhesin and cytolytic activities triggered influenza-related sequential complications.

  10. Direct Correlation Between Ligand-Induced α-Synuclein Oligomers and Amyloid-like Fibril Growth

    PubMed Central

    Nors Perdersen, Martin; Foderà, Vito; Horvath, Istvan; van Maarschalkerweerd, Andreas; Nørgaard Toft, Katrine; Weise, Christoph; Almqvist, Fredrik; Wolf-Watz, Magnus; Wittung-Stafshede, Pernilla; Vestergaard, Bente

    2015-01-01

    Aggregation of proteins into amyloid deposits is the hallmark of several neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. The suggestion that intermediate oligomeric species may be cytotoxic has led to intensified investigations of pre-fibrillar oligomers, which are complicated by their transient nature and low population. Here we investigate alpha-synuclein oligomers, enriched by a 2-pyridone molecule (FN075), and the conversion of oligomers into fibrils. As probed by leakage assays, the FN075 induced oligomers potently disrupt vesicles in vitro, suggesting a potential link to disease related degenerative activity. Fibrils formed in the presence and absence of FN075 are indistinguishable on microscopic and macroscopic levels. Using small angle X-ray scattering, we reveal that FN075 induced oligomers are similar, but not identical, to oligomers previously observed during alpha-synuclein fibrillation. Since the levels of FN075 induced oligomers correlate with the amounts of fibrils among different FN075:protein ratios, the oligomers appear to be on-pathway and modeling supports an ‘oligomer stacking model’ for alpha-synuclein fibril elongation. PMID:26020724

  11. Synthetic peptide homologous to. beta. protein from Alzheimer's disease forms amyloid-like fibrils in vitro

    SciTech Connect

    Kirschner, D.A.; Inouye, H.; Duffy, L.K.; Sinclair, A.; Lind, M.; Selkoe, D.J.

    1987-10-01

    Progressive amyloid deposition in senile plaques and cortical blood vessels may play a central role in the pathogenesis of Alzheimer's disease. The authors have used x-ray diffraction and electron microscopy to study the molecular organization and morphology of macromolecular assemblies formed by three synthetic peptides homologous to ..beta.. protein of brain amyloid: ..beta..-(1-28), residues 1-28 of the ..beta.. protein; (Ala/sup 1 -/..beta..-(1-28), ..beta..-(1-28) with alanine substituted for lysine at position 16; and ..beta..-(18-28), residues 18-28 of the ..beta.. protein. ..beta..-(1-28) readily formed fibrils in vitro that were similar in ultrastructure to the in vivo amyloid and aggregated into large bundles resembling those of senile plaque cores. X-ray patterns from partially dried, oriented pellets showed a cross-..beta..-conformation. (Ala/sup 16/)..beta..-(1-28) formed ..beta..-pleated sheet assemblies that were dissimilar to in vivo fibrils. The width of the 10-A spacing indicated stacks of about six sheets. Thus, substitution of the uncharged alanine for the positively charged lysine in the ..beta..-strand region enhances the packing of the sheets and dramatically alters the type of macromolecular aggregate formed. BETA-(18-28) formed assemblies that had even a greater number of stacked sheets. The findings on these homologous synthetic assemblies help to define the specific sequence that is required to form Alzheimer's-type amyloid fibrils, thus providing an in vitro model of age-related cerebral amyloidogenesis.

  12. Fiber diffraction of synthetic α-synuclein filaments shows amyloid-like cross-β conformation

    PubMed Central

    Serpell, Louise C.; Berriman, John; Jakes, Ross; Goedert, Michel; Crowther, R. Anthony

    2000-01-01

    Filamentous inclusions made of α-synuclein constitute the defining neuropathological characteristic of Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Rare familial cases of Parkinson's disease are associated with mutations A53T and A30P in α-synuclein. We report here the assembly properties and secondary structure characteristics of recombinant α-synuclein. Carboxy-terminally truncated human α-synuclein (1–87) and (1–120) showed the fastest rates of assembly, followed by human A53T α-synuclein, and rat and zebra finch α-synuclein. Wild-type human α-synuclein and the A30P mutant showed slower rates of assembly. Upon shaking, filaments formed within 48 h at 37°C. The related proteins β- and γ-synuclein only assembled after several weeks of incubation. Synthetic human α-synuclein filaments were decorated by an antibody directed against the carboxy-terminal 10 amino acids of α-synuclein, as were filaments extracted from dementia with Lewy bodies and multiple system atrophy brains. Circular dichroism spectroscopy indicated that α-synuclein undergoes a conformational change from random coil to β-sheet structure during assembly. X-ray diffraction and electron diffraction of the α-synuclein assemblies showed a cross-β conformation characteristic of amyloid. PMID:10781096

  13. Flavone Derivatives as Inhibitors of Insulin Amyloid-Like Fibril Formation

    PubMed Central

    Malisauskas, Ricardas; Botyriute, Akvile; Cannon, Jonathan G.; Smirnovas, Vytautas

    2015-01-01

    Several natural and synthetic flavone derivatives have been reported to inhibit formation of amyloid fibrils or to remodel existing fibrils. These studies suggest that the numbers and positions of hydroxyl groups on the flavone rings determine their effectiveness as amyloid inhibitors. In many studies the primary method for determining the effectiveness of inhibition is measuring Thioflavin T (ThT) fluorescence. This method demonstrably results in a number of false positives for inhibition. We studied the effects of 265 commercially available flavone derivatives on insulin fibril formation. We enhanced the effectiveness of ThT fluorescence measurements by fitting kinetic curves to obtain halftime of aggregation (t50). Maximal values of ThT fluorescence varied two fold or more in one third of all cases, but this did not correlate with changes in t50. Changes in t50 values were more accurate measures of inhibition of amyloid formation. We showed that without a change in an assay, but just by observing complete kinetic curves it is possible to eliminate numbers of false positive and sometimes even false negative results. Examining the data from all 265 flavones we confirmed previous observations that identified the importance of hydroxyl groups for inhibition. Our evidence suggests the importance of hydroxyl groups at locations 5, 6, 7, and 4’, and the absence of a hydroxyl group at location 3, for inhibiting amyloid formation. However, the main conclusion is that the positions are not additive. The structures and their effects must be thought of in the context of the whole molecule. PMID:25799281

  14. Synthetic peptide homologous to beta protein from Alzheimer disease forms amyloid-like fibrils in vitro.

    PubMed Central

    Kirschner, D A; Inouye, H; Duffy, L K; Sinclair, A; Lind, M; Selkoe, D J

    1987-01-01

    Progressive amyloid deposition in senile plaques and cortical blood vessels may play a central role in the pathogenesis of Alzheimer disease. We have used x-ray diffraction and electron microscopy to study the molecular organization and morphology of macromolecular assemblies formed by three synthetic peptides homologous to beta protein of brain amyloid: beta-(1-28), residues 1-28 of the beta protein; [Ala16]beta-(1-28), beta-(1-28) with alanine substituted for lysine at position 16; and beta-(18-28), residues 18-28 of the beta protein. beta-(1-28) readily formed fibrils in vitro that were similar in ultrastructure to the in vivo amyloid and aggregated into large bundles resembling those of senile plaque cores. X-ray patterns from partially dried, oriented pellets showed a cross-beta-conformation. A series of small-angle, equatorial maxima were consistent with a tubular fibril having a mean diameter of 86 A and a wall composed of pairs of cross-beta-pleated sheets. The data may also be consistent with pairs of cross-beta-sheets that are centered 71-A apart. [Ala16]beta-(1-28) formed beta-pleated sheet assemblies that were dissimilar to in vivo fibrils. The width of the 10-A spacing indicated stacks of about six sheets. Thus, substitution of the uncharged alanine for the positively charged lysine in the beta-strand region enhances the packing of the sheets and dramatically alters the type of macromolecular aggregate formed. beta-(18-28) formed assemblies that had even a greater number of stacked sheets, approximately equal to 24 per diffracting domain as indicated by the sharp intersheet reflection. Our findings on these homologous synthetic assemblies help to define the specific sequence that is required to form Alzheimer-type amyloid fibrils, thus providing an in vitro model of age-related cerebral amyloidogenesis. Images PMID:3477820

  15. Detection of fusobacterium nucleatum and fadA adhesin gene in patients with orthodontic gingivitis and non-orthodontic periodontal inflammation.

    PubMed

    Liu, Ping; Liu, Yi; Wang, Jianning; Guo, Yang; Zhang, Yujie; Xiao, Shuiqing

    2014-01-01

    Fusobacterium nucleatum is one of the most abundant gram-negative bacilli colonizing the subgingival plaque and closely associated with periodontal disease. However it is unclear whether F. nucleatum is involved in gingival inflammation under orthodontic appliance. A novel adhesin, FadA, which is unique to oral Fusobacteria, is required for F. nucleatum binding and invasion to epithelial cells and thus may play an important role in colonization of Fusobacterium in the host. In this study, we evaluated the prevalence of F. nucleatum and its virulence factor FadA adhesion gene (fadA) in 169 subgingival biofilm samples from 55 cases of gingivitis patients with orthodontic appliances, 49 cases of gingivitis patients without orthodontic treatment, 35 cases of periodontitis patients and 30 cases of periodontally healthy people via PCR. The correlations between the F. nucleatum/fadA and gingivitis index(GI)was also analyzed. The detection rate of F. nucleatum/fadA in periodontitis group and non-orthodontic gingivitis group was higher than the other two groups (p<0.01) while it was higher in orthodontic gingivitis group than in health people (p<0.05). An obviously positive correlation was observed between the prevalence of F. nucleatum/fadA and GI. F. nucleatum carrying fadA may be more closely related to the development of gingivitis and periodontal disease compared with orthodontic gingivitis.

  16. Purification, crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding region of the Streptococcus gordonii adhesin GspB

    SciTech Connect

    Pyburn, Tasia M.; Yankovskaya, Victoria; Bensing, Barbara A.; Cecchini, Gary; Sullam, Paul M.; Iverson, T.M.

    2012-07-11

    The carbohydrate-binding region of the bacterial adhesin GspB from Streptococcus gordonii strain M99 (GspB{sub BR}) was expressed in Escherichia coli and purified using affinity and size-exclusion chromatography. Separate sparse-matrix screening of GspB{sub BR} buffered in either 20 mM Tris pH 7.4 or 20 mM HEPES pH 7.5 resulted in different crystallographic behavior such that different precipitants, salts and additives supported crystallization of GspB{sub BR} in each buffer. While both sets of conditions supported crystal growth in space group P2{sub 1}2{sub 1}2{sub 1}, the crystals had distinct unit-cell parameters of a = 33.3, b = 86.7, c = 117.9 {angstrom} for crystal form 1 and a = 34.6, b = 98.3, c = 99.0 {angstrom} for crystal form 2. Additive screening improved the crystals grown in both conditions such that diffraction extended to beyond 2 {angstrom} resolution. A complete data set has been collected to 1.3 {angstrom} resolution with an overall R{sub merge} value of 0.04 and an R{sub merge} value of 0.33 in the highest resolution shell.

  17. The Actinobacillus pleuropneumoniae HMW1C-Like Glycosyltransferase Mediates N-Linked Glycosylation of the Haemophilus influenzae HMW1 Adhesin

    PubMed Central

    Choi, Kyoung-Jae; Grass, Susan; Paek, Seonghee; St. Geme, Joseph W.; Yeo, Hye-Jeong

    2010-01-01

    The Haemophilus influenzae HMW1 adhesin is an important virulence exoprotein that is secreted via the two-partner secretion pathway and is glycosylated at multiple asparagine residues in consensus N-linked sequons. Unlike the heavily branched glycans found in eukaryotic N-linked glycoproteins, the modifying glycan structures in HMW1 are mono-hexoses or di-hexoses. Recent work demonstrated that the H. influenzae HMW1C protein is the glycosyltransferase responsible for transferring glucose and galactose to the acceptor sites of HMW1. An Actinobacillus pleuropneumoniae protein designated ApHMW1C shares high-level homology with HMW1C and has been assigned to the GT41 family, which otherwise contains only O-glycosyltransferases. In this study, we demonstrated that ApHMW1C has N-glycosyltransferase activity and is able to transfer glucose and galactose to known asparagine sites in HMW1. In addition, we found that ApHMW1C is able to complement a deficiency of HMW1C and mediate HMW1 glycosylation and adhesive activity in whole bacteria. Initial structure-function studies suggested that ApHMW1C consists of two domains, including a 15-kDa N-terminal domain and a 55-kDa C-terminal domain harboring glycosyltransferase activity. These findings suggest a new subfamily of HMW1C-like glycosyltransferases distinct from other GT41 family O-glycosyltransferases. PMID:21209858

  18. An adhesin-like protein, Lam29, from Lactobacillus mucosae ME-340 binds to histone H3 and blood group antigens in human colonic mucus.

    PubMed

    Watanabe, Masamichi; Kinoshita, Hideki; Huang, I-Nung; Eguchi, Kei; Tsurumi, Takuya; Kawai, Yasushi; Kitazawa, Haruki; Kimura, Katsunori; Taketomo, Naoki; Kikuchi, Daisuke; Sase, Tomohiko; Miura, Koh; Ogawa, Hitoshi; Shibata, Chikashi; Horii, Akira; Saito, Tadao

    2012-01-01

    A cell-surface 29-kDa protein (Lam29, cysteine-binding protein of the ABC transporter) from Lactobacillus mucosae ME-340 showed an adhesin-like property for human ABO blood group antigens expressed on the gastrointestinal mucosa. In addition, here we report that Lam29 also bound to an 18-kDa protein on human colonic mucus. By ligand blot assay and N-terminal amino acid sequence of the protein, it was identified as human histone H3. By ligand blot and microplate binding assays with recombinant histone H3, binding between Lam29 and histone H3 was confirmed. The adhesion of ME-340 cells to histone H3 was significantly inhibited by 26% after the addition of 2.5 mg/mL Lam29 as compared to the absence of Lam29 (p<0.01). By GHCl extraction and transcription attenuation of ME-340 cells, binding reduction of ME340 cells against histone H3 was detected at 12% and 13% respectively, as compared to control cells by the BIACORE assay (p<0.01). These data indicate that Lam29 shows multiple binding activities to blood group antigens and histone H3 in human colonic mucus. This is the first report to indicate that lactobacilli expressing Lam29 adhere to histone H3 on gastrointestinal mucosa.

  19. Two autonomous structural modules in the fimbrial shaft adhesin FimA mediate Actinomyces interactions with streptococci and host cells during oral biofilm development

    SciTech Connect

    Mishra, Arunima; Devarajan, Bharanidharan; Reardon, Melissa E.; Dwivedi, Prabhat; Krishnan, Vengadesan; Cisar, John O.; Das, Asis; Narayana, Sthanam V.L.; Ton-That, Hung

    2011-09-06

    By combining X-ray crystallography and modelling, we describe here the atomic structure of distinct adhesive moieties of FimA, the shaft fimbrillin of Actinomyces type 2 fimbriae, which uniquely mediates the receptor-dependent intercellular interactions between Actinomyces and oral streptococci as well as host cells during the development of oral biofilms. The FimA adhesin is built with three IgG-like domains, each of which harbours an intramolecular isopeptide bond, previously described in several Gram-positive pilins. Genetic and biochemical studies demonstrate that although these isopeptide bonds are dispensable for fimbrial assembly, cell-cell interactions and biofilm formation, they contribute significantly to the proteolytic stability of FimA. Remarkably, FimA harbours two autonomous adhesive modules, which structurally resemble the Staphylococcus aureus Cna B domain. Each isolated module can bind the plasma glycoprotein asialofetuin as well as the polysaccharide receptors present on the surface of oral streptococci and epithelial cells. Thus, FimA should serve as an excellent paradigm for the development of therapeutic strategies and elucidating the precise molecular mechanisms underlying the interactions between cellular receptors and Gram-positive fimbriae.

  20. Inhibition and Reversal of Microbial Attachment by an Antibody with Parasteric Activity against the FimH Adhesin of Uropathogenic E. coli

    PubMed Central

    Friend, Della; Jalan, Aachal; Gupta, Shivani; Interlandi, Gianluca; Liu, Yan; Tchesnokova, Veronika; Rodriguez, Victoria B.; Sumida, John P.; Strong, Roland K.; Wu, Xue-Ru; Thomas, Wendy E.; Sokurenko, Evgeni V.

    2015-01-01

    Attachment proteins from the surface of eukaryotic cells, bacteria and viruses are critical receptors in cell adhesion or signaling and are primary targets for the development of vaccines and therapeutic antibodies. It is proposed that the ligand-binding pocket in receptor proteins can shift between inactive and active conformations with weak and strong ligand-binding capability, respectively. Here, using monoclonal antibodies against a vaccine target protein - fimbrial adhesin FimH of uropathogenic Escherichia coli, we demonstrate that unusually strong receptor inhibition can be achieved by antibody that binds within the binding pocket and displaces the ligand in a non-competitive way. The non-competitive antibody binds to a loop that interacts with the ligand in the active conformation of the pocket but is shifted away from ligand in the inactive conformation. We refer to this as a parasteric inhibition, where the inhibitor binds adjacent to the ligand in the binding pocket. We showed that the receptor-blocking mechanism of parasteric antibody differs from that of orthosteric inhibition, where the inhibitor replaces the ligand or allosteric inhibition where the inhibitor binds at a site distant from the ligand, and is very potent in blocking bacterial adhesion, dissolving surface-adherent biofilms and protecting mice from urinary bladder infection. PMID:25974133

  1. O-mannosylation of the Mycobacterium tuberculosis adhesin Apa is crucial for T cell antigenicity during infection but is expendable for protection.

    PubMed

    Nandakumar, Subhadra; Kannanganat, Sunil; Dobos, Karen M; Lucas, Megan; Spencer, John S; Fang, Sunan; McDonald, Melissa A; Pohl, Jan; Birkness, Kristin; Chamcha, Venkateswarlu; Ramirez, Melissa V; Plikaytis, Bonnie B; Posey, James E; Amara, Rama Rao; Sable, Suraj B

    2013-01-01

    Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

  2. The bvg-repressed gene brtA, encoding biofilm-associated surface adhesin, is expressed during host infection by Bordetella bronchiseptica.

    PubMed

    Nishikawa, Sayaka; Shinzawa, Naoaki; Nakamura, Keiji; Ishigaki, Keisuke; Abe, Hiroyuki; Horiguchi, Yasuhiko

    2016-02-01

    Bordetella species display phase modulation between Bvg(+) and Bvg(-) phases. Because expression of known virulence factors is up-regulated in the Bvg(+) phase, bacteria in this phase are considered competent for infection. However, the Bvg(-) phase is of negligible importance for infection. No studies have shown that bacterial factors specific to the Bvg(-) phase (bvg-repressed factors) are expressed in the course of Bordetella infection. In the present study, the gene brtA (Bordetella RTX-family Adhesin), which is a typical bvg-repressed gene but is expressed in B. bronchiseptica infecting hosts, was characterized. BrtA is composed of repeated pairs of the VCBS unit and dystroglycan-type cadherin-like unit, the von Willebrand Factor A domain, RTX motif and type I secretion target signal. It is herein demonstrated that BrtA is secreted by the type I secretion system and is essential for Ca(2+) -dependent bacteria-to-substrate adherence, followed by biofilm formation. Although the contribution of BrtA to bacterial colonization of the rat trachea currently remains unclear, this is the first study to present concrete evidence for the expression of a bvg-repressed gene during infection, which may provide a novel aspect for analyses of Bordetella pathogenesis. PMID:26756546

  3. Methicillin-Susceptible Staphylococcus aureus Endocarditis Isolates Are Associated With Clonal Complex 30 Genotype and a Distinct Repertoire of Enterotoxins and Adhesins

    PubMed Central

    Nienaber, Juhsien J.C.; Sharma Kuinkel, Batu K.; Clarke-Pearson, Michael; Lamlertthon, Supaporn; Park, Lawrence; Rude, Thomas H.; Barriere, Steve; Woods, Christopher W.; Chu, Vivian H.; Marín, Mercedes; Bukovski, Suzana; Garcia, Patricia; Corey, G.Ralph; Korman, Tony; Doco-Lecompte, Thanh; Murdoch, David R.; Reller, L. Barth

    2011-01-01

    Background. Using multinational collections of methicillin-susceptible Staphylococcus aureus (MSSA) isolates from infective endocarditis (IE) and soft tissue infections (STIs), we sought to (1) validate the finding that S. aureus in clonal complex (CC) 30 is associated with hematogenous complications and (2) test the hypothesis that specific genetic characteristics in S. aureus are associated with infection severity. Methods. IE and STI isolates from 2 cohorts were frequency matched by geographic origin. Isolates underwent spa typing to infer CC and multiplex polymerase chain reaction for presence of virulence genes. Results. 114 isolate pairs were genotyped. IE isolates were more likely to be CC30 (19.5% vs 6.2%; P = .005) and to contain 3 adhesins (clfB, cna, map/eap; P < .0001 for all) and 5 enterotoxins (tst, sea, sed, see, and sei; P ≤ .005 for all). CC30 isolates were more likely to contain cna, tst, sea, see, seg, and chp (P < .05 for all). Conclusions. MSSA IE isolates were significantly more likely to be CC30 and to possess a distinct repertoire of virulence genes than MSSA STI isolates from the same region. The genetic basis of this association requires further study. PMID:21844296

  4. Characterization of the importance of polysaccharide intercellular adhesin/hemagglutinin of Staphylococcus epidermidis in the pathogenesis of biomaterial-based infection in a mouse foreign body infection model.

    PubMed

    Rupp, M E; Ulphani, J S; Fey, P D; Bartscht, K; Mack, D

    1999-05-01

    The production of biofilm is thought to be crucial in the pathogenesis of prosthetic-device infections caused by Staphylococcus epidermidis. An experimental animal model was used to assess the importance of biofilm production, which is mediated by polysaccharide intercellular adhesin/hemagglutinin (PIA/HA), in the pathogenesis of a biomaterial-based infection. Mice were inoculated along the length of a subcutaneously implanted intravenous catheter with either wild-type S. epidermidis 1457 or its isogenic PIA/HA-negative mutant. The wild-type strain was significantly more likely to cause a subcutaneous abscess than the mutant strain (P < 0.01) and was significantly less likely to be eradicated from the inoculation site by host defense (P < 0.05). In addition, the wild-type strain was found to adhere to the implanted catheters more abundantly than the PIA/HA-negative mutant (P < 0.05). The reliability of the adherence assay was assessed by scanning electron microscopy. To exclude contamination or spontaneous infection, bacterial strains recovered from the experimental animals were compared to inoculation strains by analysis of restriction fragment length polymorphism patterns by pulsed-field gel electrophoresis. In vitro binding of the wild-type strain and its isogenic mutant to a fibronectin-coated surface was similar. These results confirm the importance of biofilm production, mediated by PIA/HA, in the pathogenesis of S. epidermidis experimental foreign body infection.

  5. Detection of fusobacterium nucleatum and fadA adhesin gene in patients with orthodontic gingivitis and non-orthodontic periodontal inflammation.

    PubMed

    Liu, Ping; Liu, Yi; Wang, Jianning; Guo, Yang; Zhang, Yujie; Xiao, Shuiqing

    2014-01-01

    Fusobacterium nucleatum is one of the most abundant gram-negative bacilli colonizing the subgingival plaque and closely associated with periodontal disease. However it is unclear whether F. nucleatum is involved in gingival inflammation under orthodontic appliance. A novel adhesin, FadA, which is unique to oral Fusobacteria, is required for F. nucleatum binding and invasion to epithelial cells and thus may play an important role in colonization of Fusobacterium in the host. In this study, we evaluated the prevalence of F. nucleatum and its virulence factor FadA adhesion gene (fadA) in 169 subgingival biofilm samples from 55 cases of gingivitis patients with orthodontic appliances, 49 cases of gingivitis patients without orthodontic treatment, 35 cases of periodontitis patients and 30 cases of periodontally healthy people via PCR. The correlations between the F. nucleatum/fadA and gingivitis index(GI)was also analyzed. The detection rate of F. nucleatum/fadA in periodontitis group and non-orthodontic gingivitis group was higher than the other two groups (p<0.01) while it was higher in orthodontic gingivitis group than in health people (p<0.05). An obviously positive correlation was observed between the prevalence of F. nucleatum/fadA and GI. F. nucleatum carrying fadA may be more closely related to the development of gingivitis and periodontal disease compared with orthodontic gingivitis. PMID:24416378

  6. Srr2, a multifaceted adhesin expressed by ST-17 hypervirulent Group B Streptococcus involved in binding to both fibrinogen and plasminogen.

    PubMed

    Six, Anne; Bellais, Samuel; Bouaboud, Abdelouhab; Fouet, Agnès; Gabriel, Christelle; Tazi, Asmaa; Dramsi, Shaynoor; Trieu-Cuot, Patrick; Poyart, Claire

    2015-09-01

    The Group B Streptococcus (GBS) 'hypervirulent' ST-17 clone is strongly associated with invasive neonatal meningitis. Comparative genome analyses revealed that the serine-rich repeat (Srr) glycoprotein Srr2 is a cell wall-anchored protein specific for ST-17 strains, the non-ST-17 isolates expressing Srr1. Here, we unravel the binding capacity of GBS Srr proteins to relevant components of the host fibrinolysis pathway. We demonstrate that: (i) Srr2 binds plasminogen and plasmin whereas Srr1 does not; (ii) the ability of ST-17 strains to bind fibrinogen reflects a high level surface display of Srr2 combined with a higher affinity of Srr2 than Srr1 to bind this ligand; and (iii) Srr2 binding to host plasma proteins results in the formation of bacterial aggregates that are efficiently endocytosed by phagocytes. Importantly, we show that Srr2 increased bacterial survival to phagocytic killing and bacterial persistence in a murine model of meningitis. We conclude that Srr2 is a multifaceted adhesin used by the ST-17 clone to hijack ligands of the host coagulation system, thereby contributing to bacterial dissemination and invasiveness, and ultimately to meningitis. PMID:26094503

  7. Presence of fibrinogen-binding adhesin gene in Staphylococcus epidermidis isolates from central venous catheters-associated and orthopaedic implant-associated infections.

    PubMed

    Arciola, Carla Renata; Campoccia, Davide; Gamberini, Simonetta; Donati, M Elena; Montanaro, Lucio

    2004-08-01

    Attention has recently been paid to identify and elucidate those pathogenetic mechanisms, which play a significant role in sustaining the early phases of Staphylococcus epidermidis colonisation and infection development. Several analogies with the physiology of Staphylococcus aureus, a more thoroughly investigated pathogen, have lead to carefully consider all bacterial surface components that mediate cell adhesion. This study aimed at investigating the presence of the fbe gene encoding for a fibrinogen-binding protein in a collection of 107 S. epidermidis strains isolated from orthopaedic infections and 67 from central venous catheter-associated infections. The strains isolated from orthopaedic infections were in large part associated to four different classes of orthopaedic devices, respectively: internal fixation devices, external fixation devices, knee arthroprostheses and hip arthroprostheses. The molecular epidemiology analysis performed by PCR enlightened a statistically significant difference in the prevalence of this adhesion mechanism between orthopaedic infections and catheter-related infections, respectively, of 78% and 91%. The prevalence of fbe ranged from 67% to 91%, suggesting that, even though this adhesin is not strictly necessary for the development of infection, nevertheless it represents a rather common characteristic of strains causing clinical infections, this independently on the presence or the absence of implant materials. PMID:15120529

  8. The bvg-repressed gene brtA, encoding biofilm-associated surface adhesin, is expressed during host infection by Bordetella bronchiseptica.

    PubMed

    Nishikawa, Sayaka; Shinzawa, Naoaki; Nakamura, Keiji; Ishigaki, Keisuke; Abe, Hiroyuki; Horiguchi, Yasuhiko

    2016-02-01

    Bordetella species display phase modulation between Bvg(+) and Bvg(-) phases. Because expression of known virulence factors is up-regulated in the Bvg(+) phase, bacteria in this phase are considered competent for infection. However, the Bvg(-) phase is of negligible importance for infection. No studies have shown that bacterial factors specific to the Bvg(-) phase (bvg-repressed factors) are expressed in the course of Bordetella infection. In the present study, the gene brtA (Bordetella RTX-family Adhesin), which is a typical bvg-repressed gene but is expressed in B. bronchiseptica infecting hosts, was characterized. BrtA is composed of repeated pairs of the VCBS unit and dystroglycan-type cadherin-like unit, the von Willebrand Factor A domain, RTX motif and type I secretion target signal. It is herein demonstrated that BrtA is secreted by the type I secretion system and is essential for Ca(2+) -dependent bacteria-to-substrate adherence, followed by biofilm formation. Although the contribution of BrtA to bacterial colonization of the rat trachea currently remains unclear, this is the first study to present concrete evidence for the expression of a bvg-repressed gene during infection, which may provide a novel aspect for analyses of Bordetella pathogenesis.

  9. Streptococcus gordonii DL1 adhesin SspB V-region mediates coaggregation via receptor polysaccharide of Actinomyces oris T14V.

    PubMed

    Back, C R; Douglas, S K; Emerson, J E; Nobbs, A H; Jenkinson, H F

    2015-10-01

    Streptococcus gordonii SspA and SspB proteins, members of the antigen I/II (AgI/II) family of Streptococcus adhesins, mediate adherence to cysteine-rich scavenger glycoprotein gp340 and cells of other oral microbial species. In this article we investigated further the mechanism of coaggregation between S. gordonii DL1 and Actinomyces oris T14V. Previous mutational analysis of S. gordonii suggested that SspB was necessary for coaggregation with A. oris T14V. We have confirmed this by showing that Lactococcus lactis surrogate host cells expressing SspB coaggregated with A. oris T14V and PK606 cells, while L. lactis cells expressing SspA did not. Coaggregation occurred independently of expression of A. oris type 1 (FimP) or type 2 (FimA) fimbriae. Polysaccharide was prepared from cells of A. oris T14V and found to contain 1,4-, 4,6- and 3,4-linked glucose, 1,4-linked mannose, and 2,4-linked galactose residues. When immobilized onto plastic wells this polysaccharide supported binding of L. lactis expressing SspB, but not binding of L. lactis expressing other AgI/II family proteins. Purified recombinant NAVP region of SspB, comprising amino acid (aa) residues 41-847, bound A. oris polysaccharide but the C-domain (932-1470 aa residues) did not. A site-directed deletion of 29 aa residues (Δ691-718) close to the predicted binding cleft within the SspB V-region ablated binding of the NAVP region to polysaccharide. These results infer that the V-region head of SspB recognizes an actinomyces polysaccharide ligand, so further characterizing a lectin-like coaggregation mechanism occurring between two important primary colonizers.

  10. Differential Roles of Individual Domains in Selection of Secretion Route of a Streptococcus parasanguinis Serine-Rich Adhesin, Fap1▿

    PubMed Central

    Chen, Qiang; Sun, Baiming; Wu, Hui; Peng, Zhixiang; Fives-Taylor, Paula M.

    2007-01-01

    Fimbria-associated protein 1 (Fap1) is a high-molecular-mass glycosylated surface adhesin required for fimbria biogenesis and biofilm formation in Streptococcus parasanguinis. The secretion of mature Fap1 is dependent on the presence of SecA2, a protein with some homology to, but with a different role from, SecA. The signals that direct the secretion of Fap1 to the SecA2-dependent secretion pathway rather than the SecA-dependent secretion pathway have not yet been identified. In this study, Fap1 variants containing different domains were expressed in both secA2 wild-type and mutant backgrounds and were tested for their ability to be secreted by the SecA- or SecA2-dependent pathway. The presence or absence of the cell wall anchor domain (residues 2531 to 2570) at the C terminus did not alter the selection of the Fap1 secretion route. The Fap1 signal peptide (residues 1 to 68) was sufficient to support the secretion of a heterologous protein via the SecA-dependent pathway, suggesting that the signal peptide was sufficient for recognition by the SecA-dependent pathway. The minimal sequences of Fap1 required for the SecA2-dependent pathway included the N-terminal signal peptide, nonrepetitive region I (residues 69 to 102), and part of nonrepetitive region II (residues 169 to 342). The two serine-rich repeat regions (residues 103 to 168 and 505 to 2530) were not required for Fap1 secretion. However, they were both involved in the specific inhibition of Fap1 secretion via the SecA-dependent pathway. PMID:17766425

  11. A Collagen-Binding Adhesin, Acb, and Ten Other Putative MSCRAMM and Pilus Family Proteins of Streptococcus gallolyticus subsp. gallolyticus (Streptococcus bovis Group, Biotype I)▿ §

    PubMed Central

    Sillanpää, Jouko; Nallapareddy, Sreedhar R.; Qin, Xiang; Singh, Kavindra V.; Muzny, Donna M.; Kovar, Christie L.; Nazareth, Lynne V.; Gibbs, Richard A.; Ferraro, Mary J.; Steckelberg, James M.; Weinstock, George M.; Murray, Barbara E.

    2009-01-01

    Members of the Streptococcus bovis group are important causes of endocarditis. However, factors associated with their pathogenicity, such as adhesins, remain uncharacterized. We recently demonstrated that endocarditis-derived Streptococcus gallolyticus subsp. gallolyticus isolates frequently adhere to extracellular matrix (ECM) proteins. Here, we generated a draft genome sequence of an ECM protein-adherent S. gallolyticus subsp. gallolyticus strain and found, by genome-wide analyses, 11 predicted LPXTG-type cell wall-anchored proteins with characteristics of MSCRAMMs, including a modular architecture of domains predicted to adopt immunoglobulin (Ig)-like folding. A recombinant segment of one of these, Acb, showed high-affinity binding to immobilized collagen, and cell surface expression of Acb correlated with the presence of acb and collagen adherence of isolates. Three of the 11 proteins have similarities to major pilus subunits and are organized in separate clusters, each including a second Ig-fold-containing MSCRAMM and a class C sortase, suggesting that the sequenced strain encodes three distinct types of pili. Reverse transcription-PCR demonstrated that all three genes of one cluster, acb-sbs7-srtC1, are cotranscribed, consistent with pilus operons of other gram-positive bacteria. Further analysis detected expression of all 11 genes in cells grown to mid to late exponential growth phases. Wide distribution of 9 of the 11 genes was observed among S. gallolyticus subsp. gallolyticus isolates with fewer genes present in other S. bovis group species/subspecies. The high prevalence of genes encoding putative MSCRAMMs and pili, including a collagen-binding MSCRAMM, among S. gallolyticus subsp. gallolyticus isolates may play an important role in the predominance of this subspecies in S. bovis endocarditis. PMID:19717590

  12. Strain-Specific Variation of the Decorin-Binding Adhesin DbpA Influences the Tissue Tropism of the Lyme Disease Spirochete

    PubMed Central

    Lin, Yi-Pin; Benoit, Vivian; Yang, Xiuli; Martínez-Herranz, Raúl; Pal, Utpal; Leong, John M.

    2014-01-01

    Lyme disease spirochetes demonstrate strain- and species-specific differences in tissue tropism. For example, the three major Lyme disease spirochete species, Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii, are each most commonly associated with overlapping but distinct spectra of clinical manifestations. Borrelia burgdorferi sensu stricto, the most common Lyme spirochete in the U.S., is closely associated with arthritis. The attachment of microbial pathogens to cells or to the extracellular matrix of target tissues may promote colonization and disease, and the Lyme disease spirochete encodes several surface proteins, including the decorin- and dermatan sulfate-binding adhesin DbpA, which vary among strains and have been postulated to contribute to strain-specific differences in tissue tropism. DbpA variants differ in their ability to bind to its host ligands and to cultured mammalian cells. To directly test whether variation in dbpA influences tissue tropism, we analyzed murine infection by isogenic B. burgdorferi strains that encode different dbpA alleles. Compared to dbpA alleles of B. afzelii strain VS461 or B. burgdorferi strain N40-D10/E9, dbpA of B. garinii strain PBr conferred the greatest decorin- and dermatan sulfate-binding activity, promoted the greatest colonization at the inoculation site and heart, and caused the most severe carditis. The dbpA of strain N40-D10/E9 conferred the weakest decorin- and GAG-binding activity, but the most robust joint colonization and was the only dbpA allele capable of conferring significant joint disease. Thus, dbpA mediates colonization and disease by the Lyme disease spirochete in an allele-dependent manner and may contribute to the etiology of distinct clinical manifestations associated with different Lyme disease strains. This study provides important support for the long-postulated model that strain-specific variations of Borrelia surface proteins influence tissue tropism. PMID:25079227

  13. Lysine Residue 117 of the FasG Adhesin of Enterotoxigenic Escherichia coli Is Essential for Binding of 987P Fimbriae to Sulfatide

    PubMed Central

    Choi, Byung-Kwon; Schifferli, Dieter M.

    1999-01-01

    The FasG subunit of the 987P fimbriae of enterotoxigenic strains of Escherichia coli was previously shown to mediate fimbrial binding to a glycoprotein and a sulfatide receptor on intestinal brush borders of piglets. Moreover, the 987P adhesin FasG is required for fimbrial expression, since fasG null mutants are nonfimbriated. In this study, fasG was modified by site-directed mutagenesis to study its sulfatide binding properties. Twenty single mutants were generated by replacing positively charged lysine (K) or arginine (R) residues with small, nonpolar alanine (A) residues. Reduced levels of binding to sulfatide-containing liposomes correlated with reduced fimbriation and FasG surface display in four fasG mutants (R27A, R286A, R226A, and R368). Among the 16 remaining normally fimbriated mutants with wild-type levels of surface-exposed FasG, only one mutant (K117A) did not interact at all with sulfatide-containing liposomes. Four mutants (K117A, R116A, K118A, and R200A) demonstrated reduced binding to such liposomes. Since complete phenotypic dissociation between the structure and specific function of 987P was observed only with mutant K117A, this residue is proposed to play an essential role in the FasG-sulfatide interaction, possibly communicating with the sulfate group of sulfatide by hydrogen bonding and/or salt bridge formation. Residues K17, R116, K118, and R200 may stabilize this interaction. PMID:10531225

  14. Dynamics of Lewis b Binding and Sequence Variation of the babA Adhesin Gene during Chronic Helicobacter pylori Infection in Humans

    PubMed Central

    Nell, Sandra; Kennemann, Lynn; Schwarz, Sandra; Josenhans, Christine

    2014-01-01

    ABSTRACT Helicobacter pylori undergoes rapid microevolution during chronic infection, but very little is known about how this affects host interaction factors. The best-studied adhesin of H. pylori is BabA, which mediates binding to the blood group antigen Lewis b [Le(b)]. To study the dynamics of Le(b) adherence during human infection, we analyzed paired H. pylori isolates obtained sequentially from chronically infected individuals. A complete loss or significant reduction of Le(b) binding was observed in strains from 5 out of 23 individuals, indicating that the Le(b) binding phenotype is quite stable during chronic human infection. Sequence comparisons of babA identified differences due to mutation and/or recombination in 12 out of 16 strain pairs analyzed. Most amino acid changes were found in the putative N-terminal extracellular adhesion domain. One strain pair that had changed from a Le(b) binding to a nonbinding phenotype was used to study the role of distinct sequence changes in Le(b) binding. By transformations of the nonbinding strain with a babA gene amplified from the binding strain, H. pylori strains with mosaic babA genes were generated. Recombinants were enriched for a gain of Le(b) binding by biopanning or for BabA expression on the bacterial surface by pulldown assay. With this approach, we identified several amino acid residues affecting the strength of Le(b) binding. Additionally, the data showed that the C terminus of BabA, which is predicted to encode an outer membrane β-barrel domain, plays an essential role in the biogenesis of this protein. PMID:25516619

  15. Streptococcus pneumoniae Cell-Wall-Localized Phosphoenolpyruvate Protein Phosphotransferase Can Function as an Adhesin: Identification of Its Host Target Molecules and Evaluation of Its Potential as a Vaccine.

    PubMed

    Mizrachi Nebenzahl, Yaffa; Blau, Karin; Kushnir, Tatyana; Shagan, Marilou; Portnoi, Maxim; Cohen, Aviad; Azriel, Shalhevet; Malka, Itai; Adawi, Asad; Kafka, Daniel; Dotan, Shahar; Guterman, Gali; Troib, Shany; Fishilevich, Tali; Gershoni, Jonathan M; Braiman, Alex; Mitchell, Andrea M; Mitchell, Timothy J; Porat, Nurith; Goliand, Inna; Chalifa Caspi, Vered; Swiatlo, Edwin; Tal, Michael; Ellis, Ronald; Elia, Natalie; Dagan, Ron

    2016-01-01

    In Streptococcus pneumonia, phosphoenolpyruvate protein phosphotransferase (PtsA) is an intracellular protein of the monosaccharide phosphotransferase systems. Biochemical and immunostaining methods were applied to show that PtsA also localizes to the bacterial cell-wall. Thus, it was suspected that PtsA has functions other than its main cytoplasmic enzymatic role. Indeed, recombinant PtsA and anti-rPtsA antiserum were shown to inhibit adhesion of S. pneumoniae to cultured human lung adenocarcinoma A549 cells. Screening of a combinatorial peptide library expressed in a filamentous phage with rPtsA identified epitopes that were capable of inhibiting S. pneumoniae adhesion to A549 cells. The insert peptides in the phages were sequenced, and homologous sequences were found in human BMPER, multimerin1, protocadherin19, integrinβ4, epsin1 and collagen type VIIα1 proteins, all of which can be found in A549 cells except the latter. Six peptides, synthesized according to the homologous sequences in the human proteins, specifically bound rPtsA in the micromolar range and significantly inhibited pneumococcal adhesion in vitro to lung- and tracheal-derived cell lines. In addition, the tested peptides inhibited lung colonization after intranasal inoculation of mice with S. pneumoniae. Immunization with rPtsA protected the mice against a sublethal intranasal and a lethal intravenous pneumococcal challenge. In addition, mouse anti rPtsA antiserum reduced bacterial virulence in the intravenous inoculation mouse model. These findings showed that the surface-localized PtsA functions as an adhesin, PtsA binding peptides derived from its putative target molecules can be considered for future development of therapeutics, and rPtsA should be regarded as a candidate for vaccine development.

  16. Streptococcus pneumoniae Cell-Wall-Localized Phosphoenolpyruvate Protein Phosphotransferase Can Function as an Adhesin: Identification of Its Host Target Molecules and Evaluation of Its Potential as a Vaccine

    PubMed Central

    Mizrachi Nebenzahl, Yaffa; Blau, Karin; Kushnir, Tatyana; Shagan, Marilou; Portnoi, Maxim; Cohen, Aviad; Azriel, Shalhevet; Malka, Itai; Adawi, Asad; Kafka, Daniel; Dotan, Shahar; Guterman, Gali; Troib, Shany; Fishilevich, Tali; Gershoni, Jonathan M; Braiman, Alex; Mitchell, Andrea M; Mitchell, Timothy J; Porat, Nurith; Goliand, Inna; Chalifa Caspi, Vered; Swiatlo, Edwin; Tal, Michael; Ellis, Ronald; Elia, Natalie; Dagan, Ron

    2016-01-01

    In Streptococcus pneumonia, phosphoenolpyruvate protein phosphotransferase (PtsA) is an intracellular protein of the monosaccharide phosphotransferase systems. Biochemical and immunostaining methods were applied to show that PtsA also localizes to the bacterial cell-wall. Thus, it was suspected that PtsA has functions other than its main cytoplasmic enzymatic role. Indeed, recombinant PtsA and anti-rPtsA antiserum were shown to inhibit adhesion of S. pneumoniae to cultured human lung adenocarcinoma A549 cells. Screening of a combinatorial peptide library expressed in a filamentous phage with rPtsA identified epitopes that were capable of inhibiting S. pneumoniae adhesion to A549 cells. The insert peptides in the phages were sequenced, and homologous sequences were found in human BMPER, multimerin1, protocadherin19, integrinβ4, epsin1 and collagen type VIIα1 proteins, all of which can be found in A549 cells except the latter. Six peptides, synthesized according to the homologous sequences in the human proteins, specifically bound rPtsA in the micromolar range and significantly inhibited pneumococcal adhesion in vitro to lung- and tracheal-derived cell lines. In addition, the tested peptides inhibited lung colonization after intranasal inoculation of mice with S. pneumoniae. Immunization with rPtsA protected the mice against a sublethal intranasal and a lethal intravenous pneumococcal challenge. In addition, mouse anti rPtsA antiserum reduced bacterial virulence in the intravenous inoculation mouse model. These findings showed that the surface-localized PtsA functions as an adhesin, PtsA binding peptides derived from its putative target molecules can be considered for future development of therapeutics, and rPtsA should be regarded as a candidate for vaccine development. PMID:26990554

  17. The tyrosine gate as a potential entropic lever in the receptor-binding site of the bacterial adhesin FimH.

    PubMed

    Wellens, Adinda; Lahmann, Martina; Touaibia, Mohamed; Vaucher, Jonathan; Oscarson, Stefan; Roy, René; Remaut, Han; Bouckaert, Julie

    2012-06-19

    Uropathogenic Escherichia coli (UPEC) are the major causative agents of urinary tract infections. During infection, UPEC adhere to mannosylated glycoreceptors on the urothelium via the FimH adhesin located at the tip of type 1 pili. Synthetic FimH antiadhesives such as alkyl and phenyl α-D-mannopyranosides are thus ideal candidates for the chemical interception of this crucial step in pathogenesis. The crystal structures of the FimH lectin domain in its ligand-free form and in complexes with eight medium- and high-affinity mannopyranoside inhibitors are presented. The thermodynamic profiles of the FimH-inhibitor interactions indicate that the binding of FimH to α-D-mannopyranose is enthalpy-driven and has a negative entropic change. Addition of a hydrophobic aglycon influences the binding enthalpy and can induce a favorable entropic change. The alleviation of the entropic cost is at least in part explained by increased dynamics in the tyrosine gate (Tyr48 and Tyr137) of the FimH receptor-binding site upon binding of the ligand. Ligands with a phenyl group directly linked to the anomeric oxygen of α-D-mannose introduce the largest dynamics into the Tyr48 side chain, because conjugation with the anomeric oxygen of α-D-mannose forces the aromatic aglycon into a conformation that comes into close contact (≈2.65 Å) with Tyr48. A propargyl group in this position predetermines the orientation of the aglycon and significantly decreases affinity. FimH has the highest affinity for α-D-mannopyranosides substituted with hydrophobic aglycons that are compatible in shape and electrostatic properties to the tyrosine gate, such as heptyl α-D-mannose. PMID:22657089

  18. Analysis of the genetic determinants coding for the S-fimbrial adhesin (sfa) in different Escherichia coli strains causing meningitis or urinary tract infections.

    PubMed

    Ott, M; Hacker, J; Schmoll, T; Jarchau, T; Korhonen, T K; Goebel, W

    1986-12-01

    Recently we have described the molecular cloning of the genetic determinant coding for the S-fimbrial adhesin (Sfa), a sialic acid-recognizing pilus frequently found among extraintestinal Escherichia coli isolates. Fimbriae from the resulting Sfa+ E. coli K-12 clone were isolated, and an Sfa-specific antiserum was prepared. Western blots indicate that S fimbriae isolated from different uropathogenic and meningitis-associated E. coli strains, including O83:K1 isolates, were serologically related. The Sfa-specific antibodies did not cross-react with P fimbriae, but did cross-react with F1C fimbriae. Furthermore the sfa+ recombinant DNAs and some cloned sfa-flanking regions were used as probes in Southern experiments. Chromosomal DNAs isolated from O18:K1 and O83:K1 meningitis strains with and without S fimbriae and from uropathogenic O6:K+ strains were hybridized against these sfa-specific probes. Only one copy of the sfa determinant was identified on the chromosome of these strains. No sfa-specific sequences were observed on the chromosome of E. coli K-12 strains and an O7:K1 isolate. With the exception of small alterations in the sfa-coding region the genetic determinants for S fimbriae were identical in uropathogenic O6:K+ and meningitis O18:K1 and O83:K1 strains. The sfa determinant was also detected on the chromosome of K1 isolates with an Sfa-negative phenotype, and specific cross-hybridization signals were visible after blotting against F1C-specific DNA. In addition homology among the different strains was observed in the sfa-flanking regions.

  19. Strain-specific variation of the decorin-binding adhesin DbpA influences the tissue tropism of the lyme disease spirochete.

    PubMed

    Lin, Yi-Pin; Benoit, Vivian; Yang, Xiuli; Martínez-Herranz, Raúl; Pal, Utpal; Leong, John M

    2014-07-01

    Lyme disease spirochetes demonstrate strain- and species-specific differences in tissue tropism. For example, the three major Lyme disease spirochete species, Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii, are each most commonly associated with overlapping but distinct spectra of clinical manifestations. Borrelia burgdorferi sensu stricto, the most common Lyme spirochete in the U.S., is closely associated with arthritis. The attachment of microbial pathogens to cells or to the extracellular matrix of target tissues may promote colonization and disease, and the Lyme disease spirochete encodes several surface proteins, including the decorin- and dermatan sulfate-binding adhesin DbpA, which vary among strains and have been postulated to contribute to strain-specific differences in tissue tropism. DbpA variants differ in their ability to bind to its host ligands and to cultured mammalian cells. To directly test whether variation in dbpA influences tissue tropism, we analyzed murine infection by isogenic B. burgdorferi strains that encode different dbpA alleles. Compared to dbpA alleles of B. afzelii strain VS461 or B. burgdorferi strain N40-D10/E9, dbpA of B. garinii strain PBr conferred the greatest decorin- and dermatan sulfate-binding activity, promoted the greatest colonization at the inoculation site and heart, and caused the most severe carditis. The dbpA of strain N40-D10/E9 conferred the weakest decorin- and GAG-binding activity, but the most robust joint colonization and was the only dbpA allele capable of conferring significant joint disease. Thus, dbpA mediates colonization and disease by the Lyme disease spirochete in an allele-dependent manner and may contribute to the etiology of distinct clinical manifestations associated with different Lyme disease strains. This study provides important support for the long-postulated model that strain-specific variations of Borrelia surface proteins influence tissue tropism.

  20. Characterization of Inhibitors and Monoclonal Antibodies That Modulate the Interaction between Plasmodium falciparum Adhesin PfRh4 with Its Erythrocyte Receptor Complement Receptor 1*

    PubMed Central

    Lim, Nicholas T. Y.; Harder, Markus J.; Kennedy, Alexander T.; Lin, Clara S.; Weir, Christopher; Cowman, Alan F.; Call, Melissa J.; Schmidt, Christoph Q.; Tham, Wai-Hong

    2015-01-01

    Plasmodium falciparum parasites must invade red blood cells to survive within humans. Entry into red blood cells is governed by interactions between parasite adhesins and red blood cell receptors. Previously we identified that P. falciparum reticulocyte binding protein-like homologue 4 (PfRh4) binds to complement receptor 1 (CR1) to mediate entry of malaria parasites into human red blood cells. In this report we characterize a collection of anti-PfRh4 monoclonal antibodies and CR1 protein fragments that modulate the interaction between PfRh4 and CR1. We identify an anti-PfRh4 monoclonal that blocks PfRh4-CR1 interaction in vitro, inhibits PfRh4 binding to red blood cells, and as a result abolishes the PfRh4-CR1 invasion pathway in P. falciparum. Epitope mapping of anti-PfRh4 monoclonal antibodies identified distinct functional regions within PfRh4 involved in modulating its interaction with CR1. Furthermore, we designed a set of protein fragments based on extensive mutagenesis analyses of the PfRh4 binding site on CR1 and determined their interaction affinities using surface plasmon resonance. These CR1 protein fragments bind tightly to PfRh4 and also function as soluble inhibitors to block PfRh4 binding to red blood cells and to inhibit the PfRh4-CR1 invasion pathway. Our findings can aid future efforts in designing specific single epitope antibodies to block P. falciparum invasion via complement receptor 1. PMID:26324715

  1. Repetitive sequence variations in the promoter region of the adhesin-encoding gene sabA of Helicobacter pylori affect transcription.

    PubMed

    Harvey, Vivian C; Acio, Catherine R; Bredehoft, Amy K; Zhu, Laurence; Hallinger, Daniel R; Quinlivan-Repasi, Vanessa; Harvey, Samuel E; Forsyth, Mark H

    2014-10-01

    The pathogenesis of diseases elicited by the gastric pathogen Helicobacter pylori is partially determined by the effectiveness of adaptation to the variably acidic environment of the host stomach. Adaptation includes appropriate adherence to the gastric epithelium via outer membrane protein adhesins such as SabA. The expression of sabA is subject to regulation via phase variation in the promoter and coding regions as well as repression by the two-component system ArsRS. In this study, we investigated the role of a homopolymeric thymine [poly(T)] tract -50 to -33 relative to the sabA transcriptional start site in H. pylori strain J99. We quantified sabA expression in H. pylori J99 by quantitative reverse transcription-PCR (RT-PCR), demonstrating significant changes in sabA expression associated with experimental manipulations of poly(T) tract length. Mimicking the length increase of this tract by adding adenines instead of thymines had similar effects, while the addition of other nucleotides failed to affect sabA expression in the same manner. We hypothesize that modification of the poly(T) tract changes DNA topology, affecting regulatory protein interaction(s) or RNA polymerase binding efficiency. Additionally, we characterized the interaction between the sabA promoter region and ArsR, a response regulator affecting sabA expression. Using recombinant ArsR in electrophoretic mobility shift assays (EMSA), we localized binding to a sequence with partial dyad symmetry -20 and +38 relative to the sabA +1 site. The control of sabA expression by both ArsRS and phase variation at two distinct repeat regions suggests the control of sabA expression is both complex and vital to H. pylori infection.

  2. Identification of a glycoprotein produced by enterotoxigenic Escherichia coli.

    PubMed

    Lindenthal, C; Elsinghorst, E A

    1999-08-01

    Enterotoxigenic Escherichia coli (ETEC) strain H10407 is capable of invading epithelial cell lines derived from the human ileocecum and colon in vitro. Two separate chromosomally encoded invasion loci (tia and tib) have been cloned from this strain. These loci direct nonadherent and noninvasive laboratory strains of E. coli to adhere to and invade cultured human intestinal epithelial cells. The tib locus directs the synthesis of TibA, a 104-kDa outer membrane protein that is directly correlated with the adherence and invasion phenotypes. TibA is synthesized as a 100-kDa precursor (preTibA) that must be modified for biological activity. Outer membranes of recombinant E. coli expressing TibA or preTibA were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to nitrocellulose. The presence of glycoproteins was detected by oxidization of carbohydrates with periodate and labeling with hydrazide-conjugated digoxigenin. Only TibA could be detected as a glycoprotein. Complementation experiments with tib deletion mutants of ETEC strain H10407 demonstrate that the TibA glycoprotein is expressed in H10407, that the entire tib locus is required for TibA synthesis, and that TibA is the only glycoprotein produced by H10407. Protease treatment of intact H10407 cells removes the carbohydrates on TibA, suggesting that they are surface exposed. TibA shows homology with AIDA-I from diffuse-adhering E. coli and with pertactin precursor from Bordetella pertussis. Both pertactin and AIDA-I are members of the autotransporter family of outer membrane proteins and are afimbrial adhesins that play an important role in the virulence of these organisms. Analysis of the predicted TibA amino acid sequence indicates that TibA is also an autotransporter. Analysis of the tib locus DNA sequence revealed an open reading frame with similarity to RfaQ, a glycosyltransferase. The product of this tib locus open reading frame is proposed to be responsible for Tib

  3. Identification of a Glycoprotein Produced by Enterotoxigenic Escherichia coli

    PubMed Central

    Lindenthal, Christoph; Elsinghorst, Eric A.

    1999-01-01

    Enterotoxigenic Escherichia coli (ETEC) strain H10407 is capable of invading epithelial cell lines derived from the human ileocecum and colon in vitro. Two separate chromosomally encoded invasion loci (tia and tib) have been cloned from this strain. These loci direct nonadherent and noninvasive laboratory strains of E. coli to adhere to and invade cultured human intestinal epithelial cells. The tib locus directs the synthesis of TibA, a 104-kDa outer membrane protein that is directly correlated with the adherence and invasion phenotypes. TibA is synthesized as a 100-kDa precursor (preTibA) that must be modified for biological activity. Outer membranes of recombinant E. coli expressing TibA or preTibA were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to nitrocellulose. The presence of glycoproteins was detected by oxidization of carbohydrates with periodate and labeling with hydrazide-conjugated digoxigenin. Only TibA could be detected as a glycoprotein. Complementation experiments with tib deletion mutants of ETEC strain H10407 demonstrate that the TibA glycoprotein is expressed in H10407, that the entire tib locus is required for TibA synthesis, and that TibA is the only glycoprotein produced by H10407. Protease treatment of intact H10407 cells removes the carbohydrates on TibA, suggesting that they are surface exposed. TibA shows homology with AIDA-I from diffuse-adhering E. coli and with pertactin precursor from Bordetella pertussis. Both pertactin and AIDA-I are members of the autotransporter family of outer membrane proteins and are afimbrial adhesins that play an important role in the virulence of these organisms. Analysis of the predicted TibA amino acid sequence indicates that TibA is also an autotransporter. Analysis of the tib locus DNA sequence revealed an open reading frame with similarity to RfaQ, a glycosyltransferase. The product of this tib locus open reading frame is proposed to be responsible for Tib

  4. Structural insight in histo-blood group binding by the F18 fimbrial adhesin FedF.

    PubMed

    Moonens, Kristof; Bouckaert, Julie; Coddens, Annelies; Tran, Thao; Panjikar, Santosh; De Kerpel, Maia; Cox, Eric; Remaut, Han; De Greve, Henri

    2012-10-01

    F18-positive enterotoxigenic and Shiga toxin-producing Escherichia coli are responsible for post-weaning diarrhoea and oedema disease in pigs and lead to severe production losses in the farming industry. F18 fimbriae attach to the small intestine of young piglets by latching onto glycosphingolipids with A/H blood group determinants on type 1 core. We demonstrate the N-terminal domain of the F18 fimbrial subunit FedF to be responsible for ABH-mediated attachment and present its X-ray structure in ligand-free form and bound to A and B type 1 hexaoses. The FedF lectin domain comprises a 10-stranded immunoglobulin-like β-sandwich. Three linear motives, Q(47) -N(50), H(88) -S(90) and R(117) -T(119), form a shallow glycan binding pocket near the tip of the domain that is selective for type 1 core glycans in extended conformation. In addition to the glycan binding pocket, a polybasic loop on the membrane proximal surface of FedF lectin domain is shown to be required for binding to piglet enterocytes. Although dispensable for ABH glycan recognition, the polybasic surface adds binding affinity in the context of the host cell membrane, a mechanism that is proposed to direct ABH-glycan binding to cell-bound glycosphingolipids and could allow bacteria to avoid clearance by secreted glycoproteins. PMID:22812428

  5. Virulence profiling of Shiga toxin-producing Escherichia coli recovered from domestic farm animals in Northwestern Mexico

    PubMed Central

    Amézquita-López, Bianca A.; Quiñones, Beatriz; Lee, Bertram G.; Chaidez, Cristóbal

    2013-01-01

    Shiga toxin-producing Escherichia coli (STEC) is a zoonotic enteric pathogen that causes human gastrointestinal illnesses. The present study characterized the virulence profiles of O157 and non-O157 STEC strains, recovered from domestic animals in small rural farms within the agricultural Culiacan Valley in Mexico. Virulence genes coding for adhesins, cytotoxins, proteases, subtypes of Shiga toxin (Stx), and other effectors were identified in the STEC strains by PCR. The genotyping analysis revealed the presence of the effectors nleA, nleB, nleE, and nleH1-2, espK, and espN in the O157:H7 and O111:H8 STEC strains. Furthermore, the genes encoding the autoagglutinating adhesin (Saa) and subtilase (SubA) were exclusively identified in the O8:H19 eae-negative strains. The adhesin (iha) and the silent hemolysin (sheA) genes were detected in 79% of the O157 and non-O157 strains. To examine the relative toxicities of the STEC strains, a fluorescent Vero cell line, Vero-d2EGFPs, was employed to measure the inhibition of protein synthesis by Stx. Analysis of culture supernatants from serotype O8:H19 strains with the stx gene profile stx1a, stx2a, and stx2c and serotypes O75:H8 and O146:H8 strains with the stx gene profile stx1a, stx1c, and stx2b, resulted in a significant reduction in the Vero-d2EGFP fluorescent signal. These observations suggest that these non-O157 strains may have an enhanced ability to inhibit protein synthesis in Vero cells. Interestingly, analysis of the stx2c-positive O157:H7 strains resulted in a high fluorescent signal, indicating a reduced toxicity in the Vero-d2EGFP cells. These findings indicate that the O157 and non-O157 STEC strains, recovered in the Culiacan Valley, display distinct virulence profiles and relative toxicities in mammalian cells and have provided information for evaluating risks associated with zoonotic STEC in this agricultural region in Mexico. PMID:24551599

  6. Use of purified F1845 fimbrial adhesin to study localization and expression of receptors for diffusely adhering Escherichia coli during enterocytic differentiation of human colon carcinoma cell lines HT-29 and Caco-2 in culture.

    PubMed Central

    Kerneis, S; Bilge, S S; Fourel, V; Chauviere, G; Coconnier, M H; Servin, A L

    1991-01-01

    Whole diffusely adhering Escherichia coli (DAEC) C1845 cells bearing the F1845 adhesive factor bind diffusely to differentiated human colon carcinoma cell lines HT-29 and Caco-2. By using antibodies directed against the purified fimbrial adhesin F1845 factor, the expression of the DAEC F1845-specific brush border receptors in the polarized human intestinal HT-29 and Caco-2 epithelial cells was studied by indirect immunofluorescence. A low level of DAEC F1845 receptors in undifferentiated intestinal cells was detected; they were localized in a cluster of cells. DAEC F1845 receptors were expressed at a high level in differentiated HT-29 and Caco-2 cells. DAEC F1845 receptors were expressed at a strikingly high level in the apical domains of the cells and developed during enterocytic differentiation in culture, in parallel with the apical expression of the intestinal brush border hydrolase, sucrase-isomaltase. Images PMID:1682255

  7. The membrane expression of Neisseria meningitidis adhesin A (NadA) increases the proimmune effects of MenB OMVs on human macrophages, compared with NadA- OMVs, without further stimulating their proinflammatory activity on circulating monocytes.

    PubMed

    Tavano, Regina; Franzoso, Susanna; Cecchini, Paola; Cartocci, Elena; Oriente, Francesca; Aricò, Beatrice; Papini, Emanuele

    2009-07-01

    Hypervirulent MenB causing fatal human infections frequently display the oligomeric-coiled coil adhesin NadA, a 45-kDa intrinsic outer membrane protein implicated in binding to and invasion of respiratory epithelial cells. A recombinant soluble mutant lacking the 10-kDa COOH terminal membrane domain (NadA(Delta351-405)) also activates human monocytes/macrophages/DCs. As NadA is physiologically released during sepsis as part of OMVs, in this study, we tested the hypothesis that NadA(+) OMVs have an enhanced or modified proinflammatory/proimmune action compared with NadA(-) OMVs. To do this we investigated the activity of purified free NadA(Delta351-405) and of OMVs from MenB and Escherichia coli strains, expressing or not full-length NadA. NadA(Delta351-405) stimulated monocytes and macrophages to secrete cytokines (IL-1beta, TNF-alpha, IL-6, IL-12p40, IL-12p70, IL-10) and chemokines (IL-8, MIP-1alpha, MCP-1, RANTES), and full-length NadA improved MenB OMV activity, preferentially on macrophages, and only increased cytokine release. NadA(Delta351-405) induced the lymphocyte costimulant CD80 in monocytes and macrophages, and NadA(+) OMVs induced a wider set of molecules supporting antigen presentation (CD80, CD86, HLA-DR, and ICAM-1) more efficiently than NadA(-) OMVs only in macrophages. Moreover, membrane NadA effects, unlike NadA(Delta351-405) ones, were much less IFN-gamma-sensitive. The activity of NadA-positive E. coli OMVs was similar to that of control OMVs. NadA in MenB OMVs acted at adhesin concentrations approximately 10(6) times lower than those required to stimulate cells with free NadA(Delta351-405).

  8. A Conserved PapB Family Member, TosR, Regulates Expression of the Uropathogenic Escherichia coli RTX Nonfimbrial Adhesin TosA while Conserved LuxR Family Members TosE and TosF Suppress Motility

    PubMed Central

    Engstrom, Michael D.; Alteri, Christopher J.

    2014-01-01

    A heterogeneous subset of extraintestinal pathogenic Escherichia coli (ExPEC) strains, referred to as uropathogenic E. coli (UPEC), causes most uncomplicated urinary tract infections. However, no core set of virulence factors exists among UPEC strains. Instead, the focus of the analysis of urovirulence has shifted to studying broad classes of virulence factors and the interactions between them. For example, the RTX nonfimbrial adhesin TosA mediates adherence to host cells derived from the upper urinary tract. The associated tos operon is well expressed in vivo but poorly expressed in vitro and encodes TosCBD, a predicted type 1 secretion system. TosR and TosEF are PapB and LuxR family transcription factors, respectively; however, no role has been assigned to these potential regulators. Thus, the focus of this study was to determine how TosR and TosEF regulate tosA and affect the reciprocal expression of adhesins and flagella. Among a collection of sequenced UPEC strains, 32% (101/317) were found to encode TosA, and nearly all strains (91% [92/101]) simultaneously carried the putative regulatory genes. Deletion of tosR alleviates tosA repression. The tos promoter was localized upstream of tosR using transcriptional fusions of putative promoter regions with lacZ. TosR binds to this region, affecting a gel shift. A 100-bp fragment 220 to 319 bp upstream of tosR inhibits binding, suggesting localization of the TosR binding site. TosEF, on the other hand, downmodulate motility when overexpressed by preventing the expression of fliC, encoding flagellin. Deletion of tosEF increased motility. Thus, we present an additional example of the reciprocal control of adherence and motility. PMID:24935980

  9. Prevalence and antimicrobial resistance of porcine O157 and non-O157 Shiga toxin-producing Escherichia coli from India.

    PubMed

    Rajkhowa, Swaraj; Sarma, Dilip Kumar

    2014-08-01

    The aims of this study were to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains in pigs as a possible STEC reservoir in India as well as to characterize the STEC strains and to determine the antimicrobial resistance pattern of the strains. A total of 782 E. coli isolates from clinically healthy (n = 473) and diarrhoeic piglets (309) belonging to major pig-producing states of India were screened by the polymerase chain reaction (PCR) assay for the presence of virulence genes characteristic for STEC, that is, Shiga toxin-producing gene(s) (stx1, stx2), intimin (eae), enterohemolysin (hlyA) and STEC autoagglutinating adhesin (Saa). Overall STEC were detected in 113 (14.4%) piglets, and the prevalence of E. coli O157 and non-O157 STEC were 4 (0.5%) and 109 (13.9%), respectively. None of the O157 STEC isolates carried gene encoding for H7 antigen (fliCh7). The various combinations of virulence genes present in the strains studied were stx1 in 4.6%, stx1 in combination with stx2 gene in 5.1%, stx1 in combination with stx2 and ehxA in 0.6%, stx1 in combination with stx2 and eae in 0.2% and stx2 alone in 3.7%. All STEC isolates were found negative for STEC autoagglutinating adhesin (Saa). The number of STEC isolates which showed resistance to antimicrobials such as ampicillin, tetracycline, streptomycin, lincomycin, nalidixic acid, sulfadiazine, penicillin, gentamicin, kanamycin and ceftriaxone were 100, 99, 98, 97, 95, 94, 92, 88, 85 and 85, respectively. Ninety-seven isolates showed resistance to more than 2 antimicrobials, and 8 resistance groups (R1 to R8) were observed. This study demonstrates that pigs in India harbour both O157 and non-O157 STEC, and this may pose serious public health problems in future.

  10. Platinum nanostructures via self-assembly of an amyloid-like peptide: a novel electrocatalyst for the oxygen reduction

    NASA Astrophysics Data System (ADS)

    Zhou, Binbin; Sun, Zhifang; Li, Ding; Zhang, Ting; Deng, Liu; Liu, You-Nian

    2013-03-01

    An aniline-GGAAKLVFF peptide (AFP) was prepared by solid-phase synthesis. The peptide can readily self-assemble into fibrils. Platinum nanoparticles (Pt NPs) were directly immobilized on the surface of the AFP fibrils via electrostatic interaction. Compared to other currently available techniques for the fabrication of metal-peptide fibrils, the noncovalent functionalization strategy is able to deposit nanoparticles on peptide fibrils with different morphologies and high metal loading, which is important for applications in catalysis, electronic materials and other corresponding fields. The Pt-AFP fibrils were employed to modify the electrode, which exhibits high electrocatalytic activities towards oxygen reduction. Thus, the Pt-AFP fibrils hold great potential for polymer electrolyte fuel cells and other electrochemical applications.An aniline-GGAAKLVFF peptide (AFP) was prepared by solid-phase synthesis. The peptide can readily self-assemble into fibrils. Platinum nanoparticles (Pt NPs) were directly immobilized on the surface of the AFP fibrils via electrostatic interaction. Compared to other currently available techniques for the fabrication of metal-peptide fibrils, the noncovalent functionalization strategy is able to deposit nanoparticles on peptide fibrils with different morphologies and high metal loading, which is important for applications in catalysis, electronic materials and other corresponding fields. The Pt-AFP fibrils were employed to modify the electrode, which exhibits high electrocatalytic activities towards oxygen reduction. Thus, the Pt-AFP fibrils hold great potential for polymer electrolyte fuel cells and other electrochemical applications. Electronic supplementary information (ESI) available: Materials, apparatus, synthesis of AFP and Pt NPs, scheme of AFP and Fig. S1-S3. See DOI: 10.1039/c3nr33998j

  11. Antimicrobial peptide (Cn-AMP2) from liquid endosperm of Cocos nucifera forms amyloid-like fibrillar structure.

    PubMed

    Gour, Shalini; Kaushik, Vibha; Kumar, Vijay; Bhat, Priyanka; Yadav, Subhash C; Yadav, Jay K

    2016-04-01

    Cn-AMP2 is an antimicrobial peptide derived from liquid endosperm of coconut (Cocos nucifera). It consists of 11 amino acid residues and predicted to have high propensity for β-sheet formation that disposes this peptide to be amyloidogenic. In the present study, we have examined the amyloidogenic propensities of Cn-AMP2 in silico and then tested the predictions under in vitro conditions. The in silico study revealed that the peptide possesses high amyloidogenic propensity comparable with Aβ. Upon solubilisation and agitation in aqueous buffer, Cn-AMP2 forms visible aggregates that display bathochromic shift in the Congo red absorbance spectra, strong increase in thioflavin T fluorescence and fibrillar morphology under transmission electron microscopy. All these properties are typical of an amyloid fibril derived from various proteins/peptides including Aβ. PMID:27028204

  12. Dock 'n roll: folding of a silk-inspired polypeptide into an amyloid-like beta solenoid.

    PubMed

    Zhao, Binwu; Cohen Stuart, Martien A; Hall, Carol K

    2016-04-20

    Polypeptides containing the motif ((GA)mGX)n occur in silk and have a strong tendency to self-assemble. For example, polypeptides containing (GAGAGAGX)n, where X = G or H have been observed to form filaments; similar sequences but with X = Q have been used in the design of coat proteins (capsids) for artificial viruses. The structure of the (GAGAGAGX)m filaments has been proposed to be a stack of peptides in a β roll structure with the hydrophobic side chains pointing outwards (hydrophobic shell). Another possible configuration, a β roll or β solenoid structure which has its hydrophobic side chains buried inside (hydrophobic core) was, however, overlooked. We perform ground state analysis as well as atomic-level molecular dynamics simulations, both on single molecules and on two-molecule stacks of the silk-inspired sequence (GAGAGAGQ)10, to decide whether the hydrophobic core or the hydrophobic shell configuration is the most stable one. We find that a stack of two hydrophobic core molecules is energetically more favorable than a stack of two hydrophobic shell molecules. A shell molecule initially placed in a perfect β roll structure tends to rotate its strands, breaking in-plane hydrogen bonds and forming out-of-plane hydrogen bonds, while a core molecule stays in the β roll structure. The hydrophobic shell structure has type II' β turns whereas the core configuration has type II β turns; only the latter secondary structure agrees well with solid-state NMR experiments on a similar sequence (GA)15. We also observe that the core stack has a higher number of intra-molecular hydrogen bonds and a higher number of hydrogen bonds between stack and water than the shell stack. Hence, we conclude that the hydrophobic core configuration is the most likely structure. In the stacked state, each peptide has more intra-molecular hydrogen bonds than a single folded molecule, which suggests that stacking provides the extra stability needed for molecules to reach the folded state.

  13. The architecture of amyloid-like peptide fibrils revealed by X-ray scattering, diffraction and electron microscopy.

    PubMed

    Langkilde, Annette E; Morris, Kyle L; Serpell, Louise C; Svergun, Dmitri I; Vestergaard, Bente

    2015-04-01

    Structural analysis of protein fibrillation is inherently challenging. Given the crucial role of fibrils in amyloid diseases, method advancement is urgently needed. A hybrid modelling approach is presented enabling detailed analysis of a highly ordered and hierarchically organized fibril of the GNNQQNY peptide fragment of a yeast prion protein. Data from small-angle X-ray solution scattering, fibre diffraction and electron microscopy are combined with existing high-resolution X-ray crystallographic structures to investigate the fibrillation process and the hierarchical fibril structure of the peptide fragment. The elongation of these fibrils proceeds without the accumulation of any detectable amount of intermediate oligomeric species, as is otherwise reported for, for example, glucagon, insulin and α-synuclein. Ribbons constituted of linearly arranged protofilaments are formed. An additional hierarchical layer is generated via the pairing of ribbons during fibril maturation. Based on the complementary data, a quasi-atomic resolution model of the protofilament peptide arrangement is suggested. The peptide structure appears in a β-sheet arrangement reminiscent of the β-zipper structures evident from high-resolution crystal structures, with specific differences in the relative peptide orientation. The complexity of protein fibrillation and structure emphasizes the need to use multiple complementary methods. PMID:25849399

  14. The architecture of amyloid-like peptide fibrils revealed by X-ray scattering, diffraction and electron microscopy

    SciTech Connect

    Langkilde, Annette E.; Morris, Kyle L.; Serpell, Louise C.; Svergun, Dmitri I.; Vestergaard, Bente

    2015-04-01

    The aggregation process and the fibril state of an amyloidogenic peptide suggest monomer addition to be the prevailing mechanism of elongation and a model of the peptide packing in the fibrils has been obtained. Structural analysis of protein fibrillation is inherently challenging. Given the crucial role of fibrils in amyloid diseases, method advancement is urgently needed. A hybrid modelling approach is presented enabling detailed analysis of a highly ordered and hierarchically organized fibril of the GNNQQNY peptide fragment of a yeast prion protein. Data from small-angle X-ray solution scattering, fibre diffraction and electron microscopy are combined with existing high-resolution X-ray crystallographic structures to investigate the fibrillation process and the hierarchical fibril structure of the peptide fragment. The elongation of these fibrils proceeds without the accumulation of any detectable amount of intermediate oligomeric species, as is otherwise reported for, for example, glucagon, insulin and α-synuclein. Ribbons constituted of linearly arranged protofilaments are formed. An additional hierarchical layer is generated via the pairing of ribbons during fibril maturation. Based on the complementary data, a quasi-atomic resolution model of the protofilament peptide arrangement is suggested. The peptide structure appears in a β-sheet arrangement reminiscent of the β-zipper structures evident from high-resolution crystal structures, with specific differences in the relative peptide orientation. The complexity of protein fibrillation and structure emphasizes the need to use multiple complementary methods.

  15. Fungi producing significant mycotoxins.

    PubMed

    2012-01-01

    Mycotoxins are secondary metabolites of microfungi that are known to cause sickness or death in humans or animals. Although many such toxic metabolites are known, it is generally agreed that only a few are significant in causing disease: aflatoxins, fumonisins, ochratoxin A, deoxynivalenol, zearalenone, and ergot alkaloids. These toxins are produced by just a few species from the common genera Aspergillus, Penicillium, Fusarium, and Claviceps. All Aspergillus and Penicillium species either are commensals, growing in crops without obvious signs of pathogenicity, or invade crops after harvest and produce toxins during drying and storage. In contrast, the important Fusarium and Claviceps species infect crops before harvest. The most important Aspergillus species, occurring in warmer climates, are A. flavus and A. parasiticus, which produce aflatoxins in maize, groundnuts, tree nuts, and, less frequently, other commodities. The main ochratoxin A producers, A. ochraceus and A. carbonarius, commonly occur in grapes, dried vine fruits, wine, and coffee. Penicillium verrucosum also produces ochratoxin A but occurs only in cool temperate climates, where it infects small grains. F. verticillioides is ubiquitous in maize, with an endophytic nature, and produces fumonisins, which are generally more prevalent when crops are under drought stress or suffer excessive insect damage. It has recently been shown that Aspergillus niger also produces fumonisins, and several commodities may be affected. F. graminearum, which is the major producer of deoxynivalenol and zearalenone, is pathogenic on maize, wheat, and barley and produces these toxins whenever it infects these grains before harvest. Also included is a short section on Claviceps purpurea, which produces sclerotia among the seeds in grasses, including wheat, barley, and triticale. The main thrust of the chapter contains information on the identification of these fungi and their morphological characteristics, as well as factors

  16. Vehicle gas producers

    NASA Astrophysics Data System (ADS)

    Donath, E. E.

    1980-05-01

    The present petroleum supply situation with the possibility of unscheduled interruptions and the definite expectation of continued price increases calls for an investigation of the use of solid fuels for the propulsion of vehicles. The paper reviews the use of solid fuel gas producers with high thermal efficiency on motor vehicles, especially trucks and buses. Some economic comparisons are presented for pre-World War II conditions. Suggestions are made for possible future development of vehicle gas producers. The types of producers are described, along with their performance, special problems, and the importance of fuel properties.

  17. Naturally produced carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Velasco-Santos, C.; Martínez-Hernández, A. L.; Consultchi, A.; Rodríguez, R.; Castaño, V. M.

    2003-05-01

    Carbon nanotubes represent an impressive kind of materials with diverse unexpected properties, and different methods to artificially produce them have been developed. Recently, they have also been synthesized at low temperatures, demonstrating that these materials might exist in fluids or carbon rocks of the Earth's crust. A new type of natural encapsulated carbon nanotubes found in a coal-petroleum mix is presented. These findings show that all allotropic carbon forms known up to date can be produced in Nature, where pressure, catalysts particles, shear stress and parameters other than exclusively very high temperature, seem to play an important role for producing nanotubes.

  18. METHOD OF PRODUCING NEUTRONS

    DOEpatents

    Imhoff, D.H.; Harker, W.H.

    1964-01-14

    This patent relates to a method of producing neutrons in which there is produced a heated plasma containing heavy hydrogen isotope ions wherein heated ions are injected and confined in an elongated axially symmetric magnetic field having at least one magnetic field gradient region. In accordance with the method herein, the amplitude of the field and gradients are varied at an oscillatory periodic frequency to effect confinement by providing proper ratios of rotational to axial velocity components in the motion of said particles. The energetic neutrons may then be used as in a blanket zone containing a moderator and a source fissionable material to produce heat and thermal neutron fissionable materials. (AEC)

  19. Manufacturing and producibility technology

    NASA Technical Reports Server (NTRS)

    Hankins, J. D.; Dreshfield, R. L.

    1985-01-01

    Activities of the manufacturing/producibility working group within the Advanced High-Pressure O2/H2 Technology Program are summarized. The objectives of the M/P working group are: to develop and evaluate process and manufacturing techniques for advanced propulsion hardware design and selected materials; and to optimize the producibility of (SSME) components and assemblies by improved performance, increased life, greater reliability, and/or reduced cost. The technologies being developed include: plasma arc, laser, and inertia welding; combustion chamber and turbine blade coatings; coating processes; high performance alloy electroforming; and process control technology.

  20. Producing CD-ROMs.

    ERIC Educational Resources Information Center

    Hyams, Peter, Ed.

    1992-01-01

    This issue presents 11 articles that address issues relating to the production of CD-ROMs. Highlights include current uses of CD-ROM; standards; steps involved in producing CD-ROMs, including data capture, conversion, and tagging, product design, and indexing; authoring; selecting indexing and retrieval software; costs; multimedia CD-ROMs; and…

  1. Top Hispanic Degree Producers

    ERIC Educational Resources Information Center

    Diverse: Issues in Higher Education, 2012

    2012-01-01

    This article presents a list of the top 100 producers of associate, bachelor's and graduate degrees awarded to minority students based on research conducted by Dr. Victor M.H. Borden, professor of educational leadership and policy students at the Indiana University Bloomington. For the year 2012, the listings focus on Hispanic students. Data for…

  2. Carbapenemase-Producing Enterobacteriaceae

    PubMed Central

    Doi, Yohei; Paterson, David L.

    2015-01-01

    Carbapenemase-producing Enterobacteriaceae (CPE) were almost nonexistent up to the 1990s, but are today encountered routinely in hospitals and other healthcare facilities in many countries including the United States. KPC-producing Klebsiella pneumoniae was the first to emerge and spread globally and is endemic in the United States, Israel, Greece, and Italy. Recently, NDM-producing Enterobacteriaceae and OXA-48-producing K. pneumoniae appear to be disseminating from South Asia and Northern Africa, respectively. They are almost always resistant to all β-lactams including carbapenems and many other classes. Mortality from invasive CPE infections reaches up to 40%. To obtain the maximal benefit from the limited options available, dosing of antimicrobial agents should be optimized based on pharmacokinetic data, especially for colistin and carbapenems. In addition, multiple observational studies have associated combination antimicrobial therapy with lower mortality compared with monotherapy for these infections. The outcomes appear to be especially favorable when patients are treated with a carbapenem and a second agent such as colistin, tigecycline, and gentamicin, but the best approach is yet to be defined. PMID:25643272

  3. PRODUCING HIGH CORN YIELDS.

    ERIC Educational Resources Information Center

    Illinois Univ., Urbana. Coll. of Agriculture.

    RESOURCE MATERIAL ON CORN PRODUCTION FOR HIGH SCHOOL VOCATIONAL AGRICULTURE AND ADULT FARMER CLASSES WAS DESIGNED BY A STATE LEVEL GROUP OF SUBJECT MATTER SPECIALISTS, TEACHER EDUCATORS, SUPERVISORS, AND TEACHERS TO HELP SOLVE PROBLEMS THAT CONFRONT CORN PRODUCERS AT PLANTING TIME. THE SUBJECT MATTER CONCERNS PLANTING TIME, DEPTH, ROW WIDTH,…

  4. Producing superhydrophobic roof tiles.

    PubMed

    Carrascosa, Luis A M; Facio, Dario S; Mosquera, Maria J

    2016-03-01

    Superhydrophobic materials can find promising applications in the field of building. However, their application has been very limited because the synthesis routes involve tedious processes, preventing large-scale application. A second drawback is related to their short-term life under outdoor conditions. A simple and low-cost synthesis route for producing superhydrophobic surfaces on building materials is developed and their effectiveness and their durability on clay roof tiles are evaluated. Specifically, an organic-inorganic hybrid gel containing silica nanoparticles is produced. The nanoparticles create a densely packed coating on the roof tile surface in which air is trapped. This roughness produces a Cassie-Baxter regime, promoting superhydrophobicity. A surfactant, n-octylamine, was also added to the starting sol to catalyze the sol-gel process and to coarsen the pore structure of the gel network, preventing cracking. The application of ultrasound obviates the need to use volatile organic compounds in the synthesis, thereby making a 'green' product. It was also demonstrated that a co-condensation process effective between the organic and inorganic species is crucial to obtain durable and effective coatings. After an aging test, high hydrophobicity was maintained and water absorption was completely prevented for the roof tile samples under study. However, a transition from a Cassie-Baxter to a Wenzel state regime was observed as a consequence of the increase in the distance between the roughness pitches produced by the aging of the coating.

  5. Producing superhydrophobic roof tiles

    NASA Astrophysics Data System (ADS)

    Carrascosa, Luis A. M.; Facio, Dario S.; Mosquera, Maria J.

    2016-03-01

    Superhydrophobic materials can find promising applications in the field of building. However, their application has been very limited because the synthesis routes involve tedious processes, preventing large-scale application. A second drawback is related to their short-term life under outdoor conditions. A simple and low-cost synthesis route for producing superhydrophobic surfaces on building materials is developed and their effectiveness and their durability on clay roof tiles are evaluated. Specifically, an organic-inorganic hybrid gel containing silica nanoparticles is produced. The nanoparticles create a densely packed coating on the roof tile surface in which air is trapped. This roughness produces a Cassie-Baxter regime, promoting superhydrophobicity. A surfactant, n-octylamine, was also added to the starting sol to catalyze the sol-gel process and to coarsen the pore structure of the gel network, preventing cracking. The application of ultrasound obviates the need to use volatile organic compounds in the synthesis, thereby making a ‘green’ product. It was also demonstrated that a co-condensation process effective between the organic and inorganic species is crucial to obtain durable and effective coatings. After an aging test, high hydrophobicity was maintained and water absorption was completely prevented for the roof tile samples under study. However, a transition from a Cassie-Baxter to a Wenzel state regime was observed as a consequence of the increase in the distance between the roughness pitches produced by the aging of the coating.

  6. METHOD OF PRODUCING NEUTRONS

    DOEpatents

    Imhoff, D.H.; Harker, W.H.

    1964-02-01

    A method for producing neutrons is described in which there is employed a confinement zone defined between longitudinally spaced localized gradient regions of an elongated magnetic field. Changed particles and neutralizing electrons, more specifically deuterons and tritons and neutralizng electrons, are injected into the confinement field from ion sources located outside the field. The rotational energy of the parrticles is increased at the gradients by imposing an oscillating transverse electrical field thereacross. The imposition of such oscillating transverse electrical fields improves the reflection capability of such gradient fielda so that the reactive particles are retained more effectively within the zone. With the attainment of appropriate densities of plasma particles and provided that such particles are at a sufficiently high temperature, neutron-producing reactions ensue and large quantities of neutrons emerge from the containment zone. (AEC)

  7. Process for producing silicon

    DOEpatents

    Olson, J.M.; Carleton, K.L.

    1982-06-10

    A process of producing silicon includes forming an alloy of copper and silicon and positioning the alloy in a dried, molten salt electrolyte to form a solid anode structure therein. An electrically conductive cathode is placed in the electrolyte for plating silicon thereon. The electrolyte is then purified to remove dissolved oxides. Finally, an electrical potential is applied between the anode and cathode in an amount sufficient to form substantially pure silicon on the cathode in the form of substantially dense, coherent deposits.

  8. Method of producing imines

    DOEpatents

    Sithambaram, Shanthakumar; Son, Young-Chan; Suib, Steven L.

    2008-04-08

    A method for forming an imine comprises reacting a first reactant comprising a hydroxyl functionality, a carbonyl functionality, or both a hydroxyl functionality and a carbonyl functionality with a second reactant having an amine functionality in the presence of ordered porous manganese-based octahedral molecular sieves and an oxygen containing gas at a temperature and for a time sufficient for the imine to be produced.

  9. Genome Sequencing of the Pyruvate-producing Strain Candida glabrata CCTCC M202019 and Genomic Comparison with Strain CBS138

    PubMed Central

    Xu, Nan; Ye, Chao; Chen, Xiulai; Liu, Jia; Liu, Liming; Chen, Jian

    2016-01-01

    Candida glabrata CCTCC M202019 as an industrial yeast strain that is widely used to produce α-oxocarboxylic acid. Strain M202019 has been proven to have a higher pyruvate-producing capacity than the reference strain CBS138. To characterize the genotype of the M202019 strain, we generated a draft sequence of its genome, which has a size of 12.1 Mbp and a GC content of 38.47%. Evidence accumulated during genome annotation suggests that strain M202019 has strong capacities for glucose transport and pyruvate biosynthesis, defects in pyruvate catabolism, as well as variations in genes involved in nutrient and dicarboxylic acid transport, oxidative phosphorylation, and other relevant aspects of carbon metabolism, which might promote pyruvate accumulation. In addition to differences in its central carbon metabolism, a genomic analysis revealed genetic differences in adhesion metabolism. Forty-nine adhesin-like proteins of strain M202019 were identified classified into seven subfamilies. Decreased amounts of adhesive proteins, and deletions or changes of low-complexity repeats and functional domains might lead to lower adhesion and reduced pathogenicity. Further virulence experiments validated the biological safety of strain M202019. Analysis of the C. glabrata CCTCC M202019 genome sequence provides useful insights into its genetic context, physical characteristics, and potential metabolic capacity. PMID:27713500

  10. The mannose-specific lectin domains of Flo1p from Saccharomyces cerevisiae and Lg-Flo1p from S. pastorianus: crystallization and preliminary X-ray diffraction analysis of the adhesin-carbohydrate complexes.

    PubMed

    Ielasi, Francesco S; Goyal, Parveen; Sleutel, Mike; Wohlkonig, Alexandre; Willaert, Ronnie G

    2013-07-01

    Flo1p and Lg-Flo1p are two cell-wall adhesins belonging to the Flo (flocculation) protein family from the yeasts Saccharomyces cerevisiae and S. pastorianus. The main function of these modular proteins endowed with calcium-dependent lectin activity is to mediate cell-cell adhesion events during yeast flocculation, a process which is well known at the cellular level but still not fully characterized from a molecular perspective. Recently, structural features of the N-terminal Flo lectin domains, including the N-terminal domain of Lg-Flo1p (N-Lg-Flo1p), and their interactions with carbohydrate molecules have been investigated. However, structural data concerning the N-terminal domain of Flo1p (N-Flo1p), which is the most specific among the Flo proteins, are missing and information about the N-Lg-Flo1p-carbohydrate interaction still lacks detailed structural insight. Here, the crystallization and preliminary X-ray characterization of the apo form and the mannose complex of N-Flo1p and X-ray analysis of N-Lg-Flo1p crystals soaked in α-1,2-mannobiose are reported. The N-Flo1p crystals diffracted to a resolution of 1.43 Å in the case of the apo form and to 2.12 Å resolution for the mannose complex. Both crystals were orthorhombic and belonged to space group P212121, with one molecule in the asymmetric unit. The N-Lg-Flo1p-α-1,2-mannobiose complex crystal diffracted to 1.73 Å resolution and belonged to the monoclinic space group P1211 with two molecules in the asymmetric unit.

  11. A multifaceted study of stigma/style cysteine-rich adhesin (SCA)-like Arabidopsis lipid transfer proteins (LTPs) suggests diversified roles for these LTPs in plant growth and reproduction.

    PubMed

    Chae, Keun; Gonong, Benedict J; Kim, Seung-Chul; Kieslich, Chris A; Morikis, Dimitrios; Balasubramanian, Shruthi; Lord, Elizabeth M

    2010-10-01

    Lily stigma/style cysteine-rich adhesin (SCA), a plant lipid transfer protein (LTP) which is secreted into the extracellular matrix, functions in pollen tube guidance in fertilization. A gain-of-function mutant (ltp5-1) for Arabidopsis LTP5, an SCA-like molecule, was recently shown to display defects in sexual reproduction. In the current study, it is reported that ltp5-1 plants have dwarfed primary shoots, delayed hypocotyl elongation, various abnormal tissue fusions, and display multibranching. These mutant phenotypes in vegetative growth are recessive. No abnormality was found in ltp5-1/+ plants. In a phylogenetic analysis of plant LTPs, SCA-like Arabidopsis LTPs were classified with conventional plant LTPs. Homology modelling-based electrostatic similarity index (ESI) clustering was used to show diversity in spatial distributions of electrostatic potentials of SCA-like LTPs, suggestive of their various roles in interaction in the extracellular matrix space. β-Glucuronidase (GUS) analysis showed that SCA-like Arabidopsis LTP genes are diversely present in various tissues. LTP4 was found specifically in the guard cells and LTP6 in trichomes as well as in other tissues. LTP1 levels were specifically abundant in the stigma, and both LTP3 and LTP6 in the ovules. LTP2 and LTP4 gene levels were up-regulated in whole seedlings with 20% polyethylene glycol (PEG) and 300 mM NaCl treatments, respectively. LTP5 was up-regulated in the hypocotyl with 3 d dark growth conditions. LTP6 was specifically expressed in the tip of the cotyledon under drought stress conditions. The results suggest that SCA-like Arabidopsis LTPs are multifunctional, with diversified roles in plant growth and reproduction.

  12. A multifaceted study of stigma/style cysteine-rich adhesin (SCA)-like Arabidopsis lipid transfer proteins (LTPs) suggests diversified roles for these LTPs in plant growth and reproduction

    PubMed Central

    Chae, Keun; Gonong, Benedict J.; Kim, Seung-Chul; Kieslich, Chris A.; Morikis, Dimitrios; Balasubramanian, Shruthi; Lord, Elizabeth M.

    2010-01-01

    Lily stigma/style cysteine-rich adhesin (SCA), a plant lipid transfer protein (LTP) which is secreted into the extracellular matrix, functions in pollen tube guidance in fertilization. A gain-of-function mutant (ltp5-1) for Arabidopsis LTP5, an SCA-like molecule, was recently shown to display defects in sexual reproduction. In the current study, it is reported that ltp5-1 plants have dwarfed primary shoots, delayed hypocotyl elongation, various abnormal tissue fusions, and display multibranching. These mutant phenotypes in vegetative growth are recessive. No abnormality was found in ltp5-1/+ plants. In a phylogenetic analysis of plant LTPs, SCA-like Arabidopsis LTPs were classified with conventional plant LTPs. Homology modelling-based electrostatic similarity index (ESI) clustering was used to show diversity in spatial distributions of electrostatic potentials of SCA-like LTPs, suggestive of their various roles in interaction in the extracellular matrix space. β-Glucuronidase (GUS) analysis showed that SCA-like Arabidopsis LTP genes are diversely present in various tissues. LTP4 was found specifically in the guard cells and LTP6 in trichomes as well as in other tissues. LTP1 levels were specifically abundant in the stigma, and both LTP3 and LTP6 in the ovules. LTP2 and LTP4 gene levels were up-regulated in whole seedlings with 20% polyethylene glycol (PEG) and 300 mM NaCl treatments, respectively. LTP5 was up-regulated in the hypocotyl with 3 d dark growth conditions. LTP6 was specifically expressed in the tip of the cotyledon under drought stress conditions. The results suggest that SCA-like Arabidopsis LTPs are multifunctional, with diversified roles in plant growth and reproduction. PMID:20667964

  13. Immunization with the Haemophilus ducreyi trimeric autotransporter adhesin DsrA with alum, CpG or imiquimod generates a persistent humoral immune response that recognizes the bacterial surface.

    PubMed

    Samo, Melissa; Choudhary, Neelima R; Riebe, Kristina J; Shterev, Ivo; Staats, Herman F; Sempowski, Gregory D; Leduc, Isabelle

    2016-02-24

    The Ducreyi serum resistance A (DsrA) protein of Haemophilus ducreyi belongs to a large family of multifunctional outer membrane proteins termed trimeric autotransporter adhesins responsible for resistance to the bactericidal activity of human complement (serum resistance), agglutination and adhesion. The ability of DsrA to confer serum resistance and bind extracellular matrix proteins lies in its N-terminal passenger domain. We have previously reported that immunization with a recombinant form of the passenger domain of DsrA, rNT-DsrA, in complete/incomplete Freund's adjuvant, protects against a homologous challenge in swine. We present herein the results of an immunogenicity study in mice aimed at investigating the persistence, type of immune response, and the effect of immunization route and adjuvants on surrogates of protection. Our results indicate that a 20 μg dose of rNT-DsrA administered with alum elicited antisera with comparable bacterial surface reactivity to that obtained with complete/incomplete Freund's adjuvant. At that dose, high titers and bacterial surface reactivity persisted for 211 days after the first immunization. Administration of rNT-DsrA with CpG or imiquimod as adjuvants elicited a humoral response with similar quantity and quality of antibodies (Abs) as seen with Freund's adjuvant. Furthermore, intramuscular administration of rNT-DsrA elicited high-titer Abs with significantly higher reactivity to the bacterial surface than those obtained with subcutaneous immunization. All rNT-DsrA/adjuvant combinations tested, save CpG, elicited a Th2-type response. Taken together, these findings show that a 20 μg dose of rNT-DsrA administered with the adjuvants alum, CpG or imiquimod elicits high-quality Abs with reactivity to the bacterial surface that could protect against an H. ducreyi infection. PMID:26812077

  14. 7 CFR 1250.305 - Egg producer or producer.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 10 2014-01-01 2014-01-01 false Egg producer or producer. 1250.305 Section 1250.305... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE EGG RESEARCH AND PROMOTION Egg Research and Promotion Order Definitions § 1250.305 Egg producer or producer. Egg producer or...

  15. 7 CFR 1250.305 - Egg producer or producer.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 10 2013-01-01 2013-01-01 false Egg producer or producer. 1250.305 Section 1250.305... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE EGG RESEARCH AND PROMOTION Egg Research and Promotion Order Definitions § 1250.305 Egg producer or producer. Egg producer or...

  16. 7 CFR 1250.305 - Egg producer or producer.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 10 2012-01-01 2012-01-01 false Egg producer or producer. 1250.305 Section 1250.305... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE EGG RESEARCH AND PROMOTION Egg Research and Promotion Order Definitions § 1250.305 Egg producer or producer. Egg producer or...

  17. 7 CFR 1250.305 - Egg producer or producer.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 10 2011-01-01 2011-01-01 false Egg producer or producer. 1250.305 Section 1250.305... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE EGG RESEARCH AND PROMOTION Egg Research and Promotion Order Definitions § 1250.305 Egg producer or producer. Egg producer or...

  18. 7 CFR 1250.305 - Egg producer or producer.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Egg producer or producer. 1250.305 Section 1250.305... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE EGG RESEARCH AND PROMOTION Egg Research and Promotion Order Definitions § 1250.305 Egg producer or producer. Egg producer or...

  19. ION PRODUCING MECHANISM

    DOEpatents

    Lawrence, E.O.

    1958-09-16

    Improvements are presented in calutron devices and, more specifically, dealswith an improved mounting arrangement fer the ion source of the calutron. An important feature of the invention resides in a pluraiity of insulators so mounted as to be accessible from the exterior of the calutron tank and supporting at their inner ends the ion source. These insutators are arranged in mutually parallel relation and also parallel to the flux of the nmgnetic field, whereby the strain of the supporting elements is reduced to a minimum. In addition the support assembly is secured to a removable wall portion of the task to facilitate withdrawal and examination of the ion producing mechanism.

  20. ION PRODUCING MECHANISM

    DOEpatents

    Backus, J.G.

    1958-09-01

    This patent relates to improvements in calutron devices and particularly describes a novel ion source. The unique feature of this source lies in the shaping of the ionizing electron stream to conform to the arc plasma boundary at the exit slit of the ionization chamber, thereby increasing the ion density produced at the plasma boundary. The particular structure consists of an electron source disposed at onc end of an elongated ionization chambcr and a coilimating electrode positioned to trim the electron stream to a crescent shape before entering the ionization chamber.

  1. Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics

    PubMed Central

    Drori, Ran; Celik, Yeliz; Davies, Peter L.; Braslavsky, Ido

    2014-01-01

    Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 1°C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH. PMID:25008081

  2. Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics.

    PubMed

    Drori, Ran; Celik, Yeliz; Davies, Peter L; Braslavsky, Ido

    2014-09-01

    Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 1°C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH.

  3. Consuming Research, Producing Policy?

    PubMed Central

    Evans, Robert G.; Stoddart, Greg L.

    2003-01-01

    The authors’ 1990 article “Producing Health, Consuming Health Care” presented a conceptual framework for synthesizing a rapidly growing body of findings on the nonmedical determinants of health. The article received a very positive response, and here the authors reflect on what lessons might be learned from that response about the style or content of effective interdisciplinary communication. Much substantive knowledge has been accumulated since 1990, and a number of different frameworks have been developed before and since. The authors situate theirs within this literature and consider how they might have modified it if they “knew then what they know now.” They ask what impact this article, and the much broader stream of research on the determinants of health, has had on public policy? PMID:12604475

  4. APPARATUS FOR PRODUCING SHADOWGRAPHS

    DOEpatents

    Wilson, R.R.

    1959-08-11

    An apparatus is presented for obtaining shadowgraphs or radiographs of an object exposed to x rays or the like. The device includes the combination of a cloud chamber having the interior illuminated and a portion thereof transparent to light rays and x'rays, a controlled source of x rays spaced therefrom, photographic recording disposed laterally of the linear path intermediate the source and the chamber portion in oblique angularity in aspect to the path. The object to be studied is disposed intermediate the x-ray source and chamber in the linear path to provide an x-ray transmission barrier therebetween. The shadowgraph is produced in the cloud chamber in response to initiation of the x- ray source and recorded photographically.

  5. Process for producing ethanol

    SciTech Connect

    Lantero, O.J.; Fish, J.J.

    1993-07-27

    A process is described for producing ethanol from raw materials containing a high dry solid mash level having fermentable sugars or constituents which can be converted into sugars, comprising the steps of: (a) liquefaction of the raw materials in the presence of an alpha amylase to obtain liquefied mash; (b) saccharification of the liquefied mash in the presence of a glucoamylase to obtain hydrolysed starch and sugars; (c) fermentation of the hydrolysed starch and sugars by yeast to obtain ethanol; and (d) recovering the obtained ethanol, wherein an acid fungal protease is introduced to the liquefied mash during the saccharification and/or to the hydrolysed starch and sugars during the fermentation, thereby increasing the rate of production of ethanol as compared to a substantially similar process conducted without the introduction of the protease.

  6. ION PRODUCING MECHANISMS

    DOEpatents

    Brobeck, W.M.

    1959-02-10

    Ion generating means and means for producing ions of material for isotopic separation are discussed. One feature of the invention resides in providing a heater means located in the source block approximately equidistant from a charge reservoir and an arc chamber, whereby the heat distribution in the block is such as to avoid overheating and to maintain the temperature of the various critical localities of the unit at their optimum values. Another feature consists of a pair of plates disposed on either side of the arc chamber exit opening to define a narrow slit for the egression of the ion beam. When the adjacent edges of the plates have become worn, the plates may be detached and reversed to use the opposite edges thereof to define the exit opening.

  7. Insulin-producing cells.

    PubMed

    Schroeder, Insa S; Kania, Gabriela; Blyszczuk, Przemyslaw; Wobus, Anna M

    2006-01-01

    Embryonic stem (ES) cells offer great potential for cell replacement and tissue engineering therapies because of their almost unlimited proliferation capacity and the potential to differentiate into cellular derivatives of all three primary germ layers. This chapter describes a strategy for the in vitro differentiation of mouse ES cells into insulin-producing cells. The three-step protocol does not select for nestin-expressing cells as performed in previous differentiation systems. It includes (1) the spontaneous differentiation of ES cells via embryoid bodies and (2) the formation of progenitor cells of all three primary germ layers (multilineage progenitors) followed by (3) directed differentiation into the pancreatic lineage. The application of growth and extracellular matrix factors, including laminin, nicotinamide, and insulin, leads to the development of committed pancreatic progenitors, which subsequently differentiate into islet-like clusters that release insulin in response to glucose. During differentiation, transcript levels of pancreas-specific transcription factors (i.e., Pdx1, Pax4) and of genes specific for early and mature beta cells, including insulin, islet amyloid pancreatic peptide, somatostatin, and glucagon, are upregulated. C-peptide/insulin-positive islet-like clusters are formed, which release insulin in response to high glucose concentrations at terminal stages. The differentiated cells reveal functional properties with respect to voltage-activated Na+ and ATP-modulated K+ channels and normalize blood glucose levels in streptozotocin-treated diabetic mice. In conclusion, we demonstrate the efficient differentiation of murine ES cells into insulin-producing cells, which may help in the future to establish ES cell-based therapies in diabetes mellitus.

  8. Process for thermochemically producing hydrogen

    DOEpatents

    Bamberger, Carlos E.; Richardson, Donald M.

    1976-01-01

    Hydrogen is produced by the reaction of water with chromium sesquioxide and strontium oxide. The hydrogen producing reaction is combined with other reactions to produce a closed chemical cycle for the thermal decomposition of water.

  9. Coinvasion of dentinal tubules by Porphyromonas gingivalis and Streptococcus gordonii depends upon binding specificity of streptococcal antigen I/II adhesin.

    PubMed

    Love, R M; McMillan, M D; Park, Y; Jenkinson, H F

    2000-03-01

    Cell wall-anchored polypeptides of the antigen I/II family are produced by many species of oral streptococci. These proteins mediate adhesion of streptococci to salivary glycoproteins and to other oral microorganisms and promote binding of cells to collagen type I and invasion of dentinal tubules. Since infections of the root canal system have a mixed anaerobic bacterial etiology, we investigated the hypothesis that coadhesion of anaerobic bacteria with streptococci may facilitate invasive endodontic disease. Porphyromonas gingivalis ATCC 33277 cells were able to invade dentinal tubules when cocultured with Streptococcus gordonii DL1 (Challis) but not when cocultured with Streptococcus mutans NG8. An isogenic noninvasive mutant of S. gordonii, with production of SspA and SspB (antigen I/II family) polypeptides abrogated, was deficient in binding to collagen and had a 40% reduced ability to support adhesion of P. gingivalis. Heterologous expression of the S. mutans SpaP (antigen I/II) protein in this mutant restored collagen binding and tubule invasion but not adhesion to P. gingivalis or the ability to promote P. gingivalis coinvasion of dentin. An isogenic afimbrial mutant of P. gingivalis had 50% reduced binding to S. gordonii cells but was unaffected in the ability to coinvade dentinal tubules with S. gordonii wild-type cells. Expression of the S. gordonii SspA or SspB polypeptide on the surface of Lactococcus lactis cells endowed these bacteria with the abilities to bind P. gingivalis, penetrate dentinal tubules, and promote P. gingivalis coinvasion of dentin. The results demonstrate that collagen-binding and P. gingivalis-binding properties of antigen I/II polypeptides are discrete functions. Specificity of antigen I/II polypeptide recognition accounts for the ability of P. gingivalis to coinvade dentinal tubules with S. gordonii but not with S. mutans. This provides evidence that the specificity of interbacterial coadhesion may influence directly the etiology

  10. Producing VOT contrasts

    NASA Astrophysics Data System (ADS)

    Lofqvist, Anders

    2001-05-01

    The development of voice onset time (VOT) as an acoustic index for studying and classifying stop consonants also prompted a large number of studies examining laryngeal activity and interarticulator timing related to VOT. A collaboration between the Research Institute of Logopedics and Phoniatrics at the University of Tokyo and Haskins Laboratories resulted in a long line of studies using electromyographic and other techniques that provided much of the empirical foundations for what we know about laryngeal function in speech, in particular the production of voiced and voiceless consonants. This presentation will review the articulatory control of VOT differences. To make a consonant voiceless, a speaker uses a combination of glottal abduction and vocal fold tensing. The distinction between voiceless stops with long and short VOT is basically due to a difference in the timing between the glottal abduction gesture and the oral closing and opening gestures. Variations in the size of the glottal gesture also occur. More generally, variations in interarticulator timing between glottal and oral movements are used to produce the different stop categories that occur in the languages of the world. [Work supported by NIH.

  11. Surfactant Protein A (SP-A)-mediated Clearance of Staphylococcus aureus Involves Binding of SP-A to the Staphylococcal Adhesin Eap and the Macrophage Receptors SP-A Receptor 210 and Scavenger Receptor Class A*

    PubMed Central

    Sever-Chroneos, Zvjezdana; Krupa, Agnieszka; Davis, Jeremy; Hasan, Misbah; Yang, Ching-Hui; Szeliga, Jacek; Herrmann, Mathias; Hussain, Muzafar; Geisbrecht, Brian V.; Kobzik, Lester; Chroneos, Zissis C.

    2011-01-01

    Staphylococcus aureus causes life-threatening pneumonia in hospitals and deadly superinfection during viral influenza. The current study investigated the role of surfactant protein A (SP-A) in opsonization and clearance of S. aureus. Previous studies showed that SP-A mediates phagocytosis via the SP-A receptor 210 (SP-R210). Here, we show that SP-R210 mediates binding and control of SP-A-opsonized S. aureus by macrophages. We determined that SP-A binds S. aureus through the extracellular adhesin Eap. Consequently, SP-A enhanced macrophage uptake of Eap-expressing (Eap+) but not Eap-deficient (Eap−) S. aureus. In a reciprocal fashion, SP-A failed to enhance uptake of Eap+ S. aureus in peritoneal Raw264.7 macrophages with a dominant negative mutation (SP-R210(DN)) blocking surface expression of SP-R210. Accordingly, WT mice cleared infection with Eap+ but succumbed to sublethal infection with Eap- S. aureus. However, SP-R210(DN) cells compensated by increasing non-opsonic phagocytosis of Eap+ S. aureus via the scavenger receptor scavenger receptor class A (SR-A), while non-opsonic uptake of Eap− S. aureus was impaired. Macrophages express two isoforms: SP-R210L and SP-R210S. The results show that WT alveolar macrophages are distinguished by expression of SP-R210L, whereas SR-A−/− alveolar macrophages are deficient in SP-R210L expressing only SP-R210S. Accordingly, SR-A−/− mice were highly susceptible to both Eap+ and Eap− S. aureus. The lungs of susceptible mice generated abnormal inflammatory responses that were associated with impaired killing and persistence of S. aureus infection in the lung. In conclusion, alveolar macrophage SP-R210L mediates recognition and killing of SP-A-opsonized S. aureus in vivo, coordinating inflammatory responses and resolution of S. aureus pneumonia through interaction with SR-A. PMID:21123169

  12. Immunogenicity of the Plasmodium falciparum PfEMP1-VarO Adhesin: Induction of Surface-Reactive and Rosette-Disrupting Antibodies to VarO Infected Erythrocytes.

    PubMed

    Guillotte, Micheline; Juillerat, Alexandre; Igonet, Sébastien; Hessel, Audrey; Petres, Stéphane; Crublet, Elodie; Le Scanf, Cécile; Lewit-Bentley, Anita; Bentley, Graham A; Vigan-Womas, Inès; Mercereau-Puijalon, Odile

    2015-01-01

    Adhesion of Plasmodium falciparum-infected red blood cells (iRBC) to human erythrocytes (i.e. rosetting) is associated with severe malaria. Rosetting results from interactions between a subset of variant PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) adhesins and specific erythrocyte receptors. Interfering with such interactions is considered a promising intervention against severe malaria. To evaluate the feasibility of a vaccine strategy targetting rosetting, we have used here the Palo Alto 89F5 VarO rosetting model. PfEMP1-VarO consists of five Duffy-Binding Like domains (DBL1-5) and one Cysteine-rich Interdomain Region (CIDR1). The binding domain has been mapped to DBL1 and the ABO blood group was identified as the erythrocyte receptor. Here, we study the immunogenicity of all six recombinant PfEMP1-VarO domains and the DBL1- CIDR1 Head domain in BALB/c and outbred OF1 mice. Five readouts of antibody responses are explored: ELISA titres on the recombinant antigen, VarO-iRBC immunoblot reactivity, VarO-iRBC surface-reactivity, capacity to disrupt VarO rosettes and the capacity to prevent VarO rosette formation. For three domains, we explore influence of the expression system on antigenicity and immunogenicity. We show that correctly folded PfEMP1 domains elicit high antibody titres and induce a homogeneous response in outbred and BALB/c mice after three injections. High levels of rosette-disrupting and rosette-preventing antibodies are induced by DBL1 and the Head domain. Reduced-alkylated or denatured proteins fail to induce surface-reacting and rosette-disrupting antibodies, indicating that surface epitopes are conformational. We also report limited cross-reactivity between some PfEMP1 VarO domains. These results highlight the high immunogenicity of the individual domains in outbred animals and provide a strong basis for a rational vaccination strategy targeting rosetting. PMID:26222304

  13. Method for producing capsular polysaccharides

    NASA Technical Reports Server (NTRS)

    Kern, Roger G. (Inventor); Petersen, Gene R. (Inventor); Richards, Gil F. (Inventor)

    1994-01-01

    Structurally altered capsular polysaccharides are produced by mutant bacteria. These polysaccharides are isolated by selecting a wild type bacterial strain and a phage producing degradative enzymes that have substrate specificity for the capsular polysaccharides produced by the wild type bacteria. Phage-resistant mutants producing capsular polysaccharides are selected and the structurally altered capsular polysaccharide is isolated therefrom.

  14. Methods for producing complex films, and films produced thereby

    DOEpatents

    Duty, Chad E.; Bennett, Charlee J. C.; Moon, Ji -Won; Phelps, Tommy J.; Blue, Craig A.; Dai, Quanqin; Hu, Michael Z.; Ivanov, Ilia N.; Jellison, Jr., Gerald E.; Love, Lonnie J.; Ott, Ronald D.; Parish, Chad M.; Walker, Steven

    2015-11-24

    A method for producing a film, the method comprising melting a layer of precursor particles on a substrate until at least a portion of the melted particles are planarized and merged to produce the film. The invention is also directed to a method for producing a photovoltaic film, the method comprising depositing particles having a photovoltaic or other property onto a substrate, and affixing the particles to the substrate, wherein the particles may or may not be subsequently melted. Also described herein are films produced by these methods, methods for producing a patterned film on a substrate, and methods for producing a multilayer structure.

  15. Producing Runaway Stars

    NASA Astrophysics Data System (ADS)

    Kohler, Susanna

    2016-07-01

    How are the hypervelocity stars weve observed in our galaxy produced? A recent study suggests that these escapees could be accelerated by a massive black hole in the center of the Large Magellanic Cloud.A Black Hole SlingshotSince their discovery in 2005, weve observed dozens of candidate hypervelocity stars stars whose velocity in the rest frame of our galaxy exceeds the local escape velocity of the Milky Way. These stars present a huge puzzle: how did they attain these enormous velocities?One potential explanation is known as the Hills mechanism. In this process, a stellar binary is disrupted by a close encounter with a massive black hole (like those thought to reside at the center of every galaxy). One member of the binary is flung out of the system as a result of the close encounter, potentially reaching very large velocities.A star-forming region known as LHA 120-N 11, located within the LMC. Some binary star systems within the LMC might experience close encounters with a possible massive black hole at the LMCs center. [ESA/NASA/Hubble]Blame the LMC?Usually, discussions of the Hills mechanism assume that Sagittarius A*, the supermassive black hole at the center of the Milky Way, is the object guilty of accelerating the hypervelocity stars weve observed. But what if the culprit isnt Sgr A*, but a massive black hole at the center of the Large Magellanic Cloud (LMC), one of the Milky Ways satellite galaxies?Though we dont yet have evidence of a massive black hole at the center of the LMC, the dwarf galaxy is large enough to potentially host one as large as 100,000 solar masses. Assuming that it does, two scientists at the University of Cambridge, Douglas Boubert and Wyn Evans, have now modeled how this black hole might tear apart binary star systems and fling hypervelocity stars around the Milky Way.Models for AccelerationBoubert and Evans determined that the LMCs hypothetical black hole could easily eject stars at ~100 km/s, which is the escape velocity of the

  16. Shiga toxin-producing Escherichia coli in beef retail markets from Argentina

    PubMed Central

    Brusa, Victoria; Aliverti, Virginia; Aliverti, Florencia; Ortega, Emanuel E.; de la Torre, Julian H.; Linares, Luciano H.; Sanz, Marcelo E.; Etcheverría, Analía I.; Padola, Nora L.; Galli, Lucía; Peral García, Pilar; Copes, Julio; Leotta, Gerardo A.

    2013-01-01

    Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that cause mild or serious diseases and can lead to people death. This study reports the prevalence and characteristics of STEC O157 and non-O157 in commercial ground beef and environmental samples, including meat table, knife, meat mincing machine, and manipulator hands (n = 450) obtained from 90 retail markets over a nine-month period. The STEC isolates were serotyped and virulence genes as stx (Shiga toxin), rfbO157] (O157 lipopolysaccharide), fliCH7 (H7 flagellin), eae (intimin), ehxA (enterohemolysin) and saa (STEC autoagglutinating adhesin), were determined. STEC O157 were identified in 23 (25.5%) beef samples and 16 (4.4%) environmental samples, while STEC non-O157 were present in 47 (52.2%) and 182 (50.5%), respectively. Among 54 strains isolated, 17 were STEC O157:H7 and 37 were STEC non-O157. The prevalent genotype for O157 was stx2/eae/ehxA/fliCH7 (83.4%), and for STEC non-O157 the most frequent ones were stx1/stx2/saa/ehxA (29.7%); stx2 (29.7%); and stx2/saa/ehxA (27%). None of the STEC non-O157 strains were eae-positive. Besides O157:H7, other 20 different serotypes were identified, being O8:H19, O178:H19, and O174:H28 the prevalent. Strains belonging to the same serotype could be isolated from different sources of the same retail market. Also, the same serotype could be detected in different stores. In conclusion, screening techniques are increasingly sensitive, but the isolation of STEC non-O157 is still a challenge. Moreover, with the results obtained from the present work, although more studies are needed, cross-contamination between meat and the environment could be suspected. PMID:23346554

  17. Diverse Virulence Gene Content of Shiga Toxin-Producing Escherichia coli from Finishing Swine

    PubMed Central

    Fratamico, Pina M.; Bagi, Lori; Delannoy, Sabine; Fach, Patrick; Manning, Shannon D.; Funk, Julie A.

    2014-01-01

    Shiga toxin-producing Escherichia coli (STEC) infections are a critical public health concern because they can cause severe clinical outcomes, such as hemolytic uremic syndrome, in humans. Determining the presence or absence of virulence genes is essential in assessing the potential pathogenicity of STEC strains. Currently, there is limited information about the virulence genes carried by swine STEC strains; therefore, this study was conducted to examine the presence and absence of 69 virulence genes in STEC strains recovered previously from finishing swine in a longitudinal study. A subset of STEC strains was analyzed by pulsed-field gel electrophoresis (PFGE) to examine their genetic relatedness. Swine STEC strains (n = 150) were analyzed by the use of a high-throughput real-time PCR array system, which included 69 virulence gene targets. Three major pathotypes consisted of 16 different combinations of virulence gene profiles, and serotypes were determined in the swine STEC strains. The majority of the swine STEC strains (n = 120) belonged to serotype O59:H21 and carried the same virulence gene profile, which consisted of 9 virulence genes: stx2e, iha, ecs1763, lpfAO113, estIa (STa), ehaA, paa, terE, and ureD. The eae, nleF, and nleH1-2 genes were detected in one swine STEC strain (O49:H21). Other genes encoding adhesins, including iha, were identified (n = 149). The PFGE results demonstrated that swine STEC strains from pigs raised in the same finishing barn were closely related. Our results revealed diverse virulence gene contents among the members of the swine STEC population and enhance understanding of the dynamics of transmission of STEC strains among pigs housed in the same barn. PMID:25107960

  18. Method of producing submicron size particles and product produced thereby

    DOEpatents

    Bourne, R.S.; Eichman, C.C.; Welbon, W.W.

    1988-05-11

    Submicron size particles are produced by using a sputtering process to deposit particles into a liquid. The liquid is processed to recover the particles therefrom, and the particles have sizes in the range of twenty to two hundred Angstroms. Either metallic or non-metallic particles can be produced, and the metallic particles can be used in ''metallic inks.'' 4 figs.

  19. Produce handling and processing practices.

    PubMed Central

    Beuchat, L. R.; Ryu, J. H.

    1997-01-01

    In the past decade, outbreaks of human illness associated with the consumption of raw vegetables and fruits (or unpasteurized products produced from them) have increased in the United States. Changes in agronomic, harvesting, distribution, processing, and consumption patterns and practices have undoubtedly contributed to this increase. Pathogens such as Listeria monocytogenes, Clostridium botulinum, and Bacillus cereus are naturally present in some soil, and their presence on fresh produce is not rare. Salmonella, Escherichia coli O157:H7, Campylobacter jejuni, Vibrio cholerae, parasites, and viruses are more likely to contaminate fresh produce through vehicles such as raw or improperly composted manure, irrigation water containing untreated sewage, or contaminated wash water. Contact with mammals, reptiles, fowl, insects, and unpasteurized products of animal origin offers another avenue through which pathogens can access produce. Surfaces, including human hands, which come in contact with whole or cut produce represent potential points of contamination throughout the total system of growing, harvesting, packing, processing, shipping, and preparing produce for consumption. Treatment of produce with chlorinated water reduces populations of pathogenic and other microorganisms on fresh produce but cannot eliminate them. Reduction of risk for human illness associated with raw produce can be better achieved through controlling points of potential contamination in the field; during harvesting; during processing or distribution; or in retail markets, food-service facilities, or the home. PMID:9366597

  20. Escherichia coli Common Pilus (ECP) Targets Arabinosyl Residues in Plant Cell Walls to Mediate Adhesion to Fresh Produce Plants*

    PubMed Central

    Rossez, Yannick; Holmes, Ashleigh; Lodberg-Pedersen, Henriette; Birse, Louise; Marshall, Jacqueline; Willats, William G. T.; Toth, Ian K.; Holden, Nicola J.

    2014-01-01

    Outbreaks of verotoxigenic Escherichia coli are often associated with fresh produce. However, the molecular basis to adherence is unknown beyond ionic lipid-flagellum interactions in plant cell membranes. We demonstrate that arabinans present in different constituents of plant cell walls are targeted for adherence by E. coli common pilus (ECP; or meningitis-associated and temperature-regulated (Mat) fimbriae) for E. coli serotypes O157:H7 and O18:K1:H7. l-Arabinose is a common constituent of plant cell wall that is rarely found in other organisms, whereas ECP is widespread in E. coli and other environmental enteric species. ECP bound to oligosaccharides of at least arabinotriose or longer in a glycan array, plant cell wall pectic polysaccharides, and plant glycoproteins. Recognition overlapped with the antibody LM13, which binds arabinanase-sensitive pectic epitopes, and showed a preferential affinity for (1→5)-α-linked l-arabinosyl residues and longer chains of arabinan as demonstrated with the use of arabinan-degrading enzymes. Functional adherence in planta was mediated by the adhesin EcpD in combination with the structural subunit, EcpA, and expression was demonstrated with an ecpR–GFP fusion and ECP antibodies. Spinach was found to be enriched for ECP/LM13 targets compared with lettuce. Specific recognition of arabinosyl residues may help explain the persistence of E. coli in the wider environment and association of verotoxigenic E. coli with some fresh produce plants by exploitation of a glycan found only in plant, not animal, cells. PMID:25320086

  1. Escherichia coli common pilus (ECP) targets arabinosyl residues in plant cell walls to mediate adhesion to fresh produce plants.

    PubMed

    Rossez, Yannick; Holmes, Ashleigh; Lodberg-Pedersen, Henriette; Birse, Louise; Marshall, Jacqueline; Willats, William G T; Toth, Ian K; Holden, Nicola J

    2014-12-01

    Outbreaks of verotoxigenic Escherichia coli are often associated with fresh produce. However, the molecular basis to adherence is unknown beyond ionic lipid-flagellum interactions in plant cell membranes. We demonstrate that arabinans present in different constituents of plant cell walls are targeted for adherence by E. coli common pilus (ECP; or meningitis-associated and temperature-regulated (Mat) fimbriae) for E. coli serotypes O157:H7 and O18:K1:H7. l-Arabinose is a common constituent of plant cell wall that is rarely found in other organisms, whereas ECP is widespread in E. coli and other environmental enteric species. ECP bound to oligosaccharides of at least arabinotriose or longer in a glycan array, plant cell wall pectic polysaccharides, and plant glycoproteins. Recognition overlapped with the antibody LM13, which binds arabinanase-sensitive pectic epitopes, and showed a preferential affinity for (1→5)-α-linked l-arabinosyl residues and longer chains of arabinan as demonstrated with the use of arabinan-degrading enzymes. Functional adherence in planta was mediated by the adhesin EcpD in combination with the structural subunit, EcpA, and expression was demonstrated with an ecpR-GFP fusion and ECP antibodies. Spinach was found to be enriched for ECP/LM13 targets compared with lettuce. Specific recognition of arabinosyl residues may help explain the persistence of E. coli in the wider environment and association of verotoxigenic E. coli with some fresh produce plants by exploitation of a glycan found only in plant, not animal, cells.

  2. Method for producing a borohydride

    DOEpatents

    Kong, Peter C.

    2010-06-22

    A method for producing a borohydride is described that includes the steps of providing a source of borate; providing a material that chemically reduces the source of the borate to produce a borohydride; and reacting the source of the borate and the material by supplying heat at a temperature that substantially effects the production of the borohydride.

  3. Method for producing a borohydride

    DOEpatents

    Kong, Peter C.

    2008-09-02

    A method for producing a borohydride is described and which includes the steps of providing a source of borate; providing a material which chemically reduces the source of the borate to produce a borohydride; and reacting the source of borate and the material by supplying heat at a temperature which substantially effects the production of the borohydride.

  4. Method of producing molybdenum-99

    DOEpatents

    Pitcher, Eric John

    2013-05-28

    Method of producing molybdenum-99, comprising accelerating ions by means of an accelerator; directing the ions onto a metal target so as to generate neutrons having an energy of greater than 10 MeV; directing the neutrons through a converter material comprising techentium-99 to produce a mixture comprising molybdenum-99; and, chemically extracting the molybdenum-99 from the mixture.

  5. Microbial safety of fresh produce

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The book entitled “Microbial Safety of Fresh Produce” with 23 chapters is divided into following six sections: Microbial contamination of fresh produce, Pre-harvest strategies, post-harvest interventions, Produce safety during processing and handling, Public, legal, and economic Perspectives, and Re...

  6. Prevalence and Characteristics of the Epidemic Multiresistant Escherichia coli ST131 Clonal Group among Extended-Spectrum Beta-Lactamase-Producing E. coli Isolates in Copenhagen, Denmark

    PubMed Central

    Hansen, Dennis S.; Nilsson, Frida; Frimodt-Møller, Jakob; Leihof, Rikke Fleron; Struve, Carsten; Scheutz, Flemming; Johnston, Brian; Krogfelt, Karen A.; Johnson, James R.

    2013-01-01

    We report the characteristics of 115 extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli clinical isolates, from 115 unique Danish patients, over a 1-year study interval (1 October 2008 to 30 September 2009). Forty-four (38%) of the ESBL isolates represented sequence type 131 (ST13)1, from phylogenetic group B2. The remaining 71 isolates were from phylogenetic groups D (27%), A (22%), B1 (10%), and B2 (3%). Serogroup O25 ST131 isolates (n = 42; 95% of ST131) comprised 7 different K antigens, whereas two ST131 isolates were O16:K100:H5. Compared to non-ST131 isolates, ST131 isolates were associated positively with CTX-M-15 and negatively with CTX-M-1 and CTX-M-14. They also were associated positively with 11 virulence genes, including afa and dra (Dr family adhesins), the F10 papA allele (P fimbria variant), fimH (type 1 fimbriae), fyuA (yersiniabactin receptor), iha (adhesin siderophore), iutA (aerobactin receptor), kpsM II (group 2 capsules), malX (pathogenicity island marker), ompT (outer membrane protease), sat (secreted autotransporter toxin), and usp (uropathogenicity-specific protein) and negatively with hra (heat-resistant agglutinin) and iroN (salmochelin receptor). The consensus virulence gene profile (>90% prevalence) of the ST131 isolates included fimH, fyuA, malX, and usp (100% each), ompT and the F10 papA allele (95% each), and kpsM II and iutA (93% each). ST131 isolates were also positively associated with community acquisition, extraintestinal pathogenic E. coli (ExPEC) status, and the O25, K100, and H4 antigens. Thus, among ESBL E. coli isolates in Copenhagen, ST131 was the most prevalent clonal group, was community associated, and exhibited distinctive and comparatively extensive virulence profiles, plus a greater variety of capsular antigens than reported previously. PMID:23554186

  7. Interactions of O157 and non-O157 Shiga toxin-producing Escherichia coli (STEC) recovered from bovine hide and carcass with human cells and abiotic surfaces.

    PubMed

    Matheus-Guimarães, Cecilia; Gonçalves, Evanilde Maria; Cabilio Guth, Beatriz E

    2014-03-01

    Different structures related to biofilm formation by Shiga toxin-producing Escherichia coli (STEC), particularly O157 strains, have been described, but there are few data regarding their involvement in non-O157 strains. The aim of this study was to determine the ability of 14 O157 and 8 non-O157 strains isolated from bovine hide and carcass to interact with biotic and abiotic surfaces and also to evaluate the role of different adhesins. Biofilm formation assays showed that four O157 and two non-O157 strains were able to adhere to glass, and that only one O157 strain adhered to polystyrene. Reverse transcriptase-polymerase chain reaction was carried out using biofilm-forming strains to determine the expression of antigen 43 (Ag43), curli, type 1 fimbriae, STEC autotransporter contributing to biofilm formation (Sab), calcium-binding antigen 43 homologue (Cah), and autotransporter protein of enterohemorrhagic E. coli (EhaA). Most of these structures were expressed under biofilm conditions. However, the lack of Ag43 in one non-O157 strain, as well as Cah and EhaA in two O157 strains, suggests that other adhesins are involved in biofilm formation in these strains. Despite the fact that adherence to HeLa cells was detected in 20 strains (91%), it was not possible to correlate biofilm formation with adherence patterns. Invasiveness in T84 and Caco-2 cells was observed in four and three O157 strains, respectively. Altogether, we showed that there are different sets of genes involved in the interactions of STEC with biotic and abiotic surfaces. Interestingly, one O157 strain that was able to form biofilm on both glass and polystyrene also adhered to and invaded human cells, indicating an important route for its persistence in the environment and interaction with the host. Additionally, the ability of non-O157 strains not carrying the LEE pathogenicity island to form biofilm highlights an industrial and health problem that cannot be neglected.

  8. Prevalence and characteristics of the epidemic multiresistant Escherichia coli ST131 clonal group among extended-spectrum beta-lactamase-producing E. coli isolates in Copenhagen, Denmark.

    PubMed

    Olesen, Bente; Hansen, Dennis S; Nilsson, Frida; Frimodt-Møller, Jakob; Leihof, Rikke Fleron; Struve, Carsten; Scheutz, Flemming; Johnston, Brian; Krogfelt, Karen A; Johnson, James R

    2013-06-01

    We report the characteristics of 115 extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli clinical isolates, from 115 unique Danish patients, over a 1-year study interval (1 October 2008 to 30 September 2009). Forty-four (38%) of the ESBL isolates represented sequence type 131 (ST13)1, from phylogenetic group B2. The remaining 71 isolates were from phylogenetic groups D (27%), A (22%), B1 (10%), and B2 (3%). Serogroup O25 ST131 isolates (n = 42; 95% of ST131) comprised 7 different K antigens, whereas two ST131 isolates were O16:K100:H5. Compared to non-ST131 isolates, ST131 isolates were associated positively with CTX-M-15 and negatively with CTX-M-1 and CTX-M-14. They also were associated positively with 11 virulence genes, including afa and dra (Dr family adhesins), the F10 papA allele (P fimbria variant), fimH (type 1 fimbriae), fyuA (yersiniabactin receptor), iha (adhesin siderophore), iutA (aerobactin receptor), kpsM II (group 2 capsules), malX (pathogenicity island marker), ompT (outer membrane protease), sat (secreted autotransporter toxin), and usp (uropathogenicity-specific protein) and negatively with hra (heat-resistant agglutinin) and iroN (salmochelin receptor). The consensus virulence gene profile (>90% prevalence) of the ST131 isolates included fimH, fyuA, malX, and usp (100% each), ompT and the F10 papA allele (95% each), and kpsM II and iutA (93% each). ST131 isolates were also positively associated with community acquisition, extraintestinal pathogenic E. coli (ExPEC) status, and the O25, K100, and H4 antigens. Thus, among ESBL E. coli isolates in Copenhagen, ST131 was the most prevalent clonal group, was community associated, and exhibited distinctive and comparatively extensive virulence profiles, plus a greater variety of capsular antigens than reported previously.

  9. Producing Multiculturalism in Higher Education: Who's Producing What for Whom?

    ERIC Educational Resources Information Center

    Urciuoli, Bonnie

    1999-01-01

    Explores the ways in which multiculturalism is institutionally produced in a small liberal arts college. States that administrative concerns with multiculturalism are shaped by the market in students; academic multiculturalist concerns are shaped by the market in textual production. (CMK)

  10. Methods of producing cesium-131

    SciTech Connect

    Meikrantz, David H; Snyder, John R

    2012-09-18

    Methods of producing cesium-131. The method comprises dissolving at least one non-irradiated barium source in water or a nitric acid solution to produce a barium target solution. The barium target solution is irradiated with neutron radiation to produce cesium-131, which is removed from the barium target solution. The cesium-131 is complexed with a calixarene compound to separate the cesium-131 from the barium target solution. A liquid:liquid extraction device or extraction column is used to separate the cesium-131 from the barium target solution.

  11. Cellulase producing microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1997-01-01

    Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  12. Cellulase producing microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1997-12-30

    Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  13. Microorganisms for producing organic acids

    DOEpatents

    Pfleger, Brian Frederick; Begemann, Matthew Brett

    2014-09-30

    Organic acid-producing microorganisms and methods of using same. The organic acid-producing microorganisms comprise modifications that reduce or ablate AcsA activity or AcsA homolog activity. The modifications increase tolerance of the microorganisms to such organic acids as 3-hydroxypropionic acid, acrylic acid, propionic acid, lactic acid, and others. Further modifications to the microorganisms increase production of such organic acids as 3-hydroxypropionic acid, lactate, and others. Methods of producing such organic acids as 3-hydroxypropionic acid, lactate, and others with the modified microorganisms are provided. Methods of using acsA or homologs thereof as counter-selectable markers are also provided.

  14. Jet Shockwaves Produce Gamma Rays

    NASA Video Gallery

    Theorists believe that GRB jets produce gamma rays by two processes involving shock waves. Shells of material within the jet move at different speeds and collide, generating internal shock waves th...

  15. Methods of producing transportation fuel

    DOEpatents

    Nair, Vijay; Roes, Augustinus Wilhelmus Maria; Cherrillo, Ralph Anthony; Bauldreay, Joanna M.

    2011-12-27

    Systems, methods, and heaters for treating a subsurface formation are described herein. At least one method for producing transportation fuel is described herein. The method for producing transportation fuel may include providing formation fluid having a boiling range distribution between -5.degree. C. and 350.degree. C. from a subsurface in situ heat treatment process to a subsurface treatment facility. A liquid stream may be separated from the formation fluid. The separated liquid stream may be hydrotreated and then distilled to produce a distilled stream having a boiling range distribution between 150.degree. C. and 350.degree. C. The distilled liquid stream may be combined with one or more additives to produce transportation fuel.

  16. Molecular Characterization of Enterotoxin-Producing Escherichia coli Collected in 2011–2012, Russia

    PubMed Central

    Kartsev, Nikolay N.; Fursova, Nadezhda K.; Pachkunov, Dmitry M.; Bannov, Vasiliy A.; Eruslanov, Boris V.; Svetoch, Edward A.; Dyatlov, Ivan A.

    2015-01-01

    Enterotoxin-producing Escherichia coli (ETEC) are one of the main causative agents of diarrhea in children especially in developing countries and travel diarrhoea in adults. Pathogenic properties of ETEC associated with their ability to produce a heat-stable (ST) and/or heat-labile (LT) enterotoxins, as well as adhesins providing bacterial adhesion to intestinal epithelial cells. This study presents the molecular characterization of the ETEC isolates collected from the Central and Far-Eastern regions of Russia in 2011–2012. It was shown that all ETEC under study (n=18) had the heat-labile enterotoxin-coding operon elt, and had no the genes of the heat-stable enterotoxin operon est. DNA sequencing revealed two types of nucleotide exchanges in the eltB gene coding subunit B of LT in isolates collected from Cherepovets city (Central region, Russia) and Vladivostok city (Far-East region, Russia). Only one ETEC strain carried genes cfaA, cfaB, cfaC and cfaD coding adhesion factor CFA/I. Expression of LT in four ETEC isolates in the agglutination reaction was detected using a latex test-system. The isolates were assigned to serogroups O142 (n = 6), О6 (n = 4), О25 (n = 5), О26 (n = 2), and O115 (n = 1). Genotyping showed that they belonged to an earlier described sequence-type ST4 (n = 3) as well as to 11 novel sequence-types ST1043, ST1312, ST3697, ST3707, ST3708, ST3709, ST3710, ST3755, ST3756, ST3757 and ST4509. The ETEC isolates displayed different levels of antimicrobial resistance. Eight isolates were resistant to only one drug, three isolates—to two drugs, one isolate—to three drugs, two isolates—to four antibacterials, and only one isolate to each of the five, six and ten antibacterials simultaneously. Genetic determinants of the resistance to beta-lactams and other classes of antibacterials on the ETEC genomes were identified. There are blaTEM (n = 10), blaCTX-M-15 (n = 1), class 1 integron (n = 3) carrying resistance cassettes to aminoglycosides and

  17. Molecular Characterization of Enterotoxin-Producing Escherichia coli Collected in 2011-2012, Russia.

    PubMed

    Kartsev, Nikolay N; Fursova, Nadezhda K; Pachkunov, Dmitry M; Bannov, Vasiliy A; Eruslanov, Boris V; Svetoch, Edward A; Dyatlov, Ivan A

    2015-01-01

    Enterotoxin-producing Escherichia coli (ETEC) are one of the main causative agents of diarrhea in children especially in developing countries and travel diarrhoea in adults. Pathogenic properties of ETEC associated with their ability to produce a heat-stable (ST) and/or heat-labile (LT) enterotoxins, as well as adhesins providing bacterial adhesion to intestinal epithelial cells. This study presents the molecular characterization of the ETEC isolates collected from the Central and Far-Eastern regions of Russia in 2011-2012. It was shown that all ETEC under study (n=18) had the heat-labile enterotoxin-coding operon elt, and had no the genes of the heat-stable enterotoxin operon est. DNA sequencing revealed two types of nucleotide exchanges in the eltB gene coding subunit B of LT in isolates collected from Cherepovets city (Central region, Russia) and Vladivostok city (Far-East region, Russia). Only one ETEC strain carried genes cfaA, cfaB, cfaC and cfaD coding adhesion factor CFA/I. Expression of LT in four ETEC isolates in the agglutination reaction was detected using a latex test-system. The isolates were assigned to serogroups O142 (n = 6), О6 (n = 4), О25 (n = 5), О26 (n = 2), and O115 (n = 1). Genotyping showed that they belonged to an earlier described sequence-type ST4 (n = 3) as well as to 11 novel sequence-types ST1043, ST1312, ST3697, ST3707, ST3708, ST3709, ST3710, ST3755, ST3756, ST3757 and ST4509. The ETEC isolates displayed different levels of antimicrobial resistance. Eight isolates were resistant to only one drug, three isolates-to two drugs, one isolate-to three drugs, two isolates-to four antibacterials, and only one isolate to each of the five, six and ten antibacterials simultaneously. Genetic determinants of the resistance to beta-lactams and other classes of antibacterials on the ETEC genomes were identified. There are blaTEM (n = 10), blaCTX-M-15 (n = 1), class 1 integron (n = 3) carrying resistance cassettes to aminoglycosides and

  18. Molecular Characterization of Enterotoxin-Producing Escherichia coli Collected in 2011-2012, Russia.

    PubMed

    Kartsev, Nikolay N; Fursova, Nadezhda K; Pachkunov, Dmitry M; Bannov, Vasiliy A; Eruslanov, Boris V; Svetoch, Edward A; Dyatlov, Ivan A

    2015-01-01

    Enterotoxin-producing Escherichia coli (ETEC) are one of the main causative agents of diarrhea in children especially in developing countries and travel diarrhoea in adults. Pathogenic properties of ETEC associated with their ability to produce a heat-stable (ST) and/or heat-labile (LT) enterotoxins, as well as adhesins providing bacterial adhesion to intestinal epithelial cells. This study presents the molecular characterization of the ETEC isolates collected from the Central and Far-Eastern regions of Russia in 2011-2012. It was shown that all ETEC under study (n=18) had the heat-labile enterotoxin-coding operon elt, and had no the genes of the heat-stable enterotoxin operon est. DNA sequencing revealed two types of nucleotide exchanges in the eltB gene coding subunit B of LT in isolates collected from Cherepovets city (Central region, Russia) and Vladivostok city (Far-East region, Russia). Only one ETEC strain carried genes cfaA, cfaB, cfaC and cfaD coding adhesion factor CFA/I. Expression of LT in four ETEC isolates in the agglutination reaction was detected using a latex test-system. The isolates were assigned to serogroups O142 (n = 6), О6 (n = 4), О25 (n = 5), О26 (n = 2), and O115 (n = 1). Genotyping showed that they belonged to an earlier described sequence-type ST4 (n = 3) as well as to 11 novel sequence-types ST1043, ST1312, ST3697, ST3707, ST3708, ST3709, ST3710, ST3755, ST3756, ST3757 and ST4509. The ETEC isolates displayed different levels of antimicrobial resistance. Eight isolates were resistant to only one drug, three isolates-to two drugs, one isolate-to three drugs, two isolates-to four antibacterials, and only one isolate to each of the five, six and ten antibacterials simultaneously. Genetic determinants of the resistance to beta-lactams and other classes of antibacterials on the ETEC genomes were identified. There are blaTEM (n = 10), blaCTX-M-15 (n = 1), class 1 integron (n = 3) carrying resistance cassettes to aminoglycosides and

  19. Postharvest treatments of fresh produce.

    PubMed

    Mahajan, P V; Caleb, O J; Singh, Z; Watkins, C B; Geyer, M

    2014-06-13

    Postharvest technologies have allowed horticultural industries to meet the global demands of local and large-scale production and intercontinental distribution of fresh produce that have high nutritional and sensory quality. Harvested products are metabolically active, undergoing ripening and senescence processes that must be controlled to prolong postharvest quality. Inadequate management of these processes can result in major losses in nutritional and quality attributes, outbreaks of foodborne pathogens and financial loss for all players along the supply chain, from growers to consumers. Optimal postharvest treatments for fresh produce seek to slow down physiological processes of senescence and maturation, reduce/inhibit development of physiological disorders and minimize the risk of microbial growth and contamination. In addition to basic postharvest technologies of temperature management, an array of others have been developed including various physical (heat, irradiation and edible coatings), chemical (antimicrobials, antioxidants and anti-browning) and gaseous treatments. This article examines the current status on postharvest treatments of fresh produce and emerging technologies, such as plasma and ozone, that can be used to maintain quality, reduce losses and waste of fresh produce. It also highlights further research needed to increase our understanding of the dynamic response of fresh produce to various postharvest treatments. PMID:24797137

  20. Postharvest treatments of fresh produce

    PubMed Central

    Mahajan, P. V.; Caleb, O. J.; Singh, Z.; Watkins, C. B.; Geyer, M.

    2014-01-01

    Postharvest technologies have allowed horticultural industries to meet the global demands of local and large-scale production and intercontinental distribution of fresh produce that have high nutritional and sensory quality. Harvested products are metabolically active, undergoing ripening and senescence processes that must be controlled to prolong postharvest quality. Inadequate management of these processes can result in major losses in nutritional and quality attributes, outbreaks of foodborne pathogens and financial loss for all players along the supply chain, from growers to consumers. Optimal postharvest treatments for fresh produce seek to slow down physiological processes of senescence and maturation, reduce/inhibit development of physiological disorders and minimize the risk of microbial growth and contamination. In addition to basic postharvest technologies of temperature management, an array of others have been developed including various physical (heat, irradiation and edible coatings), chemical (antimicrobials, antioxidants and anti-browning) and gaseous treatments. This article examines the current status on postharvest treatments of fresh produce and emerging technologies, such as plasma and ozone, that can be used to maintain quality, reduce losses and waste of fresh produce. It also highlights further research needed to increase our understanding of the dynamic response of fresh produce to various postharvest treatments. PMID:24797137

  1. System for producing chroma signals

    NASA Technical Reports Server (NTRS)

    Vorhaben, K. H.; Lipoma, P. C. (Inventor)

    1977-01-01

    A method for obtaining electronic chroma signals with a single scanning-type image device is described. A color multiplexed light signal is produced using an arrangement of dichroic filter stripes. In the particular system described, a two layer filter is used to color modulate external light which is then detected by an image pickup tube. The resulting time division multiplexed electronic signal from the pickup tube is converted by a decoder into a green color signal, and a single red-blue multiplexed signal, which is demultiplexed to produce red and blue color signals. The three primary color signals can be encoded as standard NTSC color signals.

  2. Producing and Detecting Correlated Atoms

    SciTech Connect

    Westbrook, C. I.; Schellekens, M.; Perrin, A.; Krachmalnicoff, V.; Viana Gomes, J.; Trebbia, J.-B.; Esteve, J.; Chang, H.; Bouchoule, I.; Boiron, D.; Aspect, A.; Jeltes, T.; McNamara, J.; Hogervorst, W.; Vassen, W.

    2006-11-07

    We discuss experiments to produce and detect atom correlations in a degenerate or nearly degenerate gas of neutral atoms. First we treat the atomic analog of the celebrated Hanbury Brown Twiss experiment, in which atom correlations result simply from interference effects without any atom interactions. We have performed this experiment for both bosons and fermions. Next we show how atom interactions produce correlated atoms using the atomic analog of spontaneous four-wave mixing. Finally, we briefly mention experiments on a one dimensional gas on an atom chip in which correlation effects due to both interference and interactions have been observed.

  3. Producing miniature threads. Final report

    SciTech Connect

    Gillespie, L.K.; Robb, J.M.

    1981-11-01

    Miniature precision actuators, timers, and switches typically utilize miniature threads to provide convenient assembly, disassembly and adjustment. Thread rolling provides high-quality external threads with greater strength and lower cost than other thread-producing techniques. Tap breakage is a significant problem when 0.5 and 0.6 Unified National Miniature (UNM) threads must be produced in hard materials such as SAE K95100 high-permeability magnetic steel. Aluminum parts can be tapped with no difficulty in these sizes. Stainless steel 0.5 UNM screws break at loads of 21 lb (53 N). Thread failure occurs at thread heights of 62% full thread or lower.

  4. Shiga Toxin Producing Escherichia coli.

    PubMed

    Bryan, Allen; Youngster, Ilan; McAdam, Alexander J

    2015-06-01

    Shiga toxin-producing Escherichia coli (STEC) is among the common causes of foodborne gastroenteritis. STEC is defined by the production of specific toxins, but within this pathotype there is a diverse group of organisms. This diversity has important consequences for understanding the pathogenesis of the organism, as well as for selecting the optimum strategy for diagnostic testing in the clinical laboratory. This review includes discussions of the mechanisms of pathogenesis, the range of manifestations of infection, and the several different methods of laboratory detection of Shiga toxin-producing E coli.

  5. METHOD FOR PRODUCING DIBORON TETRACHLORIDE

    DOEpatents

    Frazer, J.W.; Holzmann, R.T.

    1961-08-01

    A method of producing diboron tetrachloride from boron trichloride is described. Gaseous boron trichloride is passed through a cavity resonating at a microwave frequency whereby a portion of the boron trichloride is converted into diboron tetrachloride. The diboron tetrachloride may then be separated from the boron trichloride by conventional means. (AEC)

  6. Resonating feathers produce courtship song.

    PubMed

    Bostwick, Kimberly S; Elias, Damian O; Mason, Andrew; Montealegre-Z, Fernando

    2010-03-22

    Male Club-winged Manakins, Machaeropterus deliciosus (Aves: Pipridae), produce a sustained tonal sound with specialized wing feathers. The fundamental frequency of the sound produced in nature is approximately 1500 Hz and is hypothesized to result from excitation of resonance in the feathers' hypertrophied shafts. We used laser Doppler vibrometry to determine the resonant properties of male Club-winged Manakin's wing feathers, as well as those of two unspecialized manakin species. The modified wing feathers exhibit a response peak near 1500 Hz, and unusually high Q-values (a measure of resonant tuning) for biological objects (Q up to 27). The unmodified wing feathers of the Club-winged Manakin do not exhibit strong resonant properties when measured in isolation. However, when measured still attached to the modified feathers (nine feathers held adjacent by an intact ligament), they resonate together as a unit near 1500 Hz, and the wing produces a second harmonic of similar or greater amplitude than the fundamental. The feathers of the control species also exhibit resonant peaks around 1500 Hz, but these are significantly weaker, the wing does not resonate as a unit and no harmonics are produced. These results lend critical support to the resonant stridulation hypothesis of sound production in M. deliciosus. PMID:19906670

  7. Resonating feathers produce courtship song

    PubMed Central

    Bostwick, Kimberly S.; Elias, Damian O.; Mason, Andrew; Montealegre-Z, Fernando

    2010-01-01

    Male Club-winged Manakins, Machaeropterus deliciosus (Aves: Pipridae), produce a sustained tonal sound with specialized wing feathers. The fundamental frequency of the sound produced in nature is approximately 1500 Hz and is hypothesized to result from excitation of resonance in the feathers' hypertrophied shafts. We used laser Doppler vibrometry to determine the resonant properties of male Club-winged Manakin's wing feathers, as well as those of two unspecialized manakin species. The modified wing feathers exhibit a response peak near 1500 Hz, and unusually high Q-values (a measure of resonant tuning) for biological objects (Q up to 27). The unmodified wing feathers of the Club-winged Manakin do not exhibit strong resonant properties when measured in isolation. However, when measured still attached to the modified feathers (nine feathers held adjacent by an intact ligament), they resonate together as a unit near 1500 Hz, and the wing produces a second harmonic of similar or greater amplitude than the fundamental. The feathers of the control species also exhibit resonant peaks around 1500 Hz, but these are significantly weaker, the wing does not resonate as a unit and no harmonics are produced. These results lend critical support to the resonant stridulation hypothesis of sound production in M. deliciosus. PMID:19906670

  8. The Top Theological Degree Producers

    ERIC Educational Resources Information Center

    Diverse: Issues in Higher Education, 2012

    2012-01-01

    Each year, "Diverse: Issues in Higher Education" publishes a list of the Top 100 producers of associate, bachelor's and graduate degrees awarded to minority students based on research conducted by Dr. Victor M. H. Borden, professor of educational leadership and policy studies at Indiana University Bloomington. This year, for the first time, it has…

  9. The Top STEM Degree Producers

    ERIC Educational Resources Information Center

    Diverse: Issues in Higher Education, 2012

    2012-01-01

    This article presents a list of the top Science, Technology, Engineering, and Mathematics (STEM) degree producers in the U.S. This list is broken down into seven categories: (1) Total Minority Research/Scholarship and Other Doctoral: Mathematics and Statistics; (2) Total Minority Bachelors: Biological and Biomedical Sciences; (3) Total Minority…

  10. Institutional Producers of Physics Research.

    ERIC Educational Resources Information Center

    Cooper, Marianne; Watterson, Hermine M.

    In order to identify producers of physics research and to determine their relative productivity, institutional affiliations of authors as given in nine physics journals were studied. Organizations were classified and analyzed by type and geographical location, and productivity established. Findings indicate that organizations differ in their rate…

  11. Marine bacteria which produce tetrodotoxin.

    PubMed Central

    Simidu, U; Noguchi, T; Hwang, D F; Shida, Y; Hashimoto, K

    1987-01-01

    A number of type strains of marine bacteria, including members of the family Vibrionaceae, were cultured and examined for tetrodotoxin productivity by high-performance liquid chromatography and gas chromatography-mass spectrometry. Most of the Vibrionaceae strains produced tetrodotoxin, anhydrotetrodotoxin, or both. PMID:3310884

  12. Employing in vitro analysis to test the potency of methylglyoxal in inducing the formation of amyloid-like aggregates of caprine brain cystatin.

    PubMed

    Bhat, Waseem Feeroze; Bhat, Sheraz Ahmad; Khaki, Peerzada Shariq Shaheen; Bano, Bilqees

    2015-01-01

    Thiol protease inhibitors (cystatins) are implicated in various disease states from cancer to neurodegenerative conditions and immune responses. Cystatins have high amyloidogenic propensity and they are prone to form fibrillar aggregates leading to amyloidosis. Particularly challenging examples of such disorders occur in type 2 diabetes, Alzheimer's and Parkinson's diseases. The aim of the present study is to find an interaction between the compound methylglyoxal (MG) which is particularly elevated in type 2 diabetes with caprine brain cystatin (CBC). Results have shown that elevated concentration of MG forms amyloid aggregates of CBC. This was achieved by allowing slow growth in a solution containing moderate to high concentrations of MG. When analysed with microscopy, the protein aggregate present in the sample after incubation consisted of extended filaments with ordered structures. This fibrillar material possesses extensive β-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit increased Thioflavin T fluorescence.

  13. N-terminal domain of PB1-F2 protein of influenza A virus can fold into amyloid-like oligomers and damage cholesterol and cardiolipid containing membranes.

    PubMed

    Ajjaji, Dalila; Richard, Charles-Adrien; Mazerat, Sandra; Chevalier, Christophe; Vidic, Jasmina

    2016-08-12

    PB1-F2 protein is a factor of virulence of influenza A viruses which increases the mortality and morbidity associated with infection. Most seasonal H1N1 Influenza A viruses express nowadays a truncated version of PB1-F2. Here we show that truncation of PB1-F2 modified supramolecular organization of the protein in a membrane-mimicking environment. In addition, full-length PB1-F2(1-90) and C-terminal PB1-F2 domain (53-90), efficiently permeabilized various anionic liposomes while N-terminal domain PB1-F2(1-52) only lysed cholesterol and cardiolipin containing lipid bilayers. These findings suggest that the truncation of PB1-F2 may impact the pathogenicity of a given virus strain.

  14. Long-term treatment of thalidomide ameliorates amyloid-like pathology through inhibition of β-secretase in a mouse model of Alzheimer's disease.

    PubMed

    He, Ping; Cheng, Xin; Staufenbiel, Matthias; Li, Rena; Shen, Yong

    2013-01-01

    Thalidomide is a tumor necrosis factor alpha (TNFα) inhibitor which has been found to have abilities against tumor growth, angiogenesis and inflammation. Recently, it has been applied in clinic for the treatment of multiple myeloma as well as some inflammatory diseases. However, whether thalidomide has any therapeutic effects on neurodegenerative disorders, i.e. Alzheimer's disease (AD) is not clear. AD is characterized by excessive amount of amyloid β peptides (Aβ), which results in a significant release of inflammatory factors, including TNFα in the brain. Studies have shown that inhibition of TNFα reduces amyloid-associated pathology, prevents neuron loss and improves cognition. Our recent report showed that genetic inhibition of TNFα/TNF receptor signal transduction down-regulates β amyloid cleavage enzyme 1 (BACE1) activity, reduces Aβ generation and improves learning and memory deficits. However, the mechanism of thalidomide involving in the mitigation of AD neuropathological features remains unclear. Here, we chronically administrated thalidomide on human APPswedish mutation transgenic (APP23) mice from 9 months old (an onset of Aβ deposits and early stage of AD-like changes) to 12 months old. We found that, in addition of dramatic decrease in the activation of both astrocytes and microglia, thalidomide significantly reduces Aβ load and plaque formation. Furthermore, we found a significant decrease in BACE1 level and activity with long-term thalidomide application. Interestingly, these findings cannot be observed in the brains of 12-month-old APP23 mice with short-term treatment of thalidomide (3 days). These results suggest that chronic thalidomide administration is an alternative approach for AD prevention and therapeutics.

  15. Force Sensitivity in Saccharomyces cerevisiae Flocculins.

    PubMed

    Chan, Cho X J; El-Kirat-Chatel, Sofiane; Joseph, Ivor G; Jackson, Desmond N; Ramsook, Caleen B; Dufrêne, Yves F; Lipke, Peter N

    2016-01-01

    amyloid-like β-aggregation of short amino acid sequences in the adhesins. In Candida albicans adhesin Als5, shear stress from vortex mixing can unfold part of the protein to expose aggregation-prone sequences, and then adhesins aggregate into nanodomains. We therefore tested whether shear stress from mixing can increase flocculation activity by potentiating similar protein remodeling and aggregation in the flocculins. The results demonstrate the applicability of the Als adhesin model and provide a rational framework for the enhancement or inhibition of flocculation in industrial applications. PMID:27547825

  16. Force Sensitivity in Saccharomyces cerevisiae Flocculins

    PubMed Central

    Chan, Cho X. J.; El-Kirat-Chatel, Sofiane; Joseph, Ivor G.; Jackson, Desmond N.; Ramsook, Caleen B.; Dufrêne, Yves F.

    2016-01-01

    on amyloid-like β-aggregation of short amino acid sequences in the adhesins. In Candida albicans adhesin Als5, shear stress from vortex mixing can unfold part of the protein to expose aggregation-prone sequences, and then adhesins aggregate into nanodomains. We therefore tested whether shear stress from mixing can increase flocculation activity by potentiating similar protein remodeling and aggregation in the flocculins. The results demonstrate the applicability of the Als adhesin model and provide a rational framework for the enhancement or inhibition of flocculation in industrial applications. PMID:27547825

  17. Shiga toxin-producing Escherichia coli in beef retail markets from Argentina.

    PubMed

    Brusa, Victoria; Aliverti, Virginia; Aliverti, Florencia; Ortega, Emanuel E; de la Torre, Julian H; Linares, Luciano H; Sanz, Marcelo E; Etcheverría, Analía I; Padola, Nora L; Galli, Lucía; Peral García, Pilar; Copes, Julio; Leotta, Gerardo A

    2012-01-01

    Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that cause mild or serious diseases and can lead to people death. This study reports the prevalence and characteristics of STEC O157 and non-O157 in commercial ground beef and environmental samples, including meat table, knife, meat mincing machine, and manipulator hands (n = 450) obtained from 90 retail markets over a nine-month period. The STEC isolates were serotyped and virulence genes as stx (Shiga toxin), rfb(O157)] (O157 lipopolysaccharide), fliC(H7) (H7 flagellin), eae (intimin), ehxA (enterohemolysin) and saa (STEC autoagglutinating adhesin), were determined. STEC O157 were identified in 23 (25.5%) beef samples and 16 (4.4%) environmental samples, while STEC non-O157 were present in 47 (52.2%) and 182 (50.5%), respectively. Among 54 strains isolated, 17 were STEC O157:H7 and 37 were STEC non-O157. The prevalent genotype for O157 was stx(2)/eae/ehxA/fliC(H7) (83.4%), and for STEC non-O157 the most frequent ones were stx(1)/stx(2)/saa/ehxA (29.7%); stx(2) (29.7%); and stx(2)/saa/ehxA (27%). None of the STEC non-O157 strains were eae-positive. Besides O157:H7, other 20 different serotypes were identified, being O8:H19, O178:H19, and O174:H28 the prevalent. Strains belonging to the same serotype could be isolated from different sources of the same retail market. Also, the same serotype could be detected in different stores. In conclusion, screening techniques are increasingly sensitive, but the isolation of STEC non-O157 is still a challenge. Moreover, with the results obtained from the present work, although more studies are needed, cross-contamination between meat and the environment could be suspected. PMID:23346554

  18. Surface properties of the Vero cytotoxin-producing Escherichia coli O157:H7.

    PubMed Central

    Sherman, P; Soni, R; Petric, M; Karmali, M

    1987-01-01

    Strains of Escherichia coli serotype O157:H7 are Vero cytotoxin-producing enteric pathogens which have been associated with sporadic cases and outbreaks of hemorrhagic colitis and with the hemolytic uremic syndrome in humans. In addition to toxin production, adherence of many pathogenic bacteria to intestinal mucosal surfaces is a critical primary step in the pathogenesis of diarrheal diseases. Although E. coli serotype O157:H7 organisms adhere to intestinal epithelia of orally infected animals in a pattern morphologically identical to that previously described in adherent, effacing E. coli infections, the mechanisms of bacterial adherence are not known. To determine the cell surface adhesins which mediate attachment of E. coli O157:H7 to epithelial surfaces, we evaluated the surface properties of these organisms. Five strains isolated from children with the hemolytic uremic syndrome were grown both in broth cultures and on agar media. Adherence and invasion of E. coli O157:H7 in Intestine 407 and HEp-2 epithelial cell lines was quantitated using an enteroinvasive E. coli strain (serotype O164:NM) as a control. Cell surface properties of E. coli O157:H7 were evaluated by agglutination of a series of erythrocytes, transmission electron microscopy, DEAE-ion-exchange chromatography, and hydrophobic interaction chromatography. E. coli O157:H7 strains adhered to but did not invade either Intestine 407 or HEp-2 cells. Homologous O157:H7 rabbit antiserum blocked attachment of bacteria to tissue culture cells, in contrast to heterologous antiserum and preimmune rabbit serum, which did not inhibit attachment of E. coli O157:H7. None of the five O15:H7 isolates mediated mannose-resistant hemagglutination under any of the in vitro culture conditions. One isolate mediated mannose-sensitive hemagglutination after serial passage in broth cultures. Pili and fibrillae were not visualized by electron microscopy on nonhemagglutinating organisms, but pili were demonstrated on the one

  19. Environmental considerations of livestock producers.

    PubMed

    Morse, D

    1995-09-01

    Regulatory agencies and citizens have placed great emphasis on sustainability of natural resources. Individuals involved in animal husbandry should understand the basic components associated with air and water quality issues. An increase in regulations governing land, air, and water resources increases the need for educators, researchers, and producers to realize impacts of current and potential legislation. Regulations and enforcement should be based on sound scientific data. Research scientists should play an active role in designing and carrying out research. Sustainability of land and economics of production are better incentives to entice producers to change than are regulations. Educational opportunities exist to promote environmental stewardship and the importance of maintaining natural resources. This paper will review current air and water quality regulations and provide examples of current activities that focus on environmental stewardship. Individuals should understand the importance of cooperative work to ensure that realistic enforcement and future regulation development be economically feasible and environmentally sound.

  20. Method for producing carbon nanotubes

    DOEpatents

    Phillips, Jonathan; Perry, William L.; Chen, Chun-Ku

    2006-02-14

    Method for producing carbon nanotubes. Carbon nanotubes were prepared using a low power, atmospheric pressure, microwave-generated plasma torch system. After generating carbon monoxide microwave plasma, a flow of carbon monoxide was directed first through a bed of metal particles/glass beads and then along the outer surface of a ceramic tube located in the plasma. As a flow of argon was introduced into the plasma through the ceramic tube, ropes of entangled carbon nanotubes, attached to the surface of the tube, were produced. Of these, longer ropes formed on the surface portion of the tube located in the center of the plasma. Transmission electron micrographs of individual nanotubes revealed that many were single-walled.

  1. Process for producing advanced ceramics

    DOEpatents

    Kwong, Kyei-Sing

    1996-01-01

    A process for the synthesis of homogeneous advanced ceramics such as SiC+AlN, SiAlON, SiC+Al.sub.2 O.sub.3, and Si.sub.3 N.sub.4 +AlN from natural clays such as kaolin, halloysite and montmorillonite by an intercalation and heat treatment method. Included are the steps of refining clays, intercalating organic compounds into the layered structure of clays, drying the intercalated mixture, firing the treated atmospheres and grinding the loosely agglomerated structure. Advanced ceramics produced by this procedure have the advantages of homogeneity, cost effectiveness, simplicity of manufacture, ease of grind and a short process time. Advanced ceramics produced by this process can be used for refractory, wear part and structure ceramics.

  2. PROCESS FOR PRODUCING URANIUM TETRAFLUORIDE

    DOEpatents

    Harvey, B.G.

    1954-09-14

    >This patent relates to improvements in the method for producing uranium tetrafluoride by treating an aqueous solutlon of a uranyl salt at an elevated temperature with a reducing agent effective in acld solutlon in the presence of hydrofluoric acid. Uranium tetrafluoride produced this way frequentiy contains impurities in the raw material serving as the source of uranium. Uranium tetrafluoride much less contaminated with impurities than when prepared by the above method can be prepared from materials containing such impurities by first adding a small proportion of reducing agent so as to cause a small fraction, for example 1 to 5% of the uranium tetrafluoride to be precipitated, rejecting such precipitate, and then precipitating and recovering the remainder of the uranium tetrafluoride.

  3. Method for producing hydrophobic aerogels

    DOEpatents

    Hrubesh, Lawrence W.; Poco, John F.; Coronado, Paul R.

    1999-01-01

    A method for treating a dried monolithic aerogel containing non-dispersed particles, with an organometallic surface modifying agent to produce hydrophobic aerogels. The dried, porous hydrophobic aerogels contain a protective layer of alkyl groups, such as methyl groups, on the modified surfaces of the pores of the aerogel. The alkyl groups at the aerogel surface typically contain at least one carbon-metal bond per group.

  4. Electromagnetic radiation produces frame dragging

    NASA Astrophysics Data System (ADS)

    Herrera, L.; Barreto, W.

    2012-09-01

    It is shown that for a generic electrovacuum spacetime, electromagnetic radiation produces vorticity of worldlines of observers in a Bondi-Sachs frame. Such an effect (and the ensuing gyroscope precession with respect to the lattice) which is a reminiscence of generation of vorticity by gravitational radiation, may be linked to the nonvanishing of components of the Poynting and the super-Poynting vectors on the planes othogonal to the vorticity vector. The possible observational relevance of such an effect is commented upon.

  5. Method of producing cyclohexasilane compounds

    SciTech Connect

    Elangovan, Arumugasamy; Anderson, Kenneth; Boudjouk, Philip R; Schulz, Douglas L

    2015-03-10

    A method of preparing a cyclohexasilane compound from trichlorosilane is provided. The method includes contacting trichlorosilane with a reagent composition to produce a compound containing a tetradecahalocyclohexasilane dianion, such as a tetradecachlorocyclohexasilane dianion. The reagent composition typically includes (a) tertiary polyamine ligand; and (b) a deprotonating reagent, such as a tertiary amine having a pKa of at least about 10.5. Methods of converting the tetradecahalocyclohexasilane dianion-containing compound to cyclohexasilane or a dodecaorganocyclohexasilane are also provided.

  6. Method for producing monodisperse aerosols

    DOEpatents

    Ortiz, Lawrence W.; Soderholm, Sidney C.

    1990-01-01

    An aerosol generator is described which is capable of producing a monodisperse aerosol within narrow limits utilizing an aqueous solution capable of providing a high population of seed nuclei and an organic solution having a low vapor pressure. The two solutions are cold nebulized, mixed, vaporized, and cooled. During cooling, particles of the organic vapor condense onto the excess seed nuclei, and grow to a uniform particle size.

  7. PROCESS OF PRODUCING SHAPED PLUTONIUM

    DOEpatents

    Anicetti, R.J.

    1959-08-11

    A process is presented for producing and casting high purity plutonium metal in one step from plutonium tetrafluoride. The process comprises heating a mixture of the plutonium tetrafluoride with calcium while the mixture is in contact with and defined as to shape by a material obtained by firing a mixture consisting of calcium oxide and from 2 to 10% by its weight of calcium fluoride at from 1260 to 1370 deg C.

  8. Method for producing metallic nanoparticles

    DOEpatents

    Phillips, Jonathan; Perry, William L.; Kroenke, William J.

    2004-02-10

    Method for producing metallic nanoparticles. The method includes generating an aerosol of solid metallic microparticles, generating non-oxidizing plasma with a plasma hot zone at a temperature sufficiently high to vaporize the microparticles into metal vapor, and directing the aerosol into the hot zone of the plasma. The microparticles vaporize in the hot zone to metal vapor. The metal vapor is directed away from the hot zone and to the plasma afterglow where it cools and condenses to form solid metallic nanoparticles.

  9. Method for producing metallic microparticles

    DOEpatents

    Phillips, Jonathan; Perry, William L.; Kroenke, William J.

    2004-06-29

    Method for producing metallic particles. The method converts metallic nanoparticles into larger, spherical metallic particles. An aerosol of solid metallic nanoparticles and a non-oxidizing plasma having a portion sufficiently hot to melt the nanoparticles are generated. The aerosol is directed into the plasma where the metallic nanoparticles melt, collide, join, and spheroidize. The molten spherical metallic particles are directed away from the plasma and enter the afterglow where they cool and solidify.

  10. Producing Gestures Facilitates Route Learning

    PubMed Central

    So, Wing Chee; Ching, Terence Han-Wei; Lim, Phoebe Elizabeth; Cheng, Xiaoqin; Ip, Kit Yee

    2014-01-01

    The present study investigates whether producing gestures would facilitate route learning in a navigation task and whether its facilitation effect is comparable to that of hand movements that leave physical visible traces. In two experiments, we focused on gestures produced without accompanying speech, i.e., co-thought gestures (e.g., an index finger traces the spatial sequence of a route in the air). Adult participants were asked to study routes shown in four diagrams, one at a time. Participants reproduced the routes (verbally in Experiment 1 and non-verbally in Experiment 2) without rehearsal or after rehearsal by mentally simulating the route, by drawing it, or by gesturing (either in the air or on paper). Participants who moved their hands (either in the form of gestures or drawing) recalled better than those who mentally simulated the routes and those who did not rehearse, suggesting that hand movements produced during rehearsal facilitate route learning. Interestingly, participants who gestured the routes in the air or on paper recalled better than those who drew them on paper in both experiments, suggesting that the facilitation effect of co-thought gesture holds for both verbal and nonverbal recall modalities. It is possibly because, co-thought gesture, as a kind of representational action, consolidates spatial sequence better than drawing and thus exerting more powerful influence on spatial representation. PMID:25426624

  11. Producing gestures facilitates route learning.

    PubMed

    So, Wing Chee; Ching, Terence Han-Wei; Lim, Phoebe Elizabeth; Cheng, Xiaoqin; Ip, Kit Yee

    2014-01-01

    The present study investigates whether producing gestures would facilitate route learning in a navigation task and whether its facilitation effect is comparable to that of hand movements that leave physical visible traces. In two experiments, we focused on gestures produced without accompanying speech, i.e., co-thought gestures (e.g., an index finger traces the spatial sequence of a route in the air). Adult participants were asked to study routes shown in four diagrams, one at a time. Participants reproduced the routes (verbally in Experiment 1 and non-verbally in Experiment 2) without rehearsal or after rehearsal by mentally simulating the route, by drawing it, or by gesturing (either in the air or on paper). Participants who moved their hands (either in the form of gestures or drawing) recalled better than those who mentally simulated the routes and those who did not rehearse, suggesting that hand movements produced during rehearsal facilitate route learning. Interestingly, participants who gestured the routes in the air or on paper recalled better than those who drew them on paper in both experiments, suggesting that the facilitation effect of co-thought gesture holds for both verbal and nonverbal recall modalities. It is possibly because, co-thought gesture, as a kind of representational action, consolidates spatial sequence better than drawing and thus exerting more powerful influence on spatial representation. PMID:25426624

  12. Method for producing viscous hydrocarbons

    DOEpatents

    Poston, Robert S.

    1982-01-01

    A method for recovering viscous hydrocarbons and synthetic fuels from a subterranean formation by drilling a well bore through the formation and completing the well by cementing a casing means in the upper part of the pay zone. The well is completed as an open hole completion and a superheated thermal vapor stream comprised of steam and combustion gases is injected into the lower part of the pay zone. The combustion gases migrate to the top of the pay zone and form a gas cap which provides formation pressure to produce the viscous hydrocarbons and synthetic fuels.

  13. Method of producing nickel electrode

    NASA Technical Reports Server (NTRS)

    Ikeda, Y.; Ohira, T.; Kumano, Y.; Nakao, T.

    1982-01-01

    A large capacity nickel electrode is provided in which the charging efficiency and discharge utilization coefficient are improved in comparison to nickel electrodes which are produced by the conventional method. Nickel electrodes retaining nickel active material or nickel active material and cobalt compounds on a porous nickel substrate are immersed in a cobalt sulfate aqueous solution whose pH is adjusted in the range of 3.5 to 6.0, followed by crystallization of the hydroxide or oxide by pyrolysis or immersion in alkali, thereby coating the surface of the nickel active material with cobalt crystals and simultaneously promoting alloying of the nickel-cobalt.

  14. Fatty acid-producing hosts

    DOEpatents

    Pfleger, Brian F; Lennen, Rebecca M

    2013-12-31

    Described are hosts for overproducing a fatty acid product such as a fatty acid. The hosts include an exogenous nucleic acid encoding a thioesterase and, optionally, an exogenous nucleic acid encoding an acetyl-CoA carboxylase, wherein an acyl-CoA synthetase in the hosts are functionally delected. The hosts prefereably include the nucleic acid encoding the thioesterase at an intermediate copy number. The hosts are preferably recominantly stable and growth-competent at 37.degree. C. Methods of producing a fatty acid product comprising culturing such hosts at 37.degree. C. are also described.

  15. Producing biofuels using polyketide synthases

    SciTech Connect

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  16. METHOD FOR PRODUCING THORIUM TETRACHLORIDE

    DOEpatents

    Mason, E.A.; Cobb, C.M.

    1960-03-15

    A process for producing thorium tetrachloride from thorium concentrate comprises reacting thorium concentrates with a carbonaceous reducing agent in excess of 0.05 part by weight per part of thoriferous concentrate at a temperature in excess of 1300 deg C, cooling and comminuting the mass, chlorinating the resulting comminuting mass by suspending in a gaseous chlorinating agent in a fluidized reactor at a temperatare maintained between about l85 deg C and 770 deg C, and removing the resulting solid ThCl/sub 4/ from the reaction zone.

  17. Carbapenemase-producing Klebsiella pneumoniae

    PubMed Central

    Deresinski, Stan

    2014-01-01

    The continuing emergence of infections due to multidrug resistant bacteria is a serious public health problem. Klebsiella pneumoniae, which commonly acquires resistance encoded on mobile genetic elements, including ones that encode carbapenemases, is a prime example. K. pneumoniae carrying such genetic material, including both blaKPC and genes encoding metallo-β-lactamases, have spread globally. Many carbapenemase-producing K. pneumoniae are resistant to multiple antibiotic classes beyond β-lactams, including tetracyclines, aminoglycosides, and fluoroquinolones. The optimal treatment, if any, for infections due to these organisms is unclear but, paradoxically, appears to often require the inclusion of an optimally administered carbapenem. PMID:25343037

  18. Biochemical degradation treats produced water

    SciTech Connect

    1997-01-20

    In Colombia, Kelt Oil Co. employs a lined, reed-based, root-zone filtering system to remove contaminants from water produced with crude oil. The roots of the reeds absorb the contaminants. The treated water is then used for agriculture. Kelt has operated te system for over 1 year near Trinidad, in the Casanare district of eastern Colombia. After 1 year, the system removed 90% of the phenol compounds. It expects 3 years will be required for the system to achieve full efficiency.

  19. Method for producing a tube

    DOEpatents

    Peterson, Kenneth A.; Rohde, Steven B.; Pfeifer, Kent B.; Turner, Timothy S.

    2007-01-02

    A method is described for producing tubular substrates having parallel spaced concentric rings of electrical conductors that can be used as the drift tube of an Ion Mobility Spectrometer (IMS). The invention comprises providing electrodes on the inside of a tube that are electrically connected to the outside of the tube through conductors that extend between adjacent plies of substrate that are combined to form the tube. Tubular substrates are formed from flexible polymeric printed wiring board materials, ceramic materials and material compositions of glass and ceramic, commonly known as Low Temperature Co-Fired Ceramic (LTCC). The adjacent plies are sealed together around the electrode.

  20. Does salmon brain produce insulin?

    PubMed

    Plisetskaya, E M; Bondareva, V M; Duan, C; Duguay, S J

    1993-07-01

    To address the question whether fish brain can produce insulin, pink salmon (Oncorhynchus gorbusha) brains were extracted and processed according to the procedure developed for purification of pancreatic insulin (Rusakov and Bondareva, 1979). Biological and immunological activity of the resulting material was evaluated respectively by a cartilage sulfation assay and by radioimmunoassay homologous for salmon insulin. Preparations from salmon brain stimulated the [35S]sulfate uptake into salmon branchial cartilage with a potency comparable to pure mammalian or salmon insulins but lower than that of mammalian insulin-like growth factor (IGF-I). In contrast, only trace amounts of radioimmunoreactive insulin could be detected by homologous radioimmunoassay. To determine whether insulin mRNA was present in salmon brain, primers specific for salmon proinsulin and salmon prepro-IGF-I were designed to amplify corresponding cDNA regions by reverse transcriptase-PCR. Insulin mRNA was found only in the endocrine pancreas (Brockmann body) while IGF-I mRNA was detected in the brain, liver, and the Brockmann body. Our results suggest that in fish pancreatic-type insulin is most likely produced only in the endocrine pancreas and then transported to the brain through blood/cerebrospinal fluid system. However, it does not exclude a possibility that some yet unknown insulin-like substances may be expressed in the neural system of ectotherm vertebrates.

  1. Dade recovery plant producing electricity

    SciTech Connect

    Not Available

    1982-06-01

    The 3000 tons/day resource recovery plant in Dade County (Florida) is described. Following a year-long contract dispute, the plant is said to be ready to: (1) accept four types of waste; (2) produce refuse-derived fuel; and (3) generate electricity for sale to Florida Power and Light at a rate of 285 million kilowatt hrs/year. Waste accepted include: (1) regular household garbage; (2) trash wastes including commercial refuse, furniture, rubber tires, appliances, tree cuttings, and other bulky items; (3) pathological wastes from hospitals and health centers; and (4) demolition materials which are routed directly to a landfill. The plant cost $165 million to construct and occupies 120 acres including 80 acres of landfill. The hydrasposal system includes four pulpers used to produce refuse-derived fuel (4300 Btu/lb). Flotation tanks and jigs are used to separate aluminium and non-ferrous metals (which are sold for recycling). The plant is described as having the potential of disposing of 60% of all the household and commercial solid wastes generated in Dade County. (MJJ)

  2. ANTIPROTONS PRODUCED IN SUPERNOVA REMNANTS

    SciTech Connect

    Berezhko, E. G.; Ksenofontov, L. T.

    2014-08-20

    We present the energy spectrum of an antiproton cosmic ray (CR) component calculated on the basis of the nonlinear kinetic model of CR production in supernova remnants (SNRs). The model includes the reacceleration of antiprotons already existing in the interstellar medium as well as the creation of antiprotons in nuclear collisions of accelerated protons with gas nuclei and their subsequent acceleration by SNR shocks. It is shown that the production of antiprotons in SNRs produces a considerable effect in their resultant energy spectrum, making it essentially flatter above 10 GeV so that the spectrum at TeV energies increases by a factor of 5. The calculated antiproton spectrum is consistent with the PAMELA data, which correspond to energies below 100 GeV. As a consistency check, we have also calculated within the same model the energy spectra of secondary nuclei and show that the measured boron-to-carbon ratio is consistent with the significant SNR contribution.

  3. Reactor-Produced Medical Radionuclides

    SciTech Connect

    Mirzadeh, Saed; Mausner, Leonard; Garland, Marc A

    2011-01-01

    The therapeutic use of radionuclides in nuclear medicine, oncology and cardiology is the most rapidly growing use of medical radionuclides. Since most therapeutic radionuclides are neutron rich and decay by beta emission, they are reactor-produced. This chapter deals mainly with production approaches with neutrons. Neutron interactions with matter, neutron transmission and activation rates, and neutron spectra of nuclear reactors are discussed in some detail. Further, a short discussion of the neutron-energy dependence of cross sections, reaction rates in thermal reactors, cross section measurements and flux monitoring, and general equations governing the reactor production of radionuclides are presented. Finally, the chapter is concluded by providing a number of examples encompassing the various possible reaction routes for production of a number of medical radionuclides in a reactor.

  4. CVD-produced boron filaments

    NASA Technical Reports Server (NTRS)

    Wawner, F. E.; Debolt, H. E.; Suplinskas, R. D.

    1980-01-01

    A technique for producing boron filaments with an average tensile strength of 6.89 GPa has been developed which involves longitudinal splitting of the filament and core (substrate) removal by etching. Splitting is accomplished by a pinch wheel device which continuously splits filaments in lengths of 3.0 m by applying a force to the side of the filament to create a crack which is then propagated along the axis by a gentle sliding action. To facilitate the splitting, a single 10 mil tungsten substrate is used instead of the usual 0.5 mil substrate. A solution of hot 30% hydrogen peroxide is used to remove the core without attacking the boron. An alternative technique is to alter the residual stress by heavily etching the filament. Average strengths in the 4.83-5.52 GPa range have been obtained by etching an 8 mil filament to 4 mil.

  5. HFIR-produced medical radioisotopes

    SciTech Connect

    Mirzadeh, S.; Knapp, F.F. Jr.; Beets, A.L.; Alexander, C.W.

    1997-12-01

    We have experimentally determined the yields of a number of medical radioisotopes produced in the Oak Ridge National Laboratory High Flux Isotope Reactor (HFIR) Hydraulic Tube (HT) facility. The HT facility is located in the very high flux region in the flux trap of the reactor, providing on-line access capability while the reactor is operating. The HT facility consists of nine vertically stacked capsules centered just adjacent to the core horizontal midplane. HFIR operates at a nominal power level of 85 MW. The capabilities of the HFIR-HT facilities offer increased efficiency, greater availability, and optimization of radioisotope production, and, as a result, the conservation of rare or expensive target isotopes.

  6. Method of producing metallic materials

    DOEpatents

    Branagan, Daniel J.

    2004-02-10

    The invention includes a method of producing a hard metallic material by forming a mixture containing at least 55% iron and at least one of B, C, Si and P. The mixture is formed into an alloy and cooled to form a metallic material having a hardness greater than about 9.2 GPa. The invention includes a method of forming a wire by combining a metal strip and a powder. The strip and the powder are rolled to form a wire containing at least 55% iron and from 2-7 additional elements including at least one of C, Si and B. The invention also includes a method of forming a hardened surface on a substrate by processing a solid mass to form a powder, applying the powder to a surface to form a layer containing metallic glass, and converting the glass to a crystalline material having a nanocrystalline grain size.

  7. Methods for producing secreted polypeptides

    DOEpatents

    Maiyuran, Suchindra; Fidantsef, Ana; Brody, Howard

    2008-07-01

    The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a nucleic acid construct comprising a first nucleotide sequence encoding a signal peptide operably linked to a second nucleotide sequence encoding the polypeptide, wherein the first nucleotide sequence is foreign to the second nucleotide sequence and the 3' end of the first nucleotide sequence is immediately upstream of the initiator codon of the second nucleotide sequence. The present invention also relates to the isolated signal peptide sequences and to constructs, vectors, and fungal host cells comprising the signal peptide sequences operably linked to nucleotide sequences encoding polypeptides.

  8. R-body-producing bacteria.

    PubMed

    Pond, F R; Gibson, I; Lalucat, J; Quackenbush, R L

    1989-03-01

    Until 10 years ago, R bodies were known only as diagnostic features by which endosymbionts of paramecia were identified as kappa particles. They were thought to be limited to the cytoplasm of two species in the Paramecium aurelia species complex. Now, R bodies have been found in free-living bacteria and other Paramecium species. The organisms now known to form R bodies include the cytoplasmic kappa endosymbionts of P. biaurelia and P. tetraurelia, the macronuclear kappa endosymbionts of P. caudatum, Pseudomonas avenae (a free-living plant pathogen), Pseudomonas taeniospiralis (a hydrogen-oxidizing soil microorganism), Rhodospirillum centenum (a photosynthetic bacterium), and a soil bacterium, EPS-5028, which is probably a pseudomonad. R bodies themselves fall into five distinct groups, distinguished by size, the morphology of the R-body ribbons, and the unrolling behavior of wound R bodies. In recent years, the inherent difficulties in studying the organization and assembly of R bodies by the obligate endosymbiont kappa, have been alleviated by cloning and expressing genetic determinants for these R bodies (type 51) in Escherichia coli. Type 51 R-body synthesis requires three low-molecular-mass polypeptides. One of these is modified posttranslationally, giving rise to 12 polypeptide species, which are the major structural subunits of the R body. R bodies are encoded in kappa species by extrachromosomal elements. Type 51 R bodies, produced in Caedibacter taeniospiralis, are encoded by a plasmid, whereas bacteriophage genomes probably control R-body synthesis in other kappa species. However, there is no evidence that either bacteriophages or plasmids are present in P. avenae or P. taeniospiralis. No sequence homology was detected between type 51 R-body-encoding DNA and DNA from any R-body-producing species, except C. varicaedens 1038. The evolutionary relatedness of different types of R bodies remains unknown. PMID:2651865

  9. Screening For Alcohol-Producing Microbes

    NASA Technical Reports Server (NTRS)

    Schubert, Wayne W.

    1988-01-01

    Dye reaction rapidly identifies alcohol-producing microbial colonies. Method visually detects alcohol-producing micro-organisms, and distinguishes them from other microbial colonies that do not produce alcohol. Method useful for screening mixed microbial populations in environmental samples.

  10. 7 CFR 926.7 - Producer.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... REQUIREMENTS APPLICABLE TO CRANBERRIES NOT SUBJECT TO THE CRANBERRY MARKETING ORDER § 926.7 Producer. Producer is synonymous with grower and means any person who produces cranberries for market and has...

  11. 7 CFR 926.7 - Producer.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... REQUIREMENTS APPLICABLE TO CRANBERRIES NOT SUBJECT TO THE CRANBERRY MARKETING ORDER § 926.7 Producer. Producer is synonymous with grower and means any person who produces cranberries for market and has...

  12. 7 CFR 926.7 - Producer.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... REQUIREMENTS APPLICABLE TO CRANBERRIES NOT SUBJECT TO THE CRANBERRY MARKETING ORDER § 926.7 Producer. Producer is synonymous with grower and means any person who produces cranberries for market and has...

  13. 7 CFR 926.7 - Producer.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... REQUIREMENTS APPLICABLE TO CRANBERRIES NOT SUBJECT TO THE CRANBERRY MARKETING ORDER § 926.7 Producer. Producer is synonymous with grower and means any person who produces cranberries for market and has...

  14. 7 CFR 926.7 - Producer.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... REQUIREMENTS APPLICABLE TO CRANBERRIES NOT SUBJECT TO THE CRANBERRY MARKETING ORDER § 926.7 Producer. Producer is synonymous with grower and means any person who produces cranberries for market and has...

  15. 7 CFR 1280.116 - Producer.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE LAMB PROMOTION, RESEARCH, AND INFORMATION ORDER Lamb Promotion, Research, and Information Order Definitions § 1280.116 Producer. Producer means any person who owns and produces lambs in the United States for sale....

  16. 7 CFR 1230.615 - Producer.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PORK PROMOTION, RESEARCH, AND... Producer means a person who produces porcine animals in the United States for sale in commerce....

  17. 7 CFR 1230.615 - Producer.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PORK PROMOTION, RESEARCH, AND... Producer means a person who produces porcine animals in the United States for sale in commerce....

  18. 7 CFR 1230.615 - Producer.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PORK PROMOTION, RESEARCH, AND... Producer means a person who produces porcine animals in the United States for sale in commerce....

  19. Can cirrus clouds produce glories?

    PubMed

    Sassen, K; Arnott, W P; Barnett, J M; Aulenbach, S

    1998-03-20

    A vague glory display was photographed over central Utah from an airplane beginning its descent through a cirrus cloud layer with an estimated cloud top temperature of -45 and -55 degrees C. Photographic analysis reveals a single reddish-brown ring of 2.5-3.0 degrees radius around the antisolar point, although a second ring appeared visually to have been present over the brief observation period. Mie and approximate nonspherical theory scattering simulations predict a population of particles with modal diameters between 9 and 15 mum. Although it is concluded that multiple-ringed glories can be accounted for only through the backscattering of light from particles that are strictly spherical in shape, the poor glory colorization in this case could imply the presence of slightly aspherical ice particles. The location of this display over mountainous terrain suggests that it was generated by an orographic wave cloud, which we speculate produced numerous frozen cloud droplets that only gradually took on crystalline characteristics during growth.

  20. Hydrodynamic Instabilities Produced by Evaporation

    NASA Astrophysics Data System (ADS)

    Romo-Cruz, Julio Cesar Ruben; Hernandez-Zapata, Sergio; Ruiz-Chavarria, Gerardo

    2012-11-01

    When a liquid layer (alcohol in the present work) is in an environment where its relative humidity is less than 100 percent evaporation appears. When RH is above a certain threshold the liquid is at rest. If RH decreases below this threshold the flow becomes unstable, and hydrodynamic cells develop. The aim of this work is to understand the formation of those cells and its main features. Firstly, we investigate how the cell size depends on the layer width. We also study how temperature depends on the vertical coordinate when the cells are present. An inverse temperature gradient is found, that is, the bottom of liquid layer is colder than the free surface. This shows that the intuitive idea that the cells are due to a direct temperature gradient, following a Marangoni-like process, does not work. We propose the hypothesis that the evaporation produce a pressure gradient that is responsible of the cell development. On the other hand, using a Schlieren technique we study the topography of the free surface when cells are present. Finally the alcohol vapor layer adjacent to the liquid surface is explored using scattering experiments, giving some insight on the plausibility of the hypothesis described previously. Authors acknowledge support by DGAPA-UNAM under project IN116312 ``Vorticidad y ondas no lineales en fluidos.''

  1. Ion produced cometary organic crust

    NASA Technical Reports Server (NTRS)

    Baratta, G. Antonio; Strazzulla, G.

    1992-01-01

    For several years many experimental results have been obtained on the chemical and physical changes induced by ion and electron irradiation of materials with a view to their Astrophysical relevance. Among the studied effects, one of particular interest is the formation of an organic refractory residue left over after ion irradiation and warming-up at room temperature. We call this residue IPHAC (ion produced hydrogenated amorphous carbon). Although 'in situ' infrared spectroscopy points out the formation of new molecular species during bombardment at low temperature, it is not clear if IPHAC is already formed or if its formation is triggered by temperature increase during warming-up of the irradiated target. Since Raman Spectroscopy is a technique particularly suitable for the analysis of carbonaceous materials, we have thought and build-up an experimental apparatus to obtain Raman Spectra of frozen hydrocarbons during ion irradiation. The present experimental results point out clearly to the formation of IPHAC already at low T and low energy deposition (approximately equal to a few eV/C-atom).

  2. Engineering microbes to produce biofuels

    SciTech Connect

    Wackett, LP

    2011-06-01

    The current biofuels landscape is chaotic. It is controlled by the rules imposed by economic forces and driven by the necessity of finding new sources of energy, particularly motor fuels. The need is bringing forth great creativity in uncovering new candidate fuel molecules that can be made via metabolic engineering. These next generation fuels include long-chain alcohols, terpenoid hydrocarbons, and diesel-length alkanes. Renewable fuels contain carbon derived from carbon dioxide. The carbon dioxide is derived directly by a photosynthetic fuel-producing organism(s) or via intermediary biomass polymers that were previously derived from carbon dioxide. To use the latter economically, biomass depolymerization processes must improve and this is a very active area of research. There are competitive approaches with some groups using enzyme based methods and others using chemical catalysts. With the former, feedstock and end-product toxicity loom as major problems. Advances chiefly rest on the ability to manipulate biological systems. Computational and modular construction approaches are key. For example, novel metabolic networks have been constructed to make long-chain alcohols and hydrocarbons that have superior fuel properties over ethanol. A particularly exciting approach is to implement a direct utilization of solar energy to make a usable fuel. A number of approaches use the components of current biological systems, but re-engineer them for more direct, efficient production of fuels.

  3. 7 CFR 1280.117 - Producer information.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... INFORMATION ORDER Lamb Promotion, Research, and Information Order Definitions § 1280.117 Producer information. Producer information means activities designed to provide producers, feeders, and first handlers with... 7 Agriculture 10 2010-01-01 2010-01-01 false Producer information. 1280.117 Section...

  4. Molecular analysis of shiga toxin-producing Escherichia coli strains isolated from hemolytic-uremic syndrome patients and dairy samples in France.

    PubMed

    Pradel, Nathalie; Bertin, Yolande; Martin, Christine; Livrelli, Valérie

    2008-04-01

    Shiga toxin-producing Escherichia coli (STEC) has been associated with food-borne diseases ranging from uncomplicated diarrhea to hemolytic-uremic syndrome (HUS). While most outbreaks are associated with E. coli O157:H7, about half of the sporadic cases may be due to non-O157:H7 serotypes. To assess the pathogenicity of STEC isolated from dairy foods in France, 40 strains isolated from 1,130 raw-milk and cheese samples were compared with 15 STEC strains isolated from patients suffering from severe disease. The presence of genes encoding Shiga toxins (stx(1), stx(2), and variants), intimin (eae and variants), adhesins (bfp, efa1), enterohemolysin (ehxA), serine protease (espP), and catalase-peroxidase (katP) was determined by PCR and/or hybridization. Plasmid profiling, ribotyping, and pulsed-field gel electrophoresis (PFGE) were used to further compare the strains at the molecular level. A new stx(2) variant, stx(2-CH013), associated with an O91:H10 clinical isolate was identified. The presence of the stx(2), eae, and katP genes, together with a combination of several stx(2) variants, was clearly associated with human-pathogenic strains. In contrast, dairy food STEC strains were characterized by a predominance of stx(1), with a minority of isolates harboring eae, espP, and/or katP. These associations may help to differentiate less virulent STEC strains from those more likely to cause disease in humans. Only one dairy O5 isolate had a virulence gene panel identical to that of an HUS-associated strain. However, the ribotype and PFGE profiles were not identical. In conclusion, most STEC strains isolated from dairy products in France showed characteristics different from those of strains isolated from patients.

  5. PRODUCING ENERGY AND RADIOACTIVE FISSION PRODUCTS

    DOEpatents

    Segre, E.; Kennedy, J.W.; Seaborg, G.T.

    1959-10-13

    This patent broadly discloses the production of plutonium by the neutron bombardment of uranium to produce neptunium which decays to plutonium, and the fissionability of plutonium by neutrons, both fast and thermal, to produce energy and fission products.

  6. 7 CFR 1150.110 - Producer.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... and Orders; Milk), DEPARTMENT OF AGRICULTURE DAIRY PROMOTION PROGRAM Dairy Promotion and Research Order Definitions § 1150.110 Producer. Producer means any person engaged in the production of milk...

  7. 7 CFR 1150.110 - Producer.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... and Orders; Milk), DEPARTMENT OF AGRICULTURE DAIRY PROMOTION PROGRAM Dairy Promotion and Research Order Definitions § 1150.110 Producer. Producer means any person engaged in the production of milk...

  8. 29 CFR 780.213 - Produce business.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 3 2010-07-01 2010-07-01 false Produce business. 780.213 Section 780.213 Labor Regulations... Specific Situations Hatchery Operations § 780.213 Produce business. In some instances, hatcheries also engage in the produce business as such and commingle with the culled eggs and chickens other eggs...

  9. 7 CFR 1430.510 - New producers.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false New producers. 1430.510 Section 1430.510 Agriculture Regulations of the Department of Agriculture (Continued) COMMODITY CREDIT CORPORATION, DEPARTMENT OF....510 New producers. Notwithstanding other provisions of this subpart, producers who were new...

  10. 7 CFR 1430.510 - New producers.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 10 2011-01-01 2011-01-01 false New producers. 1430.510 Section 1430.510 Agriculture Regulations of the Department of Agriculture (Continued) COMMODITY CREDIT CORPORATION, DEPARTMENT OF....510 New producers. Notwithstanding other provisions of this subpart, producers who were new...

  11. 29 CFR 780.213 - Produce business.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 29 Labor 3 2013-07-01 2013-07-01 false Produce business. 780.213 Section 780.213 Labor Regulations... Specific Situations Hatchery Operations § 780.213 Produce business. In some instances, hatcheries also engage in the produce business as such and commingle with the culled eggs and chickens other eggs...

  12. 29 CFR 780.213 - Produce business.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 3 2011-07-01 2011-07-01 false Produce business. 780.213 Section 780.213 Labor Regulations... Specific Situations Hatchery Operations § 780.213 Produce business. In some instances, hatcheries also engage in the produce business as such and commingle with the culled eggs and chickens other eggs...

  13. 29 CFR 780.213 - Produce business.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 29 Labor 3 2014-07-01 2014-07-01 false Produce business. 780.213 Section 780.213 Labor Regulations... Specific Situations Hatchery Operations § 780.213 Produce business. In some instances, hatcheries also engage in the produce business as such and commingle with the culled eggs and chickens other eggs...

  14. 29 CFR 780.213 - Produce business.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 29 Labor 3 2012-07-01 2012-07-01 false Produce business. 780.213 Section 780.213 Labor Regulations... Specific Situations Hatchery Operations § 780.213 Produce business. In some instances, hatcheries also engage in the produce business as such and commingle with the culled eggs and chickens other eggs...

  15. 7 CFR 1032.13 - Producer milk.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 9 2011-01-01 2011-01-01 false Producer milk. 1032.13 Section 1032.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE CENTRAL MARKETING AREA Order Regulating Handling Definitions § 1032.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  16. 7 CFR 1001.13 - Producer milk.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 9 2014-01-01 2013-01-01 true Producer milk. 1001.13 Section 1001.13 Agriculture... AND ORDERS; MILK), DEPARTMENT OF AGRICULTURE MILK IN THE NORTHEAST MARKETING AREA Order Regulating Handling Definitions § 1001.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  17. 7 CFR 1131.13 - Producer milk.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 9 2011-01-01 2011-01-01 false Producer milk. 1131.13 Section 1131.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE ARIZONA MARKETING AREA Order Regulating Handling Definitions § 1131.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  18. 7 CFR 1001.13 - Producer milk.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 9 2012-01-01 2012-01-01 false Producer milk. 1001.13 Section 1001.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE NORTHEAST MARKETING AREA Order Regulating Handling Definitions § 1001.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  19. 7 CFR 1032.13 - Producer milk.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 9 2014-01-01 2013-01-01 true Producer milk. 1032.13 Section 1032.13 Agriculture... AND ORDERS; MILK), DEPARTMENT OF AGRICULTURE MILK IN THE CENTRAL MARKETING AREA Order Regulating Handling Definitions § 1032.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  20. 7 CFR 1006.13 - Producer milk.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 9 2011-01-01 2011-01-01 false Producer milk. 1006.13 Section 1006.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE FLORIDA MARKETING AREA Order Regulating Handling Definitions § 1006.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  1. 7 CFR 1001.13 - Producer milk.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 9 2010-01-01 2009-01-01 true Producer milk. 1001.13 Section 1001.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE NORTHEAST MARKETING AREA Order Regulating Handling Definitions § 1001.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  2. 7 CFR 1126.13 - Producer milk.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 9 2014-01-01 2013-01-01 true Producer milk. 1126.13 Section 1126.13 Agriculture... AND ORDERS; MILK), DEPARTMENT OF AGRICULTURE MILK IN THE SOUTHWEST MARKETING AREA Order Regulating Handling Definitions § 1126.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  3. 7 CFR 1033.13 - Producer milk.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 9 2011-01-01 2011-01-01 false Producer milk. 1033.13 Section 1033.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE MIDEAST MARKETING AREA Order Regulating Handling Definitions § 1033.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  4. 7 CFR 1006.13 - Producer milk.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 9 2010-01-01 2009-01-01 true Producer milk. 1006.13 Section 1006.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE FLORIDA MARKETING AREA Order Regulating Handling Definitions § 1006.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  5. 7 CFR 1033.13 - Producer milk.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 9 2012-01-01 2012-01-01 false Producer milk. 1033.13 Section 1033.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE MIDEAST MARKETING AREA Order Regulating Handling Definitions § 1033.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  6. 7 CFR 1131.13 - Producer milk.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 9 2010-01-01 2009-01-01 true Producer milk. 1131.13 Section 1131.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE ARIZONA MARKETING AREA Order Regulating Handling Definitions § 1131.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  7. 7 CFR 1126.13 - Producer milk.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 9 2010-01-01 2009-01-01 true Producer milk. 1126.13 Section 1126.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE SOUTHWEST MARKETING AREA Order Regulating Handling Definitions § 1126.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  8. 7 CFR 1033.13 - Producer milk.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 9 2013-01-01 2013-01-01 false Producer milk. 1033.13 Section 1033.13 Agriculture... AND ORDERS; MILK), DEPARTMENT OF AGRICULTURE MILK IN THE MIDEAST MARKETING AREA Order Regulating Handling Definitions § 1033.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  9. 7 CFR 1006.13 - Producer milk.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 9 2013-01-01 2013-01-01 false Producer milk. 1006.13 Section 1006.13 Agriculture... AND ORDERS; MILK), DEPARTMENT OF AGRICULTURE MILK IN THE FLORIDA MARKETING AREA Order Regulating Handling Definitions § 1006.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  10. 7 CFR 1033.13 - Producer milk.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 9 2010-01-01 2009-01-01 true Producer milk. 1033.13 Section 1033.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE MIDEAST MARKETING AREA Order Regulating Handling Definitions § 1033.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  11. 7 CFR 1131.13 - Producer milk.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 9 2014-01-01 2013-01-01 true Producer milk. 1131.13 Section 1131.13 Agriculture... AND ORDERS; MILK), DEPARTMENT OF AGRICULTURE MILK IN THE ARIZONA MARKETING AREA Order Regulating Handling Definitions § 1131.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  12. 7 CFR 1126.13 - Producer milk.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 9 2013-01-01 2013-01-01 false Producer milk. 1126.13 Section 1126.13 Agriculture... AND ORDERS; MILK), DEPARTMENT OF AGRICULTURE MILK IN THE SOUTHWEST MARKETING AREA Order Regulating Handling Definitions § 1126.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  13. 7 CFR 1131.13 - Producer milk.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 9 2012-01-01 2012-01-01 false Producer milk. 1131.13 Section 1131.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE ARIZONA MARKETING AREA Order Regulating Handling Definitions § 1131.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  14. 7 CFR 1033.13 - Producer milk.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 9 2014-01-01 2013-01-01 true Producer milk. 1033.13 Section 1033.13 Agriculture... AND ORDERS; MILK), DEPARTMENT OF AGRICULTURE MILK IN THE MIDEAST MARKETING AREA Order Regulating Handling Definitions § 1033.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  15. 7 CFR 1006.13 - Producer milk.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 9 2012-01-01 2012-01-01 false Producer milk. 1006.13 Section 1006.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE FLORIDA MARKETING AREA Order Regulating Handling Definitions § 1006.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  16. 7 CFR 1126.13 - Producer milk.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 9 2012-01-01 2012-01-01 false Producer milk. 1126.13 Section 1126.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE SOUTHWEST MARKETING AREA Order Regulating Handling Definitions § 1126.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  17. 7 CFR 1006.13 - Producer milk.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 9 2014-01-01 2013-01-01 true Producer milk. 1006.13 Section 1006.13 Agriculture... AND ORDERS; MILK), DEPARTMENT OF AGRICULTURE MILK IN THE FLORIDA MARKETING AREA Order Regulating Handling Definitions § 1006.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  18. 7 CFR 1032.13 - Producer milk.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 9 2013-01-01 2013-01-01 false Producer milk. 1032.13 Section 1032.13 Agriculture... AND ORDERS; MILK), DEPARTMENT OF AGRICULTURE MILK IN THE CENTRAL MARKETING AREA Order Regulating Handling Definitions § 1032.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  19. 7 CFR 1032.13 - Producer milk.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 9 2012-01-01 2012-01-01 false Producer milk. 1032.13 Section 1032.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE CENTRAL MARKETING AREA Order Regulating Handling Definitions § 1032.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  20. 7 CFR 1001.13 - Producer milk.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 9 2013-01-01 2013-01-01 false Producer milk. 1001.13 Section 1001.13 Agriculture... AND ORDERS; MILK), DEPARTMENT OF AGRICULTURE MILK IN THE NORTHEAST MARKETING AREA Order Regulating Handling Definitions § 1001.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  1. 7 CFR 1126.13 - Producer milk.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 9 2011-01-01 2011-01-01 false Producer milk. 1126.13 Section 1126.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE SOUTHWEST MARKETING AREA Order Regulating Handling Definitions § 1126.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  2. 7 CFR 1131.13 - Producer milk.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 9 2013-01-01 2013-01-01 false Producer milk. 1131.13 Section 1131.13 Agriculture... AND ORDERS; MILK), DEPARTMENT OF AGRICULTURE MILK IN THE ARIZONA MARKETING AREA Order Regulating Handling Definitions § 1131.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  3. 7 CFR 1001.13 - Producer milk.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 9 2011-01-01 2011-01-01 false Producer milk. 1001.13 Section 1001.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE NORTHEAST MARKETING AREA Order Regulating Handling Definitions § 1001.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  4. 7 CFR 1032.13 - Producer milk.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 9 2010-01-01 2009-01-01 true Producer milk. 1032.13 Section 1032.13 Agriculture... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE CENTRAL MARKETING AREA Order Regulating Handling Definitions § 1032.13 Producer milk. Producer milk means the skim milk (or the skim equivalent...

  5. 7 CFR 1219.20 - Producer.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions § 1219.20 Producer. Producer means any person who is engaged in the business of producing Hass avocados in the United...

  6. 7 CFR 1219.20 - Producer.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions § 1219.20 Producer. Producer means any person who is engaged in the business of producing Hass avocados in the United...

  7. 7 CFR 1434.4 - Eligible producer.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... FOR HONEY § 1434.4 Eligible producer. (a) To be eligible to receive an individual or joint loan or loan deficiency payments under this part, a person must: (1) Have produced honey in the United States... calendar year; (2) Be responsible for the risk of keeping the bees and producing honey; (3) Have...

  8. 7 CFR 1434.4 - Eligible producer.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... FOR HONEY § 1434.4 Eligible producer. (a) To be eligible to receive an individual or joint loan or loan deficiency payments under this part, a person must: (1) Have produced honey in the United States... calendar year; (2) Be responsible for the risk of keeping the bees and producing honey; (3) Have...

  9. 7 CFR 1434.4 - Eligible producer.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... FOR HONEY § 1434.4 Eligible producer. (a) To be eligible to receive an individual or joint loan or loan deficiency payments under this part, a person must: (1) Have produced honey in the United States... calendar year; (2) Be responsible for the risk of keeping the bees and producing honey; (3) Have...

  10. 7 CFR 1434.4 - Eligible producer.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... FOR HONEY § 1434.4 Eligible producer. (a) To be eligible to receive an individual or joint loan or loan deficiency payments under this part, a person must: (1) Have produced honey in the United States... calendar year; (2) Be responsible for the risk of keeping the bees and producing honey; (3) Have...

  11. 7 CFR 1219.20 - Producer.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions § 1219.20 Producer. Producer means any person who is engaged in the business of producing Hass avocados in the United...

  12. 7 CFR 1218.16 - Producer.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BLUEBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Blueberry Promotion, Research, and Information Order Definitions § 1218.16 Producer... is engaged in the business of producing, or causing to be produced for any market, blueberries...

  13. 7 CFR 1219.20 - Producer.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions § 1219.20 Producer. Producer means any person who is engaged in the business of producing Hass avocados in the United...

  14. 7 CFR 1218.16 - Producer.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BLUEBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Blueberry Promotion, Research, and Information Order Definitions § 1218.16 Producer... is engaged in the business of producing, or causing to be produced for any market, blueberries...

  15. 7 CFR 1434.4 - Eligible producer.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... FOR HONEY § 1434.4 Eligible producer. (a) To be eligible to receive an individual or joint loan or loan deficiency payments under this part, a person must: (1) Have produced honey in the United States... calendar year; (2) Be responsible for the risk of keeping the bees and producing honey; (3) Have...

  16. 7 CFR 1219.20 - Producer.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE HASS AVOCADO PROMOTION, RESEARCH, AND INFORMATION Hass Avocado Promotion, Research, and Information Order Definitions § 1219.20 Producer. Producer means any person who is engaged in the business of producing Hass avocados in the United...

  17. 7 CFR 1280.122 - Seedstock producer.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AGREEMENTS AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE LAMB PROMOTION, RESEARCH, AND INFORMATION ORDER Lamb Promotion, Research, and Information Order Definitions § 1280.122 Seedstock producer. Seedstock producer means any lamb producer in the U.S. who engages in the production and sale of...

  18. 7 CFR 1230.21 - Producer.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PORK PROMOTION, RESEARCH, AND CONSUMER INFORMATION Pork Promotion, Research, and Consumer Information Order Definitions § 1230.21 Producer. Producer means a person who produces porcine animals in the United States for sale in commerce....

  19. 7 CFR 1230.21 - Producer.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PORK PROMOTION, RESEARCH, AND CONSUMER INFORMATION Pork Promotion, Research, and Consumer Information Order Definitions § 1230.21 Producer. Producer means a person who produces porcine animals in the United States for sale in commerce....

  20. 7 CFR 1230.21 - Producer.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PORK PROMOTION, RESEARCH, AND CONSUMER INFORMATION Pork Promotion, Research, and Consumer Information Order Definitions § 1230.21 Producer. Producer means a person who produces porcine animals in the United States for sale in commerce....

  1. Produced water discharges into marine ecosystems

    SciTech Connect

    Pitre, R.L.

    1984-05-01

    Formation waters which are produced in conjunction with oil or gas are discharged into offshore areas throughout the world. The produced water volume in petroleum production can vary from 20% to 150% of the oil production volume. Salinities of produced water can vary from 1,000 to 250,000 ppm. Before produced waters are discharged to the ocean, they are passed through oil-water separators to reduce the oil content to acceptable levels. Temperatures of the produced water stream are normally about 45/sup 0/ C. The small amounts of oil released in produced water discharges will either evaporate to the atmosphere or be degraded by marine organisms. Biodegradation can be enhanced with the addition of nutrients. Within the OCS of the United States, produced waters are regulated by NPDES permits issued by the U.S. Environmental Protection Agency. Produced waters from a Cities Service platform located in an area of coral reefs offshore of the Philippine Islands have been discharged to the surrounding seas for several years with no environmental harm. Environmental impacts from produced water discharges into marine waters are very minimal and have never caused a problem. Therefore, there is no precedent for stricter governmental regulation of produced water discharges into marine ecosystems.

  2. Process for producing ethanol from syngas

    SciTech Connect

    Krause, Theodore R; Rathke, Jerome W; Chen, Michael J

    2013-05-14

    The invention provides a method for producing ethanol, the method comprising establishing an atmosphere containing methanol forming catalyst and ethanol forming catalyst; injecting syngas into the atmosphere at a temperature and for a time sufficient to produce methanol; and contacting the produced methanol with additional syngas at a temperature and for a time sufficient to produce ethanol. The invention also provides an integrated system for producing methanol and ethanol from syngas, the system comprising an atmosphere isolated from the ambient environment; a first catalyst to produce methanol from syngas wherein the first catalyst resides in the atmosphere; a second catalyst to product ethanol from methanol and syngas, wherein the second catalyst resides in the atmosphere; a conduit for introducing syngas to the atmosphere; and a device for removing ethanol from the atmosphere. The exothermicity of the method and system obviates the need for input of additional heat from outside the atmosphere.

  3. Method for producing oxygen from lunar materials

    NASA Technical Reports Server (NTRS)

    Sullivan, Thomas A. (Inventor)

    1993-01-01

    This invention is related to producing oxygen from lunar or Martian materials, particularly from lunar ilmenite in situ. The process includes producing a slurry of the minerals and hot sulfuric acid, the acid and minerals reacting to form sulfates of the metal. Water is added to the slurry to dissolve the minerals into an aqueous solution, the first aqueous solution is separated from unreacted minerals from the slurry, and the aqueous solution is electrolyzed to produce the metal and oxygen.

  4. Stable, fertile, high polyhydroxyalkanoate producing plants and methods of producing them

    DOEpatents

    Bohmert-Tatarev, Karen; McAvoy, Susan; Peoples, Oliver P.; Snell, Kristi D.

    2015-08-04

    Transgenic plants that produce high levels of polyhydroxybutyrate and methods of producing them are provided. In a preferred embodiment the transgenic plants are produced using plastid transformation technologies and utilize genes which are codon optimized. Stably transformed plants able to produce greater than 10% dwt PHS in tissues are also provided.

  5. 7 CFR 1124.12 - Producer.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ..., producer means any person who produces milk approved by a duly constituted regulatory agency for fluid consumption as Grade A milk and whose milk (or components of milk) is: (1) Received at a pool plant directly... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE PACIFIC NORTHWEST MARKETING AREA...

  6. 7 CFR 1030.12 - Producer.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... means any person who produces milk approved by a duly constituted regulatory agency for fluid consumption as Grade A milk and whose milk is: (1) Received at a pool plant directly from the producer or... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE UPPER MIDWEST MARKETING AREA Order...

  7. 7 CFR 1030.12 - Producer.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... means any person who produces milk approved by a duly constituted regulatory agency for fluid consumption as Grade A milk and whose milk is: (1) Received at a pool plant directly from the producer or... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE UPPER MIDWEST MARKETING AREA Order...

  8. 7 CFR 1033.12 - Producer.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... means any person who produces milk approved by a duly constituted regulatory agency for fluid consumption as Grade A milk and whose milk is: (1) Received at a pool plant directly from the producer or... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE MIDEAST MARKETING AREA Order...

  9. 7 CFR 1124.12 - Producer.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ..., producer means any person who produces milk approved by a duly constituted regulatory agency for fluid consumption as Grade A milk and whose milk (or components of milk) is: (1) Received at a pool plant directly... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE PACIFIC NORTHWEST MARKETING AREA...

  10. 7 CFR 1033.12 - Producer.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... means any person who produces milk approved by a duly constituted regulatory agency for fluid consumption as Grade A milk and whose milk is: (1) Received at a pool plant directly from the producer or... and Orders; Milk), DEPARTMENT OF AGRICULTURE MILK IN THE MIDEAST MARKETING AREA Order...

  11. 9 CFR 114.16 - Producing subsidiaries.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PRODUCTION REQUIREMENTS FOR BIOLOGICAL PRODUCTS § 114.16 Producing subsidiaries. A serial or subserial of a biological product may be... amount of each serial or subserial credited to each participating producer shall be determined at...

  12. 9 CFR 114.16 - Producing subsidiaries.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PRODUCTION REQUIREMENTS FOR BIOLOGICAL PRODUCTS § 114.16 Producing subsidiaries. A serial or subserial of a biological product may be... amount of each serial or subserial credited to each participating producer shall be determined at...

  13. 7 CFR 1216.22 - Producer.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order Definitions § 1216.22 Producer. Producer means any person engaged in the production and sale of peanuts and who owns, or shares the ownership...

  14. 7 CFR 1216.22 - Producer.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PEANUT PROMOTION, RESEARCH, AND INFORMATION ORDER Peanut Promotion, Research, and Information Order Definitions § 1216.22 Producer. Producer means any person engaged in the production and sale of peanuts and who owns, or shares the ownership...

  15. 7 CFR 1208.19 - Producer.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PROCESSED RASPBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Processed Raspberry Promotion, Research, and Information Order Definitions § 1208.19 Producer. Producer means any person who grows 20,000 pounds or more of raspberries...

  16. 7 CFR 1208.19 - Producer.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE PROCESSED RASPBERRY PROMOTION, RESEARCH, AND INFORMATION ORDER Processed Raspberry Promotion, Research, and Information Order Definitions § 1208.19 Producer. Producer means any person who grows 20,000 pounds or more of raspberries...

  17. Music Teacher as Writer and Producer

    ERIC Educational Resources Information Center

    Randles, Clint

    2012-01-01

    In this article I attempt to redefine the role of a music teacher as being more than a director, the more traditional term ascribed to this position. I do this by using descriptions of the role of "writer" and "producer" of student lives borrowed from music education philosophy, screenwriting, and professional music producers. This vision is…

  18. 7 CFR 985.6 - Producer.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Producer. 985.6 Section 985.6 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE MARKETING ORDER REGULATING THE HANDLING OF SPEARMINT OIL PRODUCED IN THE FAR WEST...

  19. 7 CFR 985.6 - Producer.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 8 2014-01-01 2014-01-01 false Producer. 985.6 Section 985.6 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE MARKETING ORDER REGULATING THE HANDLING OF SPEARMINT OIL PRODUCED IN THE FAR WEST...

  20. 7 CFR 1260.116 - Producer.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AND ORDERS; MISCELLANEOUS COMMODITIES), DEPARTMENT OF AGRICULTURE BEEF PROMOTION AND RESEARCH Beef Promotion and Research Order Definitions § 1260.116 Producer. Producer means any person who owns or acquires... of this subpart if (a) the person's only share in the proceeds of a sale of cattle or beef is a...