Antibacterial Peptide Protocols M E T H O D S IN M O L E C U L A R B I O L O G Y ' John M Walker, SERIES EDITOR 83. Receptor Signal Transduction Protocois, edited by 53. Veast Protocols: Methods in Cell and Molecular Biology, R. A. J. Challiss, 1997 edited by Ivor H. Evans, 1996 82. Arabadopsis Protocols, edited by Jose MMartinez-Zapaler 52. Capillary Electrophoresis: Principles, Instrumentation, and Julio Salinas, 1997 and Applications, edited by Kevin D. Altria, 1996 81. Plant Virology Protocols, edited by Gary D. Foster, 1997 51. Antibody Engineering Protocols, edited by SarfAirPflu/. 1995 80. Immunodiemical Protocols (2nd. ed.), edited by 50. Species Diagnostics Protocols: PCR and Other Nucleic John Pound, 1997 Acid Methods, edited by Justin P. Clapp, 1996 79. Polyamine Protocols, edited by David M. L Morgan, 1997 49. Plant Gene Transfer and Expression Protocols, edited by 78. Antibacterial Peptide Protocols, edited by William M. Heddwyn Jones, 1995 Shafer, 1997 48. Animal Cell Electroporation and Electrofusion Proto 77. Protein Synthesis: Methods and Protocols, edited by Robin cols, edited by Jac A. Nickoloff, 1995 Martin, 1997 47. Electroporation Protocols for Microorganisms, edited by 76. Glycoanalysis Protocols, edited by Elizabeth f. Jac A. Nickoloff, 1995 Hounsel, 1997 46. Diagnostic Bacteriology Protocols, edited by Jenny 75. Basic Cell Culture Protocols, edited by Jeffrey W. Pollard Howard and David M. Whitcombe, 1995 and John M. Walker, 1997 45. Monoclonal Antibody Protocols, edited by William C. 74. Ribozyme Protocols, edited by Philip C. Turner, 1997 Davis, 1995 73. Neuropeptide Protocols, edited by G. Brent Irvine and 44. Agrobttcterium Protocols, edited by Kevan M. A. Gartland CarvellH. Williams. 1997 and Michael R. Davey, 1995 72. Neurotransmitter Methods, edited by S/cAardC.Sayne, 1997 43. In Vitro Toxicity Testing Protocols, edited by Sheila 71. PRINS and In Situ PCR Protocols, edited by John R. O'Hare and Chris K. Atlerwill, 1995 Gosden, 1997 42. ELISA: Theory and Practice, by John R. Crowther. 1995 70. Sequence Data Analysis Guidebook, edited by Simon R. 41. Signal Transduction Protocols, edited by David A. Kendall Swindell, 1997 and Stephen J. Hill, 1995 69. cDNA Library Protocols, edited by Ian G. Cowell 40. Protein Stability and Folding: Theory and Practice, and Caroline A. Austin, 1997 tiiteiby Bret A. Shirley 1995 68. Gene Isolation and Mapping Protocols, edited by 39. Baculovlrus Expression Protocols, edited by Christopher Jacqueline Boultwood, 1997 D. Richardson. 1995 67. PCR Cloning Protocols: From Molecular Cloning to 38. Cryopreservation and Freeze-Drying Protocols, Genetic Engineering, edited by Bruce A. While, 1996 edited by John G. Day and Mark R. McLellan, 1995 66. Epitope Mapping Protocols, edited by Glenn E. Morris, 1996 37. In Vitro Transcription and Translation Protocols, 65. PCR Sequencing Protocols, edited by Ralph Rapley, 1996 edited by Martin J. Tymms, 1995 64. Protein Sequencing Protocols, edited by Bryan J. Smith, 1996 36. Peptide Analysis Protocols, edited by Ben M. Dunn and 63. Recombinant Proteins: Detection and Isolation Protocols, Michael W. Pennington, 1994 edited by Rocky S Tuan, 1996 35. Peptide Synthesis Protocols, edited by Michael W. 62. Recombinant Gene Expression Protocols, edited by Rocky Pennington and Ben M. Dunn, 1994 S Tuan, 1996 34. Immunocytochemical Methods and Protocols, edited by 61. Protein and Peptide Analysis by Mass Spectrometry, Lorette C. Javois, 1994 edited by John R Chapman, 1996 33. In Situ Hybridization Protocols, edited by K. H. Andy 60. Protein NMR Protocols, edited by David G. Reid, 1996 Choo, 1994 59. Protein Purification Protocols, edited by Shawn Doonan, 32. Basic Protein and Peptide Protocols, edited by John M. 1996 Walker, 1994 58. Basic DNA and RNA Protocols, edited by Adrian J. 31. Protocols for Gene Analysis, edited by Adrian J. Harwood, Harwood, 1996 1994 57. In Vitro Mutagenesis Protocols, edited by Michael K. 30. DNA-Protein Interactions, edited by C. Geoff Kneale, 1994 Trower, 1996 29. Chromosome Analysis Protocols, edited by John R. 56. Crystallographic Methods and Protocols, edited by Chris Gosden, 1994 topher Jones, Barbara Mulloy, and Mark Sanderson, 19962 8. Protocols for Nucleic Acid Analysis by Nonradioactive 55. Plant Cell Electroporation and Electrofusion Protocols, Probes, edited by Peter G. Isaac. 1994 edited by Jac A. Nickoloff, 1995 27. Biomembrane Protocols: //. Architecture and Function. 54. VAC Protocols, edited by/)avi<iMarfae, 1995 edited by John M. Graham and Joan A. Higgins, 1994 M E T H O D S IN M O L E C U L A R BIOLOGY™ Antibacterial Peptide Protocols Edited by William M. Shafer Emory University School of Medicine, Atlanta, GA Humana Press ^ j ^ Totowa, New Jersey © 1997 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology" is a trademark of The Humana Press Inc. All authored papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. This publication is printed on acid-free paper. C55 ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials. Cover illustration: Fig. 1 from Chapter 10, "An Approach Combining Rapid cDNA Amplification and Chemical Synthesis for the Identification of Novel, Cathelicidin-Derived, Antimicrobial Peptides," by Alessandro Tossi, Marco Scocchi, Margherita Zanetti, Renato Gennaro, Paola Storici, and Domenico Romeo. Cover design by Patricia F. Cleary. 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Library of Congress Cataloging in Publication Data Main entry under title: Methods in molecular biology'^". Antibacterial peptide protocols/edited by William M. Shafer. p. cm.—(Methods in molecular biology; 78) Includes bibliographical references and index. ISBN 0-89603-408-9 (alk. paper) 1. Peptide antibiotics—Research—^Methodology. 2. Peptide antibiotics—Analysis. I. Shafer, William M. II. Series: Methods in molecular biology (Totowa, NJ); 78. RS431.P37A57 1997 615'.329—dc21 97-24698 CIP Preface As this book. Antibacterial Peptide Protocols, will attest, my enthusi asm for the field of antibacterial peptides is based on a conviction (and I am unashamed to say, prejudice) that these substances are in essence antibiotics produced by the host that then participate in host defense against infectious agents. Because of their capacity to exert antibiotic-like action against patho genic microorganisms (bacteria, fungi, parasites, and viruses), there is reason to believe that these agents will soon be used clinically to treat infectious diseases. In fact, in recent years, biotechnology companies have been formed for the sole purpose of developing antibacterial peptides for clinical use. It should be emphasized that antibacterial peptides will likely play a major role in the treatment of infectious diseases, particularly with the increasing prob lem of multidrug-resistant microbes and the relative dearth of new antibiotics being provided by pharmaceutical companies. The topic of this volume of Methods in Molecular Biology, the diverse methods used in research on antibacterial peptides, is thus quite timely. As the subject of antibacterial peptides develops into its own discipline (something strongly suggested by the explosion in the number of papers published over the past decade), it is essential that reliable techniques and strategies be made available not only to those of us in the field, but also to the newcomers and researchers in complementary disciplines. At first glance, the compilation of such techniques might seem an easy task, but, given the number and variety of techniques employed, it would be impossible for one person to take this on singlehandedly; after all, contemporary antimicrobial peptide research requires the availability of reliable protocols in microbiology, microbial genetics, cell biology, molecular biology, biochemistry, physical chemistry, in vivo experi mentation, and histopathology. From the beginning, it was my purpose to assemble a team of experts who employ techniques in one or more of the aforementioned disciplines. Toward that end, I was extremely gratified when the numerous authors agreed to prepare chapters in their areas of expertise. Without exception, each author expressed a willingness to do so because of a general feeling that the field had reached a level of maturity that warranted the preparation of a methods-style handbook. vi Preface Antibacterial Peptide Protocols is divided into three main sections that are interconnected and preceded by a historical review of antibacterial pep tides. The first section deals w^ith the isolation and characterization of antimi crobial peptides. Herein, procedures involving the biochemical fraction of antimicrobial peptide-containing extracts from vertebrate and invertebrate sources and the physiochemical analysis of purified peptides are described in a series of chapters. Recent advances in the molecular characterization of genes encoding antimicrobial peptides, as well as procedures for using expression systems to isolate peptides, are described in the second section. Section three describes bioassays and microbial genetic techniques for studying antimicro bial peptides. It should be emphasized that the use of a reliable, standard bio- assay for evaluating antimicrobial peptide activity is the necessary foundation for all studies dealing with this class of bioactive peptides. Toward this end, many different variations of antibacterial assays have been published and are used on a routine basis in different laboratories. The reader will notice this in chapters dedicated to other topics, but the authors include an antibacterial assay as the final readout system. An evaluation of the mechanisms by which peptides exert their antibacterial action are also covered in this section. Finally, it has become increasingly obvious in the past decade that these pep tides possess activities other than their bactericidal effects. Thus, the capaci ties of certain peptides to inhibit the endotoxic effects of lipopolysaccharide and to serve as chemotactic agents for phagocytic cells have been documented, as is described in several chapters. These additional properties of antibacterial peptides may, in the final analysis, prove to be as important, if not more so, than their antibacterial action. I trust that the reader will find each chapter of Antibacterial Peptide Pro tocols beneficial and helpful for his or her own particular needs. In closing, I wish to thank each author for their enthusiastic and diligent work in the prepa ration of this book. Special thanks are extended to Professor John Walker for his encouragement and advice, and to Mr. Thomas Lanigan for allowing me an extraordinary amount of freedom in choosing topics and authors. William M. Shafer Contents Preface v Contributors ix 1 Origins and Development of Peptide Antibiotic Research: From Extracts to Abstracts to Contracts John K. Spitznagel 1 PART I. ISOLATION AND CHARACTERIZATION OF ANTIBACTERIAL PEPTIDES 2 HPLC Methods for Purification of Antimicrobial Peptides Michael E. Selsted 17 3 Strategies for the Isolation and Characterization of Antimicrobial Peptides of Invertebrates Charles Hetru and Philippe Bulet 35 4 Big Defensin and Tachylectins-1 and -2 Shun-ichiro Kawabata and Sadaaki Iwanaga 51 5 Tachyplesin and Anti-Lipopolysaccharide Factor Tatsushi Muta and Sadaaki Iwanaga 63 6 Circular Dichroism Studies of Secondary Structure of Peptides Martha M. Juban, Maryam M. Javadpour, and Mary D. Barkiey.... 73 7 Analytical Ultracentrifugation Studies of Association of Peptides Martha M. Juban, Maryam M. Javadpour, and Mary D. Barkiey.... 79 8 NMR Characterization of Amphipathic Helical Peptides Xiaotang Wang and Kathleen M. Morden 85 PART II. MOLECULAR BIOLOGY OF ANTIBACTERIAL PEPTIDES 9 Laboratory Production of Antimicrobial Peptides in Native Conformation Erika V. Valore and Tomas Ganz 115 10 An Approach Combining Rapid cDNA Amplification and Chemical Synthesis for the Identification of Novel, Cathelicidin-Derived, Antimicrobial Peptides Alessandro Tossi, Marco Scocchi, Margherita Zanetti, Renato Gennaro, Paoia StoricI, and Domenico Romeo 133 vii via Contents 11 Molecular Biological Strategies in the Analysis of Antibiotic Peptide Gene Families: Ttie Use of Oligonucleotides as Hybridization Probes Charles L. Bavins and Gill Diamond 151 PART III. ASSAY SYSTEMS FOR STUDYING ANTIBACTERIAL PEPTIDES 12 Designer Assays for Antimicrobial Peptides: Disputing ttie "One-Size-Fits-All" Theory Deborah A. Steinberg and Robert I. Lehrer 169 13 Interaction of Cationic Peptides with Bacterial Membranes Shaflque Fidai, Susan W. Farmer, and Robert E. W. Hancock... 187 14 The Genetic Basis of Microbial Resistance to Antimicrobial Peptides EduardoA. Groisman and Arden Aspedon 205 15 Assay of Antibacterial Activities of the Bactericidal/Permeability- Increasing Protein in Natural Biological Fluids Jerrold Weiss 217 16 Assay Systems for Measurement of Chemotactic Activity H. Anne Perelra 233 17 Neutralization of the In Vivo Activity of E. co//-Derived Lipopolysaccharide by Cationic Peptides Daniel J. Brackett, Megan R. Lerner, and H. Anne Perelra 247 Index 257 Contributors ARDEN ASPEDON • Department of Biology, University of Missouri-St. Louis, St. Louis, MO MARY D. BARKLEY • Department of Biochemistry, Louisiana State University, Baton Rouge, LA CHARLES L. BEVINS • Research Institute, The Cleveland Clinic Foundation, Cleveland, OH DANIEL J. BRACKETT • Departments of Surgery, Anesthesiology, and Pathology, University of Oklahoma Health Sciences Center, Department of Veterans Affairs Medical Center, Oklahoma City, OK PHILIPPE BULET • Institut de Biologie Moleculaire et Cellulaire CNRS, Strasbourg Cedex France GILL DIAMOND • Department of Anatomy, Cell Biology, and Injury Sciences, University of Medicine and Dentistry of New Jersey, Newark, NJ SUSAN W. FARMER • Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada SHAFIQUE FIDAI • Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada ToMAS GANZ • Department of Medicine, University of California, Los Angeles, CA RENATO GENNARO • Department of Biomedical Science and Technology, University ofUdine, Italy EDUARDO A. GROISMAN • Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO ROBERT E. W. HANCOCK • Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada CHARLES HETRU • Institut de Biologie Moleculaire et Cellulaire CNRS, Strasbourg Cedex, France SADAAKI IWANAGA • Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan MARYAM M. JAVADPOUR • Department of Biochemistry, Louisiana State University, Baton Rouge, LA MARTHA M. JUBAN • Department of Biochemistry, Louisiana State University, Baton Rouge, LA ix X Contributors SHUN-ICHIRO KAWABATA • Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan ROBERT I. LEHRER • Department of Medicine, University of California, Los Angeles, CA MEGAN R. LERNER • Departments of Surgery, Anesthesiology, and Pathology, University of Oklahoma Health Sciences Center, Department of Veterans Affairs Medical Center, Oklahoma City, OK KATHLEEN M. MORDEN • Department of Biochemistry, Louisiana State University, Baton Rouge, LA TATSUSHI MUTA • Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan; Present Address: Department of Biochemistry, Kyushu University School of Medicine, Fukuoka, Japan H. ANNE PEREIRA • Department of Pathology, University of Oklahoma Health Science Center, Oklahoma City, OK DoMENico ROMEO • Department of Biochemistry, Biophysics, and Macromolecular Chemistry, University of Trieste, Italy MARCO SCOCCHI • Department of Biochemistry, Biophysics, and Macromolecular Chemistry, University of Trieste, Italy; National Laboratory of the Interuniversity Biotechnology Consortium, AREA Science Park, Trieste, Italy MICHAEL E. SELSTED • Department of Pathology, College of Medicine, University of California, Irvine, CA JOHN K. SPITZNAGEL • Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA DEBORAH A. STEINBERG • IntraBiotics Pharmaceuticals, Inc., Sunnyvale, CA PAOLA STORICI • Department of Biochemistry, Biophysics, and Macromolecular Chemistry, University of Trieste, Italy ALESSANDRO TOSSI • Department of Biochemistry, Biophysics, and Macromolecular Chemistry, University of Trieste, Italy ERIKA V. VALORE • Department of Medicine, University of California, Los Angeles, CA XIAOTANG WANG • Department of Biochemistry, Louisiana State University, Baton Rouge, LA JERROLD WEISS • Department of Microbiology, New York University School of Medicine, New York MARGHERITA ZANETTI • National Laboratory of the Interuniversity Biotechnology Consortium, AREA Science Park, Trieste, Italy; Department of Biomedical Science and Technology, University ofUdine, Italy