DOCSLIB.ORG

Substrate Specificities of Outer Membrane Proteases of the Omptin Family in Escherichia Coli, Salmonella Enterica, and Citrobacter Rodentium

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Substrate Specificities of Outer Membrane Proteases of the Omptin Family in Escherichia Coli, Salmonella Enterica, and Citrobacter Rodentium Substrate specificities of outer membrane proteases of the omptin family in Escherichia coli, Salmonella enterica, and Citrobacter rodentium Andrea Portt, McGill University, Montreal October 9th, 2010 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master’s in Microbiology © Andrea Portt 2010 Table of Contents LIST OF ABBREVIATIONS 3 ABSTRACT 6 ACKNOWLEDGEMENTS 7 CONTRIBUTIONS OF AUTHORS 8 LITERATURE REVIEW 9 2. OMPTINS 9 3. OMPTIN SUBSTRATES 13 A. ANTIMICROBIAL PEPTIDES 13 B. BLOOD CLOTTING PROTEINS AND EXTRACELLULAR MATRIX 14 C. COMPLEMENT 16 D. TROPOMYOSIN 17 4. EVOLUTION OF OMPTINS 17 5. OMPTIN REGULATION 20 6. OMPTINS OF PATHOGENIC ENTEROBACTEREACEAE 22 A. Y. PESTIS 22 B. S. ENTERICA 23 C. SHIGELLA 25 D. ATTACHING AND EFFACING BACTERIA 25 INTRODUCTION 28 MATERIALS AND METHODS 29 1. BACTERIAL GROWTH 29 2. BACTERIAL STRAINS AND CONSTRUCTION OF PLASMIDS 29 A. STRAINS 29 B. PLASMIDS 31 3. DISK INHIBITION ASSAYS 34 4. MINIMUM INHIBITORY CONCENTRATION DETERMINATIONS 34 5. PROTEOLYTIC CLEAVAGE OF AMPS BY CELLS EXPRESSING OMPTINS 34 6. OM DISRUPTION ASSAY WITH 1-N-PHENYLNAPHTHYLAMINE 35 7. DEGRADATION OF TROPOMYOSIN BY E. COLI AND C. RODENTIUM STRAINS EXPRESSING OMPTINS 35 8. REAL-TIME QUANTITATIVE PCR 35 9. PURIFICATION OF NATIVE CROP 35 10. EXPRESSION, PURIFICATION, AND REFOLDING OF HIS-PGTE AND HIS-CROP 36 A. EXPRESSION 36 B. PURIFICATION 36 C. REFOLDING 36 11. CLEAVAGE OF C2 FRET SUBSTRATE BY WHOLE CELLS OR PURIFIED OMPTINS 37 A. WHOLE CELLS 37 B. PURIFIED OMPTINS 37 1 RESULTS 38 1. GROWTH INHIBITION OF E. COLI AND C. RODENTIUM STRAINS EXPRESSING OMPTINS BY C18G. 38 A. DISK INHIBITION 38 B. MINIMAL INHIBITORY CONCENTRATIONS 40 2. C18G CLEAVAGE BY OMPT, OMPP, AND CROP 42 A. C. RODENTIUM 42 B. S. ENTERICA 42 3. LL-37 CLEAVAGE BY CROP 42 4. SOME OMPTINS PREVENT OM DISRUPTION BY AMPS 46 A. NPN WITHOUT AMPS 46 B. C18G 46 C. PMB 46 5. TROPOMYOSIN 50 A. E. COLI BL21(DE3) 50 B. C. RODENTIUM 52 7. RT-QPCR 55 8. OMPTIN EXPRESSION AT THE OM 55 9. PURIFICATION AND ACTIVITY OF NATIVE CROP 58 10. PURIFICATION AND ACTIVITY OF HIS-PGTE AND HIS-CROP 58 DISCUSSION 64 1. OMPT, OMPP, AND CROP CONFERRED RESISTANCE TO C18G IN DISK INHIBITION AND MIC ASSAYS, PGTE DID NOT 64 2. OMPT, OMPP, AND CROP CLEAVE C18G, UNLIKE PGTE 65 3. SOME OMPTINS PREVENT OM DISRUPTION BY AMPS 67 4. TROPOMYOSIN 68 5. TIME COURSE OF FRET C2 SUBSTRATE CLEAVAGE 69 6. RT-QPCR 70 7. OM PREPARATIONS 70 8. PURIFICATION AND ACTIVITY OF NATIVE CROP 72 9. PURIFICATION AND ACTIVITY OF HIS-PGTE AND HIS-CROP 72 10. REFOLDED OMPTINS 73 11. FINAL CONCLUSIONS AND FUTURE DIRECTIONS 73 REFERENCES 75 ATTACHED PAPER 80 MOL. MICROBIOL 74, 98-111 (2009) 80 2 1 List of abbreviations 2 !2-AP – !2-Antiplasmin 3 A/E – Attaching and Effacing 4 AMP – Antimicrobial Peptide 5 C18G – Cysteine 18 Glycine, an 18 amino acid synthetic antimicrobial peptide 6 CFU – Colony Forming Unit 7 CHAPS – 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate, a 8 zwitterionic detergent 9 CIHR – Canadian Institutes for Health Research 10 CMI – les Concentrations Minimales Inhibitrices 11 DTT – Dithiothreitol 12 ECM – ExtraCellular Matrix 13 EDTA – Disodium ethylenediaminetetraacetate dihydrate, a chelating agent 14 EHEC – EnteroHemorrhagic E. coli 15 EPEC – EnteroPathogenic E. coli 16 HGT – Horizontal Gene Transfer 17 LB – Luria-Bertani broth 18 LD50 – Lethal Dose of a pathogen for 50 % of mice infected 19 LEE – Locus of Enterocyte Effacement pathogenicity island 3 20 LPS – Lipopolysaccharide 21 MAC – Membrane Attack Complex 22 mCRAMP – Mouse Cathelicidin-Related AntiMicrobial Peptide 23 MIC – Minimal Inhibitory Concentration 24 NPN - 1-N-PhenylNaphthylamine 25 NSERC – Natural Sciences and Engineering Research Council 26 OM – Outer Membrane 27 OMP - Outer Membrane Protein 28 PAM – les Peptides AntiMicrobiens 29 PBS – Phosophate-Buffered Saline 30 Plg – Plasminogen 31 RDEC – Rabbit Diarrhoeagenic E. coli 32 RT-qPCR – Real-Time Quantitative Polymerase Chain Reaction 33 Sarcosyl – N-Lauroyl-Sarcosine 34 SCV – Salmonella-Containing Vacuole 35 SDS-PAGE – Sodium Dodecyl Sulphate PolyAcrylamide Gel 36 T3SS – Type 3 Secretion System 37 TFPI – Tissue Factor Pathway Inhibitor 38 TSB – Tryptic Soy Broth 4 39 uPA – Urokinase Plaminogen Activator 40 UTI – Urinary Tract Infection 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 5 58 Abstract 59 Outer membrane proteins (OMPs) of the omptin family are present in some 60 Gram-negative bacteria. Omptins protect cells by cleaving antimicrobial peptides 61 (AMPs). For example, the Escherichia coli OmpT and OmpP impart resistance by 62 cleaving the AMP protamine, and PgtE of Salmonella enterica was also reported 63 to cleave AMPs. Despite identification of many in vitro substrates, few 64 physiological substrates have been tested. Our laboratory recently identified the 65 omptin CroP of Citrobacter rodentium, which cleaves the murine !-helical AMP 66 mCRAMP and the similarly !-helical synthetic AMP C18G. We examined AMPs 67 as substrates of several omptins. OmpT, OmpP, and CroP cleave the AMP C18G 68 more effectively than PgtE, leading to higher minimal inhibitory concentrations 69 (MICs). This suggests that AMP cleavage is an important function of OmpT, 70 OmpP, and CroP during infection. Based on initial results, we attempted to 71 compare and contrast specificity and catalytic activity of C18G cleavage by 72 purified CroP and PgtE. 73 74 Les protéines de la membrane externe de la famille omptin se trouvent dans 75 quelques bactéries à Gram négatif. En coupant les peptides antimicrobiens 76 (PAMs), les omptins protègent les bactéries. Par exemple, les omptins OmpT et 77 OmpP d’Escherichia coli, et l’omptin PgtE de Salmonella enterica, coupent les 78 PAMs. Malgré l’indentification de plusieurs substrats in vitro, peu de substrats 79 physiologiques ont été découverts. Dans notre laboratoire, on a identifié l’omptin 80 CroP de Citrobacter rodentium, qui coupe le PAM murin mCRAMP. On décrit ici 81 l’activité de plusieurs omptins contre les PAMs. OmpT, OmpP et CroP coupent le 82 PAM C18G plus efficacement que PgtE, ce qui mène à des concentrations 83 minimale inhibitrices (CMIs) plus haute. Ces résultats suggèrent que l’activité 84 catalytique d’OmpT, OmpP, et CroP est importante lors de l’infection. À la suite 85 des résultats préliminaires, on a tenté de comparer la spécificité et l’activité 86 catalytique contre C18G des protéases purifiées CroP et PgtE. 6 87 Acknowledgements 88 The author gratefully acknowledges her supervisor, Dr. Hervé Le Moual, for his 89 careful guidance through every stage of this work. Thanks are due to Dr. Valerie 90 Le Sage for her hard work with C. rodentium in the lab and in preparing the 91 Molecular Microbiology manuscript. Dr. Samantha Gruenheid provided guidance 92 and technical training. Dr. Benoit Cousineau offered insight as a committee 93 member. Special thanks are due to Jenny-Lee Thomassin, who produced the LL- 94 37 cleavage data in Figure 9. I would also like to recognize my labmates Charles 95 Viau for his encouragement and his wealth of biochemical knowledge, and Daniel 96 Ting for his balanced perspective in all things microbiological and philosophical. 97 Charles and Daniel also worked with Mg2+ to stabilize the outer membrane in 98 NPN assays. Under the supervision of Dr. France Daigle at the Université de 99 Montréal, Christine LePage and Sébastien Sabbagh undertook the RT-qPCR work 100 and construction of the S. enterica !pgtE mutant. Dr. James Taylor and Richard 101 Wargachuk were a consistent source of advice throughout this project. Thanks are 102 also due to my parents, Cam Portt and Rena Burkholder, as well as Allen 103 Larocque, Laura Canty, Carmen Evans, and Peter F. Woodford, author of 104 Scientific Writing for Graduate Students. 105 The Government on Ontario financially supported this work in the form of the 106 Ontario-Quebec Exchange Fellowship. The Canadian Institutes for Health 107 Research (CIHR), McGill University, and the Natural Sciences and Engineering 108 Research Council (NSERC) of Canada provided additional funding. 109 110 111 112 113 114 115 116 7 117 Contributions of Authors 118 Dr. Valerie Le Sage and our supervisor Dr. Hervé Le Moual undertook the 119 majority of the scientific direction, laboratory work, and writing of the Molecular 120 Microbiology paper attached to this thesis, see page 80. Under the supervision of 121 Dr. Samantha Gruenheid, Lei Zhu produced the C. rodentium phoPQ knockout. 122 Christine Lepage undertook RT-qPCR experiments under the supervision of Dr. 123 France Daigle. Andrea Portt produced the C. rodentium croP knockout, 124 contributed data to Figures 1c, 3a, 5a, and 6, produced Table 1, and helped edit 125 the manuscript. Charles Viau contributed "-galactosidase data for the effect of 126 AMPs on activation of PhoPQ in C. rodentium. 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 8 145 Literature review 146 1. OM proteins 147 The outer membrane (OM) of Gram-negative bacteria is the surface that interacts 148 with the environment and the eukaryotic host, and consequently many OM 149 proteins (OMPs) are crucial for bacterial virulence1. OMPs have many roles in 150 pathogenic Gram-negative bacteria. OMPs interact with the host as antigens, 151 receptors or adhesins, and proteases. For example, the pH 6 antigen, of Yersinia 152 pestis has antigenic properties, but it also promotes adhesion to human epithelial 153 cells via binding to phosphatidylcholine2. OM proteases may cleave bacterial or 154 host substrates. SepA is a Shigella OM protease of the autotransporter family. 155 Autotransporters transport and release one of their own domains, resulting in 156 secretion of the extracellular domain3. The secreted portion of SepA contributes to 157 tissue invasion4.
Load more

Recommended publications
  • Proteolytic Enzymes in Grass Pollen and Their Relationship to Allergenic Proteins
    Proteolytic Enzymes in Grass Pollen and their Relationship to Allergenic Proteins By Rohit G. Saldanha A thesis submitted in fulfilment of the requirements for the degree of Masters by Research Faculty of Medicine The University of New South Wales March 2005 TABLE OF CONTENTS TABLE OF CONTENTS 1 LIST OF FIGURES 6 LIST OF TABLES 8 LIST OF TABLES 8 ABBREVIATIONS 8 ACKNOWLEDGEMENTS 11 PUBLISHED WORK FROM THIS THESIS 12 ABSTRACT 13 1. ASTHMA AND SENSITISATION IN ALLERGIC DISEASES 14 1.1 Defining Asthma and its Clinical Presentation 14 1.2 Inflammatory Responses in Asthma 15 1.2.1 The Early Phase Response 15 1.2.2 The Late Phase Reaction 16 1.3 Effects of Airway Inflammation 16 1.3.1 Respiratory Epithelium 16 1.3.2 Airway Remodelling 17 1.4 Classification of Asthma 18 1.4.1 Extrinsic Asthma 19 1.4.2 Intrinsic Asthma 19 1.5 Prevalence of Asthma 20 1.6 Immunological Sensitisation 22 1.7 Antigen Presentation and development of T cell Responses. 22 1.8 Factors Influencing T cell Activation Responses 25 1.8.1 Co-Stimulatory Interactions 25 1.8.2 Cognate Cellular Interactions 26 1.8.3 Soluble Pro-inflammatory Factors 26 1.9 Intracellular Signalling Mechanisms Regulating T cell Differentiation 30 2 POLLEN ALLERGENS AND THEIR RELATIONSHIP TO PROTEOLYTIC ENZYMES 33 1 2.1 The Role of Pollen Allergens in Asthma 33 2.2 Environmental Factors influencing Pollen Exposure 33 2.3 Classification of Pollen Sources 35 2.3.1 Taxonomy of Pollen Sources 35 2.3.2 Cross-Reactivity between different Pollen Allergens 40 2.4 Classification of Pollen Allergens 41 2.4.1
    [Show full text]
  • Structure Prediction of Outer Membrane Protease Protein of Salmonella Typhimurium Using Computational Techniques
    INT. J. BIOAUTOMATION, 2016, 20(1), 5-18 Structure Prediction of Outer Membrane Protease Protein of Salmonella typhimurium Using Computational Techniques Rozina Tabassum*, Muhammad Haseeb, Sahar Fazal Department of Bioinformatics and Biosciences Mohammad Ali Jinnah University Islamabad, Pakistan E-mails: [email protected], [email protected], [email protected] *Corresponding author Received: April 04, 2015 Accepted: November 16, 2015 Published: March 31, 2016 Abstract: Salmonella typhimurium, a facultative gram-negative intracellular pathogen belonging to family Enterobacteriaceae, is the most frequent cause of human gastroenteritis worldwide. PgtE gene product,outer membrane protease emerges important in the intracellular phases of salmonellosis. The pgtE gene product of S. typhimurium was predicted to be capable of proteolyzing T7 RNA polymerase and localize in the outer membrane of these gram negative bacteria. PgtE product of S. enterica and OmpT of E. coli, having high sequence similarity have been revealed to degrade macrophages, causing salmonellosis and other diseases. The three-dimensional structure of the protein was not available through Protein Data Bank (PDB) creating lack of structural information about E protein. In our study, by performing Comparative model building, the three dimensional structure of outer membrane protease protein was generated using the backbone of the crystal structure of Pla of Yersinia pestis, retrieved from PDB, with MODELLER (9v8). Quality of the model was assessed by validation tool PROCHECK, web servers like ERRAT and ProSA are used to certify the reliability of the predicted model. This information might offer clues for better understanding of E protein and consequently for developmet of better therapeutic treatment against pathogenic role of this protein in salmonellosis and other diseases.
    [Show full text]
  • Handbook of Proteolytic Enzymes Second Edition Volume 1 Aspartic and Metallo Peptidases
    Handbook of Proteolytic Enzymes Second Edition Volume 1 Aspartic and Metallo Peptidases Alan J. Barrett Neil D. Rawlings J. Fred Woessner Editor biographies xxi Contributors xxiii Preface xxxi Introduction ' Abbreviations xxxvii ASPARTIC PEPTIDASES Introduction 1 Aspartic peptidases and their clans 3 2 Catalytic pathway of aspartic peptidases 12 Clan AA Family Al 3 Pepsin A 19 4 Pepsin B 28 5 Chymosin 29 6 Cathepsin E 33 7 Gastricsin 38 8 Cathepsin D 43 9 Napsin A 52 10 Renin 54 11 Mouse submandibular renin 62 12 Memapsin 1 64 13 Memapsin 2 66 14 Plasmepsins 70 15 Plasmepsin II 73 16 Tick heme-binding aspartic proteinase 76 17 Phytepsin 77 18 Nepenthesin 85 19 Saccharopepsin 87 20 Neurosporapepsin 90 21 Acrocylindropepsin 9 1 22 Aspergillopepsin I 92 23 Penicillopepsin 99 24 Endothiapepsin 104 25 Rhizopuspepsin 108 26 Mucorpepsin 11 1 27 Polyporopepsin 113 28 Candidapepsin 115 29 Candiparapsin 120 30 Canditropsin 123 31 Syncephapepsin 125 32 Barrierpepsin 126 33 Yapsin 1 128 34 Yapsin 2 132 35 Yapsin A 133 36 Pregnancy-associated glycoproteins 135 37 Pepsin F 137 38 Rhodotorulapepsin 139 39 Cladosporopepsin 140 40 Pycnoporopepsin 141 Family A2 and others 41 Human immunodeficiency virus 1 retropepsin 144 42 Human immunodeficiency virus 2 retropepsin 154 43 Simian immunodeficiency virus retropepsin 158 44 Equine infectious anemia virus retropepsin 160 45 Rous sarcoma virus retropepsin and avian myeloblastosis virus retropepsin 163 46 Human T-cell leukemia virus type I (HTLV-I) retropepsin 166 47 Bovine leukemia virus retropepsin 169 48
    [Show full text]
  • Structural Characterization of Bacterial Defense Complex Marko Nedeljković
    Structural characterization of bacterial defense complex Marko Nedeljković To cite this version: Marko Nedeljković. Structural characterization of bacterial defense complex. Biomolecules [q-bio.BM]. Université Grenoble Alpes, 2017. English. NNT : 2017GREAV067. tel-03085778 HAL Id: tel-03085778 https://tel.archives-ouvertes.fr/tel-03085778 Submitted on 22 Dec 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE Pour obtenir le grade de DOCTEUR DE LA COMMUNAUTE UNIVERSITE GRENOBLE ALPES Spécialité : Biologie Structurale et Nanobiologie Arrêté ministériel : 25 mai 2016 Présentée par Marko NEDELJKOVIĆ Thèse dirigée par Andréa DESSEN préparée au sein du Laboratoire Institut de Biologie Structurale dans l'École Doctorale Chimie et Sciences du Vivant Caractérisation structurale d'un complexe de défense bactérienne Structural characterization of a bacterial defense complex Thèse soutenue publiquement le 21 décembre 2017, devant le jury composé de : Monsieur Herman VAN TILBEURGH Professeur, Université Paris Sud, Rapporteur Monsieur Laurent TERRADOT Directeur de Recherche, Institut de Biologie et Chimie des Protéines, Rapporteur Monsieur Patrice GOUET Professeur, Université Lyon 1, Président Madame Montserrat SOLER-LOPEZ Chargé de Recherche, European Synchrotron Radiation Facility, Examinateur Madame Andréa DESSEN Directeur de Recherche, Institut de Biologie Structurale , Directeur de These 2 Contents ABBREVIATIONS ..............................................................................................................................
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 8,561,811 B2 Bluchel Et Al
    USOO8561811 B2 (12) United States Patent (10) Patent No.: US 8,561,811 B2 Bluchel et al. (45) Date of Patent: Oct. 22, 2013 (54) SUBSTRATE FOR IMMOBILIZING (56) References Cited FUNCTIONAL SUBSTANCES AND METHOD FOR PREPARING THE SAME U.S. PATENT DOCUMENTS 3,952,053 A 4, 1976 Brown, Jr. et al. (71) Applicants: Christian Gert Bluchel, Singapore 4.415,663 A 1 1/1983 Symon et al. (SG); Yanmei Wang, Singapore (SG) 4,576,928 A 3, 1986 Tani et al. 4.915,839 A 4, 1990 Marinaccio et al. (72) Inventors: Christian Gert Bluchel, Singapore 6,946,527 B2 9, 2005 Lemke et al. (SG); Yanmei Wang, Singapore (SG) FOREIGN PATENT DOCUMENTS (73) Assignee: Temasek Polytechnic, Singapore (SG) CN 101596422 A 12/2009 JP 2253813 A 10, 1990 (*) Notice: Subject to any disclaimer, the term of this JP 2258006 A 10, 1990 patent is extended or adjusted under 35 WO O2O2585 A2 1, 2002 U.S.C. 154(b) by 0 days. OTHER PUBLICATIONS (21) Appl. No.: 13/837,254 Inaternational Search Report for PCT/SG2011/000069 mailing date (22) Filed: Mar 15, 2013 of Apr. 12, 2011. Suen, Shing-Yi, et al. “Comparison of Ligand Density and Protein (65) Prior Publication Data Adsorption on Dye Affinity Membranes Using Difference Spacer Arms'. Separation Science and Technology, 35:1 (2000), pp. 69-87. US 2013/0210111A1 Aug. 15, 2013 Related U.S. Application Data Primary Examiner — Chester Barry (62) Division of application No. 13/580,055, filed as (74) Attorney, Agent, or Firm — Cantor Colburn LLP application No.
    [Show full text]
  • Crystal Structure of the Outer Membrane Protease Ompt from Escherichia Coli Suggests a Novel Catalytic Site
    The EMBO Journal Vol. 20 No. 18 pp. 5033±5039, 2001 Crystal structure of the outer membrane protease OmpT from Escherichia coli suggests a novel catalytic site Lucy Vandeputte-Rutten1, R.Arjen Kramer2, peptide that is excreted by epithelial cells of the urinary Jan Kroon1,², Niek Dekker2,3, tract (Stumpe et al., 1998). Inactivation of the gene Maarten R.Egmond2 and Piet Gros1,4 encoding Pla in Y.pestis, the causative agent of plague, increased the median lethal dose of the bacterium for mice 1 Department of Crystal and Structural Chemistry, Bijvoet Center for by 106-fold (Sodeinde et al., 1992). The role of Pla in Biomolecular Research and 2Department of Enzymology and Protein Engineering, Center for Biomembranes and Lipid Enzymology, pathogenicity might be related to its ability to activate Institute of Biomembranes, Utrecht University, Padualaan 8, plasminogen, a feature shared with OmpT (Lundrigan and 3584 CH Utrecht, The Netherlands Webb, 1992). SopA from S.¯exneri, the causative agent of 3Present address: Structural Chemistry Laboratories, AstraZeneca bacillary dysentery, cleaves the endogenous autotranspor- R&D, S-43183 MoÈlndal, Sweden ter IcsA that has an essential role in the formation of actin tails in host cells, and therefore SopA might be involved in 4Corresponding author e-mail: [email protected] actin-based motility inside infected cells (Egile et al., 1997; Shere et al., 1997). Thus, the proteolytic activity of ²Deceased the omptins is probably involved in a variety of ways in the pathogenicity of these bacteria, ranging from bacterial OmpT from Escherichia coli belongs to a family of defence and plasmin-mediated tissue in®ltration to motility highly homologous outer membrane proteases, known inside infected cells.
    [Show full text]
  • New Retroviral-Like Membrane-Associated Aspartic Proteases From
    Novas proteases aspárticas membranares do tipo retroviral de rickettsiae: caracterização bioquímica e determinação de especificidade New retroviral-Iike membrane-associated aspartic proteases from rickettsiae: biochemical characterization and specificity profiling Tese de Doutoramento apresentada à Universidade de Coimbra para obtenção do grau de Doutor em Bioquímica (especialidade de Tecnologia Bioquímica). Doctoral thesis in the scientific area of Biochemistry (specialty Biochemistry Technology) presented to the University of Coimbra. Rui Gonçalo Batista Mamede da Cruz Coimbra - 2014 Department of Life Sciences University of Coimbra UNIVERSIDADE DE COIMBRA Front cover image illustrates Rickettsia cells (in green) invading mammalian host cells. Publishing rights were granted by Michael Taylor, the original author of the image. Agradecimentos / Acknowledgments A conclusão desta etapa não teria sido possível sem o apoio incondicional da família, amigos, orientadores, colegas e de todos aqueles que foram cruzando o meu caminho e deixando o seu contributo, em particular nos últimos quatro anos que conduziram a esta dissertação. Gostaria por isso de lhes deixar algumas palavras de profundo e sincero agradecimento. Em primeiro lugar, gostaria de agradecer à Doutora Isaura Simões e ao Professor Carlos Faro, por me terem acolhido e proporcionado as excepcionais condições para a realização deste trabalho. Em especial à Doutora Isaura Simões, expresso a minha profunda gratidão pela orientação, rigor, sentido crítico e paixão com que encara a ciência. Agradeço-lhe por acreditar em mim e por me fazer ir sempre mais além. Um agradecimento muito especial a todos aqueles que fazem e fizeram parte do laboratório de Biotecnologia de Molecular do Biocant, pelo seu contributo no meu crescimento científico, profissional e pessoal.
    [Show full text]
  • Engineering of Protease Variants Exhibiting High Catalytic Activity and Exquisite Substrate Selectivity
    Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity Navin Varadarajan*†‡, Jongsik Gam†‡, Mark J. Olsen*, George Georgiou*§¶, and Brent L. Iverson*†ʈ *Institute for Cellular and Molecular Biology and Departments of †Chemistry and Biochemistry and §Chemical Engineering, University of Texas, Austin, TX 78712 Edited by Robert T. Sauer, Massachusetts Institute of Technology, Cambridge, MA, and approved April 1, 2005 (received for review January 4, 2005) The exquisite selectivity and catalytic activity of enzymes have (13–15). Similarly, the evolution of highly active variants of been shaped by the effects of positive and negative selection aspartate aminotransferases capable of accepting branched or pressure during the course of evolution. In contrast, enzyme aromatic amino acid substrates was accompanied by a relaxation variants engineered by using in vitro screening techniques to of the substrate selectivity (16, 17). accept novel substrates typically display a higher degree of cata- In nature, the evolution of enzymes occurs as the result of lytic promiscuity and lower total turnover in comparison with their positive selective pressure for turnover of physiological sub- natural counterparts. Using bacterial display and multiparameter strates, combined with simultaneous negative selective pressure flow cytometry, we have developed a novel methodology for to eliminate completely, or at least drastically suppress, delete- emulating positive and negative selective pressure in vitro for the rious activities. It follows that the engineering of enzymes isolation of enzyme variants with reactivity for desired novel exhibiting high catalytic activity and substrate selectivity for a substrates, while simultaneously excluding those with reactivity particular desired substrate should be similarly accomplished by toward undesired substrates.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2004/0120901 A1 Wu Et Al
    US 20040120901A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0120901 A1 Wu et al. (43) Pub. Date: Jun. 24, 2004 (54) DENTAL COMPOSITIONS INCLUDING (22) Filed: Dec. 20, 2002 ENZYMES AND METHODS Publication Classification (76) Inventors: Dong Wu, Woodbury, MN (US); Joel D. Oxman, Minneapolis, MN (US); (51) Int. Cl." ............................... A61K 7/28: A61C 5/00 Sumita B. Mitra, West St. Paul, MN (52) U.S. Cl. ........................................... 424/50; 433/217.1 (US); Ingo Reinhold Haberlein, Weilheim (DE) (57) ABSTRACT A hardenable dental composition that includes a polymer Correspondence Address: izable component and a therapeutic enzyme mixed within 3M INNOVATIVE PROPERTIES COMPANY the polymerizable component, wherein upon hardening the PO BOX 33427 polymerizable component to form a hardened dental mate ST. PAUL, MN 55133-3427 (US) rial having a therapeutic enzyme mixed therein, the hard ened dental material with the enzyme mixed therein is in (21) Appl. No.: 10/327,411 contact with Saliva in a Subject's mouth for at least 1 day. US 2004/O120901 A1 Jun. 24, 2004 DENTAL COMPOSITIONS INCLUDING ENZYMES form a hardened dental material having a therapeutic AND METHODS enzyme mixed therein, the hardened dental material with the enzyme mixed therein is in contact with Saliva in a Subject's BACKGROUND mouth for at least 1 day; wherein the therapeutic enzyme is Selected from the group consisting of oxidases, peroxidases, 0001 Enzymes have been used in products for the laccases, proteases, carbohydrases, lipases, and combina improvement of oral health. Such products include, for tions thereof, and wherein the polymerizable component is example, mouthwashes, toothpastes, dentrifices, and the Selected from the group consisting of (meth)acrylates, like.
    [Show full text]
  • Synergies with and Resistance to Membrane-Active Peptides
    antibiotics Review Synergies with and Resistance to Membrane-Active Peptides Adam Kmeck, Robert J. Tancer , Cristina R. Ventura and Gregory R. Wiedman * Department of Chemistry and Biochemistry, Seton Hall University, South Orange, NJ 07079, USA; [email protected] (A.K.); [email protected] (R.J.T.); [email protected] (C.R.V.) * Correspondence: [email protected] Received: 24 August 2020; Accepted: 17 September 2020; Published: 19 September 2020 Abstract: Membrane-active peptides (MAPs) have long been thought of as the key to defeating antimicrobial-resistant microorganisms. Such peptides, however, may not be sufficient alone. In this review, we seek to highlight some of the common pathways for resistance, as well as some avenues for potential synergy. This discussion takes place considering resistance, and/or synergy in the extracellular space, at the membrane, and during interaction, and/or removal. Overall, this review shows that researchers require improved definitions of resistance and a more thorough understanding of MAP-resistance mechanisms. The solution to combating resistance may ultimately come from an understanding of how to harness the power of synergistic drug combinations. Keywords: membrane-active peptides; antimicrobial-resistance; drug synergy 1. Introduction Membrane-active peptides (MAPs) are peptides ranging from about 4–40 amino acids in length that can interact with the cell membrane through permeabilization or other antimicrobial mechanisms [1,2]. They are often comprised of amino acid residues that are positively charged at pH 7. They can be grouped into four main structural categories: Linear α-helices, extended structures (usually abundant in Glycine, Arginine, Tryptophan, or Proline residues), β-sheets (often stabilized by disulfide linkages), and loops that contain both α and β moieties [3].
    [Show full text]
  • New Promising Targets for Synthetic Omptin-Based Peptide Vaccine Against Gram-Negative Pathogens
    Article New Promising Targets for Synthetic Omptin-Based Peptide Vaccine against Gram-Negative Pathogens Valentina A. Feodorova 1,*, Anna M. Lyapina 1, Sergey S. Zaitsev 1, Maria A. Khizhnyakova 1, Lidiya V. Sayapina 2, Onega V. Ulianova 1, Sergey S. Ulyanov 3 and Vladimir L. Motin 4,* 1 Laboratory for Molecular Biology and NanoBiotechnology, Federal Research Center for Virology and Microbiology, Branch in Saratov, 410028 Saratov, Russia; [email protected] (A.M.L.); zaytsev- [email protected] (S.S.Z.); [email protected] (O.V.U.) 2 Department of Vaccine Control, Scientific Center on Expertise of Medical Application Products, 127051, Moscow, Russia; [email protected] 3 Department for Medical Optics, Saratov State University, 410012, Saratov, Russia; [email protected] 4 Department of Pathology, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA; * Correspondence: [email protected] (V.A.F.); [email protected] (V.L.M.) Received: 25 February 2019; Accepted: 4 April 2019; Published: 10 April 2019 Abstract: Omptins represent a family of proteases commonly found in various Gram-negative pathogens. These proteins play an important role in host–pathogen interaction and have been recognized as key virulence factors, highlighting the possibility of developing an omptin-based broad-spectrum vaccine. The prototypical omptin, His-tagged recombinant Pla, was used as a model target antigen. In total, 46 linear and 24 conformational epitopes for the omptin family were predicted by the use of ElliPro service. Among these we selected highly conserved, antigenic, non- allergenic, and immunogenic B-cell epitopes. Five epitopes (2, 6, 8, 10, and 11 corresponding to Pla regions 52–60, 146–150, 231–234, 286–295, and 306–311, respectively) could be the first choice for the development of the new generation of target-peptide-based vaccine against plague.
    [Show full text]
  • The Conserved Mechanism of Icsa Polar Targeting Among
    UNLV Retrospective Theses & Dissertations 1-1-2008 The conserved mechanism of IcsA polar targeting among proteobacteria, characterization of the omptin family, and the roles and regulation of IcsP in Shigella flexneri Eun-Hae Kim University of Nevada, Las Vegas Follow this and additional works at: https://digitalscholarship.unlv.edu/rtds Repository Citation Kim, Eun-Hae, "The conserved mechanism of IcsA polar targeting among proteobacteria, characterization of the omptin family, and the roles and regulation of IcsP in Shigella flexneri" (2008). UNLV Retrospective Theses & Dissertations. 2377. http://dx.doi.org/10.25669/kqaw-ymty This Thesis is protected by copyright and/or related rights. It has been brought to you by Digital Scholarship@UNLV with permission from the rights-holder(s). You are free to use this Thesis in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Thesis has been accepted for inclusion in UNLV Retrospective Theses & Dissertations by an authorized administrator of Digital Scholarship@UNLV. For more information, please contact [email protected]. THE CONSERVED MECHANISM OF ICSA POLAR TARGETING AMONG PROTEOBACTERIA, CHARACTERIZATION OF THE OMPTIN FAMILY, AND THE ROLES AND REGULATION OF ICSP IN SHIGELLA FLEXNERI by Eun-Hae Kim Bachelor of Science University of Southern California 2004 A thesis submitted in partial fulfillment of the requirements for the Master of Science Degree in Biological Sciences School of Life Sciences College of Sciences Graduate College University of Nevada, Las Vegas August 2008 UMI Number: 1460534 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted.
    [Show full text]