DOCSLIB.ORG

United States Patent ( 10 ) Patent No.: US 10,676,721 B2 Collins Et Al

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
United States Patent ( 10 ) Patent No.: US 10,676,721 B2 Collins Et Al US010676721B2 United States Patent ( 10 ) Patent No.: US 10,676,721 B2 Collins et al. (45 ) Date of Patent : Jun . 9 , 2020 ( 54 ) BACTERIOPHAGES EXPRESSING 6,699,701 B1 3/2004 Sulakvelidze et al . ANTIMICROBIAL PEPTIDES AND USES 6,759,229 B2 * 7/2004 Schaak 435 /235.1 7,211,426 B2 5/2007 Bruessow et al. THEREOF 8,153,119 B2 4/2012 Collins et al. 8,182,804 B1 5/2012 Collins et al. ( 75 ) Inventors : James J. Collins, Newton , MA (US ) ; 2001/0026795 A1 10/2001 Merril et al . Michael Koeris , Natick , MA (US ) ; 2002/0013671 Al 1/2002 Ananthaiyer et al. 2004/0161141 A1 8/2004 Dewaele Timothy Kuan - Ta Lu , Charlestown, 2005/0004030 A1 1/2005 Fischetti et al . MA (US ) ; Tanguy My Chau , Palo 2007/0020240 A1 1/2007 Jayasheela et al. Alto , CA ( US ) ; Gregory 2007/0134207 Al 6/2007 Bruessow et al . Stephanopoulos , Winchester , MA (US ) ; 2007/0207209 A1 * 9/2007 Murphy A61K 9/06 Christopher Jongsoo Yoon , Seoul 424/484 (KR ) 2007/0248724 Al 10/2007 Sulakvelidze et al. 2008/0194000 Al 8/2008 Pasternack et al. ( 73 ) Assignees : Trustees of Boston University , Boston , 2008/0247997 Al 10/2008 Reber et al . MA (US ) ; Massachusetts Institute of 2008/0311643 A1 12/2008 Sulakvelidze et al. Technology , Cambridge, MA (US ) 2008/0318867 A1 12/2008 Loessner et al. FOREIGN PATENT DOCUMENTS ( * ) Notice: Subject to any disclaimer , the term of this patent is extended or adjusted under 35 EP 0403458 A1 12/1990 CO7K 7/10 WO 2002/034892 Al 5/2002 U.S.C. 154 ( b ) by 2166 days. WO 2004/046319 A2 6/2004 WO 2004/062677 Al 7/2004 (21 ) Appl. No .: 13 /224,776 WO 2005/009451 A1 2/2005 WO 2005/07 1088 A1 8/2005 ( 22 ) Filed : Sep. 2 , 2011 WO 2006/137847 A2 12/2006 (Under 37 CFR 1.47 ) WO 2008/110840 A1 9/2008 WO 2009/108406 A2 9/2009 (65 ) Prior Publication Data US 2015/0050717 A1 Feb. 19 , 2015 OTHER PUBLICATIONS Westwater et al. , Antimicrobial Agents and Chemotherapy, 2003 , Related U.S. Application Data vol. 47 , pp . 1301-1307 . * (63 ) Continuation of application No. Westwater et al. , Antimicrobial Agents and Chemotherapy, 2003 , PCT /US2010 /026357 , filed on Mar. 5 , 2010 . Vo147, pp . 1301-1307. * Horgan et al. , Applied and Environmental Microbiology, 2008, vol . (60 ) Provisional application No. 61/ 157,773 , filed on Mar. 75 , pp . 872-874 .* 5 , 2009 . Chap , Genbank accession No. YP_241096.1, published May 20 , 2007. * (51 ) Int. Ci. Clark et al ., Trends in Biotechnology , 2006 , vol. 24., pp . 212-218 . * C12N 7700 ( 2006.01 ) Takahara et al. , The Journal of Biological Chemistry , 1985 , vol . 260 , A61K 35/76 (2015.01 ) pp . 2670-2674 .* A61K 38/17 ( 2006.01) Sargent et al. , FEMS, 2006 , vol. 254 , pp. 198-207 .* COZK 14/435 ( 2006.01 ) (Continued ) COZK 14/46 ( 2006.01 ) C12N 9/50 ( 2006.01 ) Primary Examiner - Mindy G Brown (52 ) U.S. CI. ( 74 ) Attorney , Agent, or Firm - Nixon Peabody LLP CPC C12N 7700 (2013.01 ) ; A61K 35/76 ( 2013.01 ) ; A61K 38/1767 (2013.01 ) ; CO7K ( 57 ) ABSTRACT 14/43504 ( 2013.01 ) ; CO7K 14/43563 ( 2013.01) ; C07K 14/463 (2013.01 ) ; C12N The present invention is generally related to engineered 9/503 (2013.01 ) ; C12N 2795/10221 ( 2013.01 ) ; bacteriophages expressing antimicrobial peptides or lytic C12N 2795/10231 ( 2013.01 ) ; C12N enzymes or fragments thereof for targeting a broad spectrum 2795/10232 (2013.01 ) ; CI2N 2795/12032 of bacterial hosts , and for the long - term suppression of ( 2013.01) ; C12N 2795/12043 ( 2013.01 ) ; C12N bacterial phage resistance for reducing bacterial infections . 2795/14132 ( 2013.01) ; C12N 2795/14143 In some embodiments , bacteriophages express antimicrobial ( 2013.01 ) peptides or antimicrobial polypeptides ( e.g. phage lytic (58 ) Field of Classification Search enzymes ) which are secreted from the host bacteria , or None alternatively released upon lysis of the bacterial host cell . See application file for complete search history. Aspects of the present invention also relate to the use of the engineered bacteriophages for the reduction of bacterial (56 ) References Cited infections, both in a subject or for bioremediation purposes, in clinical settings and wound healing . U.S. PATENT DOCUMENTS 4,678,750 A 7/1987 Vandenbergh et al. 19 Claims, 30 Drawing Sheets 6,335,012 B1 1/2002 Fischetti et al. Specification includes a Sequence Listing . US 10,676,721 B2 Page 2 ( 56 ) References Cited Jensen et al. , Appl Environ Microbiol ., 64 ( 2 ) :575-80 ( 1998) . “ Preva lence of broad - host - range lytic bacteriophages of Sphaerotilus natans, Escherichia coli, and Pseudomonas aeruginosa .” OTHER PUBLICATIONS Keller et al. , Nat Rev Microbiol. , 4 ( 4 ): 249-58 (2006 ). " Communi Stein , et al. , “ Two Different Lantibiotic - Like Peptides Originate cation in bacteria : an ecological and evolutionary perspective .” from the Ericin Gene Cluster of Bacillus subtills A 1/3 , " J. Bacteriol Lederberg , J., Proc Natl Acad Sci US A., 93 ( 8 ) :3167-8 , ( 1996 ) . 2002 , 184 ( 6 ) :1703-1711 . “ Smaller fleas . ad infinitum : therapeutic bacteriophage redux .” Obeso , et al. , “ Lytic activity of the recombinant staphylococcal Liu et al. , Science ., 295 ( 5562 ): 2091-4 (2002 ) . “ Reverse transcriptase Bacteriophage PhiH5 endolysin active against Staphylococcus aureus mediated tropism switching in Bordetella bacteriophage . ” in milk , ” Int J Food Microbiol. 2008 , 128 ( 2 ) :212-218 . Loose et al ., Nature . , 443 ( 7113 ) : 867-9 ( 2006 ) . “ A linguistic model Becker et al ., FEMS Microbiol Lett. , 287 ( 2 ) : 185-91 (2008 ) . “ The for the rational design of antimicrobial peptides .” phage K lytic enzyme LysK and lysostaphin act synergistically to Lorch, A., Biotechnology and Development Monitor , 39 :14-17 , kill MRSA . " ( 1999 ) . “ Bacteriophages : An alternative to antibiotics ? " . Brogden , K.A . , Nat Rev Microbiol ., 3 ( 3 ) :238-50 ( 2005 ) . “ Antimi Lu et al ., Proc Natl Acad Sci US A., 104 ( 27 ) : 11197-202 (2007 ) . crobial peptides: pore formers or metabolic inhibitors in bacteria ? " . “ Dispersing biofilms with engineered enzymatic bacteriophage .” Lutz and Bujard , Nucleic Acids Res ., 25 ( 6 ) :1203-10 ( 1997 ) . “ Inde Lu et al. , Proc Natl Acad Sci US A., 106 ( 12 ) : 4629-34 , (2009 ) . pendent and tight regulation of transcriptional in units Escherichia “ Engineered bacteriophage targeting gene networks as adjuvants for coli via the LacR / 0 , the TetR / O and AraC /11-12 regulatory ele antibiotic therapy. ” ments . ” Merril et al. , Nat Rev Drug Discov ., 2 ( 6 ): 489-97 (2003 ) “ The Cegelski et al. , Nat Rev Microbiol . , 6 ( 1 ) : 17-27 (2008 ) . “ The prospect for bacteriophage therapy in Western medicine. ” biology and future prospects of antivirulence therapies. ” Movva et al ., J Biol Chem . , 255 ( 1 ) :27-9 , ( 1980 ) “ Amino acid Curtin et al ., Antimicrob Agents Chemother. , 50 ( 4 ) :1268-75 ( 2006 ) . sequence of the signal peptide of ompA protein , a major outer “Using bacteriophages to reduce formation of catheter- associated membrane protein of Escherichia coli .” biofilms by Staphylococcus epidermidis .” O'Flaherty et al ., Journal of Bacteriology , 187 (20 ) :7161-7164 , Dai et al. , Proc Natl Acad Sci US A., 107 ( 9 ): 4347-52 (2010 ) . ( 2005 ) . “ The recombinant phage lysin LysK has a broad spectrum “ Three- dimensional structure of tropism - switching Bordetella bac of lytic activity against clinically relevant staphylococci, including teriophage. " methicillin - resistant Staphylococcus aureus. Deresinski, S., Clin Infect Dis ., 48 ( 8 ) : 1096-101 (2009 ) . “ Bacterio Skurnik et al. , Int J Med Microbiol ., 296 ( 1 ) : 5-14 ( 2006 ) . " Phage phage therapy : exploiting smaller fleas. " therapy : facts and fiction ." D'Herelle , F. Comptes Rendus Hebdomadaires des Seances de Stone, R., Science . , 298( 5594 ): 728-31 (2002 ). “ Bacteriophage therapy . L'academie des Sciences ( 165 ) : 373-5 . ( 1917 ) . " An invisible Stalin's forgotten cure .” antagonist microbe of dysentery bacillus” . Twort, F. W., Lancet , 2 :1241-1243 , ( 1915 ) . “ An investigation on the Fischetti, V.A., Nat Biotechnol ., 19 ( 8 ) :734-5 (2001 ) . “ Phage anti nature of ultra -microscopic viruses. ” bacterials make a comeback . ” Westwater et al. , Antimicrob Agents Chemother. , 47 ( 4 ) : 1301-7 Hagens et al ., Microb Drug Resist ., 12 ( 3 ) : 164-8 (2006 ) . “ Augmen ( 2003 ) . “ Use of genetically engineered phage to deliver antimicro tation of the antimicrobial efficacy of antibiotics by filamentous bial agents to bacteria : an alternative therapy for treatment of phage. ” bacterial infections. " Hancock et al ., Nat Biotechnol ., 24 ( 12 ) : 1551-7 ( 2006 ) . “ Antimi Wise, R., J Antimicrob Chemother ., 54 ( 2 ) : 306-10 , (2004 ) . “ The crobial and host -defense peptides as new anti- infective therapeutic relentless rise of resistance ? " strategies. " Yacoby et al. , Antimicrob Agents Chemother. , 50 (6 ) :2087-97 , (2006 ) . Horgan et al ., Appl Environ Microbiol ., 75 ( 3 ) :872-4 (2009 ). “ Phage “ Targeting antibacterial agents by using drug -carrying filamentous lysin Lysk can be truncated to its CHAP domain and retain lytic bacteriophages .” activity against live antibiotic -resistant staphylococci. " Yacoby et al ., Antimicrob Agents Chemother. , 51 (6 ) :2156-63 , (2007 ) . Huff et al. , Poultry Science , 83: 1944-1947 , (2004 ) . “ Therapeutic “ Targeted drug - carrying bacteriophages as antibacterial nanomedicines. ” efficacy of bacteriophage and Baytril ( enrofloxacin ) individually and in combination to treat colibacillosis in broilers ." * cited by examiner U.S. Patent Jun . 9 , 2020 Sheet 1 of 30 US 10,676,721 B2 17.CecP1+OmpA 9 TIMEHOURS() 1FIG. 17.CecP1-OmpA best. 782 17.Indol-OmpA 00.00( ABSORPTION U.S. Patent Jun . 9 , 2020 Sheet 2 of 30 US 10,676,721 B2 17.Cam-Omp 1wegduo- H moment IAND ........ 10 26 8 FIG.2 terhet weet wat*** SAUREUSGROWTHCURVEFOR17 ws w 0.6 U.S. Patent Jun . 9 , 2020 Sheet 3 of 30 US 10,676,721 B2 6 Co .FIG b . 2 . 1 1 ?? 1.2 U.S. Patent Jun . 9 , 2020 Sheet 4 of 30 US 10,676,721 B2 SA.1589.41.640ug/mL2 EC.J589.7.1924g/mliEC.1589.47.192ug /mL2 ECJ589.47.640ug/mL2 $4.3589.7.192ug/mL1 SA.J589.7.192ugmL2 $4,3589.41.640ugml1 EC.J589.47.640ugml1 tristewomano betoout more times en arbetet.wetter etconceitonei see ning oleei FIG.4 Z U.S.
Load more

Recommended publications
  • Copyrighted Material
    CHAPTER 1 Viruses and Their Importance CHAPTER 1 AT A GLANCE Viruses infect: • Humans • Other vertebrates • Invertebrates Smallpox1 Bluetongue virus-infected sheep2 Tipula sp. larvae (leatherjackets) infected with invertebrate iridescent virus 1 • Plants • Fungi • Bacteria COPYRIGHTED MATERIAL Escherichia coli Delayed emergence of Damaged potato Mushroom virus X4 cell potato caused by tobacco (spraing) caused by with phage T4 attached5 rattle virus infection3 tobacco rattle virus infection3 1 cc01.indd01.indd 1 118/01/138/01/13 88:14:14 PPMM 2 CHAPTER 1 VIRUSES AND THEIR IMPORTANCE CHAPTER 1 AT A GLANCE (continued) Some viruses are useful: • Phage typing of bacteria • Sources of enzymes • Pesticides • Anti-bacterial agents • Anti-cancer agents • Gene vectors Viruses are parasites; they depend on cells for molecular building blocks, machinery, and energy. Virus particles are small; dimensions range approx. from 20–400 nm. A virus genome is composed of one of the following: double-stranded single-stranded double-stranded single-stranded DNA DNA RNA RNA Photographs reproduced with permission of 1 World Health Organization. 2 From Umeshappa et al . (2011) Veterinary Immunology and Immunopathology , 141, 230. Reproduced by permission of Elsevier and the authors. 3 MacFarlane and Robinson (2004) Chapter 11, Microbe-Vector Interactions in Vector-Borne Diseases , 63rd Symposium of the Society for General Microbiology, Cambridge University Press. Reprinted with permission. 4 University of Warwick. 5 Cornell Integrated Microscopy Center. The presence of viruses is obvious in host organ- 1.1 VIRUSES ARE UBIQUITOUS ON EARTH isms showing signs of disease. Many healthy organisms, however, are hosts of non-pathogenic virus infections, Viruses infect all cellular life forms: eukaryotes (ver- some of which are active, while some are quiescent.
    [Show full text]
  • Enantioselektive Biotransformationen Zur Synthese Von Molekülen Mit
    Untersuchung zur Produktion und gerichteten Immobilisierung einer Alginat Lyase aus Sphingomonas sp. A1 Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Naturwissenschaftlichen Fakultät I Biowissenschaften der Martin-Luther-Universität Halle-Wittenberg von Herrn Dipl.-Biol. Christian Beyerodt geboren am 15.12.1982 in Halle an der Saale Halle (Saale) 2015 Gutachter/in: 1. Prof. Dr. Markus Pietzsch 2. Prof. Dr. Reinhard Neubert 3. Prof. Dr. Christoph Syldatk Tag der öffentlichen Verteidigung: 30.03.2016 2 Selbstständigkeitserklärung Hiermit erkläre ich, Christian Beyerodt, dass ich die vorliegende Arbeit - mit Ausnahme der aufgeführten Personen, Unterlagen bzw. Literaturstellen - selbstständig und ohne fremde Hilfe angefertigt habe, _________________ Halle, Datum 3 Danksagung Die vorliegende Dissertation entstand während meiner Tätigkeit als wissenschaftlicher Mitar- beiter in der Arbeitsgruppe „Aufarbeitung biotechnischer Produkte“ des Institutes für Phar- mazie an der Martin-Luther-Universität Halle-Wittenberg. Viele Menschen haben mich wäh- rend dieser Zeit unterstützt und dazu beigetragen, dass ich diese Zeit der Promotion als ei- nen positiven Lebensabschnitt in Erinnerung behalten werde. Diesen Personen möchte ich im Folgenden danken. Zuerst möchte ich mich bei Prof. Dr. Markus Pietzsch für das entgegengebrachte Vertrauen, mir das Thema zu überlassen, bedanken. Auch danke ich für die vielen konstruktiven Dis- kussionen, Kritiken und Hilfestellungen, welche diese Arbeit erst ermöglichten. Weiterhin danke ich der Firma Lanxess, im speziellen Frau Dr. Hermsdorf und Herrn Dr. Schellenberg. Das interdisziplinäre Projekt wurde die Grundlage für meine Forschungen. Ich danke natürlich auch der gesamten Arbeitsgruppe „AG-Pietzsch“ für die schöne Zeit und die zahlreichen Aktivitäten neben der Arbeit. Die Sommerfeste waren immer ein riesen Spaß.
    [Show full text]
  • Analysis of Carbohydrates and Glycoconjugates by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry: an Update for 2011–2012
    ANALYSIS OF CARBOHYDRATES AND GLYCOCONJUGATES BY MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY: AN UPDATE FOR 2011–2012 David J. Harvey* Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, Oxford OX1 3QU, UK Received 19 June 2014; accepted 15 January 2015 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.21471 This review is the seventh update of the original article published text. As the review is designed to complement the earlier work, in 1999 on the application of MALDI mass spectrometry to the structural formulae, etc. that were presented earlier are not analysis of carbohydrates and glycoconjugates and brings repeated. However, a citation to the structure in the earlier work coverage of the literature to the end of 2012. General aspects is indicated by its number with a prefix (i.e., 1/x refers to such as theory of the MALDI process, matrices, derivatization, structure x in the first review and 2/x to structure x the second). MALDI imaging, and fragmentation are covered in the first part Books, general reviews, and review-type articles directly of the review and applications to various structural types concerned with, or including, MALDI analysis of carbohydrates constitute the remainder. The main groups of compound are and glycoconjugates to have been published during the review oligo- and poly-saccharides, glycoproteins, glycolipids, glyco- period are listed in Table 1. Reviews relating to more specific sides, and biopharmaceuticals. Much of this material is presented aspects of MALDI analysis are listed in the appropriate sections. in tabular form. Also discussed are medical and industrial applications of the technique, studies of enzyme reactions, and II.
    [Show full text]
  • Glycoside Hydrolase Dish from Desulfovibrio Vulgaris Degrades The
    Glycoside Hydrolase DisH from Desulfovibrio vulgaris Degrades the N-Acetylgalactosamine Component of Diverse Biofilms Lei Zhu1, Venkata G. Poosarla1, Sooyeon Song1, Thammajun L. Wood1, Daniel S. Miller3, Bei Yin3, and Thomas K. Wood1, 2* 1Department of Chemical Engineering and 2Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, 16802 3Dow Chemical Company, Collegeville, PA, 19426 *To whom correspondence should be addressed: [email protected] Running title: D. vulgaris DisH disperses its biofilm This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1111/1462-2920.14064 This article is protected by copyright. All rights reserved. SIGNIFICANCE The global costs of corrosion are more than $2.5 trillion every year (3.4% of the global gross domestic product), and a large part of corrosion (30%) is microbiologically influenced corrosion (MIC), which affects oil production, drinking water systems, and pipelines. MIC is commonly caused by sulfate- reducing bacteria (SRB) biofilms, and Desulfovibrio vulgaris is the model organism. The biofilm matrix of D. vulgaris consists of proteins and polysaccharides of mannose, fucose, and N-acetylgalactosamine (GalNAc). However, little is known about how to control its biofilm formation. Since bacteria must degrade their biofilm to disperse, in this work, we identified and then studied predicted secreted glycoside hydrolases from D. vulgaris. We show here that DisH (DVU2239, dispersal hexosaminidase), a previously uncharacterized protein, is an N-acetyl-β-D-hexosaminidase that disperses the D.
    [Show full text]
  • The Efficacy of Lyticase and Β-Glucosidase Enzymes on Biofilm
    Banar et al. BMC Microbiology (2019) 19:291 https://doi.org/10.1186/s12866-019-1662-9 RESEARCH ARTICLE Open Access The efficacy of lyticase and β-glucosidase enzymes on biofilm degradation of Pseudomonas aeruginosa strains with different gene profiles Maryam Banar1, Mohammad Emaneini1, Reza Beigverdi1, Rima Fanaei Pirlar1, Narges Node Farahani1, Willem B. van Leeuwen2 and Fereshteh Jabalameli1* Abstract Background: Pseudomonas aeruginosa is a nosocomial pathogen that causes severe infections in immunocompromised patients. Biofilm plays a significant role in the resistance of this bacterium and complicates the treatment of its infections. In this study, the effect of lyticase and β-glucosidase enzymes on the degradation of biofilms of P. aeruginosa strains isolated from cystic fibrosis and burn wound infections were assessed. Moreover, the decrease of ceftazidime minimum biofilm eliminating concentrations (MBEC) after enzymatic treatment was evaluated. Results: This study demonstrated the effectiveness of both enzymes in degrading the biofilms of P. aeruginosa.In contrast to the lyticase enzyme, β-glucosidase reduced the ceftazidime MBECs significantly (P < 0.05). Both enzymes had no cytotoxic effect on the A-549 human lung carcinoma epithelial cell lines and A-431 human epidermoid carcinoma cell lines. Conclusion: Considering the characteristics of the β-glucosidase enzyme, which includes the notable degradation of P. aeruginosa biofilms and a significant decrease in the ceftazidime MBECs and non-toxicity for eukaryotic cells, this enzyme can be a promising therapeutic candidate for degradation of biofilms in burn wound patients, but further studies are needed. Keywords: Pseudomonas aeruginosa, β-Glucosidase, Lyticase, Biofilm Background patients and associated with chronic infections and antibiotic Pseudomonas aeruginosa is a nosocomial pathogen that resistance [4, 8].
    [Show full text]
  • Abstract 9 10 Bacteriophages (Phages) Are Bacterial Parasites That Can Themselves Be Parasitized by Phage 11 Satellites
    bioRxiv preprint doi: https://doi.org/10.1101/2021.03.30.437493; this version posted March 30, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 To catch a hijacker: abundance, evolution and genetic diversity of 2 P4-like bacteriophage satellites 3 Jorge A. Moura de Sousa1,*& Eduardo P.C. Rocha1,* 4 5 1Microbial Evolutionary Genomics, Institut Pasteur, CNRS, UMR3525, Paris 75015, France 6 * corresponding authors: [email protected], [email protected] 7 8 Abstract 9 10 Bacteriophages (phages) are bacterial parasites that can themselves be parasitized by phage 11 satellites. The molecular mechanisms used by satellites to hijack phages are sometimes 12 understood in great detail, but the origins, abundance, distribution, and composition of these 13 elements are poorly known. Here, we show that P4-like elements are present in more than 14 10% of the genomes of Enterobacterales, and in almost half of those of Escherichia coli, 15 sometimes in multiple distinct copies. We identified over 1000 P4-like elements with very 16 conserved genetic organization of the core genome and a few hotspots with highly variable 17 genes. These elements are never found in plasmids and have very little homology to known 18 phages, suggesting an independent evolutionary origin. Instead, they are scattered across 19 chromosomes, possibly because their integrases are often exchanged with other elements. 20 The rooted phylogenies of hijacking functions are correlated and suggest longstanding co- 21 evolution.
    [Show full text]
  • Biofilms As Promoters of Bacterial Antibiotic Resistance and Tolerance
    antibiotics Review Biofilms as Promoters of Bacterial Antibiotic Resistance and Tolerance Cristina Uruén 1,† , Gema Chopo-Escuin 1,†, Jan Tommassen 2 , Raúl C. Mainar-Jaime 1 and Jesús Arenas 1,* 1 Unit of Microbiology and Immunology, Faculty of Veterinary, University of Zaragoza, Miguel Servet, 177, 50017 Zaragoza, Spain; [email protected] (C.U.); [email protected] (G.C.-E.); [email protected] (R.C.M.-J.) 2 Department of Molecular Microbiology, Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands; [email protected] * Correspondence: [email protected] † These authors contributed equally to this work. Abstract: Multidrug resistant bacteria are a global threat for human and animal health. However, they are only part of the problem of antibiotic failure. Another bacterial strategy that contributes to their capacity to withstand antimicrobials is the formation of biofilms. Biofilms are associations of microorganisms embedded a self-produced extracellular matrix. They create particular environments that confer bacterial tolerance and resistance to antibiotics by different mechanisms that depend upon factors such as biofilm composition, architecture, the stage of biofilm development, and growth condi- tions. The biofilm structure hinders the penetration of antibiotics and may prevent the accumulation of bactericidal concentrations throughout the entire biofilm. In addition, gradients of dispersion of nutrients and oxygen within the biofilm generate different metabolic states of individual cells and favor the development of antibiotic tolerance and bacterial persistence. Furthermore, antimicrobial resistance may develop within biofilms through a variety of mechanisms. The expression of efflux pumps may be induced in various parts of the biofilm and the mutation frequency is induced, while the presence of extracellular DNA and the close contact between cells favor horizontal gene transfer.
    [Show full text]
  • ROLE of in VIVO-INDUCED GENES ILVI and HFQ in the SURVIVAL and VIRULENCE of ACTINOBACILLUS PLEUROPNEUMONIAE By
    ROLE OF IN VIVO-INDUCED GENES ILVI AND HFQ IN THE SURVIVAL AND VIRULENCE OF ACTINOBACILLUS PLEUROPNEUMONIAE By Sargurunathan Subashchandrabose A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Comparative Medicine and Integrative Biology 2011 ABSTRACT ROLE OF IN VIVO-INDUCED GENES ILVI AND HFQ IN THE SURVIVAL AND VIRULENCE OF ACTINOBACILLUS PLEUROPNEUMONIAE By Sargurunathan Subashchandrabose Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a contagious and often fatal disease of pigs. Infection with this bacterium leads to the development of a fulminant pleuropneumonia resulting in severe damage to the lungs. A. pleuropneumoniae genes that are up-regulated during the infection of pig lungs were previously identified in our laboratory and designated as in vivo-induced genes. The role of two such in vivo-induced genes, ilvI and hfq, in the pathobiology of A. pleuropneumoniae is the subject of this dissertation. The gene ilvI encodes an enzyme involved in the biosynthesis of branched-chain amino acids (BCAAs). The leucine-responsive regulatory protein (Lrp) is a transcriptional regulator of the ilvIH operon. BCAA biosynthetic genes are associated with virulence in pathogens infecting the respiratory tract and blood stream. Also, twenty five percent of the A. pleuropneumoniae in vivo-induced genes were up-regulated under BCAA limitation, suggesting that these BCAAs may be found at limiting concentrations in certain sites of the mammalian body such as the respiratory tract. The concentration of amino acids in the porcine pulmonary epithelial lining fluid was determined and BCAAs were found at limiting concentration in the respiratory tract.
    [Show full text]
  • Enzyme Dynamics of Biofilm-Breaking Dispersin B
    Enzyme Dynamics of Biofilm-Breaking Dispersin B Kim, Edward (School: Kalaheo High School) Biofilm-based infections pose two major problems: extreme resistance to antibiotics and many other conventional antimicrobial agents and extreme capacity for evading the host defenses. Dispersin B is a glycoside hydrolase that hydrolyzes the linear polymers in poly-β-1,6-N-acetylglucosamine, considered the main component of the biofilm. Thus, understanding the enzyme dynamics of biofilm-breaking Dispersin B and the nature of the substrate transition state would be important to develop effective antibiotic strategies. To obtain thermodynamic activation parameters, assays of Dispersin B and a biofilm from Staphylococcus aureus were conducted. Dispersin B successfully disturbed biofilm formation due to its cleaving abilities. Additionally, various concentrations of glucose were added to bacteria media, and addition of glucose was found to improve the biofilm formation. Using the optimal concentration of 8 mg/L glucose, independent temperature-controlled assays were performed with varying degrees of substrate concentration to determine the rate constant, which was input in the Arrhenius and Eyring equations. From the resulting thermodynamic values, the Gibbs free energy of activation was determined to be 62.9 kJ/mol at 37 degrees Celsius. Molecular dynamics simulations were performed to predict the transition state structure. After chemically drawn and optimized using the program Avogadro, the substrate analog (4-Nitrophenyl N-acetyl-β-D-glucosaminide) and the potential transition state structure were docked in the active site of Dispersin B. After 1ns molecular dynamics simulations, the potential transition state was observed. This computational method could yield better models of transition-state inhibitors for drug design.
    [Show full text]
  • Book of Abstracts Of
    BOOK OF ABSTRACTS OF BOOK OF ABSTRACTS OF CEB ANNUAL MEETING 2017 6 JULY 2017, BRAGA, PORTUGAL Edited by: Eugénio Campos Ferreira Publisher: Universidade do Minho, Centro de Engenharia Biológica Campus de Gualtar, 4710-057 Braga, Portugal ISBN: 978-989-97478-8-3 DOI: 10.21814/CEBsam2017 © Universidade do Minho This publication contains research works sponsored by Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Foreword The Centre of Biological Engineering (CEB, from the Portuguese title ‘Centro de Engenharia Biológica’) is a research unit of the School of Engineering of the University of Minho, which is recognized as a strategic infrastructure for the development of the Portuguese Biotechnology and Bioengineering fields. The mission of CEB is to generate, disseminate, and apply knowledge with relevance for society in the economic, social and cultural dimensions, and to contribute to the expansion of the scientific and technological fields under its scope of activity. The research carried out at CEB covers the molecular, cellular and process scales, combining knowledge from the exact, natural, health, environmental and engineering sciences, in order to develop new products and processes as well as a wide range of bioengineering and biotechnological applications in the agro-food, environmental, energy, industrial fine chemistry, biomedical and health fields. CEB combines R&D activities with advanced training, technology transfer, consulting and services, with the aim of fostering the industrial and agro-food, health, and environmental sectors.
    [Show full text]
  • Article Differential Codon Adaptation
    Differential Codon Adaptation between dsDNA and ssDNA Phages in Escherichia coli Shivapriya Chithambaram,1 Ramanandan Prabhakaran,1 and Xuhua Xia*,1 1Department of Biology and Center for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, Ontario, Canada *Corresponding author: E-mail: [email protected]. Associate editor: Helen Piontkivska Abstract Because phages use their host translation machinery, their codon usage should evolve toward that of highly expressed host genes. We used two indices to measure codon adaptation of phages to their host, rRSCU (thecorrelationinrelative synonymous codon usage [RSCU] between phages and their host) and Codon Adaptation Index (CAI) computed with highly expressed host genes as the reference set (because phage translation depends on host translation machinery). These indices used for this purpose are appropriate only when hosts exhibit little mutation bias, so only phages para- Downloaded from sitizing Escherichia coli were included in the analysis. For double-stranded DNA (dsDNA) phages, both rRSCU and CAI decrease with increasing number of transfer RNA genes encoded by the phage genome. rRSCU is greater for dsDNA phages than for single-stranded DNA (ssDNA) phages, and the low rRSCU values are mainly due to poor concordance in RSCU values for Y-ending codons between ssDNA phages and the E. coli host, consistent with the predicted effect of C!T mutation bias in the ssDNA phages. Strong C!T mutation bias would improve codon adaptation in codon families (e.g., Gly) where U-ending codons are favored over C-ending codons (“U-friendly” codon families) by highly expressed host http://mbe.oxfordjournals.org/ genes but decrease codon adaptation in other codon families where highly expressed host genes favor C-ending codons against U-ending codons (“U-hostile” codon families).
    [Show full text]
  • Structural-Functional Analysis Reveals a Specific Domain Organization in Family GH20 Hexosaminidases
    RESEARCH ARTICLE Structural-Functional Analysis Reveals a Specific Domain Organization in Family GH20 Hexosaminidases Cristina Val-Cid, Xevi Biarnés, Magda Faijes*, Antoni Planas Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, Barcelona, Spain * [email protected] Abstract Hexosaminidases are involved in important biological processes catalyzing the hydrolysis of N-acetyl-hexosaminyl residues in glycosaminoglycans and glycoconjugates. The GH20 enzymes present diverse domain organizations for which we propose two minimal model architectures: Model A containing at least a non-catalytic GH20b domain and the catalytic α α OPEN ACCESS one (GH20) always accompanied with an extra -helix (GH20b-GH20- ), and Model B with only the catalytic GH20 domain. The large Bifidobacterium bifidum lacto-N-biosidase was Citation: Val-Cid C, Biarnés X, Faijes M, Planas A used as a model protein to evaluate the minimal functional unit due to its interest and struc- (2015) Structural-Functional Analysis Reveals a Specific Domain Organization in Family GH20 tural complexity. By expressing different truncated forms of this enzyme, we show that Hexosaminidases. PLoS ONE 10(5): e0128075. Model A architectures cannot be reduced to Model B. In particular, there are two structural doi:10.1371/journal.pone.0128075 requirements general to GH20 enzymes with Model A architecture. First, the non-catalytic Academic Editor: Elena Papaleo, University of domain GH20b at the N-terminus of the catalytic GH20 domain is required for expression Copenhagen, DENMARK and seems to stabilize it. Second, the substrate-binding cavity at the GH20 domain always Received: July 2, 2014 involves a remote element provided by a long loop from the catalytic domain itself or, when Accepted: April 23, 2015 this loop is short, by an element from another domain of the multidomain structure or from the dimeric partner.
    [Show full text]