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5TH EDITION

VIRULENCE
MECHANISMS OF
BACTERIAL PATHOGENS
5TH EDITION

VIRULENCE
MECHANISMS OF
BACTERIAL PATHOGENS
Edited by
Indira T. Kudva Tracy L. Nicholson
National Animal Disease Center National Animal Disease Center
Agricultural Research Service Agricultural Research Service
U.S. Department of Agriculture U.S. Department of Agriculture
Ames, IA 50010 Ames, IA 50010

Nancy A. Cornick John P. Bannantine

Department of Veterinary Microbiology and National Animal Disease Center
Preventive Medicine Agricultural Research Service
College of Veterinary Medicine U.S. Department of Agriculture
Iowa State University Ames, IA 50010
Ames, IA 50011 Bryan H. Bellaire
Paul J. Plummer Department of Veterinary Microbiology
Department of Veterinary Diagnostic and and Preventive Medicine
Production Animal Medicine College of Veterinary Medicine
College of Veterinary Medicine Iowa State University
Iowa State University Ames, IA 50011
Ames, IA 50011
Qijing Zhang
Department of Veterinary Microbiology and
Preventive Medicine
College of Veterinary Medicine
Iowa State University
Ames, IA 50011

Washington, DC
Copyright © 2016 American Society for Microbiology. All rights reserved. No part of this publication
may be reproduced or transmitted in whole or in part or reused in any form or by any means,
electronic or mechanical, including photocopying and recording, or by any information storage and
retrieval system, without permission in writing from the publisher.

Disclaimer: To the best of the publisher’s knowledge, this publication provides information
concerning the subject matter covered that is accurate as of the date of publication. The publisher
is not providing legal, medical, or other professional services. Any reference herein to any specific
commercial products, procedures, or services by trade name, trademark, manufacturer, or
otherwise does not constitute or imply endorsement, recommendation, or favored status by the
American Society for Microbiology (ASM). The views and opinions of the author(s) expressed in this
publication do not necessarily state or reflect those of ASM, and they shall not be used to advertise
or endorse any product.

Library of Congress Cataloging-in-Publication Data

Names: Kudva, Indira T., editor.

Title: Virulence mechanisms of bacterial pathogens / edited by Indira T.
Kudva [and six others].
Description: Fifth edition. | Washington, DC: ASM Press, [2016] | Includes
bibliographical references and index.
Identifiers: LCCN 2016014513 (print) | LCCN 2016016980 (ebook) | ISBN
9781555819279 (hardcover) | ISBN 9781555819286 ()
Subjects: LCSH: Virulence (Microbiology) | Pathogenic bacteria. |
Host-bacteria relationships.
Classification: LCC QR175 .V57 2016 (print) | LCC QR175 (ebook) | DDC
616.9/201--dc23
LC record available at https://lccn.loc.gov/2016014513

All Rights Reserved

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

Address editorial correspondence to

ASM Press, 1752 N St., N.W.,
Washington, DC 20036-2904, USA

Send orders to ASM Press, P.O. Box 605, Herndon, VA 20172, USA
Phone: 800-546-2416; 703-661-1593
Fax: 703-661-1501
E-mail: [email protected]
Online: http://www.asmscience.org

Cover: Neisseria gonorrhoeae bacteria, TEM. Credit: Dr Linda Stannard, UCT/Science Photo Library.
Contents

Contributors ix
Preface xv
Acknowledgments xvii

I. BACTERIAL-HOST INTERFACE
Section Editor: Nancy A. Cornick
1 Evolution of Bacterial Pathogens Within the Human Host 3
Kimberly A. Bliven and Anthony T. Maurelli
2 Bacterial Metabolism Shapes the Host–Pathogen Interface 15
Karla D. Passalacqua, Marie-Eve Charbonneau, and Mary X.D. O’Riordan
3 Iron Acquisition Strategies of Bacterial Pathogens 43
Jessica R. Sheldon, Holly A. Laakso, and David E. Heinrichs

II. BACTERIAL COMMUNICATION AND VIRULENCE

Section Editor: Paul J. Plummer
4 Sociomicrobiology and Pathogenic Bacteria 89
Joao B. Xavier
5 Candida-Bacteria Interactions: Their Impact on Human Disease 103
Devon L. Allison, Hubertine M. E. Willems, J.A.M.S. Jayatilake, Vincent M. Bruno,
Brian M. Peters, and Mark E. Shirtliff
6 Microbial Endocrinology in the Pathogenesis of Infectious Disease 137
Mark Lyte
7 Small RNAs in Bacterial Virulence and Communication 169
Sarah L. Svensson and Cynthia M. Sharma

III. BACTERIAL SECRETION SYSTEMS

Section Editor: Indira T. Kudva
8 Bacterial Secretion Systems: An Overview 215
Erin R. Green and Joan Mecsas
9 The Structure and Function of Type III Secretion Systems 241
Ryan Q. Notti and C. Erec Stebbins
10 Mechanism and Function of Type IV Secretion During Infection
of the Human Host 265
Christian Gonzalez-Rivera, Minny Bhatty, and Peter J. Christie

v
vi  contents

11 Type V Secretion Systems in Bacteria 305

Enguo Fan, Nandini Chauhan, D. B. R. K. Gupta Udatha,
Jack C. Leo, and Dirk Linke
12 The Versatile Type VI Secretion System 337
Christopher J. Alteri and Harry L.T. Mobley
13 Type VII Secretion: A Highly Versatile Secretion System 357
Louis S. Ates, Edith N. G. Houben, and Wilbert Bitter

IV. BACTERIAL DEFENSES

Section Editor: Qijing Zhang
14 Stress Responses, Adaptation, and Virulence of Bacterial Pathogens
During Host Gastrointestinal Colonization 387
Annika Flint, James Butcher, and Alain Stintzi
15 Bacterial Evasion of Host Antimicrobial Peptide Defenses 413
Jason N. Cole and Victor Nizet
16 Antigenic Variation in Bacterial Pathogens 445
Guy H. Palmer, Troy Bankhead, and H. Steven Seifert
17 Mechanisms of Antibiotic Resistance 481
Jose M. Munita and Cesar A. Arias

V. BACTERIAL PERSISTENCE: WITHIN AND BETWEEN HOSTS

Section Editor: Tracy L. Nicholson
18 Chronic Bacterial Pathogens: Mechanisms of Persistence 515
Mariana X. Byndloss and Renee M. Tsolis
19 The Staphylococcal Biofilm: Adhesins, Regulation,
and Host Response 529
Alexandra E. Paharik and Alexander R. Horswill
20 Surviving Between Hosts: Sporulation and Transmission 567
Michelle C. Swick, Theresa M. Koehler, and Adam Driks
21 Staying Alive: Vibrio cholerae’s Cycle of Environmental Survival,
Transmission, and Dissemination 593
Jenna G. Conner, Jennifer K. Teschler, Christopher J. Jones,
and Fitnat H. Yildiz

VI. HOST CELL ENSLAVEMENT BY INTRACELLULAR BACTERIA

Section Editor: John P. Bannantine
22 Hijacking and Use of Host Lipids by Intracellular Pathogens 637
Alvaro Toledo and Jorge L. Benach
23 Contrasting Lifestyles Within the Host Cell 667
Elizabeth Di Russo Case and James E. Samuel
 contents vii

24 Intracellular Growth of Bacterial Pathogens: The Role of Secreted

Effector Proteins in the Control of Phagocytosed Microorganisms 693
Valérie Poirier and Yossef Av-Gay
25 Cellular Exit Strategies of Intracellular Bacteria 715
Kevin Hybiske and Richard Stephens

VII. TARGETED THERAPIES

Section Editor: Bryan H. Bellaire
26 Novel Targets of Antimicrobial Therapies 741
Sarah E. Maddocks
27 Innovative Solutions to Sticky Situations: Antiadhesive Strategies for
Treating Bacterial Infections 753
Zachary T. Cusumano, Roger D. Klein, and Scott J. Hultgren
28 The Role of Biotin in Bacterial Physiology and Virulence: a Novel
Antibiotic Target for Mycobacterium tuberculosis 797
Wanisa Salaemae, Grant W. Booker, and Steven W. Polyak
29 Recent Advances and Understanding of Using Probiotic-Based
Interventions to Restore Homeostasis of the Microbiome for the
Prevention/Therapy of Bacterial Diseases 823
Jan S. Suchodolski and Albert E. Jergens

Index 843
Contributors

Devon L. Allison
Graduate Program in Life Sciences, Molecular Microbiology and Immunology,
University of Maryland-Baltimore; Department of Microbial Pathogenesis,
University of Maryland-Baltimore, Dental School, Baltimore, MD 21201
Christopher J. Alteri
Department of Microbiology and Immunology, University of Michigan
Medical School, Ann Arbor, MI 48109
Cesar A. Arias
Department of Internal Medicine, Division of Infectious Diseases,
University of Texas Medical School at Houston, Houston, TX 77030;
International Center for Microbial Genomics; Molecular Genetics and
Antimicrobial Resistance Unit, Universidad El Bosque, Bogota, Colombia
Louis S. Ates
Department of Medical Microbiology and Infection Control, VU University
Medical Center, Amsterdam, The Netherlands
Yossef Av-Gay
Division of Infectious Diseases, Department of Medicine, University of British
Columbia, Vancouver, BC V6H 3Z6 Canada
Troy Bankhead
Department of Veterinary Microbiology and Pathology, Paul G. Allen School
for Global Animal Health, Washington State University, Pullman, WA 99164
Jorge L. Benach
Department of Molecular Genetics and Microbiology, Stony Brook University,
Center for Infectious Diseases at the Center for Molecular Medicine, Stony Brook,
NY 11794
Minny Bhatty
Department of Microbiology and Molecular Genetics, University of Texas
Medical School at Houston, Houston, TX 77030
Wilbert Bitter
Department of Medical Microbiology and Infection Control, VU University
Medical Center; Section Molecular Microbiology, Amsterdam Institute of
Molecules, Medicine and Systems, Vrije Universiteit Amsterdam, 1081 BT
Amsterdam, The Netherlands

ix
x  contributors

Kimberly A. Bliven
Department of Microbiology and Immunology, F. Edward Hébert School of
Medicine, Uniformed Services University of the Health Sciences, Bethesda,
MD 20814
Grant W. Booker
Department of Molecular and Cellular Biology, School of Biological Science;
Center for Molecular Pathology, The University of Adelaide, North Terrace
Campus, Adelaide, South Australia 5005, Australia
Vincent M. Bruno
The Institute for Genomic Sciences; Department of Microbiology and
Immunology, School of Medicine, University of Maryland-Baltimore, Baltimore,
MD 21201
James Butcher
Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology
and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario,
Canada K1H 8M5
Mariana X. Byndloss
Department of Medical Microbiology and Immunology, School of Medicine,
University of California at Davis, Davis, CA 95616
Elizabeth Di Russo Case
Department of Microbial Pathogenesis and Immunology, College of Medicine,
Texas A&M Health Sciences Center, Bryan, TX 77807
Marie-Eve Charbonneau
Department of Microbiology and Immunology, University of Michigan Medical
School, Ann Arbor, MI 48109
Nandini Chauhan
Department of Biosciences, University of Oslo, Blindern, 0316 Oslo, Norway
Peter J. Christie
Department of Microbiology and Molecular Genetics, University of Texas
Medical School at Houston, Houston, TX 77030
Jason N. Cole
Department of Pediatrics, University of California San Diego, La Jolla,
CA 92093; School of Chemistry and Molecular Biosciences, Australian
Infectious Diseases Research Center, University of Queensland, St Lucia,
Queensland 4072, Australia
Jenna G. Conner
Microbiology and Environmental Toxicology, University of California Santa
Cruz, Santa Cruz, CA 95064
Zachary T. Cusumano
Department of Molecular Microbiology, Washington University
School of Medicine, St. Louis, MO 63110
 contributors xi

Adam Driks
Loyola University Chicago, Stritch School of Medicine, Maywood, IL 60153
Enguo Fan
Institute of Biochemistry and Molecular Biology, University of Freiburg,
Freiburg D-79104, Germany
Annika Flint
Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology
and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario,
Canada K1H 8M5
Christian Gonzalez-Rivera
Department of Microbiology and Molecular Genetics, University of Texas
Medical School at Houston, Houston, TX 77030
Erin R. Green
Program in Molecular Microbiology, Sackler School of Graduate Biomedical
Sciences, Tufts University, Boston, MA 02111
David E. Heinrichs
Department of Microbiology and Immunology, University of Western Ontario,
London, Ontario, Canada N6A 5C1
Alexander R. Horswill
Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine,
University of Iowa, Iowa City, IA 52242
Edith N. G. Houben
Section Molecular Microbiology, Amsterdam Institute of Molecules,
Medicine and Systems, Vrije Universiteit Amsterdam, 1081 BT Amsterdam,
The Netherlands
Scott J. Hultgren
Department of Molecular Microbiology, Washington University School of
Medicine, St. Louis, MO 63110
Kevin Hybiske
Division of Allergy and Infectious Diseases, Department of Medicine,
University of Washington, Seattle, WA 98195
J.A.M.S. Jayatilake
Department of Oral Medicine and Periodontology, Faculty of Dental Sciences,
University of Peradeniya, Sri Lanka
Albert E. Jergens
Department of Veterinary Clinical Sciences, College of Veterinary Medicine,
Iowa State University, Ames, IA 50010
Christopher J. Jones
Microbiology and Environmental Toxicology, University of California
Santa Cruz, Santa Cruz, CA 95064
xii  contributors

Roger D. Klein
Department of Molecular Microbiology, Washington University School
of Medicine, St. Louis, MO 63110
Theresa M. Koehler
University of Texas Medical School at Houston, Houston, TX 77030
Holly A. Laakso
Department of Microbiology and Immunology, University of Western Ontario,
London, Ontario, Canada N6A 5C1
Jack C. Leo
Department of Biosciences, University of Oslo, Blindern, 0316 Oslo, Norway
Dirk Linke
Department of Biosciences, University of Oslo, Blindern, 0316 Oslo, Norway
Mark Lyte
Department of Veterinary Microbiology and Preventive Medicine,
College of Veterinary Medicine, Iowa State University, Ames, IA 50011
Sarah E. Maddocks
Department of Biomedical Sciences, Cardiff School of Health Sciences,
Cardiff Metropolitan University, Western Avenue, Llandaff, Wales, CF5 2YB
Anthony T. Maurelli
Department of Microbiology and Immunology, F. Edward Hébert School of
Medicine, Uniformed Services University of the Health Sciences, Bethesda,
MD 20814
Joan Mecsas
Program in Molecular Microbiology, Sackler School of Graduate Biomedical
Sciences; Department of Molecular Biology and Microbiology, Tufts University
School of Medicine, Boston, MA 02111
Harry L.T. Mobley
Department of Microbiology and Immunology, University of Michigan Medical
School, Ann Arbor, MI 48109
Jose M. Munita
Department of Internal Medicine, Division of Infectious Diseases, University of
Texas Medical School at Houston, Houston, TX 77030; International Center for
Microbial Genomics; Clinica Alemana de Santiago, Universidad del Desarrollo
School of Medicine, Santiago, Chile
Victor Nizet
Department of Pediatrics; Skaggs School of Pharmacy and Pharmaceutical
Sciences; Center for Immunity, Infection & Inflammation, University of
California San Diego, La Jolla, CA 92093
Ryan Q. Notti
Laboratory of Structural Microbiology, Rockefeller University, New York,
NY 10065; Tri-Institutional Medical Scientist Training Program, Weill Cornell
Medical College, New York, NY 10021
 contributors xiii

Mary X.D. O’Riordan

Department of Microbiology and Immunology, University of Michigan Medical
School, Ann Arbor, MI 48109
Alexandra E. Paharik
Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine,
University of Iowa, Iowa City, IA 52242
Guy H. Palmer
Department of Veterinary Microbiology and Pathology, Paul G. Allen School for
Global Animal Health, Washington State University, Pullman, WA 99164
Karla D. Passalacqua
Department of Microbiology and Immunology, University of Michigan Medical
School, Ann Arbor, MI 48109
Brian M. Peters
Department of Clinical Pharmacy, University of Tennessee Health Science
Center, Memphis, TN 38103
Valérie Poirier
Division of Infectious Diseases, Department of Medicine, University of British
Columbia, Vancouver, BC V6H 3Z6
Steven W. Polyak
Department of Molecular and Cellular Biology, School of Biological Science;
Center for Molecular Pathology, The University of Adelaide, North Terrace
Campus, Adelaide, South Australia 5005, Australia
Wanisa Salaemae
Department of Molecular and Cellular Biology, School of Biological Science,
The University of Adelaide, North Terrace Campus, Adelaide, South Australia
5005, Australia
James E. Samuel
Department of Microbial Pathogenesis and Immunology, College of Medicine,
Texas A&M Health Sciences Center, Bryan, TX 77807
H. Steven Seifert
Department of Microbiology-Immunology, Feinberg School of Medicine,
Northwestern University, Chicago, IL 60611
Cynthia M. Sharma
Research Center for Infectious Diseases (ZINF) University of Würzburg,
Würzburg, Germany 97080
Jessica R. Sheldon
Department of Microbiology and Immunology, University of Western Ontario,
London, Ontario, Canada N6A 5C1
Mark E. Shirtliff
Department of Microbial Pathogenesis, University of Maryland-Baltimore,
Dental School; The Institute for Genomic Sciences, School of Medicine,
University of Maryland-Baltimore, Baltimore, MD 21201
xiv  contributors

C. Erec Stebbins
Laboratory of Structural Microbiology, Rockefeller University, New York,
NY 10065
Richard Stephens
Program in Infectious Diseases, School of Public Health, University of
California, Berkeley, Berkeley, CA 94720
Alain Stintzi
Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology
and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario,
Canada K1H 8M5
Jan S. Suchodolski
Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences,
College of Veterinary Medicine and Biomedical Sciences, Texas A&M
University, College Station, TX 77845
Sarah L. Svensson
Research Center for Infectious Diseases (ZINF) University of Würzburg,
Würzburg, Germany 97080
Michelle C. Swick
University of Texas Medical School at Houston, Houston, TX 77030
Jennifer K. Teschler
Microbiology and Environmental Toxicology, University of California
Santa Cruz, Santa Cruz, CA 95064
Alvaro Toledo
Department of Molecular Genetics and Microbiology, Stony Brook University,
Center for Infectious Diseases at the Center for Molecular Medicine,
Stony Brook, NY 11794
Renee M. Tsolis
Department of Medical Microbiology and Immunology, School of Medicine,
University of California at Davis, Davis, CA 95616
D. B. R. K. Gupta Udatha
Department of Biosciences, University of Oslo, Blindern, 0316 Oslo, Norway
Hubertine M. E. Willems
Department of Clinical Pharmacy, University of Tennessee Health Science
Center, Memphis, TN 38103
Joao B. Xavier
Program for Computational Biology, Memorial Sloan Kettering Cancer Center,
New York, NY 10065
Fitnat H. Yildiz
Microbiology and Environmental Toxicology, University of California Santa
Cruz, Santa Cruz, CA 95064
Preface

“Generation of new ideas and refinement or extension of established concepts are

the essence of advances in knowledge”: Dr. Carlton Gyles, Preface, Virulence Mech-
anisms of Bacterial Pathogens, 1st Edition, 1988, ASM Press. This was the driving
force behind the current fifth edition of this monograph, which essentially is a
compilation of bacterial virulence strategies and cutting-edge therapies (target-
ing these strategies) that have been unraveled in recent years and/or provide new
insights into established dogmas.
Previous editions of this book always provided interesting and timely infor-
mation on topics not always covered in textbooks making them reliable reference
sources. Traditionally these were published as follow-up to a series of Interna-
tional Symposia on Virulence Mechanisms of Bacterial Pathogens, held in Ames,
Iowa in 1987, 1994, 1999, and 2006. Hence, the last edition was published in 2007.
With all the scientific advancements made in the area of bacterial pathogenesis
since then, there was a pressing need for a more recent, updated version of this
book. To make up for the lapsed time and the inevitable financial constraints,
a general consensus was reached to initiate the publication sans a symposium.
This turned out to be quite an insightful decision as it enabled the editors and all
contributors to provide their undivided attention to weaving together a harmo-
nious and comprehensive monograph.
Sections in this edition have been organized in a systematic manner keeping in
sync with the journey a pathogen undertakes in its host. Therefore, these sections
discuss, key events occurring at the bacterial-host interface (section I) that enable
colonization, bacterial reliance on communication (section II) and secretion (sec-
tion III) to initiate/enhance virulence, bacterial defense (section IV), persistence
(section V), and host-exploitation strategies (section VI) that allow for extended
survival in the host. The concluding section (section VII) discusses novel thera-
peutic approaches being developed to target some of these virulence mechanisms.
It was our intent to deliver the science through this monograph and allow
our savvy readers the luxury of philosophizing. As such, we sought contributions
from distinguished experts, whether as authors or reviewers, making this mono-
graph a one-stop learning tool for recent advances made in the field of bacterial
virulence while stepping away from being just another “textbook”. The contents
were selected to be beneficial to diverse readership (students, faculty, scientists
in academic, clinical, corporate and/or government settings) while promoting
discussion, extrapolation, exploration and multi-dimensional thinking.

Indira T. Kudva
Executive Editor
xv
Acknowledgments

The Section Editors acknowledge the timely contributions made by all the
authors and the tremendous support provided by:
ASM Press
Editors, Microbiology Spectrum
Reviewers:
Keith M. Derbyshire, PhD
Director, Division of Genetics,
Center for Medical Science, Wadsworth Center
Professor, Biomedical Sciences, University at Albany
NYSDOH
Albany, NY 12201
Cammie Lesser, MD, PhD
Associate Professor Medicine (Microbiology and Immunobiology)
Massachusetts General Hospital/Harvard Medical School
Cambridge, MA 02139
Joseph D. Mougous, PhD
Associate Professor
Department of Microbiology
University of Washington
Seattle, WA 98195
Joseph P. Vogel, PhD
Associate Professor
Department of Molecular Microbiology
Washington University
St. Louis, MO 63110
Timothy L. Yahr, PhD
Director of Graduate Studies
Professor of Microbiology
University of Iowa, Carver College of Medicine
Iowa City, IA 52242

xvii
BACTERIAL-HOST INTERFACE
Evolution of Bacterial Pathogens
Within the Human Host

KIMBERLY A. BLIVEN1 and ANTHONY T. MAURELLI1

1
INTRODUCTION

The success or failure of a pathogen is entirely dependent on its ability to

survive, reproduce, and spread to a new host or environment. Host immune
systems, predators, microbial competitors, parasites, and environmental
resource limitations all exert selective pressures that shape the genomes
of microbial populations (1). Host fitness, meanwhile, is determined by the
ability of the host to survive and reproduce; the host must therefore effec-
tively curtail diseases that impair either of these abilities.
Dawkins and Krebs suggest that the conflicting drives between host and
pathogen have led to an evolutionary “arms race,” where an asymmetric
“attack-defense” strategy has come into play (2). At the basic level, this
concept suggests that when the host evolves new defenses to thwart the
pathogen’s attack, the pathogen is forced to adapt a more effective attack
strategy to penetrate the heightened defenses. In response, the host must
once again evolve to cope with the new attack mechanism, and the cycle
continues. Evolutionarily fit pathogens, which are able to survive, repli-
cate, and spread effectively within the host, have the most likely chance

1
Department of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services
University of the Health Sciences, Bethesda, MD 20814.
Virulence Mechanisms of Bacterial Pathogens, 5th edition
Edited by Indira T. Kudva, Nancy A. Cornick, Paul J. Plummer, Qijing Zhang, Tracy L. Nicholson,
John P. Bannantine, and Bryan H. Bellaire
© 2016 American Society for Microbiology, Washington, DC
doi:10.1128/microbiolspec.VMBF-0017-2015

3
4 BLIVEN AND MAURELLI

of passing their genes on to the next gen- about host and microbial biology or is the
eration. Similarly, host genotypes are more result of artificial laboratory-induced evo-
likely to persist within the population if lution during serial passaging of bacterial
those particular individuals are more ca- strains. Due to the sheer enormity of evo-
pable of controlling or resisting infection. lutionary timescales, defining the pre-
Evolution, therefore, is driven by positive cise origins of and factors driving natural
directional selection in the arms race model; evolutionary events is often a difficult
eventually, the most beneficial alleles will be- undertaking.
come fixed in a population. Another model
favors frequency-dependent (balancing) se-
lection, a process that maintains rare alleles ANTAGONISTIC PLEIOTROPY AND THE
and therefore preserves polymorphic diver- FITNESS COST-BENEFIT ANALYSIS
sity within a population (3). Simply put, allele
fixation is prevented in certain instances At the most basic level, the theory of natural
because different bacterial alleles confer dis- selection stipulates that within a bacterial
tinct advantages to the pathogen in the pres- population, beneficial traits will be con-
ence of different host alleles (i.e., different served (selected for) and deleterious traits
environments). Supporting evidence for both eventually discarded (selected against). The
directional and frequency-dependent selec- actual evolutionary process is considerably
tion can be found within nature, and both more complex, however, due to the exis-
types probably occur in bacterial populations. tence of genetic drift (the change in genetic
In this chapter, we explore the host- diversity of a population due to random
pathogen interface and offer examples of chance) and antagonistic pleiotropy.
pathogen adaptation in response to common Antagonistic pleiotropy is the concept
host selective pressures (Table 1). Although that a single gene may control more than
we will focus exclusively on bacterial patho- one phenotype, some of which may be bene-
gens within the human host, many of the ficial to the organism and some deleterious
concepts discussed in this review are readily (7). Therefore, a gene may confer a selective
applicable to other organisms, such as viruses, advantage within one particular environ-
parasites, and fungi, which can infect a wide ment, but its expression could be detrimental
range of hosts including plants, animals, and within a different environment. Conserva-
amoeba (4–6). tion of this gene ultimately is determined
As a final note, much of the evidence by the overall necessity of the gene to the
presented here to support presumed evolu- organism’s fitness and the timing of selection.
tionary events is either speculation based Bacterial pathogens may evolve mechanisms
on what is currently known or suspected to neutralize the deleterious effects arising

TABLE 1 Examples of pathogenic mechanisms to evade or overcome selective pressures within the
human host
Selective pressures Pathogenic mechanisms to evade or overcome these pressures

Physical barriers in host (i.e., mucosal epithelium) Mucinases Enterotoxins Exfoliative toxins Transcytosis through
M cells
Host complement Complement inhibitor protein C3 protease
Sequestration of host resources (e.g., iron) Enterobactin/aerobactin systems
Host B and T cell lymphocytes Cytotoxins T3SS-mediated apoptosis
Antibiotics, antimicrobial peptides Efflux pumps Mutations in antimicrobial targets Enzymes to
inactivate antibiotics (e.g., beta-lactamases)
Bacterial colicins Colicin immunity proteins
Bacterial T6SSs T6S immunity proteins
 CHAPTER 1 • Evolution of Bacterial Pathogens Within the Human Host 5

from antagonistic pleiotropy, while at the by the host, because patients infected with
same time conserving the beneficial ones. P. aeruginosa develop antibodies against T3SS
Temporal regulation is a powerful tool to effector proteins; conversely, biofilm produc-
ensure that specific genes are only turned on tion likely allows for the persistence of the
when required and are turned off to prevent organism in the respiratory tract (11).
detrimental expression within a particular Finally, certain bacteria simply tolerate
environment. Certain outer membrane pro- deleterious fitness costs if the benefits of
teins or systems are temporally regulated expressing the gene outweigh the negative
within the host, because they may provide a effects. Antibiotic-resistance mutations that
marker for recognition by the host immune allow bacteria to survive exposure to anti-
system. Flagellar expression, for example, is microbials often come with a significant
downregulated by Salmonella enterica sero- fitness disadvantage, for example, and sec-
var Typhi in vivo to prevent activation of ondary compensatory mutations in these
the host inflammatory response; however, strains may eventually arise to restore fit-
outside the host, motility is likely important ness rather than lose resistance (12).
for the bacterium to seek out and scavenge
nutrients from the environment (8).
Other bacteria avoid the deleterious effects THE IMPACT OF HOST-PATHOGEN
of a gene through gene inactivation; mutants INTERACTIONS ON
that lose functionality of the gene once it MICROBIAL EVOLUTION
becomes deleterious can out-compete the
wild-type parent strain, and eventually these Inside the host, a successful pathogen will
mutants will dominate the population. Pseu- pilfer resources to survive, replicate, and
domonas aeruginosa, an opportunistic patho- eventually escape; concomitantly, the host
gen of cystic fibrosis patients, often switches will attempt to recognize and subsequently
to a mucoid phenotype in vivo as a result rid the body of the intruder. Coevolution be-
of overproduction of the exopolysaccharide tween host and pathogen naturally occurs as
alginate, which allows for the production of a a result of these interactions (13). For prac-
bacterial biofilm in the lung (9, 10). MucA tical purposes, we restrict our discussion to
is a P. aeruginosa transmembrane protein that bacterial adaptation within the human host,
binds to and represses the sigma factor AlgU, but it is important to recognize that many
which acts as the transcriptional activator of of these concepts are applicable to patho-
the alginate synthesis operon. AlgU activates gens of other hosts as well, such as plants and
AlgR, a suppressor of type III secretion sys- amoeba (14–16). As novel genetic variants
tem (T3SS) expression; when mucA is ex- within the human population emerge which
pressed, therefore, so are the T3SS genes. prove more successful at preventing or over-
During acute infection, the T3SS plays an coming infection, only pathogen variants
essential role in establishment of the bac- that allow the bacteria to surmount or avoid
terium within the respiratory tract. Once in- this new response will be successful. Within
fection has been established, however, chronic the last century, these natural host defenses,
infection appears to favor loss of T3SS and a which take much longer to evolve than their
switch to biofilm production (11). Both of microbial counterparts, have been supple-
these phenotypes are at least partially driven mented by man-made developments, such
by various mutations in mucA which lead to as antibiotics and modern medical inter-
derepression of AlgU, subsequent production ventions, which place added pressures on
of alginate, and suppression of the T3SS (9). microbes to adapt (17). Host innate and adap-
Hauser speculates that loss of the T3SS pro- tive immune responses and modern medical
tects the bacterium from eventual recognition interventions are all selective pressures that
6 BLIVEN AND MAURELLI

contribute to pathogen evolution within the prokaryotes are thought to colonize the
human host. Furthermore, microbial com- entire skin surface of the human adult (20).
petition, against either other pathogens or Consequently, bacteria that exploit more
commensal bacteria, also shapes pathogen hostile and less frequently occupied niches
genomes. may gain a selective edge in survival by
Bacteria have several advantages over avoiding sites of high competition. Natural
the human host when it comes to evolution: structural barriers, however, typically pre-
first, their generation times are significantly vent pathogens from engaging deeper host
shorter, leading to more rapid selection tissues. Physical blocks to infection include
within a population. In conjunction with a the intestinal and respiratory mucosa, the
shorter generation time, bacterial popula- blood-brain barrier, the blood–cerebral spi-
tions are typically larger, which may allow nal fluid barrier, and the placental barrier
for greater genetic diversity from which to (21). Most of these structures consist of a
select. Lastly, many bacteria utilize hori- single layer of epithelial or endothelial cells
zontal gene transfer (HGT), which accounts bound closely together by tight junctions,
for the rapid spread of advantageous alleles adherens junctions, and desmosomes, which
between strains or even species (18). Viru- preclude bacteria from passively crossing
lence genes are commonly located on trans- (21, 22). Gastric and respiratory epithelia
ferred pathogenicity islands (PAIs), which support an additional protective coating of
are segments of the genome associated with mucus, which consists primarily of mucin
mobility elements, such as integrase genes glycoproteins and antimicrobial molecules
or transposons. PAIs can often be distin- (23). Mucin glycoproteins, produced by epi-
guished from the remainder of the genome thelial goblet cells and submucosal glands,
by a disparate G+C content (19). can either remain cell-associated or undergo
secretion into the mucosa, where they con-
tribute to the viscous layer of mucus that can
Host Selective Pressures: The Innate
effectively trap microbes (24). Additionally,
and Adaptive Immune Systems
nonspecific antimicrobials, such as defensins
The innate immune system is one of the first and lysozymes, and specific antimicrobials,
challenges encountered by the incoming such as IgG and secretory IgA, also limit the
pathogen following host contact. These di- growth of microbes within the mucosa (23).
verse host defenses include physical barriers Bacterial pathogens have developed numer-
such as the mucosal epithelium, activation ous mechanisms to counteract these defenses.
of the complement cascade, circulating anti- The mucosal barrier can be broken down
microbial peptides and cytokines, leukocytes, by mucinases such as the Pic enzyme of Shi-
activation of the adaptive immune system, gella and enteroaggregative Escherichia coli
and sequestration of host nutrients away (EAEC) (25, 26). The pic gene is located on a
from pathogenic bacteria. In addition to chromosomal pathogenicity island in Shi-
effective evasion of innate immune mecha- gella and flanked by insertion-like elements
nisms, bacteria must also prevent or avoid in EAEC, indicating a history of horizontal
adaptive immune responses, which include B gene transfer in these pathogens (26). This
cell antibody production and T cell–mediated potential gene transfer is intriguing because
cytotoxicity. Pathogenic bacteria have evolved mucin degradation is also important for cer-
different approaches to overcome these host tain gastrointestinal commensals, which me-
defenses. tabolize mucin glycoproteins for energy (27).
In the human colon alone, intestinal mi- It is tempting to speculate that these enzymes
crobiota concentrations average 1011 micro- first evolved within human commensal bac-
organisms per gram gut content, while 3 × 108 teria as a means of nutrient acquisition and
 CHAPTER 1 • Evolution of Bacterial Pathogens Within the Human Host 7

only later spread to emerging pathogens to (or gut-associated lymphoid tissue), enteric
confer passage through the mucosal surface. bacteria transcytosed through microfold
Such a concept would support the hypothesis cells must then contend with macrophages,
proposed by Rasko et al., who suggest that T lymphocytes, B lymphocytes, and dendritic
commensal E. coli acts as a “genetic sink” for cells.
pathogenic E. coli isolates (28). Other patho- As a putative example of counterevolution,
gens, such as Yersinia enterocolitica and the human host may have developed mech-
Vibrio cholerae, avoid the thickest layers of anisms to avoid bacterial-mediated adhesion
the mucosal layer by targeting microfold processes. Helicobacter pylori binds to the ad-
cells within the small intestine for uptake hesion decoy Muc1, a mucin expressed on the
(23, 29). These specialized epithelial cells surface of epithelial cells in the gastrointes-
sample microorganisms residing in the intes- tinal tract (36). Muc1 is subsequently shed
tinal lumen and present them to immune from the epithelial surface along with cou-
cells in the underlying lymphoid tissue. pled bacteria, precluding long-term adhesion.
Microfold cells are situated in the region of Consequently, wild type mice have a 5-fold
the epithelium known as the dome, which lower H. pylori colonization burden than
lacks mucin-secreting goblet cells (23). Muc1-/- mice. Furthermore, human epidemi-
Next, to breach the epithelial/endothelial ological studies have linked shorter Muc1
barrier, pathogens must either actively cross alleles to a higher probability of chronic gas-
using microbial-mediated processes or oppor- tritis progression, indicating that longer
tunistically cross following disruption Muc1 alleles may confer a protective advan-
of barrier integrity. Some pathogens, such as tage to the host (37). Polymorphisms between
Bacteroides fragilis and Staphylococcus aureus, human Muc1 alleles are largely restricted
directly break cell-cell junctions (30, 31). to the extracellular domain, which consists
B. fragilis, an opportunistic pathogen, encodes of a region of 30 to 90 tandem repeat units
a zinc-dependent metalloprotease toxin, BFT rich in serine and threonine. A study by Costa
(B. fragilis enterotoxin), which cleaves the et al., demonstrated a significant positive
extracellular domain of E-cadherin, a host association between the number of Muc1
zonula adherens protein (30). Like the pic tandem repeats and bacterial adherence for
genes of Shigella and EAEC, the bft gene is two strains of H. pylori in vitro (38). Longer
carried on a PAI present in all enterotoxi- Muc1 alleles probably evolved from shorter
genic B. fragilis strains (32). S. aureus induces alleles via duplication events and may have
bullous impetigo and staphylococcal scalded emerged to protect against pathogens such as
skin syndrome through the actions of three H. pylori (39).
exfoliative toxins (ETs): ETA, ETB, and ETD Complement cascade activation via the
(31). The ETs act as serine proteases which classical, lectin, and alternative pathways pre-
cleave human desmoglein 1, a transmembrane cedes the cleavage of C3 convertase into C3a,
protein of desmosomes. The genes encoding an anaphylatoxin, and C3b, which binds to
these toxins are carried on different mobile the surface of microbes (otherwise known as
genetic elements: the ETA gene is carried by a opsonization) to promote the eventual clear-
family of Sa1int phages; the ETB gene is ance of bacteria through phagocytosis. Addi-
plasmid-encoded; and the ETD gene localizes tionally, C3 convertase may convert to the
to a 9-kB PAI (33, 34). Other pathogens, such lytic C5 convertase through addition of a C3b
as Shigella, Salmonella, and Listeria, trans- molecule. Pathogens have evolved mecha-
cytose through microfold cells in the gut nisms to evade or block these processes (40).
to gain access to the basolateral surface of The S. aureus staphylococcal complement in-
the intestinal epithelium (35). Because these hibitor protein stabilizes C3 convertase, pre-
specialized host cells overlay Peyer’s patches venting its cleavage into the active C3a and
8 BLIVEN AND MAURELLI

C3b fragments and attenuating anaphylatoxin Uropathogenic E. coli strains express the
activity and bacterial opsonization (41). Like siderophore salmochelin, a glycosylated form
many of the previously described pathoge- of enterobactin resistant to the effects of
nicity factors, the gene encoding staphylo- NGAL (47).
coccal complement inhibitor (scn) is located Finally, if a pathogen manages to evade the
on a PAI (42). Rather than preventing C3 innate immune system and can successfully
cleavage, the Neisseria meningitidis serine compete with commensal bacteria, it must
protease NalP splits C3 at a unique site, gen- then elude host adaptive immune responses,
erating shorter C3a-like and longer C3b-like including B- and T-cell lymphocytes (48).
fragments (43). The C3b-like fragments are One bacterial strategy employed in this eva-
capable of binding N. meningitidis but are sion process inhibits lymphocyte prolifera-
rapidly degraded by host complement fac- tion. The VacA cytotoxin of H. pylori blocks the
tors H (fH) and I (fI). Although the activity activity of host calcineurin, leading to down-
of the C3a-like fragment has not been de- stream attenuation of interleukin-2 (IL-2) tran-
termined, this fragment lacks the conserved scription, a key mediator of T cell proliferation
C-terminal arginine residue found in wild (49). Alternatively, bacteria can avoid the
type C3a that is essential for activity, and adaptive immune response altogether by medi-
therefore this truncated version is likely ating lymphocyte cell death. For example,
inactive. Shigella induces B-cell apoptosis through the
A final example of an innate host selective actions of its T3SS (50).
pressure is the sequestration of host resources
or nutrients away from colonizing bacteria.
Host Selective Pressures:
Iron, an essential nutrient, is in short supply
Antibiotic Resistance
within the host, either sequestered away in
host cells or stored as a complex in hemoglo- The rise of adaptive antibiotic resistance in
bin, which is inaccessible to most microbes bacteria is perhaps one of the most intensely
(44). Correspondingly, pathogens have been studied examples of pathogen evolution in
forced to develop numerous mechanisms to response to a specific selective pressure(s)
scavenge host iron. Predictably, these systems (51). Blair et al. separated adaptive resistance
are often iron-regulated, and their genes mechanisms into three primary categories:
are expressed following bacterial exposure reduced drug permeability through altera-
to the low-iron environment of the human tions in the bacterial membrane or the de-
host. Certain surface-bound receptors can velopment of efflux pumps that quickly
recognize iron-bound complexes, such as expel antimicrobials; prevention of binding
heme, transferrin, or lactoferrin. Additionally, through mutation of antimicrobial targets;
secreted bacterial siderophores (aerobactin and the direct inactivation of antimicrobial
and enterobactin) steal iron away from host agents by specific enzymes (51). Well-
transferrin and lactoferrin. E. coli strains characterized efflux pumps include the mul-
can encode for both of these systems (45). tidrug exporters discovered in the common
Another putative example of arms race food-borne pathogens E. coli (ArcAB-TolC),
coevolution is the mammalian neutrophil S. enterica (EmrAB), and S. aureus (QacA/B,
gelatinase-associated lipoprotein (NGAL). NorA) (52). Linezolid, an oxazolidinone class
NGAL directly binds the catecholate-type antibiotic, binds the 23S rRNA subunit and
ferric siderophore complexed to iron, pre- blocks tRNA interactions with the A site to
venting bacterial iron sequestration and even- prevent peptide bond formation (53). Un-
tually exerting a bacteriostatic effect upon surprisingly, linezolid resistance in a num-
microbial populations (46). Some bacteria can ber of bacterial species has been linked to a
even bypass this defense mechanism, however. G2576T mutation in the 23S rRNA gene,
 CHAPTER 1 • Evolution of Bacterial Pathogens Within the Human Host 9

precluding linezolid binding at this site and (64, 65). Lastly, colicin M, a unique member
providing an example of Blair's second of the colicin family, blocks peptidoglycan
category of adaptive drug resistance (54, biosynthesis by degrading undecaprenyl
55). Finally, inactivating enzymes such as phosphate-linked peptidoglycan precursors.
beta-lactamases, aminoglycoside acyltrans- These lipid-anchored intermediates are crit-
ferases, and monooxygenases are responsi- ical for the transport of peptidoglycan sub-
ble for the hydrolysis, group transfer, or units across the cytoplasmic membrane (66,
oxidation of their respective antibiotics (56, 67). To protect their own population against
57). the harmful effects of these toxic peptides,
The rapid spread of antimicrobial resis- the producers of colicins must concomitantly
tance, and the rise of multidrug resistance, express immunity proteins, which block the
is often linked to the HGT dissemination action of their respective colicins. Immunity
of genes encoding these enzymes, because proteins of pore-forming colicins sit in the
many PAIs and plasmids have been shown inner membrane and block colicin insertion.
to carry one or more drug-resistance genes Nuclease colicin immunity proteins bind to
(58). Resistance adaptations often come with DNase or RNase colicins to prevent their en-
a fitness cost, however, which has been zymatic activity, and the immunity protein
demonstrated both in vivo and in vitro (59). Cmi binds colicin M to render it catalytically
inactive (61, 68). Competing bacteria can
acquire these immunity proteins via HGT,
Microbial Competition
providing protection against E. coli colicin
Competition between microbes undoubtedly toxicity. For example, Shigella, which does
plays a role in driving pathogen evolution, not produce the pore-forming colicin V,
although this aspect of microbial evolution nevertheless encodes an immunity protein
has not been widely studied and, except on its SHI-2 PAI, which protects against
for a few examples, is still only very poorly colicin V produced by strains of E. coli (69,
understood. Bacteria can directly eliminate 70).
potential rivals through use of toxic pep- The recently discovered T6SSs of Gram-
tides (bacteriocins) or through the utiliza- negative bacteria are responsible for the
tion of type six secretion systems (T6SSs) direct delivery of effector proteins into neigh-
(60, 61). boring eukaryotic or bacterial cells, resulting
Bacteriocins are toxic peptides produced in the death of host cells or the lysis of po-
by bacteria that can target and kill neighbor- tential microbial competitors (71). VgrG1,
ing microbes. Colicins, the most well-known an ADP-ribosyltransferase, is secreted from
members of this category, are produced by the Aeromonas hydrophila T6SS into host
strains of E. coli, although bacteriocins have cells, where it disrupts the actin cytoskeleton
been described in a wide variety of bacteria, and induces host cell apoptosis (72). Most of
including S. aureus, Pseudomonas pyogenes, the described T6SS effectors, however, have
Yersinia pestis, and Serratia marcescens (61, been shown to target other microbes. The
62). In E. coli, colicins exhibit a number of T6SS-exported proteins 1 and 3 (Tse1 and
different modes of action. Pore-forming coli- Tse3) of P. aeruginosa exhibit amidase and
cins, such as colicin A, can insert into the muramidase activity, respectively, against bac-
inner membranes of susceptible bacteria to terial peptidoglycan (73). P. aeruginosa also
create ion channels (63). Nuclease colicins, encodes type VI lipase effector (Tle) proteins,
such as colicins E9 and E3, translocate across which degrade the bacterial phospholipid
the outer and inner membranes of a sus- phosphatidylethanolamine (74). In Dickeya
ceptible bacterium to the cytoplasm, where dadantii, the Rhs (rearrangement hotspots)
they function as DNases (E9) or RNases (E3) proteins RhsA and RhsB are secreted through
10 BLIVEN AND MAURELLI

the T6SS and function as toxic endonucle- CITATION

ases in susceptible bacteria. While D. dadantii
Bliven KA, Maurelli AT. 2016. Evolution of
is a plant pathogen, the human pathogen
bacterial pathogens within the human host.
S. marcescens also expresses a T6SS-secreted
Microbiol Spectrum 4(1):VMBF-0017-2015.
Rhs-family protein, although its function is
unknown (75, 76). Similar to the colicin pro-
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Bacterial Metabolism Shapes
the Host–Pathogen Interface

KARLA D. PASSALACQUA, 1 MARIE-EVE CHARBONNEAU,1 and

MARY X.D. O’RIORDAN1
2
THE INTERFACE BETWEEN TWO ORGANISMS IS SHAPED
BY METABOLIC INTERACTIONS

Why do organisms “eat”? This core question drives the study of biochemistry—
specifically metabolism. The short answer to this seemingly simple question
is 2-fold: first, eating provides cells with the physical building blocks for the
generation of cellular components (i.e., growth of the physical cell; something
must come from something); second, eating is the way to extract energy to do
cellular work (i.e., powering the process of growth; work is never done for
free). These two processes—catabolism and anabolism—are inextricably
linked. The pathways of catabolism, such as glycolysis and the tricarboxylic
acid (TCA) cycle, which break down molecules for energy metabolism, also
branch off into anabolic pathways that generate building blocks for the cell.
Bacterial metabolism is dynamic and flexible, with different bacterial species
encoding different metabolic capacities within their genomes. Thus, the ca-
nonical TCA cycle may function fully in one bacterial species, while another
bacterium, missing a key enzyme of the cycle now uses the other TCA enzymes
in branched oxidative and reductive pathways. Moreover, depending on the
availability of carbon sources or oxygen, even if a bacterium encodes all of the
1
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109.
Virulence Mechanisms of Bacterial Pathogens, 5th edition
Edited by Indira T. Kudva, Nancy A. Cornick, Paul J. Plummer, Qijing Zhang, Tracy L. Nicholson,
John P. Bannantine, and Bryan H. Bellaire
© 2016 American Society for Microbiology, Washington, DC
doi:10.1128/microbiolspec.VMBF-0027-2015

15
16 PASSALACQUA, CHARBONNEAU, AND O’RIORDAN

enzymes for respiration, with its high-energy ogens maintain energy metabolism within
yield, the less energy-efficient but faster pro- the host environment; in other words, how
cess of fermentation may predominate. Thus, do bacterial pathogens uniquely go about the
the flexible metabolic space of rapidly evolving business of eating inside a host to meet their
bacterial genomes enables many different ways energy demands (1)? First, a brief note on
for bacteria to take advantage of nutrients in energy: it is worth noting that energy is not
complex environments for robust replication. a thing but, rather, a potential, and that the
The essential infrastructure of metabolism energy all cells work to acquire is the energy
is made more complex during bacterial in- that “lives” within the chemical bonds of
fection when one organism thrives by draw- what the cell eats. Ultimately, for cells to do
ing nutrients from the other. From a bacterial work, chemical bond energy from some food
perspective, the mammalian host is a vast source must be moved to a molecule that can
ecosystem, with some regions such as the be used directly to fuel cellular tasks—the
intestine heavily populated with competi- predominant, but not exclusive, form being
tors, while other niches are wide open for adenosine triphosphate (ATP). The idea that
exploitation. To appreciate how bacterial phosphate bond energy transfer is the way
metabolism shapes infection, it is important cells power most cellular work stands as a
to consider the localized host environment monumental paradigm shift in the natural
where the bacteria replicate, as well as the sciences (2, 3), ushering in the full eluci-
metabolic capacity of the pathogen. The host dation of “central metabolism,” the foun-
environment is not simply a source of food dational energy yielding pathways for all
for bacteria. Rather, host cells are constantly cellular life. A more intimate way of viewing
controlling their own metabolic function, central metabolism is as the process that best
using available nutrients and removing waste illustrates the close kinship of all cellular
products. In addition, host organisms actively life. Glycolysis shows us how we are related
survey their inner spaces for invading micro- to Escherichia coli. The TCA cycle unites us
organisms. Thus, bacterial pathogens must with our cousins, the fungi. And no matter
overcome constant pressure from the preda- what the genetic content, there is a common
tory immune system of the host. These de- need for ATP.
fining aspects of the host–pathogen interaction Thus, when considering the specific role
are drivers of disease, whether it is acute or of energy-yielding metabolism during bac-
chronic, inflammatory or silent, mild or deadly. terial infection, it is good to remember that
In this article, we consider three aspects of both the pathogen and the infected host are
metabolism in the host–pathogen interaction: engaging in nonstop, regulated central meta-
first, how bacteria within a host employ specific bolic activity, often competing for the same
modules of central metabolism to generate resources and usually trying to influence the
energy, second, how bacterial pathogens ex- behavior of the other. Here, we start by ex-
ploit the host for critical nutrients required for ploring how some bacterial pathogens con-
proliferation, and lastly, how these bacterial duct energy metabolism during infection.
invaders use metabolic tricks to sense their
environment and to evade host immunity.
What’s for Dinner Inside the Host?
Depending on where in the host an invading
BACTERIAL ENERGY METABOLISM bacterium takes up residence, the food sources
DURING INFECTION available within that niche will determine
whether that microbe can successfully estab-
This section focuses primarily on recent re- lish itself. Some pathogens, such as invasive
search that is revealing how bacterial path- streptococci, Salmonella enterica, and Brucella
 CHAPTER 2 • Bacterial Metabolism Shapes the Host–Pathogen Interface 17

abortus, can feed their “sweet tooth” during flexibility than other Gram-positive bacteria
infection by acquiring specific carbohydrates, (5, 6).
whereas the “low-carb” pathogen Mycobac- The phosphoenolpyruvate–sugar phos-
terium tuberculosis prefers to fuel itself with photransferase and carbohydrate catabolite
energy-rich fatty acids during a chronic in- repression systems also have expanded roles
fection that can last for many decades. Recent in the more serious and invasive Streptococ-
research has revealed some unique ways in cus pathogens, Streptococcus pneumoniae and
which pathogens go about feeding themselves Streptococcus pyogenes, by being involved in
during host colonization. controlling levels of virulence gene expres-
Pathogenic streptococci are a group of sion, such as the toxin streptolysin S (7–9).
Gram-positive bacteria that cause a range of Also, S. pneumoniae is able to use a wider
disease from mild (dental caries) to severe range of carbon sources for energy catabo-
(pneumonia and sepsis). These bacteria es- lism compared to other Gram-positive orga-
tablish themselves on extracellular surfaces nisms (10) and has the ability to obtain and
of the host, such as on the tissues of the hu- use complex carbohydrates that it is able to
man naso-pharynx or in dental biofilms. remove from host extracellular surfaces with
Here, they are positioned to be fed directly specialized enzymes (11–13). A comprehen-
by the host as the host feeds him- or herself sive review of streptococcal carbohydrate
or to acquire nutrients from the extracellu- utilization during pathogenesis can be found
lar surfaces of host tissues. Streptococcus elsewhere; these brief examples introduce
mutans, a major cause of dental caries, sets and highlight the themes that (i) the ability
itself apart from the resident, nonpathogenic of pathogens to exploit the host niche and
oral microbiota by having a large and flexible obtain preferred food sources for energy ca-
metabolic range with regard to nutrient ac- tabolism is an important aspect of bacterial
quisition. As the human diet has changed virulence, (ii) pathogens often set themselves
over time to become richer in carbohydrates apart from nonpathogens by having an ex-
(especially sucrose), so has the physiological panded and flexible capability for obtaining
capability of S. mutans evolved (4, 5). A study preferred energy sources within the site of
that compared ancient and modern bacterial infection, and (iii) virulence gene expression
communities of dental plaque suggests that is often intimately linked to the pathways of
pathogenic S. mutans has become a more carbon acquisition and energy metabolism.
common resident of the human oral cavity as Focusing in more closely on central me-
a result of this change in diet, especially in tabolism, it is essential to emphasize the key
modern, postindustrial times (4). One of the role played by glucose in energy-yielding
reasons for this coevolutionary success has catabolism. When considering energy metab-
been the expansion of the S. mutans genome olism via respiration, where glycolysis and
to contain a wide range of genes involved in the TCA cycle are both utilized, it is the
carbohydrate uptake, especially the EII per- simple sugar glucose that gives a fast and
meases of the phosphoenolpyruvate–sugar efficient energy yield in the form of ATP for
phosphotransferase system (PTS), the multi- host and pathogen alike. Thus, these path-
enzyme pathway of carbohydrate uptake ways are important for many bacterial path-
present in most bacteria. Also, the carbohy- ogens, such as Salmonella and Brucella, which
drate catabolite repression system, which have evolved to be able to feed their energy-
allows bacteria to regulate which carbohy- yielding pathways during infection, speci-
drates to use for energy catabolism by sensing fically with glucose “stolen” from the host
intracellular nutrient content, also appears (Fig. 1).
to be unique in S. mutans in that it has more The pathogenic streptococci previously
levels of carbohydrate catabolite repression mentioned reside at the surface of cell tissues
18 PASSALACQUA, CHARBONNEAU, AND O’RIORDAN

FIGURE 1 A simplified view of catabolic energy-yielding pathways in bacteria and points of relevance for
the indicated bacterial pathogens. The figure shows a simplified outline of some of the different
pathways for utilizing carbohydrates or fatty acids for the generation of ATP. These catabolic pathways
and the anabolic pathways that they feed are under extremely complex levels of control, which have
been studied mainly in noninfectious organisms (126). However, the unique metabolic strategies
employed by infectious bacteria are becoming more appreciated as important aspects of bacterial
pathogenesis (1). *ATP generated by substrate level phosphorylation; **ATP generated by oxidative
phosphorylation; solid lines, metabolic pathway; dashed lines, substrates that feed into catabolic
pathways and products generated by bacteria as a result of metabolism or required for metabolism;
dotted lines, main energy-yielding metabolites generated from pathways. Blue letters indicate
specific points of importance for the energy-yielding metabolism of select pathogens listed as
follows (see Table 1 for summary): (A) Pathogenic streptococci (Streptococcus mutans, Streptococcus
pyogenes, Streptococcus pneumoniae); (B) Salmonella enterica serovar Typhimurium; (C) Brucella abortus;
(D) Mycobacterium tuberculosis; (E) Clostridium difficile; (F) Enterohemorrhagic Escherichia coli; (G)
Aggregatibacter actinomycetemcomitans; (H) Listeria monocytogenes.
 CHAPTER 2 • Bacterial Metabolism Shapes the Host–Pathogen Interface 19

TABLE 1 Summary points of energy-yielding metabolism for select pathogenic bacteria

Select
Label Bacterium Points of interest for metabolism during infection references

A Pathogenic streptococci Extracellular pathogens 1–13

Streptococcus mutans Relies on simple and complex carbohydrates from host diet
Streptococcus pyogenes or from host cell surfaces
Streptococcus pneumoniae Genomes have expanded sugar uptake systems for dietary
flexibility
Can express enzymes that cleave host glycans for use in
energy-yielding metabolism
Sugar uptake and control is tightly linked with expression
of classic virulence factors
B Salmonella enterica Can infect both intracellularly and extracellularly 14, 15, 19,
serovar Typhimurium Intracellular infection requires glucose, glycolysis, 23, 44–47
and the TCA cycle
Intracellular bacteria can influence host cell gene expression
to favor glucose availability
Specific TCA cycle reactions that generate malate are required
for intracellular infection
Bacteria that establish extracellular infection in the gut can
engage in anaerobic respiration using resident metabolites
generated by the host during inflammation and by the
resident microbiota
C Brucella abortus Intracellular pathogen 21
Requires glucose for intracellular infection
Passively benefits from host cell metabolism for glucose
D Mycobacterium Intracellular pathogen 24, 27–29
tuberculosis Utilizes fatty acid oxidation for energy metabolism
Associates with lipid bodies in specialized cells where they
can acquire fatty acids
Glyoxylate cycle required for replenishment of TCA cycle
metabolites in the absence of glycolysis
E Clostridium difficile Extracellular pathogen and obligate anaerobe 33
Fermentation is main energy-yielding pathway
May outcompete the resident microbiota by engaging in
succinate to butyrate fermentation after antibiotic treatment
by taking advantage of a rise in succinate generated by the
gut microbiota
F Enterohemorrhagic Extracellular pathogen that can form “attaching and effacing” 34–36
Esherichia coli (EHEC) lesions in the host gut
May take advantage of colocalization with species of the
resident microbiota in the carbohydrate-poor intestinal
mucosal layer
Metabolites produced by colocalized microbiota may induce
shift from glycolysis to gluconeogenesis, increasing virulence
gene expression
G Aggregatibacter Extracellular pathogen of the oral cavity 38
actinomycetemcomitans Switches to L-lactate catabolism when colocalized with resident
microbiota generating L-lactate
Catabolic shift may favor bacterial expansion and increased
host damage
H Listeria monocytogenes Intracellular pathogen 40, 41
Specifically scavenges the essential cofactor lipoate from
host lipoyl-peptides
Host-derived lipoate essential for catabolic enzymes
20 PASSALACQUA, CHARBONNEAU, AND O’RIORDAN

where glucose concentration is low (12) cokinase), resulting in a total glucose catabolic
but where many other carbohydrates, both mutant. However, although this strain was
simple and complex, are vastly abundant. highly attenuated in tissue culture and in vivo,
S. enterica and B. abortus, on the other hand, it was still able to replicate at low levels, sug-
are intracellular pathogens and must go about gesting that although glucose is the predom-
feeding themselves from the inside of host inant energy source in this model, there likely
cells where the range and concentration of exists another, less preferred carbon source
available carbon sources are very limited. available within the host.
Therefore, rather than evolve an expanded At this point, it is important to emphasize
ability to utilize a range of carbohydrates, how fundamental a role the glycolysis path-
these bacteria are able to more efficiently way plays within the metabolic infrastruc-
steal the fewer carbon sources available to ture of the cell. This one central pathway
them from within the host cell—primarily, participates in a variety of activities which
glucose or glucose phosphate. Using a mouse include (i) providing the molecule pyruvate
model and a series of bacterial metabolic to the TCA cycle during aerobic or anaero-
deletion mutants, it has been shown that bic respiration, (ii) being a sole anaerobic
S. enterica serovar Typhimurium (S. Typhi- energy-yielding pathway called “fermenta-
murium) has a major dependence upon glu- tion,” which has a lower energy yield than
cose, glycolysis, and the TCA cycle both in respiration, and (iii) providing most of the
tissue culture and during infection in mice enzymes for gluconeogenesis, which is a
(14, 15). The S. Typhimurium mouse model glucose-generating pathway that is basically
has been used to elucidate the molecular glycolysis in reverse. So considering the
mechanism of infection for the human path- previous finding that glycolysis and glucose
ogen S. enterica serovar Typhi, which causes are of primary importance to intracellular
typhoid fever (16, 17). In their intracellular Salmonellae, one might ask what role the
niche within host macrophages (phagocytic glycolysis pathway serves for the bacterium.
cells of the innate immune system), S. Again, using a systematic series of metabolic
Typhimurium resides within a Salmonella- knockout strains, it has been shown that
containing vacuole, where it is protected S. Typhimurium requires a fully intact TCA
from host defenses but where it nonetheless cycle within its host, in this case the BALB/c
is able to obtain the nutrients needed for in- mouse, for full virulence (15). Just as impor-
tracellular replication (18). Bacteria lacking tantly, this work revealed that certain meta-
both homologs of phosphofructokinase, a key bolic pathways, including gluconeogenesis,
enzyme unique to the glycolysis pathway, fatty acid metabolism, and the glyoxylate
are unable to replicate within macrophage bypass, are not essential for S. Typhimurium
culture and are severely attenuated during during infection (15). More recent refinement
infection of BALB/c mice (14), showing a of this work has identified the specific steps
major dependence on this pathway during in the TCA cycle that are the most important
intracellular infection. for full virulence, including the conversion
Also in this study, extensive use of 15 dif- of fumarate to malate and the conversion
ferent metabolic mutants of S. Typhimurium of malate to oxaloacetate and pyruvate (19)
showed that glucose, specifically, is the pre- (Fig. 1).
ferred carbon source within macrophages But what makes this work both complex
and in the mouse host (14). Importantly, the and confusing is that different metabolic
key triple knockout strain from this study mutants show a range of virulence pheno-
lacked genes for both glucose uptake (two types, from totally avirulent to attenuated
phosphoenolpyruvate–sugar phosphotrans- to fully virulent. This observation along
ferase systems) and glucose catabolism (glu- with the wide range of metabolic flexibility
 CHAPTER 2 • Bacterial Metabolism Shapes the Host–Pathogen Interface 21

and redundancy in bacterial genomes high- not able to establish a persistent infection
lights the difficulty of pinpointing the precise in Ppar-delta−/− cells or mice (23). Host ex-
modes of metabolism during microbe–host pression of PPAR-delta is not necessary for
interactions. Regardless, this example illus- other intracellular pathogens such as Listeria
trates the modularity of metabolic pathways monocytogenes, Francisella tularensis, and
and the presence of key vulnerable choke- M. tuberculosis, illustrating the diversity in
points that may be unique to each organism. survival strategies of different pathogens. In
In this case, the vulnerability of infectious general, PPAR-delta controls host cell me-
S. Typhimurium for running a complete tabolism, moving the macrophage away from
TCA cycle is its need for malate to generate glucose metabolism and promoting fatty-
pyruvate and oxaloacetate to replenish the acid oxidation, putatively freeing up intra-
cycle. Ablating enzymatic steps upstream of cellular glucose that S. Typhimurium can
the two essential reactions for generating then use. Testing whether increased glucose
malate results in only slight attenuation, sug- availability in the PPAR-delta-expressing,
gesting that the microbe is able to use mol- fat-metabolizing macrophage was permitting
ecules potentially scavenged from the host to S. Typhimurium to replicate, the authors
generate the key malate precursor, succinate, observed that a bacterial mutant deficient
outside of those two steps. The authors in- in glucose transport was unable to replicate
triguingly hypothesize that succinate, orni- within the macrophage. Additionally, the au-
thine, or arginine scavenged from host cells thors saw that wild type bacteria were able to
may be used by the bacterium for malate acquire a fluorescent glucose analog from the
generation; however, this idea needs further macrophages and also replicated to higher
investigation. levels when wild type host cells were treated
This last point brings into focus the dy- with a PPAR-delta agonist (23). Together,
namic tension that exists between host cells these data provide strong evidence that the
and infecting microbes. To complete the dis- metabolic capacity of bacterial pathogens
cussion of the need for glucose, the follow- is strongly influenced by host metabolic
ing describes pathogens that rely on host cell function.
behavior to provide adequate glucose needed During chronic B. abortus infection in
for basic energy metabolism. Both S. Typhi- mice, M2 type macrophages in the spleen
murium and B. abortus infect and replicate harbor replicating bacterial cells and express
within host cell macrophages, which are im- the gene for the PPAR-gamma regulator (21),
mune cells that can adopt a range of behav- another PPAR protein that tilts macrophage
iors depending on which of their many jobs metabolism away from glucose usage and
they are performing. Both pathogens are able toward fatty acid oxidation. A PPAR-gamma
to establish a persistent, chronic infection agonist increased macrophage intracellular
within macrophages that are in the “M2” glucose concentration and also supported
state, a noninflammatory phenotype charac- increased bacterial colonization, whereas
terized by a certain repertoire of gene ex- blocking PPAR-gamma activity with a spe-
pression (20, 21), in particular, the regulatory cific inhibitor decreased splenic colonization
proteins of the peroxisome proliferator- of B. abortus in mice. As in the previous
activated receptors (PPAR) (22). Each path- study, mutant B. abortus deficient in glucose
ogen relies on host expression of a different transport was greatly attenuated for growth
PPAR protein to promote an intracellular in cell culture and in mice, strongly suggest-
environment with available glucose for bac- ing that glucose availability is key for this
terial metabolism. For S. Typhimurium, host microbe to establish chronic infection. Two
expression of PPAR-delta is important for important observations comparing these
persistent infection, since the bacteria are two studies are that (i) both studies eluci-
22 PASSALACQUA, CHARBONNEAU, AND O’RIORDAN

dated bacterial metabolism during persist- The M. tuberculosis bacillus establishes a

ent, chronic bacterial infections, suggesting particularly resilient, chronic infection in the
that pathogens likely implement different host lung by persisting in a dormant state
metabolic strategies over the lifetime of an within granulomas, conglomerations of host
infection, and (ii) whereas S. Typhimurium immune cells putatively working to wall off
may actively induce increased expression of the spread of infection but inadvertently
PPAR-delta in the host since infected macro- serving as a stable reservoir for bacilli. Recent
phages upregulate this gene in the absence of work has shown that oxygenated mycolic
cytokine stimulation, it appears that B. abor- acids, molecules found on the cell surface of
tus does not directly cause macrophages to certain Mycobacteria, can induce lung mac-
increase PPAR-gamma. Thus, bacterial path- rophages to transform into a phenotype
ogens may actively or passively take advan- called the “foamy macrophage,” an important
tage of host cell metabolic capacity. These component of the granuloma characterized
studies then raise the question as to why these by a high lipid content and ablation of bac-
two pathogens benefit from host expression tericidal activity (27). But does induction of
of different PPAR proteins, since both homo- this macrophage phenotype only help pro-
logs have similar roles in the macrophage. tect the bacteria from clearance, or does
Lastly, the dependence on glycolytic metabo- the foamy macrophage contribute to bacterial
lism in these two bacteria and their ability to metabolism? Despite being in a semidormant
obtain host glucose demonstrate that central state, the bacilli within the granuloma asso-
metabolism is a key determinant in virulence ciate closely with lipid droplets inside these
and infectious persistence. lipid-rich macrophages (27), and subsequent
Glucose is not the only molecule that can investigation has confirmed that M. tubercu-
be oxidized by cells for energy metabolism. losis is able to accumulate intracellular, host-
In fact, fatty acids harbor much more poten- derived triacylglycerols (28). The use of these
tial energy than glucose, and oxidation of host-derived triacylglycerols for bacterial me-
fatty acids can provide a greater yield of ATP tabolism during the dormant state is strongly
(Fig. 1). However, bacteria can only establish supported by the observation that bacterial
infection if they are able to acquire usable fatty acid oxidation genes are upregulated
food sources. A landmark study revealed that during residence within lipid-loaded macro-
the major pathogen M. tuberculosis prefers phages (28). As to exactly how the bacteria
to metabolize fatty acids rather than glucose conduct metabolism in vivo, two bacterial
during persistent infection (24). The genomic homologs of isocitrate lyase (ICL1 and ICL2)
era has expanded our view of M. tuberculosis are both needed for intracellular replica-
metabolism by identifying the range of met- tion (29). These proteins are vital for the
abolic genes that this organism harbors, in glyoxylate cycle in M. tuberculosis, a pathway
particular, a high degree of redundancy in needed for replenishment of TCA cycle
genes for fatty acid beta-oxidation enzymes intermediates during fatty acid metabolism
(25). A large-scale genetic screen has shown when glycolysis is not being utilized (Fig. 1).
that several M. tuberculosis glycolysis en- Sugar and fat and, in a pinch, amino acids
zymes are not needed for growth in mice can be fed into the central metabolism to
(26). More recent work has refined how this yield energy. The questions explored in
pathogen makes its energetic living during these examples regarding the preferred food
infection, answering key questions as to where sources and metabolic strategies of several
in the host the microbes acquire their pre- bacterial pathogens are relevant for other
ferred food source and what pathogen-specific pathogenic species as well. In the case of
pathways/proteins are key for metabolism the human body, a wide range of tissue types
during infection. and physiological environments exist where
 CHAPTER 2 • Bacterial Metabolism Shapes the Host–Pathogen Interface 23

pathogens elicit disease, each one being bacterial pathogens have found unique ways
unique in terms of carbon source content of exploiting.
and availability. One might then speculate One of the most severe pathogens of the
that pathogens that are able to infect nu- gastrointestinal system is the Gram-positive
merous tissue types, such as Staphylococcus endospore-forming bacterium Clostridium
aureus, display a range of metabolic behavior difficile. The various classical virulence
based on the variable levels of glucose, fatty factors that this microbe, an obligate anaer-
acids, and other carbon resources at different obe, produces, such as toxins, have been well
sites of infection. Also, do bacteria that infect characterized (32). But how does C. difficile
immune-privileged tissues, such as the brain make its living, metabolically speaking,
and meninges, have the ability to acquire nu- within the gut milieu? It is extremely diffi-
trients that are normally unavailable to other cult to test this question directly in hosts
microbes? These and many other questions that harbor a vastly diverse native gut micro-
about energy metabolism during infection biota. To simplify the situation, a model sys-
will reveal more surprising capabilities of tem using gnotobiotic mice (mice harboring a
bacterial pathogens. defined and limited microbiota) has helped
reveal how C. difficile benefits from the fer-
mentation products of one microbe to run its
The Host Is a Crowded and
own fermentative metabolism (33). The non-
Sometimes Bountiful Place
pathogenic, common gut microbe Bacteroides
One of the most exciting developments in thetaiotaomicron produces the metabolite
microbiology has been the launching of the succinate during fermentation of carbohy-
Human Microbiome Project (30, 31), an drates. In B. thetaiotaomicron–harboring
undertaking to identify and investigate the mice, C. difficile is able to grow to higher
myriad microorganisms that reside in and population densities when the mice are fed
on the human body. This endeavor is now a high-carbohydrate diet compared to when
possible due to the availability of new ge- they are fed a low-carbohydrate diet. When
nomic tools that allow researchers to in- the global transcriptional profile of C. difficile
vestigate metagenomes, complex microbial grown in B. thetaiotaomicron–harboring
communities composed of multiple species. mice fed a standard, carbohydrate-rich diet
But one of the most important aspects of versus a polysaccharide deficient one was
the Human Microbiome Project is that it compared, the pathogen showed increased
has increased appreciation for the ways the expression of multiple carbohydrate meta-
nonpathogenic, resident microbiota of the bolism genes under the former condition,
body can contribute to health and disease. in particular, genes involved in the succinate
In other words, our bodies are complex to butyrate fermentation pathway. When
ecosystems, and when bacterial pathogens mice are colonized with B. thetaiotaomicron
cause disease, they are often doing it within only, high levels of succinate are detectable
an environment where the growth of other in the mouse gut. In mice cocolonized with
microbes may have an influence, for better B. thetaiotaomicron and C. difficile, higher
or worse. In terms of energy metabolism, levels of butyrate are generated than in
it is important to appreciate that within a mice harboring C. difficile only. Thus, the
species-rich niche such as the gastrointesti- C. difficile appears to adapt its metabolic
nal tract or the oral cavity, a multitude of strategy to use B. thetaiotaomicron–generated
resident bacterial species are engaging in a succinate when it is present, generating bu-
wide range of metabolism, consuming dif- tyrate as an end product. But in the normal
ferent food sources and excreting different gut, succinate levels are generally kept low
waste products, creating a niche that some due to the presence of other resident bacteria
24 PASSALACQUA, CHARBONNEAU, AND O’RIORDAN

that produce other fermentation end prod- trations (36). Since Cra enhances virulence
ucts, such as the short-chain fatty acids gene expression in nutritional environments
acetate and butyrate. that suppress glycolysis but enhance gluco-
However, wild type mice treated with anti- neogenesis, the authors hypothesized that
biotics have higher levels of succinate in the in vivo, EHEC benefits from localized succi-
gut, suggesting that antibiotic treatment nate production by the resident microbiota in
changes the microbiota population distribu- the sugar-limited region of the gut epithelial
tion and thus changes the metabolite content surface, as opposed to the carbohydrate-rich
as well. In antibiotic-treated mice, wild type gut lumen (34, 36).
C. difficile greatly out-competes an isogenic A final example of the dynamic metabolic
mutant lacking the succinate transporter (33), interplay between bacterial pathogens and re-
supporting the idea that C. difficile experi- sident microbiota shows that “cross-feeding”
ences a growth environment that supports of preferred food sources can alter virulence
succinate fermentation when members of behavior. The bacterium Aggregatibacter
the microbiota that prevent the accumula- actinomycetemcomitans can colonize the
tion of succinate are absent. This defined human oral cavity and cause aggressive peri-
experimental approach greatly illuminates odontitis. A facultative anaerobe, A. actino-
our understanding of how a pathogen like mycetemcomitans can use several carbon
C. difficile can be present in the gut at non- sources but prefers to catabolize L-lactate
pathogenic levels under normal conditions over glucose, even though growth is slower
but then can expand and cause disease after using L-lactate (37). The microbe does not
antibiotic treatment, all due to a simple switch catabolize L-lactate anaerobically but, rather,
in energy-yielding metabolism. oxidizes this food source in an oxygen-
The benign gut resident B. thetaiotaomi- dependent manner (38). When grown aerobi-
cron also may assist another enteric pathogen cally with glucose during in vitro coculture
via succinate production. Enterohemorrhagic with the nonpathogenic resident of the oral
E. coli (EHEC) has a curious way of colo- microbiota, Streptococcus gordonii, A. actino-
nizing the gut by forming what are called mycetemcomitans mutants deficient in L-lactate
“attaching and effacing lesions,” causing metabolism show diminished growth, though
intestinal epithelial cells to remodel their they are able to grow well in high-glucose
cytoskeleton to form pedestal-like extensions mono-culture, suggesting that the ability to
upon which the EHEC firmly attach them- metabolize L-lactate is important specifically in
selves. EHEC exhibits lower levels of viru- the presence of S. gordonii. Using a mouse
lence gene expression in conditions that thigh abscess model (a common model system
promote glycolysis (i.e., carbohydrate rich), for examining oral bacterial pathogenesis),
while virulence expression is increased in the authors further showed that the ability
sugar-poor conditions where gluconeogene- to metabolize L-lactate is important only for
sis is needed by the microbe (34, 35). EHEC A. actinomycetemcomitans infection during
grown in vitro in the presence of B. thetaiota- coculture with S. gordonii, since mutants
omicron showed enhanced expression of lacking the ability to catabolize L-lactate estab-
virulence genes, and attaching and effacing lish mono-culture infection as well as wild
lesion formation was much higher in tissue type but are much less abundant in coculture
culture when EHEC was cocultured with abscesses (38). These data strongly suggest
B. thetaiotaomicron (36). Interestingly, viru- that L-lactate produced by S. gordonii, and
lence gene expression in the presence of also potentially by host lactate dehydrogenase
B. thetaiotaomicron was decreased in EHEC in the subgingival crevice, can enhance the
mutants lacking the protein Cra, which is a growth of this pathogen in the human oral
regulatory protein that senses sugar concen- cavity.
 CHAPTER 2 • Bacterial Metabolism Shapes the Host–Pathogen Interface 25

These examples not only further illustrate within the host cell (40). This example high-
that the ability to find and utilize preferred lights the importance of organic accessory
food sources during infection is important cofactors that bacteria need for running me-
for bacterial pathogens, but also show that tabolism during infection, and it also shows
the host resident microbial landscape can how pathogens may evolve to have an ex-
have major effects on pathogen energy panded ability obtain important cofactors that
metabolism by providing specific nutritional exist in various forms within the host.
environments. More importantly, these stud-
ies support the idea that changes to or per-
The End of the Line:
turbations in the host microbiota can create
Using a Novel Electron Sink
environments that can enhance or inhibit a
to Outpace the Competitors
pathogen’s ability to engage in its preferred
microbial metabolic pathways, thereby sup- The last example illustrating the flexible
porting or hindering damage to the host. and opportunistic logic of one bacterial
pathogen’s approach to metabolism involves
S. Typhimurium, which is able to make its
The Parts that Make It All Go
metabolic living inside the host cell by ac-
Yet another way of looking at central metab- quiring intracellular glucose and undergoing
olism for bacterial pathogens is to ask ques- standard aerobic respiration (glycolysis +
tions about the enzymatic machinery that is TCA cycle). But this bacterium is also able
essential for conducting preferred modes of to replicate within the lumen of the gas-
metabolism. Organisms vary widely in their trointestinal tract, outside of host cells and
ability to generate essential cellular com- among the intestinal microbiota, where oxy-
ponents from scratch and in their ability to gen is limiting and where competition for
acquire key components from exogenous carbon sources is fierce. The key to under-
sources when de novo synthesis is not pos- standing the significance of these findings is
sible. The intracellular pathogen L. mono- an appreciation of the differences between
cytogenes replicates efficiently within the using fermentation versus respiration for
cytoplasm of host cells, where it is able to ATP generation. Fermentation can be simple
acquire a variety of host molecules to support glycolysis run cyclically and is characterized
growth, including glucose-6-phosphate as by a low yield of ATP per oxidized carbon
a carbon source (39) and the essential thiol- source input relative to respiration (Fig. 1).
containing cofactor lipoate (40), which is Unlike fermentation, respiration capitalizes
needed for several enzymes involved in aero- on energy released during the process of
bic metabolism (41). The L. monocytogenes electron transport to create a proton gradient
genome does not contain genes necessary for that drives enzymatic ATP synthesis (oxida-
de novo lipoate biosynthesis (42). However, tive phosphorylation), resulting in a much
this bacterium does produce two functional larger yield of usable energy. But the central
lipoate ligase enzymes (LplA1 and LplA2) difference is that only respiration requires a
that are able to catalyze the covalent attach- highly oxidized, terminal electron acceptor to
ment of lipoate to the enzyme pyruvate de- act as a sink at the end of electron transport,
hydrogenase, a critical enzyme for bacterial whereas fermentation relies on the enzy-
respiration (40, 43). The LplA1 enzyme is matic reoxidation of the electron carriers that
specifically needed for L. monocytogenes were reduced along the pathway (NADH
intracellular growth and virulence in mice to NAD+), generating some waste product
(41). More importantly, LplA1 but not LplA2 in the process, often an acid such as lactate.
is needed for utilization of lipoyl-peptides, Although the electron sink that supports
a specific form of lipoate that is available the highest theoretical energy yield via
26 PASSALACQUA, CHARBONNEAU, AND O’RIORDAN

respiration is oxygen, many other molecules of tetrathionate in the inflamed gut, since
(such as nitrate or sulfate) can play the role wild type bacteria did not out-compete tetra-
of terminal electron acceptor as well. thionate metabolic mutants in mice lacking
And this brings the discussion back to the gene encoding the NADPH oxidase. The
S. Typhimurium. Infection of mice with ability to undergo anaerobic respiration with
S. Typhimurium results in acute intestinal a terminal electron acceptor generated by the
inflammation that is induced by various bac- combined activity of both the host (reactive
terial virulence factors, including two differ- oxygen) and the resident microbiota (hydro-
ent bacterial secretion systems (44). The gen sulfide) is a remarkably unique strategy
gut inflammation promotes a less hospitable to bolster a pathogenic metabolic advantage.
environment for the resident microbiota and Tetrathionate anaerobic respiration is not
allows the Salmonellae to out-compete the the only trick used by S. Typhimurium to out-
resident microbiota (45). Part of the mecha- compete the microbiota in the colon. Nitrate
nism that supports this growth advantage is an even more electronegative electron ac-
for S. Typhimurium in the inflamed gut is its ceptor than tetrathionate, and thus it pro-
unique ability to use the molecule tetrathio- motes an even stronger energy yield when
nate as a terminal electron acceptor for used for anaerobic respiration. In a mouse
anaerobic respiration. Bacteria in the mouse model of colitis, a strain of S. Typhimurium
colon, a generally anaerobic environment, lysogenized by a bacteriophage carrying the
produce hydrogen sulphide as a fermentation virulence gene sopE was able to switch to
byproduct, which is converted to the less nitrate metabolism in the gut to out-compete
toxic molecule thiosulphate by host mucosal the native microbiota (47). Like the tetrathio-
cells. Importantly, thiosulphate can be con- nate example, the generation of nitrate in
verted to tetrathionate by a strong oxidant. the gut is dependent on host inflammation.
In a mouse model of colitis, C57BL/6 mice However, unlike tetrathionate, luminal ni-
infected with S. Typhimurium show acute trate concentration rises due to the host
levels of cecal inflammation, whereas infec- expression of the inducible nitric oxide
tion with bacteria lacking the ability to use synthase, which generates nitric oxide that
tetrathionate in anaerobic respiration results then leads to nitrate. Bacteria with the sopE-
in inflammation with increased cecal tetra- containing bacteriophage did not have a
thionate concentration (46). Tetrathionate growth advantage in iNOS-deficient mice.
was not detected in the gut of mice infected Interestingly, the lysogenized bacteria sup-
with S. Typhimurium lacking the virulence press the genes for tetrathionate metabolism
factors that cause inflammation, strongly both in vitro and in vivo when nitrate is
supporting the idea that reactive oxygen available, illustrating a remarkable level of
species generated by the host during inflam- metabolic flexibility and control (47).
mation are responsible for the appearance of The examples outlined thus far clearly
tetrathionate that is usable by the bacteria. show that bacterial pathogens engage in
Bacteria able to use tetrathionate respiration energy-yielding metabolism during infection
showed a strong growth advantage compared in ways that take advantage of the specific
to tetrathionate metabolic mutants in the host environment at the site of infection. The
mouse gut, but not in the spleen, suggesting process of bacterial metabolism is extremely
that this metabolic advantage is site-specific dynamic, and pathogens exhibit a wide range
in the host (46). Importantly, generation of of metabolic control and flexibility during
reactive oxygen by the host NADPH oxidase infection. A key point to emphasize about
during inflammation, and not nitric oxide energy-yielding metabolic pathways is that,
production by the nitric oxide synthase, although we consider them mainly in terms
seems to be responsible for the generation of their importance in ATP generation, the
 CHAPTER 2 • Bacterial Metabolism Shapes the Host–Pathogen Interface 27

reality is that intermediates for both anabo- perturb cellular redox potential and can drive
lism and catabolism are constantly being fed production of highly reactive hydroxyl radi-
into and siphoned off these pathways for cals. Therefore, all organisms possess bio-
many other metabolic needs, with constant chemical systems to sense and regulate metal
regulation based on the moment-to-moment levels, and mammals have evolved strategies
needs of the cell. Thus, each of the stories to sequester free metal ions to limit toxicity
illustrated here represents a “tip of the and also to restrict availability of these metal
iceberg” situation, posing new questions ions to invading microorganisms. This con-
about how pathogenic catabolic behavior is cept of growth restriction by limiting access
connected to the anabolic needs and strate- to essential metals is called nutritional im-
gies during infection as well. munity (50, 51). As described below, patho-
gens have evolved numerous mechanisms for
metal uptake or efflux to circumvent nutri-
ION AND NUTRIENT ACQUISITION tional immunity (Table 2).
BY BACTERIAL PATHOGENS
Iron
The human body is a rich reservoir of fun- Iron (Fe) is the most abundant transition
damental nutrients for bacteria that can metal in the human body, but free iron is
exploit it, and nutrient acquisition is an essen- almost undetectable. Bacteria need concen-
tial aspect of host–pathogen interactions. The trations on the order of 10−6M iron to survive
mechanisms of energy generation described and proliferate, whereas circulation of free
in the previous section require high levels iron in human blood is approximately 10−18M.
of carbon and other building blocks, and Iron is a cofactor for many enzymes involved
bacteria must extract these nutrients from in fundamental cellular processes, including
resources found in the host, including sugars, DNA replication, transcription, metabolism,
amino acids, lipids and nitrogen-containing and energy generation through respiration.
compounds. Moreover, transition metals
such as iron, zinc and manganese are essen- Iron sequestration by the host
tial for survival and proliferation of all living In vertebrates, most iron is stored intracel-
organisms. This section will focus on strate- lularly in complex with heme, a tetrapyrrole
gies bacteria use to gain access to these ring that binds a ferrous iron (Fe2+) atom.
critical nutrients from the complex host Heme is primarily found in the oxygen-
environment. transporting protein hemoglobin, contained
within circulating erythrocytes. Moreover,
free hemoglobin or heme is bound by the
Transition Metal Ions:
serum proteins haptoglobin and hemopexin,
Precious Metals for Life
respectively (52). At physiological pH, free
Transition metals, including iron, zinc, man- Fe2+ in the extracellular environment is rap-
ganese, and copper, among others, are idly oxidized to ferric iron (Fe3+) and cap-
nutrients required for many biological pro- tured by the serum protein, transferrin, or by
cesses. These metals have unique redox lactoferrin, a glycoprotein found in human
potential because they can undergo change secretions such as saliva, tears, and breast
in their oxidation state and serve as essential milk, rendering free ferrous iron unavailable
cofactors for many enzymes (48). In bacteria, for microorganisms. In cells, Fe3+ is captured
it is estimated that 30 to 45% of enzymes by the storage protein ferritin. In phagocytic
require a metal cofactor for function (49). cells of the innate immune system, such
However, at high concentrations, these as macrophages and neutrophils, the phago-
metals are toxic for the cells because they somal membrane protein NRAMP1 (natural
28 PASSALACQUA, CHARBONNEAU, AND O’RIORDAN

TABLE 2 Summary of bacterial mechanisms for metal ion acquisition

Pathogens Infection site (model) Ion System contributing to virulence Reference

Salmonella enterica Systemic infection (mice) Fe EntC, EntB, IroN 62

serovar Typhimurium (salmochelin/enterobactin)
Streptococcus suis Systemic infection (mice) Fe FeoB, iron transporter 56
Campylobacter jejuni Gastrointestinal tract (chick) Fe FeoB, iron transporter 58
Staphylococcus aureus Systemic infection (mice) Fe Isd heme uptake system 69
Yersinia pestis Systemic infection/bubonic Fe Yef and Feo iron transporter 127
plague (mice)
S. aureus Systemic infection (mice) Mn MntABC and MntH Mn 82
transporters
Escherichia coli (UPEC) Bladder and kidney (mice) Zn ZnuABC and ZupT Zn 86
transporters
S. Typhimurium Inflammed gut (mice) Zn ZnuABC Zn uptake system 84
Acinetobacter baumannii Lungs (mice) Zn ZnuABC Zn uptake system 83
Mycobacterium tuberculosis Differentiated human Zn CtpC P1-type ATPase, 85
macrophages Zn efflux system
M. tuberculosis Lung and lymph nodes Cu MctB copper transport 95
(guinea pigs) protein B
E. coli Mouse macrophage-like cells Cu CopA ATPase, Cu+ export 89
system
E. coli (UPEC) Bladder (mice) Cu Yersinibactin 96
Pseudomonas aeruginosa Systemic infection (mice) Cu CopA1 P-type ATPase, Cu+ 94
efflux system

resistance-associated macrophage protein 1), across the cytoplasmic membrane of Gram-

pumps Fe2+ out of the phagosomal compart- negative and Gram-positive bacteria by the
ment, which physically surrounds bacteria FeoB family of transporters. Members of the
taken up by the host cell, to reduce metal FeoB family of metal transporters are im-
ion availability to intracellular bacteria (53). portant for the virulence of many pathogens,
These mechanisms of iron limitation also exist including Streptococcus suis and Campylobac-
in plants and invertebrates, which suggests ter jejuni (56–58).
a conserved and universal innate immune Siderophores are low-molecular-mass
response against invading pathogens (54, 55). iron chelators secreted by bacteria that bind
to Fe3+ with an affinity higher than mamma-
Bacterial mechanisms of iron acquisition lian Fe-binding proteins such as lactoferrin
The host mechanisms of iron sequestration and transferrin, allowing them to steal iron
discussed above efficiently limit the availabil- from these host proteins. Iron-bound sidero-
ity of iron, but bacterial pathogens possess phores are recognized at the bacterial surface
their own weapons to counteract host innate by specific bacterial receptors and trans-
defenses and to acquire Fe2+ that will be used ported into the cytoplasm where bound Fe3+
as a nutrient source. Specific strategies used is reduced into Fe2+ or released by degra-
by a given pathogen mainly depend on the dation of the siderophore. Epithelial cells
particular sites of infection and the intracel- and neutrophils can neutralize some sidero-
lular or extracellular lifestyle. Bacterial mech- phores by secreting the antimicrobial pro-
anisms for iron acquisition can be divided tein lipocalin-2 (also known as neutrophil
into three major categories: siderophores, gelatinase-associated lipocalin), during acute
heme acquisition systems, and free Fe2+ trans- infection (59). Lipocalin-2 expression is con-
porters (50). Free Fe2+ can enter the peri- trolled by signaling through Toll-like recep-
plasm of Gram-negative bacteria through tors, pattern recognition receptors of the
nonspecific porins and gets transported innate immune system responsive to micro-
 CHAPTER 2 • Bacterial Metabolism Shapes the Host–Pathogen Interface 29

bial ligands. Lipocalin-2 can sequester the quired for growth in iron-depleted environ-
catecholate siderophore enterobactin, which ments (70–72).
is produced by common pathogenic entero-
bacteria such as E. coli and S. Typhimurium, Zinc and manganese
causative agents of gastrointestinal diseases Sequestration of zinc (Zn) and manganese
(59). To circumvent this immune defense, (Mn) is also an important innate defense
some bacteria synthesize a modified sidero- strategy used by vertebrates to fight bacterial
phore, called salmochelin, which is a glu- infection. Zinc is used for its structural and
cosylated derivative of enterobactin that is catalytic roles in a large number of proteins.
not recognized by lipocalin-2 (60). Produc- In bacteria, 5 to 6% of the proteome consists
tion of salmochelin is essential for efficient of zinc-binding proteins, justifying the need
colonization and growth of S. Typhimurium for specific acquisition systems (73). Among
within the inflamed gut (61, 62). them, enzymes involved in central metabolic
Heme represents one of the most abun- pathways, DNA repair, response to oxidative
dant sources of iron, and pathogens have stress, and antibiotic resistance require zinc
evolved heme uptake systems as well as he- as an essential cofactor (74). Manganese is
mophore systems. To gain access to heme, also an important metal for bacteria, because
which is mainly bound to hemoproteins, bac- many Mn-dependent enzymes encoded by
teria secrete exotoxins to degrade hemoglo- pathogens are involved in central carbon me-
bin (63). Free heme is captured by specific tabolism and are necessary for resistance to
membrane complexes (TonB-dependent oxidative stress (75).
heme acquisition systems in Gram-negative
bacteria and Isd systems in Gram-positive Control of Mn2+ and Zn2+ levels
bacteria) and transported into the cytoplasm, by the host
where heme-catabolizing enzymes release Vertebrates secrete proteins that belong to
Fe2+ (50, 64). Heme is the preferred source the S100 family and inhibit bacterial growth
of iron of S. aureus, a member of the skin by chelating Mn2+ and Zn2+. S100A7 protein
microbiome that can cause tissue abscesses, (also called psoriasin) is secreted as a homo-
as well as more severe diseases including dimer by keratinocytes and inhibits E. coli
bacteremia and endocarditis (65–67). S. au- growth on human skin through sequestra-
reus can grow in vitro with erythrocytes as tion and chelation of Zn2+ (76). Similarly,
the sole source of iron. Hemolysins secreted calprotectin, which is a heterodimer formed
by S. aureus lyse erythrocytes, releasing he- by S100A8 and S100A9 (also known as
moglobin, which is targeted by the IsdB calgranulin A and B, respectively), represents
(iron-regulated surface determinant B) cell ∼40% of the cytosolic protein pool in neu-
wall receptor. As alternative routes for iron ac- trophils and exhibits antimicrobial activity
quisition, IsdH recognizes the hemogloblin- against bacterial pathogens through chela-
haptoglobin complex, whereas IsdA captures tion of the nutrients Mn2+ and Zn2+ (77, 78).
free heme (68). The Isd system is critical for Calprotectin is the major neutrophil-derived
iron acquisition during systemic S. aureus protein found in abscesses formed by S. aureus
infection and for efficient colonization of (77). As is true for iron, immune cells also
spleen, kidneys, and heart (69). In addition control Mn2+ and Zn2+ availability to intra-
to surface heme receptors, some bacteria also cellular bacteria by multiple mechanisms.
secrete hemophores, which are proteins that Manganese is pumped out of the phagosome
bind heme and can be reacquired by the bac- by the NRAMP1 protein, whereas zinc levels
teria. Bacillus anthracis, the causative agent are reduced by the action of members of
of anthrax, secretes two hemophores, IsdX1 the ZIP and ZnT zinc transporter family (79–
and IsdX2, which bind heme and are re- 81).
30 PASSALACQUA, CHARBONNEAU, AND O’RIORDAN

Bacterial acquisition of Mn2+ and Zn2+ state (Cu2+) and, as a consequence, is critical
The fight for metals is a continuous ongoing for proteins involved in a wide range of cel-
process between bacteria and the host. As lular processes. Copper is also used by some
described for iron, bacterial mechanisms metalloenzymes involved in electron transfer
exist that minimize the chelation of zinc reactions, such as cytochrome oxidase (87).
and manganese by calprotectin. To counter- However, in contrast to Fe, Mn, and Zn,
act this antimicrobial response, S. aureus ex- which are required for bacterial growth,
presses specialized manganese transporters, copper is mainly recognized for its antimi-
MntABC and MntH, to compete with cal- crobial benefits and its role in innate immune
protectin for Mn2+ during systemic infection defense against bacteria.
(82). Likewise, S. Typhimurium and Acineto-
bacter baumannii express the high-affinity Copper toxicity
zinc transporter ZnuABC to overcome the In mammals, the innate immune response
antimicrobial effect of calprotectin during provoked by secretion of the proinflamma-
infection (83, 84). S. Typhimurium, in addi- tory cytokine, interferon-γ, induces expres-
tion to neutralizing the effect of calpro- sion of Cu pumps, resulting in an increased
tectin, also exploits the accumulation of Cu concentration at the infection site (88).
this antimicrobial molecule during infec- In activated macrophages, the Cu+ transport
tion of the inflamed gut to advantageously protein 1 (CTR1) takes Cu+ inside the cell,
compete with the resident host microbiota whereas the P-type ATPase ATP7A pumps
(84). Cu+ inside the phagolysosome, again empha-
sizing the importance of precise regulation of
Use of zinc in host defense metal ion levels (89). Multiple mechanisms
In the constant battle to limit metal access have been described to explain copper toxic-
during infection, mammals also exploit the ity in bacteria. Macrophages use copper to
toxicity associated with elevated concentra- increase bacterial killing by oxidative dam-
tions of transition metals as an antimicrobial age. In the phagosome, Cu+ can interact with
strategy. Macrophages control zinc levels hydrogen peroxide to produce the highly
inside the bacteria-containing phagosome reactive hydroxyl anion and hydroxyl radical,
during infection by M. tuberculosis. This in- which in turn causes lethal damage to lipids,
crease in free Zn2+ results in accumula- proteins, and nucleic acids. Unligated copper
tion of the metal inside the cytoplasm of also causes disruption of iron-sulfur clusters
M. tuberculosis, reducing intracellular growth. by replacing the Fe atoms, resulting in the
Mycobacterium resolves this zinc-dependent disruption of protein structure. Enzymes that
intoxication by expressing CtpC, a metal contain an Fe-S cluster, such as dehydratases
efflux P1-type ATPase (85). Similarly, the involved in branch-chain amino acid synthe-
ZnuABC and ZupT zinc exporters are re- sis in E. coli, are targeted by Cu during in-
quired for optimal colonization of mice fection (90). An alternative mechanism has
bladders and kidneys during urinary tract in- been described during Neisseria gonorrhoeae
fection caused by E. coli (86). Thus, it is clear infection, where increased Cu levels result
that while bacteria must acquire metal ions in increased sensitivity to reactive nitrogen
for replication and metabolism, levels of these species produced by innate immune cells
key nutrients within the bacteria themselves (91).
are tightly regulated.
Copper regulation by bacteria
Copper The recurring theme of measure–counter-
Copper (Cu) is a redox-active metal ion that measure should be now apparent, because
can exist in a reduced (Cu+) or an oxidized bacteria have evolved strategies to prevent
 CHAPTER 2 • Bacterial Metabolism Shapes the Host–Pathogen Interface 31

copper toxicity imposed by the host and to anisms used by pathogenic bacteria to max-
tightly regulate the level of cytoplasmic imize nutrient acquisition in the complex
copper (92). In general, bacterial proteomes environment of the host (graphically summa-
contain only a few copper-binding proteins rized in Fig. 2).
(∼0.3%), and these proteins are mainly lo-
calized in the periplasm or in the cyoplasmic The arsenal of microbial
membrane, preventing unligated copper from degradative enzymes
accessing the cytoplasm. Bacteria can also Host macromolecules are a good source of
express Cu-binding proteins to sequester carbon and nitrogen when bacterial enzymes,
free Cu + , such as the Mycobacterium such as proteases or phospholipases, can
methallothionein, (MymT), which protects degrade these molecules to extract essential
the bacterium from copper toxicity (93). nutrients. Vibrio cholerae, the causative agent
Many pathogenic bacteria encode a P-type of the severe diarrheal disease cholera, uti-
ATPase Cu+ efflux protein that pumps copper lizes host sialic acid to generate carbon,
outside of the bacterial cytoplasm. For E. coli, nitrogen, and energy (98, 99). Sialic acids
a mutant lacking the CopA ATPase is hyper- are a family of nine-carbon amino sugars
sensitive to killing by macrophage-like cells found in abundance in the mammalian gut.
(89). Similarly, a CopA1 ATPase mutant V. cholerae encodes a sialic acid catabolism
of Pseudomonas aeruginosa is attenuated in gene cluster (the nan-nag cluster) located in
a systemic mouse model of infection (94). the VPI2 pathogenicity island, which is found
M. tuberculosis also encodes a membrane only in toxigenic strains that are the primary
copper transporter that is required to ensure cause of disease. NanH, a neuraminidase, is
resistance to copper toxicity and intracellular required for cleavage of bound sialic acid
survival in vivo (95). Lastly, uropathogenic from higher-order gangliosides found at
E. coli use an unusual method to limit copper the cell surface. The free sialic acid (mainly
toxicity. During bladder infection, uropatho- N-acetylneuraminic acid or Neu5Ac) is taken
genic E. coli secretes the siderophore yersinia- up by V. cholerae and converted to fructose-
bactin, which allows capture of essential iron, 6-P that feeds into the glycolytic pathway.
but this compound can also bind to copper This catabolic pathway plays a significant
and prevent its toxicity (96). Consistently, role for V. cholerae colonization of infant
E. coli strains that produce yersiniabactin mice, an important animal model for studying
exhibit greater resistance to copper-related human cholera (98). The nan cluster is also
toxicity. found in many other human pathogens that
colonize the gut, including E. coli, S. Typhi-
murium, Shigella spp., and Clostridium spp.,
Nutrient Acquisition
among others, and a correlation between
In the host cell, many nutrients essential for sialic acid catabolism and bacterial fitness in
bacterial growth are enclosed within complex the gut is now emerging (100). Of note, the
molecules such as proteins and higher-order use of NanH to hydrolyze host sialic acid as a
glycolipids and are not readily accessible. carbon source is not restricted to gut patho-
Moreover, depending on the particular niche gens but is also required for biofilm forma-
colonized by the pathogen or the local in- tion and lung colonization by P. aeruginosa
flammation state, nutrient supply might vary (101).
greatly during the course of infection. There- The γ-glutamyl transpeptidase, GGT, pro-
fore, nutritional restriction by the host is duced by F. tularensis is another striking
an important aspect of the innate immune example of a microbial strategy for nutrient
defense against pathogens (97). The follow- acquisition (102). F. tularensis is a highly in-
ing section will highlight some of the mech- fectious Gram-negative bacterial pathogen
Another random document with
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 Hän ei voinut hengittääkään, ja papin rauhalliset kasvot katsoivat
noita hehkuvia poskia ja palavia silmiä.

»Odota, tyttäreni, älä kiihoitu liiaksi. Sano tunnustuksesi.»

Roma koetti puhua, mutta sanat tuskin kuuluivat:

»Minä tunnustan… tunnustan… en voi, isä.»

Nuuska putosi vanhan miehen hyppysistä.

»Etkö ole kristitty?»

»En ole kastettu, mutta minut on kasvatettu luostarissa, ja…»

»Sitten en voi kuulla tunnustustasi. Kaste on ovi kirkkoon, ja ilman

sitä…»

»Mutta minä olen suuressa tuskassa. Pyhän Neitsyen tähden,

kuule minua!
Oi, kuule minua, isä, kuule minua!»

Vaikka vanha pappi oli tottunut ihmissydämen kärsimyksiin, valtasi

hänet ääretön sääli, ja hän sanoi ystävällisellä, hellällä äänellä:

»Jatka, tyttäreni. En voi antaa sinulle synninpäästöä, koska et ole

kirkon lapsi, mutta minä olen vanha mies ja jos voin auttaa
sieluparkaasi kantamaan taakkaansa, niin Jumala minua varjelkoon
työntämästä sinua pois.»

Kiihkein sanoin Roma kertoi tuskansa, peittämättä mitään,

lieventämättä mitään ja nimittämättä tai moittimatta ketään. Vihdoin
värisevä, nyyhkyttävä ääni taukosi ja syntyi hetken hiljaisuus, jolloin
äänet kirkossa tuntuivat tulevan kuin etäältä. Sitten lausui
ystävällinen ääni ristikon takaa:

»Tyttäreni, sinä et ole tässä asiassa tehnyt syntiä eikä sinulla ole
mitään kaduttavaa. Se, että tuskat sinua vaivaavat, todistaa, että
sielusi on puhdas ja että elät yhteydessä Jumalan kanssa.
Hermostuminen ja huoli on heikontanut ruumiillista terveyttäsi, ja
siksi on luonnollista, että luulet tehneesi syntiä, vaikka et ole
tehnytkään. Se on suloinen ominaisuus useimmissa naisina, mutta
valitettavasti vain harvoissa miehissä! Synti ei ollut sinun syntisi,
mene siis kotiisi rauhassa, ja Jumala sinua lohduttakoon.»

»Rakas isä… sinä olet niin hyvä, mutta oletko unohtanut…»

»Miehesikö? En! En voi sanoa, pitäisikö sinun kertoa hänelle vai ei.
Omasta puolestani olisin sitä vastaan, sillä miksi rasittaisin hänen
omaatuntoaan ja panisin alttiiksi perheen rauhan? Sinun tuskasi
siksi, että luonto itse tulee asian ilmaisemaan, on perustusta vailla ja
siis perkeleen kiusausta. Ne sinun tulee jättää. Mutta koska
avioliittosi on ainoastaan kirkollinen ja se toinen henkilö (menettelit
oikein, kun et sanonut hänen nimeään, lapseni) voi käyttää
hyväkseen olosuhteita erottaakseen teidät ja koska miehesi voisi
ikipäiviksi kadottaa luottamuksensa sinuun, jos tunnustuksesi tulee
liian myöhään, en osaa neuvoa, mikä olisi parasta turvallisuudellesi
ja mielesi rauhalle. Anna minun kysyä neuvoa viisaammalta. Anna
minun sanoa salaisuutesi korkealle henkilölle, ystävälliselle korvalle,
pyhimyksen sydämelle, vanhalle, pyhälle miehelle. Tule takaisin tai
sano minulle nimesi, jos tahdot, ja jos tuolla pyhällä miehellä on
jotain sanomista sinulle, olen sen ilmoittava. Mene nyt kotiisi
rauhassa, tyttäreni, ja Jumala ottakoon sinut isälliseen syliinsä.»
 Kun Roma nousi rippituolista, tunsi hän samaa kuin henkilö, joka
on potenut kovaa tautia ja on paranemaan päin. Koko hänen
olemuksensa oli omituisesti muuttunut. Suuri paino oli pudonnut
pois, hänellä oli uusi sielu, ja hänen ruumiinsakin tuntui kevyeltä
kuin ilma.

Saarnaaja saarnasi vielä värisevällä äänellään, ja vaimot ja tytöt

itkivät vielä kun Roma astui pois kirkosta, mutta nyt hän kuuli kaiken
tuon kuin unessa. Vasta kun hän saapui porttikäytävälle ja sokea
kerjäläinen luetteli huolensa hänelle, hän heräsi lumouksesta. Niin
äkillinen ja salaperäinen oli tuo muutos, kun hän palasi taivaasta
maan päälle.
XII.

Ensimmäisessä postissa aamulla »Sisar Angelica» sai kirjeen Davido

Rossilta.

»Rakkaani! — Kirjeesi saapui onnellisesti ja tuotti minulle suurta

iloa ja ehkä hiukan suruakin. Paitsi sitä tuskaa, jota aina tunnen
ajatellessani kansaraukkaani, olin hiukan alakuloinen lukiessani
jotain rivien välistä. Teeskenteletkö sinä onnentunnetta minun
rohkeuttani ylläpitääksesi ja estääksesi minua syöksymästä sinun
luoksesi huolimatta kaikesta? Kerran sinä olet tuleva onnelliseksi,
armaani. Silloin saan taas kuulla hopeanheleän naurusi niinkuin
tuona suloisena päivänä Campagnalla. Odotahan vain! Me olemme
nuoria vielä, ja elämä on edessämme.

Rukoile puolestani, oma sydämeni, että kätteni työ onnistuisi.

Olen työssä yöt ja päivät. Kokouksia, toimikuntia, kirjeenvaihtoa
lakkaamatta. Suuria suunnitteluja on tekeillä, armas, ja kohta saat
kuulla kaikki. Olen ylpeä, että arvostelin oikein sinun luonteesi
siveellistä voimaa ja että on mahdollista sanoa sinulle kaikki.

Olemme määränneet keskustoimikunnan ja järjestäneet

yhdistyksemme. Kaikki ovat yhtä mieltä minun kanssani siitä, että
yhteistoiminta on välttämätön. Eurooppa näyttää olevan kypsynyt
täydelliseen muutokseen, mutta ensimmäinen suuri työ on
toimitettava Roomassa. Minä saan kehoituksia kaikkialta. Kansojen
veljellinen yhdistyminen jatkuu jatkumistaan. Voima, joka on
suurempi raakuuden voimaa, leviää yli maailman.

Toisaalta lukemattomat miehet, jotka elävät ristiriidasta,

koettavat tukahduttaa luonnon ja Jumalan äänen. Kirkkokin koettaa
jakaa ihmiskuntaa. Kirjeestäsi päättäen sitä koetetaan tehdä taas
Roomassa. Se on vaarallista. Pappi-raukkoja kiusataan molemmilta
puolin. Toisaalta vallanpitäjät vaativat heitä puoltamaan heidän
valtaansa, olkoonpa se kuinka huono tahansa, ja toisaalta kirkko
vaatii heitä puoltamaan sen ajallisia oikeuksia. Liitän tähän
julistuksen papeille. Ehkä saat vanhan Albert Pellegrinon sen
painattajaksi ja levittäjäksi, kuten edelliselläkin kerralla. Jumala
suokoon, että siitä olisi apua!

Bruno-raukka! Olet epäilemättä oikeassa otaksuessasi, että häntä

kiusataan, jotta hänet saataisiin pettämään minut. En ole huolissani
ainoastaan itseni tähden. Minulle olisi ikuinen suru, jos hänen
mielensä tulisi myrkytetyksi. Koska Charles Minghelli on vankilassa
vangiksi puettuna, voi mitä tahansa tapahtua. Kun se mies tuli
luokseni tultuaan erotetuksi Lontoosta, pyysi hän apua
murhatakseen paronin. Minä epäsin, ja hän liittyi
vastapuolueeseen. Salainen tuomioistuin, jossa asioita valmistetaan
julkista käsittelyä varten, on pirullinen laitos, julmuuden ja
vääryyden pesä. Se on hävitetty melkein kaikista sivistysmaista,
mutta kauniin Italiamme tuomioistuimet ja vankilat ovat yhä
vieläkin salavehkeiden pesäpaikkoja, missä avuttomia, kurjia
olentoja peloitetaan jos jollakin tavalla kaikenlaisten apumiesten
välityksellä, jotka ovat valmiit vaikka mihin. Vanki ei ole ihminen
enää, vaan välittäjä, jonka tehtävänä on syyttää toisia. Hänen
sielunsa turmellaan, hänen petoksestaan maksetaan. Käy itse
tapaamassa Brunoa, jos mahdollista, ja pelasta hänet omalta
itseltään ja noilta ihmisiltä, joiden ainoana toimena elämässä on
saada varmuus rikoksista.

Ja nyt tahtoisin puhua ystävästäsi. Lohduta häntä. Tyttö-raukka

ei ole sen syyllisempi kuin jos veturi olisi ajanut hänen ylitsensä tai
villi peto syössyt hänen kimppuunsa häkistään. Älköön hän kiusatko
itseään enää. Se ei ole oikein, se ei ole hyvin. Ruumiimme ei ole
ainoa osa meissä, joka on taudille altis, sinun täytyy pelastaa
hänen sielunsa taudin uhalta.

Mitä siihen tulee, pitäisikö hänen kertoa siitä miehelleen, on

minulla siitä varma mielipiteeni. Kaikella muotoa hänen tulee se
tehdä. Omantunnon tuomioistuimen edessä synti ei ole ainoastaan
itse teossa. Sellainen teko on annettu anteeksi olipa se sitten
salainen tai julkinen. Jumala antoi sen anteeksi Davidille. Kristus
antoi sen anteeksi Jerusalemin vaimolle. Mutta salaaminen,
valehteleminen ja kaksikielisyys, sitä ei voi antaa anteeksi
ennenkuin se on tunnustettu.

Toinen seikka, jota sinun puhdas mielesi, armas, ei ole tullut

ajatelleeksi. Se toinen mies on olemassa. Ajattele mikä valta
hänellä on ystävääsi. Jos hän kaikesta huolimatta vielä tahtoo
omistaa tytön, hän voi peloittaa häntä ja uhata ilmaista kaiken
hänen miehelleen. Tämä voi saattaa vaimo-paran onnettomaksi ja
aikaa myöten vaimon tahto ehkä murtuu ja hän voi ehkä alistuakin.
Taikka sitten tuo mies voi todellakin kertoa kaiken hänen
miehelleen loukatakseen ja murhatakseen molempien onnen. Miten
käy vaimolle silloin? Uskooko hänen miehensä häntä silloin enää?
 Noiden vaarojen välttämiseksi on hänen parasta puhua heti.
Luottakoon hän miehensä rakkauteen ja kertokoon hänelle kaikki.
Jos mies on oikea mies, niin hän ajattelee: »Ainoastaan hänen
puhtautensa on pakottanut hänet kertomaan», ja mies rakastaa
häntä enemmän kuin ennen. Hän ehkä tuntee hetken tuskaa.
Jokainen mies tahtoo mielellään uskoa, että hänen poimimansa
kukka on tahraton. Mutta hänen parempi luontonsa on voittava
hänen turhamaisuutensa ja hän on sanova: »Vaimoni rakastaa
minua, minä rakastan häntä, hän on viaton, ja jos joku isku on
häntä kohtaava, täytyy sen ensin kohdata minua.»

Tervehdykseni sinulle, rakkaani. Ystäväsi on varmaan todellinen

nainen, ja olit oikeassa kohdellessasi häntä hellästi. Mutta olit
myöskin oikeassa ollessasi ankara ja antaessasi hänen kulkea
kiirastulen läpi. Näin hyvät naiset aina menettelevät toisia naisia
kohtaan. Se on jonkinmoinen todistus heidän puhtaudestaan ja se
on myöskin heidän vahva turvansa, vaikka ajattelemattomat voivat
puhua toisin. Minä rakastan sinua ankaruutesi tähden tuota
vääryyttä kärsinyttä kyyhkysparkaa kohtaan, armaani, juuri yhtä
paljon kuin rakastan sinua hellyytesi tähden. Se on todistuksena
minulle, kuinka oikein arvostelin sielusi ylevyyttä, sen puhtautta ja
henkesi tulta sekä sydämesi kultaa. Kunnes tapaamme jälleen, oma
armaani.

Sinun D. R.»

Myötäliitetty »Julistus papeille» oli näin kuuluva:

»Ei ainoastaan Italiassa ja Irlannissa, vaan myöskin Venäjällä,

Ranskassa, Amerikassa ja koko maailmassa katolisen kirkon papit
nousevat kansan seasta. Miksi siis papit niin usein kansan
taisteluissa valtoja vastaan asettuvat kansaa sortamaan, sen intoa
sammuttamaan ja sen toiveita tukahduttamaan?

Veljet! Vastaus ei ole kaukana. On olemassa kirkon sielu ja on

olemassa kirkon ruumis. Kirkon sielu on taivaallinen, erehtymätön,
muuttumaton ja elää iankaikkisesti. Kirkon ruumis on inhimillinen,
rajoitettu ja katoava. Kirkon sielu on nöyrä ja polvistuu ristin
juureen. Kirkon ruumis on ylpeä ja istuu ruhtinaitten valtaistuimien
ääressä.

Kirkon papit! Piispanne sanovat teille, että kansan pyrinnöt ovat

Jumalan pilkkaamista ja kymmenen käskyn rikkomista. Tuo vanha
huuto
on kohonnut kaikkien ihmiskunnan marttyyrien ajamia muutoksia
vastaan
Kristuksen päivistä alkaen.

Mutta jos kansan pyrinnöt eivät ole sopusoinnussa uskonnon

kanssa ja jos heidän johtajansa ovat jumalattomia miehiä, on
teidän velvollisuutenne pelastaa kansa noista molemmista
vaaroista. Älkää antako kenenkään enää sanoa, että kirkko on
ainoastaan vanhentunut ilmiö ihmiskunnan kehityskulussa ja kaiken
edistyksen esteenä. Tulkoon ihmiskunnan pelastus kirkon
papistosta, niin häviävät kaikki uskonnottomat ja jumalattomat.

Mutta ovatko kansan pyrinnöt ristiriidassa uskonnon kanssa?

Kuunnelkaa ääniä, jotka värähtelevät läpi maailman. Kansat
puhuvat helluntaikielillä kaikkialla maailmassa. Sosialismi,
kommunismi ja ehkä anarkismikin! Mutta nämä ovat toiveita, ei
systeemejä, ne ovat sairauden ilmaisumuotoja, ei parannuskeinoja.
Ja eräs vaatimus on yhteinen kaikille — ihmiskunnan yhteyden
vaatimus! Se on se ääni, joka kaikuu kaikkialla, ja minä pyydän
teitä miettimään, eikö se ole Jeesuksen ääni.

Jeesuksen papit! Avatkaa evankeliumi ja sanokaa, eikö Kristus

opettanut, että me olemme yksi ainoa lauma, jolla on yksi ainoa
paimen, ja että kaikki ihmiset ovat Jumalan poikia ja veljiä
Hänessä?

Taivaassako vain ihmisperheen tulee toteuttaa tätä? Tarkoittiko

hän, että maan päällä on oleva ankaraa erotusta ja kauhistuttavaa
epätasaisuutta ja että luonto ja Jumala selvästi osoittavat
tahtovansa sitä ja luovansa sitä? Miksi hän siis opetti meitä
rukoilemaan »Lähestyköön Sinun valtakuntasi niin maan päällä kuin
taivaassa?»

Mutta vaikka taivaan valtakunta maan päällä olisikin

saavuttamaton tuhatvuotinen valtakunta, ette te Jeesuksen papit
tahtone kieltää Hänen lapsiltaan tuon unelman lohdutusta. Minä
uneksin kirkosta, joka ei huoli maallisista oikeuksistaan, mitkä
houkuttelevat sen jakamaan ihmiset kahteen luokkaan, rikkaisiin ja
köyhiin, moneen kansakuntaan, ystäviin ja vihollisiin. Minä uneksin
kansojen Pyhästä Isästä, joka tehdään maailman henkiseksi
hallitsijaksi, ei siten että Pyhä Henki vaikuttaa seitsemään
kardinaaliin suljettujen ovien takana, vaan siten, että se vaikuttaa
koko maailmaan taivaan valossa. Se on se korkea kirkko ja korkea
paavi, josta uneksin, ja jos Jumala tahtoo, olen ne myöskin kerran
näkevä.

Davido Rossi.»
XIII.

»Rakas Davido Rossi! — Koko päivän olen kantanut kirjettäsi kuin

pyhimyslipasta, joskus pikkuisen kurkistaen sitä ajaessani
vaunuissa tai omnibusseissa, vieläpä joskus kadullakin. Olen juuri
palannut kirjapainosta. Vanha Albert on humbugi. Hän keksi jos
jonkinlaisia esteitä. Entisestä julistuksesta muka hänellä oli hyvin
paljon huolta. Hän on joka hetki pelännyt vangitsemista, ja se, joka
ne naulasi ilmoituspilareihin, on kärsinyt samanlaisia tuskia.

Johtopäätös: lisää rahaa. Sitä hän sai, ja kaikki on nyt hyvin.

Se, mitä sanoit Brunosta, on saattanut minut vallan kuumeeseen,

ja minä olen kirjoittanut kenraalitirehtöörille ja pyytänyt saada
tavata häntä. Asiamiehemme Napoleon on myöskin nyt sitä mieltä,
että Bruno on salaisen inkvisitsionin uhri. Ei mikään pyhä
inkvisitsioni ole koskaan enemmän ylenkatsonut keinojen
valikoimista. Asianajaja N. sanoo, että Italian viranomaiset ovat
perineet huonon hallituksen paheet. Kamalaa on, että tehdään
väärin sen varjolla, että muka estettäisiin toisia tekemästä väärin.
Mutta tässä tapauksessa tehdään väärin siksi, että estettäisiin toisia
tekemästä oikein. Olen varma, että Brunoa koetetaan houkutella
sinun pettäjäksesi. Jospa saisin olla hänen sijassaan! Voisivatkohan
heidän juonensa vaikuttaa minuun? Ennen kuolisin.

Ja nyt tahtoisin puhua siitä ystävästäni. Tuskin voin pitää kynää

sormissani, kun kirjoitan hänestä. Sinä puhuit niin hyvästi ja jalosti.
Olisihan minun pitänyt tietää mitä sinä ajattelit ja kumminkin…

Armas, kuinka minä saatan jatkaa? Etkö voi arvata, mitä tahtoisin
sanoa sinulle? Kirjeesi pakottaa minut tunnustamaan. Tulkoon mitä
tahansa, en voi vaieta enää. Etkö arvannut kuka ystäväraukkani
on? Arvelin, että muistaisit edellisen kirjeenvaihtomme, kun sinä
olit rakastavinasi jotakin toista. Sinä et nähtävästi ole ajatellut sitä,
ja se on taas todistus — katkera ja suloinen todistus rakkaudestasi
ja luottamuksestasi minuun. Sinä asetit minut niin korkealle, ettet
ollenkaan epäillytkään, että puhuisin itsestäni. Niin oli kumminkin
laita, ja ystäväraukkani olen minä itse.

Kärsin koko ajan, kun näin millaiselle puhtauden patsaalle sinä

minut asetit. Kirjeesi, jotka kirjoitit ennenkuin ilmoitit rakkautesi,
siihen aikaan, jolloin koetit vastustaa tunnottasi, saattoivat minut
häpeämään, koska tiesin, etten ansainnut kiintymystäsi. Useinkin
täytyi minun olla katsomatta silmiisi, kun sanoit minua hyväksi.
Olisin tahtonut itkeä ja huutaa »ei, ei, ei!» ja musertaa palasiksi
sinun luomasi kuvan. Mutta kuinka minä olisin hennonut? Kuka
rakastava nainen voi särkeä ihannoidun kuvansa miehen
sydämestä? Hän voi ainoastaan koettaa kohoutua tuon kuvan
tasalle. Sitä olen koettanut, eikä ole minun syyni, etten ole
onnistunut.

Minussa on paljon moitittavaa. Oli hetkiä, jolloin velvollisuuden

olisi pitänyt pakottaa minut puhumaan. Semmoinen hetki oli juuri
avioliittomme edellä. Muistatko, että koetin sanoa sinulle jotain?
Sinä olit ystävällinen etkä tahtonut kuunnella. »Mennyt on
mennyttä», sanoit, ja minä olin hyvin iloinen, kun pääsin siitä. Sinä
et tiennyt, mitä tahdoin sanoa, muutoin et olisi pyytänyt minua
vaikenemaan. Mutta minä tiesin, mitä se oli, ja siitä lähtien olen
lakkaamatta kärsinyt. Ja nyt minusta tuntuu kuin olisin pettänyt
sinut. Olen saattanut sinut puhumaan ja toimimaan toisin kuin ehkä
muutoin olisit toiminut. Anna minulle anteeksi! En tahdo pitää sinua
kiinni missään suhteessa. Ota kaikki antamasi takaisin, minulla ei
ole oikeutta valittaa.

Paitsi sitä oli omassa asiassani puolia, joista en kertonut

puhuessani »ystävästäni». Pelkäsin asian tulevan tunnetuksi.
Armas, minä en saa piiloutua sen suloisen puolustuksen taakse,
jonka sinä keksit minulle. Minä todellakin ajattelin sitä toista
miestä. Minä en pelännyt, että hän uhkauksillaan turmelisi
rakkauteni, sillä sitä ei mikään maailman mahtavuus voisi tehdä.
Mutta minä pelkäsin, että hän kertoisi kertomuksensa ennen minua
ja siten saisi sinut työntämään minut pois luotasi. Se tuotti minulle
tuskia yöt päivät, ja nyt minä tunnustan tunnustettavani, ettet
luulisi minua paremmaksi kuin olen.

On toinenkin asia, jota et tietänyt. Rakas, antaisin elämäni, jos

minun ei tarvitsisi sitä kertoa, mutta minun täytyy tunnustaa sinulle
kaikki. Sinä tiedät, kuka se mies on, ja Jumalan edessä vakuutan,
että hänessä yksin oli syy. Mutta oma syyni tuli jälestäpäin. Sen
sijaan, että olisin lopettanut kaiken yhteyden hänen kanssaan, elin
ystävällinen hänelle ja vastaanotin isäni tiloista tulevat tulot, jotka
hän minulle antoi, sekä ajattelin häntä tulevana miehenänikin. Ja
kun sinun puheesi piazzalla näytti saattavan toiveeni
vaaranalaiseksi, päätin masentaa sinut.
 Se on hirveätä. Kuinka minä voin sen kertoa sinulle kuolematta
häpeästä? Nyt tiedät, kuinka paljon petin sinua, ja tarkoitukseni
ilkeys vie minulta rohkeuden pyytää sinulta anteeksi. Voitko
ajatella, että minä en ollut ollenkaan parempi kuin Delila
tavatessani sinut ensi kerran! Mutta taivas tuli avuksi ja pelasti
sinut. Kuinka sinä vaikutit minuun! Ensin sinä loit uudestaan isäni
minulle, ja minä näin hänet sellaisena kuin hän todella oli enkä
sellaisena, jommoiseksi häntä oli minulle ennen kuvattu. Sitten sinä
annoit minulle sielun, ja minä näin itseni. Rakas, älä vihaa minua.
Sinun suuri sydämesi ei voisi olla niin julma, jos tietäisit kuinka olen
kärsinyt.

Vihdoin tuli rakkaus, ja minä tahdoin pitää siitä kiinni. Voi kuinka
hartaasti tahdoin! Siitä syystä en kertonut sinulle. Se oli
jonkinmoista peliä, se oli huumausta. Kaikkea, mitä tapahtui, pidin
rangaistuksena. Tulipa köyhyys, häpeä, kurjuus, entä sitten! Se
puhdistaisi vain syntistä entisyyttäni ja veisi minut lähemmäksi
sinua. Mutta kun vihdoin hän, joka oli loukannut minua, uhkasi
loukata sinua minun kauttani, jouduin epätoivoon. Sinä et aavista,
mitä aikeita silloin haudoin. Aioin surmatakin itseni saadakseni
kaikki loppumaan. Mutta minä en hennonut särkeä sydäntäsi siten.
Paitsi sitä tuo teko jo olisi ilmaissut sinulle jotain, ja minua hirvitti
ajatus, että sinä minun kuoltuani saisit tietää koko kurjan kohtaloni.

Nyt tiedät kaikki, armas. En ole salannut mitään. Kuten näet, en

ole ainoastaan ystävä-raukkani, vaan jotain vielä pahempaa — oma
itseni. Voitko antaa minulle anteeksi? En uskalla pyytää sitä. Mutta
älä anna minun olla epäilyksessä. Kirjoita. Tai vielä mieluummin,
sähkötä. Yksi ainoa sana vain. Siinä kyllin.
 Tahtoisin lähettää sinulle rakastavan tervehdykseni, mutta tänä
iltana en uskalla. Olen rakastanut sinua ensi hetkestä asti enkä
koskaan voi lakata sinua rakastamasta, tapahtuipa mitä tahansa.
Minusta tuntuu kuin antaisit minulle anteeksi, jos käsittäisit, että
olen maailmassa ainoastaan rakastaakseni sinua ja että pahin
rikokseni syntyi siten, että rakastin sinua enemmän kuin järkeä ja
kunniaakin. Päätä kuinka tahansa, olen sinun ja voin uhrata
elämäni ainoastaan sinulle.

Päivä koittaa ja pyhän Pietarin risti hohtaa lumivalkeana

aamusumun läpi. Onhan se toivon merkki? Päivä koittaa kaakossa,
ja se saapuisi nopeammin luoteeseen, jos se rakastaisi sinua yhtä
paljon kuin minä. Olen kirjoittanut tämän kirjeen yhä uudestaan
pitkin yötä. Muistatko sitä kirjettä, joka minun piti polttaa, koska se
sisälsi salaisuutesi? Tässä on kirje, joka sisältää minun salaisuuteni
— mutta kuinka paljon kurjempi ja tuhoisampi tämä on! — Sinun
onneton tyttö-raukkasi

Roma.»
XIV.

Roma asettui asumaan Rossin huoneistoon. Kun hän iäksi

neuvottelemaan vanhuksien kanssa ja näyttämään heille Rossin
sähkösanomaa, olivat he onnellisia kuin lapset. Vanha kuuro vaimo
puheli lakkaamatta. Elena ei ollut lähettänyt mitään tietoja, ja ajatus,
että hän oli mennyt luostariin, näytti mahdottomalta, mutta he
rukoilivat joka päivä pyhää Antoniusta. Eilen oli kulunut kuukausi
pojan kuolemasta, ja he olivat vieneet orvokkivihon Campo Santoon,
mutta siellä oli jo kaunis seppele ennestään — pyhä Neitsyt oli
muistanut pikku Giuseppea.

Brunoko? Niin, he olivat kuulleet hänestä ja käyneet häntä

tervehtimässäkin. Mutta hän oli ollut hyvin omituinen, hyvin jäykkä
ja kova. Kun he puhkesivat itkuun nähdessään hänen vankipukunsa,
oli hän käskenyt heitä vaikenemaan eikä saattamaan häntä
naurunalaiseksi. Hän tahtoi ainoastaan puhua Elenasta. Joku oli
kertonut hänelle, että Elena oli mennyt pois, ja kun he viittasivat
sinne päin, että hän ehkä oli luostarissa, niin Bruno vain nauroi ja
kirosi.

Seuraavana päivänä Roma muutti uuteen asuntoonsa. Hän ei

tuonut mukanaan muuta kuin muutamia kirstuja, joissa oli hänen
tärkeimmät tavaransa sekä hänen isänsä kuva ja Elenan antama
Madonna. Panttilainaaja eli ottanut haltuunsa loput hänen
omaisuudestaan ja maksanut niistä rahasumman. Useimmat
kipsimallit ateljeessa rikottiin ja vietiin pois. Suihkukaivo, joka oli
marmorista, pantiin pimeään kellariin vanhan garibaldilaisen asunnon
alle. Ainoastaan yksi osa kannettiin yläkertaan. Siinä oli Davido
Rossin veistokuvan malli ja marmorilohkare Kristuksen päätä varten.

Paitsi koiraansa ei Roma tuonut ketään mukanaan Piazza

Navonalle. Felice oli palannut paronin luo ja Nattalina oli erotettu.
Vanhan eukon piti siivota huoneet ja keittää ruoka, ja Roman piti itse
käydä ostoksilla. Naapurit ymmärsivät kohta asian laidan. Hän oli
Rossin vaimo. He rupesivat kutsumaan häntä signoraksi.

Romasta tuntui hyvin suloiselta asua Rossin huoneissa. Ne

ikäänkuin henkivät hänen läsnäoloaan. Vastaanottohuone, jossa oli
piano ja fonografi sekä kuvat seininä, toi Roman mieleen jokaisen
Rossin äänen värähdyksen. Makuuhuone oli ensin pyhättö, jota
Roma ei tahtonut käyttää, ennenkuin hän oli asettanut pienen
Madonnansa sinne. Sitten se muuttui hänen pieneksi majakseen, ja
kun hän nukkui siellä, tunsi hän outoa värähdystä, jommoista hän ei
koskaan ennen ollut tuntenut.

Nyt, kun hän asui Rossin ympäristössä, tuntui hänestä kuin hän
huomaisi jotain uutta Rossissa joka hetki. Katolla hyppelevät oravat
toivat mieleen Rossin pienenä poikana, ja lintuset, jotka juuri pesivät
ja siitä syystä laulelivat pitkin päivää, panivat Roman ajattelemaan
sykkivin sydämin heitä molempia. Lahjat, joita toiset naiset olivat
antaneet Rossille, herättivät Romassa melkein kuumemaista
uteliaisuutta. Muutamat olivat Englannista, toiset Amerikasta, ja
monet olivat naisilta, jotka eivät koskaan olleet Davido Rossia
nähneetkään. Ne tekivät Roman onnelliseksi ja ylpeäksi, mutta
myöskin hiukan mustasukkaiseksi.

Ensimmäisinä päivinä Piazza Navonalla hän uskotteli itselleen, että

tässä kaupunginosassa asuminen oli mitä hauskinta. Ensiksikin siellä
oli nuo taiteelliset, kapeat kadut ja vanhat pihat, veistetyt kivet ja
pienet lamput Madonnan kuvien edessä. Sitten siellä oli aina
ihmispäitä ikkunoissa, jäätelöveden myyjiä katukäytävillä, kirkuvia
aaseja ja huutavia, leikkiviä lapsia.

Kaikki oli luonnollista ja hauskaa. Roma eli ensi kertaa

ihmistoveriensa parissa, ja kun hänen hajuaistinsa joskus kärsi
likaisten ihmisten läheisyydestä, koetti hän sanoa itselleen, että se
oli vain väärää hienoutta tuo, joka saattoi hänet kärsimään.

Ennen kaikkea häntä liikutti ihmisten köyhyys, sillä se lähensi

häntä eniten Davido Rossiin. Tuolla oli majatalo, jossa köyhät miehet
saivat vuoteen kymmenestä pennistä, ja locande, jossa he viidestä
pennistä saivat nojata käsivartensa nuoraan. Tuolla oli kuninkaallinen
Monte di Pietà, valtion panttilaitos, ja kuninkaallinen Ranco del Lotto,
valtion arpajaislaitos. Tuolla oli rattaat, jotka ajoivat ympäri katuja
kooten almuja köyhille helposti liikutettujen roomalaisten heitellessä
vaatekappaleita ikkunoista. Tuolla oli sairaus ja köyhyys, pellagra,
joka tarttuu pelkällä maissilla elävään kansaan. Tuolla kulkivat
Punaisen Ristin sisaret sairaiden seassa, tohtorit jakelivat unijuomaa
kuoleville, ja kuolema nukutti heidät ikuiseen uneen.

Trinità dei Montilta Roma oli katsellut kaikkea tuota kuin aitiosta
katsellaan näyttämöä, mutta nyt hän oli itse sen keskellä. Tässä
samassa ilmanalassa Davido Rossi eli. Rossi oli ehkä pakosta
joutunut siihen, mutta hän jäi sinne vapaasta tahdosta. Nuori
arkatuntoinen, hienostunut nainen kärsi siellä tuskia, mutta hän
luulotteli olevansa tyytyväinen.

Kaikkialla oli Rossi ja yhä vain Rossi! Joka ilta, kun Roma meni
levolle köyhässä asunnossaan, hänen viimeinen ajatuksensa oli
rakkauden sanelema rukous yön pimeydessä. Tuo rukous oli hyvin
yksinkertainen ja lapsellinen ja sisälsi sen, että Rossi aina rakastaisi
häntä, olipa hän minkälainen tahansa ja sanoipa maailma mitä
tahansa ja tekivätpä pahat ihmiset mitä tahansa.

Tätä mielialaa kesti viikon verran, mutta sitten se alkoi masentua.

Kaiken onnen takana piili pelko kirjeestä. Roma laski tunnit, jotka
olivat kuluneet siitä asti, kun hän pani sen postiin, ja kuinka kauan
viipyisi, ennenkuin hän saisi vastauksen. Jos Rossi sähköttäisi, voisi
vastaus tulla kolmessa päivässä. Mutta hän ei saanut mitään
vastausta.

»Hänestä on varmaan parempi kirjoittaa», sanoi Roma itselleen.

Tietysti hän kirjoittaa heti, ja viiden päivän perästä saan vastauksen.
Viidentenä päivänä hän meni tapaamaan luostarin portinvartijaa,
mutta tällä ei ollut mitään »sisar Angelicalle».

»Alpeilla on ehkä lunta ja postijunat ovat myöhästyneet», ajatteli

hän, ja sitten hän läksi Pialelle, jossa sähkösanomat ovat yleisön
nähtävinä. Sveitsissä oli todellakin lunta. Juuri sitähän Roma oli
otaksunut, ja siis Rossin kirje saapuisi seuraavana aamuna. Mutta se
ei saapunut silloinkaan.

»Kuinka tuhma minä olen! Kirjeeni tietysti tuli sunnuntaina

Lontooseen!» Hän ei ollut ajatellut, kuinka englantilaiset ovat
järjestäneet postinkulkunsa sunnuntaina. Vielä päivä, yksi ainoa
päivä, niin hän saisi sanoman Rossilta ja tulisi onnelliseksi.
 Mutta päivä kului ja vielä toinenkin eikä mitään kirjettä kuulunut.
Roman mieli alkoi masentua, ja kirkas sateenkaari hänen elämänsä
taivaalla kalpeni. Katolta kuuluva laulu kiusasi häntä nyt. Kuinka ne
saattoivatkaan kiusata kurkkuaan noin? Ulkona satoi ja taivas oli
pimeä.

Sitten vanha garibaldilainen ja hänen vaimonsa tulivat pelokkaan

näköisinä ja asiapaperi kädessä yläkertaan. Heidät oli kutsuttu
todistajiksi Brunon asiassa. Tutkimus oli tapahtuva kolmen päivän
perästä.

»Minä olen, luojan kiitos, kuuro, eivätkä he voi paljoa minua

kiusata», sanoi vanha vaimo.

Roma pukeutui yksinkertaiseen mustaan olkihattuun, jonka sivuun

oli pistetty höyhen, ja läksi asianajaja Napoleon Fusellin toimistoon.

»Aioin juuri kirjoittaa teille, hyvä neiti», sanoi tuo suuri mies
vaipuen tuoliinsa. »Ikävä kyllä on työni ollut turha. Ei maksa vaivaa
jatkaa. Mies on tunnustanut.»

»Tunnustanut?» Roma puristi takkinsa rintamusta.

»Tunnustanut ja ilmaissut liittolaisensa.»

»Liittolaisensa?»

»Erikoisesti Rossin, jota hän on syyttänyt osallisuudesta

vaaralliseen salaliittoon.»

»Mihin salaliittoon?»

»Sitä ei vielä tiedetä. Ylihuomenna saamme kuulla siitä.»

 »Mutta miksi? Mikä on tarkoitus?»

»Anteeksi! Nähtävästi Brunolle on luvattu armo, ja siitä syystä

tarkoituksemme on tavallaan saavutettu. Tuskin tarvinnee puolustaa
miestä enää. Siitä viranomaiset pitävät huolen.»

»Mikä on oleva seuraus?»

»Luultavasti parlamentti heti lopetettuaan istuntonsa pääsiäiseksi

vaatii Rossia saapumaan kymmenen päivän kuluessa. Mutta te
saatte tietää siitä ensimmäisenä.»

»Kuinka niin?»

»Sillä kutsu naulataan sen talon ovelle, missä hän viimeksi asui, ja
jokaisen muun talon ovelle, missä tiedetään hänen käyneen.»

»Mutta ellei hän koskaan saa kuulla siitä tai ellei hän välitä siitä?»

»Hänet tuomitaan siitä huolimatta, ja hänen tuomionsa

painatetaan mustilla kirjaimilla ja naulataan samoihin paikkoihin.»

»Entä sitten?»

»Sitten Rossin elämä Roomassa on lopussa. Hänet suljetaan pois

kaikista julkisista toimista ja karkoitetaan parlamentista.»

»Entä Bruno?»

»Hänet vapautetaan seuraavana päivänä.»

Roma palasi kotiin nolona ja alakuloisena. Kirje oli odottamassa

häntä. Se oli Rooman vankilain johtajalta. Vaikka sääntöjen mukaan
ainoastaan sukulaiset saivat tavata vankeja, paitsi erikoistapauksissa,
ei johtajalla ollut mitään sitä vastaan, että Bruno Roccon entinen
emäntä tulee tätä vankia katsomaan tavalliseen, vieraille määrättyyn
aikaan huomenna, sunnuntaina, iltapäivällä.

Kello kaksi seuraavana päivänä Roma läksi Regina Cœliin.

XV.

Regina Cœlin vankila on rakennettu lohkaistun pyörän muotoiseksi,

jonka akseli on pyöreällä pihalla ja puolapuut osoittavat koppirivejä.
Se on puolittain tutkimusvankila, puolittain rangaistusvankila. Kaikki
vangit käyttävät vankilan vaatteita ja ovat yleisten sääntöjen alaisia.
Useimmat ovat kirjapainotaitureita ja heidän työpajansa on valtion
suuri kirjapaino. Parlamentin asiakirjat painetaan siellä ja samoin
virallinen lehti, jossa tarkasti kerrotaan kaikki kuninkaan toimet ja
hovin tanssiaiset, päivälliskutsut ja vastaanotot. Tästä lähteestä
vangit etupäässä ammentavat tietonsa ulkomaailman tapahtumista.
Kaikki miehet, jotka työskentelevät kirjapainossa, ovat numeroidut ja
muutamat ovat kahleilla.

Kun rautaovi, joka erottaa vankilan elävien maailmasta, oli

sulkeutunut, huomasi Bruno olevansa yksin kopissaan. Oli melkein
puoliyö, ja kellon lyödessä astui sisään kaksi vartiaa, toinen kantaen
savuavaa lyhtyä, toinen vasaraa, jolla hän tunnusteli
ikkunaristikkojen kestävyyttä. Nämä olivat battitoreja, ja kun he
olivat poistuneet Brunon kopista, saattoi hän kuulla vasaran lyönnit
heidän kulkiessaan kopista koppiin. Puoli kolmen ja viiden aikaan he
palasivat uudestaan. Se oli heidän yökäyntinsä.
 Bruno ei nukkunut. Nyt vasta hänellä oli aikaa ajatella, mitä oli
tapahtunut. Hän muisti pikku Giuseppea ja hänen sydäntään vihloi.
Viereisessä kopissa joku itkeä nyyhkytti koko yön. Se oli
seitsentoistavuotinen poika.

Kello yhdeksän seuraavana aamuna kello soi, oven pieni luukku

avautui ja vanki, jolla oli rautakahleet jaloissaan, työnsi sisään
leipäpalan ja tuopin vettä. Kello yksitoista kaksi vartijaa astui sisään
vieden hänet ulkomaailman puolella olevaan virastohuoneeseen.

»Nimi?» sanoi tuomari, ja apulainen luki kirjasta:

»Bruno Rocco, kuvanveistäjän apulainen Piazza Navonalta n:o 14,

vastustanut kiihkeästi viranomaisia ja haavoittanut muutamia
sotilaita.»

»Onko tämä sama mies, joka asui yhdessä parlamentinjäsen

Rossin kanssa?»

»Sama mies.»

»Katsopas, hyvä ystävä. Sinä näytät rehelliseltä mieheltä. Kerropas

minulle, missä ystäväsi Rossi nyt oleskelee.»

»Ottakaa itse selvä siitä», vastasi Bruno.

»Vaiti!» huusi vartija.

»Mitä sinä ajattelet, kun puhut tuolla tavalla tuomarille?» sanoi

apulainen.

»Ajattelen, että minä en ole mikään lapsi, jota houkutellaan

sokeripaloilla», sanoi Bruno.
 »Panepas hänet sitten vangin vaatteisiin ja pidä kaksi päivää
vedellä ja leivällä», sanoi tuomari.

Bruno vietiin kylpemään, hänen oma pukunsa otettiin pois ja sen

sijaan annettiin karkeasta, harmaasta, mustaraitaisesta vaatteesta
tehty puku. Sitten hänet vietiin pihalle jaloittelemaan.

Kävelypaikka, kuten itse vankilakin, oli pyörän muotoinen. Aseilla

varustettu vahti seisoi keskellä olevalla akselipuulla, ja vangit
astuivat edestakaisin kuin kahlehditut karhut häkissään. Tämä pyöriö
oli ulkoilmassa, ja väliin kuului ääniä maailmasta muurin yli. Lähellä
oli luostarikoulu, ja Bruno saattoi kuulla leikkivien lasten iloisia ääniä
sieltä. Silloin hän muisti pikku Giuseppen, ja tulinen rauta poltti
hänen sieluaan.

Myöhään iltapäivällä tirehtööri lähetti häntä hakemaan, ja hänet

vietiin pieneen, pimeään virastohuoneeseen sisimmän rautaportin
kautta, missä vankilan johtaja vastaanottaa vankien valituksia.

»Hyvä mies», sanoi tirehtööri, »minun vallassani on hiukan

lieventää oloasi, mutta jos sinä loukkaat sotilasvirkamiehiä, täytyy
minun kieltää sinulta kaikki helpotukset ja ryhtyä ankariin keinoihin».

»Pitäkööt sotilasvirkamiehet huolta itsestään», vastasi Bruno.

»Minulla on ystäviä, jotka minusta huolehtivat.»

Kun hän oli taas kopissaan, hän ajatteli Elenaa. Elena-raukka! Äiti-
raukka! Mitähän hän nyt tekisi? Hautajaiset ovat kohta, eikä hänellä
ole yhtään rahaa. Mutta herra Rossi kyllä pitää huolen rahasta. Hän
pitää huolen kaikesta. Jumala häntä siunatkoon! Jumala siunatkoon
heitä molempia!
 Battitorit tulivat yökäynnilleen, poika viereisessä kopissa oli vaiti, ja
Bruno aikoi ruveta levolle, kun hän kuuli hiljaista koputusta
vasemmanpuoliseen seinään.

»Kuka siellä?» huusi Bruno.

Syntyi hetken äänettömyys, ja sitten koputus kuului taas.

»Siellä on vanki, joka koettaa puhua minulle», ajatteli Bruno. Siellä

oli joku, jota oli pahasti kohdeltu ja joka tarvitsi apua. Bruno muisti,
mitä hän oli kuullut vankien kielestä. Koputukset vastasivat tietysti
eri kirjaimia, ja hypähtäen ylös Bruno vastasi puhutteluun. Hetkeä
myöhemmin hän puhui helposti viereisen kopin asukkaan kanssa.

»Kauan eläköön anarkia», koputti hänen naapurinsa, ja Bruno

vastasi:
»Kauan eläköön vallankumous.»

»Oletteko Bruno Rocco?»

»Olen. Tunnenko teidät?»

»Ette.»

»Kuka olette?»

»Olen… numero 333, rangaistusvanki.»

»Vankiko?»

»Niin. Korehtuurin lukija virallisessa lehdessä. Tunsin teidät

koppinne numerosta. Tiedän kaikki. Luin lapsestanne. Säälin teitä.
Ette saa nähdä poikanne hautaustakaan.»
 »Joku toinen pitää huolta siitä.»

»Hyvä. Voinko auttaa teitä mitenkään?»

»Ottakaa selvä, mitä Rossi puuhaa.»

»Otan. Hyvää yötä.»

»Hyvää yötä.»

Sananvaihto loppui moneen hyvästelykoputukseen, ja Bruno vaipui

uneen.
Seuraavana aamuna hänet vietiin taas tuomarin eteen.

»Bruno Rocco», sanoi tuomari, »sinä tiedät olevasi syyllinen, ja jos

sinut tuomitaan kymmenen vuoden vankeuteen, pääset hyvin
vähällä. Mutta sinä et kuulunut johtavaan joukkoon ja jos nyt olet
järkevä mies ja autat meitä vaikeassa tehtävässämme, voimme ehkä
kuninkaan armon nojalla tehdä jotain hyväksesi. Sanopas nyt,
tiedätkö, kuka oli pääsyynä meteliin?»

»Tietysti tiedän», vastasi Bruno.

»Hyvä! Kuka se oli?»

»Herra Nälkä!… Ette taida tuntea häntä? Ja kumminkin hän on

teidän naapurinne ja asuu vallan viereisessä talossa.»

Bruno nauroi niin että huone kajahti. Tuomari puri huultaan.

»Hyvä mies, sinä et näy muistavan, ettet nyt ole vertaistesi

vankien parissa ja että me sotaoikeuden nojalla voimme tuomita
sinut piiskattavaksi.»
 »Ja te ette näy muistavan, että minä en ole mikään sieni, josta voi
puristaa ulos mitä tahansa ja ettei teidän piiskanne voi avata minun
suutani.»

»Koetetaanpa», sanoi tuomari, ja vastoin sääntöjä Bruno vietiin

ulos piiskattavaksi. Hän ei koko ajalla virkkanut mitään.

»Rohkeutta!» kuiskasi kappalainen, kun Bruno puolipyörryksissä

katsoi ylös taivasta kohti ja hoiperteli uhkaavan näköisenä takaisin
koppiinsa.
XVI.

Oli kolmas päivä metelin jälkeen. Battitorit olivat käyneet

ensimmäisellä yökäynnillään, ja Bruno oli vielä valveilla. Hän odotti
koputusta viereisestä kopista. Se kuului vihdoin vallan hiljaa:

»Oletteko siellä?»

»Olen.»

»Piiskasivat, niinkö?»

»Ei tee mitään! Kerran saavat sen maksaa.»

»Oletteko kuullut uutista?»

»Mitä?»

»Rossi on paennut.»

»Paennut?»

»Paennut Englantiin… Tosi… Tämän päivän lehdissä. Koko vankila

tietää sen.»
 »Mutta juuri siihen mieheen luotin…»

»Sitä arvelin.»

»Kuka hautaa nyt poika-parkani?»

»Kunnan hautaajat pitävät siitä huolen.»

Käytävästä kuuluvat askeleet keskeyttivät koputukset, ja

keskustelu lakkasi. Bruno ei nukkunut. Katkerat, synkät ajatukset
valtasivat hänen mielensä, ja hän käveli edestakaisin pimeässä.
Vähää jälkeen kello 5:n naapuri koputti taas.

»Ei ollut kaunista, että Rossi lähti.»

»Hyvä se oli. Hyvä, että pääsi pakoon.»

»Mitenkä pakoon? Parlamentinjäsenenä hän ei ollut missään

vaarassa. Muu syy siihen lienee ollut.»

»Mikä muu syy?»

»Itse tiedätte kai parhaiten. Joku nainen?»

»Eikö mitä.»

»Kuulin semmoista. Säälin teitä, hyvä mies. Voinko auttaa teitä

mitenkään? Tahdotteko uutisia ulkoa?»

»Tahdon. Missä vaimoni on ja onko hänellä millä elää ja miten

hautajaiset suoritettiin?»

»Otan selkoa siitä… Nyt kuuluu Jumalan ääni.»

 »Jumalan ääni» oli kello, joka ilmaisi yhdeksän lyönnin ja leivän
sekä veden jakamishetken.

Myrkky oli syöpynyt Brunon sieluun, ja alhaisia ajatuksia Elenasta

alkoi hiipiä hänen mieleensä. Kello yksitoista vartija tuli sanomaan,
että joku tahtoi kysellä hänen asiaansa. Se oli asianajaja Napoleon
Fuselli. Bruno tapasi hänet virastossa pyöreän pihan luona. Siellä oli
ovessa soikea lasilevy, ja koko ajan keskustelun kestäessä vartija
kulki edestakaisin oven ulkopuolella, katsoen tuontuostakin
huoneeseen.

»Minua on pyydetty puolustamaan teitä.»

»Kuka on pyytänyt?»

»Donna Roma, teidän entinen emäntänne.»

Mutta myrkky oli tehnyt tehtävänsä, ja Brunon sielu oli täynnä

epäluuloa. Miksi Donna Roma? Tahtooko hän saada hänet, Brunon,
vankilasta pois vahtimaan Elenaa? Asianajaja ei saanut mitään
selville hänestä.

»Minä tulen uudestaan tapaamaan teitä», sanoi asianajaja, ja

Bruno vietiin takaisin koppiinsa. Siellä hän hautoi ajatusta, joka oli
syöpynyt hänen mieleensä, kunnes jokainen hellä sana, jonka Rossi
oli lausunut Elenalle, johtui hänen muistiinsa. Hän vihasi itseään
pahojen ajatustensa tähden, mutta hän ei voinut ajaa niitä pois.
»Minä olen hullu, he ovat kuin veli ja sisar», sanoi hän itselleen,
mutta se ei rauhoittanut häntä. Sinä yönä koputus kuului taas.

»Nukutteko?»

»En.»