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Sample records for adhesin protein p1

  1. Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy.

    PubMed

    Tang, Wenxing; Bhatt, Avni; Smith, Adam N; Crowley, Paula J; Brady, L Jeannine; Long, Joanna R

    2016-02-01

    The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ~57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

  2. Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy

    PubMed Central

    Tang, Wenxing; Bhatt, Avni; Smith, Adam N.; Crowley, Paula J.; Brady, L. Jeannine; Long, Joanna R.

    2016-01-01

    The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ~57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to 1) globally characterize cell walls isolated from a Gram-positive bacterium and 2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin. PMID:26837620

  3. Identification of a supramolecular functional architecture of Streptococcus mutans adhesin P1 on the bacterial cell surface.

    PubMed

    Heim, Kyle P; Sullan, Ruby May A; Crowley, Paula J; El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Tang, Wenxing; Besingi, Richard; Dufrene, Yves F; Brady, L Jeannine

    2015-04-03

    P1 (antigen I/II) is a sucrose-independent adhesin of Streptococcus mutans whose functional architecture on the cell surface is not fully understood. S. mutans cells subjected to mechanical extraction were significantly diminished in adherence to immobilized salivary agglutinin but remained immunoreactive and were readily aggregated by fluid-phase salivary agglutinin. Bacterial adherence was restored by incubation of postextracted cells with P1 fragments that contain each of the two known adhesive domains. In contrast to untreated cells, glutaraldehyde-treated bacteria gained reactivity with anti-C-terminal monoclonal antibodies (mAbs), whereas epitopes recognized by mAbs against other portions of the molecule were masked. Surface plasmon resonance experiments demonstrated the ability of apical and C-terminal fragments of P1 to interact. Binding of several different anti-P1 mAbs to unfixed cells triggered release of a C-terminal fragment from the bacterial surface, suggesting a novel mechanism of action of certain adherence-inhibiting antibodies. We also used atomic force microscopy-based single molecule force spectroscopy with tips bearing various mAbs to elucidate the spatial organization and orientation of P1 on living bacteria. The similar rupture lengths detected using mAbs against the head and C-terminal regions, which are widely separated in the tertiary structure, suggest a higher order architecture in which these domains are in close proximity on the cell surface. Taken together, our results suggest a supramolecular organization in which additional P1 polypeptides, including the C-terminal segment originally identified as antigen II, associate with covalently attached P1 to form the functional adhesive layer.

  4. Salicylic acid enhances Staphylococcus aureus extracellular adhesin protein expression.

    PubMed

    Alvarez, Lucía P; Barbagelata, María S; Cheung, Ambrose L; Sordelli, Daniel O; Buzzola, Fernanda R

    2011-11-01

    One of the virulence factors required by Staphylococcus aureus at the early stages of infection is Eap, a secreted adhesin that binds many host proteins and is upregulated by the two-component regulatory system saeRS. The S. aureus Newman strain harbors a mutation in saeS that is thought to be responsible for the high level of Eap expression in this strain. This study was designed to ascertain whether salicylic acid (SAL) affects the expression of Eap and the internalization of S. aureus into epithelial cells. The strain Newman treated with SAL exhibited increased levels of eap transcription and protein expression. Furthermore, SAL treatment increased the eap promoter activity. SAL treatment enhanced Eap expression in the Newman and in other S. aureus strains that do not carry the mutation in saeS. Internalization of S. aureus eap and sae mutants into the MAC-T epithelial cells was significantly decreased compared with the wild-type counterparts. In conclusion, we demonstrated that a low concentration of SAL increased S. aureus Eap expression possibly due to enhancement of sae. SAL may create the conditions for S. aureus persistence in the host, not only by decreasing the capsular polysaccharide expression as shown before, but also by enhancing Eap expression.

  5. Differentiation of salivary agglutinin-mediated adherence and aggregation of mutans streptococci by use of monoclonal antibodies against the major surface adhesin P1.

    PubMed Central

    Brady, L J; Piacentini, D A; Crowley, P J; Oyston, P C; Bleiweis, A S

    1992-01-01

    The ability to adhere to salivary agglutinin-coated hydroxyapatite beads and to aggregate in the presence of fluid-phase salivary agglutinin was tested by using 25 isolates of mutants streptococci representing eight serotypes. Both adherence and aggregation activity correlated with expression of the Mr-185,000 cell surface antigen P1 on Streptococcus mutans serotype c, e, and f strains. In addition, it was shown that the P1 molecule itself served as the adhesin of S. mutans serotype c, since adherence was significantly inhibited by the presence of recombinant-specified Mr-150,000 P1. The ability of S. sobrinus strains to adhere or aggregate did not correlate with expression of the P1 cross-reactive antigen SpaA. There was also evidence for interaction with salivary agglutinin, as manifested by aggregation but not adherence of S. rattus serotype b, which does not express a P1 cross-reactive antigen. To understand the interaction of P1 with salivary agglutinin at the molecular level, a panel of 11 anti-P1 monoclonal antibodies was tested for inhibitory activity in adherence and aggregation inhibition assays. Overlapping, but not identical, subsets of monoclonal antibodies were found to inhibit adherence and aggregation, indicating that the interactions of P1 with salivary agglutinin which mediate these two phenomena are different. The localization of functional domains of P1 which may mediate the aggregation and adherence reactions is discussed. PMID:1541515

  6. 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

  7. 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.

  8. 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

  9. 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

  10. Autocatalytic association of proteins by covalent bond formation: a Bio Molecular Welding toolbox derived from a bacterial adhesin

    PubMed Central

    Bonnet, J.; Cartannaz, J.; Tourcier, G.; Contreras-Martel, C.; Kleman, J. P.; Morlot, C.; Vernet, T.; Di Guilmi, A. M.

    2017-01-01

    Unusual intramolecular cross-links present in adhesins from Gram-positive bacteria have been used to develop a generic process amenable to biotechnology applications. Based on the crystal structure of RrgA, the Streptococcus pneumoniae pilus adhesin, we provide evidence that two engineered protein fragments retain their ability to associate covalently with high specificity, in vivo and in vitro, once isolated from the parent protein. We determined the optimal conditions for the assembly of the complex and we solved its crystal structure at 2 Å. Furthermore, we demonstrate biotechnological applications related to antibody production, nanoassembly and cell-surface labeling based on this process we named Bio Molecular Welding. PMID:28252635

  11. The Streptococcus gordonii Adhesin CshA Protein Binds Host Fibronectin via a Catch-Clamp Mechanism*♦

    PubMed Central

    Back, Catherine R.; Sztukowska, Maryta N.; Till, Marisa; Lamont, Richard J.; Jenkinson, Howard F.; Nobbs, Angela H.; Race, Paul R.

    2017-01-01

    Adherence of bacteria to biotic or abiotic surfaces is a prerequisite for host colonization and represents an important step in microbial pathogenicity. This attachment is facilitated by bacterial adhesins at the cell surface. Because of their size and often elaborate multidomain architectures, these polypeptides represent challenging targets for detailed structural and functional characterization. The multifunctional fibrillar adhesin CshA, which mediates binding to both host molecules and other microorganisms, is an important determinant of colonization by Streptococcus gordonii, an oral commensal and opportunistic pathogen of animals and humans. CshA binds the high-molecular-weight glycoprotein fibronectin (Fn) via an N-terminal non-repetitive region, and this protein-protein interaction has been proposed to promote S. gordonii colonization at multiple sites within the host. However, the molecular details of how these two proteins interact have yet to be established. Here we present a structural description of the Fn binding N-terminal region of CshA, derived from a combination of X-ray crystallography, small angle X-ray scattering, and complementary biophysical methods. In vitro binding studies support a previously unreported two-state “catch-clamp” mechanism of Fn binding by CshA, in which the disordered N-terminal domain of CshA acts to “catch” Fn, via formation of a rapidly assembled but also readily dissociable pre-complex, enabling its neighboring ligand binding domain to tightly clamp the two polypeptides together. This study presents a new paradigm for target binding by a bacterial adhesin, the identification of which will inform future efforts toward the development of anti-adhesive agents that target S. gordonii and related streptococci. PMID:27920201

  12. Dimeric and Trimeric Fusion Proteins Generated with Fimbrial Adhesins of Uropathogenic Escherichia coli.

    PubMed

    Luna-Pineda, Víctor M; Reyes-Grajeda, Juan Pablo; Cruz-Córdova, Ariadnna; Saldaña-Ahuactzi, Zeus; Ochoa, Sara A; Maldonado-Bernal, Carmen; Cázares-Domínguez, Vicenta; Moreno-Fierros, Leticia; Arellano-Galindo, José; Hernández-Castro, Rigoberto; Xicohtencatl-Cortes, Juan

    2016-01-01

    Urinary tract infections (UTIs) are associated with high rates of morbidity and mortality worldwide, and uropathogenic Escherichia coli (UPEC) is the main etiologic agent. Fimbriae assembled on the bacterial surface are essential for adhesion to the urinary tract epithelium. In this study, the FimH, CsgA, and PapG adhesins were fused to generate biomolecules for use as potential target vaccines against UTIs. The fusion protein design was generated using bioinformatics tools, and template fusion gene sequences were synthesized by GenScript in the following order fimH-csgA-papG-fimH-csgA (fcpfc) linked to the nucleotide sequence encoding the [EAAAK]5 peptide. Monomeric (fimH, csgA, and papG), dimeric (fimH-csgA), and trimeric (fimH-csgA-papG) genes were cloned into the pLATE31 expression vector and generated products of 1040, 539, 1139, 1442, and 2444 bp, respectively. Fusion protein expression in BL21 E. coli was induced with 1 mM IPTG, and His-tagged proteins were purified under denaturing conditions and refolded by dialysis using C-buffer. Coomassie blue-stained SDS-PAGE gels and Western blot analysis revealed bands of 29.5, 11.9, 33.9, 44.9, and 82.1 kDa, corresponding to FimH, CsgA, PapG, FC, and FCP proteins, respectively. Mass spectrometry analysis by MALDI-TOF/TOF revealed specific peptides that confirmed the fusion protein structures. Dynamic light scattering analysis revealed the polydispersed state of the fusion proteins. FimH, CsgA, and PapG stimulated the release of 372-398 pg/mL IL-6; interestingly, FC and FCP stimulated the release of 464.79 pg/mL (p ≤ 0.018) and 521.24 pg/mL (p ≤ 0.002) IL-6, respectively. In addition, FC and FCP stimulated the release of 398.52 pg/mL (p ≤ 0.001) and 450.40 pg/mL (p ≤ 0.002) IL-8, respectively. High levels of IgA and IgG antibodies in human sera reacted against the fusion proteins, and under identical conditions, low levels of IgA and IgG antibodies were detected in human urine. Rabbit polyclonal antibodies

  13. Dimeric and Trimeric Fusion Proteins Generated with Fimbrial Adhesins of Uropathogenic Escherichia coli

    PubMed Central

    Luna-Pineda, Víctor M.; Reyes-Grajeda, Juan Pablo; Cruz-Córdova, Ariadnna; Saldaña-Ahuactzi, Zeus; Ochoa, Sara A.; Maldonado-Bernal, Carmen; Cázares-Domínguez, Vicenta; Moreno-Fierros, Leticia; Arellano-Galindo, José; Hernández-Castro, Rigoberto; Xicohtencatl-Cortes, Juan

    2016-01-01

    Urinary tract infections (UTIs) are associated with high rates of morbidity and mortality worldwide, and uropathogenic Escherichia coli (UPEC) is the main etiologic agent. Fimbriae assembled on the bacterial surface are essential for adhesion to the urinary tract epithelium. In this study, the FimH, CsgA, and PapG adhesins were fused to generate biomolecules for use as potential target vaccines against UTIs. The fusion protein design was generated using bioinformatics tools, and template fusion gene sequences were synthesized by GenScript in the following order fimH-csgA-papG-fimH-csgA (fcpfc) linked to the nucleotide sequence encoding the [EAAAK]5 peptide. Monomeric (fimH, csgA, and papG), dimeric (fimH-csgA), and trimeric (fimH-csgA-papG) genes were cloned into the pLATE31 expression vector and generated products of 1040, 539, 1139, 1442, and 2444 bp, respectively. Fusion protein expression in BL21 E. coli was induced with 1 mM IPTG, and His-tagged proteins were purified under denaturing conditions and refolded by dialysis using C-buffer. Coomassie blue-stained SDS-PAGE gels and Western blot analysis revealed bands of 29.5, 11.9, 33.9, 44.9, and 82.1 kDa, corresponding to FimH, CsgA, PapG, FC, and FCP proteins, respectively. Mass spectrometry analysis by MALDI-TOF/TOF revealed specific peptides that confirmed the fusion protein structures. Dynamic light scattering analysis revealed the polydispersed state of the fusion proteins. FimH, CsgA, and PapG stimulated the release of 372–398 pg/mL IL-6; interestingly, FC and FCP stimulated the release of 464.79 pg/mL (p ≤ 0.018) and 521.24 pg/mL (p ≤ 0.002) IL-6, respectively. In addition, FC and FCP stimulated the release of 398.52 pg/mL (p ≤ 0.001) and 450.40 pg/mL (p ≤ 0.002) IL-8, respectively. High levels of IgA and IgG antibodies in human sera reacted against the fusion proteins, and under identical conditions, low levels of IgA and IgG antibodies were detected in human urine. Rabbit polyclonal antibodies

  14. Spiroplasma eriocheiris Adhesin-Like Protein (ALP) Interacts with Epidermal Growth Factor (EGF) Domain Proteins to Facilitate Infection

    PubMed Central

    Hou, Libo; Liu, Yuhan; Gao, Qi; Xu, Xuechuan; Ning, Mingxiao; Bi, Jingxiu; Liu, Hui; Liu, Min; Gu, Wei; Wang, Wen; Meng, Qingguo

    2017-01-01

    Spiroplasma eriocheiris is a novel pathogen found in recent years, causing the tremor disease (TD) of Chinese mitten crab Eriocheir sinensis. Like Spiroplasma mirum, S. eriocheiris infects the newborn mouse (adult mice are not infected) and can cause cataract. Adhesion-related protein is an important protein involved in the interaction between pathogen and host. In this study, the Adhesin-like Protein (ALP) of S. eriocheiris was detected on its outer membrane by using immune electron microscopy, and was found to be involved in the bacterium's infection of mouse embryo fibroblasts (3T6-Swiss albino). Yeast two-hybrid analysis demonstrated that ALP interacts with a diverse group of mouse proteins. The interactions between recombinant partial fibulin7 (FBLN7; including two epidermal growth factor [EGF] domains) and ALP were confirmed by Far-western blotting and colocalization. We synthetized the domains of FBLN7 [EGF domain: amino acids 136–172 and complement control protein (CCP) domain: 81–134 amino acids], and demonstrated that only EGF domain of FBLN7 can interact with ALP. Because the EGF domain has high degree of similarity to EGF, it can activate the downstream EGFR signaling pathway, in key site amino acids. The EGFR pathway in 3T6 cells was restrained after rALP stimulation resulting from competitive binding of ALP to EGF. The unborn mouse, newborn mouse, and the adult mouse with cataract have a small amount of expressed FBLN7; however, none was detected in the brain and very little expression was seen in the eye of normal adult mice. In short, ALP as a S. eriocheiris surface protein, is critical for infection and further supports the role of ALP in S. eriocheiris infection by competitive effection of the EGF/EGFR axis of the target cells. PMID:28184355

  15. Cluster analysis identifies aminoacid compositional features that indicate Toxoplasma gondii adhesin proteins

    PubMed Central

    Arenas, Ailan F; Salcedo, Gladys E; Moncada, Diego M; Erazo, Diego A; Osorio, Juan F; Gomez-Marin, Jorge E

    2012-01-01

    Toxoplasma gondii invade host cells using a multi-step process that depends on the regulated secretion of adhesions. To identify key primary sequence features of adhesins in this parasite, we analyze the relative frequency of individual amino acids, their dipeptide frequencies, and the polarity, polarizability and Van der Waals volume of the individual amino acids by using cluster analysis. This method identified cysteine as a key amino acid in the Toxoplasma adhesin group. The best vector algorithm of non-concatenated features was for 2 attributes: the single amino acid relative frequency and the dipeptide frequency. Polarity, polarizability and Van der Waals volume were not good classificatory attributes. Single amino acid attributes clustered unambiguously 67 apicomplexan hypothetical adhesins. This algorithm was also useful for clustering hypothetical Toxoplasma target host receptors. All of the cluster performances had over 70% sensitivity and 80% specificity. Compositional aminoacid data can be useful for improving machine learning-based prediction software when homology and structural data are not sufficient. PMID:23144551

  16. Cohesive Properties of the Caulobacter crescentus Holdfast Adhesin Are Regulated by a Novel c-di-GMP Effector Protein

    PubMed Central

    Sprecher, Kathrin S.; Hug, Isabelle; Nesper, Jutta; Potthoff, Eva; Mahi, Mohamed-Ali; Sangermani, Matteo; Kaever, Volkhard; Schwede, Torsten; Vorholt, Julia

    2017-01-01

    ABSTRACT When encountering surfaces, many bacteria produce adhesins to facilitate their initial attachment and to irreversibly glue themselves to the solid substrate. A central molecule regulating the processes of this motile-sessile transition is the second messenger c-di-GMP, which stimulates the production of a variety of exopolysaccharide adhesins in different bacterial model organisms. In Caulobacter crescentus, c-di-GMP regulates the synthesis of the polar holdfast adhesin during the cell cycle, yet the molecular and cellular details of this control are currently unknown. Here we identify HfsK, a member of a versatile N-acetyltransferase family, as a novel c-di-GMP effector involved in holdfast biogenesis. Cells lacking HfsK form highly malleable holdfast structures with reduced adhesive strength that cannot support surface colonization. We present indirect evidence that HfsK modifies the polysaccharide component of holdfast to buttress its cohesive properties. HfsK is a soluble protein but associates with the cell membrane during most of the cell cycle. Coincident with peak c-di-GMP levels during the C. crescentus cell cycle, HfsK relocalizes to the cytosol in a c-di-GMP-dependent manner. Our results indicate that this c-di-GMP-mediated dynamic positioning controls HfsK activity, leading to its inactivation at high c-di-GMP levels. A short C-terminal extension is essential for the membrane association, c-di-GMP binding, and activity of HfsK. We propose a model in which c-di-GMP binding leads to the dispersal and inactivation of HfsK as part of holdfast biogenesis progression. PMID:28325767

  17. 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

  18. Characterization of the P1 protein and coding region of the zucchini yellow mosaic virus.

    PubMed

    Wisler, G C; Purcifull, D E; Hiebert, E

    1995-01-01

    The nucleotide sequence of the 5'-terminal P1 coding region of an aphid-transmissible isolate of zucchini yellow mosaic virus (ZYMV; strain FL/AT), a mild isolate (strain MD) and a severe isolate (strain SV), all from Florida, were compared with two other ZYMV isolates. The ZYMV MD and SV isolates and an isolate from California (ZYMV CA) had 95-98% sequence similarities to FL/AT, whereas an isolate from Reunion Island (ZYMV RU) had a 60% sequence similarity to FL/AT. ZYMV MD had an 18 nucleotide insert following the start codon of the P1 coding region. The P1 proteins of all ZYMV isolates shared conserved amino acids in areas of the C terminus similar to those reported for other potyviruses. Polyclonal antisera were prepared to the P1 proteins of ZYMV FL/AT and RU expressed in Escherichia coli. The FL/AT and RU P1 antisera showed varying degrees of reactivity in Western blots with extracts of pumpkin (Cucurbita pepo L.) singly infected with a number of distinct ZYMV isolates. The reaction of the FL/AT P1 antiserum with isolate RU-infected tissue extracts was very weak compared to the homologous reaction. Neither antiserum reacted with extracts from plants singly infected with three other potyviruses, a potexvirus, or a cucumovirus. The P1 proteins of ZYMV isolates ranged in molecular mass from 33 kDa to 35 kDa. The P1 protein of strain MD was larger (35 kDa) than that of FL/AT (34 kDa). Indirect immunofluorescence tests with FL/AT P1 antiserum indicated that the P1 protein aggregates in ZYMV-infected tissues. The antisera to the ZYMV P1 proteins have potential as serological probes for identifying ZYMV and for distinguishing ZYMV isolates by immunoblotting.

  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. The malate synthase of Paracoccidioides brasiliensis is a linked surface protein that behaves as an anchorless adhesin

    PubMed Central

    2009-01-01

    Background The pathogenic fungus Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM). This is a pulmonary mycosis acquired by inhalation of fungal airborne propagules that can disseminate to several organs and tissues leading to a severe form of the disease. Adhesion and invasion to host cells are essential steps involved in the internalization and dissemination of pathogens. Inside the host, P. brasiliensis may use the glyoxylate cycle for intracellular survival. Results Here, we provide evidence that the malate synthase of P. brasiliensis (PbMLS) is located on the fungal cell surface, and is secreted. PbMLS was overexpressed in Escherichia coli, and polyclonal antibody was obtained against this protein. By using Confocal Laser Scanning Microscopy, PbMLS was detected in the cytoplasm and in the cell wall of the mother, but mainly of budding cells of the P. brasiliensis yeast phase. PbMLSr and its respective polyclonal antibody produced against this protein inhibited the interaction of P. brasiliensis with in vitro cultured epithelial cells A549. Conclusion These observations indicated that cell wall-associated PbMLS could be mediating the binding of fungal cells to the host, thus contributing to the adhesion of fungus to host tissues and to the dissemination of infection, behaving as an anchorless adhesin. PMID:20034376

  1. Serotype 3 pneumococci sequester platelet-derived human thrombospondin-1 via the adhesin and immune evasion protein Hic.

    PubMed

    Binsker, Ulrike; Kohler, Thomas P; Krauel, Krystin; Kohler, Sylvia; Habermeyer, Johanna; Schwertz, Hansjörg; Hammerschmidt, Sven

    2017-04-07

    Streptococcus pneumoniae serotype 3 strains emerge frequently within clinical isolates of invasive diseases. Bacterial invasion into deeper tissues is associated with colonization and immune evasion mechanisms. Thus, pneumococci express a versatile repertoire of surface proteins sequestering and interacting specifically with components of the human extracellular matrix and serum. Hic, a PspC-like pneumococcal surface protein, possesses vitronectin and factor H binding activity. Here, we show that heterologously expressed Hic domains interact, similar to the classical PspC molecule, with human matricellular thrombospondin-1 (hTSP-1). Binding studies with isolated human thrombospondin-1 and various Hic domains suggest that the interaction between hTSP-1 and Hic differs from binding to vitronectin and factor H. Binding of Hic to hTSP-1 is inhibited by heparin and chondroitin sulfate A, indicating binding to the N-terminal globular domain or type I repeats of hTSP-1. Competitive inhibition experiments with other pneumococcal hTSP-1 adhesins demonstrated that PspC and PspC-like Hic recognize similar domains, whereas PavB and Hic can bind simultaneously to hTSP-1. In conclusion, Hic binds specifically hTSP-1; however, truncation in the N-terminal part of Hic decreases the binding activity, suggesting that the full length of the α-helical regions of Hic is required for an optimal interaction.

  2. Trimeric Autotransporter Adhesins in Members of the Burkholderia Cepacia Complex: A Multifunctional Family of Proteins Implicated in Virulence

    PubMed Central

    Mil-Homens, Dalila; Fialho, Arsénio M.

    2011-01-01

    Trimeric autotransporter adhesins (TAAs) are multimeric surface proteins exclusively found in bacteria. They are involved in various biological traits of pathogenic Gram-negative bacteria including adherence, biofilm formation, invasion, survival within eukaryotic cells, serum resistance, and cytotoxicity. TAAs have a modular architecture composed by a conserved membrane-anchored C-terminal domain and a variable number of stalk and head domains. In this study, a bioinformatic approach has been used to analyze the distribution and architecture of TAAs among Burkholderia cepacia complex (Bcc) genomes. Fifteen genomes were probed revealing a total of 74 encoding sequences. Compared with other bacterial species, the Bcc genomes contain a large number of TAAs (two genes to up to eight genes, such as in B. cenocepacia). Phylogenetic analysis showed that the TAAs grouped into at least eight distinct clusters. TAAs with serine-rich repeats are clearly well separated from others, thereby representing a different evolutionary lineage. Comparative gene mapping across Bcc genomes reveals that TAA genes are inserted within conserved synteny blocks. We further focused our analysis on the epidemic strain B. cenocepacia J2315 in which seven TAAs were annotated. Among these, three TAA-encoding genes (BCAM019, BCAM0223, and BCAM0224) are organized into a cluster and are candidates for multifunctional virulence factors. Here we review the current insights into the functional role of BCAM0224 as a model locus. PMID:22919579

  3. Oligosaccharide ligands for NKR-P1 protein activate NK cells and cytotoxicity

    NASA Astrophysics Data System (ADS)

    Bezouška, Karel; Yuen, Chun-Ting; O'Brien, Jacqui; Childs, Robert A.; Chai, Wengang; Lawson, Alexander M.; Drbal, Karel; Fišerová, Anna; Posíšil, Miloslav; Feizi, Ten

    1994-11-01

    A diversity of high-affinity Oligosaccharide ligands are identified for NKR-P1, a membrane protein on natural killer (NK) cells which contains an extracellular Ca2+-dependent lectin domain. Interactions of such oligosaccharides on the target cell surface with NKR-P1 on the killer cell surface are crucial both for target cell recognition and for delivery of stimulatory or inhibitory signals linked to the NK cytolytic machinery. NK-resistant tumour cells are rendered susceptible by preincubation with liposomes expressing NKR-P1 ligands, suggesting that purging of tumour or virally infected cells in vivo may be a therapeutic possibility.

  4. 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.

  5. The Bfp60 surface adhesin is an extracellular matrix and plasminogen protein interacting in Bacteroides fragilis

    PubMed Central

    de Oliveira Ferreira, Eliane; Teixeira, Felipe; Cordeiro, Fabiana; Lobo, Leandro Araujo; Rocha, Edson R.; Smith, Jeffrey C.; Domingues, Regina M C P

    2014-01-01

    Plasminogen (Plg) is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by several pathogenic species of bacteria to manipulate the host plasminogen system and facilitate invasion of tissues during infection by modifying the activation of this process through the binding of Plg at their surface. Bacteroides fragilis is the most commonly isolated Gram-negative obligate anaerobe from human clinical infections, such as intra-abdominal abscesses and anaerobic bacteraemia. The ability of B. fragilis to convert plasminogen (Plg) into plasmin has been associated with an outer membrane protein named Bfp60. In this study, we characterized the function of Bfp60 protein in B. fragilis 638R by constructing the bfp60 defective strain and comparing its with that of the wild type regarding binding to laminin-1 (LMN-1) and activation of Plg into plasmin. Although the results showed in this study indicate that Bfp60 surface protein of B. fragilis is important for the recognition of LMN-1 and Plg activation, a significant slow activation of Plg into plasmin was observed in the mutant strain. For that reason, the possibility of another unidentified mechanism activating Plg is also present in B. fragilis can not be discarded. The results demonstrate that Bfp60 protein is responsible for the recognition of laminin and Plg-plasmin activation. Although the importance of this protein is still unclear in the pathogenicity of the species, it is accepted that since other pathogenic bacteria use this mechanism to disseminate through the extracellular matrix during the infection, it should also contribute to the virulence of B. fragilis. PMID:23850366

  6. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.

    PubMed

    Zakeri, Bijan; Fierer, Jacob O; Celik, Emrah; Chittock, Emily C; Schwarz-Linek, Ulrich; Moy, Vincent T; Howarth, Mark

    2012-03-20

    Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.

  7. 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

  8. Targeted protein engineering provides insights into binding mechanism and affinities of bacterial collagen adhesins.

    PubMed

    Ross, Caná L; Liang, Xiaowen; Liu, Qing; Murray, Barbara E; Höök, Magnus; Ganesh, Vannakambadi K

    2012-10-05

    The collagen-binding bacterial proteins, Ace and Cna, are well characterized on the biochemical and structural level. Despite overall structural similarity, recombinant forms of the Ace and Cna ligand-binding domains exhibit significantly different affinities and binding kinetics for collagen type I (CI) in vitro. In this study, we sought to understand, in submolecular detail, the bases for these differences. Using a structure-based approach, we engineered Cna and Ace variants by altering specific structural elements within the ligand-binding domains. Surface plasmon resonance-based binding analysis demonstrated that mutations that are predicted to alter the orientation of the Ace and Cna N(1) and N(2) subdomains significantly affect the interaction between the MSCRAMM (microbial surface components recognizing adhesive matrix molecule) and CI in vitro, including affinity, association/dissociation rates and binding ratio. Moreover, we utilized this information to engineer an Ace variant with an 11,000-fold higher CI affinity than the parent protein. Finally, we noted that several engineered proteins that exhibited a weak interaction with CI recognized more sites on CI, suggesting an inverse correlation between affinity and specificity.

  9. Protein E of Haemophilus influenzae is a ubiquitous highly conserved adhesin.

    PubMed

    Singh, Birendra; Brant, Marta; Kilian, Mogens; Hallström, Björn; Riesbeck, Kristian

    2010-02-01

    Protein E (PE) of nontypeable Haemophilus influenzae (NTHi) is involved in adhesion and activation of epithelial cells. A total of 186 clinical NTHi isolates, encapsulated H. influenzae, and culture collection strains were analyzed. PE was highly conserved in both NTHi and encapsulated H. influenzae (96.9%-100% identity without the signal peptide). PE also existed in other members of the genus Pasteurellaceae. The epithelial cell binding region (amino acids 84-108) was completely conserved. Phylogenetic analysis of the pe sequence separated Haemophilus species into 2 separate clusters. Importantly, PE was expressed in 98.4% of all NTHi (126 isolates) independently of the growth phase.

  10. Anaplasma marginale Outer Membrane Protein A Is an Adhesin That Recognizes Sialylated and Fucosylated Glycans and Functionally Depends on an Essential Binding Domain.

    PubMed

    Hebert, Kathryn S; Seidman, David; Oki, Aminat T; Izac, Jerilyn; Emani, Sarvani; Oliver, Lee D; Miller, Daniel P; Tegels, Brittney K; Kannagi, Reiji; Marconi, Richard T; Carlyon, Jason A

    2017-03-01

    Anaplasma marginale causes bovine anaplasmosis, a debilitating and potentially fatal tick-borne infection of cattle. Because A. marginale is an obligate intracellular organism, its adhesins that mediate entry into host cells are essential for survival. Here, we demonstrate that A. marginale outer membrane protein A (AmOmpA; AM854) contributes to the invasion of mammalian and tick host cells. AmOmpA exhibits predicted structural homology to OmpA of A. phagocytophilum (ApOmpA), an adhesin that uses key lysine and glycine residues to interact with α2,3-sialylated and α1,3-fucosylated glycan receptors, including 6-sulfo-sialyl Lewis x (6-sulfo-sLe(x)). Antisera against AmOmpA or its predicted binding domain inhibits A. marginale infection of host cells. Residues G55 and K58 are contributory, and K59 is essential for recombinant AmOmpA to bind to host cells. Enzymatic removal of α2,3-sialic acid and α1,3-fucose residues from host cell surfaces makes them less supportive of AmOmpA binding. AmOmpA is both an adhesin and an invasin, as coating inert beads with it confers adhesiveness and invasiveness. Recombinant forms of AmOmpA and ApOmpA competitively antagonize A. marginale infection of host cells, but a monoclonal antibody against 6-sulfo-sLe(x) fails to inhibit AmOmpA adhesion and A. marginale infection. Thus, the two OmpA proteins bind related but structurally distinct receptors. This study provides a detailed understanding of AmOmpA function, identifies its essential residues that can be targeted by blocking antibody to reduce infection, and determines that it binds to one or more α2,3-sialylated and α1,3-fucosylated glycan receptors that are unique from those targeted by ApOmpA.

  11. Piracy of adhesins: attachment of superinfecting pathogens to respiratory cilia by secreted adhesins of Bordetella pertussis.

    PubMed

    Tuomanen, E

    1986-12-01

    Two proteins secreted by Bordetella pertussis are known to mediate adherence of these bacteria to mammalian respiratory cilia. When either ciliated cells or other pathogenic bacteria were pretreated with these adhesins, Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus acquired the ability to adhere to cilia in vitro and in vivo. Such piracy of adhesins may contribute to superinfection in mucosal diseases such as whooping cough.

  12. Phylogeny of replication initiator protein TrfA reveals a highly divergent clade of incompatibility group P1 plasmids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Incompatibility group P-1 (incP-1) includes broad host range plasmids of Gram negative bacteria and are classified into five subgroups (alpha, beta, gamma, delta, and epsilon). The incP-1 replication module consists of the trfA gene, encoding the replication initiator protein TrfA, and the origin o...

  13. Purification of the Escherichia coli type 1 pilin and minor pilus proteins and partial characterization of the adhesin protein.

    PubMed Central

    Hanson, M S; Hempel, J; Brinton, C C

    1988-01-01

    Type 1 pili of Escherichia coli contain three integral minor proteins with apparent molecular weights (Mr) of 28,000 (28K protein), 16,500, and 14,500 attached to rods composed of Mr-17,000 pilin subunits (Hanson and Brinton, Nature [London] 322:265-268). We describe here an improvement on our earlier method of pilus purification, which gives higher yields and higher purity. Also reported are methods allowing fractionation of intact type 1 pili into rods of pure pilin and free minor proteins, as well as fractionation of the 28K tip adhesion protein from the 16.5K and 14.5K proteins. We have determined the amino acid composition and amino-terminal sequence of the adhesion protein. This sequence shows limited homology with the amino-terminal sequences of several E. coli pilins, including type 1. Images PMID:2900235

  14. Pyrolysis products from amino acids and protein: highest mutagenicity requires cytochrome P1-450.

    PubMed

    Nebert, D W; Bigelow, S W; Okey, A B; Yahagi, T; Mori, Y; Nagao, M; Sugimura, T

    1979-11-01

    Pyrolysis products of proteins and amino acids are highly mutagenic, but metabolism of these chemicals by rat liver subcellular fractions is known to be required for production of the mutagenic intermediates. We examined the mutagenesis of seven purified pyrolysis products from tryptophan, lysine, glutamic acid, and soybean globulin with Salmonella typhimurium strain TA98 in the presence of liver fractions from genetically "responsive" C57BL/6N and Ah(b)/Ah(d) or "nonresponsive" DBA/2N and Ah(d)/Ah(d) mice that had been pretreated in vivo with benzo[a]pyrene. For all pyrolysis products tested, mutagenesis is 2-fold to more than 1000-fold greater with C57BL/6N and Ah(b)/Ah(d) than with DBA/2N or Ah(d)/Ah(d) liver fractions. A sucrose density gradient assay for detecting the Ah regulatory gene product, the receptor, was studied with C57BL/6N hepatic cytosol. At levels 100 times in excess of [1,6-(3)H]2,3,7,8-tetrachlorodibenzo-p-dioxin, nonlabeled 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methylcholanthrene, and beta-naphthoflavone (inducers of cytochrome P(1)-450) are able to displace the radioligand from its hepatic cytosolic receptor; four pyrolysates from tryptophan, glutamic acid, and soybean globulin did not have this capacity. These data indicate that the pyrolysis products tested, although not effective as inducers of cytochrome P(1)-450, are most mutagenic when metabolized by P(1)-450. Potent P(1)-450 inducers-present in pyrolysates during the combustion process-might be present in quantities insufficient to initiate mutagenesis or carcinogenesis but might have a synergistic action, or act as "comutagens" or "cocarcinogens," with the N-containing heterocyclic pyrolysis products. A quantitative relationship between mutagenic and carcinogenic potency of these pyrolysis products remains, however, to be demonstrated.

  15. Vaccine efficacy of the attenuated Erysipelothrix rhusiopathiae YS-19 expressing a recombinant protein of Mycoplasma hyopneumoniae P97 adhesin against mycoplasmal pneumonia of swine.

    PubMed

    Shimoji, Yoshihiro; Oishi, Eiji; Muneta, Yoshihiro; Nosaka, Hideji; Mori, Yasuyuki

    2003-01-17

    The attenuated Erysipelothrix rhusiopathiae YS-19 strain was constructed for the purpose of delivering the C-terminal portion of the Mycoplasma hyopneumoniae P97 adhesin to the mucosal surface of the respiratory tract of pigs. In this study, the efficacy of the YS-19 vaccine against mycoplasmal pneumonia of swine was evaluated. Animal experiments revealed that intranasal immunization of pigs with the YS-19 strain significantly reduced the severity of pneumonic lung lesions caused by M. hyopneumoniae infection. In YS-19-immunized pigs, P97-specific serum antibodies were not detected. However, when stimulated with the P97 protein, peripheral blood mononuclear cells from the YS-19-immunized pigs had a significantly higher stimulation index (P<0.05) than that of cells from control pigs at 7 days post-challenge.

  16. Carbohydrate Receptors of Bacterial Adhesins: Implications and Reflections

    NASA Astrophysics Data System (ADS)

    Ohlsen, K.; Oelschlaeger, T. A.; Hacker, J.; Khan, A. S.

    Bacteria entering a host depend on adhesins to achieve colonization. Adhesins are bacterial surface structures mediating binding to host surficial areas. Most adhesins are composed of one or several proteins. Usually a single bacterial strain is able to express various adhesins. The adhesion type expressed may influence host-, tissue or even cell tropism of Gram-negative and of Gram-positive bacteria. The binding of fimbrial as well as of afimbrial adhesins of Gram-negative bacteria to host carbohydrate structures (=receptors) has been elucidated in great detail. In contrast, in Gram-positives, most well studied adhesins bind to proteinaceous partners. Nevertheless, for both bacterial groups the binding of bacterial adhesins to eukaryotic carbohydrate receptors is essential for establishing colonization or infection. The characterization of this interaction down to the submolecular level provides the basis for strategies to interfere with this early step of infection which should lead to the prevention of subsequent disease. However, this goal will not be achieved easily because bacterial adherence is not a monocausal event but rather mediated by a variety of adhesins.

  17. A high-molecular-weight outer membrane protein of Xanthomonas oryzae pv. oryzae exhibits similarity to non-fimbrial adhesins of animal pathogenic bacteria and is required for optimum virulence.

    PubMed

    Ray, Suvendra K; Rajeshwari, R; Sharma, Yogendra; Sonti, Ramesh V

    2002-11-01

    Transposon insertions in a novel 3.798 kb open reading frame (ORF) of the rice pathogen, Xanthomonas oryzae pv. oryzae (Xoo) cause virulence deficiency and altered colony/lawn morphology. This ORF encodes a protein, XadA, of 1,265 amino acids that exhibits significant similarity to non-fimbrial adhesins of animal pathogenic bacteria such as Yersinia YadA and Moraxella UspA1. An interesting feature is that the YadA similarity region is repeated six times within the XadA sequence and encompasses almost the entire length of the protein. Anti-XadA antibodies identified a 110 kDa outer membrane protein that was sensitive to protease treatment of whole cells. XadA expression is induced in minimal medium. Homology modelling suggests that XadA adopts a beta-helix conformation-like pertactin, a non-fimbrial adhesin of Bordetella pertussis. This work is the first characterization of a non-fimbrial adhesin-like molecule in a plant pathogenic bacterium. It extends our knowledge about the repertoire of homologous virulence factors that are deployed by animal and plant pathogenic bacteria to include functions potentially involved in adhesion.

  18. The Fusobacterium nucleatum Outer Membrane Protein RadD Is an Arginine-Inhibitable Adhesin Required for Inter-Species Adherence and the Structured Architecture of Multi-Species Biofilm

    PubMed Central

    Kaplan, Christopher W.; Lux, Renate; Haake, Susan Kinder; Shi, Wenyuan

    2009-01-01

    Summary A defining characteristic of the suspected periodontal pathogen Fusobacterium nucleatum is its ability to adhere to a plethora of oral bacteria. This distinguishing feature is suggested to play an important role in oral biofilm formation and pathogenesis, with fusobacteria proposed to serve as central “bridging organisms” in the architecture of the oral biofilm bringing together species which would not interact otherwise. Previous studies indicate that these bacterial interactions are mediated by galactose- or arginine-inhibitable adhesins although genetic evidence for the role and nature of these proposed adhesins remains elusive. To characterize these adhesins at the molecular level, the genetically transformable F. nucleatum strain ATCC 23726 was screened for adherence properties, and arginine inhibitable adhesion was evident, while galactose-inhibitable adhesion was not detected. Six potential arginine binding proteins were isolated from the membrane fraction of F. nucleatum ATCC 23726 and identified via mass spectroscopy as members of the outer membrane family of proteins in F. nucleatum. Inactivation of the genes encoding these six candidates for arginine-inhibitable adhesion and two additional homologues revealed that only a mutant derivative carrying an insertion in Fn1526 (now designated as radD) demonstrated significantly decreased co-aggregation with representatives of the Gram-positive “early oral colonizers”. Lack of the 350 kDa outer membrane protein encoded by radD resulted in the failure to form the extensive structured biofilm observed with the parent strain when grown in the presence of Streptococcus sanguinis ATCC 10556. These findings indicate that radD is responsible for arginine-inhibitable adherence of F. nucleatum and provides definitive molecular evidence that F. nucleatum adhesins play a vital role in inter-species adherence and multispecies biofilm formation. PMID:19007407

  19. The structure of E. coli IgG-binding protein D suggests a general model for bending and binding in trimeric autotransporter adhesins.

    PubMed

    Leo, Jack C; Lyskowski, Andrzej; Hattula, Katarina; Hartmann, Marcus D; Schwarz, Heinz; Butcher, Sarah J; Linke, Dirk; Lupas, Andrei N; Goldman, Adrian

    2011-07-13

    The Escherichia coli Ig-binding (Eib) proteins are trimeric autotransporter adhesins (TAAs) and receptors for IgG Fc. We present the structure of a large fragment of the passenger domain of EibD, the first TAA structure to have both a YadA-like head domain and the entire coiled-coil stalk. The stalk begins as a right-handed superhelix, but switches handedness halfway down. An unexpected β-minidomain joins the two and inserts a ∼120° rotation such that there is no net twist between the beginning and end of the stalk. This may be important in folding and autotransport. The surprisingly large cavities we found in EibD and other TAAs may explain how TAAs bend to bind their ligands. We identified how IgA and IgG bind and modeled the EibD-IgG Fc complex. We further show that EibD promotes autoagglutination and biofilm formation and forms a fibrillar layer covering the cell surface making zipper-like contacts between cells.

  20. 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

  1. Interaction of Candida albicans cell wall Als3 protein with Streptococcus gordonii SspB adhesin promotes development of mixed-species communities.

    PubMed

    Silverman, Richard J; Nobbs, Angela H; Vickerman, M Margaret; Barbour, Michele E; Jenkinson, Howard F

    2010-11-01

    Candida albicans colonizes human mucosa and prosthetic surfaces associated with artificial joints, catheters, and dentures. In the oral cavity, C. albicans coexists with numerous bacterial species, and evidence suggests that bacteria may modulate fungal growth and biofilm formation. Streptococcus gordonii is found on most oral cavity surfaces and interacts with C. albicans to promote hyphal and biofilm formation. In this study, we investigated the role of the hyphal-wall protein Als3p in interactions of C. albicans with S. gordonii. Utilizing an ALS3 deletion mutant strain, it was shown that cells were not affected in initial adherence to the salivary pellicle or in hyphal formation in the planktonic phase. However, the Als3(-) mutant was unable to form biofilms on the salivary pellicle or deposited S. gordonii DL1 wild-type cells, and after initial adherence, als3Δ/als3Δ (ΔALS3) cells became detached concomitant with hyphal formation. In coaggregation assays, S. gordonii cells attached to, and accumulated around, hyphae formed by C. albicans wild-type cells. However, streptococci failed to attach to hyphae produced by the ΔALS3 mutant. Saccharomyces cerevisiae S150-2B cells expressing Als3p, but not control cells, supported binding of S. gordonii DL1. However, S. gordonii Δ(sspA sspB) cells deficient in production of the surface protein adhesins SspA and SspB showed >50% reduced levels of binding to S. cerevisiae expressing Als3p. Lactococcus lactis cells expressing SspB bound avidly to S. cerevisiae expressing Als3p, but not to S150-2B wild-type cells. These results show that recognition of C. albicans by S. gordonii involves Als3 protein-SspB protein interaction, defining a novel mechanism in fungal-bacterial communication.

  2. DNA bending by the silencer protein NeP1 is modulated by TR and RXR.

    PubMed Central

    Arnold, R; Burcin, M; Kaiser, B; Muller, M; Renkawitz, R

    1996-01-01

    NeP1 binds to the F1 silencer element of the chicken lysozyme gene and, in the presence of TR, v-ERBA or RAR, synergistically represses transcriptional activity. This repression involves a silencing mechanism acting independently of the relative promoter position. Here we show that NeP1 alone can induce a significant directed bend on DNA. The chicken homologue of human NeP1, CTCF, shows identical binding and bending properties. In contrast, the isolated DNA binding domain of CTCF efficiently binds DNA, but fails to confer bending. Similarly, the TR-RXR hetero- or homodimer, binding adjacent to NeP1 at the F2 sequence, do not show significant DNA bending. The binding of the T3 ligand to TR changes neither the magnitude nor the direction of the NeP1 induced bend. However, when all factors are bound simultaneously as a quaternary complex, the TR-RXR heterodimer changes the location of the bend center, the flexure angle and the bending direction. PMID:8758989

  3. Alfalfa mosaic virus replicase proteins, P1 and P2, localize to the tonoplast in the presence of virus RNA

    SciTech Connect

    Ibrahim, Amr; Hutchens, Heather M.; Howard Berg, R.; Sue Loesch-Fries, L.

    2012-11-25

    To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed with P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.

  4. 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

  5. Photodynamics of blue-light-regulated phosphodiesterase BlrP1 protein from Klebsiella pneumoniae and its photoreceptor BLUF domain

    NASA Astrophysics Data System (ADS)

    Tyagi, A.; Penzkofer, A.; Griese, J.; Schlichting, I.; Kirienko, Natalia V.; Gomelsky, Mark

    2008-12-01

    The BlrP1 protein from the enteric bacterium Klebsiella pneumoniae consists of a BLUF and an EAL domain and may activate c-di-GMP phosphodiesterase by blue-light. The full-length protein, BlrP1, and its BLUF domain, BlrP1_BLUF, are characterized by optical absorption and emission spectroscopy. The cofactor FAD in its oxidized redox state (FAD ox) is brought from the dark-adapted receptor state to the 10-nm red-shifted putative signalling state by violet light exposure. The recovery to the receptor state occurs with a time constant of about 1 min. The quantum yield of signalling state formation is about 0.17 for BlrP1_BLUF and about 0.08 for BlrP1. The fluorescence efficiency of the FAD ox cofactor is small due to photo-induced reductive electron transfer. Prolonged light exposure converts FAD ox in the signalling state to the fully reduced hydroquinone form FAD redH - and causes low-efficient chromophore release with subsequent photo-degradation. The photo-cycle and photo-reduction dynamics in the receptor state and in the signalling state are discussed.

  6. Differential regulation of the parathyroid hormone-related protein gene P1 and P3 promoters by cAMP.

    PubMed

    Chilco, P J; Leopold, V; Zajac, J D

    1998-03-16

    The role of calcitonin, and other agonists which activate the cAMP pathway, in regulating transcription of the human parathyroid hormone-related protein (PTHrP) gene was investigated in a human lung cancer cell line (BEN). Both calcitonin and forskolin caused a 5-6-fold increase in transcription initiated from both the P1 and P3 promoters, but with no observed effect on the P2 promoter. Maximal 6-fold activation of the P1 promoter occurred at 16 h post-stimulation and effects of calcitonin were observed within the pM range. The PKC agonist, phorbol 12-myristate 13-acetate diester (PMA), did not modulate transcription initiated from the P1 promoter. The ionophore ionomycin had a small effect on transcription of the P1 promoter, and transcriptional control may involve an interaction between the cAMP and intracellular calcium second messenger pathways. Deletion mapping studies indicated that increases in transcription of the human PTHrP gene is being mediated via a CRE element situated at -3313 to -3306 upstream of the P1 promoter. Mutational analysis of this CRE element confirmed a role for this sequence in mediating the increase in transcription effected by cAMP. Consistent with these transfection studies, RT-PCR of PTHrP mRNA also indicated a significant increase in transcripts generated from the P1 promoter. Gel retardation assays utilising a fragment of the P1 promoter region, encompassing the putative CRE, determined that nuclear proteins were binding to this region. Competition binding studies with labelled probe and cold competitors determined that the binding was specific for this sequence. A wild-type CRE consensus oligonucleotide also competed for binding with this sequence.

  7. Structural Context for Protein N-glycosylation in Bacteria: The Structure of PEB3, an Adhesin from Campylobacter Jejuni

    SciTech Connect

    Rangarajan,E.; Bhatia, S.; Watson, D.; Munger, C.; Cygler, M.; Matte, A.; Young, N.

    2007-01-01

    Campylobacter jejuni is unusual among bacteria in possessing a eukaryotic-like system for N-linked protein glycosylation at Asn residues in sequons of the type Asp/Glu-Xaa-Asn-Xaa-Ser/Thr. However, little is known about the structural context of the glycosylated sequons, limiting the design of novel recombinant glycoproteins. To obtain more information on sequon structure, we have determined the crystal structure of the PEB3 (Cj0289c) dimer. PEB3 has the class II periplasmic-binding protein fold, with each monomer having two domains with a ligand-binding site containing citrate located between them, and overall resembles molybdate- and sulfate-binding proteins. The sequon around Asn90 is located within a surface-exposed loop joining two structural elements. The three key residues are well exposed on the surface; hence, they may be accessible to the PglB oligosaccharyltransferase in the folded state.

  8. 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

  9. Quality Control of Fungus-specific Glucosylceramide in Cryptococcus neoformans by Endoglycoceramidase-related Protein 1 (EGCrP1)*

    PubMed Central

    Ishibashi, Yohei; Ikeda, Kazutaka; Sakaguchi, Keishi; Okino, Nozomu; Taguchi, Ryo; Ito, Makoto

    2012-01-01

    A fungus-specific glucosylceramide (GlcCer), which contains a unique sphingoid base possessing two double bonds and a methyl substitution, is essential for pathogenicity in fungi. Although the biosynthetic pathway of the GlcCer has been well elucidated, little is known about GlcCer catabolism because a GlcCer-degrading enzyme (glucocerebrosidase) has yet to be identified in fungi. We found a homologue of endoglycoceramidase tentatively designated endoglycoceramidase-related protein 1 (EGCrP1) in several fungal genomic databases. The recombinant EGCrP1 hydrolyzed GlcCer but not other glycosphingolipids, whereas endoglycoceramidase hydrolyzed oligosaccharide-linked glycosphingolipids but not GlcCer. Disruption of egcrp1 in Cryptococcus neoformans, a typical pathogenic fungus causing cryptococcosis, resulted in the accumulation of fungus-specific GlcCer and immature GlcCer that possess sphingoid bases without a methyl substitution concomitant with a dysfunction of polysaccharide capsule formation. These results indicated that EGCrP1 participates in the catabolism of GlcCer and especially functions to eliminate immature GlcCer in vivo that are generated as by-products due to the broad specificity of GlcCer synthase. We conclude that EGCrP1, a glucocerebrosidase identified for the first time in fungi, controls the quality of GlcCer by eliminating immature GlcCer incorrectly generated in C. neoformans, leading to accurate processing of fungus-specific GlcCer. PMID:22072709

  10. The Afa/Dr adhesins of diffusely adhering Escherichia coli stimulate interleukin-8 secretion, activate mitogen-activated protein kinases, and promote polymorphonuclear transepithelial migration in T84 polarized epithelial cells.

    PubMed

    Bétis, Fréderic; Brest, Patrick; Hofman, Véronique; Guignot, Julie; Bernet-Camard, Marie-Françoise; Rossi, Bernard; Servin, Alain; Hofman, Paul

    2003-03-01

    Afa/Dr diffusely adhering Escherichia coli (Afa/Dr DAEC) strains cause symptomatic urinary tract and intestinal infections. The proinflammatory effects of Afa/Dr DAEC strains in vitro have been not investigated to date. In the present study, we used confluent polarized monolayers of intestinal cell line T84 to evaluate the consequences of epithelial infection by Afa/Dr DAEC strains in terms of proinflammatory response. Polymorphonuclear leukocyte (PMNL) migration across the epithelial barrier was induced after incubation of the T84 monolayers with the wild-type Afa/Dr DAEC strain C1845 harboring the fimbrial F1845 adhesin and strain IH11128 harboring the Dr hemagglutinin, and the E. coli laboratory strain HB101 was transformed with the pSSS1 plasmid, producing Afa/Dr F1845 adhesin. PMNL migrations were correlated with a basolateral secretion of interleukin-8 by T84 cells and were abolished after incubation of epithelial cells with an anti-decay accelerating factor (DAF) antibody that recognized the short consensus repeat 3 domain of DAF (monoclonal antibody 1H4). Moreover, Afa/Dr DAEC strains induced tyrosine phosphorylation of several T84 proteins and activated the mitogen-activated protein kinases (ERK1/2 mitogen-activated protein, P38, and Jun-C kinases). These data demonstrated for the first time that, in vitro, Afa/Dr DAEC strains exert a proinflammatory signal in intestinal epithelial cells.

  11. Fibronectin Binding to the Treponema pallidum Adhesin Protein Fragment rTp0483 on Functionalized Self-Assembled Monolayers

    PubMed Central

    Dickerson, Matthew T.; Abney, Morgan B.; Cameron, Caroline E.; Knecht, Marc; Bachas, Leonidas G.; Anderson, Kimberly W.

    2012-01-01

    Past work has shown that Treponema pallidum, the causative agent of syphilis, binds host fibronectin (FN). FN and other host proteins are believed to bind to rare outer membrane proteins (OMPs) of T. pallidum, and it is postulated that this interaction may facilitate cell attachment and mask antigenic targets on the surface. This research seeks to prepare a surface capable of mimicking the FN binding ability of T. pallidum in order to investigate the impact of FN binding with adsorbed Tp0483 on the host response to the surface. By understanding this interaction it may be possible to develop more effective treatments for infection and possibly mimic the stealth properties of the bacteria. Functionalized self-assembled monolayers (SAMs) on0 gold were used to investigate rTp0483 and FN adsorption. Using a quartz crystal microbalance (QCM) rTp0483 adsorption and subsequent FN adsorption onto rTp0483 was determined to be higher on negatively charged carboxylate-terminated self-assembled monolayers (−COO− SAMs) compared to the other surfaces analyzed. Kinetic analysis of rTp0483 adsorption using surface plasmon resonance (SPR) supported this finding. Kinetic analysis of FN adsorption using SPR revealed a multi-step event, where the concentration of immobilized rTp0483 plays a role in FN binding. An examination of relative QCM dissipation energy compared to the shift in frequency showed a correlation between the physical properties of adsorbed rTp0483 and SAM surface chemistry. In addition, AFM images of rTp0483 on selected SAMs illustrated a preference of rTp0483 to bind as aggregates. Adsorption on −COO− SAMs was more uniform across the surface, which may help further explain why FN bound more strongly. rTp0483 antibody studies suggested the involvement of amino acids 274–289 and 316–333 in binding between rTp0483 to FN, while a peptide blocking study only showed inhibition of binding with amino acids 316–333. Finally, surface adsorbed rTp0483 with FN

  12. Interaction of the trans-frame potyvirus protein P3N-PIPO with host protein PCaP1 facilitates potyvirus movement.

    PubMed

    Vijayapalani, Paramasivan; Maeshima, Masayoshi; Nagasaki-Takekuchi, Nahoko; Miller, W Allen

    2012-01-01

    A small open reading frame (ORF), pipo, overlaps with the P3 coding region of the potyviral polyprotein ORF. Previous evidence suggested a requirement for pipo for efficient viral cell-to-cell movement. Here, we provide immunoblotting evidence that the protein PIPO is expressed as a trans-frame protein consisting of the amino-terminal half of P3 fused to PIPO (P3N-PIPO). P3N-PIPO of Turnip mosaic virus (TuMV) fused to GFP facilitates its own cell-to-cell movement. Using a yeast two-hybrid screen, co-immunoprecipitation assays, and bimolecular fluorescence complementation (BiFC) assays, we found that P3N-PIPO interacts with host protein PCaP1, a cation-binding protein that attaches to the plasma membrane via myristoylation. BiFC revealed that it is the PIPO domain of P3N-PIPO that binds PCaP1 and that myristoylation of PCaP1 is unnecessary for interaction with P3N-PIPO. In PCaP1 knockout mutants (pcap1) of Arabidopsis, accumulation of TuMV harboring a GFP gene (TuMV-GFP) was drastically reduced relative to the virus level in wild-type plants, only small localized spots of GFP were visible, and the plants showed few symptoms. In contrast, TuMV-GFP infection in wild-type Arabidopsis yielded large green fluorescent patches, and caused severe stunting. However, viral RNA accumulated to high level in protoplasts from pcap1 plants indicating that PCaP1 is not required for TuMV RNA synthesis. In contrast to TuMV, the tobamovirus Oilseed rape mosaic virus did not require PCaP1 to infect Arabidopsis plants. We conclude that potyviral P3N-PIPO interacts specifically with the host plasma membrane protein PCaP1 to participate in cell-to-cell movement. We speculate that PCaP1 links a complex of viral proteins and genomic RNA to the plasma membrane by binding P3N-PIPO, enabling localization to the plasmodesmata and cell-to-cell movement. The PCaP1 knockout may contribute to a new strategy for recessive resistance to potyviruses.

  13. Interaction of the Trans-Frame Potyvirus Protein P3N-PIPO with Host Protein PCaP1 Facilitates Potyvirus Movement

    PubMed Central

    Vijayapalani, Paramasivan; Maeshima, Masayoshi; Nagasaki-Takekuchi, Nahoko; Miller, W. Allen

    2012-01-01

    A small open reading frame (ORF), pipo, overlaps with the P3 coding region of the potyviral polyprotein ORF. Previous evidence suggested a requirement for pipo for efficient viral cell-to-cell movement. Here, we provide immunoblotting evidence that the protein PIPO is expressed as a trans-frame protein consisting of the amino-terminal half of P3 fused to PIPO (P3N-PIPO). P3N-PIPO of Turnip mosaic virus (TuMV) fused to GFP facilitates its own cell-to-cell movement. Using a yeast two-hybrid screen, co-immunoprecipitation assays, and bimolecular fluorescence complementation (BiFC) assays, we found that P3N-PIPO interacts with host protein PCaP1, a cation-binding protein that attaches to the plasma membrane via myristoylation. BiFC revealed that it is the PIPO domain of P3N-PIPO that binds PCaP1 and that myristoylation of PCaP1 is unnecessary for interaction with P3N-PIPO. In PCaP1 knockout mutants (pcap1) of Arabidopsis, accumulation of TuMV harboring a GFP gene (TuMV-GFP) was drastically reduced relative to the virus level in wild-type plants, only small localized spots of GFP were visible, and the plants showed few symptoms. In contrast, TuMV-GFP infection in wild-type Arabidopsis yielded large green fluorescent patches, and caused severe stunting. However, viral RNA accumulated to high level in protoplasts from pcap1 plants indicating that PCaP1 is not required for TuMV RNA synthesis. In contrast to TuMV, the tobamovirus Oilseed rape mosaic virus did not require PCaP1 to infect Arabidopsis plants. We conclude that potyviral P3N-PIPO interacts specifically with the host plasma membrane protein PCaP1 to participate in cell-to-cell movement. We speculate that PCaP1 links a complex of viral proteins and genomic RNA to the plasma membrane by binding P3N-PIPO, enabling localization to the plasmodesmata and cell-to-cell movement. The PCaP1 knockout may contribute to a new strategy for recessive resistance to potyviruses. PMID:22511869

  14. Variability of P1 protein of zucchini yellow mosaic virus for strain differentiation and phylogenetic analysis with other potyviruses.

    PubMed

    Lee, K C; Wong, S M

    1998-01-01

    The complete nucleotide sequence of a Singapore isolate of zucchini yellow mosaic potyvirus (ZYMV-S) was determined from viral cDNA clones. The complete genome is 9603 nucleotides in length excluding the poly (A) tail. Computer analysis of the sequence revealed a single large open reading frame (ORF) that presumably encodes a polyprotein of 3082 amino acids with a calculated molecular weight of 350 kDa. Analysis of the helper component (HC) protein showed that the highly conserved motif K-I-T-C which is involved in aphid transmission appeared as K-L-S-C. There is also a change of D-A-G to G-A-G triplet near the N-terminal of the coat protein (CP). Amino acid sequence identity comparison of ZYMV-S gene products with the California and Reunion Island isolates of ZYMV revealed a minimum range of 65-75% to a maximum range of 95-98%. Comparison with other distinct potyviruses showed a low degree of identity from 19-74%. The 5' untranslated region (UTR) of ZYMV-S showed 67% and 72% identity when compared with the California and Reunion Island isolates, respectively. The sequence variability in the 5' UTR of ZYMV can be exploited for strain differentiation and phylogenetic analysis. ZYMV-S shared 94% and 82% identity in the 3' UTR as compared to the California and Reunion Island isolates, respectively. The P1 protein of ZYMV-S shared moderate sequence variability among ZYMV isolates but high sequence variability among all potyviruses. In addition, phylogenetic analysis using the P1 protein indicated that highly variable proteins in the viral genome could also be employed in the study of potyvirus taxonomy and used for strain differentiation.

  15. Comparative analysis of the structures of the outer membrane protein P1 genes from major clones of Haemophilus influenzae type b.

    PubMed Central

    Munson, R; Grass, S; Einhorn, M; Bailey, C; Newell, C

    1989-01-01

    P1 outer membrane proteins from Haemophilus influenzae type b are heterogeneous antigenically and with respect to apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For determination of the molecular basis for the differences in the P1 proteins, the genes for the P1 proteins from strain 1613, representative of outer membrane protein subtype 3L, and strain 8358, representative of outer membrane protein subtype 6U, were cloned, sequenced, and compared with the previously reported gene for the P1 protein from strain MinnA, a strain with the outer membrane protein subtype 1H. These prototype strains are representatives of the three major clonal families of H. influenzae type b responsible for invasive disease in diverse areas of the world. The nucleotide sequences of the P1 genes from strains 1613 and 8358 were 94 and 90% identical to the MinnA sequence, respectively. The derived amino acid sequences were 91 and 86% identical, respectively. Heterogeneity between the MinnA and 1613 proteins was largely localized to two short variable regions; the protein from strain 8538 contained a third variable region not observed in the other P1 proteins. Thus, the outer membrane protein P1 genes are highly conserved; the variable regions may code for the previously demonstrated strain-specific antigenic determinants. Images PMID:2572549

  16. Identification of a Collagen Type I Adhesin of Bacteroides fragilis

    PubMed Central

    Galvão, Bruna P. G. V.; Weber, Brandon W.; Rafudeen, Mohamed S.; Ferreira, Eliane O.; Patrick, Sheila; Abratt, Valerie R.

    2014-01-01

    Bacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The ability to adhere to components of the extracellular matrix, including collagen, is related to bacterial host colonisation. Collagen Far Western analysis of the B. fragilis outer membrane protein (OMP) fraction revealed the presence two collagen adhesin bands of ∼31 and ∼34 kDa. The collagen adhesins in the OMP fraction were separated and isolated by two-dimensional SDS-PAGE and also purified by collagen affinity chromatography. The collagen binding proteins isolated by both these independent methods were subjected to tandem mass spectroscopy for peptide identification and matched to a single hypothetical protein encoded by B. fragilis NCTC 9343 (BF0586), conserved in YCH46 (BF0662) and 638R (BF0633) and which is designated in this study as cbp1 (collagen binding protein). Functionality of the protein was confirmed by targeted insertional mutagenesis of the cbp1 gene in B. fragilis GSH18 which resulted in the specific loss of both the ∼31 kDa and the ∼34 kDa adhesin bands. Purified his-tagged Cbp1, expressed in a B. fragilis wild-type and a glycosylation deficient mutant, confirmed that the cbp1 gene encoded the observed collagen adhesin, and showed that the 34 kDa band represents a glycosylated version of the ∼31 kDa protein. Glycosylation did not appear to be required for binding collagen. This study is the first to report the presence of collagen type I adhesin proteins in B. fragilis and to functionally identify a gene encoding a collagen binding protein. PMID:24618940

  17. Afa, a diffuse adherence fibrillar adhesin associated with enteropathogenic Escherichia coli.

    PubMed

    Keller, Rogéria; Ordoñez, Juana G; de Oliveira, Rosana R; Trabulsi, Luiz R; Baldwin, Thomas J; Knutton, Stuart

    2002-05-01

    O55 is one of the most frequent enteropathogenic Escherichia coli (EPEC) O serogroups implicated in infantile diarrhea in developing countries. Multilocus enzyme electrophoresis analysis showed that this serogroup includes two major electrophoretic types (ET), designated ET1 and ET5. ET1 corresponds to typical EPEC, whilst ET5 comprises strains with different combinations of virulence genes, including those for localized adherence (LA) and diffuse adherence (DA). Here we report that ET5 DA strains possess a DA adhesin, designated EPEC Afa. An 11.6-kb chromosomal region including the DA adhesin operon from one O55:H(-) ET5 EPEC strain was sequenced and found to encode a protein with 98% identity to AfaE-1, an adhesin associated with uropathogenic E. coli. Although described as an afimbrial adhesin, we show that both AfaE-1 and EPEC Afa possess fine fibrillar structures. This is the first characterization and demonstration of an Afa adhesin associated with EPEC.

  18. 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.

  19. A novel P(1B)-type Mn2+-transporting ATPase is required for secreted protein metallation in mycobacteria.

    PubMed

    Padilla-Benavides, Teresita; Long, Jarukit E; Raimunda, Daniel; Sassetti, Christopher M; Argüello, José M

    2013-04-19

    Transition metals are central for bacterial virulence and host defense. P(1B)-ATPases are responsible for cytoplasmic metal efflux and play roles either in limiting cytosolic metal concentrations or in the maturation of secreted metalloproteins. The P(1B)-ATPase, CtpC, is required for Mycobacterium tuberculosis survival in a mouse model (Sassetti, C. M., and Rubin, E. J. (2003) Genetic requirements for mycobacterial survival during infection. Proc. Natl. Acad. Sci. U.S.A. 100, 12989-12994). CtpC prevents Zn(2+) toxicity, suggesting a role in Zn(2+) export from the cytosol (Botella, H., Peyron, P., Levillain, F., Poincloux, R., Poquet, Y., Brandli, I., Wang, C., Tailleux, L., Tilleul, S., Charriere, G. M., Waddell, S. J., Foti, M., Lugo-Villarino, G., Gao, Q., Maridonneau-Parini, I., Butcher, P. D., Castagnoli, P. R., Gicquel, B., de Chastellièr, C., and Neyrolles, O. (2011) Mycobacterial P1-type ATPases mediate resistance to zinc poisoning in human macrophages. Cell Host Microbe 10, 248-259). However, key metal-coordinating residues and the overall structure of CtpC are distinct from Zn(2+)-ATPases. We found that isolated CtpC has metal-dependent ATPase activity with a strong preference for Mn(2+) over Zn(2+). In vivo, CtpC is unable to complement Escherichia coli lacking a functional Zn(2+)-ATPase. Deletion of M. tuberculosis or Mycobacterium smegmatis ctpC leads to cytosolic Mn(2+) accumulation but no alterations in other metals levels. Whereas ctpC-deficient M. tuberculosis is sensitive to extracellular Zn(2+), the M. smegmatis mutant is not. Both ctpC mutants are sensitive to oxidative stress, which might explain the Zn(2+)-sensitive phenotype of the M. tuberculosis ctpC mutant. CtpC is a high affinity/slow turnover ATPase, suggesting a role in protein metallation. Consistent with this hypothesis, mutation of CtpC leads to a decrease of Mn(2+) bound to secreted proteins and of the activity of secreted Fe/Mn-superoxide dismutase, particularly in M. smegmatis

  20. Encapsidation of poliovirus replicons encoding the complete human immunodeficiency virus type 1 gag gene by using a complementation system which provides the P1 capsid protein in trans.

    PubMed Central

    Porter, D C; Ansardi, D C; Morrow, C D

    1995-01-01

    Poliovirus genomes which contain small regions of the human immunodeficiency virus type 1 (HIV-1) gag, pol, and env genes substituted in frame for the P1 capsid region replicate and express HIV-1 proteins as fusion proteins with the P1 capsid precursor protein upon transfection into cells (W. S. Choi, R. Pal-Ghosh, and C. D. Morrow, J. Virol. 65:2875-2883, 1991). Since these genomes, referred to as replicons, do not express capsid proteins, a complementation system was developed to encapsidate the genomes by providing P1 capsid proteins in trans from a recombinant vaccinia virus, VV-P1. Virus stocks of encapsidated replicons were generated after serial passage of the replicon genomes into cells previously infected with VV-P1 (D. C. Porter, D. C. Ansardi, W. S. Choi, and C. D. Morrow, J. Virol. 67:3712-3719, 1993). Using this system, we have further defined the role of the P1 region in viral protein expression and RNA encapsidation. In the present study, we constructed poliovirus replicons which contain the complete 1,492-bp gag gene of HIV-1 substituted for the entire P1 region of poliovirus. To investigate whether the VP4 coding region was required for the replication and encapsidation of poliovirus RNA, a second replicon in which the complete gag gene was substituted for the VP2, VP3, and VP1 capsid sequences was constructed. Transfection of replicon RNA with and without the VP4 coding region into cells resulted in similar levels of expression of the HIV-1 Gag protein and poliovirus 3CD protein, as indicated by immunoprecipitation using specific antibodies. Northern (RNA) blot analysis of RNA from transfected cells demonstrated comparable levels of RNA replication for each replicon. Transfection of the replicon genomes into cells infected with VV-P1 resulted in the encapsidation of the genomes; serial passage in the presence of VV-P1 resulted in the generation of virus stocks of encapsidated replicons. Analysis of the levels of protein expression and encapsidated

  1. Localization and characterization of Xylella fastidiosa haemagglutinin adhesins.

    PubMed

    Voegel, Tanja M; Warren, Jeremy G; Matsumoto, Ayumi; Igo, Michele M; Kirkpatrick, Bruce C

    2010-07-01

    Xylella fastidiosa is a gram-negative, xylem-inhabiting, plant-pathogenic bacterium responsible for several important diseases including Pierce's disease (PD) of grapevines. The bacteria form biofilms in grapevine xylem that contribute to the occlusion of the xylem vessels. X. fastidiosa haemagglutinin (HA) proteins are large afimbrial adhesins that have been shown to be crucial for biofilm formation. Little is known about the mechanism of X. fastidiosa HA-mediated cell-cell aggregation or the localization of the adhesins on the cell. We generated anti-HA antibodies and show that X. fastidiosa HAs are present in the outer membrane and secreted both as soluble proteins and in membrane vesicles. Furthermore, the HA pre-proteins are processed from the predicted molecular mass of 360 kDa to a mature 220 kDa protein. Based on this information, we are evaluating a novel form of potential resistance against PD by generating HA-expressing transgenic grapevines.

  2. 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

  3. A plasma membrane-associated protein of Arabidopsis thaliana AtPCaP1 binds copper ions and changes its higher order structure.

    PubMed

    Nagasaki-Takeuchi, Nahoko; Miyano, Masashi; Maeshima, Masayoshi

    2008-10-01

    PCaP1, a hydrophilic cation-binding protein, is bound to the plasma membrane in Arabidopsis thaliana. We focused on the physicochemical properties of PCaP1 to understand its uniqueness in terms of structure and binding of metal ions. On fluorescence analysis, PCaP1 showed a signal of structural change in the presence of Cu(2+). The near-UV CD spectra showed a marked change of PCaP1 in CuCl(2) solution. The far-UV CD spectra showed the presence of alpha-helices and the intrinsically unstructured region. However, addition of Cu(2+) gave no change in the far-UV CD spectra. These results indicate that Cu(2+) induced a change in the tertiary structure without changing the secondary structure. The protein was sensitive to proteinase in the presence of Cu(2+), supporting that Cu(2+) is involved in the structural change. The PCaP1 solution was titrated with CuCl(2) and the change in the fluorescence spectrum was monitored to characterize Cu(2+)-binding properties. The obtained values of K(d) for Cu(2+) and the ligand-binding number were 10 microM and six ions per molecule, respectively. These findings indicate that PCaP1 has a high Cu(2+)-binding capacity with a relatively high affinity. PCaP1 lacks cysteine and histidine residues. A large number of glutamate residues may be involved in the Cu(2+) binding.

  4. The Cation-Responsive Protein NhaR of Escherichia coli Activates pgaABCD Transcription, Required for Production of the Biofilm Adhesin Poly-β-1,6-N-Acetyl-d-Glucosamine▿

    PubMed Central

    Goller, Carlos; Wang, Xin; Itoh, Yoshikane; Romeo, Tony

    2006-01-01

    The pgaABCD operon of Escherichia coli is required for production of the biofilm adhesin poly-β-1,6-N-acetyl-d-glucosamine (PGA). We establish here that NhaR, a DNA-binding protein of the LysR family of transcriptional regulators, activates transcription of this operon. Disruption of the nhaR gene decreased biofilm formation without affecting planktonic growth. PGA production was undetectable in an nhaR mutant strain. Expression of a pgaA′-′lacZ translational fusion was induced by NaCl and alkaline pH, but not by CaCl2 or sucrose, in an nhaR-dependent fashion. Primer extension and quantitative real-time reverse transcription-PCR analyses further revealed that NhaR affects the steady-state level of pga mRNA. A purified recombinant NhaR protein bound specifically and with high affinity within the pgaABCD promoter region; one apparent binding site overlaps the −35 element, and a second site lies immediately upstream of the first. This protein was necessary and sufficient for activation of in vitro transcription from the pgaA promoter. These results define a novel mechanism for regulation of biofilm formation in response to environmental conditions and suggest an expanded role for NhaR in promoting bacterial survival. PMID:16997959

  5. 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

    2015-11-07

    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.

  6. Lipoprotein CD0873 is a novel adhesin of Clostridium difficile.

    PubMed

    Kovacs-Simon, Andrea; Leuzzi, Rosanna; Kasendra, Magdalena; Minton, Nigel; Titball, Richard W; Michell, Stephen L

    2014-07-15

    Clostridium difficile is a cause of antibiotic-associated diarrhea and colitis, a healthcare-associated intestinal disease. Colonization of the gut is a critical step in the course of infection. The C. difficile lipoprotein CD0873 was identified as a putative adhesin through a bioinformatics approach. Surface exposure of CD0873 was confirmed and a CD0873 mutant was generated. The CD0873 mutant showed a significant reduction in adherence to Caco-2 cells and wild-type bacteria preincubated with anti-CD0873 antibodies showed significantly decreased adherence to Caco-2 cells. In addition, we demonstrated that purified recombinant CD0873 protein alone associates with Caco-2 cells. This is the first definitive identification of a C. difficile adhesin, which now allows work to devise improved measures for preventing and treating disease.

  7. The afimbrial adhesive sheath encoded by the afa-3 gene cluster of pathogenic Escherichia coli is composed of two adhesins.

    PubMed

    Garcia, M I; Gounon, P; Courcoux, P; Labigne, A; Le Bouguenec, C

    1996-02-01

    The afa-3 gene cluster determines the formation of an afimbrial adhesive sheath that is expressed by uropathogenic as well as diarrhoea-associated Escherichia coli strains. It contains six genes (afaA-afaF), among which the afaE3 gene is known to code for the structural AfaE-III adhesin (previously designated AFA-III), whereas no role has yet been identified for the afaD gene product. The afa-3 gene cluster is closely related to the daa operon that codes for an adhesin, the F1845 adhesin, which is highly related to the AfaE-III adhesin; however, unlike the AfaE-III adhesin, F1845 is a fimbrial adhesin. Reported in this work is the construction of chimeras between the afa-3 and daa operons. Analyses of the phenotypes conferred by these afa-3/daa chimeric clusters allowed us to conclude that the biogenesis of a fimbrial or an afimbrial adhesin is fully determined by the amino acid sequence of the AfaE-III and F1845 adhesins. Moreover, the role of the AfaD product in the biosynthesis of the afimbrial sheath was assessed by immunogold and immunofluorescence experiments. The AfaD and the AfaE-III products were purified and used to raise rabbit and mouse antisera. Similar to AfaE-III, AfaD was found to be a surface-exposed protein as well as an adhesin; both AfaD and AfaE-III are concomittantly expressed by the bacterial cell. These results demonstrate, for the first time, that the afimbrial adhesive sheath expressed by pathogenic E. coli is composed of two adhesins.

  8. The Human Disease-Associated Aβ Amyloid Core Sequence Forms Functional Amyloids in a Fungal Adhesin

    PubMed Central

    Rameau, Rachele D.; Jackson, Desmond N.; Beaussart, Audrey; Dufrêne, Yves F.

    2016-01-01

    ABSTRACT There is increasing evidence that many amyloids in living cells have physiological functions. On the surfaces of fungal cells, amyloid core sequences in adhesins can aggregate into 100- to 1,000-nm-wide patches to form high-avidity adhesion nanodomains on the cell surface. The nanodomains form through interactions that have amyloid-like properties: binding of amyloid dyes, perturbation by antiamyloid agents, and interaction with homologous sequences. To test whether these functional interactions are mediated by typical amyloid interactions, we substituted an amyloid core sequence, LVFFA, from human Aβ protein for the native sequence IVIVA in the 1,419-residue Candida albicans adhesin Als5p. The chimeric protein formed cell surface nanodomains and mediated cellular aggregation. The native sequence and chimeric adhesins responded similarly to the amyloid dye thioflavin T and to amyloid perturbants. However, unlike the native protein, the nanodomains formed by the chimeric protein were not force activated and formed less-robust aggregates under flow. These results showed the similarity of amyloid interactions in the amyloid core sequences of native Als5p and Aβ, but they also highlighted emergent properties of the native sequence. Also, a peptide composed of the Aβ amyloid sequence flanked by amino acids from the adhesin formed two-dimensional sheets with sizes similar to the cell surface patches of the adhesins. These results inform an initial model for the structure of fungal cell surface amyloid nanodomains. PMID:26758179

  9. Structures of the Toxoplasma gliding motility adhesin

    PubMed Central

    Song, Gaojie; Springer, Timothy A.

    2014-01-01

    Micronemal protein 2 (MIC2) is the key adhesin that supports gliding motility and host cell invasion by Toxoplasma gondii. With a von Willebrand factor A (VWA) domain and six thrombospondin repeat domains (TSR1–6) in its ectodomain, MIC2 connects to the parasite actomyosin system through its cytoplasmic tail. MIC2-associated protein (M2AP) binds noncovalently to the MIC2 ectodomain. MIC2 and M2AP are stored in micronemes as proforms. We find that the MIC2–M2AP ectodomain complex is a highly elongated 1:1 monomer with M2AP bound to the TSR6 domain. Crystal structures of N-terminal fragments containing the VWA and TSR1 domains for proMIC2 and MIC2 reveal a closed conformation of the VWA domain and how it associates with the TSR1 domain. A long, proline-rich, disulfide-bonded pigtail loop in TSR1 overlaps the VWA domain. Mannose α-C-linked to Trp-276 in TSR1 has an unusual 1C4 chair conformation. The MIC2 VWA domain includes a mobile α5-helix and a 22-residue disordered region containing two disulfide bonds in place of an α6-helix. A hydrophobic residue in the prodomain binds to a pocket adjacent to the α7-helix that pistons in opening of the VWA domain to a putative high-affinity state. PMID:24639528

  10. The mouse Mcmd gene for DNA replication protein P1MCM3 maps to bands A3-A5 on chromosome 1 by fluorescence in situ hybridization

    SciTech Connect

    Yoshida, Ikuya; Kimura, Hiroshi; Takagi, Nobuo

    1996-03-05

    This report describes the localization of the mouse Mcmd gene for DNA replication to mouse chromosome 1, bands A3-A5 using fluorescence in situ hybridization. This finding supports the recent mapping of the human MCM3 gene to human chromosome 6p12, which shows synteny with mouse chromosome 1. The mouse Mcmd gene encodes the protein P1MCM3 which is essential for DNA replication. 13 refs., 1 fig.

  11. EnP1 and EnP2, two proteins associated with the Encephalitozoon cuniculi endospore, the chitin-rich inner layer of the microsporidian spore wall.

    PubMed

    Peuvel-Fanget, Isabelle; Polonais, Valérie; Brosson, Damien; Texier, Catherine; Kuhn, Lauriane; Peyret, Pierre; Vivarès, Christian; Delbac, Frédéric

    2006-03-01

    Microsporidia are obligate intracellular parasites forming environmentally resistant spores that harbour a rigid cell wall. This wall comprises an outer layer or exospore and a chitin-rich inner layer or endospore. So far, only a chitin deacetylase-like protein has been shown to localize to the Encephalitozoon cuniculi endospore and either one or two proteins have been clearly assigned to the exospore in two Encephalitozoon species: SWP1 in E. cuniculi, SWP1 and SWP2 in Encephalitozoon intestinalis. Here, we report the identification of two new spore wall proteins in E. cuniculi, EnP1 and EnP2, the genes of which are both located on chromosome I (ECU01_0820 and ECU01_1270, respectively) and have no known homologue. Detected by immunoscreening of an E. cuniculi cDNA library, enp1 is characterized by small-sized 5' and 3' untranslated regions and is highly expressed throughout the whole intracellular cycle. The encoded basic 40 kDa antigen displays a high proportion of cysteine residues, arguing for a significant role of disulfide bridges in spore wall assembly. EnP2 is a 22 kDa serine-rich protein that is predicted to be O-glycosylated and glycosylated phosphatidyl inositol-anchored. Although having been identified by mass spectrometry of a dithiothreitol-soluble fraction, this protein contains only two cysteine residues. Mouse polyclonal antibodies were raised against EnP1 and EnP2 recombinant proteins produced in Escherichia coli Our immunolocalisation data indicate that EnP1 and EnP2 are targeted to the cell surface as early as the onset of sporogony and are finally associated with the chitin-rich layer of the wall in mature spores.

  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. 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.

  14. XadM, a novel adhesin of Xanthomonas oryzae pv. oryzae, exhibits similarity to Rhs family proteins and is required for optimum attachment, biofilm formation, and virulence.

    PubMed

    Pradhan, Binod B; Ranjan, Manish; Chatterjee, Subhadeep

    2012-09-01

    By screening a transposon-induced mutant library of Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, we have identified a novel 5.241-kb open reading frame (ORF) named xadM that is required for optimum virulence and colonization. This ORF encodes a protein, XadM, of 1,746 amino acids that exhibits significant similarity to Rhs family proteins. The XadM protein contains several repeat domains similar to a wall-associated surface protein of Bacillus subtilis, which has been proposed to be involved in carbohydrate binding. The role of XadM in X. oryzae pv. oryzae adhesion was demonstrated by the impaired ability of an xadM mutant strain to attach and form biofilms. Furthermore, we show that XadM is exposed on the cell surface and its expression is regulated by growth conditions and plays an important role in the early attachment and entry inside rice leaves. Interestingly, XadM homologs are present in several diverse bacteria, including many Xanthomonas spp. and animal-pathogenic bacteria belonging to Burkholderia spp. This is the first report of a role for XadM, an Rhs family protein, in adhesion and virulence of any pathogenic bacteria.

  15. 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.

  16. 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

  17. Preliminary structural studies of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi, a part of the P0/P1/P2 complex.

    PubMed

    Ayub, Maximiliano Juri; Barroso, Juan A; Levin, Mariano J; Aguilar, Carlos F

    2005-08-01

    The Trypanosoma cruzi ribosomal P0 protein (TcP0) is part of the ribosomal stalk, which is an elongated lateral protuberance of the large ribosomal subunit involved in the translocation step of protein synthesis. The TcP0 C-terminal peptide is highly antigenic and a major target of the antibody response in patients with systemic lupus erythematosus and patients suffering chronic heart disease produced by Trypanosoma cruzi infection. The structural properties of TcP0 have been explored by circular dichroism, tryptophan fluorescence and limited proteolysis experiments. These studies were complemented by secondary structure consensus prediction analysis. The results suggest that the tertiary structure of TcP0 could be described as a compact, stable, trypsin-resistant, 200 residues long N-terminal domain belonging to the alpha/beta class and a more flexible, degradable, helical, 123 residues long C-terminal domain which could be involved in the formation of an unusual hydrophobic zipper with the ribosomal P1/P2 proteins to form the P0/P1/P2 complex.

  18. Sequence TTKF↓QE Defines the Site of Proteolytic Cleavage in Mhp683 Protein, a Novel Glycosaminoglycan and Cilium Adhesin of Mycoplasma hyopneumoniae*

    PubMed Central

    Bogema, Daniel R.; Scott, Nichollas E.; Padula, Matthew P.; Tacchi, Jessica L.; Raymond, Benjamin B. A.; Jenkins, Cheryl; Cordwell, Stuart J.; Minion, F. Chris; Walker, Mark J.; Djordjevic, Steven P.

    2011-01-01

    Mycoplasma hyopneumoniae colonizes the ciliated respiratory epithelium of swine, disrupting mucociliary function and inducing chronic inflammation. P97 and P102 family members are major surface proteins of M. hyopneumoniae and play key roles in colonizing cilia via interactions with glycosaminoglycans and mucin. The p102 paralog, mhp683, and homologs in strains from different geographic origins encode a 135-kDa pre-protein (P135) that is cleaved into three fragments identified here as P45683, P48683, and P50683. A peptide sequence (TTKF↓QE) was identified surrounding both cleavage sites in Mhp683. N-terminal sequences of P48683 and P50683, determined by Edman degradation and mass spectrometry, confirmed cleavage after the phenylalanine residue. A similar proteolytic cleavage site was identified by mass spectrometry in another paralog of the P97/P102 family. Trypsin digestion and surface biotinylation studies showed that P45683, P48683, and P50683 reside on the M. hyopneumoniae cell surface. Binding assays of recombinant proteins F1683–F5683, spanning Mhp683, showed saturable and dose-dependent binding to biotinylated heparin that was inhibited by unlabeled heparin, fucoidan, and mucin. F1683–F5683 also bound porcine epithelial cilia, and antisera to F2683 and F5683 significantly inhibited cilium binding by M. hyopneumoniae cells. These data suggest that P45683, P48683, and P50683 each display cilium- and proteoglycan-binding sites. Mhp683 is the first characterized glycosaminoglycan-binding member of the P102 family. PMID:21969369

  19. Characterization of two new putative adhesins of Leptospira interrogans.

    PubMed

    Figueredo, Jupciana M; Siqueira, Gabriela H; de Souza, Gisele O; Heinemann, Marcos B; Vasconcellos, Silvio A; Chapola, Erica G B; Nascimento, Ana L T O

    2017-01-01

    We here report the characterization of two novel proteins encoded by the genes LIC11122 and LIC12287, identified in the genome sequences of Leptospira interrogans, annotated, respectively, as a putative sigma factor and a hypothetical protein. The CDSs LIC11122 and LIC12287 have signal peptide SPII and SPI and are predicted to be located mainly at the cytoplasmic membrane of the bacteria. The genes were cloned and the proteins expressed using Escherichia coli. Proteinase K digestion showed that both proteins are surface exposed. Evaluation of interaction of recombinant proteins with extracellular matrix components revealed that they are laminin binding and they were called Lsa19 (LIC11122) and Lsa14 (LIC12287), for Leptospiral-surface adhesin of 19 and 14 kDa, respectively. The bindings were dose-dependent on protein concentration, reaching saturation, fulfilling the ligand-binding criteria. Reactivity of the recombinant proteins with leptospirosis human sera has shown that Lsa19 and, to a lesser extent, Lsa14, are recognized by antibodies, suggesting that, most probably, Lsa19 is expressed during infection. The proteins interact with plasminogen and generate plasmin in the presence of urokinase-type plasminogen activator. Plasmin generation in Leptospira has been associated with tissue penetration and immune evasion strategies. The presence of a sigma factor on the cell surface playing a secondary role, probably mediating host -pathogen interaction, suggests that LIC11122 is a moonlighting protein candidate. Although the biological significance of these putative adhesins will require the generation of mutants, our data suggest that Lsa19 is a potential candidate for future evaluation of its role in adhesion/colonization activities during L. interrogans infection.

  20. Mapping the tail fiber as the receptor binding protein responsible for differential host specificity of Pseudomonas aeruginosa bacteriophages PaP1 and JG004.

    PubMed

    Le, Shuai; He, Xuesong; Tan, Yinling; Huang, Guangtao; Zhang, Lin; Lux, Renate; Shi, Wenyuan; Hu, Fuquan

    2013-01-01

    The first step in bacteriophage infection is recognition and binding to the host receptor, which is mediated by the phage receptor binding protein (RBP). Different RBPs can lead to differential host specificity. In many bacteriophages, such as Escherichia coli and Lactococcal phages, RBPs have been identified as the tail fiber or protruding baseplate proteins. However, the tail fiber-dependent host specificity in Pseudomonas aeruginosa phages has not been well studied. This study aimed to identify and investigate the binding specificity of the RBP of P. aeruginosa phages PaP1 and JG004. These two phages share high DNA sequence homology but exhibit different host specificities. A spontaneous mutant phage was isolated and exhibited broader host range compared with the parental phage JG004. Sequencing of its putative tail fiber and baseplate region indicated a single point mutation in ORF84 (a putative tail fiber gene), which resulted in the replacement of a positively charged lysine (K) by an uncharged asparagine (N). We further demonstrated that the replacement of the tail fiber gene (ORF69) of PaP1 with the corresponding gene from phage JG004 resulted in a recombinant phage that displayed altered host specificity. Our study revealed the tail fiber-dependent host specificity in P. aeruginosa phages and provided an effective tool for its alteration. These contributions may have potential value in phage therapy.

  1. 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

    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

    2011-02-11

    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-R210(L) and SP-R210(S). The results show that WT alveolar macrophages are distinguished by expression of SP-R210(L), whereas SR-A(-/-) alveolar macrophages are deficient in SP-R210(L) expressing only SP-R210(S). 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-R210(L) 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.

  2. Yersinia adhesins: An arsenal for infection.

    PubMed

    Chauhan, Nandini; Wrobel, Agnieszka; Skurnik, Mikael; Leo, Jack C

    2016-10-01

    The Yersiniae are a group of Gram-negative coccobacilli inhabiting a wide range of habitats. The genus harbors three recognized human pathogens: Y. enterocolitica and Y. pseudotuberculosis, which both cause gastrointestinal disease, and Y. pestis, the causative agent of plague. These three organisms have served as models for a number of aspects of infection biology, including adhesion, immune evasion, evolution of pathogenic traits, and retracing the course of ancient pandemics. The virulence of the pathogenic Yersiniae is heavily dependent on a number of adhesin molecules. Some of these, such as the Yersinia adhesin A and invasin of the enteropathogenic species, and the pH 6 antigen of Y. pestis, have been extensively studied. However, genomic sequencing has uncovered a host of other adhesins present in these organisms, the functions of which are only starting to be investigated. Here, we review the current state of knowledge on the adhesin molecules present in the Yersiniae, and their functions and putative roles in the infection process.

  3. Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1

    SciTech Connect

    Gruenig, Marielle C.; Lu, Duo; Won, Sang Joon; Dulberger, Charles L.; Manlick, Angela J.; Keck, James L.; Cox, Michael M.

    2012-03-16

    The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosen DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 {angstrom} resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded {beta}-hairpin that is sandwiched between several {alpha}-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.

  4. Antibodies from multiple sclerosis patients preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus influenzae

    PubMed Central

    Walvoort, Marthe T. C.; Testa, Chiara; Eilam, Raya; Aharoni, Rina; Nuti, Francesca; Rossi, Giada; Real-Fernandez, Feliciana; Lanzillo, Roberta; Brescia Morra, Vincenzo; Lolli, Francesco; Rovero, Paolo; Imperiali, Barbara; Papini, Anna Maria

    2016-01-01

    In autoimmune diseases, there have been proposals that exogenous “molecular triggers”, i.e., specific this should be ‘non-self antigens’ accompanying infectious agents, might disrupt control of the adaptive immune system resulting in serious pathologies. The etiology of the multiple sclerosis (MS) remains unclear. However, epidemiologic data suggest that exposure to infectious agents may be associated with increased MS risk and progression may be linked to exogenous, bacterially-derived, antigenic molecules, mimicking mammalian cell surface glycoconjugates triggering autoimmune responses. Previously, antibodies specific to a gluco-asparagine (N-Glc) glycopeptide, CSF114(N-Glc), were identified in sera of an MS patient subpopulation. Since the human glycoproteome repertoire lacks this uniquely modified amino acid, we turned our attention to bacteria, i.e., Haemophilus influenzae, expressing cell-surface adhesins including N-Glc, to establish a connection between H. influenzae infection and MS. We exploited the biosynthetic machinery from the opportunistic pathogen H. influenzae (and the homologous enzymes from A. pleuropneumoniae) to produce a unique set of defined glucosylated adhesin proteins. Interestingly we revealed that a hyperglucosylated protein domain, based on the cell-surface adhesin HMW1A, is preferentially recognized by antibodies from sera of an MS patient subpopulation. In conclusion the hyperglucosylated adhesin is the first example of an N-glucosylated native antigen that can be considered a relevant candidate for triggering pathogenic antibodies in MS. PMID:28008952

  5. Structural and functional studies of ReP1-NCXSQ, a protein regulating the squid nerve Na+/Ca2+ exchanger.

    PubMed

    Cousido-Siah, Alexandra; Ayoub, Daniel; Berberián, Graciela; Bollo, Mariana; Van Dorsselaer, Alain; Debaene, François; DiPolo, Reinaldo; Petrova, Tatiana; Schulze-Briese, Clemens; Olieric, Vincent; Esteves, Adriana; Mitschler, André; Sanglier-Cianférani, Sarah; Beaugé, Luis; Podjarny, Alberto

    2012-09-01

    The protein ReP1-NCXSQ was isolated from the cytosol of squid nerves and has been shown to be required for MgATP stimulation of the squid nerve Na(+)/Ca(2+) exchanger NCXSQ1. In order to determine its mode of action and the corresponding biologically active ligand, sequence analysis, crystal structures and mass-spectrometric studies of this protein and its Tyr128Phe mutant are reported. Sequence analysis suggests that it belongs to the CRABP family in the FABP superfamily. The X-ray structure at 1.28 Å resolution shows the FABP β-barrel fold, with a fatty acid inside the barrel that makes a relatively short hydrogen bond to Tyr128 and shows a double bond between C9 and C10 but that is disordered beyond C12. Mass-spectrometric studies identified this fatty acid as palmitoleic acid, confirming the double bond between C9 and C10 and establishing a length of 16 C atoms in the aliphatic chain. This acid was caught inside during the culture in Escherichia coli and therefore is not necessarily linked to the biological activity. The Tyr128Phe mutant was unable to activate the Na(+)/Ca(2+) exchanger and the corresponding crystal structure showed that without the hydrogen bond to Tyr128 the palmitoleic acid inside the barrel becomes disordered. Native mass-spectrometric analysis confirmed a lower occupancy of the fatty acid in the Tyr128Phe mutant. The correlation between (i) the lack of activity of the Tyr128Phe mutant, (ii) the lower occupancy/disorder of the bound palmitoleic acid and (iii) the mass-spectrometric studies of ReP1-NCXSQ suggests that the transport of a fatty acid is involved in regulation of the NCXSQ1 exchanger, providing a novel insight into the mechanism of its regulation. In order to identify the biologically active ligand, additional high-resolution mass-spectrometric studies of the ligands bound to ReP1-NCXSQ were performed after incubation with squid nerve vesicles both with and without MgATP. These studies clearly identified palmitic acid as the

  6. Heterologous Expression of Bartonella Adhesin A in Escherichia coli by Exchange of Trimeric Autotransporter Adhesin Domains Results in Enhanced Adhesion Properties and a Pathogenic Phenotype

    PubMed Central

    Schmidgen, Thomas; Kaiser, Patrick O.; Ballhorn, Wibke; Franz, Bettina; Göttig, Stephan; Linke, Dirk

    2014-01-01

    Human-pathogenic Bartonella henselae causes cat scratch disease and vasculoproliferative disorders. An important pathogenicity factor of B. henselae is the trimeric autotransporter adhesin (TAA) Bartonella adhesin A (BadA), which is modularly constructed, consisting of a head, a long and repetitive neck-stalk module, and a membrane anchor. BadA is involved in bacterial autoagglutination, binding to extracellular matrix proteins and host cells, and in proangiogenic reprogramming. The slow growth of B. henselae and limited tools for genetic manipulation are obstacles for detailed examination of BadA and its domains. Here, we established a recombinant expression system for BadA mutants in Escherichia coli allowing functional analysis of particular BadA domains. Using a BadA mutant lacking 21 neck-stalk repeats (BadA HN23), the BadA HN23 signal sequence was exchanged with that of E. coli OmpA, and the BadA membrane anchor was additionally replaced with that of Yersinia adhesin A (YadA). Constructs were cloned in E. coli, and hybrid protein expression was detected by immunoblotting, fluorescence microscopy, and flow cytometry. Functional analysis revealed that BadA hybrid proteins mediate autoagglutination and binding to collagen and endothelial cells. In vivo, expression of this BadA construct correlated with higher pathogenicity of E. coli in a Galleria mellonella infection model. PMID:24682330

  7. The influence of cis-acting P1 protein and translational elements on the expression of Potato virus Y helper-component proteinase (HCPro) in heterologous systems and its suppression of silencing activity.

    PubMed

    Tena Fernández, Fátima; González, Inmaculada; Doblas, Paula; Rodríguez, César; Sahana, Nandita; Kaur, Harpreet; Tenllado, Francisco; Praveen, Shelly; Canto, Tomas

    2013-06-01

    In the Potyvirus genus, the P1 protein is the first N-terminal product processed from the viral polyprotein, followed by the helper-component proteinase (HCPro). In silencing suppression patch assays, we found that Potato virus Y (PVY) HCPro expressed from a P1-HCPro sequence increased the accumulation of a reporter gene, whereas protein expressed from an HCPro sequence did not, even with P1 supplied in trans. This enhancing effect of P1 has been noted in other potyviruses, but has remained unexplained. We analysed the accumulation of PVY HCPro in infiltrated tissues and found that it was higher when expressed from P1-HCPro than from HCPro sequences. Co-expression of heterologous suppressors increased the steady-state level of mRNA expressed from the HCPro sequence, but not that of protein. This suggests that, in the absence of P1 upstream, either HCPro acquires a conformation that affects negatively its activity or stability, or that its translation is reduced. To test these options, we purified HCPro expressed in the presence or absence of upstream P1, and found no difference in purification pattern and final soluble state. By contrast, alteration of the Kozak context in the HCPro mRNA sequence to favour translation increased partially suppressor accumulation and activity. Furthermore, protein activity was not lower than in protein expressed from P1-HCPro sequences. Thus, a direct role for P1 on HCPro suppressor activity or stability, by influencing its conformation during translation, can be excluded. However, P1 could still have an indirect effect favouring HCPro accumulation. Our data highlight the relevance of cis-acting translational elements in the heterologous expression of HCPro.

  8. A novel-type phosphatidylinositol phosphate-interactive, Ca-binding protein PCaP1 in Arabidopsis thaliana: stable association with plasma membrane and partial involvement in stomata closure.

    PubMed

    Nagata, Chisako; Miwa, Chika; Tanaka, Natsuki; Kato, Mariko; Suito, Momoe; Tsuchihira, Ayako; Sato, Yori; Segami, Shoji; Maeshima, Masayoshi

    2016-05-01

    The Ca(2+)-binding protein-1 (PCaP1) of Arabidopsis thaliana is a new type protein that binds to phosphatidylinositol phosphates and Ca(2+)-calmodulin complex as well as free Ca(2+). Although biochemical properties, such as binding to ligands and N-myristoylation, have been revealed, the intracellular localization, tissue and cell specificity, integrity of membrane association and physiological roles of PCaP1 are unknown. We investigated the tissue and intracellular distribution of PCaP1 by using transgenic lines expressing PCaP1 linked with a green fluorescence protein (GFP) at the carboxyl terminus of PCaP1. GFP fluorescence was obviously detected in most tissues including root, stem, leaf and flower. In these tissues, PCaP1-GFP signal was observed predominantly in the plasma membrane even under physiological stress conditions but not in other organelles. The fluorescence was detected in the cytosol when the 25-residue N-terminal sequence was deleted from PCaP1 indicating essential contribution of N-myristoylation to the plasma membrane anchoring. Fluorescence intensity of PCaP1-GFP in roots was slightly decreased in seedlings grown in medium supplemented with high concentrations of iron for 1 week and increased in those grown with copper. In stomatal guard cells, PCaP1-GFP was strictly, specifically localized to the plasma membrane at the epidermal-cell side but not at the pore side. A T-DNA insertion mutant line of PCaP1 did not show marked phenotype in a life cycle except for well growth under high CO2 conditions. However, stomata of the mutant line did not close entirely even in high osmolarity, which usually induces stomata closure. These results suggest that PCaP1 is involved in the stomatal movement, especially closure process, in leaves and response to excessive copper in root and leaf as a mineral nutrient as a physiological role.

  9. AFA and F17 adhesins produced by pathogenic Escherichia coli strains in domestic animals.

    PubMed

    Le Bouguénec, C; Bertin, Y

    1999-01-01

    AFA and F17 are afimbrial and fimbrial adhesins, respectively, produced by pathogenic Escherichia coli strains in domestic animals. F17-related fimbriae are mainly detected on bovine and ovine E. coli associated with diarrhoea or septicaemia. The F17-G adhesin subunits recognize N-acetyl-D-glucosamine (GlcNAc) receptors present on bovine intestinal cells. Some F17 subtypes also bind to GlcNAc receptors present on human uroepithelial and intestinal Caco-2 cells or to the laminin contained in the basement of mammalian membranes. F17 is often associated with other virulence factors (aerobactin, serum resistance, CNF2 toxin, K99, CS31A or AFA adhesins) on pathogenic E. coli. A cluster of only four genes is required to synthesize functional F17-related fimbrial structures. The hypothesis of multifunctional F17 fimbrial subunits is supported by the fact that: i) the N-terminal part of the adhesin subunit participates in receptor recognition, whereas the C-terminal part is required for biogenesis of the fimbrial filament; and ii) the interaction between structural and adhesin subunits seems to be crucial for the initiation of monomer polymerization. Recently, determinants related to the afa gene clusters from human pathogenic E. coli associated with intestinal and extra-intestinal infections were identified in strains isolated from calves and piglets with diarrhoea and septicaemia. Two afa-related gene clusters, designated afa-7 and afa-8, that encode afimbrial adhesins were cloned and characterized from bovine pathogenic E. coli. These animal afa gene clusters were plasmid and chromosome borne and were expressed by strains that produced other virulence factors such as CNF toxins, F17, PAP and CS31A adhesins. A high frequency of afa-8 and a low prevalence of afa-7 among bovine E. coli isolates were suggested by preliminary epidemiological studies. As with the human afa gene clusters, the animal ones encode an adhesive structure composed of two proteins: AfaE which

  10. Characterization of invasive Neisseria meningitidis from Atlantic Canada, 2009 to 2013: With special reference to the nonpolysaccharide vaccine targets (PorA, factor H binding protein, Neisseria heparin-binding antigen and Neisseria adhesin A)

    PubMed Central

    Tsang, Raymond SW; Law, Dennis KS; Gad, Rita R; Mailman, Tim; German, Gregory; Needle, Robert

    2015-01-01

    BACKGROUND: Serogroup B Neisseria meningitidis (MenB) has always been a major cause of invasive meningococcal disease (IMD) in Canada. With the successful implementation of a meningitis C conjugate vaccine, the majority of IMD in Canada is now caused by MenB. OBJECTIVE: To investigate IMD case isolates in Atlantic Canada from 2009 to 2013. Data were analyzed to determine the potential coverage of the newly licensed MenB vaccine. METHODS: Serogroup, serotype and serosubtype antigens were determined from IMD case isolates. Clonal analysis was performed using multilocus sequence typing. The protein-based vaccine antigen genes were sequenced and the predicted peptides were investigated. RESULTS: The majority of the IMD isolates were MenB (82.5%, 33 of 40) and, in particular, sequence type (ST)-154 B:4:P1.4 was responsible for 47.5% (19 of 40) of all IMD case isolates in Atlantic Canada. Isolates of this clone expressed the PorA antigen P1.4 and possessed the nhba genes encoding for Neisseria heparin-binding antigen peptide 2, which together matched exactly with two of the four components of the new four-component meningococcal B vaccine. Nineteen MenB isolates had two antigenic matches, another five MenB and one meningitis Y isolate had one antigenic match. This provided 75.8% (25 of 33) potential coverage for MenB, or a 62.5% (25 of 40) overall potential coverage for IMD. CONCLUSION: From 2009 to 2013, IMD in Atlantic Canada was mainly caused by MenB and, in particular, the B:4:P1.4 ST-154 clone, which accounted for 47.5% of all IMD case isolates. The new four-component meningococcal B vaccine appeared to offer adequate coverage against MenB in Atlantic Canada. PMID:26744586

  11. Oxygen promotes biofilm formation of Shewanella putrefaciens CN32 through a diguanylate cyclase and an adhesin

    PubMed Central

    Wu, Chao; Cheng, Yuan-Yuan; Yin, Hao; Song, Xiang-Ning; Li, Wen-Wei; Zhou, Xian-Xuan; Zhao, Li-Ping; Tian, Li-Jiao; Han, Jun-Cheng; Yu, Han-Qing

    2013-01-01

    Although oxygen has been reported to regulate biofilm formation by several Shewanella species, the exact regulatory mechanism mostly remains unclear. Here, we identify a direct oxygen-sensing diguanylate cyclase (DosD) and reveal its regulatory role in biofilm formation by Shewanella putrefaciens CN32 under aerobic conditions. In vitro and in vivo analyses revealed that the activity of DosD culminates to synthesis of cyclic diguanylate (c-di-GMP) in the presence of oxygen. DosD regulates the transcription of bpfA operon which encodes seven proteins including a large repetitive adhesin BpfA and its cognate type I secretion system (TISS). Regulation of DosD in aerobic biofilms is heavily dependent on an adhesin BpfA and the TISS. This study offers an insight into the molecular mechanism of oxygen-stimulated biofilm formation by S. putrefaciens CN32. PMID:23736081

  12. Genome of Bacteriophage P1

    PubMed Central

    Łobocka, Małgorzata B.; Rose, Debra J.; Plunkett, Guy; Rusin, Marek; Samojedny, Arkadiusz; Lehnherr, Hansjörg; Yarmolinsky, Michael B.; Blattner, Frederick R.

    2004-01-01

    P1 is a bacteriophage of Escherichia coli and other enteric bacteria. It lysogenizes its hosts as a circular, low-copy-number plasmid. We have determined the complete nucleotide sequences of two strains of a P1 thermoinducible mutant, P1 c1-100. The P1 genome (93,601 bp) contains at least 117 genes, of which almost two-thirds had not been sequenced previously and 49 have no homologs in other organisms. Protein-coding genes occupy 92% of the genome and are organized in 45 operons, of which four are decisive for the choice between lysis and lysogeny. Four others ensure plasmid maintenance. The majority of the remaining 37 operons are involved in lytic development. Seventeen operons are transcribed from σ70 promoters directly controlled by the master phage repressor C1. Late operons are transcribed from promoters recognized by the E. coli RNA polymerase holoenzyme in the presence of the Lpa protein, the product of a C1-controlled P1 gene. Three species of P1-encoded tRNAs provide differential controls of translation, and a P1-encoded DNA methyltransferase with putative bifunctionality influences transcription, replication, and DNA packaging. The genome is particularly rich in Chi recombinogenic sites. The base content and distribution in P1 DNA indicate that replication of P1 from its plasmid origin had more impact on the base compositional asymmetries of the P1 genome than replication from the lytic origin of replication. PMID:15489417

  13. Architecture and adhesive activity of the Haemophilus influenzae Hsf adhesin.

    PubMed

    Cotter, Shane E; Yeo, Hye-Jeong; Juehne, Twyla; St Geme, Joseph W

    2005-07-01

    Haemophilus influenzae type b is an important cause of meningitis and other serious invasive diseases and initiates infection by colonizing the upper respiratory tract. Among the major adhesins in H. influenzae type b is a nonpilus protein called Hsf, a large protein that forms fiber-like structures on the bacterial surface and shares significant sequence similarity with the nontypeable H. influenzae Hia autotransporter. In the present study, we characterized the structure and adhesive activity of Hsf. Analysis of the predicted amino acid sequence of Hsf revealed three regions with high-level homology to the HiaBD1 and HiaBD2 binding domains in Hia. Based on examination of glutathione S-transferase fusion proteins corresponding to these regions, two of the three had adhesive activity and one was nonadhesive in assays with cultured epithelial cells. Structural modeling demonstrated that only the two regions with adhesive activity harbored an acidic binding pocket like the binding pocket identified in the crystal structure of HiaBD1. Consistent with these results, disruption of the acidic binding pockets in the adhesive regions eliminated adhesive activity. These studies advance our understanding of the architecture of Hsf and the family of trimeric autotransporters and provide insight into the structural determinants of H. influenzae type b adherence.

  14. Mapping the Binding Domain of the F18 Fimbrial Adhesin

    PubMed Central

    Smeds, A.; Pertovaara, M.; Timonen, T.; Pohjanvirta, T.; Pelkonen, S.; Palva, A.

    2003-01-01

    F18 fimbrial Esherichia coli strains are associated with porcine postweaning diarrhea and pig edema disease. Recently, the FedF subunit was identified as the adhesin of the F18 fimbriae. In this study, adhesion domains of FedF were further studied by constructing deletions within the fedF gene and expressing FedF proteins with deletions either together with the other F18 fimbrial subunits or as fusion proteins tagged with maltose binding protein. The region essential for adhesion to porcine intestinal epithelial cells was mapped between amino acid residues 60 and 109 of FedF. To map the binding domain even more closely, all eight charged amino acid residues within this region were independently replaced by alanine. Three of these single point mutants expressing F18 fimbriae exhibited significantly diminished capabilities to adhere to porcine epithelial cells in vitro. In addition, a triple point mutation and a double point mutation completely abolished receptor adhesiveness. The result further confirmed that the region between amino acid residues 60 and 109 is essential for the binding of F18 fimbriae to their receptor. In addition, the adhesion capability of the binding domain was eliminated after treatment with iodoacetamide, suggesting the formation of a disulfide bridge between Cys-63 and Cys-83, whereas Cys-111 and Cys-116 could be deleted without affecting the binding ability of FedF. PMID:12654838

  15. Interaction of Mycobacterium leprae with human airway epithelial cells: adherence, entry, survival, and identification of potential adhesins by surface proteome analysis.

    PubMed

    Silva, Carlos A M; Danelishvili, Lia; McNamara, Michael; Berredo-Pinho, Márcia; Bildfell, Robert; Biet, Franck; Rodrigues, Luciana S; Oliveira, Albanita V; Bermudez, Luiz E; Pessolani, Maria C V

    2013-07-01

    This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.

  16. Ribosomal acidic phosphoproteins P1 and P2 are not required for cell viability but regulate the pattern of protein expression in Saccharomyces cerevisiae.

    PubMed Central

    Remacha, M; Jimenez-Diaz, A; Bermejo, B; Rodriguez-Gabriel, M A; Guarinos, E; Ballesta, J P

    1995-01-01

    Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1 alpha, YP1 beta, YP2 alpha, and YP2 beta, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23 degrees C. Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes. These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently. PMID:7651393

  17. Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: a component of the P1/P2/P0 complex.

    PubMed

    Juri Ayub, M; Levin, M J; Aguilar, C F

    2001-07-01

    The P0 protein is part of the ribosomal eukaryotic stalk, which is an elongated lateral protuberance of the large ribosomal subunit involved in the translocation step of protein synthesis. P0 is the minimal portion of the stalk that is able to support accurate protein synthesis. The P0 C-terminal peptide is highly antigenic and a major target of the antibody response in patients with systemic lupus erythematosus and patients suffering chronic heart disease produced by the Trypanosoma cruzi parasite. The T. cruzi P0 (TcP0) protein was cloned into the pRSET A vector and expressed in Escherichia coli fused to a His-tag. The identity of the protein was confirmed by immunoblotting. Due to the formation of inclusion bodies the protein was purified using the following steps: (i) differential centrifugation to separate the inclusion bodies from soluble proteins and (ii) affinity chromatography under denaturing conditions. TcP0 showed high tendency to aggregation during refolding assays. However, TcP0 could be efficiently folded in the presence of a low concentration of SDS. The folding of the protein was confirmed using urea gradient electrophoresis, limited proteolysis, circular dichroism, and tryptophan fluorescence. Native electrophoresis showed that the folded TcP0 (and not a folding intermediate) was the cause of aggregation in the absence of SDS. The protocol described here permitted us to obtain large amounts (up to 30 mg per culture liter) of pure and folded TcP0, a very hydrophobic protein with a high tendency to aggregation.

  18. 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

  19. Identification of glycoprotein receptors within the human salivary proteome for the lectin-like BabA and SabA adhesins of Helicobacter pylori by fluorescence-based 2-D bacterial overlay.

    PubMed

    Walz, Anke; Odenbreit, Stefan; Stühler, Kai; Wattenberg, Andreas; Meyer, Helmut E; Mahdavi, Jafar; Borén, Thomas; Ruhl, Stefan

    2009-03-01

    Because gastric infection by Helicobacter pylori takes place via the oral route, possible interactions of this bacterium with human salivary proteins could occur. By using modified 1- and 2-D bacterial overlay, binding of H. pylori adhesins BabA and SabA to the whole range of salivary proteins was explored. Bound salivary receptor molecules were identified by MALDI-MS and by comparison to previously established proteome maps of whole and glandular salivas. By use of adhesin-deficient mutants, binding of H. pylori to MUC7 and gp-340 could be linked to the SabA and BabA adhesins, respectively, whereas binding to MUC5B was associated with both adhesins. Binding of H. pylori to the proline-rich glycoprotein was newly detected and assigned to BabA adhesin whereas the SabA adhesin was found to mediate binding to newly detected receptor molecules, including carbonic anhydrase VI, secretory component, heavy chain of secretory IgA1, parotid secretory protein and zinc-alpha(2)-glycoprotein. Some of these salivary glycoproteins are known to act as scavenger molecules or are involved in innate immunity whereas others might come to modify the pathogenetic properties of this organism. In general, this 2-D bacterial overlay technique represents a useful supplement in adhesion studies of bacteria with complex protein mixtures.

  20. Structure of a Burkholderia pseudomallei Trimeric Autotransporter Adhesin Head

    PubMed Central

    Edwards, Thomas E.; Phan, Isabelle; Abendroth, Jan; Dieterich, Shellie H.; Masoudi, Amir; Guo, Wenjin; Hewitt, Stephen N.; Kelley, Angela; Leibly, David; Brittnacher, Mitch J.; Staker, Bart L.; Miller, Samuel I.; Van Voorhis, Wesley C.; Myler, Peter J.; Stewart, Lance J.

    2010-01-01

    Background Pathogenic bacteria adhere to the host cell surface using a family of outer membrane proteins called Trimeric Autotransporter Adhesins (TAAs). Although TAAs are highly divergent in sequence and domain structure, they are all conceptually comprised of a C-terminal membrane anchoring domain and an N-terminal passenger domain. Passenger domains consist of a secretion sequence, a head region that facilitates binding to the host cell surface, and a stalk region. Methodology/Principal Findings Pathogenic species of Burkholderia contain an overabundance of TAAs, some of which have been shown to elicit an immune response in the host. To understand the structural basis for host cell adhesion, we solved a 1.35 Å resolution crystal structure of a BpaA TAA head domain from Burkholderia pseudomallei, the pathogen that causes melioidosis. The structure reveals a novel fold of an intricately intertwined trimer. The BpaA head is composed of structural elements that have been observed in other TAA head structures as well as several elements of previously unknown structure predicted from low sequence homology between TAAs. These elements are typically up to 40 amino acids long and are not domains, but rather modular structural elements that may be duplicated or omitted through evolution, creating molecular diversity among TAAs. Conclusions/Significance The modular nature of BpaA, as demonstrated by its head domain crystal structure, and of TAAs in general provides insights into evolution of pathogen-host adhesion and may provide an avenue for diagnostics. PMID:20862217

  1. Bartonella henselae Pap31, an Extracellular Matrix Adhesin, Binds the Fibronectin Repeat III13 Module

    PubMed Central

    Dabo, S. M.; Confer, A. W.; Anderson, B. E.; Gupta, Snehalata

    2006-01-01

    Bartonella henselae wound-associated infections suggest involvement of extracellular matrix molecules in adhesion and invasion. Pap31 was previously identified as a hemin-binding protein. Our recent studies suggest the protein is an adhesin that is recognized by the host's immune systems. In this study we examined the interactions of B. henselae Pap31 with fibronectin (Fn), heparin (Hep), and human umbilical vein endothelial cells (HUVECs). The cloned gene was expressed in Escherichia coli, and the purified Pap31 protein elicited strong antibody responses in mice and was reactive with rabbit anti-live B. henselae and mouse anti-Pap31 antibodies by Western blotting. Pap31 bound to immobilized Fn and to HUVECs in a dose-dependent manner and to Hep. Fn fragment-binding assays identified the Hep-1 and Hep-2 binding domains of human Fn and in particular the 12-13FnIII repeat module as primary binding sites for this adhesin. Furthermore, Pap31 binding to the above Fn fragments could be inhibited by Hep, suggesting a common binding site involving the 13FnIII repeat module on the Hep-2 domain of Fn. Adherence of intact B. henselae to HUVECs was inhibited by increasing concentrations of anti-Pap31 antibodies. In addition, purified Pap31 coprecipitated effectively with Fn and anti-Fn antibodies. Taken together, these data suggest that Pap31 is an Fn-binding protein mediating the B. henselae-host interaction(s), and they implicate the 13FnIII repeat module as an important binding site for this adhesin on the Fn molecule. These interactions may be important initial steps leading to bacterial attachment and colonization that promote the establishment of B. henselae infections in vivo. PMID:16622186

  2. 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

  3. Biodiversity-Based Identification and Functional Characterization of the Mannose-Specific Adhesin of Lactobacillus plantarum

    PubMed Central

    Pretzer, Gabriele; Snel, Johannes; Molenaar, Douwe; Wiersma, Anne; Bron, Peter A.; Lambert, Jolanda; de Vos, Willem M.; van der Meer, Roelof; Smits, Mari A.; Kleerebezem, Michiel

    2005-01-01

    Lactobacillus plantarum is a frequently encountered inhabitant of the human intestinal tract, and some strains are marketed as probiotics. Their ability to adhere to mannose residues is a potentially interesting characteristic with regard to proposed probiotic features such as colonization of the intestinal surface and competitive exclusion of pathogens. In this study, the variable capacity of 14 L. plantarum strains to agglutinate Saccharomyces cerevisiae in a mannose-specific manner was determined and subsequently correlated with an L. plantarum WCFS1-based genome-wide genotype database. This led to the identification of four candidate mannose adhesin-encoding genes. Two genes primarily predicted to code for sortase-dependent cell surface proteins displayed a complete gene-trait match. Their involvement in mannose adhesion was corroborated by the finding that a sortase (srtA) mutant of L. plantarum WCFS1 lost the capacity to agglutinate S. cerevisiae. The postulated role of these two candidate genes was investigated by gene-specific deletion and overexpression in L. plantarum WCFS1. Subsequent evaluation of the mannose adhesion capacity of the resulting mutant strains showed that inactivation of one candidate gene (lp_0373) did not affect mannose adhesion properties. In contrast, deletion of the other gene (lp_1229) resulted in a complete loss of yeast agglutination ability, while its overexpression quantitatively enhanced this phenotype. Therefore, this gene was designated to encode the mannose-specific adhesin (Msa; gene name, msa) of L. plantarum. Domain homology analysis of the predicted 1,000-residue Msa protein identified known carbohydrate-binding domains, further supporting its role as a mannose adhesin that is likely to be involved in the interaction of L. plantarum with its host in the intestinal tract. PMID:16109954

  4. Influence of the poly-3-hydroxybutyrate (PHB) granule-associated proteins (PhaP1 and PhaP2) on PHB accumulation and symbiotic nitrogen fixation in Sinorhizobium meliloti Rm1021.

    PubMed

    Wang, Chunxia; Sheng, Xiaoyan; Equi, Raymie C; Trainer, Maria A; Charles, Trevor C; Sobral, Bruno W S

    2007-12-01

    Sinorhizobium meliloti cells store excess carbon as intracellular poly-3-hydroxybutyrate (PHB) granules that assist survival under fluctuating nutritional conditions. PHB granule-associated proteins (phasins) are proposed to regulate PHB synthesis and granule formation. Although the enzymology and genetics of PHB metabolism in S. meliloti have been well characterized, phasins have not yet been described for this organism. Comparison of the protein profiles of the wild type and a PHB synthesis mutant revealed two major proteins absent from the mutant. These were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as being encoded by the SMc00777 (phaP1) and SMc02111 (phaP2) genes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins associated with PHB granules followed by MALDI-TOF confirmed that PhaP1 and PhaP2 were the two major phasins. Double mutants were defective in PHB production, while single mutants still produced PHB, and unlike PHB synthesis mutants that have reduced exopolysaccharide, the double mutants had higher exopolysaccharide levels. Medicago truncatula plants inoculated with the double mutant exhibited reduced shoot dry weight (SDW), although there was no corresponding reduction in nitrogen fixation activity. Whether the phasins are involved in a metabolic regulatory response or whether the reduced SDW is due to a reduction in assimilation of fixed nitrogen rather than a reduction in nitrogen fixation activity remains to be established.

  5. Identification and Characterization of an Escorter for Two Secretory Adhesins in Toxoplasma gondii

    PubMed Central

    Reiss, Matthias; Viebig, Nicola; Brecht, Susan; Fourmaux, Marie-Noelle; Soete, Martine; Di Cristina, Manlio; Dubremetz, Jean François; Soldati, Dominique

    2001-01-01

    The intracellular protozoan parasite Toxoplasma gondii shares with other members of the Apicomplexa a common set of apical structures involved in host cell invasion. Micronemes are apical secretory organelles releasing their contents upon contact with host cells. We have identified a transmembrane micronemal protein MIC6, which functions as an escorter for the accurate targeting of two soluble proteins MIC1 and MIC4 to the micronemes. Disruption of MIC1, MIC4, and MIC6 genes allowed us to precisely dissect their contribution in sorting processes. We have mapped domains on these proteins that determine complex formation and targeting to the organelle. MIC6 carries a sorting signal(s) in its cytoplasmic tail whereas its association with MIC1 involves a lumenal EGF-like domain. MIC4 binds directly to MIC1 and behaves as a passive cargo molecule. In contrast, MIC1 is linked to a quality control system and is absolutely required for the complex to leave the early compartments of the secretory pathway. MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act. We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite. PMID:11157983

  6. UpaG, a New Member of the Trimeric Autotransporter Family of Adhesins in Uropathogenic Escherichia coli▿ †

    PubMed Central

    Valle, Jaione; Mabbett, Amanda N.; Ulett, Glen C.; Toledo-Arana, Alejandro; Wecker, Karine; Totsika, Makrina; Schembri, Mark A.; Ghigo, Jean-Marc; Beloin, Christophe

    2008-01-01

    The ability of Escherichia coli to colonize both intestinal and extraintestinal sites is driven by the presence of specific virulence factors, among which are the autotransporter (AT) proteins. Members of the trimeric AT adhesin family are important virulence factors for several gram-negative pathogens and mediate adherence to eukaryotic cells and extracellular matrix (ECM) proteins. In this study, we characterized a new trimeric AT adhesin (UpaG) from uropathogenic E. coli (UPEC). Molecular analysis of UpaG revealed that it is translocated to the cell surface and adopts a multimeric conformation. We demonstrated that UpaG is able to promote cell aggregation and biofilm formation on abiotic surfaces in CFT073 and various UPEC strains. In addition, UpaG expression resulted in the adhesion of CFT073 to human bladder epithelial cells, with specific affinity to fibronectin and laminin. Prevalence analysis revealed that upaG is strongly associated with E. coli strains from the B2 and D phylogenetic groups, while deletion of upaG had no significant effect on the ability of CFT073 to colonize the mouse urinary tract. Thus, UpaG is a novel trimeric AT adhesin from E. coli that mediates aggregation, biofilm formation, and adhesion to various ECM proteins. PMID:18424525

  7. The anticoagulant activated protein C (aPC) promotes metaplasticity in the hippocampus through an EPCR-PAR1-S1P1 receptors dependent mechanism.

    PubMed

    Maggio, Nicola; Itsekson, Zeev; Ikenberg, Benno; Strehl, Andreas; Vlachos, Andreas; Blatt, Ilan; Tanne, David; Chapman, Joab

    2014-08-01

    Thrombin and other clotting factors regulate long-term potentiation (LTP) in the hippocampus through the activation of the protease activated receptor 1 (PAR1) and consequent potentiation of N-methyl-d-aspartate receptor (NMDAR) functions. We have recently shown that the activation of PAR1 either by thrombin or the anticoagulant factor activated protein C (aPC) has differential effects on LTP. While thrombin activation of PAR1 induces an NMDAR-mediated slow onset LTP, which saturates the ability to induce further LTP in the exposed network, aPC stimulation of PAR1 enhances tetanus induced LTP through a voltage-gated calcium channels mediated mechanism. In this study, we addressed the mechanisms by which aPC enhances LTP in hippocampal slices. Using extracellular recordings, we show that a short tetanic stimulation, which does not induce LTP, is able to enhance plasticity in the presence of aPC through a mechanism that requires the activation of sphingosine-1 phosphate receptor 1 and intracellular Ca(2+) stores. These data identify aPC as a "metaplastic molecule", capable of shifting the threshold of LTP towards further potentiation. Our findings propose novel strategies to enhance plasticity in neurological diseases associated with the breakdown of the blood brain barrier and alterations in synaptic plasticity.

  8. Neisseria Adhesin A Variation and Revised Nomenclature Scheme

    PubMed Central

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

    2014-01-01

    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/. PMID:24807056

  9. Identical strength of the T cell responses against E2, nsP1 and capsid CHIKV proteins in recovered and chronic patients after the epidemics of 2005-2006 in La Reunion Island.

    PubMed

    Hoarau, Jean-Jacques; Gay, Frederick; Pellé, Olivier; Samri, Assia; Jaffar-Bandjee, Marie-Christine; Gasque, Philippe; Autran, Brigitte

    2013-01-01

    To characterize the immunity developed by patients infected by chikungunya virus (CHIKV), we studied the intensity and specificity of CHIKV-specific T cells mediated responses in chronic and recovered patients at 12 to 24 months post-infection. T cells were challenged in vitro against CHIKV synthetic peptides covering the length of three viral proteins, capsid, E2 and nsP1 proteins as well as all inactivated virus particles. Cytokine production was assessed by ELISPOT and intracellular labeling. T cells producing IFN-γ were detected against CHIKV in 85% patient's cells either by direct ELISPOT assay (69% of patients) or after expansion of memory T cells allowing the detection of both CD4 and CD8 specific-T cells in 16% additional cases. The IFN-γ response was mainly engaged in response to nsP1 or E2 (52% and 46% cases, respectively) but in only 27% cases against the capsid. The anti-E2 response represented half the magnitude of the total CHIKV IFN-γ production and was mainly directed against the C-terminal half part of the protein. Almost all patients had conserved a T cell specific response against CHIKV with a clear hierarchy of T cell responses (CD8 > CD4) engaged against E2 > nsP1 > capsid. More importantly, the intensity of responses was not significantly different between recovered and chronic patients. These findings constitute key elements to a better understanding of patient T cell immunoreactivity against CHIKV and argue against a possible defect of T cell immunoresponse in the chronicity post-CHIKV infection.

  10. Isolation of lactic acid bacteria bound to the porcine intestinal mucosa and an analysis of their moonlighting adhesins

    PubMed Central

    KINOSHITA, Hideki; OHUCHI, Satoko; ARAKAWA, Kensuke; WATANABE, Masamichi; KITAZAWA, Haruki; SAITO, Tadao

    2016-01-01

    The adhesion of lactic acid bacteria (LAB) to the intestinal mucosa is one of the criteria in selecting for probiotics. Eighteen LAB were isolated from porcine intestinal mucin (PIM): ten strains of Lactobacillus, six strains of Weissella, and two strains of Streptococcus. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for phosphate-buffered saline (PBS) extracts from the LAB, many bands were detected in half of the samples, while a few and/or no clear bands were detected in the other half. All six of the selected LAB showed adhesion to PIM. L. johnsonii MYU 214 and MYU 221 showed adhesion at more than 10%. W. viridescens MYU 208, L. reuteri MYU 213, L. mucosae MYU 225, and L. agilis MYU 227 showed medium levels of adhesion at 5.9–8.3%. In a comprehensive analysis for the adhesins in the PBS extracts using a receptor overlay analysis, many moonlighting proteins were detected and identified as candidates for adhesins: GroEL, enolase, and elongation factor Tu in MYU 208; peptidase C1, enolase, formyl-CoA transferase, phosphoglyceromutase, triosephosphate isomerase, and phosphofructokinase in MYU 221; and DnaK, enolase, and phosphoglycerate kinase in MYU 227. These proteins in the PBS extracts, which included such things as molecular chaperones and glycolytic enzymes, may play important roles as adhesins. PMID:27867805

  11. Differential recognition of members of the carcinoembryonic antigen family by Afa/Dr adhesins of diffusely adhering Escherichia coli (Afa/Dr DAEC).

    PubMed

    Berger, Cedric N; Billker, Oliver; Meyer, Thomas F; Servin, Alain L; Kansau, Imad

    2004-05-01

    Little is known about the molecular bases underlying the virulence of diffusely adhering Escherichia coli (DAEC) harbouring the Afa/Dr family of adhesins. These adhesins recognize as receptors the GPI-anchored proteins CD55 (decay-accelerating factor, DAF) and CD66e (carcinoembryonic antigen, CEA). CD66e is a member of the CEA-related cell adhesion molecules (CEACAM) family, comprising seven members. We analysed the interactions of Afa/Dr DAEC with the CEACAMs using CEACAM-expressing CHO and HeLa cells. The results demonstrate that only E. coli expressing a subfamily of Afa/Dr adhesins, named here Afa/Dr-I, including Dr, F1845 and AfaE-III adhesins, bound onto CHO cells expressing CEACAM1, CEA or CEACAM6. Whereas all the Afa/Dr adhesins elicit recruitment of CD55 around adhering bacteria, only the Afa/Dr-I subfamily elicits the recruitment of CEACAM1, CEA and CEACAM6. In addition, although CEACAM3 is not recognized as a receptor by the subfamily of Afa/Dr adhesins, it is recruited around bacteria in HeLa cells. The recruited CEACAM1, CEA and CEACAM6 around adhering bacteria resist totally or in part a detergent extraction, whereas the recruited CEACAM3 does not. Finally, the results show that recognition of CEA and CEACAM6, but not CEACAM1, is accompanied by tight attachment to bacteria of cell surface microvilli-like extensions, which are elongated. Moreover, recognition of CEA is accompanied by an activation of the Rho GTPase Cdc42 and by a phosphorylation of ERM, which in turn elicit the observed cell surface microvilli-like extensions.

  12. 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

  13. Role of Staphylococcus aureus surface adhesins in orthopaedic device infections: are results model-dependent?

    PubMed

    Darouiche, R O; Landon, G C; Patti, J M; Nguyen, L L; Fernau, R C; McDevitt, D; Greene, C; Foster, T; Klima, M

    1997-01-01

    Bacterial colonisation of prosthetic material can lead to clinical infection or implant failure, or both, often requiring removal of the device. Adherence of Staphylococcus aureus to bioprosthetic materials is mediated by adhesins belonging to the MSCRAMM (microbial surface components recognising adhesive matrix molecules) family of microbial cell surface proteins. The objective of this study was to compare the virulence of a mutant strain of S. aureus Newman that possesses all three fibrinogen-, fibronectin- and collagen-binding MSCRAMMs (MSCRAMM-positive strain) with that of a mutant strain that lacks all three types of MSCRAMMs (MSCRAMM-negative strain) in a rabbit model of orthopaedic device-related infection. After a hole was drilled into the knee joint of each animal, a group of 10 rabbits was inoculated with the MSCRAMM-positive strain and another group of 10 rabbits received the MSCRAMM-negative strain. A stainless steel screw was then placed into the drilled hole. Two weeks later, the rabbits were killed and serum samples, bone tissue and implants were harvested for bacteriological and histopathological evaluation. No significant difference in infection rates was demonstrated between the two groups. The ability to delineate the role of S. aureus surface adhesins in causing orthopaedic device-related infection could be model-dependent.

  14. 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

  15. The Haemophilus ducreyi trimeric autotransporter adhesin DsrA protects against an experimental infection in the swine model of chancroid.

    PubMed

    Fusco, William G; Choudhary, Neelima R; Routh, Patty A; Ventevogel, Melissa S; Smith, Valerie A; Koch, Gary G; Almond, Glen W; Orndorff, Paul E; Sempowski, Gregory D; Leduc, Isabelle

    2014-06-24

    Adherence of pathogens to cellular targets is required to initiate most infections. Defining strategies that interfere with adhesion is therefore important for the development of preventative measures against infectious diseases. As an adhesin to host extracellular matrix proteins and human keratinocytes, the trimeric autotransporter adhesin DsrA, a proven virulence factor of the Gram-negative bacterium Haemophilus ducreyi, is a potential target for vaccine development. A recombinant form of the N-terminal passenger domain of DsrA from H. ducreyi class I strain 35000HP, termed rNT-DsrAI, was tested as a vaccine immunogen in the experimental swine model of H. ducreyi infection. Viable homologous H. ducreyi was not recovered from any animal receiving four doses of rNT-DsrAI administered with Freund's adjuvant at two-week intervals. Control pigs receiving adjuvant only were all infected. All animals receiving the rNT-DsrAI vaccine developed antibody endpoint titers between 3.5 and 5 logs. All rNT-DsrAI antisera bound the surface of the two H. ducreyi strains used to challenge immunized pigs. Purified anti-rNT-DsrAI IgG partially blocked binding of fibrinogen at the surface of viable H. ducreyi. Overall, immunization with the passenger domain of the trimeric autotransporter adhesin DsrA accelerated clearance of H. ducreyi in experimental lesions, possibly by interfering with fibrinogen binding.

  16. Breast cancer resistance protein (Bcrp1/Abcg2) reduces systemic exposure of the dietary carcinogens aflatoxin B1, IQ and Trp-P-1 but also mediates their secretion into breast milk.

    PubMed

    van Herwaarden, Antonius E; Wagenaar, Els; Karnekamp, Barbara; Merino, Gracia; Jonker, Johan W; Schinkel, Alfred H

    2006-01-01

    The breast cancer resistance protein (BCRP/ABCG2) usually protects the body from a wide variety of environmental and dietary xenotoxins by reducing their net uptake from intestine and by increasing their hepatobiliary, intestinal and renal elimination. BCRP is also highly expressed in lactating mammary glands in mice, and this expression is conserved in cows and humans. As a result, BCRP substrates can be secreted into milk. We investigated whether different classes of dietary carcinogens are substrates of Bcrp1/BCRP and the implications for systemic exposure and breast milk contamination. Using polarized cell lines, we found that Bcrp1 transports the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and the potent human hepatocarcinogen aflatoxin B1, and decreases their cellular accumulation up to 10-fold. In vivo pharmacokinetic studies showed that [14C]IQ, [14C]Trp-P-1 and [3H]aflatoxin B1 plasma levels were substantially lower in wild-type compared with Bcrp1-/- mice, after both oral and intravenous administration, demonstrating that Bcrp1 restricts systemic exposure to these carcinogens. Moreover, Bcrp1 mediates transfer of [14C]IQ, [14C]Trp-P-1 and [3H]aflatoxin into milk, with 3.4+/-0.6, 2.6+/-0.3 and 3.8+/-0.5-fold higher milk to plasma ratios, respectively, in lactating wild-type versus Bcrp1-/- mice. We have thus identified Bcrp1/BCRP as one of the molecular mechanisms by which heterocyclic amines and aflatoxin are transferred into milk, thereby posing a health risk to breast-fed infants and dairy consumers. Paradoxically, Bcrp1/BCRP appears to have both protective and adverse roles with respect to exposure to dietary carcinogens.

  17. Design of HIV-1 protease inhibitors with pyrrolidinones and oxazolidinones as novel P1'-ligands to enhance backbone-binding interactions with protease: synthesis, biological evaluation, and protein-ligand X-ray studies

    SciTech Connect

    Ghosh, Arun K.; Leshchenko-Yashchuk, Sofiya; Anderson, David D.; Baldridge, Abigail; Noetzel, Marcus; Miller, Heather B.; Tie, Yunfeng; Wang, Yuan-Fang; Koh, Yasuhiro; Weber, Irene T.; Mitsuya, Hiroaki

    2009-09-02

    Structure-based design, synthesis, and biological evaluation of a series of novel HIV-1 protease inhibitors are described. In an effort to enhance interactions with protease backbone atoms, we have incorporated stereochemically defined methyl-2-pyrrolidinone and methyl oxazolidinone as the P1{prime}-ligands. These ligands are designed to interact with Gly-27{prime} carbonyl and Arg-8 side chain in the S1{prime}-subsite of the HIV protease. We have investigated the potential of these ligands in combination with our previously developed bis-tetrahydrofuran (bis-THF) and cyclopentanyltetrahydrofuran (Cp-THF) as the P2-ligands. Inhibitor 19b with a (R)-aminomethyl-2-pyrrolidinone and a Cp-THF was shown to be the most potent compound. This inhibitor maintained near full potency against multi-PI-resistant clinical HIV-1 variants. A high resolution protein-ligand X-ray crystal structure of 19b-bound HIV-1 protease revealed that the P1{prime}-pyrrolidinone heterocycle and the P2-Cp-ligand are involved in several critical interactions with the backbone atoms in the S1{prime} and S2 subsites of HIV-1 protease.

  18. Antibodies to the HMW1/HMW2 and Hia Adhesins of Nontypeable Haemophilus influenzae Mediate Broad-Based Opsonophagocytic Killing of Homologous and Heterologous Strains

    PubMed Central

    Winter, Linda E.

    2014-01-01

    The HMW1/HMW2 and Hia proteins are highly immunogenic surface adhesins of nontypeable Haemophilus influenzae (NTHi). Approximately 75% of NTHi strains express HMW1/HMW2 adhesins, and most of the remaining 25% express an Hia adhesin. Our objective in this study was to assess the ability of antisera raised against purified HMW1/HMW2 proteins or recombinant Hia proteins to mediate opsonophagocytic killing of a large panel of unrelated NTHi strains. Native HMW1/HMW2 proteins were purified from three HMW1/HMW2-expressing NTHi strains. Recombinant fusion proteins expressing surface-exposed segments of either of two prototype Hia proteins were purified from Escherichia coli transformants. Immune sera raised in guinea pigs were assessed for their ability to mediate killing of NTHi in an opsonophagocytic assay with the HL-60 phagocytic cell line. The three HMW1/HMW2 antisera mediated killing of 22 of 65, 43 of 65, and 28 of 65 unrelated HMW1/HMW2-expressing NTHi strains, respectively. As a group, the three sera mediated killing of 48 of 65 HMW1/HMW2-expressing strains. The two Hia immune sera mediated killing of 12 of 24 and 13 of 24 unrelated Hia-expressing NTHi strains, respectively. Together, they mediated killing of 15 of 24 Hia-expressing strains. Neither the HMW1/HMW2 nor the Hia antisera mediated killing of NTHi expressing the alternative adhesin type. Antibodies directed against native HMW1/HMW2 proteins and recombinant Hia proteins are capable of mediating broad-based opsonophagocytic killing of homologous and heterologous NTHi strains. A vaccine formulated with a limited number of HMW1/HMW2 and Hia proteins might provide protection against disease caused by most NTHi strains. PMID:24574538

  19. 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

  20. The Mycobacterium tuberculosis cell-surface glycoprotein apa as a potential adhesin to colonize target cells via the innate immune system pulmonary C-type lectin surfactant protein A.

    PubMed

    Ragas, Aude; Roussel, Lucie; Puzo, Germain; Rivière, Michel

    2007-02-23

    Tuberculosis is still a major health problem, and understanding the mechanism by which Mycobacterium tuberculosis (Mtb) invades and colonizes its host target cells remains an important issue for the control of infection. The innate immune system C-type lectins (C-TLs), including the human pulmonary surfactant protein A (PSP-A), have been recently identified as determinant players in the early recognition of the invading pathogen and in mounting the host defense response. Although the antigenic lipoglycan mannosylated lipoarabinomannan is currently considered to be the major C-TL target on the mycobacterial surface, the recognition by some C-TLs of the only mycobacterial species composing the "Mtb complex" indicates that mannosylated lipoarabinomannan cannot account alone for this specificity. Thus, we searched for the mycobacterial molecules targeted by human PSP-A, focusing our attention on the Mtb surface glycoproteins. We developed an original functional proteomic approach based on a lectin blot assay using crude human bronchoalveolar lavage fluid as a source of physiological PSP-A. Combined with selective cell-surface protein extraction and mass spectrometry peptide mapping, this strategy allowed us to identify the Apa (alanine- and proline-rich antigenic) glycoprotein as new potential target for PSP-A. This result was supported by direct binding of PSP-A to purified Apa. Moreover, EDTA addition or deglycosylation of purified Apa samples completely abolished the interaction, demonstrating that the interaction is calcium- and mannose-dependent, as expected. Finally, we provide convincing evidence that Apa, formerly considered as mainly secreted, is associated with the cell wall for a sufficiently long time to aid in the attachment of PSP-A. Because, to date, Apa seems to be restricted to the Mtb complex strains, we propose that it may account for the selective recognition of those strains by PSP-A and other immune system C-TLs containing homologous functional

  1. The Three-dimensional Structure of the Extracellular Adhesion Domain of the Sialic Acid-binding Adhesin SabA from Helicobacter pylori

    PubMed Central

    Pang, Siew Siew; Nguyen, Stanley Thai Son; Perry, Andrew J.; Day, Christopher J.; Panjikar, Santosh; Tiralongo, Joe; Whisstock, James C.; Kwok, Terry

    2014-01-01

    The gastric pathogen Helicobacter pylori is a major cause of acute chronic gastritis and the development of stomach and duodenal ulcers. Chronic infection furthermore predisposes to the development of gastric cancer. Crucial to H. pylori survival within the hostile environment of the digestive system are the adhesins SabA and BabA; these molecules belong to the same protein family and permit the bacteria to bind tightly to sugar moieties LewisB and sialyl-LewisX, respectively, on the surface of epithelial cells lining the stomach and duodenum. To date, no representative SabA/BabA structure has been determined, hampering the development of strategies to eliminate persistent H. pylori infections that fail to respond to conventional therapy. Here, using x-ray crystallography, we show that the soluble extracellular adhesin domain of SabA shares distant similarity to the tetratricopeptide repeat fold family. The molecule broadly resembles a golf putter in shape, with the head region featuring a large cavity surrounded by loops that vary in sequence between different H. pylori strains. The N-terminal and C-terminal helices protrude at right angles from the head domain and together form a shaft that connects to a predicted outer membrane protein-like β-barrel trans-membrane domain. Using surface plasmon resonance, we were able to detect binding of the SabA adhesin domain to sialyl-LewisX and LewisX but not to LewisA, LewisB, or LewisY. Substitution of the highly conserved glutamine residue 159 in the predicted ligand-binding pocket abrogates the binding of the SabA adhesin domain to sialyl-LewisX and LewisX. Taken together, these data suggest that the adhesin domain of SabA is sufficient in isolation for specific ligand binding. PMID:24375407

  2. SgrA, a nidogen-binding LPXTG surface adhesin implicated in biofilm formation, and EcbA, a collagen binding MSCRAMM, are two novel adhesins of hospital-acquired Enterococcus faecium.

    PubMed

    Hendrickx, Antoni P A; van Luit-Asbroek, Miranda; Schapendonk, Claudia M E; van Wamel, Willem J B; Braat, Johanna C; Wijnands, Lucas M; Bonten, Marc J M; Willems, Rob J L

    2009-11-01

    Hospital-acquired Enterococcus faecium isolates responsible for nosocomial outbreaks and invasive infections are enriched in the orf2351 and orf2430 genes, encoding the SgrA and EcbA LPXTG-like cell wall-anchored proteins, respectively. These two surface proteins were characterized to gain insight into their function, since they may have favored the rapid emergence of this nosocomial pathogen. We are the first to identify a surface adhesin among bacteria (SgrA) that binds to the extracellular matrix molecules nidogen 1 and nidogen 2, which are constituents of the basal lamina. EcbA is a novel E. faecium MSCRAMM (microbial surface component recognizing adhesive matrix molecules) that binds to collagen type V. In addition, both SgrA and EcbA bound to fibrinogen; however, SgrA targeted the alpha and beta chains, whereas EcbA bound to the gamma chain of fibrinogen. An E. faecium sgrA insertion mutant displayed reduced binding to both nidogens and fibrinogen. SgrA did not mediate binding of E. faecium cells to biotic materials, such as human intestinal epithelial cells, human bladder cells, and kidney cells, while this LPXTG surface adhesin is implicated in E. faecium biofilm formation. The acm and scm genes, encoding two other E. faecium MSCRAMMs, were expressed at the mRNA level together with sgrA during all phases of growth, whereas ecbA was expressed only in exponential and late exponential phase, suggesting orchestrated expression of these adhesins. Expression of these surface proteins, which bind to extracellular matrix proteins and are involved in biofilm formation (SgrA), may contribute to the pathogenesis of hospital-acquired E. faecium infections.

  3. Defining Potential Vaccine Targets of Haemophilus ducreyi Trimeric Autotransporter Adhesin DsrA.

    PubMed

    Fusco, William G; Choudhary, Neelima R; Stewart, Shelley M; Alam, S Munir; Sempowski, Gregory D; Elkins, Christopher; Leduc, Isabelle

    2015-04-01

    Haemophilus ducreyi is the causative agent of the sexually transmitted genital ulcer disease chancroid. Strains of H. ducreyi are grouped in two classes (I and II) based on genotypic and phenotypic differences, including those found in DsrA, an outer membrane protein belonging to the family of multifunctional trimeric autotransporter adhesins. DsrA is a key serum resistance factor of H. ducreyi that prevents binding of natural IgM at the bacterial surface and functions as an adhesin to fibronectin, fibrinogen, vitronectin, and human keratinocytes. Monoclonal antibodies (MAbs) were developed to recombinant DsrA (DsrA(I)) from prototypical class I strain 35000HP to define targets for vaccine and/or therapeutics. Two anti-DsrAI MAbs bound monomers and multimers of DsrA from genital and non-genital/cutaneous H. ducreyi strains in a Western blot and reacted to the surface of the genital strains; however, these MAbs did not recognize denatured or native DsrA from class II strains. In a modified extracellular matrix protein binding assay using viable H. ducreyi, one of the MAbs partially inhibited binding of fibronectin, fibrinogen, and vitronectin to class I H. ducreyi strain 35000HP, suggesting a role for anti-DsrA antibodies in preventing binding of H. ducreyi to extracellular matrix proteins. Standard ELISA and surface plasmon resonance using a peptide library representing full-length, mature DsrAI revealed the smallest nominal epitope bound by one of the MAbs to be MEQNTHNINKLS. Taken together, our findings suggest that this epitope is a potential target for an H. ducreyi vaccine.

  4. A Processed Multidomain Mycoplasma hyopneumoniae Adhesin Binds Fibronectin, Plasminogen, and Swine Respiratory Cilia

    PubMed Central

    Seymour, Lisa M.; Deutscher, Ania T.; Jenkins, Cheryl; Kuit, Tracey A.; Falconer, Linda; Minion, F. Chris; Crossett, Ben; Padula, Matthew; Dixon, Nicholas E.; Djordjevic, Steven P.; Walker, Mark J.

    2010-01-01

    Porcine enzootic pneumonia is a chronic respiratory disease that affects swine. The etiological agent of the disease, Mycoplasma hyopneumoniae, is a bacterium that adheres to cilia of the swine respiratory tract, resulting in loss of cilia and epithelial cell damage. A M. hyopneumoniae protein P116, encoded by mhp108, was investigated as a potential adhesin. Examination of P116 expression using proteomic analyses observed P116 as a full-length protein and also as fragments, ranging from 17 to 70 kDa in size. A variety of pathogenic bacterial species have been shown to bind the extracellular matrix component fibronectin as an adherence mechanism. M. hyopneumoniae cells were found to bind fibronectin in a dose-dependent and saturable manner. Surface plasmon resonance was used to show that a recombinant C-terminal domain of P116 bound fibronectin at physiologically relevant concentrations (KD 24 ± 6 nm). Plasmin(ogen)-binding proteins are also expressed by many bacterial pathogens, facilitating extracellular matrix degradation. M. hyopneumoniae cells were found to also bind plasminogen in a dose-dependent and saturable manner; the C-terminal domain of P116 binds to plasminogen (KD 44 ± 5 nm). Plasminogen binding was abolished when the C-terminal lysine of P116 was deleted, implicating this residue as part of the plasminogen binding site. P116 fragments adhere to the PK15 porcine kidney epithelial-like cell line and swine respiratory cilia. Collectively these data suggest that P116 is an important adhesin and virulence factor of M. hyopneumoniae. PMID:20813843

  5. A processed multidomain mycoplasma hyopneumoniae adhesin binds fibronectin, plasminogen, and swine respiratory cilia.

    PubMed

    Seymour, Lisa M; Deutscher, Ania T; Jenkins, Cheryl; Kuit, Tracey A; Falconer, Linda; Minion, F Chris; Crossett, Ben; Padula, Matthew; Dixon, Nicholas E; Djordjevic, Steven P; Walker, Mark J

    2010-10-29

    Porcine enzootic pneumonia is a chronic respiratory disease that affects swine. The etiological agent of the disease, Mycoplasma hyopneumoniae, is a bacterium that adheres to cilia of the swine respiratory tract, resulting in loss of cilia and epithelial cell damage. A M. hyopneumoniae protein P116, encoded by mhp108, was investigated as a potential adhesin. Examination of P116 expression using proteomic analyses observed P116 as a full-length protein and also as fragments, ranging from 17 to 70 kDa in size. A variety of pathogenic bacterial species have been shown to bind the extracellular matrix component fibronectin as an adherence mechanism. M. hyopneumoniae cells were found to bind fibronectin in a dose-dependent and saturable manner. Surface plasmon resonance was used to show that a recombinant C-terminal domain of P116 bound fibronectin at physiologically relevant concentrations (K(D) 24 ± 6 nm). Plasmin(ogen)-binding proteins are also expressed by many bacterial pathogens, facilitating extracellular matrix degradation. M. hyopneumoniae cells were found to also bind plasminogen in a dose-dependent and saturable manner; the C-terminal domain of P116 binds to plasminogen (K(D) 44 ± 5 nm). Plasminogen binding was abolished when the C-terminal lysine of P116 was deleted, implicating this residue as part of the plasminogen binding site. P116 fragments adhere to the PK15 porcine kidney epithelial-like cell line and swine respiratory cilia. Collectively these data suggest that P116 is an important adhesin and virulence factor of M. hyopneumoniae.

  6. A hydrophilic cation-binding protein of Arabidopsis thaliana, AtPCaP1, is localized to plasma membrane via N-myristoylation and interacts with calmodulin and the phosphatidylinositol phosphates PtdIns(3,4,5)P(3) and PtdIns(3,5)P(2).

    PubMed

    Nagasaki, Nahoko; Tomioka, Rie; Maeshima, Masayoshi

    2008-05-01

    A hydrophilic cation-binding protein, PCaP1, was found to be stably bound to the plasma membrane in Arabidopsis thaliana. PCaP1 was quantified to account for 0.03-0.08% of the crude membrane fractions from roots and shoots. Its homologous protein was detected in several plant species. We investigated the mechanism of membrane association of PCaP1 by transient expression of fusion protein with green fluorescent protein. The amino-terminal sequence of 27 residues of PCaP1 had a potential to localize the fusion protein with green fluorescent protein to the plasma membrane, and the substitution of Gly at position 2 with Ala resulted in the cytoplasmic localization of PCaP1. When PCaP1 was expressed in the in vitro transcription/translation system with [(3)H]myristic acid, the label was incorporated into PCaP1, but not into a mutant PCaP1 with Gly2 replaced by Ala. These results indicate that PCaP1 tightly binds to the plasma membrane via N-myristoylation at Gly2. We examined the binding capacity with phosphatidylinositol phosphates (PtdInsPs), and found that PCaP1 selectively interacts with phosphatidylinositol 3,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate. Competition assay with the N-terminal peptide and mutational analysis revealed that PCaP1 interacts with these two PtdInsPs at the N-terminal part. Interaction of PCaP1 with the membrane and PtdInsPs was not altered in the presence of Ca(2+) at physiological concentrations. Furthermore, calmodulin associated with PCaP1 in a Ca(2+)-dependent manner, and its association weakened the interaction of PCaP1 with PtdInsPs. These results indicate that the N-terminal part is essential for both N-myristoylation and interaction with PtdInsPs, and that PCaP1 may be involved in intracellular signalling through interaction with PtdInsPs and calmodulin.

  7. Trichomonas vaginalis: the adhesins AP51 and AP65 bind heme and hemoglobin.

    PubMed

    Ardalan, Shahed; Lee, B Craig; Garber, Gary E

    2009-04-01

    Trichomonas vaginalis is the cause of human trichomoniasis, the most common non-viral sexually transmitted disease worldwide. Although acquisition of iron by binding to host hemoglobin through distinct receptor(s) has been described, no specific heme- or hemoglobin-binding site has been reported in this parasite. To determine the presence of hemoglobin-binding protein(s), membrane proteins were subjected to hemoglobin-affinity chromatography. Eluted proteins were analysed by SDS-PAGE. Two protein bands of 48 and 63 kDa were detected. Competition assay with an excess amount of hemoglobin or hemin in hemoglobin-affinity chromatography could block the 63- and 48-kDa bands, respectively. Further analysis by mass spectrometry indicated that the 48- and 63-kDa proteins had identity with two T. vaginalis adhesins: AP51 and AP65, respectively. This study confirms the existence of multifunctional proteins in T. vaginalis, and suggested that AP51 and AP65, besides serving as adhesion molecules, could also act as heme- and hemoglobin-binding proteins.

  8. Atomic force and super-resolution microscopy support a role for LapA as a cell-surface biofilm adhesin of Pseudomonas fluorescens

    PubMed Central

    Ivanov, Ivan E.; Boyd, Chelsea D.; Newell, Peter D.; Schwartz, Mary E.; Turnbull, Lynne; Johnson, Michael S.; Whitchurch, Cynthia B.; O’Toole, George A.; Camesano, Terri A.

    2012-01-01

    Pseudomonas fluorescence Pf0-1 requires the large repeat protein LapA for stable surface attachment. This study presents direct evidence that LapA is a cell-surface-localized adhesin. Atomic force microscopy (AFM) revealed a significant twofold reduction in adhesion force for mutants lacking the LapA protein on the cell surface compared to the wild-type strain. Deletion of lapG, a gene encoding a periplasmic cysteine protease that functions to release LapA from the cell surface, resulted in a twofold increase in the force of adhesion. Three-dimensional structured illumination microscopy (3D-SIM) revealed the presence of the LapA protein on the cell surface, consistent with its role as an adhesin. The protein is only visualized in the cytoplasm for a mutant of the ABC transporter responsible for translocating LapA to the cell surface. Together, these data highlight the power of combining the use of AFM and 3D-SIM with genetic studies to demonstrate that LapA, a member of a large group of RTX-like repeat proteins, is a cell-surface adhesin. PMID:23064158

  9. Expression and purification of the mannose recognition domain of the FimH adhesin.

    PubMed

    Schembri, M A; Hasman, H; Klemm, P

    2000-07-15

    Type 1 fimbriae have been shown to be specifically required for Escherichia coli colonisation and pathogenesis of the urinary tract. These structural organelles mediate specific adhesion to alpha-D-mannosides by virtue of the FimH adhesin. FimH is a two-domain protein in which the N-terminal domain contains the receptor-binding site and the C-terminal domain is required for organelle integration. To date, FimH has only been isolated as a complex with the system-specific chaperone FimC. Here we report that a functional form of the FimH receptor-binding domain can be readily isolated and characterised by replacing the C-terminal domain with a histidine tag.

  10. Biorecognition of Escherichia coli K88 adhesin for glycated porcine albumin.

    PubMed

    Sarabia-Sainz, Andre-i; Ramos-Clamont, Gabriela; Candia-Plata, Ma María del Carmen; Vázquez-Moreno, Luz

    2009-03-01

    Escherichia coli (E. coli) that expresses galactose-reactive lectins, like K88 adhesin, causes high mortality among piglets. Carbohydrates that compete for adhesion could serve as an alternative for disease prevention. Porcine serum albumin (PSA) was modified by non-enzymatic glycation with lactose to produce PSA-Lac or PSA-Glc beta (1-4) Gal, as confirmed by reduction of available free amino groups, increased molecular mass and by Ricinus communis lectin recognition. E. coli K88 binds to PSA-Lac treatments containing three and four lactoses, respectively. In addition, PSA-Lac partially inhibited K88 strain adherence to mucins. These results suggest that neoglycoconjugates obtained by non-enzymatic glycation of proteins may serve in the prophylaxis of piglets' diarrhea.

  11. Characterization of plasmid-borne afa-3 gene clusters encoding afimbrial adhesins expressed by Escherichia coli strains associated with intestinal or urinary tract infections.

    PubMed

    Le Bouguenec, C; Garcia, M I; Ouin, V; Desperrier, J M; Gounon, P; Labigne, A

    1993-12-01

    The afa gene clusters encode afimbrial adhesins (AFA) that are expressed by uropathogenic and diarrhea-associated Escherichia coli strains and belong to a family of hemagglutinins recognizing the Dr blood group antigen as a receptor. This family so far includes AFA-I and AFA-III as well as the Dr and F1845 adhesins (B. Nowicki, A. Labigne, S. Moseley, R. Hull, S. Hull, and J. Moulds, Infect. Immun. 58:279-281, 1990). Reported in this work is the genetic organization of the afa-3 gene cluster cloned from a uropathogenic E. coli strain (A30) which expressed a subtype of AFA designated AFA-III. The amino acid sequence of AFA-III was deduced from the nucleotide sequence of the afaE3 gene and was found to be highly homologous to that of the Dr adhesin (98.1% identity). A polymerase chain reaction assay was developed to detect the presence of afa-3 gene clusters in E. coli strains. Study of the genetic support of the afa-3 gene clusters in the strains which showed positive amplification revealed that they were always located on large, 100-kb plasmids whether the strains originated from patients with cystitis or with diarrhea. Moreover, the cloned afa-3 gene clusters from A30 and from the diarrhea-associated strain AL845 appeared to be carried by 9-kb plasmid regions which displayed a similar genetic organization. Chloramphenicol was reported to be a potent inhibitor of receptor binding by the Dr adhesin (Nowicki et al., Infect. Immun. 58:279-281, 1990). AFA-III expressed by strains AL845 and AL847 appeared to mediate, like the Dr adhesin, chloramphenicol-sensitive hemagglutination, whereas AFA-III produced by A30 conferred chloramphenicol-resistant adherence. A comparison of the sequences of these four proteins indicated that the amino acid at position 52 of the processed AFA could be part of the receptor-binding domain.

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

    PubMed

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

    2014-07-04

    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.

  13. 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

  14. Identification of the mucin-binding adhesin of Pseudomonas cepacia isolated from patients with cystic fibrosis.

    PubMed Central

    Sajjan, S U; Forstner, J F

    1992-01-01

    In previous experiments, we have shown that isolates of Pseudomonas cepacia from sputa of patients with cystic fibrosis (CF), particularly those with severe lung infection, exhibited specific binding to purified respiratory or intestinal mucins (U. Sajjan, M. Corey, M. Karmali, and J. Forstner, J. Clin. Invest. 89:648-656, 1992). The present report describes the identification of the adhesin as a protein located on fimbriae of mucin-binding P. cepacia. From a total of 53 isolates available (from 22 patients with CF), we used three mucin-binding and three non-mucin-binding isolates for our experiments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude P. cepacia homogenates was performed, the separated proteins were blotted onto nitrocellulose and overlaid with purified mucin, and mucin-binding components were detected with an antimucin antibody and then a second-antibody-alkaline phosphatase conjugate system. Only mucin-binding isolates exhibited a positively stained band at an Mr of 22,000. The 22-kDa protein was purified, and a polyclonal antibody specific for it was developed in rabbits. By electron microscopy and immunogold labelling, both the antibody and mucin (separately) were localized to pili present over the entire surface of the bacterial cells. Non-mucin-binding isolates did not have (or had very few) pili and did not stain with either mucin or the antibody to the 22-kDa protein. The purified 22-kDa protein and its antibody were each able to inhibit piliated P. cepacia binding to mucin. The amino acid composition of the 22-kDa protein was dissimilar to those of the major pilin proteins of Escherichia coli (type 1 pilus) and P. aeruginosa (PAK and PAO1 strains). Both the pili of P. aeruginosa PAK and PAO1 and antibodies to these pili failed to inhibit P. cepacia binding to mucin. Thus, P. cepacia adhesion to mucin is mediated by a pilin-associated 22-kDa protein which differs from epithelial-cell-binding pilin proteins of P. aeruginosa

  15. Combined expression of multidrug resistance protein (MRP) and glutathione S-transferase P1-1 (GSTP1-1) in MCF7 cells and high level resistance to the cytotoxicities of ethacrynic acid but not oxazaphosphorines or cisplatin.

    PubMed

    Morrow, C S; Smitherman, P K; Townsend, A J

    1998-10-15

    We tested the hypothesis that combined increased expression of human glutathione S-transferase P1-1 (GSTP1-1), an enzyme that catalyzes the conjugation with glutathione of several toxic electrophiles, and the glutathione-conjugate efflux pump, multidrug resistance protein (MRP), confers high level resistance to the cytotoxicities of anticancer and other drugs. To accomplish this, we developed MCF7 breast carcinoma cell derivatives that express high levels of GSTP1-1 and MRP, alone and in combination. Parental MCF7 cells, which express no GSTP1-1 and negligible MRP, served as control cells. We found that either MRP or GSTP1-1 alone conferred significant resistance to ethacrynic acid cytotoxicity. Moreover, combined expression of GSTP1-1 and MRP conferred a high level of resistance to ethacrynic acid that was greater than resistance conferred by either protein alone. Increased MRP was also associated with modest resistance to the oxazaphosphorine compounds mafosfamide, 4-hydroxycyclophosphamide, and 4-hydroperoxycyclophosphamide. However, coordinated expression of GSTP1-1 with MRP failed to augment this modest resistance. Similarly, GSTP1-1 had no effect on the sensitivities to cisplatin of MCF7 cells regardless of MRP expression. These results establish that coordinated expression of MRP and GSTP1-1 can confer high level resistance to the cytotoxicities of some drugs, including ethacrynic acid, but that such resistance is variable and does not apply to all toxic drugs that can potentially form glutathione conjugates in either spontaneous or GSTP1-1-catalyzed reactions.

  16. 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

  17. Biochemical and immunological characterization of recombinant allergen Lol p 1.

    PubMed

    Tamborini, E; Faccini, S; Lidholm, J; Svensson, M; Brandazza, A; Longhi, R; Groenlund, H; Sidoli, A; Arosio, P

    1997-11-01

    Pollen from perennial rye grass (Lolium perenne), a major cause of type-I allergy worldwide, contains a complex mixture of allergenic proteins among which Lol p 1 is one of the most important. We describe the expression, purification and characterization of a recombinant Lol p 1 overproduced in Escherichia coli. The recombinant allergen, expressed in high yields and purified in milligram amounts, bound to specific IgE antibodies from human sera, induced histamine release from sensitized human basophils, and elicited rabbit antisera that recognize specifically recombinant Lol p 1 and natural Lol p 1 of pollen extract. Recombinant Lol p 1 was used to develop ImmunoCAP assays for analysis of 150 sera that were Radioallergosorbent test positive to L. perenne pollen. In 130 of them (87%) the assay detected a significant level of IgE antibodies to Lol p 1, reaching on average 37% of the level obtained with a test for IgE to the whole grass pollen extract. To map epitopes on Lol p 1, we produced three deletion mutants [des-(116-240)-Lol p 1, des-(1-88)-Lol p 1 and des-(133-189)-Lol p 1], which were efficiently expressed in bacteria. These all showed a strong reactivity with the specific rabbit IgG antibodies, but lacked most or all the allergenic properties of recombinant Lol p 1. A study of the antigenic structure of Lol p 1 was performed using the three deletion mutants and a set of 17-18-residue overlapping synthetic peptides covering the whole allergen sequence. The results indicate that human IgE and rabbit IgG antibodies bind to distinct regions of Lol p 1, and that at least some important IgE epitopes are mainly conformational. The findings suggest that recombinant allergens constitute useful reagents for further development of serological diagnosis of allergy, and that it should be possible to produce immunogenic fragments of allergenic proteins without allergenic properties.

  18. The role of filamentous hemagglutinin adhesin in adherence and biofilm formation in Acinetobacter baumannii ATCC19606(T).

    PubMed

    Darvish Alipour Astaneh, Shakiba; Rasooli, Iraj; Mousavi Gargari, Seyed Latif

    2014-09-01

    Filamentous hemagglutinin adhesins (FHA) are key factors for bacterial attachment and subsequent cell accumulation on substrates. Here an FHA-like Outer membrane (OM) adhesin of Acinetobacter baumannii ATCC19606(T) was displayed on Escherichia coli. The candidate autotransporter (AT) genes were identified in A. baumannii ATCC19606(T) genome. The exoprotein (FhaB1) and transporter (FhaC1) were produced independently within the same cell (FhaB1C1). The fhaC1 was mutated. In vitro adherence to epithelial cells of the recombinant FhaB1C1 and the mutant strains were compared with A. baumanni ATCC19606(T). A bivalent chimeric protein (K) composed of immunologically important portions of fhaB1 (B) and fhaC1 (C) was constructed. The mice vaccinated with chimeric protein were challenged with A. baumannii ATCC19606(T) and FhaB1C1 producing recombinant E. coli. Mutations in the fhaC1 resulted in the absence of FhaB1 in the OM. Expression of FhaB1C1 enhanced the adherence of recombinant bacteria to A546 bronchial cell line. The results revealed association of FhaB1 with bacterial adhesion and biofilm formation. Immunization with a combination of recombinant B and K proteins proved protective against A. baumanni ATCC19606(T). The findings may be applied in active and passive immunization strategies against A. baumannii.

  19. 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

  20. Natural proteoglycan receptor analogs determine the dynamics of Opa adhesin-mediated gonococcal infection of Chang epithelial cells.

    PubMed Central

    van Putten, J P; Hayes, S F; Duensing, T D

    1997-01-01

    Many bacterial pathogens possess a complex machinery for the induction and/or secretion of factors that promote their uptake by mammalian cells. We searched for the molecular basis of the 60- to 90-min lag time in the interaction of Neisseria gonorrhoeae carrying the heparin-binding Opa adhesin with Chang epithelial cells. Infection assays in the presence of chloramphenicol demonstrated that the Opa-mediated gonococcal infection of Chang cells required bacterial protein synthesis when the microorganisms were derived from GC agar but not when grown in liquid media. Further analysis indicated that contact with agar ingredients rather than the growth state of the microorganisms determined the infection dynamics. DEAE chromatography of GC agar extracts and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and testing of collected fractions in infection assays identified negatively charged high-molecular-weight polysaccharides in the agar as inhibitors of the cellular infection. Electron microscopy showed that agar-grown gonococci were surrounded by a coat of alcian blue-positive material, probably representing accreted polysaccharides. Similar antiphagocytic material was isolated from bovine serum, indicating that in biological fluids gonococci producing the heparin-binding Opa adhesin may become covered with externally derived polysaccharides as well. Binding assays with gonococci and epithelial proteoglycan receptors revealed that polysaccharides derived from agar or serum compete with the proteoglycans for binding of the heparin-binding Opa adhesin and thus act as receptor analogs. Growth of gonococci in a polysaccharide-free environment resulted in optimal proteoglycan receptor binding and rapid bacterial entry into Chang cells. The recognition that gonococci with certain phenotypes can recruit surface polysaccharides that determine in vitro infection dynamics adds a different dimension to the well-recognized biological significance of genetic

  1. Cooperative role for tetraspanins in adhesin-mediated attachment of bacterial species to human epithelial cells.

    PubMed

    Green, Luke R; Monk, Peter N; Partridge, Lynda J; Morris, Paul; Gorringe, Andrew R; Read, Robert C

    2011-06-01

    The tetraspanins are a superfamily of transmembrane proteins with diverse functions and can form extended microdomains within the plasma membrane in conjunction with partner proteins, which probably includes receptors for bacterial adhesins. Neisseria meningitidis, the causative agent of meningococcal disease, attaches to host nasopharyngeal epithelial cells via type IV pili and opacity (Opa) proteins. We examined the role of tetraspanin function in Neisseria meningitidis adherence to epithelial cells. Tetraspanins CD9, CD63, and CD151 were expressed by HEC-1-B and DETROIT 562 cells. Coincubation of cells with antibodies against all three tetraspanin molecules used individually or in combination, with recombinant tetraspanin extracellular domains (EC2), or with small interfering RNAs (siRNAs) significantly reduced adherence of Neisseria meningitidis. In contrast, recombinant CD81, a different tetraspanin, had no effect on meningococcal adherence. Antitetraspanin antibodies reduced the adherence to epithelial cells of Neisseria meningitidis strain derivatives expressing Opa and pili significantly more than isogenic strains lacking these determinants. Adherence to epithelial cells of strains of Staphylococcus aureus, Neisseria lactamica, Escherichia coli, and Streptococcus pneumoniae was also reduced by pretreatment of cells with tetraspanin antibodies and recombinant proteins. These data suggest that tetraspanins are required for optimal function of epithelial adhesion platforms containing specific receptors for Neisseria meningitidis and potentially for multiple species of bacteria.

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

    PubMed

    Kern, Justin; Schneewind, Olaf

    2010-01-01

    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.

  3. High resolution studies of the Afa/Dr adhesin DraE and its interaction with chloramphenicol.

    PubMed

    Pettigrew, David; Anderson, Kirstine L; Billington, Jason; Cota, Ernesto; Simpson, Peter; Urvil, Petri; Rabuzin, Filip; Roversi, Pietro; Nowicki, Bogdan; du Merle, Laurence; Le Bouguénec, Chantal; Matthews, Stephen; Lea, Susan M

    2004-11-05

    Pathogenic Escherichia coli expressing Afa/Dr adhesins are able to cause both urinary tract and diarrheal infections. The Afa/Dr adhesins confer adherence to epithelial cells via interactions with the human complement regulating protein, decay accelerating factor (DAF or CD55). Two of the Afa/Dr adhesions, AfaE-III and DraE, differ from each other by only three residues but are reported to have several different properties. One such difference is disruption of the interaction between DraE and CD55 by chloramphenicol, whereas binding of AfaE-III to CD55 is unaffected. Here we present a crystal structure of a strand-swapped trimer of wild type DraE. We also present a crystal structure of this trimer in complex with chloramphenicol, as well as NMR data supporting the binding position of chloramphenicol within the crystal. The crystal structure reveals the precise atomic basis for the sensitivity of DraE-CD55 binding to chloramphenicol and demonstrates that in contrast to other chloramphenicol-protein complexes, drug binding is mediated via recognition of the chlorine "tail" rather than via intercalation of the benzene rings into a hydrophobic pocket.

  4. 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

  5. Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix.

    PubMed

    Borlee, Bradley R; Goldman, Aaron D; Murakami, Keiji; Samudrala, Ram; Wozniak, Daniel J; Parsek, Matthew R

    2010-02-01

    Pseudomonas aeruginosa, the principal pathogen of cystic fibrosis patients, forms antibiotic-resistant biofilms promoting chronic colonization of the airways. The extracellular (EPS) matrix is a crucial component of biofilms that provides the community multiple benefits. Recent work suggests that the secondary messenger, cyclic-di-GMP, promotes biofilm formation. An analysis of factors specifically expressed in P. aeruginosa under conditions of elevated c-di-GMP, revealed functions involved in the production and maintenance of the biofilm extracellular matrix. We have characterized one of these components, encoded by the PA4625 gene, as a putative adhesin and designated it cdrA. CdrA shares structural similarities to extracellular adhesins that belong to two-partner secretion systems. The cdrA gene is in a two gene operon that also encodes a putative outer membrane transporter, CdrB. The cdrA gene encodes a 220 KDa protein that is predicted to be rod-shaped protein harbouring a beta-helix structural motif. Western analysis indicates that the CdrA is produced as a 220 kDa proprotein and processed to 150 kDa before secretion into the extracellular medium. We demonstrated that cdrAB expression is minimal in liquid culture, but is elevated in biofilm cultures. CdrAB expression was found to promote biofilm formation and auto-aggregation in liquid culture. Aggregation mediated by CdrA is dependent on the Psl polysaccharide and can be disrupted by adding mannose, a key structural component of Psl. Immunoprecipitation of Psl present in culture supernatants resulted in co-immunoprecipitation of CdrA, providing additional evidence that CdrA directly binds to Psl. A mutation in cdrA caused a decrease in biofilm biomass and resulted in the formation of biofilms exhibiting decreased structural integrity. Psl-specific lectin staining suggests that CdrA either cross-links Psl polysaccharide polymers and/or tethers Psl to the cells, resulting in increased biofilm structural

  6. X-ray crystal structures of Staphylococcus aureus collagen adhesin and sortases

    NASA Astrophysics Data System (ADS)

    Zong, Yinong

    For many gram-positive bacteria, adhesion to host tissues is the first critical step in developing an infection. The adhesion is mediated by a superfamily of bacterial surface proteins, called MSCRAMM (microbial surface components recognizing adhesive matrix molecules), which in most cases are covalently attached to the cell wall peptidoglycan. Collagen adhesin (CNA) from Staphylococcus aureus, one of the MSCRAMMs, is responsible for bacterial binding to collagen molecules. CNA and other MSCRAMMs are anchored to the cell wall by a transpeptidase, sortase. The knowledge about how bacterial surface proteins adhere to host molecules and how they are sorted onto the cell wall is crucial for the design of novel antibiotics against bacterial infections. The crystal structures of CNA31--344 (residue 31 to 334), a truncation of CNA's collagen binding region, and CNA31--344 in complex with a collagen peptide were determined. CNA31--344 contains two domains, and between them is a big hole formed by a loop connecting the two domains. In the structure of CNA31--344-collagen complex, the collagen peptide is locked in the hole formed by the two domains of CNA 31--344. We reason that the two domains of CNA31--344 are open in the physiological condition, and close up when binding to collagen. This binding mechanism may be common for other bacterial collagen adhesins. There are two known sortases in Staphylococcus aureus. Sortase A is responsible for anchoring most MSCRAMMs that have a LPXTG (X represents any amino acid) sorting motif and sortase B for a bacterial ion acquisition protein. The crystal structures of both sortases indicate that they share a common catalytic mechanism. Unlike typical cysteine transpeptidases, sortases may use a novel Cys-Arg catalytic dyad instead of a Cys-His pair. All other sortases found in gram-positive bacteria may have similar active site architecture and employ the same catalytic dyad because the critical residues are all conserved among them

  7. 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.

  8. Use of Atomic Force Microscopy to Study the Multi-Modular Interaction of Bacterial Adhesins to Mucins

    PubMed Central

    Gunning, A. Patrick; Kavanaugh, Devon; Thursby, Elizabeth; Etzold, Sabrina; MacKenzie, Donald A.; Juge, Nathalie

    2016-01-01

    The mucus layer covering the gastrointestinal (GI) epithelium is critical in selecting and maintaining homeostatic interactions with our gut bacteria. However, the molecular details of these interactions are not well understood. Here, we provide mechanistic insights into the adhesion properties of the canonical mucus-binding protein (MUB), a large multi-repeat cell–surface adhesin found in Lactobacillus inhabiting the GI tract. We used atomic force microscopy to unravel the mechanism driving MUB-mediated adhesion to mucins. Using single-molecule force spectroscopy we showed that MUB displayed remarkable adhesive properties favouring a nanospring-like adhesion model between MUB and mucin mediated by unfolding of the multiple repeats constituting the adhesin. We obtained direct evidence for MUB self-interaction; MUB–MUB followed a similar binding pattern, confirming that MUB modular structure mediated such mechanism. This was in marked contrast with the mucin adhesion behaviour presented by Galectin-3 (Gal-3), a mammalian lectin characterised by a single carbohydrate binding domain (CRD). The binding mechanisms reported here perfectly match the particular structural organization of MUB, which maximizes interactions with the mucin glycan receptors through its long and linear multi-repeat structure, potentiating the retention of bacteria within the outer mucus layer. PMID:27834807

  9. Mycobacterium tuberculosis adhesins: potential biomarkers as anti-tuberculosis therapeutic and diagnostic targets.

    PubMed

    Govender, Viveshree S; Ramsugit, Saiyur; Pillay, Manormoney

    2014-09-01

    Adhesion to host cells is a precursor to host colonization and evasion of the host immune response. Conversely, it triggers the induction of the immune response, a process vital to the host's defence against infection. Adhesins are microbial cell surface molecules or structures that mediate the attachment of the microbe to host cells and thus the host-pathogen interaction. They also play a crucial role in bacterial aggregation and biofilm formation. In this review, we discuss the role of adhesins in the pathogenesis of the aetiological agent of tuberculosis, Mycobacterium tuberculosis. We also provide insight into the structure and characteristics of some of the characterized and putative M. tuberculosis adhesins. Finally, we examine the potential of adhesins as targets for the development of tuberculosis control strategies.

  10. Molecular cloning and characterization of the afa-7 and afa-8 gene clusters encoding afimbrial adhesins in Escherichia coli strains associated with diarrhea or septicemia in calves.

    PubMed

    Lalioui, L; Jouve, M; Gounon, P; Le Bouguenec, C

    1999-10-01

    The afa gene clusters, which encode proteins involved in adhesion to epithelial cells, from Escherichia coli strains associated with urinary and intestinal infections in humans have been characterized. Pathogenic isolates of bovine and porcine origin that possess afa-related sequences have recently been described. We report in this work the cloning and characterization of the afa-7 and afa-8 gene clusters from bovine isolates. Hybridization and sequencing experiments revealed that despite similarity in genetic organization, the afa-7 and afa-8 genes, and the well-characterized afa-3 operon expressed by human-pathogenic isolates, correspond to three different members of the afa family of gene clusters. However, like the afa-3 gene cluster, both the afa-7 and afa-8 gene clusters were found to encode an afimbrial adhesin (AfaE) and an invasin (AfaD). The AfaD peptides encoded by the three gene clusters were only 45% identical, but functional complementation experiments indicated that they belong to the same family of invasins. Hemagglutination and adhesion assays demonstrated that the AfaE-VII and AfaE-VIII adhesins bind to different receptors and that these receptors are not the human decay-accelerating factor recognized to be the receptor of all previously described AfaE adhesins. The AfaE-VIII adhesin is very similar to the M agglutinin of human-uropathogenic strains. We used PCR assays to screen 25 bovine strains for afaD and afaE genes of either the afa-7 or afa-8 gene cluster. The afa-8 gene cluster was highly prevalent in bovine isolates previously reported to carry afa-related sequences (23 of 24 strains), particularly in strains producing cytotoxic necrotizing factors (16 of 16 strains). The location of the afa-8 gene cluster on the plasmids or chromosome of these isolates suggests that it could be carried by a mobile element, facilitating its dissemination among bovine-pathogenic E. coli strains.

  11. 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

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

    PubMed

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

    1994-12-01

    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

  13. The Translocation Domain in Trimeric Autotransporter Adhesins Is Necessary and Sufficient for Trimerization and Autotransportation

    PubMed Central

    Mikula, Kornelia M.; Leo, Jack C.; Łyskowski, Andrzej; Kedracka-Krok, Sylwia; Pirog, Artur

    2012-01-01

    Trimeric autotransporter adhesins (TAAs) comprise one of the secretion pathways of the type V secretion system. The mechanism of their translocation across the outer membrane remains unclear, but it most probably occurs by the formation of a hairpin inside the β-barrel translocation unit, leading to transportation of the passenger domain from the C terminus to the N terminus through the lumen of the β-barrel. We further investigated the phenomenon of autotransportation and the rules that govern it. We showed by coexpressing different Escherichia coli immunoglobulin-binding (Eib) proteins that highly similar TAAs could form stochastically mixed structures (heterotrimers). We further investigated this phenomenon by coexpressing two more distantly related TAAs, EibA and YadA. These, however, did not form heterotrimers; indeed, coexpression was lethal to the cells, leading to elimination of one or another of the genes. However, substituting in either protein the barrel of the other one so that the barrels were identical led to formation of heterotrimers as for Eibs. Our work shows that trimerization of the β-barrel, but not the passenger domain, is necessary and sufficient for TAA secretion while the passenger domain is not. PMID:22155776

  14. Cooperation of Adhesin Alleles in Salmonella-Host Tropism

    PubMed Central

    De Masi, Leon; Yue, Min; Hu, Changmin; Rakov, Alexey V.; Rankin, Shelley C.

    2017-01-01

    ABSTRACT Allelic combinations and host specificities for three fimbrial adhesins, FimH, BcfD, and StfH, were compared for 262 strains of Salmonella enterica serovar Newport, a frequent human and livestock pathogen. Like FimH, BcfD had two major alleles (designated A and B), whereas StfH had two allelic groups, each with two alleles (subgroup A1 and A2 and subgroup B1 and B2). The most prevalent combinations of FimH/BcfD/StfH alleles in S. Newport were A/A/A1 and B/B/B1. The former set was most frequently found in bovine and porcine strains, whereas the latter combination was most frequently found in environmental and human isolates. Bacteria genetically engineered to express Fim, Bcf, or Stf fimbriae on their surface were tested with the different alleles for binding to human, porcine, and bovine intestinal epithelial cells. The major allelic combinations with bovine and porcine strains (A/A/A1) or with human isolates (B/B/B1) provided at least two alleles capable of binding significantly better than the other alleles to an intestinal epithelial cell line from the respective host(s). However, each combination of alleles kept at least one allele mediating binding to an intestinal epithelial cell from another host. These findings indicated that allelic variation in multiple adhesins of S. Newport contributes to bacterial adaptation to certain preferential hosts without losing the capacity to maintain a broad host range. IMPORTANCE Salmonella enterica remains a leading foodborne bacterial pathogen in the United States; infected livestock serve often as the source of contaminated food products. A study estimated that over a billion Salmonella gastroenteritis cases and up to 33 million typhoid cases occur annually worldwide, with 3.5 million deaths. Although many Salmonella strains with a broad host range present preferential associations with certain host species, it is not clear what determines the various levels of host adaptation. Here, causal properties of host

  15. Catch-bond mechanism of the bacterial adhesin FimH.

    PubMed

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

    2016-03-07

    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.

  16. 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.

  17. 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

  18. XacFhaB adhesin, an important Xanthomonas citri ssp. citri virulence factor, is recognized as a pathogen-associated molecular pattern.

    PubMed

    Garavaglia, Betiana S; Zimaro, Tamara; Abriata, Luciano A; Ottado, Jorgelina; Gottig, Natalia

    2016-12-01

    Adhesion to host tissue is one of the key steps of the bacterial pathogenic process. Xanthomonas citri ssp. citri possesses a non-fimbrial adhesin protein, XacFhaB, required for bacterial attachment, which we have previously demonstrated to be an important virulence factor for the development of citrus canker. XacFhaB is a 4753-residue-long protein with a predicted β-helical fold structure, involved in bacterial aggregation, biofilm formation and adhesion to the host. In this work, to further characterize this protein and considering its large size, XacFhaB was dissected into three regions based on bioinformatic and structural analyses for functional studies. First, the capacity of these protein regions to aggregate bacterial cells was analysed. Two of these regions were able to form bacterial aggregates, with the most amino-terminal region being dispensable for this activity. Moreover, XacFhaB shows features resembling pathogen-associated molecular patterns (PAMPs), which are recognized by plants. As PAMPs activate plant basal immune responses, the role of the three XacFhaB regions as elicitors of these responses was investigated. All adhesin regions were able to induce basal immune responses in host and non-host plants, with a stronger activation by the carboxyl-terminal region. Furthermore, pre-infiltration of citrus leaves with XacFhaB regions impaired X. citri ssp. citri growth, confirming the induction of defence responses and restraint of citrus canker. This work reveals that adhesins from plant pathogens trigger plant defence responses, opening up new pathways for the development of protective strategies for disease control.

  19. 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

  20. Sticky Matrix: Adhesion Mechanism of the Staphylococcal Polysaccharide Intercellular Adhesin.

    PubMed

    Formosa-Dague, Cécile; Feuillie, Cécile; Beaussart, Audrey; Derclaye, Sylvie; Kucharíková, Soňa; Lasa, Iñigo; Van Dijck, Patrick; Dufrêne, Yves F

    2016-03-22

    The development of bacterial biofilms on surfaces leads to hospital-acquired infections that are difficult to fight. In Staphylococci, the cationic polysaccharide intercellular adhesin (PIA) forms an extracellular matrix that connects the cells together during biofilm formation, but the molecular forces involved are unknown. Here, we use advanced force nanoscopy techniques to unravel the mechanism of PIA-mediated adhesion in a clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) strain. Nanoscale multiparametric imaging of the structure, adhesion, and elasticity of bacteria expressing PIA shows that the cells are surrounded by a soft and adhesive matrix of extracellular polymers. Cell surface softness and adhesion are dramatically reduced in mutant cells deficient for the synthesis of PIA or under unfavorable growth conditions. Single-cell force spectroscopy demonstrates that PIA promotes cell-cell adhesion via the multivalent electrostatic interaction with polyanionic teichoic acids on the S. aureus cell surface. This binding mechanism rationalizes, at the nanoscale, the well-known ability of PIA to strengthen intercellular adhesion in staphylococcal biofilms. Force nanoscopy offers promising prospects for understanding the fundamental forces in antibiotic-resistant biofilms and for designing anti-adhesion compounds targeting matrix polymers.

  1. Transcriptional Auto-Regulation of RUNX1 P1 Promoter.

    PubMed

    Martinez, Milka; Hinojosa, Marcela; Trombly, Daniel; Morin, Violeta; Stein, Janet; Stein, Gary; Javed, Amjad; Gutierrez, Soraya E

    2016-01-01

    RUNX1 a member of the family of runt related transcription factors (RUNX), is essential for hematopoiesis. The expression of RUNX1 gene is controlled by two promoters; the distal P1 promoter and the proximal P2 promoter. Several isoforms of RUNX1 mRNA are generated through the use of both promoters and alternative splicing. These isoforms not only differs in their temporal expression pattern but also exhibit differences in tissue specificity. The RUNX1 isoforms derived from P2 are expressed in a variety of tissues, but expression of P1-derived isoform is restricted to cells of hematopoietic lineage. However, the control of hematopoietic-cell specific expression is poorly understood. Here we report regulation of P1-derived RUNX1 mRNA by RUNX1 protein. In silico analysis of P1 promoter revealed presence of two evolutionary conserved RUNX motifs, 0.6kb upstream of the transcription start site, and three RUNX motifs within 170bp of the 5'UTR. Transcriptional contribution of these RUNX motifs was studied in myeloid and T-cells. RUNX1 genomic fragment containing all sites show very low basal activity in both cell types. Mutation or deletion of RUNX motifs in the UTR enhances basal activity of the RUNX1 promoter. Chromatin immunoprecipitation revealed that RUNX1 protein is recruited to these sites. Overexpression of RUNX1 in non-hematopoietic cells results in a dose dependent activation of the RUNX1 P1 promoter. We also demonstrate that RUNX1 protein regulates transcription of endogenous RUNX1 mRNA in T-cell. Finally we show that SCL transcription factor is recruited to regions containing RUNX motifs in the promoter and the UTR and regulates activity of the RUNX1 P1 promoter in vitro. Thus, multiple lines of evidence show that RUNX1 protein regulates its own gene transcription.

  2. 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.

  3. Serine-Rich Repeat Adhesins Contribute to Streptococcus gordonii-Induced Maturation of Human Dendritic Cells

    PubMed Central

    Ko, Eun Byeol; Kim, Sun Kyung; Seo, Ho Seong; Yun, Cheol-Heui; Han, Seung Hyun

    2017-01-01

    Dendritic cells (DCs) play a pivotal role in the induction of immunity by recognition, capture, process, and presentation of antigens from infectious microbes. Streptococcus gordonii is able to cause life-threatening systemic diseases such as infective endocarditis. Serine-rich repeat (SRR) glycoproteins of S. gordonii are sialic acid-binding adhesins mediating the bacterial adherence to the host and the development of infective endocarditis. Thus, the SRR adhesins are potentially involved in the bacterial adherence to DCs and the maturation and activation of DCs required for the induction of immunity to S. gordonii. Here, we investigated the phenotypic and functional changes of human monocyte-derived DCs treated with wild-type S. gordonii or the SRR adhesin-deficient mutant. The mutant poorly bound to DCs and only weakly increased the expression of CD83, CD86, MHC class II, and PD-L1 on DCs compared with the wild-type. In addition, the mutant induced lower levels of TNF-α, IL-6, and IL-12 than the wild-type in DCs. When DCs sensitized with the mutant were co-cultured with autologous T cells, they induced weaker proliferation and activation of T cells than DCs stimulated with the wild-type. Blockade of SRR adhesin with 3′-sialyllactose markedly reduced S. gordonii binding and internalization, causing attenuation of the bacterial immunostimulatory potency in DC maturation. Collectively, our results suggest that SRR adhesins of S. gordonii are important for maturation and activation of DCs.

  4. Dra/AfaE adhesin of uropathogenic Dr/Afa+ Escherichia coli mediates mortality in pregnant rats.

    PubMed

    Wroblewska-Seniuk, K; Selvarangan, R; Hart, A; Pladzyk, R; Goluszko, P; Jafari, A; du Merle, L; Nowicki, S; Yallampalli, C; Le Bouguénec, C; Nowicki, B

    2005-11-01

    Escherichia coli bearing adhesins of the Dr/Afa family frequently causes urogenital infections during pregnancy in humans and has been associated with mortality in pregnant rats. Two components of the adhesin, Dra/AfaE and Dra/AfaD, considered virulence factors, are responsible for bacterial binding and internalization. We hypothesize that gestational mortality caused by Dr/Afa+ E. coli is mediated by one of these two proteins, Dra/AfaE or Dra/AfaD. In this study, using afaE and/or afaD mutants, we investigated the role of the afaE and afaD genes in the mortality of pregnant rats from intrauterine infection. Sprague-Dawley rats, on the 17th day of pregnancy, were infected with the E. coli afaE+ afaD and afaE afaD+ mutants. The clinical E. coli strain (afaE+ afaD+) and the afaE afaD double mutant were used as positive and negative controls, respectively. The mortality rate was evaluated 24 h after infection. The highest maternal mortality was observed in the group infected with the afaE+ afaD+ strain, followed by the group infected with the afaE+ afaD strain. The mortality was dose dependent. The afaE afaD double mutant did not cause maternal mortality, even with the highest infection dose. The in vivo studies corresponded with the invasion assay, where the afaE+ strains were the most invasive (afaE+ afaD strain > afaE+ afaD+ strain), while the afaE mutant strains (afaE afaD+ and afaE afaD strains) seemed to be noninvasive. This study shows for the first time that the afaE gene coding for the AfaE subunit of Dr/Afa adhesin is involved in the lethal outcome of gestational infection in rats. This lethal effect associated with AfaE correlates with the invasiveness of afaE+ E. coli strains in vitro.

  5. 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.

  6. Dra/AfaE Adhesin of Uropathogenic Dr/Afa+ Escherichia coli Mediates Mortality in Pregnant Rats

    PubMed Central

    Wroblewska-Seniuk, K.; Selvarangan, R.; Hart, A.; Pladzyk, R.; Goluszko, P.; Jafari, A.; du Merle, L.; Nowicki, S.; Yallampalli, C.; Le Bouguénec, C.; Nowicki, B.

    2005-01-01

    Escherichia coli bearing adhesins of the Dr/Afa family frequently causes urogenital infections during pregnancy in humans and has been associated with mortality in pregnant rats. Two components of the adhesin, Dra/AfaE and Dra/AfaD, considered virulence factors, are responsible for bacterial binding and internalization. We hypothesize that gestational mortality caused by Dr/Afa+ E. coli is mediated by one of these two proteins, Dra/AfaE or Dra/AfaD. In this study, using afaE and/or afaD mutants, we investigated the role of the afaE and afaD genes in the mortality of pregnant rats from intrauterine infection. Sprague-Dawley rats, on the 17th day of pregnancy, were infected with the E. coli afaE+ afaD and afaE afaD+ mutants. The clinical E. coli strain (afaE+ afaD+) and the afaE afaD double mutant were used as positive and negative controls, respectively. The mortality rate was evaluated 24 h after infection. The highest maternal mortality was observed in the group infected with the afaE+ afaD+ strain, followed by the group infected with the afaE+ afaD strain. The mortality was dose dependent. The afaE afaD double mutant did not cause maternal mortality, even with the highest infection dose. The in vivo studies corresponded with the invasion assay, where the afaE+ strains were the most invasive (afaE+ afaD strain > afaE+ afaD+ strain), while the afaE mutant strains (afaE afaD+ and afaE afaD strains) seemed to be noninvasive. This study shows for the first time that the afaE gene coding for the AfaE subunit of Dr/Afa adhesin is involved in the lethal outcome of gestational infection in rats. This lethal effect associated with AfaE correlates with the invasiveness of afaE+ E. coli strains in vitro. PMID:16239563

  7. Effects of orally applied Fes p1-displaying L. plantarum WCFS1 on Fes p1 induced allergy in mice.

    PubMed

    Minic, Rajna; Gavrovic-Jankulovic, Marija; Petrusic, Vladimir; Zivkovic, Irena; Eijsink, Vincent G H; Dimitrijevic, Ljiljana; Mathiesen, Geir

    2015-04-10

    Group I grass pollen allergens are major contributors to grass pollen-related seasonal allergic rhinitis, and as such a primary target for allergen specific immunotherapy. In this study the potential therapeutic role of oral application of Lactobacillus plantarum WCFS1, directing cell wall attachment of the recombinant Fes p1 allergen, from Festuca pratensis was tested in a mouse model of Fes p1 allergy. For surface expression of Fes p1 allergen in L. plantarum WCFS1 pSIP system with inducible expression was used. Balb/c mice were sensitized with Fes p1 protein in alum and subsequently received live recombinant L. plantarum orally. Antibody levels (IgE, total IgG, IgG1, IgG2a, and IgA) were determined by ELISA. Differential eosinophil count in peripheral blood was performed. Reduced peripheral blood eosinophilia and increased serum IgG2A levels was detected in both groups which received live L. plantarum orally. Specific serum IgA levels were increased only in mice treated with the recombinant bacteria. Oral application of L. plantarum WCFS1 has a beneficial therapeutic effect in a mouse model of Fes p1 allergy. Cell surface expression of Fes p1 allergen potentiates this phenomenon in an allergen specific way.

  8. 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

    2016-01-04

    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.

  9. Receptor affinity purification of a lipid-binding adhesin from Helicobacter pylori.

    PubMed Central

    Lingwood, C A; Wasfy, G; Han, H; Huesca, M

    1993-01-01

    Our previous work has shown that Helicobacter pylori specifically recognizes gangliotetraosylceramide, gangliotriaosylceramide, and phosphatidylethanolamine in vitro. This binding specificity is shared by exoenzyme S from Pseudomonas aeruginosa, and monoclonal antibodies against this adhesin prevent the attachment of H. pylori to its lipid receptors. We now report the use of a novel, versatile affinity matrix to purify a 63-kDa exoenzyme S-like adhesin from H. pylori which is responsible for the lipid-binding specificity of this organism. Images PMID:8500882

  10. Sphingosine 1-phosphate receptor 1 (S1P(1)) upregulation and amelioration of experimental autoimmune encephalomyelitis by an S1P(1) antagonist.

    PubMed

    Cahalan, Stuart M; Gonzalez-Cabrera, Pedro J; Nguyen, Nhan; Guerrero, Miguel; Cisar, Elizabeth A George; Leaf, Nora B; Brown, Steven J; Roberts, Edward; Rosen, Hugh

    2013-02-01

    Sphingosine 1-phosphate receptor 1 (S1P(1)) is a G protein-coupled receptor that is critical for proper lymphocyte development and recirculation. Agonists to S1P(1) are currently in use clinically for the treatment of multiple sclerosis, and these drugs may act on both S1P(1) expressed on lymphocytes and S1P(1) expressed within the central nervous system. Agonists to S1P(1) and deficiency in S1P(1) both cause lymphocyte sequestration in the lymph nodes. In the present study, we show that S1P(1) antagonism induces lymphocyte sequestration in the lymph nodes similar to that observed with S1P(1) agonists while upregulating S1P(1) on lymphocytes and endothelial cells. Additionally, we show that S1P(1) antagonism reverses experimental autoimmune encephalomyelitis in mice without acting on S1P(1) expressed within the central nervous system, demonstrating that lymphocyte sequestration via S1P(1) antagonism is sufficient to alleviate autoimmune pathology.

  11. Detection of pap, sfa and afa adhesin-encoding operons in uropathogenic Escherichia coli strains: relationship with expression of adhesins and production of toxins.

    PubMed

    Blanco, M; Blanco, J E; Alonso, M P; Mora, A; Balsalobre, C; Muñoa, F; Juárez, A; Blanco, J

    1997-12-01

    A total of 243 Escherichia coli strains isolated from patients with urinary tract infections (UTI) were investigated for the presence of pap, sfa and afa adhesin-encoding operons by using the polymerase chain reaction. It was found that 54%, 53% and 2% of the strains exhibited the pap, sfa and afa genotypes, respectively. Pap+ and/or sfa+ strains were more frequent in cases of acute pyelonephritis (94%) than in cases of cystitis (67%) (P < 0.001) and asymptomatic bacteriuria (57%) (P < 0.001). The pap and/or sfa operons were found in 90% of strains expressing mannose-resistant haemagglutination (MRHA) versus 37% of MRHA-negative strains (P < 0.001). The presence of pap and sfa operons was especially significant in strains belonging to MRHA types III (100%) (without P adhesins) and IVa (97%) (expressing the specific Gal-Gal binding typical of P adhesins). Both pap and sfa operons were closely associated with toxigenic E. coli producing alpha-haemolysin (Hly+) and/or the cytotoxic necrotizing factor type 1. There was an apparent correlation between the pap and sfa operons and the O serogroups of the strains. Thus, 93% of strains belonging to O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 and O83 possessed pap and/or sfa operons, versus only 32% of strains belonging to other serogroups (P < 0.001). The results obtained in this study confirm the usefulness of our MRHA typing system for presumptive identification of pathogenic E. coli exhibiting different virulence factors. Thus, 85% of strains that possessed both pap and sfa adhesin-encoding operons showed MRHA types III or IVa previously associated with virulence of E. coli strains that cause UTI and bacteraemia.

  12. Intervention with polyphenol-rich fruit juices results in an elevation of glutathione S-transferase P1 (hGSTP1) protein expression in human leucocytes of healthy volunteers.

    PubMed

    Hofmann, Thomas; Liegibel, Ute; Winterhalter, Peter; Bub, Achim; Rechkemmer, Gerhard; Pool-Zobel, Beatrice Louise

    2006-12-01

    Polyphenols are probably antigenotoxic on account of their antioxidant activities and might alter phase I and II enzymes in a way that results in chemoprotection. We investigated the hypothesis that polyphenols enhance expression of glutathione S-transferases (GSTs), which increases carcinogen detoxification and thereby provides protection against oxidative stress. HGSTP1 protein expression and GST polymorphisms were determined in leucocytes obtained during an intervention study with healthy subjects consuming two fruit juices in an 8 wk trial (polyphenol-free run in phase, juice intervention phase, washout phase, second juice intervention phase, each treatment regime lasted for 2 wk). The study had originally shown that juice intervention significantly reduced oxidative DNA damage in leucocytes at week 8 (Bub, A., Watzl, B., Blockhaus, M., Briviba, K. et al., J. Nutr. Biochem. 2003, 14, 90-98). We reanalysed the levels of DNA damage based on GST genotypes. We also treated leucocytes in vitro with mixtures of polyphenols and determined cytotoxicity and expression of 96 genes related to drug metabolism. Key results with leucocytes of the intervention study were that the initial content of hGSTP1 protein was first suppressed at weeks 4 and 6. At week 8, however, hGSTP1 protein expression was significantly increased. HGSTP1 protein levels and DNA damage were inversely correlated (p = 0.005), but there was no difference for cells obtained from subjects with hGSTM1*1 and hGSTM1*0 genotypes, nor was there any difference between cells from subjects consuming the two different juices. The treatment of leucocytes with polyphenol mixtures in vitro did not result in modulated GST gene expression or total GST activity, but in an up-regulation of other biotransformation enzymes (e. g., members of the cytochrome P450 and the sulphotransferase family). In conclusion, in vitro treatment of leucocytes led to a modulated mRNA expression of selected genes, not directly related to

  13. P-1 truss arrival at KSC

    NASA Technical Reports Server (NTRS)

    2000-01-01

    The P-1 truss, a component of the International Space Station, is moved from the Shuttle Landing Facility toward the newly constructed RLV hangar (viewed here from inside the hangar) as precaution against bad weather approaching the Center (background). The truss will eventually be transferred to the Operations and Checkout Building for processing. In the background is the Super Guppy transport that brought it to KSC. The P-1 truss, scheduled to fly in spring of 2002, is part of a total 10-truss, girder-like structure on the Station that will ultimately extend the length of a football field. Astronauts will attach the 14-by-15 foot structure to the port side of the center truss, S0, during the spring assembly flight. The 33,000-pound P- 1 will house the thermal radiator rotating joint (TRRJ) that will rotate the Station's radiators away from the sun to increase their maximum cooling efficiency.

  14. P-1 truss arrival at KSC

    NASA Technical Reports Server (NTRS)

    2000-01-01

    The P-1 truss, a component of the International Space Station, arrives inside the RLV hangar, located near the Shuttle Landing Facility at KSC. Approaching bad weather caused the detour as a precaution. The truss will eventually be transferred to the Operations and Checkout Building for processing. The P-1 truss, scheduled to fly in spring of 2002, is part of a total 10-truss, girder-like structure on the Station that will ultimately extend the length of a football field. Astronauts will attach the 14-by- 15 foot structure to the port side of the center truss, S0, during the spring assembly flight. The 33,000-pound P-1 will house the thermal radiator rotating joint (TRRJ) that will rotate the Station's radiators away from the sun to increase their maximum cooling efficiency.

  15. 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

  16. Cooperative Role for Tetraspanins in Adhesin-Mediated Attachment of Bacterial Species to Human Epithelial Cells ▿ †

    PubMed Central

    Green, Luke R.; Monk, Peter N.; Partridge, Lynda J.; Morris, Paul; Gorringe, Andrew R.; Read, Robert C.

    2011-01-01

    The tetraspanins are a superfamily of transmembrane proteins with diverse functions and can form extended microdomains within the plasma membrane in conjunction with partner proteins, which probably includes receptors for bacterial adhesins. Neisseria meningitidis, the causative agent of meningococcal disease, attaches to host nasopharyngeal epithelial cells via type IV pili and opacity (Opa) proteins. We examined the role of tetraspanin function in Neisseria meningitidis adherence to epithelial cells. Tetraspanins CD9, CD63, and CD151 were expressed by HEC-1-B and DETROIT 562 cells. Coincubation of cells with antibodies against all three tetraspanin molecules used individually or in combination, with recombinant tetraspanin extracellular domains (EC2), or with small interfering RNAs (siRNAs) significantly reduced adherence of Neisseria meningitidis. In contrast, recombinant CD81, a different tetraspanin, had no effect on meningococcal adherence. Antitetraspanin antibodies reduced the adherence to epithelial cells of Neisseria meningitidis strain derivatives expressing Opa and pili significantly more than isogenic strains lacking these determinants. Adherence to epithelial cells of strains of Staphylococcus aureus, Neisseria lactamica, Escherichia coli, and Streptococcus pneumoniae was also reduced by pretreatment of cells with tetraspanin antibodies and recombinant proteins. These data suggest that tetraspanins are required for optimal function of epithelial adhesion platforms containing specific receptors for Neisseria meningitidis and potentially for multiple species of bacteria. PMID:21464080

  17. 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

  18. Leptospira surface adhesin (Lsa21) induces Toll like receptor 2 and 4 mediated inflammatory responses in macrophages

    PubMed Central

    Faisal, Syed M.; Varma, Vivek P.; Subathra, M.; Azam, Sarwar; Sunkara, Anil K.; Akif, Mohd; Baig, Mirza. S.; Chang, Yung-Fu

    2016-01-01

    Leptospirosis is zoonotic and emerging infectious disease of global importance. Little is understood about Leptospira pathogenesis and host immune response. In the present work we have investigated how Leptospira modulates the host innate immune response mediated by Toll-like receptors (TLRs) via surface exposed proteins. We screened Leptospira outer membrane/surface proteins for their ability to activate/inhibit TLR2/4 signaling in HEK293 cell lines. Of these the 21 kDa Leptospira surface adhesin, Lsa21 had strong TLR2 and TLR4 activity leading to production of proinflammatory cytokines and expression of costimulatory molecules in mouse macrophages. This activity of Lsa21 on innate response was dependent on activation of mitogen activated protein kinases (MAPKs) via stimulating the rapid phosphorylation of p38, JNK and activation of transcription factor NF-κB. Additionally, neutralizing antibodies against TLR2 and TLR4 significantly inhibited cytokine secretion and attenuated Lsa21 induced phosphorylation of p38 and JNK. Furthermore, Lsa21 induced cytokine levels were significantly lower in TLR2−/− and TLR4−/− than in wild type mouse macrophage cell lines. Confocal microscopy and molecular docking confirmed that Lsa21 interacted with both TLR2 and TLR4. These results indicate that Lsa21 is a potent TLR2 and TLR4 agonist that induces strong innate response and may play important role in Leptospira pathogenesis. PMID:27996041

  19. 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.

  20. 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

  1. Role of Ca²⁺ in folding the tandem β-sandwich extender domains of a bacterial ice-binding adhesin.

    PubMed

    Guo, Shuaiqi; Garnham, Christopher P; Karunan Partha, Sarathy; Campbell, Robert L; Allingham, John S; Davies, Peter L

    2013-11-01

    A Ca(2+) -dependent 1.5-MDa antifreeze protein present in an Antarctic Gram-negative bacterium, Marinomonas primoryensis (MpAFP), has recently been reassessed as an ice-binding adhesin. The non-ice-binding region II (RII), one of five distinct domains in MpAFP, constitutes ~ 90% of the protein. RII consists of ~ 120 tandem copies of an identical 104-residue sequence. We used the Protein Homology/analogy Recognition Engine server to define the boundaries of a single 104-residue RII construct (RII monomer). CD demonstrated that Ca(2+) is required for RII monomer folding, and that the monomer is fully structured at a Ca(2+) /protein molar ratio of 10 : 1. The crystal structure of the RII monomer was solved to a resolution of 1.35 Å by single-wavelength anomalous dispersion and molecular replacement methods with Ca(2+) as the heavy atom to obtain phase information. The RII monomer folds as a Ca(2+) -bound immunoglobulin-like β-sandwich. Ca(2+) ions are coordinated at the interfaces between each RII monomer and its symmetry-related molecules, suggesting that these ions may be involved in the stabilization of the tandemly repeated RII. We hypothesize that > 600 Ca(2+) ions help to rigidify the chain of 104-residue repeats in order to project the ice-binding domain of MpAFP away from the bacterial cell surface. The proposed role of RII is to help the strictly aerobic bacterium bind surface ice in an Antarctic lake for better access to oxygen and nutrients. This work may give insights into other bacterial proteins that resemble MpAFP, especially those of the large repeats-in-toxin family that have been characterized as adhesins exported via the type I secretion pathway.

  2. Intramolecular isopeptide but not internal thioester bonds confer proteolytic and significant thermal stability to the S. pyogenes pilus adhesin Spy0125.

    PubMed

    Walden, Miriam; Crow, Allister; Nelson, Miles D; Banfield, Mark J

    2014-03-01

    Streptococcus pyogenes and other Gram-positive bacterial pathogens present long macromolecular filaments known as pili on their surface that mediate adhesion and colonization. These pili are covalent polymers, assembled by sortases. Typically, they comprise a putative adhesin at their tip, a backbone subunit present in multiple copies and a basal subunit that is covalently anchored to the peptidoglycan layer of the cell surface. The crystal structures of pilin subunits revealed the presence of unusual covalent linkages in these proteins, including intramolecular isopeptide and internal thioester bonds. The intramolecular isopeptide bonds in backbone pilins are important for protein stability. Here, using both the wild-type protein and a set of mutants, we assessed the proteolytic and thermal stability of the S. pyogenes pilus tip adhesin Spy0125, in the presence and absence of its intramolecular isopeptide and internal thioester bonds. We also determined a crystal structure of the internal thioester bond variant Spy0125(Cys426Ala). We find that mutations in the intramolecular isopeptide bonds compromise the stability of Spy0125. Using limited proteolysis and thermal denaturation assays, we could separate the contribution of each intramolecular isopeptide bond to Spy0125 stability. In contrast, mutation in the internal thioester bond had a lesser effect on protein stability and the crystal structure is essentially identical to wild type. This work suggests that the internal thioester in Spy0125, although having a minor contributory role, is not required for protein stability and must have a different primary function, most likely mediating a covalent interaction with host cell ligands.

  3. The EPA2 adhesin encoding gene is responsive to oxidative stress in the opportunistic fungal pathogen Candida glabrata.

    PubMed

    Juárez-Cepeda, Jacqueline; Orta-Zavalza, Emmanuel; Cañas-Villamar, Israel; Arreola-Gómez, Jorge; Pérez-Cornejo, Gloria Patricia; Hernández-Carballo, Carmen Yudith; Gutiérrez-Escobedo, Guadalupe; Castaño, Irene; De Las Peñas, Alejandro

    2015-11-01

    Candida glabrata has emerged as an important opportunistic pathogen in both mucosal and bloodstream infections. C. glabrata contains 67 adhesin-like glycosylphosphatidylinositol-cell-wall proteins (GPI-CWPs), which are classified into seven groups and the largest is the Epa family. Epa proteins are very diverse and their expression is differentially regulated. Like many of the EPA genes, EPA2 is localized in a subtelomeric region where it is subject to chromatin-based transcriptional silencing and its role remains largely unexplored. In this study, we show that EPA2 gene is induced specifically in vitro in the presence of oxidative stress generated by H2O2. This induction is dependent on both Yap1 and Skn7, whereas Msn4 represses EPA2 expression. Interestingly, EPA2 is not induced during phagocytosis, but its expression can be identified in the liver in a murine model of systemic infection. Epa2 has no effect on the virulence of C. glabrata. The work presented herein provides a foundation for future studies to dissect the molecular mechanism(s) by which EPA2 of C. glabrata can be induced in the presence of oxidative stress in a region subject to subtelomeric silencing.

  4. 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.

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

    PubMed Central

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

    1998-01-01

    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. PMID:9637697

  6. 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

  7. 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.

  8. Programming controlled adhesion of E. coli to target surfaces, cells, and tumors with synthetic adhesins.

    PubMed

    Piñero-Lambea, Carlos; Bodelón, Gustavo; Fernández-Periáñez, Rodrigo; Cuesta, Angel M; Álvarez-Vallina, Luis; Fernández, Luis Ángel

    2015-04-17

    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.

  9. 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.

  10. 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.

  11. Identification of amino acids in the Dr adhesin required for binding to decay-accelerating factor.

    PubMed

    Van Loy, Cristina P; Sokurenko, Evgeni V; Samudrala, Ram; Moseley, Steve L

    2002-07-01

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

  12. Plectin isoform P1b and P1d deficiencies differentially affect mitochondrial morphology and function in skeletal muscle.

    PubMed

    Winter, Lilli; Kuznetsov, Andrey V; Grimm, Michael; Zeöld, Anikó; Fischer, Irmgard; Wiche, Gerhard

    2015-08-15

    Plectin, a versatile 500-kDa cytolinker protein, is essential for muscle fiber integrity and function. The most common disease caused by mutations in the human plectin gene, epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is characterized by severe skin blistering and progressive muscular dystrophy. Besides displaying pathological desmin-positive protein aggregates and degenerative changes in the myofibrillar apparatus, skeletal muscle specimens of EBS-MD patients and plectin-deficient mice are characterized by massive mitochondrial alterations. In this study, we demonstrate that structural and functional alterations of mitochondria are a primary aftermath of plectin deficiency in muscle, contributing to myofiber degeneration. We found that in skeletal muscle of conditional plectin knockout mice (MCK-Cre/cKO), mitochondrial content was reduced, and mitochondria were aggregated in sarcoplasmic and subsarcolemmal regions and were no longer associated with Z-disks. Additionally, decreased mitochondrial citrate synthase activity, respiratory function and altered adenosine diphosphate kinetics were characteristic of plectin-deficient muscles. To analyze a mechanistic link between plectin deficiency and mitochondrial alterations, we comparatively assessed mitochondrial morphology and function in whole muscle and teased muscle fibers of wild-type, MCK-Cre/cKO and plectin isoform-specific knockout mice that were lacking just one isoform (either P1b or P1d) while expressing all others. Monitoring morphological alterations of mitochondria, an isoform P1b-specific phenotype affecting the mitochondrial fusion-fission machinery and manifesting with upregulated mitochondrial fusion-associated protein mitofusin-2 could be identified. Our results show that the depletion of distinct plectin isoforms affects mitochondrial network organization and function in different ways.

  13. Characterization of Three Novel Adhesins of Leptospira interrogans

    PubMed Central

    Siqueira, Gabriela H.; Atzingen, Marina V.; Alves, Ivy J.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Nascimento, Ana L. T. O.

    2013-01-01

    We report cloning, expression, purification, and characterization of three predicted leptospiral membrane proteins (LIC11360, LIC11009, and LIC11975). In silico analysis and proteinase K accessibility data suggest that these proteins might be surface exposed. We show that proteins encoded by LIC11360, LIC11009 and LIC11975 genes interact with laminin in a dose-dependent and saturable manner. The proteins are referred to as leptospiral surface adhesions 23, 26, and 36 (Lsa23, Lsa26, and Lsa36), respectively. These proteins also bind plasminogen and generate active plasmin. Attachment of Lsa23 and Lsa36 to fibronectin occurs through the involvement of the 30-kDa and 70-kDa heparin-binding domains of the ligand. Dose-dependent, specific-binding of Lsa23 to the complement regulator C4BP and to a lesser extent, to factor H, suggests that this protein may interfere with the complement cascade pathways. Leptospira spp. may use these interactions as possible mechanisms during the establishment of infection. PMID:23958908

  14. 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

  15. 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.

  16. Characterization and functional analysis of AatB, a novel autotransporter adhesin and virulence factor of avian pathogenic Escherichia coli.

    PubMed

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

    2013-07-01

    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.

  17. 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

  18. Onion yellow phytoplasma P38 protein plays a role in adhesion to the hosts.

    PubMed

    Neriya, Yutaro; Maejima, Kensaku; Nijo, Takamichi; Tomomitsu, Tatsuya; Yusa, Akira; Himeno, Misako; Netsu, Osamu; Hamamoto, Hiroshi; Oshima, Kenro; Namba, Shigetou

    2014-12-01

    Adhesins are microbial surface proteins that mediate the adherence of microbial pathogens to host cell surfaces. In Mollicutes, several adhesins have been reported in mycoplasmas and spiroplasmas. Adhesins P40 of Mycoplasma agalactiae and P89 of Spiroplasma citri contain a conserved amino acid sequence known as the Mollicutes adhesin motif (MAM), whose function in the host cell adhesion remains unclear. Here, we show that phytoplasmas, which are plant-pathogenic mollicutes transmitted by insect vectors, possess an adhesion-containing MAM that was identified in a putative membrane protein, PAM289 (P38), of the 'Candidatus Phytoplasma asteris,' OY strain. P38 homologs and their MAMs were highly conserved in related phytoplasma strains. While P38 protein was expressed in OY-infected insect and plant hosts, binding assays showed that P38 interacts with insect extract, and weakly with plant extract. Interestingly, the interaction of P38 with the insect extract depended on MAM. These results suggest that P38 is a phytoplasma adhesin that interacts with the hosts. In addition, the MAM of adhesins is important for the interaction between P38 protein and hosts.

  19. Expression of human protamine P1 in sperm of transgenic mice

    SciTech Connect

    Wyrobek, A.J.; Keith, C.; Stilwell, J.; Lowe, X.; Anderson, G.

    1994-12-31

    Transgenic mice were produced by pronuclear injection with DNA constructs containing human protamine P1 cDNA recombined with a murine protamine P1 promoter, and were identified by PCR. Expression of human P1 was investigated using huplm, a monoclonal antibody specific for human P1, applied to murine testicular cells, smears of epididymal sperm, and smears of detergent-isolated sperm nuclei. Various antibodies and nontransgenic littermates were used as controls. Two male founders (T3 and T7) sired more than five generations of transgenic offspring each with continued expression of human P1 in their sperm. Transgenic animals appear of normal fertility with sperm of typical nuclear morphology. The human P1 transgene was expressed postmeioticly in both lines, as expected. Nearly 100% of sperm of T3 and T7 hemizygotes labeled with huplm, consistent with complete diffusion of human P1 protein through the intercellular bridge of spermatogenic cells. Human P1 labeling of sperm nuclei was not visibly affected by sonication or by treatment with the detergent MATAB or the reducing agent DTT. A third founder female (T5) showed a transmission pattern consistent with insertion of the transgene into an X chromosome; her transgenic offspring expressed human P1 in only a small fraction of sperm. Human P1 transgenes may serve as efficient targets for germinal mutations and transgenicmice may provide promising models for investigating the DNA complexes.

  20. Identification and characterization of a novel Fusobacterium nucleatum adhesin involved in physical interaction and biofilm formation with Streptococcus gordonii.

    PubMed

    Lima, Bruno P; Shi, Wenyuan; Lux, Renate

    2017-02-07

    To successfully colonize the oral cavity, bacteria must directly or indirectly adhere to available oral surfaces. Fusobacterium nucleatum plays an important role in oral biofilm community development due to its broad adherence abilities, serving as a bridge between members of the oral biofilm that cannot directly bind to each other. In our efforts to characterize the molecular mechanisms utilized by F. nucleatum to physically bind to key members of the oral community, we investigated the involvement of F. nucleatum outer membrane proteins in its ability to bind to the pioneer biofilm colonizer, Streptococcus gordonii. Here, we present evidence that in addition to the previously characterized fusobacterial adhesin RadD, the interaction between F. nucleatum ATCC 23726 and S. gordonii V288 involves a second outer membrane protein, which we named coaggregation mediating protein A (CmpA). We also characterized the role of CmpA in dual-species biofilm formation with S. gordonii V288, evaluated growth-phase-dependent as well as biofilm expression profiles of radD and cmpA, and confirmed an important role for CmpA, especially under biofilm growth conditions. Our findings underscore the complex set of specific interactions involved in physical binding and thus community integration of interacting bacterial species. This complex set of interactions could have critical implications for the formation and maturation of the oral biofilms in vivo, and could provide clues to the mechanism behind the distribution of organisms inside the human oral cavity.

  1. 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

  2. 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.

  3. The Collagen-Binding Adhesin Is a Virulence Factor in Staphylococcus aureus Keratitis

    PubMed Central

    Rhem, Marcus N.; Lech, Elizabeth M.; Patti, Joseph M.; McDevitt, Damien; Höök, Magnus; Jones, Dan B.; Wilhelmus, Kirk R.

    2000-01-01

    A collagen-binding strain of Staphylococcus aureus produced suppurative inflammation in a rabbit model of soft contact lens-associated bacterial keratitis more often than its collagen-binding-negative isogenic mutant. Reintroduction of the cna gene on a multicopy plasmid into the mutant helped it regain its corneal adherence and infectivity. The topical application of a collagen-binding peptide before bacterial challenge decreased S. aureus adherence to deepithelialized corneas. These data suggest that the collagen-binding adhesin is involved in the pathogenesis of S. aureus infection of the cornea. PMID:10816547

  4. Filamentous hemagglutinin of Bordetella pertussis: a key adhesin with immunomodulatory properties?

    PubMed

    Villarino Romero, Rodrigo; Osicka, Radim; Sebo, Peter

    2014-01-01

    The filamentous hemagglutinin of pathogenic Bordetellae is a prototype of a large two-partner-system-secreted and β-structure-rich bacterial adhesin. It exhibits several binding activities that may facilitate bacterial adherence to airway mucosa and host phagocytes in the initial phases of infection. Despite three decades of research on filamentous hemagglutinin, there remain many questions on its structure-function relationships, integrin interactions and possible immunomodulatory signaling capacity. Here we review the state of knowledge on this important virulence factor and acellular pertussis vaccine component. Specific emphasis is placed on outstanding questions that are yet to be answered.

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

    PubMed Central

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

    2011-01-01

    Summary 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. PMID:22053824

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 26 CFR 31.3402(p)-1 - Voluntary withholding agreements.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 15 2010-04-01 2010-04-01 false Voluntary withholding agreements. 31.3402(p)-1 Section 31.3402(p)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SOURCE Collection of Income Tax at Source § 31.3402(p)-1 Voluntary withholding agreements. (a) In...

  11. 26 CFR 31.3402(p)-1 - Voluntary withholding agreements.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 15 2014-04-01 2014-04-01 false Voluntary withholding agreements. 31.3402(p)-1 Section 31.3402(p)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SOURCE Collection of Income Tax at Source § 31.3402(p)-1 Voluntary withholding agreements. (a) In...

  12. 26 CFR 31.3402(p)-1 - Voluntary withholding agreements.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 15 2012-04-01 2012-04-01 false Voluntary withholding agreements. 31.3402(p)-1 Section 31.3402(p)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SOURCE Collection of Income Tax at Source § 31.3402(p)-1 Voluntary withholding agreements. (a) In...

  13. 26 CFR 31.3402(p)-1 - Voluntary withholding agreements.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 15 2013-04-01 2013-04-01 false Voluntary withholding agreements. 31.3402(p)-1 Section 31.3402(p)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SOURCE Collection of Income Tax at Source § 31.3402(p)-1 Voluntary withholding agreements. (a) In...

  14. 26 CFR 31.3402(p)-1 - Voluntary withholding agreements.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 15 2011-04-01 2011-04-01 false Voluntary withholding agreements. 31.3402(p)-1 Section 31.3402(p)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SOURCE Collection of Income Tax at Source § 31.3402(p)-1 Voluntary withholding agreements. (a) In...

  15. Pseudomonas aeruginosa Minor Pilins Prime Type IVa Pilus Assembly and Promote Surface Display of the PilY1 Adhesin*

    PubMed Central

    Nguyen, Ylan; Sugiman-Marangos, Seiji; Harvey, Hanjeong; Bell, Stephanie D.; Charlton, Carmen L.; Junop, Murray S.; Burrows, Lori L.

    2015-01-01

    Type IV pili (T4P) contain hundreds of major subunits, but minor subunits are also required for assembly and function. Here we show that Pseudomonas aeruginosa minor pilins prime pilus assembly and traffic the pilus-associated adhesin and anti-retraction protein, PilY1, to the cell surface. PilV, PilW, and PilX require PilY1 for inclusion in surface pili and vice versa, suggestive of complex formation. PilE requires PilVWXY1 for inclusion, suggesting that it binds a novel interface created by two or more components. FimU is incorporated independently of the others and is proposed to couple the putative minor pilin-PilY1 complex to the major subunit. The production of small amounts of T4P by a mutant lacking the minor pilin operon was traced to expression of minor pseudopilins from the P. aeruginosa type II secretion (T2S) system, showing that under retraction-deficient conditions, T2S minor subunits can prime T4P assembly. Deletion of all minor subunits abrogated pilus assembly. In a strain lacking the minor pseudopilins, PilVWXY1 and either FimU or PilE comprised the minimal set of components required for pilus assembly. Supporting functional conservation of T2S and T4P minor components, our 1.4 Å crystal structure of FimU revealed striking architectural similarity to its T2S ortholog GspH, despite minimal sequence identity. We propose that PilVWXY1 form a priming complex for assembly and that PilE and FimU together stably couple the complex to the major subunit. Trafficking of the anti-retraction factor PilY1 to the cell surface allows for production of pili of sufficient length to support adherence and motility. PMID:25389296

  16. Structural Basis for Toughness and Flexibility in the C-terminal Passenger Domain of an Acinetobacter Trimeric Autotransporter Adhesin*

    PubMed Central

    Koiwai, Kotaro; Hartmann, Marcus D.; Linke, Dirk; Lupas, Andrei N.; Hori, Katsutoshi

    2016-01-01

    Trimeric autotransporter adhesins (TAAs) on the cell surface of Gram-negative pathogens 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. It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain (TM), and the passenger domain contains an N-terminal head, N-terminal stalk, C-terminal head (Chead), and C-terminal stalk (Cstalk). The Chead-Cstalk-TM fragment, which is conserved in many Acinetobacter TAAs, has by itself the head-stalk-anchor architecture of a complete TAA. Here, we show the crystal structure of the Chead-Cstalk fragment, AtaA_C-terminal passenger domain (CPSD), providing the first view of several conserved TAA domains. The YadA-like head (Ylhead) of the fragment is capped by a unique structure (headCap), composed of three β-hairpins and a connector motif; it also contains a head insert motif (HIM1) before its last inner β-strand. The headCap, Ylhead, and HIM1 integrally form a stable Chead structure. Some of the major domains of the CPSD fragment are inherently flexible and provide bending sites for the fiber between segments whose toughness is ensured by topological chain exchange and hydrophobic core formation inside the trimer. Thus, although adherence assays using in-frame deletion mutants revealed that the characteristic adhesive sites of AtaA reside in its N-terminal part, the flexibility and toughness of the CPSD part provide the resilience that enables the adhesive properties of the full-length fiber across a wide range of conditions. PMID:26698633

  17. Characterization of AfaE adhesins produced by extraintestinal and intestinal human Escherichia coli isolates: PCR assays for detection of Afa adhesins that do or do not recognize Dr blood group antigens.

    PubMed

    Le Bouguénec, C; Lalioui, L; du Merle, L; Jouve, M; Courcoux, P; Bouzari, S; Selvarangan, R; Nowicki, B J; Germani, Y; Andremont, A; Gounon, P; Garcia, M I

    2001-05-01

    Operons of the afa family are expressed by pathogenic Escherichia coli strains associated with intestinal and extraintestinal infections in humans and animals. The recently demonstrated heterogeneity of these operons (L. Lalioui, M. Jouve, P. Gounon, and C. Le Bouguénec, Infect. Immun. 67:5048-5059, 1999) was used to develop a new PCR assay for detecting all the operons of the afa family with a single genetic tool. This PCR approach was validated by investigating three collections of human E. coli isolates originating from the stools of infants with diarrhea (88 strains), the urine of patients with pyelonephritis (97 strains), and the blood of cancer patients (115 strains). The results obtained with this single test and those previously obtained with several PCR assays were closely correlated. The AfaE adhesins encoded by the afa operons are variable, particularly with respect to the primary sequence encoded by the afaE gene. The receptor binding specificities have not been determined for all of these adhesins; some recognize the Dr blood group antigen (Afa/Dr(+) adhesins) on the human decay-accelerating factor (DAF) as a receptor, and others (Afa/Dr(-) adhesins) do not. Thus, the afa operons detected in this study were characterized by subtyping the afaE gene using specific PCRs. In addition, the DAF-binding capacities of as-yet-uncharacterized AfaE adhesins were tested by various cellular approaches. The afaE8 subtype (Afa/Dr(-) adhesin) was found to predominate in afa-positive isolates from sepsis patients (75%); it was frequent in afa-positive pyelonephritis E. coli (55.5%) and absent from diarrhea-associated strains. In contrast, Afa/Dr(+) strains (regardless of the afaE subtype) were associated with both diarrhea (100%) and extraintestinal infections (44 and 25% in afa-positive pyelonephritis and sepsis strains, respectively). These data suggest that there is an association between the subtype of AfaE adhesin and the physiological site of the infection

  18. A tobacco etch virus protease with increased substrate tolerance at the P1' position.

    PubMed

    Renicke, Christian; Spadaccini, Roberta; Taxis, Christof

    2013-01-01

    Site-specific proteases are important tools for in vitro and in vivo cleavage of proteins. They are widely used for diverse applications, like protein purification, assessment of protein-protein interactions or regulation of protein localization, abundance or activity. Here, we report the development of a procedure to select protease variants with altered specificity based on the well-established Saccharomyces cerevisiae adenine auxotrophy-dependent red/white colony assay. We applied this method on the tobacco etch virus (TEV) protease to obtain a protease variant with altered substrate specificity at the P1' Position. In vivo experiments with tester substrates showed that the mutated TEV protease still efficiently recognizes the sequence ENLYFQ, but has almost lost all bias for the amino acid at the P1' Position. Thus, we generated a site-specific protease for synthetic approaches requiring in vivo generation of proteins or peptides with a specific N-terminal amino acid.

  19. The mRNA Decay Pathway Regulates the Expression of the Flo11 Adhesin and Biofilm Formation in Saccharomyces cerevisiae

    PubMed Central

    Lo, Tricia L.; Qu, Yue; Uwamahoro, Nathalie; Quenault, Tara; Beilharz, Traude H.; Traven, Ana

    2012-01-01

    Regulation of the FLO11 adhesin is a model for gene expression control by extracellular signals and developmental switches. We establish that the major mRNA decay pathway regulates FLO11 expression. mRNA deadenylation of transcriptional repressors of FLO11 by the exonuclease Ccr4 keeps their levels low, thereby allowing FLO11 transcription. PMID:22595243

  20. Molecular and Genomic Analysis of Genes Encoding Surface-Anchored Proteins from Clostridium difficile

    PubMed Central

    Karjalainen, Tuomo; Waligora-Dupriet, Anne-Judith; Cerquetti, Marina; Spigaglia, Patrizia; Maggioni, Andrea; Mauri, Pierluigi; Mastrantonio, Paola

    2001-01-01

    The gene slpA, encoding the S-layer precursor protein in the virulent Clostridium difficile strains C253 and 79–685, was identified. The precursor protein carries a C-terminal highly conserved anchoring domain, similar to the one found in the Cwp66 adhesin (previously characterized in strain 79–685), an SLH domain, and a variable N-terminal domain mediating cell adherence. The genes encoding the S-layer precursor proteins and the Cwp66 adhesin are present in a genetic locus carrying 17 open reading frames, 11 of which encode a similar two-domain architecture, likely to include surface-anchored proteins. PMID:11292772

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

    PubMed

    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; Shirtliff, Mark E

    2012-12-01

    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

  2. Impact of OmpR on the membrane proteome of Yersinia enterocolitica in different environments: repression of major adhesin YadA and heme receptor HemR.

    PubMed

    Nieckarz, Marta; Raczkowska, Adrianna; Dębski, Janusz; Kistowski, Michał; Dadlez, Michał; Heesemann, Jürgen; Rossier, Ombeline; Brzostek, Katarzyna

    2016-03-01

    Enteropathogenic Yersinia enterocolitica is able to grow within or outside the mammalian host. Previous transcriptomic studies have indicated that the regulator OmpR plays a role in the expression of hundreds of genes in enterobacteria. Here, we have examined the impact of OmpR on the production of Y. enterocolitica membrane proteins upon changes in temperature, osmolarity and pH. Proteomic analysis indicated that the loss of OmpR affects the production of 120 proteins, a third of which are involved in uptake/transport, including several that participate in iron or heme acquisition. A set of proteins associated with virulence was also affected. The influence of OmpR on the abundance of adhesin YadA and heme receptor HemR was examined in more detail. OmpR was found to repress YadA production and bind to the yadA promoter, suggesting a direct regulatory effect. In contrast, the repression of hemR expression by OmpR appears to be indirect. These findings provide new insights into the role of OmpR in remodelling the cell surface and the adaptation of Y. enterocolitica to different environmental niches, including the host.

  3. Burkholderia cenocepacia K56-2 trimeric autotransporter adhesin BcaA binds TNFR1 and contributes to induce airway inflammation.

    PubMed

    Mil-Homens, Dalila; Pinto, Sandra N; Matos, Rute G; Arraiano, Cecília; Fialho, Arsenio M

    2017-04-01

    Chronic lung disease caused by persistent bacterial infections is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). CF pathogens acquire antibiotic resistance, overcome host defenses, and impose uncontrolled inflammation that ultimately may cause permanent damage of lungs' airways. Among the multiple CF-associated pathogens, Burkholderia cenocepacia and other Burkholderia cepacia complex bacteria have become prominent contributors of disease progression. Here, we demonstrate that BcaA, a trimeric autotransporter adhesin (TAA) from the epidemic strain B. cenocepacia K56-2, is a tumor necrosis factor receptor 1-interacting protein able to regulate components of the tumor necrosis factor signaling pathway and ultimately leading to a significant production of the proinflammatory cytokine IL-8. Notably, this study is the first to demonstrate that a protein belonging to the TAA family is involved in the induction of the inflammatory response during B. cenocepacia infections, contributing to the success of the pathogen. Moreover, our results reinforce the relevance of the TAA BcaA as a multifunctional protein with a major role in B. cenocepacia virulence.

  4. 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

  5. The Hypervariable Amino-Terminus of P1 Protease Modulates Potyviral Replication and Host Defense Responses

    PubMed Central

    Pasin, Fabio; Simón-Mateo, Carmen; García, Juan Antonio

    2014-01-01

    The replication of many RNA viruses involves the translation of polyproteins, whose processing by endopeptidases is a critical step for the release of functional subunits. P1 is the first protease encoded in plant potyvirus genomes; once activated by an as-yet-unknown host factor, it acts in cis on its own C-terminal end, hydrolyzing the P1-HCPro junction. Earlier research suggests that P1 cooperates with HCPro to inhibit host RNA silencing defenses. Using Plum pox virus as a model, we show that although P1 does not have a major direct role in RNA silencing suppression, it can indeed modulate HCPro function by its self-cleavage activity. To study P1 protease regulation, we used bioinformatic analysis and in vitro activity experiments to map the core C-terminal catalytic domain. We present evidence that the hypervariable region that precedes the protease domain is predicted as intrinsically disordered, and that it behaves as a negative regulator of P1 proteolytic activity in in vitro cleavage assays. In viral infections, removal of the P1 protease antagonistic regulator is associated with greater symptom severity, induction of salicylate-dependent pathogenesis-related proteins, and reduced viral loads. We suggest that fine modulation of a viral protease activity has evolved to keep viral amplification below host-detrimental levels, and thus to maintain higher long-term replicative capacity. PMID:24603811

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

    PubMed

    Rowe, Helen A; Griffiths, Natalie J; Hill, Darryl J; Virji, Mumtaz

    2007-01-01

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

  7. 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.

  8. Dual function of a bacterial protein as an adhesin and extracellular effector of host GTPase signaling

    PubMed Central

    Stones, Daniel Henry; Krachler, Anne Marie

    2015-01-01

    Bacterial pathogens often target conserved cellular mechanisms within their hosts to rewire signaling pathways and facilitate infection. Rho GTPases are important nodes within eukaryotic signaling networks and thus constitute a common target of pathogen-mediated manipulation. A diverse array of microbial mechanisms exists to interfere with Rho GTPase signaling. While targeting of GTPases by secreted bacterial effectors is a well-known strategy bacterial pathogens employ to interfere with the host, we have recently described pathogen adhesion as a novel extracellular stimulus that hijacks host GTPase signaling. The Multivalent Adhesion Molecule MAM7 from Vibrio parahaemolyticus directly binds host cell membrane lipids. The ensuing coalescence of phosphatidic acid ligands in the host membrane leads to downstream activation of RhoA and actin rearrangements. Herein, we discuss mechanistic models of lipid-mediated Rho activation and the implications from the infected host's and the pathogen's perspective. PMID:26156628

  9. Evolution of Salmonella enterica Virulence via Point Mutations in the Fimbrial Adhesin

    PubMed Central

    Kisiela, Dagmara I.; Chattopadhyay, Sujay; Libby, Stephen J.; Karlinsey, Joyce E.; Fang, Ferric C.; Tchesnokova, Veronika; Kramer, Jeremy J.; Beskhlebnaya, Viktoriya; Samadpour, Mansour; Grzymajlo, Krzysztof; Ugorski, Maciej; Lankau, Emily W.; Mackie, Roderick I.; Clegg, Steven; Sokurenko, Evgeni V.

    2012-01-01

    Whereas the majority of pathogenic Salmonella serovars are capable of infecting many different animal species, typically producing a self-limited gastroenteritis, serovars with narrow host-specificity exhibit increased virulence and their infections frequently result in fatal systemic diseases. In our study, a genetic and functional analysis of the mannose-specific type 1 fimbrial adhesin FimH from a variety of serovars of Salmonella enterica revealed that specific mutant variants of FimH are common in host-adapted (systemically invasive) serovars. We have found that while the low-binding shear-dependent phenotype of the adhesin is preserved in broad host-range (usually systemically non-invasive) Salmonella, the majority of host-adapted serovars express FimH variants with one of two alternative phenotypes: a significantly increased binding to mannose (as in S. Typhi, S. Paratyphi C, S. Dublin and some isolates of S. Choleraesuis), or complete loss of the mannose-binding activity (as in S. Paratyphi B, S. Choleraesuis and S. Gallinarum). The functional diversification of FimH in host-adapted Salmonella results from recently acquired structural mutations. Many of the mutations are of a convergent nature indicative of strong positive selection. The high-binding phenotype of FimH that leads to increased bacterial adhesiveness to and invasiveness of epithelial cells and macrophages usually precedes acquisition of the non-binding phenotype. Collectively these observations suggest that activation or inactivation of mannose-specific adhesive properties in different systemically invasive serovars of Salmonella reflects their dynamic trajectories of adaptation to a life style in specific hosts. In conclusion, our study demonstrates that point mutations are the target of positive selection and, in addition to horizontal gene transfer and genome degradation events, can contribute to the differential pathoadaptive evolution of Salmonella. PMID:22685400

  10. New adhesin of enteroaggregative Escherichia coli related to the Afa/Dr/AAF family.

    PubMed

    Boisen, Nadia; Struve, Carsten; Scheutz, Flemming; Krogfelt, Karen A; Nataro, James P

    2008-07-01

    Enteroaggregative Escherichia coli (EAEC) is an important cause of diarrhea worldwide. We analyzed 17 Danish EAEC strains, isolated in the course of a case control study, for phenotypic and genotypic properties. The strains belonged to at least 14 different serotypes. Using PCR to investigate the prevalence of various putative virulence genes, we found that all but two strains were typical EAEC, as they harbored all or part of the previously described AggR regulon. The majority of the strains harbored genes encoding aggregative adherence fimbriae (AAF). The most common was AAF/I, found in nine strains; eight strains carried no known AAF-related genes. We utilized TnphoA mutagenesis to localize the aggregative adherence (AA) adhesin from one typical EAEC strain, C1010-00, which lacked a known AAF. We identified a TnphoA insertion in a hypothetical Dr-related pilin deposited in GenBank as HdaA. Four additional Danish strains harbored HdaA, and all but one displayed AA to HEp-2 cells. By using PCR primers derived from the pilins and ushers from the three AAF and Hda, we found that 16 of 17 strains exhibited evidence of one of these factors; importantly, the one negative strain also lacked the aggR gene. Cloning of the complete Hda gene cluster and expression in E. coli DH5alpha resulted in AA and complementation of the C1010-00 nonadherent mutant. Four related adhesins have now been found to confer AA in typical EAEC strains; our data suggest that, together, these variants may account for AA in the large majority of strains.

  11. Efficacy of Leishmania donovani ribosomal P1 gene as DNA vaccine in experimental visceral leishmaniasis.

    PubMed

    Masih, Shet; Arora, Sunil K; Vasishta, Rakesh K

    2011-09-01

    The acidic ribosomal proteins of the protozoan parasites have been described as prominent antigens during human disease. We present here data showing the molecular cloning and protective efficacy of P1 gene of Leishmania donovani as DNA vaccine. The PCR amplified complete ORF cloned in either pQE or pVAX vector was used either as peptide or DNA vaccine against experimentally induced visceral leishmaniasis in hamsters. The recombinant protein rLdP1 was given along with Freund's adjuvant and the plasmid DNA vaccine, pVAX-P1 was used alone either as single dose or double dose (prime and boost) in different groups of hamsters which were subsequently challenged with a virulent dose of 1×10(7) L. donovani (MHOM/IN/DD8/1968 strain) promastigotes by intra-cardiac route. While the recombinant protein rLdP1 or DNA vaccine pVAX-P1 in single dose format were not found to be protective, DNA vaccine in a prime-boost mode was able to induce protection with reduced mortality, a significant (75.68%) decrease in splenic parasite burden and increased expression of Th1 type cytokines in immunized hamsters. Histopathology of livers and spleens from these animals showed formation of mature granulomas with compact arrangement of lymphocytes and histiocytes, indicating its protective potential as vaccine candidate.

  12. 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

  13. 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

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

    PubMed

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

    2016-01-01

    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. IMPORTANCEStaphylococcus epidermidis is a leading cause of infections related

  15. 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.

  16. Permeabilization of mitochondria and red blood cells by polycationic peptides BTM-P1 and retro-BTM-P1.

    PubMed

    Lemeshko, Victor V

    2011-10-01

    Mitochondrial and plasma membrane permeabilization by polycationic peptides BTM-P1 and retro-BTM-P1 were studied. BTM-P1 was more active than its retro-analog. In the sucrose medium, the capacity of BTM-P1 to permeabilize mitochondria was lower than in salt media. In contrast, retro-BTM-P1 showed the lowest activity in the KCl medium. The efficacy of both peptides to permeabilize red blood cells was higher in the sucrose medium and depended on the nature of salt in high ionic strength media. BTM-P1, but not retro-BTM-P1, induced biphasic change in light dispersion of red blood cells with artificially generated high transmembrane potential: the initial phase of fast cell shrinkage preceded the subsequent phase of cell swelling. The shrunken red blood cells demonstrated increased sensitivity to BTM-P1 that might be explained by the cell suicide mechanism via phosphatidylserine exposure at the cell surface. As a working hypothesis, we assume that some peptide topology characteristics, such as the orientation and values of the total and local electrical dipole moments, interacting with the membrane dipole potential, as well as the asymmetric distribution of polar and non-polar side chains are important factors affecting the membrane-permeabilizing activity of polycationic peptides.

  17. P1 Ref Endonuclease: A Molecular Mechanism for Phage-Enhanced Antibiotic Lethality

    PubMed Central

    Ronayne, Erin A.; Wan, Y. C. Serena; Boudreau, Beth A.; Landick, Robert; Cox, Michael M.

    2016-01-01

    Ref is an HNH superfamily endonuclease that only cleaves DNA to which RecA protein is bound. The enigmatic physiological function of this unusual enzyme is defined here. Lysogenization by bacteriophage P1 renders E. coli more sensitive to the DNA-damaging antibiotic ciprofloxacin, an example of a phenomenon termed phage-antibiotic synergy (PAS). The complementary effect of phage P1 is uniquely traced to the P1-encoded gene ref. Ref is a P1 function that amplifies the lytic cycle under conditions when the bacterial SOS response is induced due to DNA damage. The effect of Ref is multifaceted. DNA binding by Ref interferes with normal DNA metabolism, and the nuclease activity of Ref enhances genome degradation. Ref also inhibits cell division independently of the SOS response. Ref gene expression is toxic to E. coli in the absence of other P1 functions, both alone and in combination with antibiotics. The RecA proteins of human pathogens Neisseria gonorrhoeae and Staphylococcus aureus serve as cofactors for Ref-mediated DNA cleavage. Ref is especially toxic during the bacterial SOS response and the limited growth of stationary phase cultures, targeting aspects of bacterial physiology that are closely associated with the development of bacterial pathogen persistence. PMID:26765929

  18. The P-1 truss in the O&C

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Part of the P-1 truss is seen as it rests in a workstand in the Operations and Checkout Building. Scheduled to fly in spring of 2002, the P-1 is part of a total 10-truss, girder-like structure that will ultimately extend the length of a football field. Astronauts will attach the 14- by 15-foot structure to the port side of the center truss, S0, during the spring assembly flight. The 33,000-pound P-1 will house the thermal radiator rotating joint (TRRJ) that will rotate the International Space Station's radiators away from the sun to increase their maximum cooling efficiency.

  19. Lactobacillus reuteri Surface Mucus Adhesins Upregulate Inflammatory Responses Through Interactions With Innate C-Type Lectin Receptors

    PubMed Central

    Bene, Krisztián P.; Kavanaugh, Devon W.; Leclaire, Charlotte; Gunning, Allan P.; MacKenzie, Donald A.; Wittmann, Alexandra; Young, Ian D.; Kawasaki, Norihito; Rajnavolgyi, Eva; Juge, Nathalie

    2017-01-01

    The vertebrate gut symbiont Lactobacillus reuteri exhibits strain-specific adhesion and health-promoting properties. Here, we investigated the role of the mucus adhesins, CmbA and MUB, upon interaction of L. reuteri ATCC PTA 6475 and ATCC 53608 strains with human monocyte-derived dendritic cells (moDCs). We showed that mucus adhesins increased the capacity of L. reuteri strains to interact with moDCs and promoted phagocytosis. Our data also indicated that mucus adhesins mediate anti- and pro-inflammatory effects by the induction of interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α), IL-1β, IL-6, and IL-12 cytokines. L. reuteri ATCC PTA 6475 and ATCC 53608 were exclusively able to induce moDC-mediated Th1 and Th17 immune responses. We further showed that purified MUB activates moDCs and induces Th1 polarized immune responses associated with increased IFNγ production. MUB appeared to mediate these effects via binding to C-type lectin receptors (CLRs), as shown using cell reporter assays. Blocking moDCs with antibodies against DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) or Dectin-2 did not affect the uptake of the MUB-expressing strain, but reduced the production of TNF-α and IL-6 by moDCs significantly, in line with the Th1 polarizing capacity of moDCs. The direct interaction between MUB and CLRs was further confirmed by atomic force spectroscopy. Taken together these data suggest that mucus adhesins expressed at the cell surface of L. reuteri strains may exert immunoregulatory effects in the gut through modulating the Th1-promoting capacity of DCs upon interaction with C-type lectins. PMID:28326063

  20. Association of IS1016 with the hia adhesin gene and biotypes V and I in invasive nontypeable Haemophilus influenzae.

    PubMed

    Satola, Sarah W; Napier, Brooke; Farley, Monica M

    2008-11-01

    A subset of invasive nontypeable Haemophilus influenzae (NTHI) strains has evidence of IS1016, an insertion element associated with division I H. influenzae capsule serotypes. We examined IS1016-positive invasive NTHI isolates collected as part of Active Bacterial Core Surveillance within the Georgia Emerging Infections Program for the presence or absence of hmw1 and hmw2 (two related adhesin genes that are common in NTHI but absent in encapsulated H. influenzae) and hia (homologue of hsf, an encapsulated H. influenzae adhesin gene). Isolates were serotyped using slide agglutination, confirmed as NTHI strains using PCR capsule typing, and biotyped. Two hundred twenty-nine invasive NTHI isolates collected between August 1998 and December 2006 were screened for IS1016; 22/229 (9.6%) were positive. Nineteen of 201 previously identified IS1016-positive invasive NTHI isolates collected between January 1989 and July 1998 were also examined. Forty-one IS1016-positive and 56 randomly selected IS1016-negative invasive NTHI strains were examined. The hia adhesin was present in 39 of 41 (95%) IS1016-positive NTHI strains and 1 of 56 (1.8%) IS1016-negative NTHI strains tested; hmw (hmw1, hmw2, or both) was present in 50 of 56 (89%) IS1016-negative NTHI isolates but in only 5 of 41 (12%; all hmw2) IS1016-positive NTHI isolates. IS1016-positive NTHI strains were more often biotype V (P < 0.001) or biotype I (P = 0.04) than IS1016-negative NTHI strains, which were most often biotype II. Pulsed-field gel electrophoresis revealed the expected genetic diversity of NTHI with some clustering based on IS1016, hmw or hia, and biotypes. A significant association of IS1016 with biotypes V and I and the presence of hia adhesins was found among invasive NTHI. IS1016-positive NTHI strains may represent a unique subset of NTHI strains, with characteristics more closely resembling those of encapsulated H. influenzae.

  1. 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

  2. Pathogenicity of porcine enterotoxigenic Escherichia coli that do not express K88, K99, F41, or 987P adhesins.

    PubMed

    Casey, T A; Nagy, B; Moon, H W

    1992-09-01

    Three-week-old weaned and colostrum-deprived neonatal (less than 1 day old) pigs were inoculated to determine the pathogenicity of 2 enterotoxigenic Escherichia coli isolates that do not express K88, K99, F41, or 987P adhesins (strains 2134 and 2171). Strains 2134 and 2171 were isolated from pigs that had diarrhea after weaning attributable to enterotoxigenic E coli infection. We found that both strains of E coli adhered in the ileum and caused diarrhea in pigs of both age groups. In control experiments, adherent bacteria were not seen in the ileum of pigs less than 1 day old or 3 weeks old that were noninoculated or inoculated with a nonpathogenic strain of E coli. These control pigs did not develop diarrhea. Antisera raised against strains 2134 and 2171 and absorbed with the autologous strain, grown at 18 C, were used for bacterial-agglutination and colony-immunoblot assays. Both absorbed antisera reacted with strains 2134 and 2171, but not with strains that express K99, F41, or 987P adhesins. A cross-reaction was observed with 2 wild-type K88 strains, but not with a K12 strain that expresses K88 pili. Indirect immunofluorescence with these absorbed antisera revealed adherent bacteria in frozen sections of ileum from pigs infected with either strain. We concluded that these strains are pathogenic and express a common surface antigen that may be a novel adhesin in E coli strains that cause diarrhea in weaned pigs.

  3. 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.

  4. Evidence for the Sialylation of PilA, the PI-2a Pilus-Associated Adhesin of Streptococcus agalactiae Strain NEM316

    PubMed Central

    Morello, Eric; Mallet, Adeline; Konto-Ghiorghi, Yoan; Chaze, Thibault; Mistou, Michel-Yves; Oliva, Giulia; Oliveira, Liliana; Di Guilmi, Anne-Marie; Trieu-Cuot, Patrick; Dramsi, Shaynoor

    2015-01-01

    Streptococcus agalactiae (or Group B Streptococcus, GBS) is a commensal bacterium present in the intestinal and urinary tracts of approximately 30% of humans. We and others previously showed that the PI-2a pilus polymers, made of the backbone pilin PilB, the tip adhesin PilA and the cell wall anchor protein PilC, promote adhesion to host epithelia and biofilm formation. Affinity-purified PI-2a pili from GBS strain NEM316 were recognized by N-acetylneuraminic acid (NeuNAc, also known as sialic acid) specific lectins such as Elderberry Bark Lectin (EBL) suggesting that pili are sialylated. Glycan profiling with twenty different lectins combined with monosaccharide composition by HPLC suggested that affinity-purified PI-2a pili are modified by N-glycosylation and decorated with sialic acid attached to terminal galactose. Analysis of various relevant mutants in the PI-2a pilus operon by flow-cytometry and electron microscopy analyses pointed to PilA as the pilus subunit modified by glycosylation. Double labeling using PilB antibody and EBL lectin, which specifically recognizes N-acetylneuraminic acid attached to galactose in α-2, 6, revealed a characteristic binding of EBL at the tip of the pilus structures, highly reminiscent of PilA localization. Expression of a secreted form of PilA using an inducible promoter showed that this recombinant PilA binds specifically to EBL lectin when produced in the native GBS context. In silico search for potentially glycosylated asparagine residues in PilA sequence pointed to N427 and N597, which appear conserved and exposed in the close homolog RrgA from S. pneumoniae, as likely candidates. Conversion of these two asparagyl residues to glutamyl resulted in a higher instability of PilA. Our results provide the first evidence that the tip PilA adhesin can be glycosylated, and suggest that this modification is critical for PilA stability and may potentially influence interactions with the host. PMID:26407005

  5. Structure and Function of a Fungal Adhesin that Binds Heparin and Mimics Thrombospondin-1 by Blocking T Cell Activation and Effector Function

    PubMed Central

    Brandhorst, T. Tristan; Roy, René; Wüthrich, Marcel; Nanjappa, Som; Filutowicz, Hanna; Galles, Kevin; Tonelli, Marco; McCaslin, Darrell R.; Satyshur, Kenneth; Klein, Bruce

    2013-01-01

    Blastomyces adhesin-1 (BAD-1) is a 120-kD surface protein on B. dermatitidis yeast. We show here that BAD-1 contains 41 tandem repeats and that deleting even half of them impairs fungal pathogenicity. According to NMR, the repeats form tightly folded 17-amino acid loops constrained by a disulfide bond linking conserved cysteines. Each loop contains a highly conserved WxxWxxW motif found in thrombospondin-1 (TSP-1) type 1 heparin-binding repeats. BAD-1 binds heparin specifically and saturably, and is competitively inhibited by soluble heparin, but not related glycosaminoglycans. According to SPR analysis, the affinity of BAD-1 for heparin is 33 nM±14 nM. Putative heparin-binding motifs are found both at the N-terminus and within each tandem repeat loop. Like TSP-1, BAD-1 blocks activation of T cells in a manner requiring the heparan sulfate-modified surface molecule CD47, and impairs effector functions. The tandem repeats of BAD-1 thus confer pathogenicity, harbor motifs that bind heparin, and suppress T-cell activation via a CD47-dependent mechanism, mimicking mammalian TSP-1. PMID:23853587

  6. Structural basis for the differential binding affinities of the HsfBD1 and HsfBD2 domains in the Haemophilus influenzae Hsf adhesin.

    PubMed

    Radin, Jana N; Grass, Susan A; Meng, Guoyu; Cotter, Shane E; Waksman, Gabriel; St Geme, Joseph W

    2009-08-01

    Haemophilus influenzae is a human-specific gram-negative coccobacillus that causes a variety of human infections ranging from localized respiratory infections to invasive diseases. Hsf is the major nonpilus adhesin in encapsulated strains of H. influenzae and belongs to the trimeric autotransporter family of proteins. The Hsf protein contains two highly homologous binding domains, designated HsfBD1 and HsfBD2. In this study we characterized the differential binding properties of HsfBD1 and HsfBD2. In assays using HeLa cells, we found that bacteria expressing either full-length Hsf or HsfBD1 by itself adhered at high levels, while bacteria expressing HsfBD2 by itself adhered at low levels. Immunofluorescence microscopy and a cellular enzyme-linked immunosorbent assay using purified proteins revealed that the binding affinity was significantly higher for HsfBD1 than for HsfBD2. Purified HsfBD1 was able to completely block adherence by bacteria expressing either HsfBD1 or HsfBD2, while purified HsfBD2 was able to block adherence by bacteria expressing HsfBD2 but had minimal activity against bacteria expressing HsfBD1. Conversion of the residue at position 1935 in the HsfBD1 binding pocket from Asp to Glu resulted in HsfBD2-like binding properties, and conversion of the residue at position 569 in the HsfBD2 binding pocket from Glu to Asp resulted in HsfBD1-like binding properties, as assessed by adherence assays with recombinant bacteria and by immunofluorescence microscopy with purified proteins. This work demonstrates the critical role of a single amino acid in the core of the binding pocket in determining the relative affinities of the HsfBD1 and HsfBD2 binding domains.

  7. 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

  8. 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

  9. Antibody Response to Fibronectin-Binding Adhesin FnbpA in Patients with Staphylococcus aureus Infections

    PubMed Central

    Casolini, Fabrizia; Visai, Livia; Joh, Danny; Conaldi, Pier Giulio; Toniolo, Antonio; Höök, Magnus; Speziale, Pietro

    1998-01-01

    We have analyzed antibody reactivity to a fibronectin-binding microbial surface component that recognizes adhesive matrix molecules (MSCRAMM) in blood plasma collected from patients with staphylococcal infections. All patients had elevated levels of anti-MSCRAMM antibodies compared to those of young children who, presumably, had not been exposed to staphylococcal infections. The anti-MSCRAMM antibodies preferentially reacted with the ligand-binding repeat domain of the adhesin. However, these antibodies did not inhibit fibronectin binding. Essentially, all patients had antibodies which specifically recognized the fibronectin-MSCRAMM complex but not the isolated components. Epitopes recognized by these anti-ligand-induced binding sites antibodies were found in each repeat unit of the MSCRAMM. These results demonstrate that staphylococci have bound fibronectin some time during infection and that each repeat unit in the MSCRAMM can engage in ligand binding. Furthermore, our previously proposed model, suggesting that an unordered structure in the MSCRAMM undergoes a conformational change upon ligand binding (K. House-Pompeo, Y. Xu, D. Joh, P. Speziale, and M. Höök, J. Biol. Chem. 271:1379–1384, 1996), is presumably operational in patients during infections. PMID:9784554

  10. Single-Molecule Imaging and Functional Analysis of Als Adhesins and Mannans during Candida albicans Morphogenesis

    PubMed Central

    Beaussart, Audrey; Alsteens, David; El-Kirat-Chatel, Sofiane; Lipke, Peter N.; Kucharíková, Sona; Van Dijck, Patrick; Dufrêne, Yves F.

    2012-01-01

    Cellular morphogenesis in the fungal pathogen Candida albicans is associated with changes in cell wall composition that play important roles in biofilm formation and immune responses. Yet, how fungal morphogenesis modulates the biophysical properties and interactions of the cell surface molecules is poorly understood, mainly owing to the paucity of high-resolution imaging techniques. Here, we use single-molecule atomic force microscopy to localize and analyze the key components of the surface of living C. albicans cells during morphogenesis. We show that the yeast-to-hypha transition leads to a major increase in the distribution, adhesion, unfolding and extension of Als adhesins and their associated mannans on the cell surface. We also find that morphogenesis dramatically increases cell surface hydrophobicity. These molecular changes are critical for microbe-host interactions, including adhesion, colonization, and biofilm formation. The single-molecule experiments presented here offer promising prospects for understanding how microbial pathogens use cell surface molecules to modulate biofilm and immune interactions. PMID:23145462

  11. 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.

  12. The urease enzyme of Helicobacter pylori does not function as an adhesin.

    PubMed

    Clyne, M; Drumm, B

    1996-07-01

    Helicobacter pylori urease is essential for colonization of the gastric mucosa irrespective of whether the stomach is acidic or hypochlorhydric. It has therefore been speculated that the enzyme functions as an adhesin. The aim of this study was to compare the adherence of H. pylori N6 with the adherence of an isogenic urease-negative mutant, strain N6(ureB::TnKm), to gastric cells. Strain N6 originated from a patient with gastritis. Strain N6(ureB::TnKm) is specifically modified in the gene which encodes the large subunit of urease, UreB, and hence does not form a UreA-UreB enzyme complex. We have used flow cytometry to assess the adherence of H. pylori to the cells. We have also used phase-contrast microscopy to assess the adherence of the organism to Kato III cells. In the absence of urea both strains bound to Kato III cells and to primary gastric cells. Binding of both strains to the cells occurred rapidly. The presence of urea in the incubation medium decreased the binding of strain N6 to the cells. This was due to a rise in the pH of the incubation medium, which caused loss of viability of the organism. Urea had no effect on the adherence of strain N6(ureB::TnKm). We conclude that the urease of H. pylori does not play a role in the adherence of the organism to gastric cells.

  13. P-1 truss moved to O&C Building

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Workers oversee the placement of the P-1 truss, a component of the International Space Station, onto the bed of a transport vehicle that will move it to the Operations and Checkout Building for processing. The P-1 truss, scheduled to fly in spring of 2002, is part of a total 10-truss, girder-like structure on the Station that will ultimately extend the length of a football field. Astronauts will attach the 14-by-15 foot structure to the port side of the center truss, S0, during the spring assembly flight. The 33,000-pound P-1 will house the thermal radiator rotating joint (TRRJ) that will rotate the Station's radiators away from the sun to increase their maximum cooling efficiency.

  14. P-1 truss arrives at O&C Building

    NASA Technical Reports Server (NTRS)

    2000-01-01

    The P-1 truss, a component of the International Space Station, arrives in the parking lot outside the Operations and Checkout Building where it will undergo processing. The P-1 truss, scheduled to fly in spring of 2002, is part of a total 10-truss, girder-like structure on the Space Station that will ultimately extend the length of a football field. Astronauts will attach the 14-by-15 foot structure to the port side of the center truss, S0, during the spring assembly flight. The 33,000-pound P-1 will house the thermal radiator rotating joint (TRRJ) that will rotate the Station's radiators away from the sun to increase their maximum cooling efficiency.

  15. P-1 truss moved to O&C Building

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Cranes place the P-1 truss, a component of the International Space Station, on a transport vehicle that will move it to the Operations and Checkout Building for processing. The truss had been temporarily stored in the RLV hangar in the background as a precaution against approaching bad weather. The P-1 truss, scheduled to fly in spring of 2002, is part of a total 10-truss, girder-like structure on the Station that will ultimately extend the length of a football field. Astronauts will attach the 14-by- 15 foot structure to the port side of the center truss, S0, during the spring assembly flight. The 33,000-pound P-1 will house the thermal radiator rotating joint (TRRJ) that will rotate the Station's radiators away from the sun to increase their maximum cooling efficiency.

  16. PstS-1, the 38-kDa Mycobacterium tuberculosis glycoprotein, is an adhesin, which binds the macrophage mannose receptor and promotes phagocytosis.

    PubMed

    Esparza, M; Palomares, B; García, T; Espinosa, P; Zenteno, E; Mancilla, R

    2015-01-01

    Mycobacterium tuberculosis, the primary causative agent of tuberculosis, infects macrophages and transforms the hostile intracellular environment into a permissive niche. M. tuberculosis infects macrophages using a variety of microbial ligand/cell receptor systems. In this study, binding assays with biotin-labelled mycobacterial cell wall proteins revealed five Concanavalin A-reactive proteins that bind macrophages. Among these proteins, we identified PstS-1, a 38-kDa M. tuberculosis mannosylated glycolipoprotein, and characterized it as an adhesin. Inhibition assays with mannan and immunoprecipitation demonstrated that PstS-1 binds the mannose receptor. We purified PstS-1 to 95.9% purity using ion exchange chromatography. The presence of mannose in purified PstS-1 was demonstrated by Concanavalin A interaction, which was abolished in the presence of sodium m-periodate and α-D-mannosidase. Gas chromatography revealed that purified PstS-1 contained 1% of carbohydrates by weight, which was mainly mannose. Finally, we used fluorescent microbeads coated with purified PstS-1 in phagocytosis assays and discovered that microbead uptake was inhibited by the pre-incubation of cells with GlcNAc, mannan and α-methyl mannoside. The interaction of PstS-1 coated beads with the mannose receptor was confirmed by confocal colocalization studies that showed high Pearson and Manders's colocalization coefficients. Our findings contribute to a better understanding of the strategies M. tuberculosis uses to infect host cells, the critical first step in the pathogenesis of tuberculosis.

  17. P-1 truss moves into O&C Building

    NASA Technical Reports Server (NTRS)

    2000-01-01

    The P-1 truss, a component of the International Space Station, sits inside the Operations and Checkout Building where it will undergo processing. The truss, scheduled to fly in spring of 2002, is part of a total 10-truss, girder-like structure on the Station that will ultimately extend the length of a football field. Astronauts will attach the 14-by-15 foot structure to the port side of the center truss, S0, during the spring assembly flight. The 33,000-pound P-1 will house the thermal radiator rotating joint (TRRJ) that will rotate the Station's radiators away from the sun to increase their maximum cooling efficiency.

  18. STS-113 P1 Truss paylad in Payload Changeout Room

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. - From the Payload Changeout Room on Launch Pad 39A, the P1 truss payload, plus the Crew and Equipment Translation Aid (CETA) cart B, are moved into the payload bay of Space Shuttle Endeavour. Scheduled to launch Nov. 10 on mission STS-113, Endeavour will make the 16th assembly flight to the International Space Station. Once delivered, the P1 truss will remain stowed until flight 12A.1 in 2003 when it will be attached to the central truss segment, S0, on the Space Station. The mission will also deliver the Expedition 6 crew to the Station and return Expedition 5 to Earth.

  19. 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

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

    2002-10-01

    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 hecATn7 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.

  20. Measurement of adhesive forces between bacteria and protein-coated surfaces using optical tweezers

    NASA Astrophysics Data System (ADS)

    Simpson, Kathryn H.; Bowden, Gabriela; Hook, Magnus; Anvari, Bahman

    2002-05-01

    Bacterial adhesion is a primary cause of failure in implanted medical devices. Bacteria commonly found in device-related infections, such as S. aureus, have multiple cell surface adhesins which mediate specific adhesion to molecules found in extracellular matrix and blood plasma. Adhesins recognizing fibrinogen, fibronectin, collagen, and elastin molecules have been isolated in S. aureus. We have used optical tweezers to measure the adhesive force between a single bacterium and a protein-coated surface. Various concentrations of fibronectin, fibrinogen, or whole plasma were immobilized onto 10-micrometers diameter polystyrene microspheres. We optically trapped a bacterium with a titanium-sapphire laser tuned to 830 nm and contacted the cell with a coated bead. We determined the minimum force necessary to separate the cell and bead. For beads coated with fibronectin and fibrinogen, detachment force values occurred as approximate integer multiples of an estimated single bond detachment force. With plasma-coated beads, only cells lacking the fibrinogen adhesin could be detached; therefore, we believe that either this adhesin is prevalent on wilde-type cells, or it is preferentially adsorbed onto the beads. Additionally, the whole plasma detachment forces appeared random; therefore, we believe that many adhesins participate in boding to plasma.

  1. Contribution of the collagen adhesin Acm to pathogenesis of Enterococcus faecium in experimental endocarditis.

    PubMed

    Nallapareddy, Sreedhar R; Singh, Kavindra V; Murray, Barbara E

    2008-09-01

    Enterococcus faecium is a multidrug-resistant opportunist causing difficult-to-treat nosocomial infections, including endocarditis, but there are no reports experimentally demonstrating E. faecium virulence determinants. Our previous studies showed that some clinical E. faecium isolates produce a cell wall-anchored collagen adhesin, Acm, and that an isogenic acm deletion mutant of the endocarditis-derived strain TX0082 lost collagen adherence. In this study, we show with a rat endocarditis model that TX0082 Deltaacm::cat is highly attenuated versus wild-type TX0082, both in established (72 h) vegetations (P < 0.0001) and for valve colonization 1 and 3 hours after infection (P or=50-fold reduction relative to an Acm producer) were found in three of these five nonadherent isolates, including the sequenced strain TX0016, by quantitative reverse transcription-PCR, indicating that acm transcription is downregulated in vitro in these isolates. However, examination of TX0016 cells obtained directly from infected rat vegetations by flow cytometry showed that Acm was present on 40% of cells grown during infection. Finally, we demonstrated a significant reduction in E. faecium collagen adherence by affinity-purified anti-Acm antibodies from E. faecium endocarditis patient sera, suggesting that Acm may be a potential immunotarget for strategies to control this emerging pathogen.

  2. Moraxella catarrhalis adhesin UspA1-derived recombinant fragment rD-7 induces monocyte differentiation to CD14+CD206+ phenotype.

    PubMed

    Xie, Qi; Brackenbury, Louise S; Hill, Darryl J; Williams, Neil A; Qu, Xun; Virji, Mumtaz

    2014-01-01

    Circulating monocytes in the bloodstream typically migrate to other tissues and differentiate into tissue resident macrophages, the process being determined by the constituents of the microenvironments encountered. These may include microbes and their products. In this study, we investigated whether Moraxella catarrhalis Ubiquitous Surface Protein A1 (UspA1), known to bind to a widely expressed human cell surface receptor CEACAM1, influences monocyte differentiation as receptor engagement has been shown to have profound effects on monocytes. We used the recombinant molecules corresponding to the regions of UspA1 which either bind (rD-7; UspA1527-665) or do not bind (r6-8; UspA1659-863) to CEACAM1 and investigated their effects on CD206, CD80 and CD86 expression on freshly isolated human CD14+ monocytes from peripheral blood mononuclear cells (PBMC). Exposure to rD-7, but not r6-8, biased monocyte differentiation towards a CD14+CD206+ phenotype, with reduced CD80 expression. Monocytes treated with rD-7 also secreted high levels of IL-1ra and chemokine IL-8 but not IL-10 or IL-12p70. The effects of rD-7 were independent of any residual endotoxin. Unexpectedly, these effects of rD-7 were also independent of its ability to bind to CEACAM1, as monocyte pre-treatment with the anti-CEACAM antibody A0115 known to inhibit rD-7 binding to the receptor, did not affect rD-7-driven differentiation. Further, another control protein rD-7/D (a mutant form of rD-7, known not to bind to CEACAMs), also behaved as the parent molecule. Our data suggest that specific regions of M. catarrhalis adhesin UspA1 may modulate inflammation during infection through a yet unknown receptor on monocytes.

  3. 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.

  4. Biofilm Matrix Proteins

    PubMed Central

    Fong, Jiunn N. C.; Yildiz, Fitnat H.

    2015-01-01

    Proteinaceous components of the biofilm matrix include secreted extracellular proteins, cell surface adhesins and protein subunits of cell appendages such as flagella and pili. Biofilm matrix proteins play diverse roles in biofilm formation and dissolution. They are involved in attaching cells to surfaces, stabilizing the biofilm matrix via interactions with exopolysaccharide and nucleic acid components, developing three-dimensional biofilm architectures, and dissolving biofilm matrix via enzymatic degradation of polysaccharides, proteins, and nucleic acids. In this chapter, we will review functions of matrix proteins in a selected set of microorganisms, studies of the matrix proteomes of Vibrio cholerae and Pseudomonas aeruginosa, and roles of outer membrane vesicles and of nucleoid-binding proteins in biofilm formation. PMID:26104709

  5. STS-113 P1 Truss paylad in Payload Changeout Room

    NASA Technical Reports Server (NTRS)

    2002-01-01

    KENNEDY SPACE CENTER, FLA. -- A worker in the Payload Changeout Room on Launch Pad 39A watches as the P1 truss payload, plus the Crew and Equipment Translation Aid (CETA) cart B, move into the payload bay of Space Shuttle Endeavour. Scheduled to launch Nov. 10 on mission STS-113, Endeavour will make the 16th assembly flight to the International Space Station. Once delivered, the P1 truss will remain stowed until flight 12A.1 in 2003 when it will be attached to the central truss segment, S0, on the Space Station. The mission will also deliver the Expedition 6 crew to the Station and return Expedition 5 to Earth.

  6. Bidirectional Bacterial Gliding Motility Powered by the Collective Transport of Cell Surface Proteins

    NASA Astrophysics Data System (ADS)

    Wada, Hirofumi; Nakane, Daisuke; Chen, Hsuan-Yi

    2013-12-01

    The gliding motility of Flavobacterium johnsoniae is driven by moving surface adhesive proteins. Recently, these motility components were observed to travel along a closed loop on the cell surface. The mechanism by which such moving surface adhesins give rise to cell motion remains unknown. On the basis of the unique motility properties of F. johnsoniae, we present a generic model for bidirectional motion of rigidly coupled adhesins, which are propelled in opposite directions. Using analytical and numerical methods, we demonstrate that, for a sufficiently large adhesin speed, bidirectional motion arises from spontaneous symmetry breaking. The model also predicts that, close to the bifurcation point, a weak asymmetry in the binding dynamics is sufficient to facilitate directed motility, indicating that the direction of motion could be sensitively regulated internally in response to inhomogeneity of the environment.

  7. Safety evaluation of nuclease P1 from Penicillium citrinum.

    PubMed

    Okado, Nobuo; Hasegawa, Kazushige; Mizuhashi, Fukutaro; Lynch, Barry S; Vo, Trung D; Roberts, Ashley S

    2016-02-01

    Nuclease P1 has been widely used in the food industry to enhance or create flavor. One commercial source of this enzyme is Penicillium citrinum, an anamorphic mesophilic fungus with a long history of safe use in Europe and Asia as a fermentation organism used in the production of ribonucleases. Given the intended use in food for human consumption, and noting its potential presence at trace levels in finished products, a series of safety studies including an in vitro Ames and chromosome aberration assay, an in vivo rat erythrocyte micronucleus assay and a 90-day oral toxicity study in rats were conducted. No mutagenic activity was observed in the Ames assay. Equivocal activity in the chromosome aberration assay was not replicated in the micronucleus assay at doses of up to 1007 mg total organic solids (TOS)/kg body weight (bw)/day. Following oral administration of nuclease P1 at dosages of 10.1, 101 or 1007 mg TOS/kg bw/day to Sprague-Dawley rats, no adverse effects on any study parameter were observed. The no-observed-adverse-effect level was considered to be 1007 mg TOS/kg bw/day. The results of the genotoxicity studies and subchronic rat study support the safe use in food production of nuclease P1 produced from P. citrinum.

  8. RELATIVE PHOTOMETRY OF HAT-P-1b OCCULTATIONS

    SciTech Connect

    Beky, Bence; Holman, Matthew J.; Noyes, Robert W.; Sasselov, Dimitar D.; Gilliland, Ronald L.; Bakos, Gaspar A.; Winn, Joshua N.

    2013-06-01

    We present Hubble Space Telescope (HST) Space Telescope Imaging Spectrograph observations of two occultations of the transiting exoplanet HAT-P-1b. By measuring the planet to star flux ratio near opposition, we constrain the geometric albedo of the planet, which is strongly linked to its atmospheric temperature gradient. An advantage of HAT-P-1 as a target is its binary companion ADS 16402 A, which provides an excellent photometric reference, simplifying the usual steps in removing instrumental artifacts from HST time-series photometry. We find that without this reference star, we would need to detrend the lightcurve with the time of the exposures as well as the first three powers of HST orbital phase, and this would introduce a strong bias in the results for the albedo. However, with this reference star, we only need to detrend the data with the time of the exposures to achieve the same per-point scatter, therefore we can avoid most of the bias associated with detrending. Our final result is a 2{sigma} upper limit of 0.64 for the geometric albedo of HAT-P-1b between 577 and 947 nm.

  9. Role of ARF6, Rab11 and External Hsp90 in the Trafficking and Recycling of Recombinant-Soluble Neisseria meningitidis Adhesin A (rNadA) in Human Epithelial Cells

    PubMed Central

    Montanari, Paolo; Benucci, Barbara; Biancucci, Marco; Nardi-Dei, Vincenzo; Caproni, Elena; Barrile, Riccardo; Picciani, Benedetta; Savino, Silvana; Aricò, Beatrice; Rappuoli, Rino; Pizza, Mariagrazia; Luini, Alberto; Sallese, Michele; Merola, Marcello

    2014-01-01

    Neisseria meningitidis adhesin A (NadA) is a meningococcus surface protein thought to assist in the adhesion of the bacterium to host cells. We have previously shown that NadA also promotes bacterial internalization in a heterologous expression system. Here we have used the soluble recombinant NadA (rNadA) lacking the membrane anchor region to characterize its internalization route in Chang epithelial cells. Added to the culture medium, rNadA internalizes through a PI3K-dependent endocytosis process not mediated by the canonical clathrin or caveolin scaffolds, but instead follows an ARF6-regulated recycling pathway previously described for MHC-I. The intracellular pool of rNadA reaches a steady state level within one hour of incubation and colocalizes in endocytic vesicles with MHC-I and with the extracellularly labeled chaperone Hsp90. Treatment with membrane permeated and impermeable Hsp90 inhibitors 17-AAG and FITC-GA respectively, lead to intracellular accumulation of rNadA, strongly suggesting that the extracellular secreted pool of the chaperone is involved in rNadA intracellular trafficking. A significant number of intracellular vesicles containing rNadA recruit Rab11, a small GTPase associated to recycling endosomes, but do not contain transferrin receptor (TfR). Interestingly, cell treatment with Hsp90 inhibitors, including the membrane-impermeable FITC-GA, abolished Rab11-rNadA colocalization but do not interfere with Rab11-TfR colocalization. Collectively, these results are consistent with a model whereby rNadA internalizes into human epithelial cells hijacking the recycling endosome pathway and recycle back to the surface of the cell via an ARF6-dependent, Rab11 associated and Hsp90-regulated mechanism. The present study addresses for the first time a meningoccoccal adhesin mechanism of endocytosis and suggests a possible entry pathway engaged by N. meningitidis in primary infection of human epithelial cells. PMID:25347845

  10. 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).

  11. 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

  12. 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

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

    2008-10-01

    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 the 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 analysed the draft genome of strain TX0016 for potential microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Genome-based bioinformatics identified 22 predicted cell-wall-anchored E. faecium surface proteins (Fms), of which 15 (including Acm) had characteristics typical of MSCRAMMs, including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one [Fms10; redesignated second collagen adhesin of E. faecium (Scm)] revealed that recombinant Scm(65) (A- and B-domains) and Scm(36) (A-domain) bound to collagen type V efficiently 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 measurements of recombinant Scm(36) and of Acm(37) indicated that these proteins were rich in beta-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; nine 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 EbpC(fm)), detected a 'ladder' pattern of high-molecular-mass protein bands in a

  13. Evaluation of the role of Mycobacterium tuberculosis pili (MTP) as an adhesin, invasin, and cytokine inducer of epithelial cells.

    PubMed

    Ramsugit, Saiyur; Pillay, Balakrishna; Pillay, Manormoney

    2016-01-01

    This study was undertaken in order to assess the involvement of Mycobacterium tuberculosis pili (MTP) as an adhesin, invasin, and cytokine inducer in the M. tuberculosis-epithelial cell interaction. A MTP-deficient strain of M. tuberculosis demonstrated a significant reduction of 69.39% (p=0.047) and 56.20% (p=0.033) in its ability to adhere to and invade A549 pulmonary epithelial cells, respectively, in comparison with the wild-type strain. Complementation of the MTP-deficient mutant restored its adhesion and invasion capacity back to the wild-type levels. Overall, it was found that similar concentrations of IL-1β, IL-4, IL-6, IL-8, G-CSF, IFN-γ, MCP-1, and TNF-α were induced in A549 cells infected with the MTP-proficient and MTP-deficient strains. However, at 48h post-infection, the MTP-deficient mutant induced significantly lower levels of TNF-α than the wild-type strain (p=0.033). Furthermore, at 72h post-infection, the mutant induced significantly higher levels of IL-8 than the wild-type (p=0.005). We conclude that MTP is an adhesin/invasin of epithelial cells and, while playing a role in M. tuberculosis entry, they do not appear to largely influence the epithelial cell cytokine response.

  14. Dynamics of Agglutinin-Like Sequence (ALS) Protein Localization on the Surface of Candida Albicans

    ERIC Educational Resources Information Center

    Coleman, David Andrew

    2009-01-01

    The ALS gene family encodes large cell-surface glycoproteins associated with "C. albicans" pathogenesis. Als proteins are thought to act as adhesin molecules binding to host tissues. Wide variation in expression levels among the ALS genes exists and is related to cell morphology and environmental conditions. "ALS1," "ALS3," and "ALS4" are three of…

  15. Immune responses induced by replication-defective adenovirus expressing the C-terminal portion of the Mycoplasma hyopneumoniae P97 adhesin.

    PubMed

    Okamba, F R; Moreau, E; Cheikh Saad Bouh, K; Gagnon, C A; Massie, B; Arella, M

    2007-06-01

    Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, colonizes the respiratory cilia of affected swine, causing significant economic losses to swine production worldwide. Vaccination is the most cost-effective strategy for the control and prevention of this disease. The goal of this study was to design and evaluate a replication-defective recombinant adenovirus, rAdP97c, expressing the C-terminal portion of P97 adhesin (P97c), an important pathogenesis-associated protein of M. hyopneumoniae, as a new vaccine candidate against M. hyopneumoniae infection. P97c-specific immune responses were evaluated in BALB/c mice following intranasal and intramuscular inoculation with rAdP97c. Mice inoculated by both routes of immunization produced significant levels of specific immunoglobulin G (IgG) antibodies in the serum and in bronchoalveolar lavage fluids (BALs). Animals immunized intranasally also produced a significant level of P97c-specific IgA in BALs. Intramuscular inoculation of rAdP97c induced a systemic and mucosal Th1-type biased response, evidenced by the predominance of IgG2a in the serum and BALs, whereas intranasal inoculation resulted in a mixed Th1/Th2-type response (balanced levels of IgG1 and IgG2a) in both sytemic and mucosal compartments. P97c-specific antibodies were able to inhibit the growth of M. hyopneumoniae cells in vitro. These data suggest that rAdP97c vaccine may represent a new strategy for controlling infection by M. hyopneumoniae.

  16. Point Mutations in FimH Adhesin of Crohn's Disease-Associated Adherent-Invasive Escherichia coli Enhance Intestinal Inflammatory Response

    PubMed Central

    Dreux, Nicolas; Denizot, Jérémy; Martinez-Medina, Margarita; Mellmann, Alexander; Billig, Maria; Kisiela, Dagmara; Chattopadhyay, Sujay; Sokurenko, Evgeni; Neut, Christel; Gower-Rousseau, Corinne; Colombel, Jean-Frédéric; Bonnet, Richard; Darfeuille-Michaud, Arlette; Barnich, Nicolas

    2013-01-01

    Adherent-invasive Escherichia coli (AIEC) are abnormally predominant on Crohn's disease (CD) ileal mucosa. AIEC reference strain LF82 adheres to ileal enterocytes via the common type 1 pili adhesin FimH and recognizes CEACAM6 receptors abnormally expressed on CD ileal epithelial cells. The fimH genes of 45 AIEC and 47 non-AIEC strains were sequenced. The phylogenetic tree based on fimH DNA sequences indicated that AIEC strains predominantly express FimH with amino acid mutations of a recent evolutionary origin - a typical signature of pathoadaptive changes of bacterial pathogens. Point mutations in FimH, some of a unique AIEC-associated nature, confer AIEC bacteria a significantly higher ability to adhere to CEACAM-expressing T84 intestinal epithelial cells. Moreover, in the LF82 strain, the replacement of fimHLF82 (expressing FimH with an AIEC-associated mutation) with fimHK12 (expressing FimH of commensal E. coli K12) decreased the ability of bacteria to persist and to induce severe colitis and gut inflammation in infected CEABAC10 transgenic mice expressing human CEACAM receptors. Our results highlight a mechanism of AIEC virulence evolution that involves selection of amino acid mutations in the common bacterial traits, such as FimH protein, and leads to the development of chronic inflammatory bowel disease (IBD) in a genetically susceptible host. The analysis of fimH SNPs may be a useful method to predict the potential virulence of E. coli isolated from IBD patients for diagnostic or epidemiological studies and to identify new strategies for therapeutic intervention to block the interaction between AIEC and gut mucosa in the early stages of IBD. PMID:23358328

  17. A functional collagen adhesin gene, acm, in clinical isolates of Enterococcus faecium correlates with the recent success of this emerging nosocomial pathogen.

    PubMed

    Nallapareddy, Sreedhar R; Singh, Kavindra V; Okhuysen, Pablo C; Murray, Barbara E

    2008-09-01

    Enterococcus faecium recently evolved from a generally avirulent commensal into a multidrug-resistant health care-associated pathogen causing difficult-to-treat infections, but little is known about the factors responsible for this change. We previously showed that some E. faecium strains express a cell wall-anchored collagen adhesin, Acm. Here we analyzed 90 E. faecium isolates (99% acm(+)) and found that the Acm protein was detected predominantly in clinically derived isolates, while the acm gene was present as a transposon-interrupted pseudogene in 12 of 47 isolates of nonclinical origin. A highly significant association between clinical (versus fecal or food) origin and collagen adherence (P

  18. Structural Study of MPN387, an Essential Protein for Gliding Motility of a Human-Pathogenic Bacterium, Mycoplasma pneumoniae

    PubMed Central

    Kawakita, Yoshito; Kinoshita, Miki; Furukawa, Yukio; Tulum, Isil; Tahara, Yuhei O.; Katayama, Eisaku

    2016-01-01

    ABSTRACT Mycoplasma pneumoniae is a human pathogen that glides on host cell surfaces with repeated catch and release of sialylated oligosaccharides. At a pole, this organism forms a protrusion called the attachment organelle, which is composed of surface structures, including P1 adhesin and the internal core structure. The core structure can be divided into three parts, the terminal button, paired plates, and bowl complex, aligned in that order from the front end of the protrusion. To elucidate the gliding mechanism, we focused on MPN387, a component protein of the bowl complex which is essential for gliding but dispensable for cytadherence. The predicted amino acid sequence showed that the protein features a coiled-coil region spanning residue 72 to residue 290 of the total of 358 amino acids in the protein. Recombinant MPN387 proteins were isolated with and without an enhanced yellow fluorescent protein (EYFP) fusion tag and analyzed by gel filtration chromatography, circular dichroism spectroscopy, analytical ultracentrifugation, partial proteolysis, and rotary-shadowing electron microscopy. The results showed that MPN387 is a dumbbell-shaped homodimer that is about 42.7 nm in length and 9.1 nm in diameter and includes a 24.5-nm-long central parallel coiled-coil part. The molecular image was superimposed onto the electron micrograph based on the localizing position mapped by fluorescent protein tagging. A proposed role of this protein in the gliding mechanism is discussed. IMPORTANCE Human mycoplasma pneumonia is caused by a pathogenic bacterium, Mycoplasma pneumoniae. This tiny, 2-μm-long bacterium is suggested to infect humans by gliding on the surface of the trachea through binding to sialylated oligosaccharides. The mechanism underlying mycoplasma “gliding motility” is not related to any other well-studied motility systems, such as bacterial flagella and eukaryotic motor proteins. Here, we isolated and analyzed the structure of a key protein which is

  19. In planta expression of a mature Der p 1 allergen isolated from an Italian strain of Dermatophagoides pteronyssinus.

    PubMed

    Marconi, Gianpiero; Albertini, Emidio; Mari, Adriano; Palazzo, Paola; Porceddu, Andrea; Raggi, Lorenzo; Bolis, Luigi; Lancioni, Hovirag; Palomba, Antonella; Lucentini, Livia; Lanfaloni, Luisa; Marcucci, Francesco; Falcinelli, Mario; Panara, Fausto

    2012-06-01

    European (Dermatophagoides pteronyssinus) and American (Dermatophagoides farinae) house dust mite species are considered the most common causes of asthma and allergic symptoms worldwide. Der p 1 protein, one of the main allergens of D. pteronyssinus, is found in high concentration in mites faecal pellets, which can became easily airborne and, when inhaled, can cause perennial rhinitis and bronchial asthma. Here we report the isolation of the Der p 1 gene from an Italian strain of D. pteronyssinus and the PVX-mediated expression of its mature form (I-rDer p 1) in Nicotiana benthamiana plants. Human sera from characterized allergic patients were used for IgE binding inhibition assays to test the immunological reactivity of I-rDer p 1 produced in N. benthamiana plants. The binding properties of in planta produced I-rDer p 1 versus the IgE of patients sera were comparable to those obtained on Der p 1 preparation immobilized on a microarray. In this paper we provide a proof of concept for the production of an immunologically active form of Der p 1 using a plant viral vector. These results pave the way for the development of diagnostic allergy tests based on in planta produced allergens.

  20. Mycobacterium tuberculosis MycP1 protease plays a dual role in regulation of ESX-1 secretion and virulence.

    PubMed

    Ohol, Yamini M; Goetz, David H; Chan, Kaman; Shiloh, Michael U; Craik, Charles S; Cox, Jeffery S

    2010-03-18

    Mycobacterium tuberculosis uses the ESX-1 secretion system to deliver virulence proteins during infection of host cells. Here we report a mechanism of posttranscriptional control of ESX-1 mediated by MycP1, a M. tuberculosis serine protease. We show that MycP1 is required for ESX-1 secretion but that, unexpectedly, genetic inactivation of MycP1 protease activity increases secretion of ESX-1 substrates. We demonstrate that EspB, an ESX-1 substrate required for secretion, is a target of MycP1 in vitro and in vivo. During macrophage infection, an inactive MycP1 protease mutant causes hyperactivation of ESX-1-stimulated innate signaling pathways. MycP1 is required for growth in mice during acute infection, while loss of its protease activity leads to attenuated virulence during chronic infection. As the key ESX-1 substrates ESAT-6 and CFP-10 are highly immunogenic, fine-tuning of their secretion by MycP1 may balance virulence and immune detection and be essential for successful maintenance of long-term M. tuberculosis infection.

  1. Bactericide, Immunomodulating, and Wound Healing Properties of Transgenic Kalanchoe pinnata Synergize with Antimicrobial Peptide Cecropin P1 In Vivo

    PubMed Central

    Lebedeva, A. A.; Zakharchenko, N. S.; Trubnikova, E. V.; Medvedeva, O. A.; Masgutova, G. A.; Zylkova, M. V.; Buryanov, Y. I.; Belous, A. S.

    2017-01-01

    Procedure of manufacturing K. pinnata water extracts containing cecropin P1 (CecP1) from the formerly described transgenic plants is established. It included incubation of leaves at +4°C for 7 days, mechanical homogenization of leaves using water as extraction solvent, and heating at +70°C for inactivating plant enzymes. Yield of CecP1 (after heating and sterilizing filtration) was 0.3% of total protein in the extract. The water extract of K. pinnata + CecP1 exhibits favorable effect on healing of wounds infected with S. aureus (equal to Cefazolin) and with a combination of S. aureus with P. aeruginosa (better than Cefazolin). Wild-type K. pinnata extract exhibited evident microbicide activity against S. aureus with P. aeruginosa but it was substantially strengthened in K. pinnata + CecP1 extract. K. pinnata extracts (both wild-type and transgenic) did not exhibit general toxicity and accelerated wound recovery. Due to immunomodulating activity, wild-type K. pinnata extract accelerated granulation of the wound bed and marginal epithelialization even better than K. pinnata + CecP1 extract. Immunomodulating and microbicide activity of K. pinnata synergizes with microbicide activity of CecP1 accelerating elimination of bacteria. PMID:28326334

  2. CRP represses the CRISPR/Cas system in Escherichia coli: evidence that endogenous CRISPR spacers impede phage P1 replication.

    PubMed

    Yang, Chi-Dung; Chen, Yen-Hua; Huang, Hsi-Yuan; Huang, Hsien-Da; Tseng, Ching-Ping

    2014-06-01

    The CRISPR/Cas system is an important aspect in bacterial immunology. The anti-phage activity of the CRISPR system has been established using synthetic CRISPR spacers, but in vivo studies of endogenous CRISPR spacers are relatively scarce. Here, we showed that bacteriophage P1 titre in Escherichia coli decreased in the glucose-containing medium compared with that in the absence of glucose. This glucose effect of E. coli against phage P1 infection disappeared in cse3 deletion mutants. The effect on the susceptibility to phage P1 was associated with cAMP receptor protein (CRP)-mediated repression of cas genes transcription and crRNA maturation. Analysis of the regulatory element in the cse1 promoter region revealed a novel CRP binding site, which overlapped with a LeuO binding site. Furthermore, the limited sequence identity between endogenous spacers and the phage P1 genome was necessary and sufficient for CRISPR-mediated repression of phage P1 replication. Trans-expression of the third and seventh spacers in the CRISPR I region or third and sixth spacers in the CRISPR II region effectively reduced phage P1 titres in the CRISPR deletion mutants. These results demonstrate a novel regulatory mechanism for cas repression by CRP and provide evidence that endogenous spacers can repress phage P1 replication in E. coli.

  3. Integrated P1 Hohlraum/Capsule Simulations for NIF Experiments

    NASA Astrophysics Data System (ADS)

    Eder, David; Spears, Brian; Town, Richard; Jones, Oggie; Ma, Tammy; Pak, Arthur; Benedetti, Robin; Hatchett, Steve; Knauer, James; MacKinnon, Andrew; Yeamans, Charles; McNaney, James; Casey, Daniel

    2014-10-01

    We discuss integrated hohlraum/capsule post-shot simulations of two full-scale cryogenic NIF experiments that drove a DT symcap capsule downward/upward by having the peak power in the upper laser beams 16% greater/less than the lower beams. This laser asymmetry results in a radiation drive P1/P0 at the capsule ablation surface of ~2% and a downward/upward capsule velocity of order 100 microns/ns in agreement with the data. The experimental velocity is determined by comparing measurements at different locations of both the arrival times of DD and DT neutrons at time-of-flight detectors, and by zirconium activation measurements that are a function of neutron energy. We compare these two shots to a control shot for the same target with no specified laser asymmetries. We also discuss simulations of planned sub-scale warm symcap experiments that have a goal of measuring DT and DD ion temperatures and the electron temperature as a function of the imposed P1 to characterize the role of non-thermal velocity on temperature measurements. This work performed under the auspices of the U.S. Department of Energy by LLNL under Contract DE-AC52-07NA27344. LLNL-ABS-656659.

  4. 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

  5. A FaeG-FedF-LT192 fusion elicits protective anti-adhesin and antitoxin antibodies against porcine enterotoxigenic Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 or F18 fimbriae and heat-labile (LT) and/or heat-stable (ST) toxins are the major cause of diarrhea in young pigs. Effective vaccines inducing anti-adhesin (anti-K88 & anti-F18) and antitoxin (anti-LT & anti-ST) 5 immunity would provide ...

  6. Catalytic activity of nuclease P1: Experiment and theory

    SciTech Connect

    Miller, J.H.; Falcone, J.M.; Shibata, M.; Box, H.C.

    1994-10-01

    Nuclease P1 from Penicillium citrinum is a zinc dependent glyco-enzyme that recognizes single stranded DNA and RNA as substrates and hydrolyzes the phosphate ester bond. Nuclease Pl seems to recognize particular conformations of the phosphodiester backbone and shows significant variation in the rate of hydrolytic activity depending upon which nucleosides are coupled by the phosphodiester bond. The efficiency of nuclease Pl in hydrolyzing the phosphodiester bonds of a substrate can be altered by modifications to one of the substrate bases induced by ionizing radiation or oxidative stress. Measurements have been made of the effect of several radiation induced lesions on the catalytic rate of nuclease Pl. A model of the structure of the enzyme has been constructed in order to better understand the binding and activity of this enzyme on various ssDNA substrates.

  7. Proteus mirabilis uroepithelial cell adhesin (UCA) fimbria plays a role in the colonization of the urinary tract.

    PubMed

    Pellegrino, Rafael; Scavone, Paola; Umpiérrez, Ana; Maskell, Duncan J; Zunino, Pablo

    2013-03-01

    Urinary tract infections (UTIs) are among the most common bacterial infections in humans. Proteus mirabilis is an opportunistic pathogen, capable of causing severe UTIs, with serious kidney damage that may even lead to death. Several virulence factors are involved in the pathogenicity of this bacterium. Among these, adherence to the uroepithelium mediated by fimbriae appears to be a significant bacterial attribute related to urovirulence. Proteus mirabilis expresses several types of fimbriae that could be involved in the pathogenesis of UTI, including uroepithelial cell adhesin (UCA). In this report, we used an uropathogenic P. mirabilis wild-type strain and an isogenic ucaA mutant unable to express UCA to study the pathogenic role of this fimbria in UTI. Ability of the mutant to adhere to desquamated uroepithelial cells and to infect mice using different experimental UTI models was significantly impaired. These results allow us to conclude that P. mirabilis UCA plays an important role in the colonization of the urinary tract.

  8. 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

  9. 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.

  10. 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.

  11. Metal Selectivity of a Cd-, Co-, and Zn-Transporting P1B-type ATPase

    PubMed Central

    Smith, Aaron T.; Ross, Matthew O.; Hoffman, Brian M.; Rosenzweig, Amy C.

    2017-01-01

    The P1B-ATPases, a family of transmembrane metal transporters important for transition metal homeostasis in all organisms, are subdivided into classes based on sequence conservation and metal specificity. The multifunctional P1B-4-ATPase CzcP is part of the cobalt, zinc, and cadmium resistance system from the metal-tolerant, model organism Cupriavidus metallidurans. Previous work revealed the presence of an unusual soluble metal-binding domain (MBD) at the CzcP N-terminus, but the nature, extent, and selectivity of the transmembrane metal-binding site (MBS) of CzcP have not been resolved. Using homology modeling, we show that four wholly conserved amino acids from the transmembrane (TM) domain (Met254, Ser474, Cys476, and His807) are logical candidates for the TM MBS, which may communicate with the MBD via interactions with the first TM helix. Metal-binding analyses indicate that wild-type (WT) CzcP has three MBSs, and data on N-terminally truncated (ΔMBD) CzcP suggest the presence of a single TM MBS. Electronic absorption and electron paramagnetic resonance spectroscopic analyses of ΔMBD CzcP and variant proteins thereof provide insight into the details of Co2+ coordination by the TM MBS. These spectroscopic data, combined with in vitro functional studies of WT and variant CzcP proteins, show that the side chains of Met254, Cys476, and His807 contribute to Cd2+, Co2+, and Zn2+ binding and transport, whereas the side chain of Ser474 appears to play a minimal role. By comparison to other P1B-4-ATPases, we suggest that an evolutionarily adapted flexibility in the TM region likely afforded CzcP the ability to transport Cd2+ and Zn2+ in addition to Co2+. PMID:28001366

  12. The Shank3 Interaction Partner ProSAPiP1 Regulates Postsynaptic SPAR Levels and the Maturation of Dendritic Spines in Hippocampal Neurons

    PubMed Central

    Reim, Dominik; Weis, Tobias M.; Halbedl, Sonja; Delling, Jan Philipp; Grabrucker, Andreas M.; Boeckers, Tobias M.; Schmeisser, Michael J.

    2016-01-01

    The postsynaptic density or PSD is a submembranous compartment containing a wide array of proteins that contribute to both morphology and function of excitatory glutamatergic synapses. In this study, we have analyzed functional aspects of the Fezzin ProSAP-interacting protein 1 (ProSAPiP1), an interaction partner of the well-known PSD proteins Shank3 and SPAR. Using lentiviral-mediated overexpression and knockdown of ProSAPiP1, we found that this protein is dispensable for the formation of both pre- and postsynaptic specializations per se. We further show that ProSAPiP1 regulates SPAR levels at the PSD and the maturation of dendritic spines. In line with previous findings on the ProSAPiP1 homolog PSD-Zip70, we conclude that Fezzins essentially contribute to the maturation of excitatory spine synapses. PMID:27252646

  13. mRNA Capping by Venezuelan Equine Encephalitis Virus nsP1: Functional Characterization and Implications for Antiviral Research

    PubMed Central

    Li, Changqing; Guillén, Jaime; Rabah, Nadia; Blanjoie, Alexandre; Debart, Françoise; Vasseur, Jean-Jacques; Canard, Bruno; Decroly, Etienne

    2015-01-01

    ABSTRACT Alphaviruses are known to possess a unique viral mRNA capping mechanism involving the viral nonstructural protein nsP1. This enzyme harbors methyltransferase (MTase) and nsP1 guanylylation (GT) activities catalyzing the transfer of the methyl group from S-adenosylmethionine (AdoMet) to the N7 position of a GTP molecule followed by the formation of an m7GMP-nsP1 adduct. Subsequent transfer of m7GMP onto the 5′ end of the viral mRNA has not been demonstrated in vitro yet. Here we report the biochemical characterization of Venezuelan equine encephalitis virus (VEEV) nsP1. We have developed enzymatic assays uncoupling the different reactions steps catalyzed by nsP1. The MTase and GT reaction activities were followed using a nonhydrolyzable GTP (GIDP) substrate and an original Western blot assay using anti-m3G/m7G-cap monoclonal antibody, respectively. The GT reaction is stimulated by S-adenosyl-l-homocysteine (Ado-Hcy), the product of the preceding MTase reaction, and metallic ions. The covalent linking between nsP1 and m7GMP involves a phosphamide bond between the nucleotide and a histidine residue. Final guanylyltransfer onto RNA was observed for the first time with an alphavirus nsP1 using a 5′-diphosphate RNA oligonucleotide whose sequence corresponds to the 5′ end of the viral genome. Alanine scanning mutagenesis of residues H37, H45, D63, E118, Y285, D354, R365, N369, and N375 revealed their respective roles in MT and GT reactions. Finally, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA), and ribavirin triphosphate on MTase and capping reactions were investigated, providing possible avenues for antiviral research. IMPORTANCE Emergence or reemergence of alphaviruses represents a serious health concern, and the elucidation of their replication mechanisms is a prerequisite for the development of specific inhibitors targeting viral enzymes. In particular, alphaviruses are able, through an original reaction sequence, to add to their

  14. 26 CFR 31.3402(p)-1T - Voluntary Withholding Agreements (temporary).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...). 31.3402(p)-1T Section 31.3402(p)-1T Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... COLLECTION OF INCOME TAX AT SOURCE Collection of Income Tax at Source § 31.3402(p)-1T Voluntary Withholding Agreements (temporary). (a)-(b) For further guidance, see § 31.3402(p)-1(a) and (b). (c) Other payments....

  15. Isolation and characterisation of putative adhesins from Helicobacter pylori with affinity for heparan sulphate proteoglycan.

    PubMed

    Ruiz-Bustos, E; Ochoa, J L; Wadström, T; Ascencio, F

    2001-03-01

    A pool of heparan sulphate-binding proteins (HSBPs) from Helicobacter pylori culture supernates was obtained by sequential ammonium sulphate precipitation and affinity chromatography on heparin-Sepharose. The chromatographic procedure yielded one major fraction that contained proteins with heparan sulphate affinity as revealed by inhibition studies of heparan sulphate binding to H. pylori cells. Preparative iso-electric focusing, SDS-PAGE and blotting experiments, with peroxidase(POD)-labelled heparan sulphate as a probe, indicated the presence of two major extracellular proteins with POD-heparan sulphate affinity. One protein had a molecular mass of 66.2 kDa and a pI of 5.4, whilst the second protein had a molecular mass of 71.5 kDa and a pI of 5.0. The N-terminal amino acid sequence of the 71.5-kDa HSBP did not show homology to any other heparin-binding protein, nor to known proteins of H. pylori, whereas the 66.2-kDa HSBP showed a high homology to an Escherichia coli chaperon protein and equine haemoglobin. A third HSBP was isolated from an outer-membrane protein (OMP) fraction of H. pylori cells with a molecular mass of 47.2 kDa. The amino acid sequence of an internal peptide of the OMP-HSBP did not show homology to the extracellular HSBP of H. pylori, or to another microbial HSBP.

  16. H-deficient Bombay and para-Bombay red blood cells are most strongly agglutinated by the galactophilic lectins of Aplysia and Pseudomonas aeruginosa that detect I and P1 antigens.

    PubMed

    Gilboa-Garber, N; Sudakevitz, D; Levene, C; Rahimi-Levene, N; Yahalom, V

    2006-01-01

    The galactophilic lectins Aplysia gonad lectin (AGL) and Pseudomonas aeruginosa lectin (PA-IL), which detect human I and P1 RBC antigens, were examined for hemagglutination of H+ (group O and B) and H-deficient (Bombay and para-Bombay phenotype) RBCs. The results were compared with those obtained using two other galactophilic lectins, Maclura pomifera lectin (MPL) and Arachis hypogaea (peanut) agglutinin (PNA), which share T-antigen affinity, and two fucose-binding H-specific lectins, Ulex europaeus (UEA-I) and Pseudomonas aeruginosa lectin (PA-IIL), as well as with those achieved with anti-I serum. The results revealed that, in contrast to UEA-I and PA-IIL, which preferentially agglutinated H+ RBCs, and to MPL and PNA, which similarly agglutinated all examined RBCs, AGL, PA-IL, and the anti-I serum agglutinated the H-deficient RBCs more strongly than did the H+ RBCs. These findings could be attributed to increased levels of I and P1 antigens on those RBCs resulting from the use of the free common H-type 2 precursor for their synthesis. Since both PA-IL and PA-IIL are regarded as potential pathogen adhesins, it would be interesting to statistically compare the sensitivities of individuals of H+ and H-deficient RBC populations to P. aeruginosa infections.

  17. P(1B)-ATPases--an ancient family of transition metal pumps with diverse functions in plants.

    PubMed

    Williams, Lorraine E; Mills, Rebecca F

    2005-10-01

    P(1B)-ATPases form a distinct evolutionary sub-family of P-type ATPases, transporting transition metals such as Cu, Zn, Cd, Pb and Co across membranes in a wide range of organisms, including plants. Structurally they are distinct from other P-types, possessing eight transmembrane helices, a CPx/SPC motif in transmembrane domain six, and putative transition metal-binding domains at the N- and/or C-termini. Arabidopsis has eight P(1B)-ATPases (AtHMA1-AtHMA8), which differ in their structure, function and regulation. They perform a variety of important physiological tasks relating to transition metal transport and homeostasis. The crucial roles of plant P(1B)-ATPases in micronutrient nutrition, delivery of essential metals to target proteins, and toxic metal detoxification are discussed.

  18. Characterization of emb, a Gene Encoding the Major Adhesin of Streptococcus defectivus

    PubMed Central

    Manganelli, Riccardo; van de Rijn, Ivo

    1999-01-01

    Streptococcus defectivus is one of the nutritionally variant streptococci, a class of viridans group streptococci first isolated from patients with endocarditis and otitis media. In previous studies, NVS-47, a clinical isolate of S. defectivus, was shown to bind to the extracellular matrix. A high-molecular-weight surface protein was identified and proposed to be responsible for mediating this binding. In the present study, the gene encoding this protein was identified by transposon mutagenesis and characterized. The gene (emb) was found to be larger than 14 kb and was partially sequenced. It encodes a protein containing at least 50 repeats of 77 amino acids predicted to assume an alternating coiled-coil conformation. The domain responsible for extracellular matrix binding was mapped to the N terminus of the protein. From sequence analysis, Emb is proposed to be the prototype of a new family of streptococcal fibrillar proteins. PMID:9864195

  19. Pathogenesis of human diffusely adhering Escherichia coli expressing Afa/Dr adhesins (Afa/Dr DAEC): current insights and future challenges.

    PubMed

    Servin, Alain L

    2014-10-01

    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.

  20. 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

  1. Bartonella henselae trimeric autotransporter adhesin BadA expression interferes with effector translocation by the VirB/D4 type IV secretion system.

    PubMed

    Lu, Yun-Yueh; Franz, Bettina; Truttmann, Matthias C; Riess, Tanja; Gay-Fraret, Jérémie; Faustmann, Marco; Kempf, Volkhard A J; Dehio, Christoph

    2013-05-01

    The Gram-negative, zoonotic pathogen Bartonella henselae is the aetiological agent of cat scratch disease, bacillary angiomatosis and peliosis hepatis in humans. Two pathogenicity factors of B. henselae - each displaying multiple functions in host cell interaction - have been characterized in greater detail: the trimeric autotransporter Bartonella adhesin A (BadA) and the type IV secretion system VirB/D4 (VirB/D4 T4SS). BadA mediates, e.g. binding to fibronectin (Fn), adherence to endothelial cells (ECs) and secretion of vascular endothelial growth factor (VEGF). VirB/D4 translocates several Bartonella effector proteins (Beps) into the cytoplasm of infected ECs, resulting, e.g. in uptake of bacterial aggregates via the invasome structure, inhibition of apoptosis and activation of a proangiogenic phenotype. Despite this knowledge of the individual activities of BadA or VirB/D4 it is unknown whether these major virulence factors affect each other in their specific activities. In this study, expression and function of BadA and VirB/D4 were analysed in a variety of clinical B. henselae isolates. Data revealed that most isolates have lost expression of either BadA or VirB/D4 during in vitro passages. However, the phenotypic effects of coexpression of both virulence factors was studied in one clinical isolate that was found to stably coexpress BadA and VirB/D4, as well as by ectopic expression of BadA in a strain expressing VirB/D4 but not BadA. BadA, which forms a dense layer on the bacterial surface, negatively affected VirB/D4-dependent Bep translocation and invasome formation by likely preventing close contact between the bacterial cell envelope and the host cell membrane. In contrast, BadA-dependent Fn binding, adhesion to ECs and VEGF secretion were not affected by a functional VirB/D4 T4SS. The obtained data imply that the essential virulence factors BadA and VirB/D4 are likely differentially expressed during different stages of the infection cycle of

  2. DNA looping-dependent autorepression of LEE1 P1 promoters by Ler in enteropathogenic Escherichia coli (EPEC)

    PubMed Central

    Bhat, Abhayprasad; Shin, Minsang; Jeong, Jae-Ho; Kim, Hyun-Ju; Lim, Hyung-Ju; Rhee, Joon Haeng; Paik, Soon-Young; Takeyasu, Kunio; Tobe, Toru; Yen, Hilo; Lee, Gwangrog; Choy, Hyon E.

    2014-01-01

    Ler, a homolog of H-NS in enteropathogenic Escherichia coli (EPEC), plays a critical role in the expression of virulence genes encoded by the pathogenic island, locus of enterocyte effacement (LEE). Although Ler acts as an antisilencer of multiple LEE operons by alleviating H-NS–mediated silencing, it represses its own expression from two LEE1 P1 promoters, P1A and P1B, that are separated by 10 bp. Various in vitro biochemical methods were used in this study to elucidate the mechanism underlying transcription repression by Ler. Ler acts through two AATT motifs, centered at position −111.5 on the coding strand and at +65.5 on the noncoding strand, by simultaneously repressing P1A and P1B through DNA-looping. DNA-looping was visualized using atomic force microscopy. It is intriguing that an antisilencing protein represses transcription, not by steric exclusion of RNA polymerase, but by DNA-looping. We propose that the DNA-looping prevents further processing of open promoter complex (RPO) at these promoters during transcription initiation. PMID:24920590

  3. Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity.

    PubMed

    Akopian, Tatos; Kandror, Olga; Tsu, Christopher; Lai, Jack H; Wu, Wengen; Liu, Yuxin; Zhao, Peng; Park, Annie; Wolf, Lisa; Dick, Lawrence R; Rubin, Eric J; Bachovchin, William; Goldberg, Alfred L

    2015-04-24

    The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X3X2X1-aminomethylcoumarin) and by determining specificity constants (kcat/Km), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X1 position, basic residues or Trp in the X2 position, and Pro ≫ Ala > Trp in the X3 position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents.

  4. Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity*

    PubMed Central

    Akopian, Tatos; Kandror, Olga; Tsu, Christopher; Lai, Jack H.; Wu, Wengen; Liu, Yuxin; Zhao, Peng; Park, Annie; Wolf, Lisa; Dick, Lawrence R.; Rubin, Eric J.; Bachovchin, William; Goldberg, Alfred L.

    2015-01-01

    The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X3X2X1-aminomethylcoumarin) and by determining specificity constants (kcat/Km), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X1 position, basic residues or Trp in the X2 position, and Pro ≫ Ala > Trp in the X3 position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents. PMID:25759383

  5. Human diffusely adhering Escherichia coli expressing Afa/Dr adhesins that use human CD55 (decay-accelerating factor) as a receptor does not bind the rodent and pig analogues of CD55.

    PubMed

    Hudault, Sylvie; Spiller, O Brad; Morgan, B Paul; Servin, Alain L

    2004-08-01

    Afa/Dr diffusely adhering Escherichia coli (DAEC) bacteria that are responsible for recurrent urinary tract and gastrointestinal infections recognized as a receptor the glycosylphosphatidylinositol (GPI)-anchored protein decay-accelerating factor (DAF; CD55) at the brush border of cultured human intestinal cells. Results show that Afa/Dr DAEC C1845 bacteria were poorly associated with the mucosa of the gastrointestinal tract of infected mice. We conducted experiments with Chinese hamster ovary (CHO) cells stably transfected with mouse (GPI or transmembrane forms), pig, or human CD55 or mouse Crry cDNAs or transfected with empty vector pDR2EF1 alpha. Recombinant E. coli AAEC185 bacteria expressing Dr or F1845 adhesins bound strongly to CHO cells expressing human CD55 but not to the CHO cells expressing mouse (transmembrane and GPI anchored), rat, or pig CD55 or mouse Crry. Positive clustering of CD55 around Dr-positive bacteria was observed in human CD55-expressing CHO cells but not around the rarely adhering Dr-positive bacteria randomly distributed at the cell surface of CHO cells expressing mouse, rat, or pig CD55.

  6. Enhanced immunogenicity of pneumococcal surface adhesin A (PsaA) in mice via fusion to recombinant human B lymphocyte stimulator (BLyS)

    PubMed Central

    2011-01-01

    Background B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor superfamily of ligands that mediates its action through three known receptors. BLyS has been shown to enhance the production of antibodies against heterologous antigens when present at elevated concentrations, supporting an immunostimulatory role for BLyS in vivo. Methods We constructed a fusion protein consisting of human BLyS and Pneumococcal Surface Adhesin A (PsaA) and used this molecule to immunize mice. The immunostimulatory attributes mediated by BLyS in vivo were evaluated by characterizing immune responses directed against PsaA. Results The PsaA-BLyS fusion protein was able to act as a co-stimulant for murine spleen cell proliferation induced with F(ab')2 fragments of anti-IgM in vitro in a fashion similar to recombinant BLyS, and immunization of mice with the PsaA-BLyS fusion protein resulted in dramatically elevated serum antibodies specific for PsaA. Mice immunized with PsaA admixed with recombinant BLyS exhibited only modest elevations in PsaA-specific responses following two immunizations, while mice immunized twice with PsaA alone exhibited undetectable PsaA-specific serum antibody responses. Sera obtained from PsaA-BLyS immunized mice exhibited high titers of IgG1, IgG2a, IgG2b, and IgG3, but no IgA, while mice immunized with PsaA admixed with BLyS exhibited only elevated titers of IgG1 following two immunizations. Splenocytes from PsaA-BLyS immunized mice exhibited elevated levels of secretion of IL-2, IL-4 and IL-5, and a very modest but consistent elevation of IFN-γ following in vitro stimulation with PsaA. In contrast, mice immunized with either PsaA admixed with BLyS or PsaA alone exhibited modestly elevated to absent PsaA-specific recall responses for the same cytokines. Mice deficient for one of the three receptors for BLyS designated Transmembrane activator, calcium modulator, and cyclophilin ligand [CAML] interactor (TACI) exhibited attenuated Psa

  7. A native Zn/Cd transporting P1B ATPase from natural overexpression in a hyperaccumulator plant reveals post-translational processing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    TcHMA4 is a P1B-type ATPase that is highly expressed in the Cd/Zn hyperaccumulator plant Thlaspi caerulescens and contains a C-terminal 9-histidine repeat. After isolation from roots, we purified TcHMA4 protein via metal affinity chromatography. The purified protein exhibited Cd- and Zn activated AT...

  8. A genome-wide association study identifies a horizontally transferred bacterial surface adhesin gene associated with antimicrobial resistant strains

    PubMed Central

    Suzuki, Masato; Shibayama, Keigo; Yahara, Koji

    2016-01-01

    Carbapenems are a class of last-resort antibiotics; thus, the increase in bacterial carbapenem-resistance is a serious public health threat. Acinetobacter baumannii is one of the microorganisms that can acquire carbapenem-resistance; it causes severe nosocomial infection, and is notoriously difficult to control in hospitals. Recently, a machine-learning approach was first used to analyze the genome sequences of hundreds of susceptible and resistant A. baumannii strains, including those carrying commonly acquired resistant mechanisms, to build a classifier that can predict strain resistance. A complementary approach is to explore novel genetic elements that could be associated with the antimicrobial resistance of strains, independent of known mechanisms. Therefore, we carefully selected A. baumannii strains, spanning various genotypes, from public genome databases, and conducted the first genome-wide association study (GWAS) of carbapenem resistance. We employed a recently developed method, capable of identifying any kind of genetic variation and accounting for bacterial population structure, and evaluated its effectiveness. Our study identified a surface adhesin gene that had been horizontally transferred to an ancestral branch of A. baumannii, as well as a specific region of that gene that appeared to accumulate multiple individual variations across the different branches of carbapenem-resistant A. baumannii strains. PMID:27892531

  9. Assessment of Pasteurella multocida A Lipopolysaccharide, as an Adhesin in an In Vitro Model of Rabbit Respiratory Epithelium

    PubMed Central

    Romero, Stefany; Esquinas, Paula; Patiño, Pilar; Martínez, Nhora

    2017-01-01

    The role of the P. multocida lipopolysaccharide (LPS) as a putative adhesin during the early stages of infection with this bacterium in the respiratory epithelium of rabbits was investigated. By light microscopy and double enzyme labeling of nasal septa tissues, the amount of bacteria attached to the respiratory epithelium and the amount of LPS present in goblet cells at different experimental times were estimated. Transmission electron microscopy (TEM) and LPS labeling with colloidal gold particles were also used to determine the exact location of LPS in the cells. Septa that were challenged with LPS of P. multocida and 30 minutes later with P. multocida showed more adherent bacteria and more severe lesions than the other treatments. Free LPS was observed in the lumen of the nasal septum, forming bilamellar structures and adhering to the cilia, microvilli, cytoplasmic membrane, and cytoplasm of epithelial ciliated and goblet cells. The above findings suggest that P. multocida LPS plays an important role in the process of bacterial adhesion and that it has the ability of being internalized into host cells. PMID:28251016

  10. 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.

  11. An introduction to protein moonlighting.

    PubMed

    Jeffery, Constance J

    2014-12-01

    Moonlighting proteins comprise a class of multifunctional proteins in which a single polypeptide chain performs multiple physiologically relevant biochemical or biophysical functions. Almost 300 proteins have been found to moonlight. The known examples of moonlighting proteins include diverse types of proteins, including receptors, enzymes, transcription factors, adhesins and scaffolds, and different combinations of functions are observed. Moonlighting proteins are expressed throughout the evolutionary tree and function in many different biochemical pathways. Some moonlighting proteins can perform both functions simultaneously, but for others, the protein's function changes in response to changes in the environment. The diverse examples of moonlighting proteins already identified, and the potential benefits moonlighting proteins might provide to the organism, such as through coordinating cellular activities, suggest that many more moonlighting proteins are likely to be found. Continuing studies of the structures and functions of moonlighting proteins will aid in predicting the functions of proteins identified through genome sequencing projects, in interpreting results from proteomics experiments, in understanding how different biochemical pathways interact in systems biology, in annotating protein sequence and structure databases, in studies of protein evolution and in the design of proteins with novel functions.

  12. Highly selective and potent agonists of sphingosine-1-phosphate 1 (S1P1) receptor.

    PubMed

    Vachal, Petr; Toth, Leslie M; Hale, Jeffrey J; Yan, Lin; Mills, Sander G; Chrebet, Gary L; Koehane, Carol A; Hajdu, Richard; Milligan, James A; Rosenbach, Mark J; Mandala, Suzanne

    2006-07-15

    Novel series of sphingosine-1-phosphate (S1P) receptor agonists were developed through a systematic SAR aimed to achieve high selectivity for a single member of the S1P family of receptors, S1P1. The optimized structure represents a highly S1P1-selective and efficacious agonist: S1P1/S1P2, S1P1/S1P3, S1P1/S1P4>10,000-fold, S1P1/S1P5>600-fold, while EC50 (S1P1) <0.2 nM. In vivo experiments are consistent with S1P1 receptor agonism alone being sufficient for achieving desired lymphocyte-lowering effect.

  13. Differential FoxP2 and FoxP1 expression in a vocal learning nucleus of the developing budgerigar

    PubMed Central

    Whitney, Osceola; Voyles, Tawni; Hara, Erina; Chen, Qianqian; White, Stephanie A.; Wright, Timothy F.

    2014-01-01

    The forkhead domain FOXP2 and FOXP1 transcription factors are implicated in several cognitive disorders with language deficits, notably autism, and thus play a central role in learned vocal motor behavior in humans. Although a similar role for FoxP2 and FoxP1 is proposed for other vertebrate species, including songbirds, the neurodevelopmental expression of these genes are unknown in a species with lifelong vocal learning abilities. Like humans, budgerigars (Melopsittacus undulatus) learn new vocalizations throughout their entire lifetime. Like songbirds, budgerigars have distinct brain nuclei for vocal learning, which include the magnocellular nucleus of the medial striatum (MMSt), a basal ganglia region that is considered developmentally and functionally analogous to Area X in songbirds. Here we used in situ hybridization and immunohistochemistry to investigate FoxP2 and FoxP1 expression in the MMSt of juvenile and adult budgerigars. We found FoxP2 mRNA and protein expression levels in the MMSt that were lower than the surrounding striatum throughout development and adulthood. In contrast, FoxP1 mRNA and protein had an elevated MMSt/striatum expression ratio as birds matured, regardless of their sex. These results show that life-long vocal plasticity in budgerigars is associated with persistent low-level FoxP2 expression in the budgerigar MMSt, and suggests the possibility that FoxP1 plays an organizational role in the neurodevelopment of vocal motor circuitry. Thus, developmental regulation of the FoxP2 and FoxP1 genes in the basal ganglia appears essential for vocal mimicry in a range of species that possess this relatively rare trait. PMID:25407828

  14. Differential FoxP2 and FoxP1 expression in a vocal learning nucleus of the developing budgerigar.

    PubMed

    Whitney, Osceola; Voyles, Tawni; Hara, Erina; Chen, Qianqian; White, Stephanie A; Wright, Timothy F

    2015-07-01

    The forkhead domain FOXP2 and FOXP1 transcription factors are implicated in several cognitive disorders with language deficits, notably autism, and thus play a central role in learned vocal motor behavior in humans. Although a similar role for FoxP2 and FoxP1 is proposed for other vertebrate species, including songbirds, the neurodevelopmental expression of these genes are unknown in a species with lifelong vocal learning abilities. Like humans, budgerigars (Melopsittacus undulatus) learn new vocalizations throughout their entire lifetime. Like songbirds, budgerigars have distinct brain nuclei for vocal learning, which include the magnocellular nucleus of the medial striatum (MMSt), a basal ganglia region that is considered developmentally and functionally analogous to Area X in songbirds. Here, we used in situ hybridization and immunohistochemistry to investigate FoxP2 and FoxP1 expression in the MMSt of juvenile and adult budgerigars. We found FoxP2 mRNA and protein expression levels in the MMSt that were lower than the surrounding striatum throughout development and adulthood. In contrast, FoxP1 mRNA and protein had an elevated MMSt/striatum expression ratio as birds matured, regardless of their sex. These results show that life-long vocal plasticity in budgerigars is associated with persistent low-level FoxP2 expression in the budgerigar MMSt, and suggests the possibility that FoxP1 plays an organizational role in the neurodevelopment of vocal motor circuitry. Thus, developmental regulation of the FoxP2 and FoxP1 genes in the basal ganglia appears essential for vocal mimicry in a range of species that possess this relatively rare trait.

  15. Functional divergence between the two P1-P2 stalk dimers on the ribosome in their interaction with ricin A chain.

    PubMed

    Grela, Przemysław; Li, Xiao-Ping; Tchórzewski, Marek; Tumer, Nilgun E

    2014-05-15

    The eukaryotic stalk, which is responsible for the recruitment of translation factors, is a pentamer containing two P1-P2 dimers with unclear modes of action. In Saccharomyces cerevisiae, P1/P2 proteins (individual P1 and P2 proteins) are organized into two distinct dimers, P1A-P2B and P1B-P2A. To investigate the functional contribution of each dimer on the ribosome, RTA (ricin A chain), which binds to the stalk to depurinate the SRL (sarcin/ricin loop), was used as a molecular probe in yeast mutants in which the binding site for one or the other dimer on P0 was deleted. Ribosome depurination and toxicity of RTA were greatly reduced in mutants containing only P1A-P2B on the ribosome, whereas those with only P1B-P2A were reduced less in depurination and were unaffected in toxicity. Ribosomes bearing P1B-P2A were depurinated by RTA at a similar level as wild-type, but ribosomes bearing P1A-P2B were depurinated at a much lower level in vitro. The latter ribosomes showed the lowest association and almost no dissociation with RTA by surface plasmon resonance. These results indicate that the P1B-P2A dimer is more critical for facilitating the access of RTA to the SRL, providing the first in vivo evidence for functional divergence between the two stalk dimers on the ribosome.

  16. An internal thioester in a pathogen surface protein mediates covalent host binding.

    PubMed

    Walden, Miriam; Edwards, John M; Dziewulska, Aleksandra M; Bergmann, Rene; Saalbach, Gerhard; Kan, Su-Yin; Miller, Ona K; Weckener, Miriam; Jackson, Rosemary J; Shirran, Sally L; Botting, Catherine H; Florence, Gordon J; Rohde, Manfred; Banfield, Mark J; Schwarz-Linek, Ulrich

    2015-06-02

    To cause disease and persist in a host, pathogenic and commensal microbes must adhere to tissues. Colonization and infection depend on specific molecular interactions at the host-microbe interface that involve microbial surface proteins, or adhesins. To date, adhesins are only known to bind to host receptors non-covalently. Here we show that the streptococcal surface protein SfbI mediates covalent interaction with the host protein fibrinogen using an unusual internal thioester bond as a 'chemical harpoon'. This cross-linking reaction allows bacterial attachment to fibrin and SfbI binding to human cells in a model of inflammation. Thioester-containing domains are unexpectedly prevalent in Gram-positive bacteria, including many clinically relevant pathogens. Our findings support bacterial-encoded covalent binding as a new molecular principle in host-microbe interactions. This represents an as yet unexploited target to treat bacterial infection and may also offer novel opportunities for engineering beneficial interactions.

  17. Sphingosine-1-phosphate (S1P) displays sustained S1P1 receptor agonism and signaling through S1P lyase-dependent receptor recycling.

    PubMed

    Gatfield, John; Monnier, Lucile; Studer, Rolf; Bolli, Martin H; Steiner, Beat; Nayler, Oliver

    2014-07-01

    The sphingosine-1-phosphate (S1P) type 1 receptor (S1P1R) is a novel therapeutic target in lymphocyte-mediated autoimmune diseases. S1P1 receptor desensitization caused by synthetic S1P1 receptor agonists prevents T-lymphocyte egress from secondary lymphoid organs into the circulation. The selective S1P1 receptor agonist ponesimod, which is in development for the treatment of autoimmune diseases, efficiently reduces peripheral lymphocyte counts and displays efficacy in animal models of autoimmune disease. Using ponesimod and the natural ligand S1P, we investigated the molecular mechanisms leading to different signaling, desensitization and trafficking behavior of S1P1 receptors. In recombinant S1P1 receptor-expressing cells, ponesimod and S1P triggered Gαi protein-mediated signaling and β-arrestin recruitment with comparable potency and efficiency, but only ponesimod efficiently induced intracellular receptor accumulation. In human umbilical vein endothelial cells (HUVEC), ponesimod and S1P triggered translocation of the endogenous S1P1 receptor to the Golgi compartment. However, only ponesimod treatment caused efficient surface receptor depletion, receptor accumulation in the Golgi and degradation. Impedance measurements in HUVEC showed that ponesimod induced only short-lived Gαi protein-mediated signaling followed by resistance to further stimulation, whereas S1P induced sustained Gαi protein-mediated signaling without desensitization. Inhibition of S1P lyase activity in HUVEC rendered S1P an efficient S1P1 receptor internalizing compound and abrogated S1P-mediated sustained signaling. This suggests that S1P lyase - by facilitating S1P1 receptor recycling - is essential for S1P-mediated sustained signaling, and that synthetic agonists are functional antagonists because they are not S1P lyase substrates.

  18. 26 CFR 1.72(p)-1 - Loans treated as distributions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 2 2010-04-01 2010-04-01 false Loans treated as distributions. 1.72(p)-1 Section 1.72(p)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Items Specifically Included in Gross Income § 1.72(p)-1...

  19. 26 CFR 31.3306(p)-1 - Employees of related corporations.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 15 2013-04-01 2013-04-01 false Employees of related corporations. 31.3306(p)-1 Section 31.3306(p)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SOURCE Federal Unemployment Tax Act (Chapter 23, Internal Revenue Code of 1954) § 31.3306(p)-1...

  20. 26 CFR 1.409(p)-1 - Prohibited allocation of securities in an S corporation.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... corporation. 1.409(p)-1 Section 1.409(p)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... Plans, Etc. § 1.409(p)-1 Prohibited allocation of securities in an S corporation. (a) Organization of this section and definition—(1) Organization of this section. Section 409(p) applies if a...

  1. 26 CFR 31.3306(p)-1 - Employees of related corporations.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 15 2011-04-01 2011-04-01 false Employees of related corporations. 31.3306(p)-1 Section 31.3306(p)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SOURCE Federal Unemployment Tax Act (Chapter 23, Internal Revenue Code of 1954) § 31.3306(p)-1...

  2. 26 CFR 1.72(p)-1 - Loans treated as distributions.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 2 2011-04-01 2011-04-01 false Loans treated as distributions. 1.72(p)-1 Section 1.72(p)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Items Specifically Included in Gross Income § 1.72(p)-1...

  3. 26 CFR 1.409(p)-1 - Prohibited allocation of securities in an S corporation.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... corporation. 1.409(p)-1 Section 1.409(p)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... Plans, Etc. § 1.409(p)-1 Prohibited allocation of securities in an S corporation. (a) Organization of this section and definition—(1) Organization of this section. Section 409(p) applies if a...

  4. 26 CFR 31.3306(p)-1 - Employees of related corporations.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 15 2012-04-01 2012-04-01 false Employees of related corporations. 31.3306(p)-1 Section 31.3306(p)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SOURCE Federal Unemployment Tax Act (Chapter 23, Internal Revenue Code of 1954) § 31.3306(p)-1...

  5. 26 CFR 31.3306(p)-1 - Employees of related corporations.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 15 2014-04-01 2014-04-01 false Employees of related corporations. 31.3306(p)-1 Section 31.3306(p)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SOURCE Federal Unemployment Tax Act (Chapter 23, Internal Revenue Code of 1954) § 31.3306(p)-1...

  6. 26 CFR 1.409(p)-1 - Prohibited allocation of securities in an S corporation.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... corporation. 1.409(p)-1 Section 1.409(p)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... Plans, Etc. § 1.409(p)-1 Prohibited allocation of securities in an S corporation. (a) Organization of this section and definition—(1) Organization of this section. Section 409(p) applies if a...

  7. Identification of Lysine Residues in the Borrelia burgdorferi DbpA Adhesin Required for Murine Infection

    PubMed Central

    Fortune, Danielle E.; Lin, Yi-Pin; Deka, Ranjit K.; Groshong, Ashley M.; Moore, Brendan P.; Hagman, Kayla E.; Leong, John M.; Tomchick, Diana R.

    2014-01-01

    Decorin-binding protein A (DbpA) of Borrelia burgdorferi mediates bacterial adhesion to heparin and dermatan sulfate associated with decorin. Lysines K82, K163, and K170 of DbpA are known to be important for in vitro interaction with decorin, and the DbpA structure, initially solved by nuclear magnetic resonance (NMR) spectroscopy, suggests these lysine residues colocalize in a pocket near the C terminus of the protein. In the current study, we solved the structure of DbpA from B. burgdorferi strain 297 using X-ray crystallography and confirmed the existing NMR structural data. In vitro binding experiments confirmed that recombinant DbpA proteins with mutations in K82, K163, or K170 did not bind decorin, which was due to an inability to interact with dermatan sulfate. Most importantly, we determined that the in vitro binding defect observed upon mutation of K82, K163, or K170 in DbpA also led to a defect during infection. The infectivity of B. burgdorferi expressing individual dbpA lysine point mutants was assessed in mice challenged via needle inoculation. Murine infection studies showed that strains expressing dbpA with mutations in K82, K163, and K170 were significantly attenuated and could not be cultured from any tissue. Proper expression and cellular localization of the mutated DbpA proteins were examined, and NMR spectroscopy determined that the mutant DbpA proteins were structurally similar to wild-type DbpA. Taken together, these data showed that lysines K82, K163, and K170 potentiate the binding of DbpA to dermatan sulfate and that an interaction(s) mediated by these lysines is essential for B. burgdorferi murine infection. PMID:24842928

  8. Amino Acid Features of P1B-ATPase Heavy Metal Transporters Enabling Small Numbers of Organisms to Cope with Heavy Metal Pollution

    PubMed Central

    Ashrafi, E.; Alemzadeh, A.; Ebrahimi, M.; Ebrahimie, E.; Dadkhodaei, N.; Ebrahimi, M.

    2011-01-01

    Phytoremediation refers to the use of plants for extraction and detoxification of pollutants, providing a new and powerful weapon against a polluted environment. In some plants, such as Thlaspi spp, heavy metal ATPases are involved in overall metal ion homeostasis and hyperaccumulation. P1B-ATPases pump a wide range of cations, especially heavy metals, across membranes against their electrochemical gradients. Determination of the protein characteristics of P1B-ATPases in hyperaccumulator plants provides a new opportuntity for engineering of phytoremediating plants. In this study, using diverse weighting and modeling approaches, 2644 protein characteristics of primary, secondary, and tertiary structures of P1B-ATPases in hyperaccumulator and nonhyperaccumulator plants were extracted and compared to identify differences between proteins in hyperaccumulator and nonhyperaccumulator pumps. Although the protein characteristics were variable in their weighting, tree and rule induction models; glycine count, frequency of glutamine-valine, and valine-phenylalanine count were the most important attributes highlighted by 10, five, and four models, respectively. In addition, a precise model was built to discriminate P1B-ATPases in different organisms based on their structural protein features. Moreover, reliable models for prediction of the hyperaccumulating activity of unknown P1B-ATPase pumps were developed. Uncovering important structural features of hyperaccumulator pumps in this study has provided the knowledge required for future modification and engineering of these pumps by techniques such as site-directed mutagenesis. PMID:21573033

  9. 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.

  10. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  11. Role of dorsal root ganglion K2P1.1 in peripheral nerve injury-induced neuropathic pain

    PubMed Central

    Mao, Qingxiang; Yuan, Jingjing; Xiong, Ming; Wu, Shaogen; Chen, Liyong; Bekker, Alex; Yang, Tiande

    2017-01-01

    Peripheral nerve injury-caused hyperexcitability and abnormal ectopic discharges in the primary sensory neurons of dorsal root ganglion (DRG) play a key role in neuropathic pain development and maintenance. The two-pore domain background potassium (K2P) channels have been identified as key determinants of the resting membrane potential and neuronal excitability. However, whether K2P channels contribute to neuropathic pain is still elusive. We reported here that K2P1.1, the first identified mammalian K2P channel, was highly expressed in mouse DRG and distributed in small-, medium-, and large-sized DRG neurons. Unilateral lumbar (L) 4 spinal nerve ligation led to a significant and time-dependent reduction of K2P1.1 mRNA and protein in the ipsilateral L4 DRG, but not in the contralateral L4 or ipsilateral L3 DRG. Rescuing this reduction through microinjection of adeno-associated virus-DJ expressing full-length K2P1.1 mRNA into the ipsilateral L4 DRG blocked spinal nerve ligation-induced mechanical, thermal, and cold pain hypersensitivities during the development and maintenance periods. This DRG viral microinjection did not affect acute pain and locomotor function. Our findings suggest that K2P1.1 participates in neuropathic pain development and maintenance and may be a potential target in the management of this disorder. PMID:28326939

  12. Intracellular Signaling and Desmoglein 2 Shedding Triggered by Human Adenoviruses Ad3, Ad14, and Ad14P1

    PubMed Central

    Wang, Hongjie; Ducournau, Corinne; Saydaminova, Kamola; Richter, Maximilian; Yumul, Roma; Ho, Martin; Carter, Darrick; Zubieta, Chloé

    2015-01-01

    ABSTRACT We recently discovered that desmoglein 2 (DSG2) is a receptor for human adenovirus species B serotypes Ad3, Ad7, Ad11, and Ad14. Ad3 is considered to be a widely distributed human pathogen. Ad3 binding to DSG2 triggers the transient opening of epithelial junctions. Here, we further delineate the mechanism that leads to DSG2-mediated epithelial junction opening in cells exposed to Ad3 and recombinant Ad3 fiber proteins. We identified an Ad3 fiber knob-dependent pathway that involves the phosphorylation of mitogen-activated protein (MAP) kinases triggering the activation of the matrix-metalloproteinase ADAM17. ADAM17, in turn, cleaves the extracellular domain of DSG2 that links epithelial cells together. The shed DSG2 domain can be detected in cell culture supernatant and also in serum of mice with established human xenograft tumors. We then extended our studies to Ad14 and Ad14P1. Ad14 is an important research and clinical object because of the recent appearance of a new, more pathogenic strain (Ad14P1). In a human epithelial cancer xenograft model, Ad14P1 showed more efficient viral spread and oncolysis than Ad14. Here, we tested the hypothesis that a mutation in the Ad14P1 fiber knob could account for the differences between the two strains. While our X-ray crystallography studies suggested an altered three-dimensional (3D) structure of the Ad14P1 fiber knob in the F-G loop region, this did not significantly change the fiber knob affinity to DSG2 or the intracellular signaling and DSG2 shedding in epithelial cancer cells. IMPORTANCE A number of widely distributed adenoviruses use the epithelial junction protein DSG2 as a receptor for infection and lateral spread. Interaction with DSG2 allows the virus not only to enter cells but also to open epithelial junctions which form a physical barrier to virus spread. Our study elucidates the mechanism beyond virus-triggered junction opening with a focus on adenovirus serotype 3. Ad3 binds to DSG2 with its fiber

  13. Protein

    MedlinePlus

    ... Search for: Harvard T.H. Chan School of Public Health Email People Departments Calendar Careers Give my.harvard ... Nutrition Source Harvard T.H. Chan School of Public Health > The Nutrition Source > What Should I Eat? > Protein ...

  14. Protein

    MedlinePlus

    ... Go lean with protein. • Choose lean meats and poultry. Lean beef cuts include round steaks (top loin, ... main dishes. • Use nuts to replace meat or poultry, not in addition to meat or poultry (i. ...

  15. Streptococcus mutans extracellular DNA is upregulated during growth in biofilms, actively released via membrane vesicles, and influenced by components of the protein secretion machinery.

    PubMed

    Liao, Sumei; Klein, Marlise I; Heim, Kyle P; Fan, Yuwei; Bitoun, Jacob P; Ahn, San-Joon; Burne, Robert A; Koo, Hyun; Brady, L Jeannine; Wen, Zezhang T

    2014-07-01

    Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA.

  16. Synthesis of chitooligomer-based glycoconjugates and their binding to the rat natural killer cell activation receptor NKR-P1.

    PubMed

    Semenuk, T; Krist, P; Pavlícek, J; Bezouska, K; Kuzma, M; Novák, P; Kren, V

    2001-10-01

    NKR-P1 protein is an important activating receptor at the surface of the rat natural killer cells. GlcNAc and chitooligomers were identified as strong activation ligands in vitro and in vivo. Their clustering brings about increase of their affinity to the NKR-P1 by 3-6 orders. Here we describe novel methodology for preparation of neoglycoproteins based on BSA carrying the chitooligomers (n = 2-5). Further on we developed novel methodology of the coupling of glycosylamines via aromatic-SCN activated linker both to protein or synthetic cores. Inhibition studies of chitooligomer glycoconjugates with the NKR-P1 receptor show that our neoglycoproteins are very strong ligands with high binding affinity (-log IC(50) = 13-15). In analogy with our previous observations with GlcNAc clustered on protein or PAMAM backbones the synthetic chitooligomer clusters should provide considerably better ligands in the in vivo antitumor treatment.

  17. A native Zn/Cd pumping P(1B) ATPase from natural overexpression in a hyperaccumulator plant.

    PubMed

    Parameswaran, Aravind; Leitenmaier, Barbara; Yang, Mingjie; Kroneck, Peter M H; Welte, Wolfram; Lutz, Gabriela; Papoyan, Ashot; Kochian, Leon V; Küpper, Hendrik

    2007-11-09

    We report here the first purification of a P(1B) type ATPase, a group of transporters that occurs in bacteria, plants and animals incl. humans, from a eukaryotic organism in native state. TcHMA4 is a P(1B) type ATPase that is highly expressed in the Cd/Zn hyperaccumulator plant Thlaspi caerulescens and contains a C-terminal 9-histidine repeat. After isolation from roots, we purified TcHMA4 protein via metal affinity chromatography. The purified protein exhibited Cd- and Zn-activated ATPase activity after reconstitution into lipid vesicles, showing that it was in its native state. Gels of crude root extract and of the purified protein revealed TcHMA4-specific bands of about 50 and 60kDa, respectively, while the TcHMA4 mRNA predicts a single protein with a size of 128kDa. This indicates the occurrence of post-translational processing; the properties of the two bands were characterised by their activity and binding properties.

  18. A Repetitive DNA Element Regulates Expression of the Helicobacter pylori Sialic Acid Binding Adhesin by a Rheostat-like Mechanism

    PubMed Central

    Vallström, Anna; Olofsson, Annelie; Öhman, Carina; Rakhimova, Lena; Borén, Thomas; Engstrand, Lars; Brännström, Kristoffer; Arnqvist, Anna

    2014-01-01

    During persistent infection, optimal expression of bacterial factors is required to match the ever-changing host environment. The gastric pathogen Helicobacter pylori has a large set of simple sequence repeats (SSR), which constitute contingency loci. Through a slipped strand mispairing mechanism, the SSRs generate heterogeneous populations that facilitate adaptation. Here, we present a model that explains, in molecular terms, how an intergenically located T-tract, via slipped strand mispairing, operates with a rheostat-like function, to fine-tune activity of the promoter that drives expression of the sialic acid binding adhesin, SabA. Using T-tract variants, in an isogenic strain background, we show that the length of the T-tract generates multiphasic output from the sabA promoter. Consequently, this alters the H. pylori binding to sialyl-Lewis x receptors on gastric mucosa. Fragment length analysis of post-infection isolated clones shows that the T-tract length is a highly variable feature in H. pylori. This mirrors the host-pathogen interplay, where the bacterium generates a set of clones from which the best-fit phenotypes are selected in the host. In silico and functional in vitro analyzes revealed that the length of the T-tract affects the local DNA structure and thereby binding of the RNA polymerase, through shifting of the axial alignment between the core promoter and UP-like elements. We identified additional genes in H. pylori, with T- or A-tracts positioned similar to that of sabA, and show that variations in the tract length likewise acted as rheostats to modulate cognate promoter output. Thus, we propose that this generally applicable mechanism, mediated by promoter-proximal SSRs, provides an alternative mechanism for transcriptional regulation in bacteria, such as H. pylori, which possesses a limited repertoire of classical trans-acting regulatory factors. PMID:24991812

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

    PubMed Central

    Luo, Qingwei; Vickers, Tim J.

    2016-01-01

    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

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

    PubMed

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

    2014-01-01

    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.

  1. The glycosphingolipid P1 is an ovarian cancer-associated carbohydrate antigen involved in migration

    PubMed Central

    Jacob, F; Anugraham, M; Pochechueva, T; Tse, B W C; Alam, S; Guertler, R; Bovin, N V; Fedier, A; Hacker, N F; Huflejt, M E; Packer, N; Heinzelmann-Schwarz, V A

    2014-01-01

    Background: The level of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P1 trisaccharide has previously been shown to significantly discriminate between ovarian cancer patients and healthy women. Here we aim to identify the Ig class that causes this discrimination, to identify on cancer cells the corresponding P1 antigen recognised by circulating anti-P1 antibodies and to shed light into the possible function of this glycosphingolipid. Methods: An independent Australian cohort was assessed for the presence of anti-P1 IgG and IgM class antibodies using suspension array. Monoclonal and human derived anti-glycan antibodies were verified using three independent glycan-based immunoassays and flow cytometry-based inhibition assay. The P1 antigen was detected by LC-MS/MS and flow cytometry. FACS-sorted cell lines were studied on the cellular migration by colorimetric assay and real-time measurement using xCELLigence system. Results: Here we show in a second independent cohort (n=155) that the discrimination of cancer patients is mediated by the IgM class of anti-P1 antibodies (P=0.0002). The presence of corresponding antigen P1 and structurally related epitopes in fresh tissue specimens and cultured cancer cells is demonstrated. We further link the antibody and antigen (P1) by showing that human naturally circulating and affinity-purified anti-P1 IgM isolated from patients ascites can bind to naturally expressed P1 on the cell surface of ovarian cancer cells. Cell-sorted IGROV1 was used to obtain two study subpopulations (P1-high, 66.1% and P1-low, 33.3%) and observed that cells expressing high P1-levels migrate significantly faster than those with low P1-levels. Conclusions: This is the first report showing that P1 antigen, known to be expressed on erythrocytes only, is also present on ovarian cancer cells. This suggests that P1 is a novel tumour-associated carbohydrate antigen recognised by the immune system in patients and may have a role in cell

  2. Dose of house dust mite antigen (P1) inhaled by infants aged one month

    SciTech Connect

    Carswell, F.; Clark, J.; Robinson, P.; Platts-Mills, T.A.

    1983-11-01

    A survey of the habitats occupied by 12 infants of one month of age revealed that approximately 10% of their day was spent in conditions of potential exposure to the major (P1) allergen of the house dust mite, Dermatophagoides pteronyssinus. A respiratory pump which reproduced the minute ventilation of an infant was placed in representative infant habitats. The P1 allergen trapped by the filter in this pump was measured as an estimate of infants' allergen intake. Detectable P1 intake was only present when there was active air disturbance (bed making and vacuuming). The average P1 intake was approximately 3 ng P1/24 hours. Comparison of this P1 intake with that which sensitizes in other situations suggests that it is usually inadequate to sensitize infants.

  3. Discovery of a novel series of potent S1P1 agonists.

    PubMed

    Crosignani, Stefano; Bombrun, Agnes; Covini, David; Maio, Maurizio; Marin, Delphine; Quattropani, Anna; Swinnen, Dominique; Simpson, Don; Sauer, Wolfgang; Françon, Bernard; Martin, Thierry; Cambet, Yves; Nichols, Anthony; Martinou, Isabelle; Burgat-Charvillon, Fabienne; Rivron, Delphine; Donini, Cristina; Schott, Olivier; Eligert, Valerie; Novo-Perez, Laurence; Vitte, Pierre-Alain; Arrighi, Jean-François

    2010-03-01

    The discovery of a novel series of S1P1 agonists is described. Starting from a micromolar HTS positive, iterative optimization gave rise to several single-digit nanomolar S1P1 agonists. The compounds were able to induce internalization of the S1P1 receptor, and a selected compound was shown to be able to induce lymphopenia in mice after oral dosing.

  4. Biochemical characterization of three distinct polygalacturonases from Neosartorya fischeri P1.

    PubMed

    Pan, Xia; Li, Ke; Ma, Rui; Shi, Pengjun; Huang, Huoqing; Yang, Peilong; Meng, Kun; Yao, Bin

    2015-12-01

    Polygalacturonase is one of the most important industrial pectinases. To enrich the genetic resources and develop new enzyme candidates, three polygalacturonase genes (Nfpg I-III) of glycosyl hydrolase family 28 were cloned from Neosartorya fischeri P1 and functionally expressed in Pichia pastoris. The purified recombinant proteins exhibited some distinguished properties. In comparison with other counterparts, NfPG I showed the highest specific activity (40, 123 U/mg), NfPG II had the highest temperature optimum (65 °C), and the pH optimum of NfPG III was the lowest (3.5). The orders of their thermostability and resistance to chemicals tested were NfPG II>NfPG III>NfPG I and NfPG II>NfPG I>NfPG III, respectively. Combinations of these enzymes showed better performance than individuals in the processing and clarification of apple and strawberry juice. These results suggest that N. fischeri polygalacturonases have great application potentials in the food industry for juice production.

  5. 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

  6. 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.

  7. Engineering and Dissecting the Glycosylation Pathway of a Streptococcal Serine-rich Repeat Adhesin*

    PubMed Central

    Zhu, Fan; Zhang, Hua; Yang, Tiandi; Haslam, Stuart M.; Dell, Anne; Wu, Hui

    2016-01-01

    Serine-rich repeat glycoproteins (SRRPs) are conserved in Gram-positive bacteria. They are crucial for modulating biofilm formation and bacterial-host interactions. Glycosylation of SRRPs plays a pivotal role in the process; thus understanding the glycosyltransferases involved is key to identifying new therapeutic drug targets. The glycosylation of Fap1, an SRRP of Streptococcus parasanguinis, is mediated by a gene cluster consisting of six genes: gtf1, gtf2, gly, gtf3, dGT1, and galT2. Mature Fap1 glycan possesses the sequence of Rha1–3Glc1-(Glc1–3GlcNAc1)-2,6-Glc1–6GlcNAc. Gtf12, Gtf3, and dGT1 are responsible for the first four steps of the Fap1 glycosylation, catalyzing the transfer of GlcNAc, Glc, Glc, and GlcNAc residues to the protein backbone sequentially. The role of GalT2 and Gly in the Fap1 glycosylation is unknown. In the present study, we synthesized the fully modified Fap1 glycan in Escherichia coli by incorporating all six genes from the cluster. This study represents the first reconstitution of an exogenous stepwise O-glycosylation synthetic pathway in E. coli. In addition, we have determined that GalT2 mediates the fifth step of the Fap1 glycosylation by adding a rhamnose residue, and Gly mediates the final glycosylation step by transferring glucosyl residues. Furthermore, inactivation of each glycosyltransferase gene resulted in differentially impaired biofilms of S. parasanguinis, demonstrating the importance of Fap1 glycosylation in the biofilm formation. The Fap1 glycosylation system offers an excellent model to engineer glycans using different permutations of glycosyltransferases and to investigate biosynthetic pathways of SRRPs because SRRP genetic loci are highly conserved. PMID:28039332

  8. Structural basis of Lewisb antigen binding by the Helicobacter pylori adhesin BabA

    PubMed Central

    Hage, Naim; Howard, Tina; Phillips, Chris; Brassington, Claire; Overman, Ross; Debreczeni, Judit; Gellert, Paul; Stolnik, Snow; Winkler, G. Sebastiaan; Falcone, Franco H.

    2015-01-01

    Helicobacter pylori is a leading cause of peptic ulceration and gastric cancer worldwide. To achieve colonization of the stomach, this Gram-negative bacterium adheres to Lewisb (Leb) antigens in the gastric mucosa using its outer membrane protein BabA. Structural information for BabA has been elusive, and thus, its molecular mechanism for recognizing Leb antigens remains unknown. We present the crystal structure of the extracellular domain of BabA, from H. pylori strain J99, in the absence and presence of Leb at 2.0- and 2.1-Å resolutions, respectively. BabA is a predominantly α-helical molecule with a markedly kinked tertiary structure containing a single, shallow Leb binding site at its tip within a β-strand motif. No conformational change occurs in BabA upon binding of Leb, which is characterized by low affinity under acidic [KD (dissociation constant) of ~227 μM] and neutral (KD of ~252 μM) conditions. Binding is mediated by a network of hydrogen bonds between Leb Fuc1, GlcNAc3, Fuc4, and Gal5 residues and a total of eight BabA amino acids (C189, G191, N194, N206, D233, S234, S244, and T246) through both carbonyl backbone and side-chain interactions. The structural model was validated through the generation of two BabA variants containing N206A and combined D233A/S244A substitutions, which result in a reduction and complete loss of binding affinity to Leb, respectively. Knowledge of the molecular basis of Leb recognition by BabA provides a platform for the development of therapeutics targeted at inhibiting H. pylori adherence to the gastric mucosa. PMID:26601230

  9. Investigation of engineered bacterial adhesins for opportunity to interface cells with abiotic materials

    NASA Astrophysics Data System (ADS)

    Terrell, Jessica L.; Dong, Hong; Holthoff, Ellen L.; Small, Meagan C.; Sarkes, Deborah A.; Hurley, Margaret M.; Stratis-Cullum, Dimitra N.

    2016-05-01

    The convenience of cellular genetic engineering has afforded the power to build `smart' synthetic biological tools with novel applications. Here, we have explored opportunities to hybridize engineered cells with inorganic materials toward the development of 'living' device-compatible systems. Cellular structural biology is engineerable based on the ability to rewrite genetic code to generate recombinant, foreign, or even unnatural proteins. With this capability on the biological end, it should be possible to achieve superior abio-compatibility with the inorganic materials that compose current microfabricated technology. This work investigated the hair-like appendages of Escherichia coli known as Type 1 fimbriae that enable natural adhesion to glycosylated substrates. Sequence alterations within the fimbrial gene cluster were found to be well-tolerated, evidenced by tagging the fimbriae with peptide-based probes. As a further development, fimbriae tips could be reconfigured to, in turn, alter cell binding. In particular, the fimbriae were fused with a genetically optimized peptide-for-inorganics to enable metal binding. This work established methodologies to systematically survey cell adhesion properties across a suite of fimbriae-modified cell types as well as to direct patterned cell adhesion. Cell types were further customized for added complexity including turning on secondary gene expression and binding to gold surfaces. The former demonstrates potential for programmable gene switches and the latter for interfacing biology with inorganic materials. In general, the incorporation of 'programmed' cells into devices can be used to provide the feature of dynamic and automated cell response. The outcomes of this study are foundational toward the critical feature of deliberate positioning of cells as configurable biocomponentry. Overall, cellular integration into bioMEMs will yield advanced sensing and actuation.

  10. Nutrient sequestration from wastewater by using zeolite Na-P1 synthesized from coal fly ash.

    PubMed

    He, Hongfu; Xu, Suyun; Han, Runqi; Wang, Qiuchen

    2017-04-01

    The objective of this study is to investigate the cation exchange property of the product zeolite Na-P1 (Z-P1) synthesized from coal fly ash (FA) by the alkali hydrothermal reaction, and to evaluate the water purification ability for the simultaneous removal of ammonium and phosphate. High-purity Z-P1 was obtained by optimizing the reaction conditions of aging time and crystallization temperature, and using FA particles of uniform particle size. Kinetic ammonium sorption experiments with Z-P1 were well described by both the Langmuir and Freundlich models, and the maximum adsorption capacity of the Z-P1 was 23.15 mg/g. Moreover, in order to determine the effect of magnesium intervention on the ammonium and phosphate removal from simulated swine wastewater, two forms of magnesium were studied, that is Mg-saturated Z-P1 and direct use of Mg(2+) source with Z-P1, as compared with the control (sole Z-P1). Results showed that Mg(2+) addition could improve phosphate removal efficiency significantly by forming struvite. Furthermore, dosing Z-P1 with dissolved Mg(2+) was better than Mg-saturated Z-P1 in terms of ammonium and phosphate removal efficiencies, and the preparation cost. When dosing 20 g/L Z-P1 with 4 mM Mg(2+), ammonium and phosphate removal efficiencies reached 65.2% and 92.3% after 30 min.

  11. Genome sequence of the phage clP1, which infects the beer spoilage bacterium Pediococcus damnosus.

    PubMed

    Kelly, David; O'Sullivan, Orla; Mills, Susan; McAuliffe, Olivia; Ross, R Paul; Neve, Horst; Coffey, Aidan

    2012-08-01

    Pediococcus damnosus (P. damnosus) bacteriophage (phage) clP1 is a novel virulent phage isolated from a municipal sewage sample collected in Southern Ireland. This phage infects the beer spoilage strain P. damnosus P82 which was isolated from German breweries. Sequencing of the phage has revealed a linear double stranded DNA genome of 38,013 base pairs (bp) with an overall GC content of 47.6%. Fifty seven open reading frames (ORFs) were identified of which 30 showed homology to previously sequenced proteins, and as a consequence 20 of these were assigned predicted functions. The majority of genes displayed homology with genes from the Lactobacillus plantarum phage phiJL-1. All genes were located on the same coding strand and in the same orientation. Morphological characterisation placed phage clP1 as a member of the Siphoviridae family with an isometric head (59 nm diameter) and non-contractile tail (length 175 nm; diameter 10nm. Interestingly, the phage clP1 genome was found to share very limited identity with other phage genome sequences in the database, and was hence considered unique. This was highlighted by the genome organisation which differed slightly to the consensus pattern of genomic organisation usually found in Siphoviridae phages. With the genetic machinery present for a lytic lifecycle and the absence of potential endotoxin factors, this phage may have applications in the biocontrol of beer spoilage bacteria. To our knowledge, this study represents the first reported P. damnosus phage genome sequence.

  12. The viral capping enzyme nsP1: a novel target for the inhibition of chikungunya virus infection

    PubMed Central

    Delang, L.; Li, C.; Tas, A.; Quérat, G.; Albulescu, I. C.; De Burghgraeve, T.; Guerrero, N. A. Segura; Gigante, A.; Piorkowski, G.; Decroly, E.; Jochmans, D.; Canard, B.; Snijder, E. J.; Pérez-Pérez, M. J.; van Hemert, M. J.; Coutard, B.; Leyssen, P.; Neyts, J.

    2016-01-01

    The chikungunya virus (CHIKV) has become a substantial global health threat due to its massive re-emergence, the considerable disease burden and the lack of vaccines or therapeutics. We discovered a novel class of small molecules ([1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones) with potent in vitro activity against CHIKV isolates from different geographical regions. Drug-resistant variants were selected and these carried a P34S substitution in non-structural protein 1 (nsP1), the main enzyme involved in alphavirus RNA capping. Biochemical assays using nsP1 of the related Venezuelan equine encephalitis virus revealed that the compounds specifically inhibit the guanylylation of nsP1. This is, to the best of our knowledge, the first report demonstrating that the alphavirus capping machinery is an excellent antiviral drug target. Considering the lack of options to treat CHIKV infections, this series of compounds with their unique (alphavirus-specific) target offers promise for the development of therapy for CHIKV infections. PMID:27545976

  13. Complete Genome Sequence of the Pyrene-Degrading Bacterium Cycloclasticus sp. Strain P1

    PubMed Central

    Lai, Qiliang; Li, Weiwei; Wang, Baojiang; Yu, Zhiwei

    2012-01-01

    Cycloclasticus sp. strain P1 was isolated from deep-sea sediments of the Pacific Ocean and characterized as a unique bacterium in the degradation of pyrene, a four-ring polycyclic aromatic hydrocarbon (PAH). Here we report the complete genome of P1 and genes associated with PAH degradation. PMID:23144416

  14. A genome-wide regulatory framework identifies maize Pericarp Color1 (P1) controlled genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    P1 encodes an R2R3-MYB transcription factor responsible for the accumulation of insecticidal flavones in maize silks and red phlobaphene pigments in pericarps and other floral tissues. Using genome-wide expression analyses (RNA-Seq) in pericarps and silks of plants with contrasting P1 alleles combin...

  15. Including an Exam P/1 Prep Course in a Growing Actuarial Science Program

    ERIC Educational Resources Information Center

    Wakefield, Thomas P.

    2014-01-01

    The purpose of this article is to describe the actuarial science program at our university and the development of a course to enhance students' problem solving skills while preparing them for Exam P/1 of the Society of Actuaries (SOA) and the Casualty Actuary Society (CAS). The Exam P/1 prep course, formally titled Mathematical Foundations of…

  16. A multi-repeat adhesin of the phytopathogen, Pectobacterium atrosepticum, is secreted by a Type I pathway and is subject to complex regulation involving a non-canonical diguanylate cyclase.

    PubMed

    Pérez-Mendoza, Daniel; Coulthurst, Sarah J; Humphris, Sonia; Campbell, Emma; Welch, Martin; Toth, Ian K; Salmond, George P C

    2011-11-01

    Cyclic diguanylate (c-di-GMP) is a second messenger controlling many important bacterial processes. The phytopathogen Pectobacterium atrosepticum SCRI1043 (Pba1043) possesses a Type I secretion system (T1SS) essential for the secretion of a proteinaceous multi-repeat adhesin (MRP) required for binding to the host plant. The genes encoding the MRP and the T1SS are tightly linked to genes encoding several putative c-di-GMP regulatory components. We show that c-di-GMP regulates secreted MRP levels in Pba1043 through the action of two genes encoding predicted diguanylate cyclase (DGC) and phosphodiesterase proteins (ECA3270 and ECA3271). Phenotypic analyses and quantification of c-di-GMP levels demonstrated that ECA3270 and ECA3271 regulate secreted MRP levels by increasing and decreasing, respectively, the intracellular levels of c-di-GMP. Moreover, ECA3270 represents the first active DGC reported to have an alternative active-site motif from the 'canonical' GG[D/E]EF. ECA3270 has an A-site motif of SGDEF and analysis of single amino acid replacements demonstrated that the first position of this motif can tolerate functional substitution. Serine in position one of the A-site is also observed in many other DGCs. Finally, another T1SS-linked regulator (ECA3265) also plays an important role in regulating secreted MRP, with an altered localization of MRP observed in an ECA3265 mutant background. Mutants defective in these three T1SS-linked regulators exhibit a reduction in root binding and virulence, confirming that this complex, finely tuned regulation system is crucial in the interaction with host plants.

  17. [Structure and function of heavy metal transporter P(1B)-ATPase in plant: a review].

    PubMed

    Zhang, Yuxiu; Zhang, Yuanya; Sun, Tao; Chai, Tuanyao

    2010-06-01

    The regulation of the heavy-metal accumulation in vivo for plant survival is very complex. The metal cation transporter plays key roles in the metabolic process. P(1B)-ATPases are the only subgroup of P-ATPases that contribute to heavy metal homeostasis presented in most organisms. Arabidopsis thaliana contains eight genes encoding P(1B)-ATPases. The current reports show that the functions of P(1B)-ATPases are involved in maintaining metal homeostasis, transporting and detoxification in plants. P(1B)-ATPases not only mediated metal ion mobilization and uptake in roots, but also contribute to the metal transport, storage and tolerance in shoots, especially in heavy metal hyperaccumulators. In this paper, we reviewed and discussed the evolution, classification, structure and function of P(1B)-ATPases in plants. HMAs-transgenic manipulation could be a feasible approach for phytoremediation and mineral nutrition fortification.

  18. Pathophysiological Consequences of a Break in S1P1-Dependent Homeostasis of Vascular Permeability Revealed by S1P1 Competitive Antagonism

    PubMed Central

    Bigaud, Marc; Dincer, Zuhal; Bollbuck, Birgit; Dawson, Janet; Beckmann, Nicolau; Beerli, Christian; Fishli-Cavelti, Gina; Nahler, Michaela; Angst, Daniela; Janser, Philipp; Otto, Heike; Rosner, Elisabeth; Hersperger, Rene; Bruns, Christian; Quancard, Jean

    2016-01-01

    Rational Homeostasis of vascular barriers depends upon sphingosine 1-phosphate (S1P) signaling via the S1P1 receptor. Accordingly, S1P1 competitive antagonism is known to reduce vascular barrier integrity with still unclear pathophysiological consequences. This was explored in the present study using NIBR-0213, a potent and selective S1P1 competitive antagonist. Results NIBR-0213 was tolerated at the efficacious oral dose of 30 mg/kg BID in the rat adjuvant-induced arthritis (AiA) model, with no sign of labored breathing. However, it induced dose-dependent acute vascular pulmonary leakage and pleural effusion that fully resolved within 3–4 days, as evidenced by MRI monitoring. At the supra-maximal oral dose of 300 mg/kg QD, NIBR-0213 impaired lung function (with increased breathing rate and reduced tidal volume) within the first 24 hrs. Two weeks of NIBR-0213 oral dosing at 30, 100 and 300 mg/kg QD induced moderate pulmonary changes, characterized by alveolar wall thickening, macrophage accumulation, fibrosis, micro-hemorrhage, edema and necrosis. In addition to this picture of chronic inflammation, perivascular edema and myofiber degeneration observed in the heart were also indicative of vascular leakage and its consequences. Conclusions Overall, these observations suggest that, in the rat, the lung is the main target organ for the S1P1 competitive antagonism-induced acute vascular leakage, which appears first as transient and asymptomatic but could lead, upon chronic dosing, to lung remodeling with functional impairments. Hence, this not only raises the question of organ specificity in the homeostasis of vascular barriers, but also provides insight into the pre-clinical evaluation of a potential safety window for S1P1 competitive antagonists as drug candidates. PMID:28005953

  19. Features of Two New Proteins with OmpA-Like Domains Identified in the Genome Sequences of Leptospira interrogans

    PubMed Central

    Teixeira, Aline F.; de Morais, Zenaide M.; Kirchgatter, Karin; Romero, Eliete C.; Vasconcellos, Silvio A.; Nascimento, Ana Lucia T. O.

    2015-01-01

    Leptospirosis is an acute febrile disease caused by pathogenic spirochetes of the genus Leptospira. It is considered an important re-emerging infectious disease that affects humans worldwide. The knowledge about the mechanisms by which pathogenic leptospires invade and colonize the host remains limited since very few virulence factors contributing to the pathogenesis of the disease have been identified. Here, we report the identification and characterization of two new leptospiral proteins with OmpA-like domains. The recombinant proteins, which exhibit extracellular matrix-binding properties, are called Lsa46 - LIC13479 and Lsa77 - LIC10050 (Leptospiral surface adhesins of 46 and 77 kDa, respectively). Attachment of Lsa46 and Lsa77 to laminin was specific, dose dependent and saturable, with KD values of 24.3 ± 17.0 and 53.0 ± 17.5 nM, respectively. Lsa46 and Lsa77 also bind plasma fibronectin, and both adhesins are plasminogen (PLG)-interacting proteins, capable of generating plasmin (PLA) and as such, increase the proteolytic ability of leptospires. The proteins corresponding to Lsa46 and Lsa77 are present in virulent L. interrogans L1-130 and in saprophyte L. biflexa Patoc 1 strains, as detected by immunofluorescence. The adhesins are recognized by human leptospirosis serum samples at the onset and convalescent phases of the disease, suggesting that they are expressed during infection. Taken together, our data could offer valuable information to the understanding of leptospiral pathogenesis. PMID:25849456

  20. HAT-P-1: A Direct Glimpse into the Atmosphere of a Hot Jupiter

    NASA Astrophysics Data System (ADS)

    Holman, Matthew

    2009-07-01

    The HATNet project has discovered a transiting planet that is an extremely valuable target for reflected light observations {Bakos et al. 2006}. HAT-P-1b, with mass M_p=0.53 +/- 0.04 M_Jup, and radius R_p=1.20 +/- 0.05 R_Jup {Winn et al. 2007}, has a density comparable to that of HD 209458b. However, HAT-P-1b's P=4.46536 day orbital period is longer than that of HD 209458b. It is expected that the cloud composition and particulate size distribution of HAT-P-1b will differ from that of HD 209458b, due to the larger semimajor axis and lower effective temperature of HAT-P-1b. The resulting geometric albedo for HAT-P-1b should be larger than that of HD 209458b. Furthermore, HAT-P-1 orbits one component of a wide binary {ADS 16402A and ADS 16402B are G0V stars with 11.2" at 1.39 pc}, making this an ideal target for ultra-precise differential photometry. Therefore, we propose ACS/HRC slitless grism photometry near times of its secondary eclipse to make the first detection of reflected light from an extrasolar planet.Note: this program was awarded DD time in Cycle 15 but the observations were not executed before the ACS failed.Holman and Bakos are co-PIs on this proposal.

  1. Differences in the P1' substrate specificities of pepsin A and chymosin.

    PubMed

    Kageyama, Hakuto; Ueda, Hiroshi; Tezuka, Takafumi; Ogasawara, Asami; Narita, Yuichi; Kageyama, Takashi; Ichinose, Masao

    2010-02-01

    Porcine pepsin A and bovine chymosin are typical models of aspartic proteinases. The hydrolytic specificities of these proteinases, along with those of human pepsin A and monkey chymosin, were investigated with 29 peptide substrates that included various P1' variants of seven parent peptides. From these peptides, AFPLEF downward arrow FREL was preferred by pepsin A and chymosin, while its P1' variant, AFPLEF downward arrow EREL was preferred by bovine chymosin. Porcine and human pepsin A showed similar hydrolytic specificities, strongly preferring a hydrophobic/aromatic residue at P1' of any type of peptide. This specificity is well explained by the very hydrophobic nature of the S1' subsite that consists of Tyr(189), Ile(213), Ile(300), Met(289), Val/Leu(291) and Leu(298). The first three residues are well conserved in pepsin family enzymes. Although bovine and monkey chymosin showed similar P1' specificity, bovine chymosin preferred peptides having Glu at P1', while monkey chymosin preferred peptides having Lys at P1'. The dual characteristics of chymosin are due to the occurrence of polar/charged residues in the S1' subsite, such as Glu/Asp(289), Gln(298) and Lys/Gln(299), which are different from the S1' subsite of pepsin A. Molecular models suggest that Glu in position 289 of bovine chymosin and Asp in position 289 of monkey chymosin are responsible for the difference in P1' specificities between the chymosins.

  2. Backup Expression of the PhaP2 Phasin Compensates for phaP1 Deletion in Herbaspirillum seropedicae, Maintaining Fitness and PHB Accumulation

    PubMed Central

    Alves, Luis P. S.; Teixeira, Cícero S.; Tirapelle, Evandro F.; Donatti, Lucélia; Tadra-Sfeir, Michelle Z.; Steffens, Maria B. R.; de Souza, Emanuel M.; de Oliveira Pedrosa, Fabio; Chubatsu, Leda S.; Müller-Santos, Marcelo

    2016-01-01

    Phasins are important proteins controlling poly-3-hydroxybutyrate (PHB) granules formation, their number into the cell and stability. The genome sequencing of the endophytic and diazotrophic bacterium Herbaspirillum seropedicae SmR1 revealed two homologous phasin genes. To verify the role of the phasins on PHB accumulation in the parental strain H. seropedicae SmR1, isogenic strains defective in the expression of phaP1, phaP2 or both genes were obtained by gene deletion and characterized in this work. Despite of the high sequence similarity between PhaP1 and PhaP2, PhaP1 is the major phasin in H. seropedicae, since its deletion reduced PHB accumulation by ≈50% in comparison to the parental and ΔphaP2. Upon deletion of phaP1, the expression of phaP2 was sixfold enhanced in the ΔphaP1 strain. The responsive backup expression of phaP2 partially rescued the ΔphaP1 mutant, maintaining about 50% of the parental PHB level. The double mutant ΔphaP1.2 did not accumulate PHB in any growth stage and showed a severe reduction of growth when glucose was the carbon source, a clear demonstration of negative impact in the fitness. The co-occurrence of phaP1 and phaP2 homologous in bacteria relatives of H. seropedicae, including other endophytes, indicates that the mechanism of phasin compensation by phaP2 expression may be operating in other organisms, showing that PHB metabolism is a key factor to adaptation and efficiency of endophytic bacteria. PMID:27242754

  3. Backup Expression of the PhaP2 Phasin Compensates for phaP1 Deletion in Herbaspirillum seropedicae, Maintaining Fitness and PHB Accumulation.

    PubMed

    Alves, Luis P S; Teixeira, Cícero S; Tirapelle, Evandro F; Donatti, Lucélia; Tadra-Sfeir, Michelle Z; Steffens, Maria B R; de Souza, Emanuel M; de Oliveira Pedrosa, Fabio; Chubatsu, Leda S; Müller-Santos, Marcelo

    2016-01-01

    Phasins are important proteins controlling poly-3-hydroxybutyrate (PHB) granules formation, their number into the cell and stability. The genome sequencing of the endophytic and diazotrophic bacterium Herbaspirillum seropedicae SmR1 revealed two homologous phasin genes. To verify the role of the phasins on PHB accumulation in the parental strain H. seropedicae SmR1, isogenic strains defective in the expression of phaP1, phaP2 or both genes were obtained by gene deletion and characterized in this work. Despite of the high sequence similarity between PhaP1 and PhaP2, PhaP1 is the major phasin in H. seropedicae, since its deletion reduced PHB accumulation by ≈50% in comparison to the parental and ΔphaP2. Upon deletion of phaP1, the expression of phaP2 was sixfold enhanced in the ΔphaP1 strain. The responsive backup expression of phaP2 partially rescued the ΔphaP1 mutant, maintaining about 50% of the parental PHB level. The double mutant ΔphaP1.2 did not accumulate PHB in any growth stage and showed a severe reduction of growth when glucose was the carbon source, a clear demonstration of negative impact in the fitness. The co-occurrence of phaP1 and phaP2 homologous in bacteria relatives of H. seropedicae, including other endophytes, indicates that the mechanism of phasin compensation by phaP2 expression may be operating in other organisms, showing that PHB metabolism is a key factor to adaptation and efficiency of endophytic bacteria.

  4. Purification and Concentration of Nanoparticles Using Diafiltration: Scientific Operating Procedure Series: SOP-P-1

    DTIC Science & Technology

    2015-07-01

    Diafiltration Scientific Operating Procedure Series: SOP-P-1 En vi ro nm en ta l L ab or at or y Lesley F. Miller and Mark A. Chappell July 2015... Scientific Operating Procedure Series: SOP-P-1 Lesley F. Miller and Mark A. Chappell Environmental Laboratory U.S. Army Engineer Research and Development...describes a Scientific Operating Procedure (SOP) and outlines the recommended steps to cleanse and purify nanomaterial solutions for use in laboratory

  5. Synthesis of the Bycroft-Gowland structure of micrococcin P1.

    PubMed

    Ciufolini, M A; Shen, Y C

    1999-12-02

    [formula: see text] We describe the chemical synthesis of the accepted structure of micrococcin P1, a member of the thiostrepton group of antibiotics, and we show that this architecture does not correspond to that of the natural product. Methods developed during the present study should greatly facilitate ongoing efforts centering on the determination of the actual structure of microccin P1, in addition to being applicable to the synthesis of more complex thiostrepton congeners.

  6. Design and Synthesis of Hydroxyethylene-Based BACE-1 Inhibitors Incorporating Extended P1 Substituents

    PubMed Central

    Sandgren, Veronica; Bäck, Marcus; Kvarnström, Ingemar; Dahlgren, Anders

    2013-01-01

    Novel BACE-1 inhibitors with a hydroxyethylene central core have been developed. Modified P1´ and extended P1 substituents were incorporated with the aim to explore potential interactions with the S1´ and the S1-S3 pocket, respectively, of BACE-1. Inhibitors were identified displaying IC50 values in the nanomolar range, i.e. 69 nM for the most potent compound. Possible inhibitor interactions with the enzyme are also discussed. PMID:23585822

  7. One SUMO is sufficient to silence the dimeric potassium channel K2P1

    PubMed Central

    Plant, Leigh D.; Dementieva, Irina S.; Kollewe, Astrid; Olikara, Sonia; Marks, Jeremy D.; Goldstein, Steve A. N.

    2010-01-01

    Small ubiquitin modifier 1 (SUMO1) is shown to regulate K2P1 background channels in the plasma membrane (PM) of live mammalian cells. Confocal microscopy reveals native SUMO1, SAE1, and Ubc9 (the enzymes that activate and conjugate SUMO1) at PM where SUMO1 and expressed human K2P1 are demonstrated to colocalize. Silent K2P1 channels in excised PM patches are activated by SUMO isopeptidase (SENP1) and resilenced by SUMO1. K2P1-Lys274 is crucial: when mutated to Gln, Arg, Glu, Asp, Cys, or Ala, the channels are constitutively active and insensitive to SUMO1 and SENP1. Tandem mass spectrometry confirms conjugation of SUMO1 to the ε-amino group of Lys274 in vitro. FRET microscopy shows that assembly of K2P1 and SUMO1 requires Lys274. Single-particle TIRF microscopy shows that wild-type channels in PM have two K2P1 subunits and assemble with two SUMO1 monomers. Although channels engineered with one Lys274 site carry just one SUMO1 they are activated and silenced by SENP1 and SUMO1 like wild-type channels. PMID:20498050

  8. Comparison of buffers for extraction of mite allergen der p 1 from dust.

    PubMed

    Prester, Ljerka; Kovačić, Jelena; Macan, Jelena

    2012-09-01

    Der p 1 is the main allergen of house dust mite Dermatophagoides pteronyssinus, which has routinely been detected in residential dust. However, the procedure for extracting Der p 1 from reservoir dust has not been well defined. The aim of this study was to compare Der p 1 mass fractions in dust extracts prepared using the following extraction buffers: phosphate (pH 7.4), borate (pH 8.0), and ammonium bicarbonate (pH 8.0), all with 0.05 % Tween 20. Twenty-eight dust samples were divided into three aliquots and each portion was extracted with one of the three buffers at room temperature. Der p 1 mass fractions were measured in a total of 84 dust extracts using the enzyme immunoassay (range: 0.1 μg g-1 to 7.53 μg g-1). Statistical methods including intraclass correlation showed a high agreement between Der p 1 mass fractions irrespective of the extracting medium. Our results suggest that all three buffers are suitable for the extraction of mite allergens and routine Der p 1 analysis in dust.

  9. Recombination between plasmids of incompatibility groups P-1 and P-2.

    PubMed Central

    Jacoby, G A; Jacob, A E; Hedges, R W

    1976-01-01

    R plasmids of incompatibility group P-2 are readily transmissible between Pseudomonas strains, but not to Escherichia coli or other enterobacteria, whereas those of group P-1 have a broad host range. Pseudomonas aeruginosa donor strains carrying both a P-1 plasmid (RP1, RP4, or R751) and a P-2 plasmid (pMG1, pMG2, pMG5, or RPL11) were mated with E. coli K-12, and selection was imposed for resistance markers on the P-2 plasmids. Transconjugants were obtained at a low frequency, in which P-2 markers were expressed and were serially transmissible in E. coli together with P-1 markers. These plasmids had P-1 incompatibility properties, conferred susceptibility to phages active on P-1 carrying strains, and behaved on sucrose gradient centrifugation as unimolecular species of higher molecular weights than the P-1 parent. Recombinant plasmid formation was independent of a functional Rec gene in both donor and recipient and, with R751, had a preferred site leading to loss of trimethoprim resistance. Interaction between insertion sequences may be involved. Thus, plasmids of group P-2 can recombine with R factors of another group quite separate in compatibility properties, host range, and pilus type. Formation of such recombinants provides one pathway by which the genetic diversity of plasmids may have evolved. PMID:821925

  10. Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae

    PubMed Central

    Tacchi, Jessica L.; Raymond, Benjamin B. A.; Haynes, Paul A.; Berry, Iain J.; Widjaja, Michael; Bogema, Daniel R.; Woolley, Lauren K.; Jenkins, Cheryl; Minion, F. Chris; Padula, Matthew P.; Djordjevic, Steven P.

    2016-01-01

    Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC–MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity. PMID:26865024

  11. Cloning and expression of diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase from Lupinus angustifolius L.

    PubMed Central

    Maksel, D; Guranowski, A; Ilgoutz, S C; Moir, A; Blackburn, M G; Gayler, K R

    1998-01-01

    The first isolation, cloning and expression of cDNA encoding an asymmetric diadenosine 5',5'''P1,P4-tetraphosphate pyrophosphohydrolase (Ap4A hydrolase) from a higher plant is described. Ap4A hydrolase protein was purified from seeds of both Lupinus luteus and Lupinus angustifolius and partially sequenced. The Ap4A hydrolase cDNA was cloned from L. angustifolius cotyledonary polyadenylated RNA using reverse transcription and PCR with primers based on the amino acid sequence. The cDNA encoded a protein of 199 amino acids, molecular mass 22982Da. When expressed in Escherichia coli fused to a maltose-binding protein, the enzyme catalysed asymmetric cleavage of Ap4A to AMP and ATP which was inhibited at concentrations of F- as low as 3 microM. These are properties characteristic of Ap4A hydrolase (asymmetrical) (EC 3.6.1. 17). Comparison of the Ap4A hydrolase sequences derived from the four known cDNAs from pig, human, lupin and fission yeast showed that, like the mammalian hydrolase, the lupin enzyme possesses a Mut T motif but no other significant similarities. No sequence similarity to the human fragile histidine triad protein, as found in the Ap4A hydrolase from Schizosaccharomyces pombe, was detected in the Ap4A hydrolase from lupin. PMID:9425114

  12. 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

  13. Mannose-inhibitable adhesins and T3-T7 receptors of Klebsiella pneumoniae inhibit phagocytosis and intracellular killing by human polymorphonuclear leukocytes.

    PubMed Central

    Pruzzo, C; Debbia, E; Satta, G

    1982-01-01

    It has recently been shown that Klebsiella pneumoniae strains adhere to human epithelial cells and that adherence is mediated by mannose-inhibitable adhesins which are also receptors for coliphages T3 and T7. We have now found that Klebsiella strain K59, which adheres to human epithelial cells and carries the receptors for coliphages T3 and T7, adheres to human polymorphonuclear leukocytes (PMN) at 4 degrees C. Strains KRTT1 and KRTT2, which are spontaneous mutants unable to adsorb coliphages T3 and T7 and adhere to human epithelial cells, at this temperature did not adhere to PMN. Adherence of K59 cells to PMN at 4 degrees C was inhibited by D-mannose, by UV-inactivated T7 phages, and by pepsin-digested anti-K59 antibodies absorbed with KRTT1 cells. At 37 degrees C the number of PMN with KRTT bacteria associated was fourfold higher than at 4 degrees C. On the contrary, the number of PMN with K59 bacteria associated at this temperature was fourfold lower than at 4 degrees C. Phagocytosis and intracellular killing experiments performed at 37 degrees C showed that KRTT1 and KRTT2 were phagocytized and killed at a higher rate than K59. After blocking of the mannose-inhibitable adhesins and T3-T7 receptors (MIAT) by D-mannose, UV-inactivated bacteriophage T7, or specific antibodies, K59 cells became more sensitive to phagocytosis and intracellular killing at 37 degrees C. K59 cells lysogenic for prophage AP3 were approximately as sensitive to phagocytosis and intracellular killing by human PMN as strains KRTT1 and KRTT2. Unencapsulated Klebsiella strains isolated from clinical specimens were found to carry MIAT most often. Four such strains were found much more resistant to phagocytosis and intracellular killing than their spontaneous mutants resistant to bacteriophages T3 and T7. PMID:7047402

  14. Mutation of Tyr137 of the universal Escherichia coli fimbrial adhesin FimH relaxes the tyrosine gate prior to mannose binding

    PubMed Central

    Rabbani, Said; Krammer, Eva-Maria; Roos, Goedele; Zalewski, Adam; Preston, Roland; Eid, Sameh; Zihlmann, Pascal; Prévost, Martine; Lensink, Marc F.; Thompson, Andrew; Ernst, Beat; Bouckaert, Julie

    2017-01-01

    The most prevalent diseases manifested by Escherichia coli are acute and recurrent bladder infections and chronic inflammatory bowel diseases such as Crohn’s disease. E. coli clinical isolates express the FimH adhesin, which consists of a mannose-specific lectin domain connected via a pilin domain to the tip of type 1 pili. Although the isolated FimH lectin domain has affinities in the nanomolar range for all high-mannosidic glycans, differentiation between these glycans is based on their capacity to form predominantly hydrophobic interactions within the tyrosine gate at the entrance to the binding pocket. In this study, novel crystal structures of tyrosine-gate mutants of FimH, ligand-free or in complex with heptyl α-d-O-mannopyranoside or 4-biphenyl α-d-O-mannopyranoside, are combined with quantum-mechanical calculations and molecular-dynamics simulations. In the Y48A FimH crystal structure, a large increase in the dynamics of the alkyl chain of heptyl α-d-O-mannopyranoside attempts to compensate for the absence of the aromatic ring; however, the highly energetic and stringent mannose-binding pocket of wild-type FimH is largely maintained. The Y137A mutation, on the other hand, is the most detrimental to FimH affinity and specificity: (i) in the absence of ligand the FimH C-terminal residue Thr158 intrudes into the mannose-binding pocket and (ii) ethylenediaminetetraacetic acid interacts strongly with Glu50, Thr53 and Asn136, in spite of multiple dialysis and purification steps. Upon mutation, pre-ligand-binding relaxation of the backbone dihedral angles at position 137 in the tyrosine gate and their coupling to Tyr48 via the interiorly located Ile52 form the basis of the loss of affinity of the FimH adhesin in the Y137A mutant. PMID:28250938

  15. Identification of a Latin American-specific BabA adhesin variant through whole genome sequencing of Helicobacter pylori patient isolates from Nicaragua

    DOE PAGES

    Thorell, Kaisa; Hosseini, Shaghayegh; Palacios Gonzales, Reyna Victoria Palacios; ...

    2016-02-29

    In this study, Helicobacter pylori (H. pylori) is one of the most common bacterial infections in humans and this infection can lead to gastric ulcers and gastric cancer. H. pylori is one of the most genetically variable human pathogens and the ability of the bacterium to bind to the host epithelium as well as the presence of different virulence factors and genetic variants within these genes have been associated with disease severity. Nicaragua has particularly high gastric cancer incidence and we therefore studied Nicaraguan clinical H. pylori isolates for factors that could contribute to cancer risk. The complete genomes ofmore » fifty-two Nicaraguan H. pylorii isolates were sequenced and assembled de novo, and phylogenetic and virulence factor analyses were performed. The Nicaraguan isolates showed phylogenetic relationship with West African isolates in whole-genome sequence comparisons and with Western and urban South-and Central American isolates using MLSA (Multi-locus sequence analysis). A majority, 77 % of the isolates carried the cancer-associated virulence gene cagA and also the s1/i1/m1 vacuolating cytotoxin, vacA allele combination, which is linked to increased severity of disease. Specifically, we also found that Nicaraguan isolates have a blood group-binding adhesin (BabA) variant highly similar to previously reported BabA sequences from Latin America, including from isolates belonging to other phylogenetic groups. These BabA sequences were found to be under positive selection at several amino acid positions that differed from the global collection of isolates. In conclusion, the discovery of a Latin American BabA variant, independent of overall phylogenetic background, suggests hitherto unknown host or environmental factors within the Latin American population giving H. pylori isolates carrying this adhesin variant a selective advantage, which could affect pathogenesis and risk for sequelae through specific adherence properties.« less

  16. Identification of a Latin American-specific BabA adhesin variant through whole genome sequencing of Helicobacter pylori patient isolates from Nicaragua

    SciTech Connect

    Thorell, Kaisa; Hosseini, Shaghayegh; Palacios Gonzales, Reyna Victoria Palacios; Chaotham, Chatchai; Graham, David Y.; Paszat, Lawrence; Rabeneck, Linda; Lundin, Samuel B.; Nookaew, Intawat; Sjoling, Asa

    2016-02-29

    In this study, Helicobacter pylori (H. pylori) is one of the most common bacterial infections in humans and this infection can lead to gastric ulcers and gastric cancer. H. pylori is one of the most genetically variable human pathogens and the ability of the bacterium to bind to the host epithelium as well as the presence of different virulence factors and genetic variants within these genes have been associated with disease severity. Nicaragua has particularly high gastric cancer incidence and we therefore studied Nicaraguan clinical H. pylori isolates for factors that could contribute to cancer risk. The complete genomes of fifty-two Nicaraguan H. pylorii isolates were sequenced and assembled de novo, and phylogenetic and virulence factor analyses were performed. The Nicaraguan isolates showed phylogenetic relationship with West African isolates in whole-genome sequence comparisons and with Western and urban South-and Central American isolates using MLSA (Multi-locus sequence analysis). A majority, 77 % of the isolates carried the cancer-associated virulence gene cagA and also the s1/i1/m1 vacuolating cytotoxin, vacA allele combination, which is linked to increased severity of disease. Specifically, we also found that Nicaraguan isolates have a blood group-binding adhesin (BabA) variant highly similar to previously reported BabA sequences from Latin America, including from isolates belonging to other phylogenetic groups. These BabA sequences were found to be under positive selection at several amino acid positions that differed from the global collection of isolates. In conclusion, the discovery of a Latin American BabA variant, independent of overall phylogenetic background, suggests hitherto unknown host or environmental factors within the Latin American population giving H. pylori isolates carrying this adhesin variant a selective advantage, which could affect pathogenesis and risk for sequelae through specific adherence

  17. Transitional structures of the TiO 2(110) surface from p(1×1) to p(1×2) studied by impact collision ion scattering spectroscopy

    NASA Astrophysics Data System (ADS)

    Asari, E.; Hayami, W.; Souda, R.

    2000-10-01

    The transitional states of surface structure of TiO 2(110) from p(1×1) to p(1×2) on low reduced to heavily reduced surfaces were studied using Impact Collision Ion Scattering Spectroscopy (ICISS). We found that (i) bridging oxygen rows were formed on low reduced surface at anneal temperature of 650°C for 2.5 h, (ii) Ti 2O unit rows were formed between the bridging oxygen rows on low reduced surface at anneal temperature of 930°C for 100 min, (iii) five-fold titanium atom rows on the surface and titanium atom rows of the Ti 2O rows are imperfectly covered with oxygen atoms on heavily reduced surface at anneal temperature of 600°C for 1 h and followed by 670°C annealing for 5 min after ion sputtering, and (iv) Ti 2O 3 unit rows proposed by Onishi and Iwasawa [H. Onishi and Y. Iwasawa, Surf. Sci. Lett., 313, 1994, L783] were completed by covering with oxygen atoms on all of the titanium atom rows on the Ti 2O rows on heavily reduced surface at higher anneal temperature of 1050°C for 40 min after ion sputtering. It is noticeable that the p(1×2) surface structures on low reduced surface is different from that of heavily reduced surface, viz., each has Ti 2O unit rows and Ti 2O 3 unit rows, respectively. The result contradicts "missing unit model" proposed by Bennett et al. [R.A. Bennett, P. Stone, N.J. Price, M. Bowker, Phys. Rev. Lett., 82, 1999, 3831].

  18. Immunogenicity of the Plasmodium falciparum PfEMP1-VarO Adhesin: Induction of Surface-Reactive and Rosette-Disrupting Antibodies to VarO Infected Erythrocytes

    PubMed Central

    Guillotte, Micheline; Juillerat, Alexandre; Igonet, Sébastien; Hessel, Audrey; Petres, Stéphane; Crublet, Elodie; Le Scanf, Cécile; Lewit-Bentley, Anita; Bentley, Graham A.

    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

  19. STS-113 Astronauts Work on Port One (P1) Truss on International Space Station

    NASA Technical Reports Server (NTRS)

    2002-01-01

    The 16th American assembly flight and 112th overall American flight to the International Space Station (ISS) launched on November 23, 2002 from Kennedy's launch pad 39A aboard the Space Shuttle Orbiter Endeavor STS-113. Mission objectives included the delivery of the Expedition Six Crew to the ISS, the return of Expedition Five crew back to Earth, and the installation and activation of the Port 1 Integrated Truss Assembly (P1). The first major component installed on the left side of the Station, the P1 truss provides an additional three External Thermal Control System radiators. Weighing in at 27,506 pounds, the P1 truss is 45 feet (13.7 meters) long, 15 feet (4.6 meters) wide, and 13 feet (4 meters) high. Three space walks, aided by the use of the Robotic Manipulator Systems of both the Shuttle and the Station, were performed in the installation of P1. In this photograph, astronauts Michael E. Lopez-Alegria (above) and John B. Herrington (below) work on the newly installed P1 truss during the mission's second scheduled session of extravehicular activity. The space walk lasted 6 hours, 10 minutes. The end effector of the Canadarm2 or Space Station Remote Manipulator System (SSRMS) and Earth's horizon are visible in the bottom of frame.

  20. FoxP1 orchestration of ASD-relevant signaling pathways in the striatum

    PubMed Central

    Araujo, Daniel J.; Anderson, Ashley G.; Berto, Stefano; Runnels, Wesley; Harper, Matthew; Ammanuel, Simon; Rieger, Michael A.; Huang, Hung-Chung; Rajkovich, Kacey; Loerwald, Kristofer W.; Dekker, Joseph D.; Tucker, Haley O.; Dougherty, Joseph D.; Gibson, Jay R.; Konopka, Genevieve

    2015-01-01

    Mutations in the transcription factor Forkhead box p1 (FOXP1) are causative for neurodevelopmental disorders such as autism. However, the function of FOXP1 within the brain remains largely uncharacterized. Here, we identify the gene expression program regulated by FoxP1 in both human neural cells and patient-relevant heterozygous Foxp1 mouse brains. We demonstrate a role for FoxP1 in the transcriptional regulation of autism-related pathways as well as genes involved in neuronal activity. We show that Foxp1 regulates the excitability of striatal medium spiny neurons and that reduction of Foxp1 correlates with defects in ultrasonic vocalizations. Finally, we demonstrate that FoxP1 has an evolutionarily conserved role in regulating pathways involved in striatal neuron identity through gene expression studies in human neural progenitors with altered FOXP1 levels. These data support an integral role for FoxP1 in regulating signaling pathways vulnerable in autism and the specific regulation of striatal pathways important for vocal communication. PMID:26494785

  1. P1 interneurons promote a persistent internal state that enhances inter-male aggression in Drosophila

    PubMed Central

    Hoopfer, Eric D; Jung, Yonil; Inagaki, Hidehiko K; Rubin, Gerald M; Anderson, David J

    2015-01-01

    How brains are hardwired to produce aggressive behavior, and how aggression circuits are related to those that mediate courtship, is not well understood. A large-scale screen for aggression-promoting neurons in Drosophila identified several independent hits that enhanced both inter-male aggression and courtship. Genetic intersections revealed that 8-10 P1 interneurons, previously thought to exclusively control male courtship, were sufficient to promote fighting. Optogenetic experiments indicated that P1 activation could promote aggression at a threshold below that required for wing extension. P1 activation in the absence of wing extension triggered persistent aggression via an internal state that could endure for minutes. High-frequency P1 activation promoted wing extension and suppressed aggression during photostimulation, whereas aggression resumed and wing extension was inhibited following photostimulation offset. Thus, P1 neuron activation promotes a latent, internal state that facilitates aggression and courtship, and controls the overt expression of these social behaviors in a threshold-dependent, inverse manner. DOI: http://dx.doi.org/10.7554/eLife.11346.001 PMID:26714106

  2. Long intergenic non-coding RNA APOC1P1-3 inhibits apoptosis by decreasing α-tubulin acetylation in breast cancer

    PubMed Central

    Liao, X-H; Wang, J-G; Li, L-Y; Zhou, D-M; Ren, K-H; Jin, Y-T; Lv, L; Yu, J-G; Yang, J-Y; Lu, Q; Zou, Q; Yu, J; Liu, X-P; Zhou, P

    2016-01-01

    Increasing evidence indicates that long non-coding RNAs (lncRNAs) act as important regulatory factors in tumor progression. However, their roles in breast cancer remain largely unknown. In present studies, we identified aberrantly expressed long intergenic non-coding RNA APOC1P1-3 (lincRNA-APOC1P1-3) in breast cancer by microarray, verified it by quantitative real-time PCR, and assessed methylation status in the promoter region by pyrosequencing. We also investigated the biological functions with plasmid transfection and siRNA silencing experiments, and further explored their mechanisms by RNA pull-down and RNA immunoprecipitation to identify binding proteins. We found that 224 lncRNAs were upregulated in breast cancer, whereas 324 were downregulated. The lincRNA-APOC1P1-3 was overexpressed in breast cancer, which was related to tumor size and hypomethylation in its promoter region. We also found that APOC1P1-3 could directly bind to tubulin to decrease α-tubulin acetylation, to inactivate caspase-3, and to inhibit apoptosis. This study demonstrates that overexpression of APOC1P1-3 can inhibit breast cancer apoptosis. PMID:27228351

  3. A Sensitive and Robust High-Throughput Screening Assay for Inhibitors of the Chikungunya Virus nsP1 Capping Enzyme

    PubMed Central

    Bullard-Feibelman, Kristen M.; Fuller, Benjamin P.; Geiss, Brian J.

    2016-01-01

    Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus that causes severe and debilitating disease symptoms. Alarmingly, transmission rates of CHIKV have increased dramatically over the last decade resulting in 1.7 million suspected cases in the Western hemisphere alone. There are currently no antivirals for treatment of CHIKV infection and novel anti-alphaviral compounds are badly needed. nsP1 is the alphavirus protein responsible for the methyltransferase and guanylyltransferase activities necessary for formation of the 5’ type 0 cap structure added to newly formed viral RNA. Formation of this cap depends on nsP1 binding GTP and transferring a methylated GMP to nascent viral RNA. We have developed a fluorescence polarization-based assay that monitors displacement of a fluorescently-labeled GTP analog in real time. Determining the relative affinities of 15 GTP analogs for nsP1 GTP revealed important structural aspects of GTP that will inform identification of inhibitors able to outcompete GTP for the nsP1 binding site. Validation of the assay for HTS was completed and a secondary orthogonal assay that measures guanylation activity was developed in order to evaluate hits from future drug screens. This platform provides an avenue for identification of potent nsP1 inhibitors, which would potentially provide compounds capable of treating disease caused by CHIKV infection. PMID:27427769

  4. The proteins secreted by Trichomonas vaginalis and vaginal epithelial cell response to secreted and episomally expressed AP65

    PubMed Central

    Kucknoor, Ashwini S.; Mundodi, Vasanthakrishna; Alderete, John F.

    2007-01-01

    Summary We showed recently that contact of human vaginal epithelial cells (VECs) by Trichomonas vaginalis and incubation with trichomonad proteins in conditioned medium induced expression of VEC genes. We performed 2-D SDS-PAGE followed by MALDI-TOF to identify the major secreted proteins. Based on protein abundance and separation of spots in 2-D gels, 32 major secreted proteins were examined, which gave 19 proteins with accession numbers. These proteins included known secreted cysteine proteinases. In addition, other secreted proteins were enzymes of carbohydrate metabolism, adhesin protein AP65, heat shock proteins, thioredoxin reductase and coronins. We confirmed that the secreted trichomonad proteins induced expression of VEC genes, including interleukin 8 (IL-8), COX-2 and fibronectin. Purified AP65 added to VECs had a pronounced effect only on IL-8 gene expression, which was inhibited in the presence of 12G4 monoclonal antibody to AP65. Moreover, AP65 expressed episomally within epithelial cells was found to enhance the expression of IL-8 and COX-2. This may be the first report of analysis of the secreted proteins of T. vaginalis and of the host epithelial cell response to these proteins and to the prominent adhesin AP65. PMID:17590165

  5. The Complete Sequence of the Acacia ligulata Chloroplast Genome Reveals a Highly Divergent clpP1 Gene.

    PubMed

    Williams, Anna V; Boykin, Laura M; Howell, Katharine A; Nevill, Paul G; Small, Ian

    2015-01-01

    Legumes are a highly diverse angiosperm family that include many agriculturally important species. To date, 21 complete chloroplast genomes have been sequenced from legume crops confined to the Papilionoideae subfamily. Here we report the first chloroplast genome from the Mimosoideae, Acacia ligulata, and compare it to the previously sequenced legume genomes. The A. ligulata chloroplast genome is 174,233 bp in size, comprising inverted repeats of 38,225 bp and single-copy regions of 92,798 bp and 4,985 bp [corrected]. Acacia ligulata lacks the inversion present in many of the Papilionoideae, but is not otherwise significantly different in terms of gene and repeat content. The key feature is its highly divergent clpP1 gene, normally considered essential in chloroplast genomes. In A. ligulata, although transcribed and spliced, it probably encodes a catalytically inactive protein. This study provides a significant resource for further genetic research into Acacia and the Mimosoideae. The divergent clpP1 gene suggests that Acacia will provide an interesting source of information on the evolution and functional diversity of the chloroplast Clp protease complex.

  6. The Complete Sequence of the Acacia ligulata Chloroplast Genome Reveals a Highly Divergent clpP1 Gene

    PubMed Central

    Williams, Anna V.; Boykin, Laura M.; Howell, Katharine A.; Nevill, Paul G.; Small, Ian

    2015-01-01

    Legumes are a highly diverse angiosperm family that include many agriculturally important species. To date, 21 complete chloroplast genomes have been sequenced from legume crops confined to the Papilionoideae subfamily. Here we report the first chloroplast genome from the Mimosoideae, Acacia ligulata, and compare it to the previously sequenced legume genomes. The A. ligulata chloroplast genome is 158,724 bp in size, comprising inverted repeats of 25,925 bp and single-copy regions of 88,576 bp and 18,298 bp. Acacia ligulata lacks the inversion present in many of the Papilionoideae, but is not otherwise significantly different in terms of gene and repeat content. The key feature is its highly divergent clpP1 gene, normally considered essential in chloroplast genomes. In A. ligulata, although transcribed and spliced, it probably encodes a catalytically inactive protein. This study provides a significant resource for further genetic research into Acacia and the Mimosoideae. The divergent clpP1 gene suggests that Acacia will provide an interesting source of information on the evolution and functional diversity of the chloroplast Clp protease complex. PMID:25955637

  7. The collectins CL-L1, CL-K1 and CL-P1, and their roles in complement and innate immunity.

    PubMed

    Hansen, Soren W K; Ohtani, Katsuki; Roy, Nitai; Wakamiya, Nobutaka

    2016-10-01

    Both the complement system and collectins play important roles in our innate immune system. The collectins, which are characterized by their inclusion of a collagen-like region and a calcium-dependent carbohydrate recognition domain, are pattern recognition molecules and include the well characterized proteins mannan-binding lectin (MBL) and the surfactant proteins SP-A/-D. Collectin liver 1 (CL-L1), collectin kidney 1 (CL-K1) and collectin placenta 1 (CL-P1) are the most recently discovered collectins. Although their function is still under investigation, accumulating information suggests that CL-L1, CL-K1 and CL-P1 play important roles in host defense by recognizing a variety of microorganisms and interacting with effector proteins, including complement components. The recent establishment of the existence of CL-K1 in the circulation in form of heteromeric complexes with CL-L1 (known as CL-LK) and its activation of the lectin pathway via MASPs, drew new attention in the complement biology, which was further strengthened by the observed interactions between CL-P1 and CRP-C1q-factor H or properdin. Deficiency of either CL-K1 or MASP-3 has been demonstrated in 3MC syndrome patients with developmental abnormalities, showing that lectin pathway components, regulation and/or activation are essential during the embryonic development; another feature that they most likely share CL-P1. Herein, we discuss the recent characteristics and roles of the collectins CL-L1, CL-K1 and CL-P1 in the complement system, in innate immunity and their possible association with disease development and pathogenesis.

  8. P-1 truss moved to work stand in O&C Building

    NASA Technical Reports Server (NTRS)

    2000-01-01

    The P-1 truss, a component of the International Space Station, is moved the length of the Operations and Checkout Building to its work stand where it will undergo processing. Scheduled to fly in spring of 2002, the P-1 is part of a total 10-truss, girder-like structure on the Station that will ultimately extend the length of a football field. Astronauts will attach the 14-by-15 foot structure to the port side of the center truss, S0, during the spring assembly flight. The 33,000-pound P-1 will house the thermal radiator rotating joint (TRRJ) that will rotate the Station's radiators away from the sun to increase their maximum cooling efficiency.

  9. P-1 truss moved to work stand in O&C Building

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Inside the Operations and Checkout Building, the P-1 truss, a component of the International Space Station, is lifted out of its canister to move to a work stand where it will undergo processing. Scheduled to fly in spring of 2002, the P-1 is part of a total 10-truss, girder-like structure on the Station that will ultimately extend the length of a football field. Astronauts will attach the 14-by-15 foot structure to the port side of the center truss, S0, during the spring assembly flight. The 33,000- pound P-1 will house the thermal radiator rotating joint (TRRJ) that will rotate the Station's radiators away from the sun to increase their maximum cooling efficiency.

  10. P-1 truss moved to work stand in O&C Building

    NASA Technical Reports Server (NTRS)

    2000-01-01

    The P-1 truss (top of photo), a component of the International Space Station, nears its work stand in the Operations and Checkout Building where it will undergo processing. Scheduled to fly in spring of 2002, the P-1 is part of a total 10-truss, girder-like structure on the Station that will ultimately extend the length of a football field. Astronauts will attach the 14-by- 15 foot structure to the port side of the center truss, S0, during the spring assembly flight. The 33,000-pound P-1 will house the thermal radiator rotating joint (TRRJ) that will rotate the Station's radiators away from the sun to increase their maximum cooling efficiency.

  11. P-1 truss moved to work stand in O&C Building

    NASA Technical Reports Server (NTRS)

    2000-01-01

    The P-1 truss, a component of the International Space Station, is lowered into a work stand in the Operations and Checkout Building where it will undergo processing. Scheduled to fly in spring of 2002, the P-1 is part of a total 10-truss, girder-like structure on the Station that will ultimately extend the length of a football field. Astronauts will attach the 14-by-15 foot structure to the port side of the center truss, S0, during the spring assembly flight. The 33,000-pound P-1 will house the thermal radiator rotating joint (TRRJ) that will rotate the Station's radiators away from the sun to increase their maximum cooling efficiency.

  12. P1 and N170 components distinguish human-like and animal-like makeup stimuli.

    PubMed

    Luo, Shuwei; Luo, Wenbo; He, Weiqi; Chen, Xu; Luo, Yuejia

    2013-06-19

    This study used event-related potentials to investigate the sensitivity of P1 and N170 components to human-like and animal-like makeup stimuli, which were derived from pictures of Peking opera characters. As predicted, human-like makeup stimuli elicited larger P1 and N170 amplitudes than did animal-like makeup stimuli. Interestingly, a right hemisphere advantage was observed for human-like but not for animal-like makeup stimuli. Dipole source analyses of 130-200-ms window showed that the bilateral fusiform face area may contribute to the differential sensitivity of the N170 component in response to human-like and animal-like makeup stimuli. The present study suggests that the amplitudes of both the P1 and the N170 are sensitive for the mouth component of face-like stimuli.

  13. Visualization of bacteriophage P1 infection by cryo-electron tomography of tiny Escherichia coli

    SciTech Connect

    Liu Jun; Chen Chengyen; Shiomi, Daisuke; Niki, Hironori; Margolin, William

    2011-09-01

    Bacteriophage P1 has a contractile tail that targets the conserved lipopolysaccharide on the outer membrane surface of the host for initial adsorption. The mechanism by which P1 DNA enters the host cell is not well understood, mainly because the transient molecular interactions between bacteriophage and bacteria have been difficult to study by conventional approaches. Here, we engineered tiny E. coli host cells so that the initial stages of P1-host interactions could be captured in unprecedented detail by cryo-electron tomography. Analysis of three-dimensional reconstructions of frozen-hydrated specimens revealed three predominant configurations: an extended tail stage with DNA present in the phage head, a contracted tail stage with DNA, and a contracted tail stage without DNA. Comparative analysis of various conformations indicated that there is uniform penetration of the inner tail tube into the E. coli periplasm and a significant movement of the baseplate away from the outer membrane during tail contraction.

  14. BEAM EXTRACTION FROM THE RECYCLER RING TO P1 LINE AT FERMILAB

    SciTech Connect

    Xiao, M.; Capista, D.; Adams, P.; Morris, D.; Yang, M. J.; Hazewood, K.

    2016-10-03

    The transfer line for beam extraction from the Recycler ring to P1 line provides a way to deliver 8 GeV kinetic energy protons from the Booster to the Delivery ring, via the Recycler, using existing beam transport lines, and without the need for new civil construction. It was designed in 2012. The kicker magnets at RR520 and the lambertson magnet at RR522 in the RR were installed in 2014 Summer Shutdown, the elements of RR to P1 Stub (permanent quads, trim quads, correctors, BPMs, the toroid at 703 and vertical bending dipole at V703 (ADCW) were installed in 2015 Summer Shutdown. On Tuesday, June 21, 2016, beam line from the Recycler Ring to P1 line was commissioned. The detailed results will be presented in this report.

  15. Functional Heterogeneity of the UpaH Autotransporter Protein from Uropathogenic Escherichia coli

    PubMed Central

    Allsopp, Luke P.; Beloin, Christophe; Moriel, Danilo Gomes; Totsika, Makrina; Ghigo, Jean-Marc

    2012-01-01

    Uropathogenic Escherichia coli (UPEC) is responsible for the majority of urinary tract infections (UTI). To cause a UTI, UPEC must adhere to the epithelial cells of the urinary tract and overcome the shear flow forces of urine. This function is mediated primarily by fimbrial adhesins, which mediate specific attachment to host cell receptors. Another group of adhesins that contributes to UPEC-mediated UTI is autotransporter (AT) proteins. AT proteins possess a range of virulence properties, such as adherence, aggregation, invasion, and biofilm formation. One recently characterized AT protein of UPEC is UpaH, a large adhesin-involved-in-diffuse-adherence (AIDA-I)-type AT protein that contributes to biofilm formation and bladder colonization. In this study we characterized a series of naturally occurring variants of UpaH. We demonstrate that extensive sequence variation exists within the passenger-encoding domain of UpaH variants from different UPEC strains. This sequence variation is associated with functional heterogeneity with respect to the ability of UpaH to mediate biofilm formation. In contrast, all of the UpaH variants examined retained a conserved ability to mediate binding to extracellular matrix (ECM) proteins. Bioinformatic analysis of the UpaH passenger domain identified a conserved region (UpaHCR) and a hydrophobic region (UpaHHR). Deletion of these domains reduced biofilm formation but not the binding to ECM proteins. Despite variation in the upaH sequence, the transcription of upaH was repressed by a conserved mechanism involving the global regulator H-NS, and mutation of the hns gene relieved this repression. Overall, our findings shed new light on the regulation and functions of the UpaH AT protein. PMID:22904291

  16. ON DIFFERENTIABILITY OF FUNCTIONS IN L^p, 0 < p < 1

    NASA Astrophysics Data System (ADS)

    Krotov, V. G.

    1983-02-01

    In this paper the author studies the connection between smoothness, expressed in terms of the integral modulus of continuity, and the existence of a derivative, understood in some sense, for functions in L^p, 0 < p < 1; an analogous question is considered for boundary values of analytic functions in the Hardy classes H^p, 0 < p < 1. A connection is established between the derivatives of an analytic function in H^p and the derivatives of its boundary value; both global and pointwise derivatives are considered.Bibliography: 25 titles.

  17. Synthesis and P1' SAR exploration of potent macrocyclic tissue factor-factor VIIa inhibitors

    SciTech Connect

    Ladziata, Vladimir; Glunz, Peter W.; Zou, Yan; Zhang, Xiaojun; Jiang, Wen; Jacutin-Porte, Swanee; Cheney, Daniel L.; Wei, Anzhi; Luettgen, Joseph M.; Harper, Timothy M.; Wong, Pancras C.; Seiffert, Dietmar; Wexler, Ruth R.; Priestley, E. Scott

    2016-10-01

    Selective tissue factor-factor VIIa complex (TF-FVIIa) inhibitors are viewed as promising compounds for treating thrombotic disease. In this contribution, we describe multifaceted exploratory SAR studies of S1'-binding moieties within a macrocyclic chemotype aimed at replacing cyclopropyl sulfone P1' group. Over the course of the optimization efforts, the 1-(1H-tetrazol-5-yl)cyclopropane P1' substituent emerged as an improved alternative, offering increased metabolic stability and lower clearance, while maintaining excellent potency and selectivity.

  18. SUMOylation silences heterodimeric TASK potassium channels containing K2P1 subunits in cerebellar granule neurons.

    PubMed

    Plant, Leigh D; Zuniga, Leandro; Araki, Dan; Marks, Jeremy D; Goldstein, Steve A N

    2012-11-20

    The standing outward K(+) current (IKso) governs the response of cerebellar granule neurons to natural and medicinal stimuli including volatile anesthetics. We showed that SUMOylation silenced half of IKso at the surface of cerebellar granule neurons because the underlying channels were heterodimeric assemblies of K2P1, a subunit subject to SUMOylation, and the TASK (two-P domain, acid-sensitive K(+)) channel subunits K2P3 or K2P9. The heterodimeric channels comprised the acid-sensitive portion of IKso and mediated its response to halothane. We anticipate that SUMOylation also influences sensation and homeostatic mechanisms in mammals through TASK channels formed with K2P1.

  19. Biological Functions of the Novel Collectins CL-L1, CL-K1, and CL-P1

    PubMed Central

    Ohtani, Katsuki; Suzuki, Yasuhiko; Wakamiya, Nobutaka

    2012-01-01

    Collectins are characterized by a collagen-like sequence and a carbohydrate recognition domain and are members of the vertebrate C-type lectin superfamily. Recently, “novel collectins”, different from “classical collectins” consisting of mannan-binding lectin (MBL) and surfactant proteins A and D (SP-A and SP-D), have been found by reverse genetics. These “novel collectins” consist of collectin liver 1 (CL-L1), collectin kidney 1 (CL-K1), and collectin placenta 1 (CL-P1) and are encoded by three separate genes. Experimental findings on human and animal collectins have shown that both novel collectins and classical collectins play an important role in innate immunity. Based on our recent results and those of others, in this paper, we summarize the new biological functions of these novel collectins in embryonic morphogenesis and development. PMID:22570530

  20. Structure and Functional Properties of the Active Form of the Proteolytic Complex, ClpP1P2, from Mycobacterium tuberculosis*

    PubMed Central

    Li, Mi; Kandror, Olga; Akopian, Tatos; Dharkar, Poorva; Wlodawer, Alexander; Maurizi, Michael R.; Goldberg, Alfred L.

    2016-01-01

    The ClpP protease complex and its regulatory ATPases, ClpC1 and ClpX, in Mycobacterium tuberculosis (Mtb) are essential and, therefore, promising drug targets. The Mtb ClpP protease consists of two heptameric rings, one composed of ClpP1 and the other of ClpP2 subunits. Formation of the enzymatically active ClpP1P2 complex requires binding of N-blocked dipeptide activators. We have found a new potent activator, benzoyl-leucine-leucine (Bz-LL), that binds with higher affinity and promotes 3–4-fold higher peptidase activity than previous activators. Bz-LL-activated ClpP1P2 specifically stimulates the ATPase activity of Mtb ClpC1 and ClpX. The ClpC1P1P2 and ClpXP1P2 complexes exhibit 2–3-fold enhanced ATPase activity, peptide cleavage, and ATP-dependent protein degradation. The crystal structure of ClpP1P2 with bound Bz-LL was determined at a resolution of 3.07 Å and with benzyloxycarbonyl-Leu-Leu (Z-LL) bound at 2.9 Å. Bz-LL was present in all 14 active sites, whereas Z-LL density was not resolved. Surprisingly, Bz-LL adopts opposite orientations in ClpP1 and ClpP2. In ClpP1, Bz-LL binds with the C-terminal leucine side chain in the S1 pocket. One C-terminal oxygen is close to the catalytic serine, whereas the other contacts backbone amides in the oxyanion hole. In ClpP2, Bz-LL binds with the benzoyl group in the S1 pocket, and the peptide hydrogen bonded between parallel β-strands. The ClpP2 axial loops are extended, forming an open axial channel as has been observed with bound ADEP antibiotics. Thus occupancy of the active sites of ClpP allosterically alters sites on the surfaces thereby affecting the association of ClpP1 and ClpP2 rings, interactions with regulatory ATPases, and entry of protein substrates. PMID:26858247

  1. Complete Genome Sequence of Rhodococcus sp. Strain IcdP1 Shows Diverse Catabolic Potential

    PubMed Central

    Qu, Jie; Miao, Li-Li; Liu, Ying

    2015-01-01

    The complete genome sequence of Rhodococcus sp. strain IcdP1 is presented here. This organism was shown to degrade a broad range of high-molecular-weight polycyclic aromatic hydrocarbons and organochlorine pesticides. The sequence data can be used to predict genes for xenobiotic biodegradation and metabolism. PMID:26139718

  2. Local structural investigation of buried InAs(x)P(1-x)/InP interfaces

    NASA Astrophysics Data System (ADS)

    Lamberti, C.; Bordiga, S.; Boscherini, F.; Pascarelli, S.; Schiavini, G. M.; Ferari, C.; Lazzarini, L.; Salviati, G.

    1994-10-01

    A local structural investigation has been carried out on the 10 A InAs(x)P(1-x) layer in ad hoc grown InAs(x)P(1-x)/InP epitaxal multistructures deposited by low pressure metallorganic chemical vapor deposition by means of extended x-ray absorption fine structure spectroscopy, high resolution transmission electron microscopy, and high resolution x-ray diffraction analyses. The goal was to characterize the local structure of the unwanted, strained, interface layers of InAs(x)P(1-x) produced by the exposure of the InP surface to AsH3 as occurs during the growth of InP/In(0.53)Ga(0.47)As heterostructures optimized for photonics. High resolution x-ray diffraction and high resolution transmission electron microscopy confirm the high crystalline perfection of the investigated interfaces. As K-edge extended x-ray absorption fine structure analysis shows, the first shell environment of As at these interfaces is similar to that found in bulk InAs(x)P(1-X) alloys of similar composition, as determined experimentally and by comparison with recent theories of bond lengths in semiconductor alloys. In particular we measure an As-In bond length which varies at most 0.02 A with As concentration at the interface; this implies that epitaxy with InP is accompanied by local structural distortions, such as bond angle variations, which accommodate the nearly constant As-In bond length.

  3. Heterogeneity of Human Neutrophil CD177 Expression Results from CD177P1 Pseudogene Conversion

    PubMed Central

    Liang, Rong; Ohnesorg, Thomas; Cho, Vicky; Abhayaratna, Walter P.; Gatenby, Paul A.; Perera, Chandima; Zhang, Yafei; Whittle, Belinda; Sinclair, Andrew; Goodnow, Christopher C.; Field, Matthew; Andrews, T. Daniel; Cook, Matthew C.

    2016-01-01

    Most humans harbor both CD177neg and CD177pos neutrophils but 1–10% of people are CD177null, placing them at risk for formation of anti-neutrophil antibodies that can cause transfusion-related acute lung injury and neonatal alloimmune neutropenia. By deep sequencing the CD177 locus, we catalogued CD177 single nucleotide variants and identified a novel stop codon in CD177null individuals arising from a single base substitution in exon 7. This is not a mutation in CD177 itself, rather the CD177null phenotype arises when exon 7 of CD177 is supplied entirely by the CD177 pseudogene (CD177P1), which appears to have resulted from allelic gene conversion. In CD177 expressing individuals the CD177 locus contains both CD177P1 and CD177 sequences. The proportion of CD177hi neutrophils in the blood is a heritable trait. Abundance of CD177hi neutrophils correlates with homozygosity for CD177 reference allele, while heterozygosity for ectopic CD177P1 gene conversion correlates with increased CD177neg neutrophils, in which both CD177P1 partially incorporated allele and paired intact CD177 allele are transcribed. Human neutrophil heterogeneity for CD177 expression arises by ectopic allelic conversion. Resolution of the genetic basis of CD177null phenotype identifies a method for screening for individuals at risk of CD177 isoimmunisation. PMID:27227454

  4. 26 CFR 31.3306(p)-1 - Employees of related corporations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 15 2010-04-01 2010-04-01 false Employees of related corporations. 31.3306(p)-1... of related corporations. (a) In general. For purposes of sections 3301, 3302, and 3306(b)(1), when two or more related corporations concurrently employ the same individual and compensate...

  5. 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.

  6. Radiative transport in the delta-P1 approximation for semi-infinite turbid media

    PubMed Central

    Seo, InSeok; Hayakawa, Carole K.; Venugopalan, Vasan

    2012-01-01

    We have developed an analytic solution for spatially resolved diffuse reflectance within the δ-P1 approximation to the radiative transport equation for a semi-infinite homogeneous turbid medium. We evaluate the performance of this solution by comparing its predictions with those provided by Monte Carlo simulations and the standard diffusion approximation. We demonstrate that the δ-P1 approximation provides accurate estimates for spatially resolved diffuse reflectance in both low and high scattering media. We also develop a multi-stage nonlinear optimization algorithm in which the radiative transport estimates provided by the δ-P1 approximation are used to recover the optical absorption (μa), reduced scattering ( μs′), and single-scattering asymmetry coefficients (g1) of liquid and solid phantoms from experimental measurements of spatially resolved diffuse reflectance. Specifically, the δ-P1 approximation can be used to recover μa, μs′, and g1 with errors within ±22%, ±18%, and ±17%, respectively, for both intralipid-based and siloxane-based tissue phantoms. These phantoms span the optical property range 4<(μs′/μa)<117. Using these same measurements, application of the standard diffusion approximation resulted in the recovery of μa and μs′ with errors of ±29% and ±25%, respectively. Collectively, these results demonstrate that the δ-P1 approximation provides accurate radiative transport estimates that can be used to determine accurately the optical properties of biological tissues, particularly in spectral regions where tissue may display moderate/low ratios of reduced scattering to absorption ( μs′/μa). PMID:18383690

  7. The variant hepatocyte nuclear factor 1 activates the P1 promoter of the human alpha-folate receptor gene in ovarian carcinoma.

    PubMed

    Tomassetti, Antonella; Mangiarotti, Fabio; Mazzi, Mimma; Sforzini, Sabrina; Miotti, Silvia; Galmozzi, Enrico; Elwood, Patrick C; Canevari, Silvana

    2003-02-01

    The alpha folate receptor (alpha FR) is a membrane glycoprotein that binds folates, and mediates their uptake and that of antifolate drugs. alpha FR is absent on ovarian surface epithelium (OSE) but is detectable during early transforming events in this epithelium, with increasing expression levels in association with tumor progression. Analysis of transcriptional regulation of the alpha FR gene have revealed two promoter regions, P1 and P4, flanking exons 1 and 4, respectively, and a requirement for three SP1 sites and an INR element for optimal P4 activity. Here, we focused on the P1 transcription regulation in ovarian carcinoma cells. RNase protection assay indicated that the 5'-untranslated region is heterogeneous because of different start sites and alternative splicing of exon 3. A core region of the P1 promoter was sufficient for maximal promoter activity in ovarian carcinoma cell lines but not in OSE cells or in alpha FR-nonexpressing cell lines. Deletion and mutation analysis of this core promoter identified a cis-regulatory element at position +27 to +33 of the untranslated exon 1, which is responsible for maximum P1 activity. This element formed an abundant DNA-protein complex with nuclear proteins from ovarian cancer cells but not from other cell lines or OSE cells. Competition experiments and supershift assays demonstrated binding of the P1 cis-regulatory element by a transcription factor involved in embryonic development, the variant hepatocyte nuclear factor-1 (vHNF1). Analysis of RNA from various cell lines and surgical specimens confirmed that vHNF1 is expressed in ovarian carcinomas. Thus, vHNF1 regulates tissue-specific transcription in ovarian carcinoma.

  8. Afa/Dr diffusely adhering Escherichia coli infection in T84 cell monolayers induces increased neutrophil transepithelial migration, which in turn promotes cytokine-dependent upregulation of decay-accelerating factor (CD55), the receptor for Afa/Dr adhesins.

    PubMed

    Bétis, Fréderic; Brest, Patrick; Hofman, Véronique; Guignot, Julie; Kansau, Imad; Rossi, Bernard; Servin, Alain; Hofman, Paul

    2003-04-01

    Ulcerative colitis and Crohn's disease are inflammatory bowel diseases thought to involve strains of Escherichia coli. We report here that two wild-type Afa/Dr diffusely adhering E. coli (DAEC) strains, C1845 and IH11128, which harbor the fimbrial F1845 adhesin and the Dr hemagglutinin, respectively, and the E. coli laboratory strain HB101, transformed with the pSSS1 plasmid to produce Afa/Dr F1845 adhesin, all induced interleukin-8 (IL-8) production and transepithelial migration of polymorphonuclear leukocytes (PMNL) in polarized monolayers of the human intestinal cell line T84 grown on semipermeable filters. We observed that after PMNL migration, expression of decay-accelerating factor (DAF, or CD55), the brush border-associated receptor for Afa/Dr adhesins, was strongly enhanced, increasing the adhesion of Afa/Dr DAEC bacteria. When examining the mechanism by which DAF expression was enhanced, we observed that the PMNL transepithelial migration induced epithelial synthesis of tumor necrosis factor alpha and IL-1beta, which in turn promoted the upregulation of DAF.

  9. 26 CFR 1.409(p)-1T - Prohibited allocations of securities in an S corporation (temporary).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... corporation (temporary). 1.409(p)-1T Section 1.409(p)-1T Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT... Plans, Etc. § 1.409(p)-1T Prohibited allocations of securities in an S corporation (temporary). (a) Organization of this section. Section 409(p) applies if a nonallocation year occurs in an employee...

  10. 26 CFR 1.409(p)-1T - Prohibited allocations of securities in an S corporation (temporary).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... corporation (temporary). 1.409(p)-1T Section 1.409(p)-1T Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT..., Stock Bonus Plans, Etc. § 1.409(p)-1T Prohibited allocations of securities in an S corporation (temporary). (a) Organization of this section. Section 409(p) applies if a nonallocation year occurs in...

  11. 26 CFR 1.409(p)-1T - Prohibited allocations of securities in an S corporation (temporary).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... corporation (temporary). 1.409(p)-1T Section 1.409(p)-1T Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT..., Stock Bonus Plans, Etc. § 1.409(p)-1T Prohibited allocations of securities in an S corporation (temporary). (a) Organization of this section. Section 409(p) applies if a nonallocation year occurs in...

  12. 26 CFR 1.409(p)-1T - Prohibited allocations of securities in an S corporation (temporary).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... corporation (temporary). 1.409(p)-1T Section 1.409(p)-1T Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT..., Stock Bonus Plans, Etc. § 1.409(p)-1T Prohibited allocations of securities in an S corporation (temporary). (a) Organization of this section. Section 409(p) applies if a nonallocation year occurs in...

  13. 26 CFR 1.409(p)-1T - Prohibited allocations of securities in an S corporation (temporary).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... corporation (temporary). 1.409(p)-1T Section 1.409(p)-1T Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT..., Stock Bonus Plans, Etc. § 1.409(p)-1T Prohibited allocations of securities in an S corporation (temporary). (a) Organization of this section. Section 409(p) applies if a nonallocation year occurs in...

  14. Intravital Imaging of Vascular Transmigration by the Lyme Spirochete: Requirement for the Integrin Binding Residues of the B. burgdorferi P66 Protein

    PubMed Central

    Kumar, Devender; Ristow, Laura C.; Shi, Meiqing; Mukherjee, Priyanka; Caine, Jennifer A.; Lee, Woo-Yong; Kubes, Paul; Coburn, Jenifer; Chaconas, George

    2015-01-01

    Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins) that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living Cd1d-/-mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of B. burgdorferi involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of B. burgdorferi mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration. PMID:26684456

  15. P-1 truss moved to work stand in O&C Building

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Inside the Operations and Checkout Building, an overhead crane lifts the top of the canister containing the P-1 truss, a component of the International Space Station. The truss, scheduled to fly in spring of 2002, is part of a total 10-truss, girder-like structure on the Station that will ultimately extend the length of a football field. Astronauts will attach the 14-by- 15 foot structure to the port side of the center truss, S0, during the spring assembly flight. The 33,000-pound P-1 will house the thermal radiator rotating joint (TRRJ) that will rotate the Station's radiators away from the sun to increase their maximum cooling efficiency.

  16. Geohydrologic data for test well UE-25p1, Yucca Mountain area, Nye County, Nevada

    USGS Publications Warehouse

    Craig, R.W.; Johnson, K.A.

    1984-01-01

    This report presents the following data for test well UE-25p 1 in Nye County, Nevada: drilling operations, lithology, availability of borehole geophysical logs, water levels, future availability of core analyses, water chemistry, pumping tests, borehole-flow surveys, and packer-injection tests. The well is one of a series of test wells drilled in and near Yucca Mountain adjacent to the Nevada Test Site in cooperation with the U.S. Department of Energy. These investigations are part of the Nevada Nuclear Waste Storage Investigations to identify suitable sites for underground storage of high-level radioactive wastes. Test well UE-25p 1 was the first in the Yucca Mountain area to penetrate rocks of Paleozoic age. To a depth of 1,244 meters, the rocks are predominantly ash-flow tuffs of Tertiary age. From 1,244 meters to a total depth of 1,805 meters, the rock is dolomite of Paleozoic age. (USGS)

  17. Prestimulus amplitudes modulate P1 latencies and evoked traveling alpha waves

    PubMed Central

    Himmelstoss, Nicole A.; Brötzner, Christina P.; Zauner, Andrea; Kerschbaum, Hubert H.; Gruber, Walter; Lechinger, Julia; Klimesch, Wolfgang

    2015-01-01

    Traveling waves have been well documented in the ongoing, and more recently also in the evoked EEG. In the present study we investigate what kind of physiological process might be responsible for inducing an evoked traveling wave. We used a semantic judgment task which already proved useful to study evoked traveling alpha waves that coincide with the appearance of the P1 component. We found that the P1 latency of the leading electrode is significantly correlated with prestimulus amplitude size and that this event is associated with a transient change in alpha frequency. We assume that cortical background excitability, as reflected by an increase in prestimulus amplitude, is responsible for the observed change in alpha frequency and the initiation of an evoked traveling trajectory. PMID:26074804

  18. Sumoylation Silences Heterodimeric TASK Potassium Channels Containing K2P1 Subunits in Cerebellar Granule Neurons

    PubMed Central

    Plant, Leigh D.; Zuniga, Leandro; Araki, Dan; Marks, Jeremy D.; Goldstein, Steve A. N.

    2013-01-01

    The standing outward K+ current (IKso) governs the response of cerebellar granule neurons to natural and medicinal stimuli including volatile anesthetics. In this study, we showed that sumoylation silenced half of IKso at the surface of cerebellar granule neurons because the underlying channels were heterodimeric assemblies of K2P1, a subunit subject to sumoylation, and the two P domain, acid-sensitive K+ (TASK) channel subunits, K2P3 or K2P9. The heteromeric channels comprised the acid-sensitive portion of IKso and mediated its response to halothane. We anticipate that sumoylation also influences sensation and homeostatic mechanisms in mammals through TASK channels formed with K2P1. PMID:23169818

  19. Development of Quasi-3DOF upper limb rehabilitation system using ER brake: PLEMO-P1

    NASA Astrophysics Data System (ADS)

    Kikuchi, T.; Fukushima, K.; Furusho, J.; Ozawa, T.

    2009-02-01

    In recent years, many researchers have studied the potential of using robotics technology to assist and quantify the motor functions for neuron-rehabilitation. Some kinds of haptic devices have been developed and evaluated its efficiency with clinical tests, for example, upper limb training for patients with spasticity after stroke. However, almost all the devices are active-type (motor-driven) haptic devices and they basically require high-cost safety system compared to passive-type (brake-based) devices. In this study, we developed a new practical haptic device 'PLEMO-P1'; this system adopted ER brakes as its force generators. In this paper, the mechanism of PLEMO-P1 and its software for a reaching rehabilitation are described.

  20. Product Plan of New Generation System Camera "OLYMPUS PEN E-P1"

    NASA Astrophysics Data System (ADS)

    Ogawa, Haruo

    "OLYMPUS PEN E-P1", which is new generation system camera, is the first product of Olympus which is new standard "Micro Four-thirds System" for high-resolution mirror-less cameras. It continues good sales by the concept of "small and stylish design, easy operation and SLR image quality" since release on July 3, 2009. On the other hand, the half-size film camera "OLYMPUS PEN" was popular by the concept "small and stylish design and original mechanism" since the first product in 1959 and recorded sale number more than 17 million with 17 models. By the 50th anniversary topic and emotional value of the Olympus pen, Olympus pen E-P1 became big sales. I would like to explain the way of thinking of the product plan that included not only the simple functional value but also emotional value on planning the first product of "Micro Four-thirds System".

  1. 8-Methoxypsoralen is a competitive inhibitor of glutathione S-transferase P1-1

    PubMed Central

    de Oliveira, Diêgo Madureira; de Farias, Marcel Tavares; Teles, André Lacerda Braga; dos Santos Junior, Manoelito Coelho; de Cerqueira, Martins Dias; Lima, Rute Maria Ferreira; El-Bachá, Ramon Santos

    2014-01-01

    The blood-brain barrier (BBB) is known to protect healthy brain cells from potentially dangerous chemical agents, but there are many evidences supporting the idea that this protective action is extended to tumor cells. Since the process of angiogenesis in brain tumors leads to BBB breakdown, biochemical characteristics of the BBB seem to be more relevant than physical barriers to protect tumor cells from chemotherapy. In fact, a number of resistance related factors were already demonstrated to be component of both BBB and tumor cells. The enzyme glutathione S-transferases (GST) detoxify electrophilic xenobiotics and endogenous secondary metabolites formed during oxidative stress. A role has been attributed to GST in the resistance of cancer cells to chemotherapeutic agents. This study characterized 8-methoxypsoralen (8-MOP) as a human GST P1-1 (hGST P1-1) inhibitor. To identify and characterize the potential inhibitory activity of 8-MOP, we studied the enzyme kinetics of the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with GSH catalyzed by hGST P1-1. We report here that 8-MOP competitively inhibited hGST P1-1 relative to CDNB, but there was an uncompetitive inhibition relative to GSH. Chromatographic analyses suggest that 8-MOP is not a substrate. Molecular docking simulations suggest that 8-MOP binds to the active site, but its position prevents the GSH conjugation. Thus, we conclude that 8-MOP is a promising prototype for new GST inhibitors pharmacologically useful in the treatment of neurodegenerative disorders and the resistance of cancer to chemotherapy. PMID:25324722

  2. Expression of Dual-Specificity Phosphatase 5 Pseudogene 1 (DUSP5P1) in Tumor Cells

    PubMed Central

    Staege, Martin S.; Müller, Katja; Kewitz, Stefanie; Volkmer, Ines; Mauz-Körholz, Christine; Bernig, Toralf; Körholz, Dieter

    2014-01-01

    Sequencing of individual clones from a newly established cDNA library from the chemoresistant Hodgkin's lymphoma cell line L-1236 led to the isolation of a cDNA clone corresponding to a short sequence from chromosome 1. Reverse transcriptase-polymerase chain reaction indicated high expression of this sequence in Hodgkin's lymphoma derived cell lines but not in normal blood cells. Further characterization of this sequence and the surrounding genomic DNA revealed that this sequence is part of a human endogenous retrovirus locus. The sequence of this endogenous retrovirus is interrupted by a pseudogene of the dual specificity phosphatase 5 (DUSP5). Reverse transcriptase-polymerase chain reaction revealed high expression of this pseudogene (DUSP5P1) in HL cell lines but not in normal blood cells or Epstein-Barr virus-immortalized B cells. Cells from other tumor types (Burkitt's lymphoma, leukemia, neuroblastoma, Ewing sarcoma) also showed a higher DUSP5P1/DUSP5 ratio than normal cells. Furthermore, we observed that higher expression of DUSP5 in relation to DUSP5P1 correlated with the expression of the pro-apoptotic factor B cell leukemia/lymphoma 2-like 11 (BCL2L11) in peripheral blood cells and HL cells. Knock-down of DUSP5 in HL cells resulted in down-regulation of BCL2L11. Thus, the DUSP5/DUSP5P1 system could be responsible for regulation of BCL2L11 leading to inhibition of apoptosis in these tumor cells. PMID:24651368

  3. Lamb shift in the hydrogen atom: Lifetime of the 2p{1/2}level

    SciTech Connect

    Karshenboim, S.G.

    1995-05-01

    Corrections of order {alpha}(Z{alpha}){sup 2} to the width of the 2p{1/2} level in the hydrogen atom are considered in the logarithmic approximation. The ratio of this width to the splitting of n=2 states can be measured to high accuracy. With the aid of the experimental data available for this ratio, the new value for the Lamb splitting is found to be 1057.8576(21) MHz. 17 refs.

  4. Slow-binding inhibitors of prolyl oligopeptidase with different functional groups at the P1 site.

    PubMed

    Venäläinen, Jarkko I; Juvonen, Risto O; Garcia-Horsman, J Arturo; Wallén, Erik A A; Christiaans, Johannes A M; Jarho, Elina M; Gynther, Jukka; Männistö, Pekka T

    2004-09-15

    POP (prolyl oligopeptidase) specifically hydrolyses a number of small proline-containing peptides at the carboxy end of the proline residue and POP inhibitors have been shown to have cognition-enhancing properties. It has been noted that certain functional groups at the P1 site of the inhibitor, which correspond to the substrate residue on the N-terminal side of the bond to be cleaved, increase the inhibitory potency. However, detailed mechanistic and kinetic analysis of the inhibition has not been studied. In the present study, we examined the effect of different functional groups at the P1 site of the parent inhibitor isophthalic acid bis-(L-prolylpyrrolidine) amide on the binding kinetics to POP. Addition of CHO, CN or COCH(2)OH groups to the P1 site increased the inhibitory potency by two orders of magnitude (K(i)=11.8-0.1 nM) and caused a clear slow-binding inhibition. The inhibitor containing a CHO group had the lowest association rate constant, k(on)=(2.43+/-0.12) x 10(5) M(-1) x s(-1), whereas the inhibitor with a CN group exhibited the fastest binding, k(on)=(12.0+/-0.08)x10(5) M(-1) x s(-1). In addition, the dissociation rate was found to be crucially dependent on the type of the functional group. Compounds with COCH(2)OH and CHO groups had much longer half-lives of dissociation (over 5 h) compared with the compound with the CN group (25 min), although the K(i) values of the compounds were relatively similar. A possibility to optimize the duration of inhibition by changing the functional group at the P1 site is important when planning therapeutically useful POP inhibitors.

  5. Pin p 1 is a major allergen in pine nut and the first food allergen described in the plant group of gymnosperms.

    PubMed

    Cabanillas, Beatriz; Crespo, Jesus F; Maleki, Soheila J; Rodriguez, Julia; Novak, Natalija

    2016-11-01

    This study aimed to report the complete sequence of a 2S albumin purified from pine nut and to analyze its allergenic properties. Individual recognition of this protein by serum IgE from pine nut-allergic patients was assessed. IgE cross-linking capacity was analyzed in a basophil activation test. Inhibition of IgE-binding and stability to heating was also assessed. The complete nucleotide sequence was obtained and a phylogenetic study was carried out. 2S albumin from pine nut (registered as Pin p 1.0101) was recognized by IgE of 75% of sera. The allergen was heat-stable and had a robust capacity to inhibit IgE-binding to whole pine nut extract. The IgE cross-linking capacity of Pin p 1 on basophils was also demonstrated. Despite the low homology of Pin p 1 sequence with other allergenic 2S albumins from angiosperms, Pin p 1 contains the typical skeleton of 8 cysteine residues, important for its α-helixes enriched structure.

  6. Lateral supraorbital approach to ipsilateral PCA-P1 and ICA-PCoA aneurysms

    PubMed Central

    Goehre, Felix; Jahromi, Behnam Rezai; Elsharkawy, Ahmed; Lehto, Hanna; Shekhtman, Oleg; Andrade-Barazarte, Hugo; Munoz, Francisco; Hijazy, Ferzat; Makhkamov, Makhkam; Hernesniemi, Juha

    2015-01-01

    Background: Aneurysms of the posterior cerebral artery (PCA) are rare and often associated with anterior circulation aneurysms. The lateral supraorbital approach allows for a very fast and safe approach to the ipsilateral lesions Circle of Willis. A technical note on the successful clip occlusion of two aneurysms in the anterior and posterior Circle of Willis via this less invasive approach has not been published before. The objective of this technical note is to describe the simultaneous microsurgical clip occlusion of an ipsilateral PCA-P1 and an internal carotid artery - posterior communicating artery (ICA-PCoA) aneurysm via the lateral supraorbital approach. Case Description: The authors present a technical report of successful clip occlusions of ipsilateral located PCA-P1 and ICA-PCoA aneurysms. A 59-year-old female patient was diagnosed with a PCA-P1 and an ipsilateral ICA-PCoA aneurysm by computed tomography angiography (CTA) after an ischemic stroke secondary to a contralateral ICA dissection. The patient underwent microsurgical clipping after a lateral supraorbital craniotomy. The intraoperative indocyanine green (ICG) videoangiography and the postoperative CTA showed a complete occlusion of both aneurysms; the parent vessels (ICA and PCA) were patent. The patient presents postoperative no new neurologic deficit. Conclusion: The lateral supraorbital approach is suitable for the simultaneous microsurgical treatment of proximal anterior circulation and ipsilateral proximal PCA aneurysms. Compared to endovascular treatment, direct visual control of brainstem perforators is possible. PMID:26060600

  7. Direct Band Gap Gallium Antimony Phosphide (GaSbxP1−x) Alloys

    PubMed Central

    Russell, H. B.; Andriotis, A. N.; Menon, M.; Jasinski, J. B.; Martinez-Garcia, A.; Sunkara, M. K.

    2016-01-01

    Here, we report direct band gap transition for Gallium Phosphide (GaP) when alloyed with just 1–2 at% antimony (Sb) utilizing both density functional theory based computations and experiments. First principles density functional theory calculations of GaSbxP1−x alloys in a 216 atom supercell configuration indicate that an indirect to direct band gap transition occurs at x = 0.0092 or higher Sb incorporation into GaSbxP1−x. Furthermore, these calculations indicate band edge straddling of the hydrogen evolution and oxygen evolution reactions for compositions ranging from x = 0.0092 Sb up to at least x = 0.065 Sb making it a candidate for use in a Schottky type photoelectrochemical water splitting device. GaSbxP1−x nanowires were synthesized by reactive transport utilizing a microwave plasma discharge with average compositions ranging from x = 0.06 to x = 0.12 Sb and direct band gaps between 2.21 eV and 1.33 eV. Photoelectrochemical experiments show that the material is photoactive with p-type conductivity. This study brings attention to a relatively uninvestigated, tunable band gap semiconductor system with tremendous potential in many fields. PMID:26860470

  8. Chemocyanin, a small basic protein from the lily stigma, induces pollen tube chemotropism

    PubMed Central

    Kim, Sunran; Mollet, Jean-Claude; Dong, Juan; Zhang, Kangling; Park, Sang-Youl; Lord, Elizabeth M.

    2003-01-01

    In plant reproduction, pollination is an essential process that delivers the sperm through specialized extracellular matrices (ECM) of the pistil to the ovule. Although specific mechanisms of guidance for pollen tubes through the pistil are not known, the female tissues play a critical role in this event. Many studies have documented the existence of diffusible chemotropic factors in the lily stigma that can induce pollen tube chemotropism in vitro, but no molecules have been isolated to date. In this study, we identified a chemotropic compound from the stigma by use of biochemical methods. We purified a lily stigma protein that is active in an in vitro chemotropism assay by using cation exchange, gel filtration, and HPLC. Tryptic digestion of the protein yielded peptides that identified the protein as a plantacyanin (basic blue protein), and this was confirmed by cloning the cDNA from the lily stigma. Plantacyanins are small cell wall proteins of unknown function. The measured molecular mass by electrospray ionization ion source MS is 9,898 Da, and the molecular mass of the mature protein (calculated from the cDNA) is 9,900.2 Da. Activity of the lily plantacyanin (named chemocyanin) is enhanced in the presence of stigma/stylar cysteine-rich adhesin, previously identified as a pollen tube adhesin in the lily style. PMID:14671326

  9. Self-generated covalent cross-links in the cell-surface adhesins of Gram-positive bacteria.

    PubMed

    Baker, Edward N; Squire, Christopher J; Young, Paul G

    2015-10-01

    The ability of bacteria to adhere to other cells or to surfaces depends on long, thin adhesive structures that are anchored to their cell walls. These structures include extended protein oligomers known as pili and single, multi-domain polypeptides, mostly based on multiple tandem Ig-like domains. Recent structural studies have revealed the widespread presence of covalent cross-links, not previously seen within proteins, which stabilize these domains. The cross-links discovered so far are either isopeptide bonds that link lysine side chains to the side chains of asparagine or aspartic acid residues or ester bonds between threonine and glutamine side chains. These bonds appear to be formed by spontaneous intramolecular reactions as the proteins fold and are strategically placed so as to impart considerable mechanical strength.

  10. Human glutathione S-transferase P1-1 functions as an estrogen receptor α signaling modulator

    SciTech Connect

    Liu, Xiyuan; An, Byoung Ha; Kim, Min Jung; Park, Jong Hoon; Kang, Young Sook; Chang, Minsun

    2014-09-26

    Highlights: • GSTP induces the classical ERα signaling event. • The functional GSTP is a prerequisite for GSTP-induced ERα transcription activity. • The expression of RIP140, a transcription cofactor, was inhibited by GSTP protein. • We propose the novel non-enzymatic role of GSTP. - Abstract: Estrogen receptor α (ERα) plays a crucial role in estrogen-mediated signaling pathways and exerts its action as a nuclear transcription factor. Binding of the ligand-activated ERα to the estrogen response element (ERE) is a central part of ERα-associated signal transduction pathways and its aberrant modulation is associated with many disease conditions. Human glutathione S-transferase P1-1 (GSTP) functions as an enzyme in conjugation reactions in drug metabolism and as a regulator of kinase signaling pathways. It is overexpressed in tumors following chemotherapy and has been associated with a poor prognosis in breast cancer. In this study, a novel regulatory function of GSTP has been proposed in which GSTP modulates ERE-mediated ERα signaling events. Ectopic expression of GSTP was able to induce the ERα and ERE-mediated transcriptional activities in ERα-positive but GSTP-negative MCF7 human breast cancer cells. This inductive effect of GSTP on the ERE-transcription activity was diminished when the cells express a mutated form of the enzyme or are treated with a GSTP-specific chemical inhibitor. It was found that GSTP inhibited the expression of the receptor interacting protein 140 (RIP140), a negative regulator of ERα transcription, at both mRNA and protein levels. Our study suggests a novel non-enzymatic role of GSTP which plays a significant role in regulating the classical ERα signaling pathways via modification of transcription cofactors such as RIP140.

  11. 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.

  12. 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.

  13. 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.

  14. More than a marine propeller--the flagellum of the probiotic Escherichia coli strain Nissle 1917 is the major adhesin mediating binding to human mucus.

    PubMed

    Troge, Anja; Scheppach, Wolfgang; Schroeder, Bjoern O; Rund, Stefan A; Heuner, Klaus; Wehkamp, Jan; Stange, Eduard F; Oelschlaeger, Tobias A

    2012-12-01

    The flagellum of the probiotic Escherichia coli strain Nissle 1917 (EcN) is not just responsible for motility, but also for EcN's ability to induce the production of human β-defensin 2. Here, we report a third function of this EcN organell. In this study we investigated the role of the EcN flagellum in adhesion to different host tissues by ex vivo and in vitro studies. Ex vivo studies with cryosections of human gut biopsies revealed that the flagellum of EcN is most likely important for efficient adhesion to the human intestinal tract. These results and in vitro studies with different epithelial cells indicated that the presence of mucus is important for efficient mediation of adhesion by the flagellum of EcN. We observed direct interaction between isolated flagella from EcN wild type and porcine mucin 2 as well as human mucus. However, we could not observe any interaction of the flagella with murine mucus. For the first time, we identified the mucus component gluconate as one receptor for the binding of flagella from EcN and were able to exclude the flagellin domain D3 as a responsible interaction partner. We propose that the flagellum of EcN is its major adhesin in vivo, which enables this probiotic strain to compete efficiently for binding sites on host tissue with several bacterial pathogens.

  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. Expression analysis of the speech-related genes FoxP1 and FoxP2 and their relation to singing behavior in two songbird species

    PubMed Central

    Chen, Qianqian; Heston, Jonathan B.; Burkett, Zachary D.; White, Stephanie A.

    2013-01-01

    SUMMARY Humans and songbirds are among the rare animal groups that exhibit socially learned vocalizations: speech and song, respectively. These vocal-learning capacities share a reliance on audition and cortico-basal ganglia circuitry, as well as neurogenetic mechanisms. Notably, the transcription factors Forkhead box proteins 1 and 2 (FoxP1, FoxP2) exhibit similar expression patterns in the cortex and basal ganglia of humans and the zebra finch species of songbird, among other brain regions. Mutations in either gene are associated with language disorders in humans. Experimental knock-down of FoxP2 in the basal ganglia song control region Area X during song development leads to imprecise copying of tutor songs. Moreover, FoxP2 levels decrease naturally within Area X when zebra finches sing. Here, we examined neural expression patterns of FoxP1 and FoxP2 mRNA in adult Bengalese finches, a songbird species whose songs exhibit greater sequence complexity and increased reliance on audition for maintaining their quality. We found that FoxP1 and FoxP2 expression in Bengalese finches is similar to that in zebra finches, including strong mRNA signals for both factors in multiple song control nuclei and enhancement of FoxP1 in these regions relative to surrounding brain tissue. As with zebra finches, when Bengalese finches sing, FoxP2 is behaviorally downregulated within basal ganglia Area X over a similar time course, and expression negatively correlates with the amount of singing. This study confirms that in multiple songbird species, FoxP1 expression highlights song control regions, and regulation of FoxP2 is associated with motor control of song. PMID:24006346

  17. VizieR Online Data Catalog: Vilnius photometry of TGU H994 P1 (Straizys+, 2016)

    NASA Astrophysics Data System (ADS)

    Straizys, V.; Cepas, V.; Boyle, R. P.; Munari, U.; Zdanavicius, J.; Maskoliunas, M.; Kazlauskas, A.; Zdanavicius, K.

    2015-11-01

    Table 2 contains the results of photometry of 855 stars down to V~20mag in the Vilnius seven-color system in the three areas of dark cloud TGU H994 P1. The identification numbers, coordinates, V magnitudes, six color indices in the Vilnius system and photometric spectral types are given. The identification numbers start from 1001 to avoid confusion with the catalogue of Cepas et al. (2013BaltA..22..243C, Cat. J/BaltA/22/243). The Coordinates are from PPMXL catalog (Roeser et al. 2010AJ....139.2440R, Cat. I/317). (1 data file).

  18. GALLIUM INDIUM ARSENIDE PHOSPHIDE (GaxIn1-xASyP1-y)

    NASA Astrophysics Data System (ADS)

    Goldberg, Yu. A.; Shmidt, Natalya M.

    The following sections are included: * Basic Parameters at 300 K * Basic Properties of GaxIn1-xASyP1-y Compositions Lattice-Matched to InP at 300 K(x≅0.47y) * Band Structure and Carrier Concentration * Temperature Dependences * Dependence on Hydrostatic Pressure * Band Discontinuities at GaInAsP/InP Heterostructure * Effective Masses * Donors and Acceptors * Electrical Properties * Mobility and Hall Effect * Transport Properties in High Electric Field * Impact Ionization * Recombination Parameters * Optical Properties * Thermal Properties * Mechanical Properties, Elastic Constants, Lattice Vibrations, Other Properties * References

  19. Engineering the substrate specificity of rhizopuspepsin: the role of Asp 77 of fungal aspartic proteinases in facilitating the cleavage of oligopeptide substrates with lysine in P1.

    PubMed Central

    Lowther, W. T.; Majer, P.; Dunn, B. M.

    1995-01-01

    Rhizopuspepsin and other fungal aspartic proteinases are distinct from the mammalian enzymes in that they are able to cleave substrates with lysine in the P1 position. Sequence and structural comparisons suggest that two aspartic acid residues, Asp 30 and Asp 77 (pig pepsin numbering), may be responsible for generating this unique specificity. Asp 30 and Asp 77 were changed to the corresponding residues in porcine pepsin, Ile 30 and Thr 77, to create single and double mutants. The zymogen forms of the wild-type and mutant enzymes were overexpressed in Escherichia coli as inclusion bodies. Following solubilization, denaturation, refolding, activation, and purification to homogeneity, structural and kinetic comparisons were made. The mutant enzymes exhibited a high degree of structural similarity to the wild-type recombinant protein and a native isozyme. The catalytic activities of the recombinant proteins were analyzed with chromogenic substrates containing lysine in the P1, P2, or P3 positions. Mutation of Asp 77 resulted in a loss of 7 kcal mol-1 of transition-state stabilization energy in the hydrolysis of the substrate containing lysine in P1. An inhibitor containing the positively charged P1-lysine side chain inhibited only the enzymes containing Asp 77. Inhibition of the Asp 77 mutants of rhizopuspepsin and several mammalian enzymes was restored upon acetylation of the lysine side chain. These results suggest that an exploitation of the specific electrostatic interaction of Asp 77 in the active site of fungal enzymes may lead to the design of compounds that preferentially inhibit a variety of related Candida proteinases in immunocompromised patients. PMID:7613467

  20. Neural FoxP2 and FoxP1 expression in the budgerigar, an avian species with adult vocal learning.

    PubMed

    Hara, Erina; Perez, Jemima M; Whitney, Osceola; Chen, Qianqian; White, Stephanie A; Wright, Timothy F

    2015-04-15

    Vocal learning underlies acquisition of both language in humans and vocal signals in some avian taxa. These bird groups and humans exhibit convergent developmental phases and associated brain pathways for vocal communication. The transcription factor FoxP2 plays critical roles in vocal learning in humans and songbirds. Another member of the forkhead box gene family, FoxP1 also shows high expression in brain areas involved in vocal learning and production. Here, we investigate FoxP2 and FoxP1 mRNA and protein in adult male budgerigars (Melopsittacus undulatus), a parrot species that exhibits vocal learning as both juveniles and adults. To examine these molecules in adult vocal learners, we compared their expression patterns in the budgerigar striatal nucleus involved in vocal learning, magnocellular nucleus of the medial striatum (MMSt), across birds with different vocal states, such as vocalizing to a female (directed), vocalizing alone (undirected), and non-vocalizing. We found that both FoxP2 mRNA and protein expressions were consistently lower in MMSt than in the adjacent striatum regardless of the vocal states, whereas previous work has shown that songbirds exhibit down-regulation in the homologous region, Area X, only after singing alone. In contrast, FoxP1 levels were high in MMSt compared to the adjacent striatum in all groups. Taken together these results strengthen the general hypothesis that FoxP2 and FoxP1 have specialized expression in vocal nuclei across a range of taxa, and suggest that the adult vocal plasticity seen in budgerigars may be a product of persistent down-regulation of FoxP2 in MMSt.

  1. Neural FoxP2 and FoxP1 expression in the budgerigar, an avian species with adult vocal learning

    PubMed Central

    Hara, Erina; Perez, Jemima M.; Whitney, Osceola; Chen, Qianqian; White, Stephanie A.; Wright, Timothy F.

    2015-01-01

    Vocal learning underlies acquisition of both language in humans and vocal signals in some avian taxa. These bird groups and humans exhibit convergent developmental phases and associated brain pathways for vocal communication. The transcription factor FoxP2 plays critical roles in vocal learning in humans and songbirds. Another member of the forkhead box gene family, FoxP1 also shows high expression in brain areas involved in vocal learning and production. Here, we investigate FoxP2 and FoxP1 mRNA and protein in adult male budgerigars (Melopsittacus undulatus), a parrot species that exhibits vocal learning as both juveniles and adults. To examine these molecules in adult vocal learners, we compared their expression patterns in the budgerigar striatal nucleus involved in vocal learning, magnocellular nucleus of the medial striatum (MMSt), across birds with different vocal states, such as vocalizing to a female (directed), vocalizing alone (undirected), and non-vocalizing. We found that both FoxP2 mRNA and protein expressions were consistently lower in MMSt than in the adjacent striatum regardless of the vocal states, whereas previous work has shown that songbirds exhibit downregulation in the homologous region, Area X, only after singing alone. In contrast, FoxP1 levels were high in MMSt compared to the adjacent striatum in all groups. Taken together these results strengthen the general hypothesis that FoxP2 and FoxP1 have specialized expression in vocal nuclei across a range of taxa, and suggest that the adult vocal plasticity seen in budgerigars may be a product of persistent down-regulation of FoxP2 in MMSt. PMID:25601574

  2. ON BOUNDARY VALUES IN L_p, p > 1, OF SOLUTIONS OF ELLIPTIC EQUATIONS

    NASA Astrophysics Data System (ADS)

    Guščin, A. K.; Mihaĭlov, V. P.

    1980-02-01

    The behavior near the boundary of generalized solutions of a second order elliptic equation \\displaystyle \\sum_{i,j=1}^n\\frac{\\partial}{\\partial x_i}\\biggl(a_{ij}(x)\\fra......artial x_j}\\biggr)=f,\\qquad x\\in Q=\\{\\vert x\\vert < 1\\}\\subset\\mathbf{R}_n,in W_p^1(\\mathcal{Q}), p > 1, is studied. It is shown that under a certain condition on the right side of the equation, the boundedness of the function \\Vert x\\Vert_{L_p(\\vert x\\vert=r)}, \\frac{1}{2}\\le r < 1, is necessary and sufficient for the existence of a limit for the solution u(rw), \\frac{1}{2}\\le r < 1, \\vert w\\vert=1, in L_p(\\vert w\\vert=1) as r\\to 1 - 0. Moreover, the summability of the function \\displaystyle (1-\\vert x\\vert)\\vert u(x)\\vert^{p - 2}\\vert\

  3. Quenching of I(2P 1/2) by O 3 and O( 3P)

    NASA Astrophysics Data System (ADS)

    Azyazov, V. N.; Antonov, I. O.; Ruffner, S.; Heaven, M. C.

    2006-02-01

    Oxygen-iodine lasers that utilize electrical or microwave discharges to produce singlet oxygen are currently being developed. The discharge generators differ from conventional chemical singlet oxygen generators in that they produce significant amounts of atomic oxygen. Post-discharge chemistry includes channels that lead to the formation of ozone. Consequently, removal of I(2P 1/2) by O atoms and O 3 may impact the efficiency of discharge driven iodine lasers. In the present study we have measured the rate constants for quenching of I(2P 1/2) by O( 3P) atoms and O 3 using pulsed laser photolysis techniques. The rate constant for quenching by O 3, 1.8x10 -12 cm 3 s -1, was found to be a factor of five smaller than the literature value. The rate constant for quenching by O( 3P) was 1.2x10 -11 cm 3 s -1. This was six times larger than a previously reported upper bound, but consistent with estimates obtained by modeling the kinetics of discharge-driven laser systems.

  4. DSC deconvolution of the structural complexity of c-MYC P1 promoter G-quadruplexes.

    PubMed

    Dettler, Jamie M; Buscaglia, Robert; Le, Vu H; Lewis, Edwin A

    2011-03-16

    We completed a biophysical characterization of the c-MYC proto-oncogene P1 promoter quadruplex and its interaction with a cationic porphyrin, 5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrin (TMPyP4), using differential scanning calorimetry, isothermal titration calorimetry, and circular dichroism spectroscopy. We examined three different 24-mer oligonucleotides, including the wild-type (WT) sequence found in the c-MYC P(1) promoter and two mutant G→T sequences that are known to fold into single 1:2:1 and 1:6:1 loop isomer quadruplexes. Biophysical experiments were performed on all three oligonucleotide sequences at two different ionic strengths (30 mM [K(+)] and 130 mM [K(+)]). Differential scanning calorimetry experiments demonstrated that the WT quadruplex consists of a mixture of at least two different folded conformers at both ionic strengths, whereas both mutant sequences exhibit a single two-state melting transition at both ionic strengths. Isothermal titration calorimetry experiments demonstrated that both mutant sequences bind 4 mols of TMPyP4 to 1 mol of DNA, in similarity to the WT sequence. The circular dichroism spectroscopy signatures for all three oligonucleotides at both ionic strengths are consistent with an intramolecular parallel stranded G-quadruplex structure, and no change in quadruplex structure is observed upon addition of saturating amounts of TMPyP4 (i.e., 4:1 TMPyP4/DNA).

  5. FURTHER CONSTRAINTS ON THE OPTICAL TRANSMISSION SPECTRUM OF HAT-P-1b

    SciTech Connect

    Montalto, M.; Santos, N. C.; Martins, J. H. C.; Figueira, P.; Alonso, R.; Iro, N.; Desidera, S.

    2015-09-20

    We report on novel observations of HAT-P-1 aimed at constraining the optical transmission spectrum of the atmosphere of its transiting hot-Jupiter exoplanet. Ground-based differential spectrophotometry was performed over two transit windows using the DOLORES spectrograph at the Telescopio Nazionale Galileo. Our measurements imply an average planet to star radius ratio equal to R{sub p}/R{sub *} = (0.1159 ± 0.0005). This result is consistent with the value obtained from recent near-infrared measurements of this object, but differs from previously reported optical measurements, being lower by around 4.4 exoplanet scale heights. Analyzing the data over five different spectral bins of ∼600 Å wide, we observed a single peaked spectrum (3.7 σ level) with a blue cutoff corresponding to the blue edge of the broad absorption wing of sodium and an increased absorption in the region in-between 6180 and 7400 Å. We also infer that the width of the broad absorption wings due to alkali metals is likely narrower than the one implied by solar abundance clear atmospheric models. We interpret the result as evidence that HAT-P-1b has a partially clear atmosphere at optical wavelengths with a more modest contribution from an optical absorber than previously reported.

  6. Integrated P1 Hohlraum/Capsule Simulations with Comparison to Neutron and X-Ray Measurements

    NASA Astrophysics Data System (AD