DOCSLIB.ORG

The Haemophilus Ducreyi Sap Transporter Contributes

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Haemophilus Ducreyi Sap Transporter Contributes THE HAEMOPHILUS DUCREYI SAP TRANSPORTER CONTRIBUTES TO ANTIMICROBIAL PEPTIDE RESISTANCE KristyLee BeaversMount SubmittedtothefacultyoftheUniversityGraduateSchool inpartialfulfillmentoftherequirements forthedegreeof DoctorofPhilosophy intheDepartmentofMicrobiologyandImmunology, IndianaUniversity August2009 AcceptedbytheFacultyofIndianaUniversity,inpartial fulfillmentoftherequirementsforthedegreeof DoctorofPhilosophy. ________________________________ MargaretE.Bauer,PhD(Chair) ________________________________ StanleyM.Spinola,MD ________________________________ DoctoralCommittee XiaofengF.Yang,PhD ________________________________ May26th,2009MaryC.Dinauer,MD/PhD ________________________________ MaureenA.Harrington,PhD ii ACKNOWLEDGEMENTS I wouldlike tothankmy friends andmyfamilyfor supportingme throughthis endeavor. It would not have been possible for me to have made it as far as I have without their unwaveringsupportandencouragement. Inparticular,I wouldlike tothankmyparents for providingme witha strongfoundation upon which to build and for their utter confidence that I would succeed, even in the darkestoftimes. I wouldlike tothankJosie HoulihanandJeff Altenburgfor their friendship throughout graduateschool.Iwillrememberthemalways. Most of all I wouldlike tothankmyhusbandfor his love andsupport. He has beenthe rockuponwhichI have dependedandI would not have beenable toachieve mygoals withouthim bymyside.MyPhD belongstohimasmuchas itdoes tome. Finally, I would like to thank my son for continuously reminding me what is most important inlife. Myworst days inthe lab were erasedbyhis simple smile. I am a better personforknowinghimanditistohimthatIdedicateallofmyfuturesuccess. iii ABSTRACT KristyLee BeaversMount THE HAEMOPHILUS DUCREYI SAPTRANSPORTERCONTRIBUTESTO ANTIMICROBIALPEPTIDERESISTANCE Haemophilus ducreyi is the causative agent of the genital ulcer disease chancroid,which has been shown to facilitate the transmission of HIV. H. ducreyi is likely exposed to multiple sources of antimicrobial peptides in vivo. APs are small, cationic molecules withbothbactericidal andimmunomodulatoryfunctions. Because H. ducreyi is able to establishandmaintainaninfection inanenvironment rich withantimicrobial peptides, we hypothesized that the bacterium was resistant to the bactericidal effects of these peptides. Usinga 96well AP bactericidal assay,we examinedH. ducreyi susceptibility to eight human APs likely to be encountered at the site of infection, including the α- defensins human neutrophil peptide-1, human neutrophil peptide-2, human neutrophil peptide-3, and human defensin 5, the β-defensins human β defensin2, human beta defensin3,andhumanbeta defensin4,andthe humancathelicidin,LL-37. H. ducreyi survival was comparedtothe survival of Escherichia coli ML35,a strainknowntobe susceptible toseveral antimicrobial peptides. H. ducreyi was significantlymore resistant than E. coli ML35 to the bactericidal effects of all peptides tested. Furthermore, we foundthat representative class I andclass II strains of H. ducreyi were eachresistant to APs of eachfunctional category,indicatingthat resistance toantimicrobial peptides could iv representaconservedmethodof pathogenesisfor H. ducreyi asaspecies.The H. ducreyi genome contains a homologfor the Sap influx transporter. Tostudy the role of the H. ducreyi Sap transporter inAP resistance,we generatedanisogenic sapA mutant andused the 96well AP bactericidal assayto compare the AP susceptibilityprofiles of wildtype H. ducreyi, the sapA mutant andthe sapA trans-complement toα-defensins,β-defensins, andLL-37. We observeda 25% decrease inthe survival of the sapA mutant whenit was exposedtoLL-37. These findings suggest that the H. ducreyi Sap transporter plays a role in H. ducreyi resistance to LL-37, but it is likely that other AP resistance mechanismscoexistwithinthe bacterium. MargaretE.Bauer,PhD v TABLE OF CONTENTS LIST OF TABLES ……………………………………………………………………....ix LIST OF FIGURES ……..………………………………………………………..…..….x LIST OF ABBREVIATIONS ……………………...………………………………...…xi LITERATURE REVIEW SECTION I: HAEMOPHILUS DUCREI HistoryofChancroid…………………………………….……..………....1 TaxonomyandGenetics of H. ducreyi ……………………………………1 ClinicalFeaturesofChancroid……………………………………………2 DiagnosisandTreatmentofChancroid…….…………..…………………2 EpidemiologyofChancroid………………………………….………...….4 H. ducreyi andHIV………………………………………………………..5 ModelsofChancroidInfection……………………………………………6 HostPathogenInteractions………………………………………………..9 VirulenceDeterminants of H. ducreyi …………………………………...10 TwoClassesof H. ducreyi ……………………………………………….16 SECTION II: ANTIMICROBIAL PEPTIDES HumanAntimicrobialPeptides……………………………………..........18 AntimicrobialPeptidesintheChancroidEnvironment………………….19 EvidenceofAPActivity In vivo ………………………………………....20 AntimicrobialPeptideBactericidal Activity……………………………..22 AntimicrobialPeptideImmunoregulatoryActivity……………………...24 BacterialResistancetoAntimicrobialPeptides………………………….25 SECTION III: SAP TRANSPORTER TheSapTransporter……………………………………………………..28 Salmonella typhimurium SapTransporter…………………………….....28 Proteus mirabilis SapTransporter……………………………………….31 vi Haemophilus influenzae SapTransporter………………………………..31 Erwinia chrysanthemi SapTransporter………………………………….32 Vibrio fisheri SapTransporter………………………………………...…33 Homologyof H. ducreyi SaptoOtherSapTransporters………………...34 OtherRoles oftheSapTransporter……………………………………...37 GeneticRegulationoftheSapTransporter……………………………....38 The H. ducreyi SapTransporterisExpressedduringHumanInfection…38 HYPOTHESIS ……………………………………………………………………….…40 METHODS BacterialStrainsandGrowthConditions………………………………………..41 SourcesofPeptides………………………………………………………………41 96wellAPBactericidal Assay………………………………………………..…42 RadialDiffusionAssay………………………………………………………..…43 PolymeraseChainReaction…………………………………………………...…43 ReverseTranscriptionPCR………………………………………………………44 QuantitativeRT-PCR………………………………………………………….…44 SouthernBlotAnalysis………………………………………………………..…45 DNA Sequencing………………………………………………………………...45 ComparativeGrowthCurves…………………………………………………….47 Constructionof35000HP sapA ………………………………………………..…47 Generationof35000HP sapA /p sapA …………………………………………..…48 RESULTS SECTION I: H. DUCREYI IS RESISTANT TO HUMAN APS Validationof96wellAPBactericidalAssay……………………………53 H. ducreyi Susceptibilityto Humanα-defensins………………………...53 H. ducreyi SusceptibilitytoHumanβ-defensins……………………...…54 H. ducreyi SusceptibilitytotheHumanCathelicidin,LL-37……………57 ClassIandClassII H. ducreyi StrainsareResistanttoAPs…………….57 vii MinimumInhibitoryConcentrationofAntimicrobialPeptides………….59 APResistanceisConservedwithintheSpecies………………………....62 SECTION II: THE H. DUCREYI SAP TRANSPORTER PLAYS A ROLE IN AP RESISTANCE Constructionofa sapA Mutant in H. ducreyi ……………………………63 The H. ducreyi SapTransporterConfersResistancetoLL-37…………..70 The H. ducreyi SapTransporterdoes notConfer resistancetoβ-defensins………..…………..………………………70 The H. ducreyi SapTransporterDoesnotConferResistanceto α-defensins……………..…...……………………………………….71 The sapA GeneisConservedAmongClassI/ClassII H. ducreyi .............71 DISCUSSION …………………………………………………………………………...75 FUTURE DIRECTIONS …………………………………………………………….....83 APPENDIX: MTR EFFLUX TRANSPORTER MUTATGENESIS ……………......89 REFERENCES …………..…………………………………………………………...…95 CURRICULUM VITAE viii LIST OF TABLES Table1. H. ducreyi virulencefactorsasdefinedbythehumanchallengemodel…….…11 Table2. H. ducreyi structures thatdidnotcontributetovirulenceinthe HCM……...…16 Table3.Antimicrobial peptide secretingcelltypesinchancroid………………………..20 Table4.Saptransporter mutantsandphenotypes………………………………….……35 Table5.Homologybetween H. ducreyi Sap proteins andotherSap proteins…………..36 Table6.Bacterialstrainsusedinthis study……………………………………………...42 Table7.PrimersusedinPCR,RT-PCR,qRT-PCR,southern,andsequencing………...46 Table8.Primersusedin sapA mutagenesis……………...……………………………....49 Table9.Plasmidsusedinstudy………………………………………………………....52 Table10.Survivalof E. coli ML35and H. ducreyi 35000HPexposedtoPG-1………..53 Table11.Minimumeffective concentrationofAPs…………………………………….62 Table12.APresistance isconservedwithinthespecies………………………………...62 Table13.Primersusedin mtrC mutagenesisandRT-PCR……………………………...94 Table14.Plasmidsusedin mtrC mutagenesis…………………………………………...94 ix LIST OF FIGURES Figure1.ModelofSaptransporter………………………………………………………30 Figure2.Geneticmapof sap-containinglociin H. ducreyi 35000HP…………………..37 Figure3.Generationofthe sapA mutant…………….………………………………..…50 Figure4.Generationof sapA complement………………………………………………51 Figure5. H. ducreyi isresistanttoα-defensins…………………………………………..55 Figure6. H. ducreyi isresistanttoβ-defensins…………………………………………..56 Figure7. H. ducreyi isresistanttothehumancathelicidin,LL-37……………………...58 Figure8.Representative class IandclassII H. ducreyi strains areresistant toAPs…….59 Figure9.AP-mediatedinhibitionof bacterialgrowth…………………………………...61 Figure10.The sap-containinglocus of H. ducreyi istranscribedasanoperon………....65 Figure11.PCRconfirmationof35000HP sapA …............................................................66 Figure12.Southernanalysisof35000HP sapA ……………………………………….…67 Figure13.The35000HP sapA mutationdidnotaffect H. ducreyi growth in vitro ……...68 Figure14.RT-PCRconfirmationof putative35000HP sapA mutant……………………69 Figure15.The H. ducreyi Saptransporter confersresistancetoLL-37………….……...72 Figure16.The H. ducreyi Saptransporterdoes notconferresistance againstβ-defensins…….………………………………………………...73 Figure17.The H. ducreyi Saptransporterdoes notconferresistancetoα-defensins…..74 Figure18. mtr effluxpumpoperon………………………………………………………91 Figure19.mtrC mutagenicstrategy…………………………………………………...…92
Load more

Recommended publications
  • Associate Professor Yiannis N. Kaznessis
    Yiannis N. Kaznessis Associate Professor Department of Chemical Engineering and Materials Science, and Digital Technology Center, University of Minnesota 421 Washington Ave. S.E., Minneapolis, MN 55455-0132 Tel: (612) 624-4197, Fax: (612) 626-7246, E-mail: [email protected] URL: http://www.cems.umn.edu/research/kaznessis ______________________________________________________________________ Professor Kaznessis’ research interests focus on computer modeling of biological matter, synthetic biology and on statistical mechanical modeling of biomolecular recognition phenomena. Professor Kaznessis teaches undergraduate “Chemical Engineering Thermodynamics”, undergraduate “Process Dynamics and Control” and the graduate course “Statistical Thermodynamics and Kinetics”. Professor Kaznessis is also the Director of the University of Minnesota Summer Bioinformatics Institute. Education Diploma, Chemical Engineering, Aristotle University of Thessaloniki, Greece, 1994. Ph.D., Chemical Engineering, University of Notre Dame, 2000. Postdoctoral Fellowship, University of Michigan and Pfizer Global Research and Development, 08/99-08/01 Appointments ASSOCIATE PROFESSOR, 08/01/07 - present Department of Chemical Engineering and Materials Science, University of Minnesota Digital Technology Center, University of Minnesota DIRECTOR OF GRADUATE STUDIES IN CHEMICAL ENGINEERING, 01/2010 – present Department of Chemical Engineering and Materials Science, University of Minnesota DIRECTOR, 01/01/03-present University of Minnesota Summer Bioinformatics Institute ASSISTANT PROFESSOR, 08/23/01-07/31/07 Department of Chemical Engineering and Materials Science, University of Minnesota Digital Technology Center, University of Minnesota POSTDOCTORAL FELLOW, 08/99-08/01 Biomolecular Structure and Drug Design, Pfizer Global Research and Development. Department of Chemical Engineering, University of Michigan. RESEARCH ASSISTANT, 09/94-08/99 Department of Chemical Engineering, University of Notre Dame, Ph.D. (2000). PROJECT MANAGER ASSISTANT, 12/93-08/94 Euroconsultants S.A., Thessaloniki, Greece.
    [Show full text]
  • Multidimensional Signatures in Antimicrobial Peptides
    Multidimensional signatures in antimicrobial peptides Nannette Y. Yount*† and Michael R. Yeaman*†‡§ *Division of Infectious Diseases, Harbor–UCLA Medical Center, and †St. John’s Cardiovascular Research Center, Research and Education Institute at Harbor–UCLA, Torrance, CA 90502; and ‡David Geffen School of Medicine, University of California, Los Angeles, CA 90024 Communicated by H. Ronald Kaback, University of California, Los Angeles, CA, March 5, 2004 (received for review January 7, 2004) Conventional analyses distinguish between antimicrobial peptides by (iii) published antimicrobial activity, and (iv) up to 75 aa in length. differences in amino acid sequence. Yet structural paradigms common The initial dataset contained over 500 known antimicrobial pep- to broader classes of these molecules have not been established. The tides (see supporting information, which is published on the PNAS current analyses examined the potential conservation of structural web site). From this initial cohort, representatives of specific classes themes in antimicrobial peptides from evolutionarily diverse organ- [e.g., ␣-defensins, ␤-defensins, insect defensins, plant defensins, isms. Using proteomics, an antimicrobial peptide signature was dis- cysteine-stabilized (CS)-␣␤ peptides] were identified on the basis of covered to integrate stereospecific sequence patterns and a hallmark their being well characterized in the literature, having a known 3D three-dimensional motif. This striking multidimensional signature is structure, and being prototypic of their
    [Show full text]
  • Binding of Protegrin-1 to Pseudomonas Aeruginosa and Burkholderia Cepacia Mark T Albrecht1, Wei Wang2, Olga Shamova2, Robert I Lehrer2,3 and Neal L Schiller1
    Available online http://respiratory-research/content/3/1/18 Research3/1/18 RespirVol 3 No Res 1 article Respiratory Research Binding of protegrin-1 to Pseudomonas aeruginosa and Burkholderia cepacia Mark T Albrecht1, Wei Wang2, Olga Shamova2, Robert I Lehrer2,3 and Neal L Schiller1 1Division of Biomedical Sciences, University of California, Riverside, California 92521, USA. 2Department of Medicine, University of California, Los Angeles, Los Angeles, California 90095, USA. 3Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095, USA. Correspondence: Neal L Schiller - [email protected] Received: 1 October 2001 Respir Res 2002, 3:18 Revisions requested: 19 November 2001 Revisions received: 29 January 2002 Accepted: 31 January 2002 © 2002 Albrecht et al, licencee BioMed Central Ltd Published: 14 March 2002 (Print ISSN 1465-9921; Online ISSN 1465-993X) Abstract Background: Pseudomonas aeruginosa and Burkholderia cepacia infections of cystic fibrosis patients' lungs are often resistant to conventional antibiotic therapy. Protegrins are antimicrobial peptides with potent activity against many bacteria, including P. aeruginosa. The present study evaluates the correlation between protegrin-1 (PG-1) sensitivity/resistance and protegrin binding in P. aeruginosa and B. cepacia. Methods: The PG-1 sensitivity/resistance and PG-1 binding properties of P. aeruginosa and B. cepacia were assessed using radial diffusion assays, radioiodinated PG-1, and surface plasmon resonance (BiaCore). Results: The six P. aeruginosa strains examined were very sensitive to PG-1, exhibiting minimal active concentrations from 0.0625–0.5 µg/ml in radial diffusion assays. In contrast, all five B. cepacia strains examined were greater than 10-fold to 100-fold more resistant, with minimal active concentrations ranging from 6–10 µg/ml.
    [Show full text]
  • The Addition of a Synthetic LPS-Targeting Domain Improves
    biomolecules Article The Addition of a Synthetic LPS-Targeting Domain Improves Serum Stability While Maintaining Antimicrobial, Antibiofilm, and Cell Stimulating Properties of an Antimicrobial Peptide Anna Maystrenko, Yulong Feng, Nadeem Akhtar and Julang Li * Animal Biosciences, University of Guelph, Guelph, ON N1G 2W1, Canada; [email protected] (A.M.); [email protected] (Y.F.); [email protected] (N.A.) * Correspondence: [email protected]; Tel.: 519-824-4120 Received: 10 June 2020; Accepted: 6 July 2020; Published: 8 July 2020 Abstract: Multi-drug resistant (MDR) bacteria and their biofilms are a concern in veterinary and human medicine. Protegrin-1 (PG-1), a potent antimicrobial peptide (AMP) with antimicrobial and immunomodulatory properties, is considered a potential alternative for conventional antibiotics. AMPs are less stable and lose activity in the presence of physiological fluids, such as serum. To improve stability of PG-1, a hybrid peptide, SynPG-1, was designed. The antimicrobial and antibiofilm properties of PG-1 and the PG-1 hybrid against MDR pathogens was analyzed, and activity after incubation with physiological fluids was compared. The effects of these peptides on the IPEC-J2 cell line was also investigated. While PG-1 maintained some activity in 25% serum for 2 h, SynPG-1 was able to retain activity in the same condition for up to 24 h, representing a 12-fold increase in stability. Both peptides had some antibiofilm activity against Escherichia coli and Salmonella typhimurium. While both peptides prevented biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA), neither could destroy MRSA’s pre-formed biofilms. Both peptides maintained activity after incubation with trypsin and porcine gastric fluid, but not intestinal fluid, and stimulated IPEC-J2 cell migration.
    [Show full text]
  • Beta-Hairpin Protein Epitope Mimetic Inhibitors of the P53-HDM2 Protein-Protein Interaction
    Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2012 ฀-Hairpin protein epitope mimetic technology in drug discovery Obrecht, Daniel ; Chevalier, Eric ; Moehle, Kerstin ; Robinson, John A Abstract: Epitopes involved in protein–protein and protein–nucleic acid interactions provide ideal starting points for rational structure-based inhibitor design. The process of design and optimization of epitope mimetics is now emerging as an innovative new approach in drug discovery. Although often derided as unsuitable for drug development, we provide examples to show how peptidomimetics can provide a new generation of drug candidates to tackle some of the most challenging targets in pharmaceutical research, and address some of the most pressing current threats to human health. DOI: https://doi.org/10.1016/j.ddtec.2011.07.006 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-69031 Journal Article Accepted Version Originally published at: Obrecht, Daniel; Chevalier, Eric; Moehle, Kerstin; Robinson, John A (2012). ฀-Hairpin protein epitope mimetic technology in drug discovery. Drug Discovery Today: Technologies, 9(1):e63-e69. DOI: https://doi.org/10.1016/j.ddtec.2011.07.006 1 ß-Hairpin Protein Epitope Mimetic Technology in Drug Discovery Daniel Obrecht1 Eric Chevalier1 Kerstin Moehle2 and John A. Robinson2 1 Polyphor AG, Hegenheimermattweg 125, 4123-Allschwil, Switzerland (www.polyphor.com) 2 Chemistry Department, University of Zurich, Winterthurerstrasse 190, 8057-Zurich, Switzerland. (www.oci.uzh.ch) Corresponding author: Prof. J. A. Robinson Chemistry Department University of Zurich Winterthurerstrasse 190 8057 Zurich, Switzerland Phone: +41-44-6354242 Fax: +41-44-6356812 e-mail: [email protected] AND e-mail: [email protected] 2 Abstract Epitopes involved in protein-protein and protein-nucleic acid interactions provide ideal starting points for rational structure-based inhibitor design.
    [Show full text]
  • Structure and Dynamics of Cationic Membrane Peptides and Proteins: Insights from Solid-State NMR
    REVIEW Structure and dynamics of cationic membrane peptides and proteins: Insights from solid-state NMR Mei Hong* and Yongchao Su Department of Chemistry, Iowa State University, Ames, Iowa Received 30 December 2010; Revised 14 January 2011; Accepted 18 January 2011 DOI: 10.1002/pro.600 Published online 22 February 2011 proteinscience.org Abstract: Many membrane peptides and protein domains contain functionally important cationic Arg and Lys residues, whose insertion into the hydrophobic interior of the lipid bilayer encounters significant energy barriers. To understand how these cationic molecules overcome the free energy barrier to insert into the lipid membrane, we have used solid-state NMR spectroscopy to determine the membrane-bound topology of these peptides. A versatile array of solid-state NMR experiments now readily yields the conformation, dynamics, orientation, depth of insertion, and site-specific protein–lipid interactions of these molecules. We summarize key findings of several Arg-rich membrane peptides, including b-sheet antimicrobial peptides, unstructured cell-penetrating peptides, and the voltage-sensing helix of voltage-gated potassium channels. Our results indicate the central role of guanidinium-phosphate and guanidinium-water interactions in dictating the structural topology of these cationic molecules in the lipid membrane, which in turn account for the mechanisms of this functionally diverse class of membrane peptides. Keywords: membrane protein; solid-state NMR; antimicrobial peptides; cell penetrating peptides; structure; dynamics; topology Introduction tides (AMPs)1 and the family of delivery molecules In this review, we will explore the structure and dy- called cell-penetrating peptides (CPPs),2 which con- namics of cationic membrane peptides and proteins tain 30 to 100% cationic Arg and Lys residues.
    [Show full text]
  • Β-Hairpin Antimicrobial Peptides
    β-hairpin antimicrobial peptides: structure, function and mode of action Ingrid Alexia Edwards MSc Chemistry and chemical engineering A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 Institute for Molecular Bioscience Abstract Abstract A ‘state of emergency’ was declared by the World Health Organization three years ago to combat the increasing rate of resistance arising in bacteria to all currently available antibiotics on the market. Antimicrobial resistance is now a worldwide concern, and renewed efforts are needed in the search for new replacement drugs for obsolete antibiotics. Antimicrobial peptides (AMPs) have been discovered and studied over decades; importantly very limited bacterial resistance has been reported to date. The work here aims to better characterize and understand the structure-function relationships of select β-hairpin AMPs, leading to the design of novel, optimized and potentially therapeutically valuable peptides. Chapter 1 reviews the field of β-hairpin AMPs and provides a background for the specific AMPs studied in this thesis. Chapter 2 consists of an original data set that strengthens the current knowledge of β-hairpin AMPs by comparing their activity profile under similar conditions. This work analysed the contribution of amphipathicity and hydrophobicity to antimicrobial activity and cytotoxicity of β- hairpin peptides, concluding that a very fine balance between charge, hydrophobicity, amphipathicity, secondary and tertiary structure and mode of action is needed for a peptide to be therapeutically valuable. From this study, two distinct but linked areas of further investigation were identified (i) a structure activity and function relationship study and (ii) a determination of the mode of action of select β-hairpin AMPs.
    [Show full text]
  • Investigations of Cationic Peptides in Lipid Membranes by Solid-State NMR Timothy Franklin Doherty Iowa State University
    Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2009 Investigations of cationic peptides in lipid membranes by solid-state NMR Timothy Franklin Doherty Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Chemistry Commons Recommended Citation Doherty, Timothy Franklin, "Investigations of cationic peptides in lipid membranes by solid-state NMR" (2009). Graduate Theses and Dissertations. 10675. https://lib.dr.iastate.edu/etd/10675 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Investigations of cationic peptides in lipid membranes by solid-state NMR by Timothy Franklin Doherty A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Analytical Chemistry Program of Study Committee: Mei Hong, Major Professor Klaus Schmidt-Rohr Amy Andreotti Emily Smith Edward Yu Iowa State University Ames, Iowa 2009 Copyright © Timothy Franklin Doherty, 2009. All rights reserved. ii Table of contents Acknowledgement vi Abstract vii Chapter 1 Introduction 1 Dynamics in lipid bilayers 1 Energetics of amphipathic peptide-membrane insertion 3 Antimicrobial peptides
    [Show full text]
  • Structures of Β-Hairpin Antimicrobial Protegrin Peptides
    ARTICLE pubs.acs.org/biochemistry Structures of β-Hairpin Antimicrobial Protegrin Peptides in Lipopolysaccharide Membranes: Mechanism of Gram Selectivity Obtained from Solid-State Nuclear Magnetic Resonance † ‡ ‡ † Yongchao Su, Alan J. Waring, ,§ Piotr Ruchala, and Mei Hong*, † Department of Chemistry, Iowa State University, Ames, Iowa 50011, United States ‡ Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, California 90095, United States §Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, California 92697-4560, United States bS Supporting Information ABSTRACT: The structural basis for the Gram selectivity of two disulfide-bonded β-hairpin antimicrobial peptides (AMPs) is investigated using solid-state nuclear magnetic resonance (NMR) spectroscopy. The hexa-arginine PG-1 exhibits potent activities against both Gram-positive and Gram-negative bac- teria, while a mutant of PG-1 with only three cationic residues maintains Gram-positive activity but is 30-fold less active against Gram-negative bacteria. We determined the topological structure and lipid interactions of these two peptides in a lipopolysaccharide (LPS)-rich membrane that mimics the outer membrane of Gram-negative bacteria and in the POPE/POPG membrane, which mimics the membrane of Gram-positive bacteria. 31P NMR line shapes indicate that both peptides cause less orientational disorder in the LPS-rich membrane than in the POPE/POPG membrane. 13C chemical shifts and 13C-1H dipolar couplings show that both peptides maintain their β-hairpin conformation in these membranes and are largely immobilized, but the mutant exhibits noticeable intermediate-time scale motion in the LPS membrane at physiological temperature, suggesting shallow insertion.
    [Show full text]
  • Protective and Anti-Inflammatory Effects of Protegrin-1 on Citrobacter Rodentium Intestinal Infection in Mice
    Protective and Anti-inflammatory Effects of Protegrin-1 on Citrobacter rodentium Intestinal Infection in Mice By Celina N. Osakowicz A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Animal Biosciences Guelph, Ontario, Canada © Celina N. Osakowicz, May, 2018 ABSTRACT PROTECTIVE AND ANTI-INFLAMMATORY EFFECTS OF PROTEGRIN-1 ON Citrobacter rodentium INTESTINAL INFECTION IN MICE Celina N. Osakowicz Advisor: University of Guelph, 2018 Dr. Julang Li Intestinal disorders and colitis affect millions of humans and food-animals world-wide. Antimicrobial peptides (AMPs) and their broad-spectrum antimicrobial activity represent a valuable potential therapeutic solution. Specifically, the potent pig-originated protegrin-1 (PG-1) has previously been shown to reduce the pathological effects of chemically induced digestive tract inflammation (colitis) along with modulation of immune responses and tissue-repair. This study aimed to extend these findings by investigating the potential protective effects of PG-1 on pathogen-induced intestinal colitis. We found that oral administration of PG-1 reduced Citrobacter rodentium intestinal infection in mice evidenced by; reduced histopathologic change in the colon, prevention of body weight loss, milder clinical signs of disease, and ultimately more effective clearance of bacterial infection relative to challenged mice. Additionally, PG-1 treatment altered the expression of various inflammatory mediators during infection to resolve inflammation and re-establish intestinal homeostasis. Interestingly, PG-1 administered in its mature form was most effective relative to the pro-form (ProPG-1). Acknowledgements I would like to acknowledge a great number of individuals who have been invaluable to this research project and whom the completion of this degree would not be possible without their support.
    [Show full text]
  • Interaction of Antimicrobial Peptide Protegrin with Biomembranes
    Interaction of antimicrobial peptide protegrin with biomembranes David Gidalevitz*†, Yuji Ishitsuka†‡, Adrian S. Muresan§, Oleg Konovalov¶, Alan J. Waringʈ**, Robert I. Lehrerʈ, and Ka Yee C. Lee‡†† *Department of Chemical Engineering, University of Leeds, Leeds LS2 9JT, United Kingdom; Departments of ‡Chemistry and §Physics, Institute for Biophysical Dynamics and the James Franck Institute, University of Chicago, Chicago, IL 60637; ¶European Synchrotron Radiation Facility, B.P. 220, 38043 Grenoble Cedex 9, France; ʈDepartment of Medicine, University of California, Los Angeles, CA 90095; and **Department of Pediatrics, University of California School of Medicine, Harbor-University of California Los Angeles Medical Center, Torrance, CA 90502 Edited by William F. DeGrado, University of Pennsylvania School of Medicine, Philadelphia, PA, and approved March 5, 2003 (received for review August 6, 2002) The antimicrobial peptide protegrin-1 (PG-1) interacts with mem- antimicrobial peptides is not always caused merely by the break- branes in a manner that strongly depends on membrane lipid down of the permeability barrier in microbes (14, 18). Other composition. In this research we use an approach representing the membrane-dependent processes, such as translocation of cytotoxic outer layers of bacterial and red blood cell membranes with lipid peptides across the membrane to the cytoplasm and facilitation of monolayers and using a combination of insertion assay, epifluo- transbilayer lipid diffusion (which leads to the loss of lipid asym- rescence microscopy, and surface x-ray scattering to gain a better metry in the membrane), may also be involved in the action of some understanding of antimicrobial peptide’s mechanism of action. We of these peptides (19).
    [Show full text]
  • Structure-Dependent Immune Modulatory Activity of Protegrin-1 Analogs
    HHS Public Access Author manuscript Author Manuscript Author ManuscriptAntibiotics Author Manuscript (Basel). Author Author Manuscript manuscript; available in PMC 2015 June 18. Published in final edited form as: Antibiotics (Basel). 2014 December ; 3(4): 694–713. doi:10.3390/antibiotics3040694. Structure-Dependent Immune Modulatory Activity of Protegrin-1 Analogs Susu M. Zughaier1,2,*,†, Pavel Svoboda3,4,†, and Jan Pohl3,4 Pavel Svoboda: [email protected]; Jan Pohl: [email protected] 1Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA 2Laboratories of Microbial Pathogenesis, Atlanta Department of Veterans Affairs Medical Center, Atlanta, GA 30033, USA 3Microchemical and Proteomics Facility, Emory University School of Medicine, Atlanta, GA 30322, USA 4Biotechnology Core Facility Branch, Division of Scientific Resources, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA Abstract Protegrins are porcine antimicrobial peptides (AMPs) that belong to the cathelicidin family of host defense peptides. Protegrin-1 (PG-1), the most investigated member of the protegrin family, is an arginine-rich peptide consisting of 18 amino acid residues, its main chain adopting a β-hairpin structure that is linked by two disulfide bridges. We report on the immune modulatory activity of PG-1 and its analogs in neutralizing bacterial endotoxin and capsular polysaccharides, consequently inhibiting inflammatory mediators’ release from macrophages. We demonstrate that the β-hairpin structure motif stabilized with at least one disulfide bridge is a prerequisite for the immune modulatory activity of this type of AMP. Keywords protegrin-1 (PG-1); Toll-like receptor (TLR) ligands; cytokines; macrophage; innate immunity © 2014 by the authors; licensee MDPI, Basel, Switzerland This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http:// creativecommons.org/licenses/by/4.0/).
    [Show full text]