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 R. A. J. Challiss, 1997

82. Arabadopsis Protocols, edited by Jose MMartinez-Zapaler and Julio Salinas, 1997

81. Plant Virology Protocols, edited by Gary D. Foster, 1997 80. Immunodiemical Protocols (2nd. ed.), edited by

John Pound, 1997 79. Polyamine Protocols, edited by David M. L Morgan, 1997 78. Antibacterial Peptide Protocols, edited by William M.

Shafer, 1997 77. Protein Synthesis: Methods and Protocols, edited by Robin

Martin, 1997 76. Glycoanalysis Protocols, edited by Elizabeth f.

Hounsel, 1997 75. Basic Cell Culture Protocols, edited by Jeffrey W. Pollard

and John M. Walker, 1997 74. Ribozyme Protocols, edited by Philip C. Turner, 1997 73. Neuropeptide Protocols, edited by G. Brent Irvine and

CarvellH. Williams. 1997 72. Neurotransmitter Methods, edited by S/cAardC.Sayne, 1997 71. PRINS and In Situ PCR Protocols, edited by John R.

Gosden, 1997 70. Sequence Data Analysis Guidebook, edited by Simon R.

Swindell, 1997 69. cDNA Library Protocols, edited by Ian G. Cowell

and Caroline A. Austin, 1997 68. Gene Isolation and Mapping Protocols, edited by

Jacqueline Boultwood, 1997 67. PCR Cloning Protocols: From Molecular Cloning to

Genetic Engineering, edited by Bruce A. While, 1996 66. Epitope Mapping Protocols, edited by Glenn E. Morris, 1996 65. PCR Sequencing Protocols, edited by Ralph Rapley, 1996 64. Protein Sequencing Protocols, edited by Bryan J. Smith, 1996 63. Recombinant Proteins: Detection and Isolation Protocols,

edited by Rocky S Tuan, 1996 62. Recombinant Gene Expression Protocols, edited by Rocky

S Tuan, 1996 61. Protein and Peptide Analysis by Mass Spectrometry,

edited by John R Chapman, 1996 60. Protein NMR Protocols, edited by David G. Reid, 1996 59. Protein Purification Protocols, edited by Shawn Doonan,

1996 58. Basic DNA and RNA Protocols, edited by Adrian J.

Harwood, 1996 57. In Vitro Mutagenesis Protocols, edited by Michael K.

Trower, 1996 56. Crystallographic Methods and Protocols, edited by Chris­

topher Jones, Barbara Mulloy, and Mark Sanderson, 1996 55. Plant Cell Electroporation and Electrofusion Protocols,

edited by Jac A. Nickoloff, 1995 54. VAC Protocols, edited by/)avi<iMarfae, 1995

53. Veast Protocols: Methods in Cell and Molecular Biology, edited by Ivor H. Evans, 1996

52. Capillary Electrophoresis: Principles, Instrumentation, and Applications, edited by Kevin D. Altria, 1996

51. Antibody Engineering Protocols, edited by SarfAirPflu/. 1995 50. Species Diagnostics Protocols: PCR and Other Nucleic

Acid Methods, edited by Justin P. Clapp, 1996 49. Plant Gene Transfer and Expression Protocols, edited by

Heddwyn Jones, 1995 48. Animal Cell Electroporation and Electrofusion Proto­

cols, edited by Jac A. Nickoloff, 1995 47. Electroporation Protocols for Microorganisms, edited by

Jac A. Nickoloff, 1995 46. Diagnostic Bacteriology Protocols, edited by Jenny

Howard and David M. Whitcombe, 1995 45. Monoclonal Antibody Protocols, edited by William C.

Davis, 1995 44. Agrobttcterium Protocols, edited by Kevan M. A. Gartland

and Michael R. Davey, 1995 43. In Vitro Toxicity Testing Protocols, edited by Sheila

O'Hare and Chris K. Atlerwill, 1995 42. ELISA: Theory and Practice, by John R. Crowther. 1995 41. Signal Transduction Protocols, edited by David A. Kendall

and Stephen J. Hill, 1995 40. Protein Stability and Folding: Theory and Practice,

tiiteiby Bret A. Shirley 1995 39. Baculovlrus Expression Protocols, edited by Christopher

D. Richardson. 1995 38. Cryopreservation and Freeze-Drying Protocols,

edited by John G. Day and Mark R. McLellan, 1995 37. In Vitro Transcription and Translation Protocols,

edited by Martin J. Tymms, 1995 36. Peptide Analysis Protocols, edited by Ben M. Dunn and

Michael W. Pennington, 1994 35. Peptide Synthesis Protocols, edited by Michael W.

Pennington and Ben M. Dunn, 1994 34. Immunocytochemical Methods and Protocols, edited by

Lorette C. Javois, 1994 33. In Situ Hybridization Protocols, edited by K. H. Andy

Choo, 1994 32. Basic Protein and Peptide Protocols, edited by John M.

Walker, 1994 31. Protocols for Gene Analysis, edited by Adrian J. Harwood,

1994 30. DNA-Protein Interactions, edited by C. Geoff Kneale, 1994 29. Chromosome Analysis Protocols, edited by John R.

Gosden, 1994 28. Protocols for Nucleic Acid Analysis by Nonradioactive

Probes, edited by Peter G. Isaac. 1994 27. Biomembrane Protocols: //. Architecture and Function.

edited by John M. Graham and Joan A. Higgins, 1994

M E T H O D S I N M O L E C U L A R B I O L O G Y ™

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.

For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel: 201-256-1699; Fax: 201-256-8341; E-mail: humana@mindspring.com, or visit our Website at www.humanapress.com

Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $8.00 per copy, plus US $00.25 per page, is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [0-89603-408-9/97 $8.00 + $00.25].

Printed in the United States of America. 1 0 9 8 7 6 5 4 3 2 !

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 enthusi­asm 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 patho­genic 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 prob­lem 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 experi­mentation, 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 pep­tides. The first section deals w ith the isolation and characterization of antimi­crobial 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 antimicro­bial 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 pep­tides possess activities other than their bactericidal effects. Thus, the capaci­ties 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 Pro­tocols 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 prepa­ration 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