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The activities of bacterial pathogens in vivo based on
contributions to a Royal Society discussion meeting
London UK meeting held on 20 21 October 1999 1st
Edition Charles J. Dorman Digital Instant Download
Author(s): Charles J. Dorman, G. Dougan, D. W. Holden, P. Williams, Harry
Smith
ISBN(s): 9781860942723, 1860942725
Edition: 1st
File Details: PDF, 15.55 MB
Year: 2002
Language: english
 THE ROYAL
SOCIETY

The Activities of
Bacterial Pathogens
Vivo
Based on contributions to a
Royal Society Discussion Meeting

Editors
H. Smith, C. J. Dorman, G. Dougan, D. W. Holden & P. William

Imperial College Press

The Activities of
Bacterial Pathogens in Vivo
Based on contributions to a
Royal Society Discussion Meeting
 THE ROYAL
SOCIETY

The Activities of
Bacterial Pathogens in Vivo
Based on contributions to a
Royal Society Discussion Meeting
London, UK Meeting held on 20-21 October 1999

Editors
H. Smith
Birmingham University, UK

C. J. Dorman
University of Dublin, UK

G. Dougan
Imperial College, UK

D. W. Holden
Imperial College, UK

P. Williams
University of Nottingham, UK

Imperial College Press

Published by
Imperial College Press
57 Shelton Street
Covent Garden
London WC2H 9HE

Distributed by
World Scientific Publishing Co. Pte. Ltd.
P O Box 128, Farrer Road, Singapore 912805
USA office: Suite IB, 1060 Main Street, River Edge, NJ 07661
UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library.

THE ACTIVITIES OF BACTERIAL PATHOGENS IN VIVO

First published in the Philosophical Transactions of The Royal Society series B in 2000
First published by Imperial College Press in 2001
Copyright © 2000 The Royal Society
The moral rights of the authors have been asserted.

All rights reserved.

ISBN 1-86094-272-5

Printed in Singapore.
 PREFACE

Pathogenic bacteria, i.e. those that produce disease, have unique biolog-
ical properties, which enable them to invade a host and produce sickness.
The molecular bases of these biological properties are the determinants of
pathogenicity and research objectives are to recognize them, identify them
chemically and relate their structure to function. Most of our present knowl-
edge comes from studies with cultures in vitro. However, there is a rising
interest in bacterial behaviour in the infected host and new methods have
been developed for studying it. The objective of the Discussion Meeting was
to describe these methods and to show how they, and a recent surge in con-
ventional studies, are shedding light on the activities of bacterial pathogens
in vivo. Participants were asked to enquire about bacterial and host factors
that operate in vivo to bring about sickness, to show how phenomena rec-
ognized in vitro relate to behaviour in vivo and, if evidence of relevance is
not available now, to indicate how it might be obtained.
There are two introductory papers. The first, by Smith, outlines the
new methods, poses questions about the behaviour of bacterial pathogens
in vivo and indicates how answers may be obtained. Growth in vivo and
the underpinning processes of nutrition and metabolism are given special
emphasis because new methods are highlighting their importance. The sec-
ond, Marshall et al, describes how the cellular environment can affect gene
expression. It deals with the expression of genes coding for determinants
of DNA topology (DNA gyrase, integration host factor and the nucleoid-
associated protein H-NS) during adaptation of Salmonella typhimurium to
the intracellular environment of macrophages. These global systems influ-
ence the transcription of genes involved in virulence, e.g. the spv locus.
Next, five papers describe the new methods and their use in understand-
ing hostpathogen interactions. The first, by Philpott et al, shows how a
combination of studies with cell cultures and those with various animal
models (infections of macaques, rabbit intestinal loops and murine lungs)
have defined the molecular basis for mucosal invasion and the stimulation
of inflammation by Shigella flexneri, which may apply to dysentry in man.
In the second, Merrell & Camilli describe the use of recombinase-based in
vivo expression technology (IVET) to detect genes that are transcription-
vi Preface

ally induced during infection, including those expressed transiently or at

low levels. Spatial and temporal expression of specific genes, e.g. for the
toxin co-regulated pilus (tcpA) and cholera toxin (cxtA) can be monitored
during the course of infection. Hautefort & Hinton discuss many techniques,
other than IVET, for detecting gene expression in vivo, e.g. differential flu-
orescence induction and in vivo antigen technology (IVIAT). Some of these
approaches can determine whether genes are expressed constitutively or
in an organ-specific or cell-type-specific fashion. The paper by Unsworth
& Holden describes signature-tagged mutagenesis and its use for S. ty-
phimurium in a mouse model to identify many virulence genes required for
growth in vivo, including several clustered on a chromosomal pathogenicity
island. It also shows how the use of a temperature-sensitive, non-replicating
plasmid and competitive index tests can demonstrate that virulence gene
function in vivo may differ from that predicted from in vitro studies. Finlay
& Brumell describe the interaction of S. typhimurium with relevant host
cells both in vitro and in various animal models. Sophisticated imaging and
molecular genetic tools are being used to monitor gene expression in both
the pathogen and the host cell during infection. Tissue culture results have
been confirmed and new questions evoked.
Three papers discuss the impact of the new methods. The first of these,
by Heithoff et al, describes identification by IVET of many housekeeping
and virulence genes of S. typhimurium, which are induced only in vivo. Some
of these genes are expressed in vitro if regulatory genes of the DNA adenine
methylase system (Dam) are mutated. Dam-negative mutants illustrate how
the loss of a single enzyme can completely block the ability of a pathogen
to cause disease yet fully elicit a protective immune response. The paper
by Moxon & Tang shows how a combination of genomics and methods
for detecting gene expression in vivo are identifying genes that relate to
virulence. It discusses practical and semantic difficulties in distinguishing
between classical virulence factors and those that promote survival and
growth in the host. It underlines the problem of obtaining animal models
that reflect disease in the natural host. The paper by DiRita et al. relates
knowledge of virulence gene regulation gained from studies in vitro to what
occurs in vivo for two pathogens. For Vibrio cholerae, the ToxR regulon is
active in vivo but the environmental factors that activate it are not clear.
For Streptococcus pyogenes, capsule production occurs in an animal model
of necrotizing skin infection. It is critical for virulence but dependent on
 Preface vii

mutation in a two-component regulatory system CsrR and CsrS, i.e. on the

loss of the regulation that occurs in vitro.
The next three papers discuss important aspects of bacterial pathogenic-
ity and the evidence for their operation in vivo. Williams et al. de-
scribes quorum sensing, i.e. the regulation of bacterial processes in a cell-
density-dependent manner through cell-to-cell communication by signalling
molecules. Relevant signalling molecules have been detected in animal mod-
els and human infections. These molecules not only control bacterial gene
expression but can also modulate host-cell responses. The paper by Cor-
nells describes the Yop virulon of Yersinia species as an archetype for type
III secretion systems, which are activated by contact with eukaryotic cells.
They allow bacteria to inject their proteins across two bacterial membranes
and the host-cell membrane to destroy or subvert target cells. Studies with
macrophages are described, but proof that they operate during animal in-
fections has not yet been obtained. Morschhauser et al. shows that point
mutation, genetic rearrangements and horizontal gene transfer processes
contribute to macroevolution, long-term processes leading to new species,
and microevolution, short-term developments occurring in days or weeks.
Microevolution occurs in vivo; genome variability of pathogenic microbes
leads to new phenotypes, which are important in acute development of an
infectious disease. Horizontal transfer in vivo of genes by plasmids, bacte-
riophages and pathogenicity islands is more important for macroevolution.
The final paper, by Dougan et al., raises practical implications of the
new knowledge. It deals with the handling of mucosally delivered antigens
in attempts to design effective vaccines. Studies are needed of the mecha-
nism of pathogenicity employed by microbial pathogens, of the combined
mucosal and systemic immune response associated with infection and recov-
ery, and of the mechanism of action of known good mucosal immunogens.
The importance of studies in the natural host or whole animal systems is
emphasized.
Some important aspects that emerged during the Meeting are summa-
rized. Many of the new methods for studying bacterial behaviour in vivo
require the pathogen to have robust genetics capable of easy manipulation.
This is not always so, for example for Campylobacter jejuni, and it is fortu-
nate that some methods, such as IVIAT, can be applied to such pathogens.
The current surge in knowledge of bacterial behaviour in vivo comes as
much from application of conventional methods (chemical and biological
viii Preface

comparison of in vivo- and in vitro-grown organisms, mutation, virulence

tests and complementation) as from new methods. A serious problem in
applying both the new and conventional methods is the frequent lack of
realistic animal models for human infections. This may severely limit our
ability to get to the molecular basis of pathogenicity in humans. The prob-
lem may be mitigated in the future by the use of transgenic animals and
the design of non-invasive methods for possible use in humans. Even when
satisfactory animal models are available, better methods are needed for fol-
lowing the progress of infection spatially and in real time in situ. Because
of its convenience, infection of macrophages in culture has been used as
a halfway house between in vitro and in vivo conditions. Although these
experiments may not reflect all the nuances of infection in animals, much
useful information has been obtained from them, some of which has been
confirmed by experiments with animals. At present, attention is largely
concentrated on bacterial activities in vivo rather than the host factors
that affect them and the interaction between the two. Most references to
host factors are made in relation to the environment in macrophages or
animals as a whole, rather than to specific factors and their changes during
infection.
In the future, host DNA microarrays may be used to investigate global
changes in eukaryotic gene expression in response to bacterial infection.
Bacterial pathogens and their exoproducts are excellent probes for host
cell biology. Some of the assumptions about the behaviour of pathogens
in vivo based on research in vitro have been confirmed, particularly with
regard to virulence determinants and regulatory systems. However, other
assumptions have been shown to be too simplistic, e.g. operation of the
ToxR regulatory system. Many of the genes expressed in vivo detected by
the new methods are involved with nutrition, growth, metabolism and sur-
vival in the tissues of the host. Some well-known traditional virulence de-
terminants — aggressins and toxins — have not been detected. Pathogens
that are fully host adapted employ slip-strand mispairing to generate pop-
ulation diversity and have fewer transcription regulators than pathogens
with both host and environmental lifestyles. We have moved from an era of
the gene to the era of the genome and can now undertake 'top-down' ap-
proaches to problems of pathogenicity. Application of the new knowledge
to the design of novel approaches to preventive therapeutic medicine has
begun and will accelerate. Ways of inducing pathogen attenuation rather
 Preface ix

than death may be derived. Many of the genes detected by the new meth-
ods are not known to be involved in metabolism, stress response, regulation
or virulence-determinant production by the pathogen under consideration,
nor of any other bacterial pathogen. Thus, vast areas of the behaviour of
pathogens in vivo remain unexplained. A major challenge for the future
will be integration of the vast amount of information that will accumulate
from genomics with equally voluminous data derived from intensive use of
the new methodologies for studying bacterial behaviour in vivo.

March 2000
C. J. Dorman 1
G. Dougan 2
D. W. Holden2
P. Williams 3
H. Smith 4

1
Department of Microbiology, Moyne Institute of Preventive Medicine,
University of Dublin, Trinity College, Dublin 2, Republic of Ireland
2
Department of Biochemistry, Wolfson Laboratories, Imperial College
of Science, Technology and Medicine, London SW7 2AY, UK
3
School of Pharmaceutical Sciences, University of Nottingham, Univer-
sity Park, Nottingham NG7 2RD, UK
4
The Medical School, University of Birmingham, Birmingham B15 2TT,
UK
 CONTENTS

Preface v

Introduction

Questions about the Behaviour of Bacterial Pathogens in Vivo 3

H. Smith

DNA Topology and Adaptation of Salmonella typhimurium

to an Intracellular Environment 39
D. G. Marshall, C. J. Dorman, F. Bowe, C. Hale
and G. Dougan

N e w Methods for Studying Bacterial Behaviour in Vivo 61

The Pathogenesis of Shigella flexneri Infection: Lessons from

in Vitro and in Vivo Studies 63
D. J. Philpott, J. D. Edgeworth and P. J. Sansonetti

Detection and Analysis of Gene Expression During Infection

by in Vivo Expression Technology 95
D. S. Merrell and A. Camilli

Measurement of Bacterial Gene Expression in Vivo 127

J. Hautefort and J. C. D. Hinton

Identification and Analysis of Bacterial Virulence Genes in Vivo 155

K. E. Unsworth and D. W. Holden

Salmonella Interactions with Host Cells: In Vitro to in Vivo 180

B. B. Finlay and J. H. Brumell

XI
xii Contents

Impact of the N e w Methods 199

In Vivo Gene Expression and the Adaptive Response: From

Pathogenesis to Vaccines and Antimicrobials 201
D. M. Heithoff, R. L. Sinsheimer, D. A. Low and M. J. Mahan

Challenge of Investigating Biologically Relevant Functions

of Virulence Factors in Bacterial Pathogens 226
R. Moxon and C. Tang

Virulence Gene Regulation Inside and Outside 260

V. J. DiRita, N. C. Engleberg, A. Heath, A. Miller,
J. A. Crawford and R. Yu

Evidence for Operation in Vivo of Aspects of

Pathogenicity Revealed by Recent Work in Vitro:
Potential Use of N e w Methods 281

Quorum Sensing and the Population-dependent Control of Virulence 283

P. Williams, M. Camara, A. Hardman, S. Swift, V. J. Hope,
K. Winzer, B. Middleton, D. I. Pritchard, B. W. Bycroft
and D. Milton

Type III Secretion: A Bacterial Device for Close Combat with

Cells of Their Eukaryotic Host 317
G. R. Cornelis

Evolution of Microbial Pathogens 349

J. Morschhauser, G. Kohler, W. Ziebuhr, G. Blum-Oehler,
U. Dobrindt and J. Hacker

The Immune Responses to Bacterial Antigens Encountered in Vivo

at Mucosal Surfaces 374
G. Dougan, M. Ghaem-Maghami, D. Pickard, G. Frankel,
G. Douce, S. Clare, S. Dunstan and C. Simmons

Index 395
Introduction
QUESTIONS ABOUT THE BEHAVIOUR OF BACTERIAL
PATHOGENS IN VIVO

HARRY SMITH
The Medical School, University of Birmingham,
Birmingham B15 2TT, UK

Bacterial pathogens cause disease in man and animals. They have unique biolog-
ical properties, which enable them to colonize mucous surfaces, penetrate them,
grow in the environment of the host, inhibit or avoid host defences and damage
the host. The bacterial products responsible for these five biological requirements
are the determinants of pathogenicity (virulence determinants). Current knowledge
comes from studies in vitro, but now interest is increasing in how bacteria behave
and produce virulence determinants within the infected host. There are three as-
pects to elucidate: bacterial activities, the host factors that affect them and the
metabolic interactions between the two. The first is relatively easy to accomplish
and, recently, new methods for doing this have been devised. The second is not
easy because of the complexity of the environment in vivo and its ever-changing
face. Nevertheless, some information can be gained from the literature and by new
methodology. The third aspect is very difficult to study effectively unless some
events in vivo can be simulated in vitro.
The objectives of the Discussion Meeting were to describe the new methods
and to show how they, and conventional studies, are revealing the activities of
bacterial pathogens in vivo. This paper sets the scene by raising some questions
and suggesting, with examples, how they might be answered.
Bacterial growth in vivo is the primary requirement for pathogenicity. Without
growth, determinants of the other four requirements are not formed. Results from
the new methods are underlining this point. The important questions are as follows.
What is the pattern of a developing infection and the growth rates and population
sizes of the bacteria at different stages? What nutrients are present in vivo and how
do they change as infection progresses and relate to growth rates and population
sizes? How are these nutrients metabolized and by what bacterial mechanisms?
Which bacterial processes handle nutrient deficiencies and antagonistic conditions
that may arise? Conventional and new methods can answer the first question and
part of the second; examples are described. The difficulties of trying to answer the
last two are discussed.
Turning to production in vivo of determinants of mucosal colonization, pene-
tration, interference with host defence and damage to the host, here are the crucial
questions. Are putative determinants, which have been recognized by studies in
vitro, produced in vivo and are they relevant to virulence? Can hitherto unknown
virulence determinants be recognized by examining bacteria grown in vivo! Does
the complement of virulence determinants change as infection proceeds? Are reg-
ulatory processes recognized in vitro, such as ToxR/ToxS, P h o P / P h o Q , quorum
sensing and type III secretion, operative in vivo? What environmental factors af-
fect virulence determinant production in vivo and by what metabolic processes?
Examples indicate that the answers to the first four questions are 'yes' in most

3
4 H. Smith

but not all cases. Attempts to answer the last, and most difficult, question are also
described.
Finally, sialylation of the lipopolysaccharide of gonococci in vivo by host-
derived cytidine 5'-monophospho-N-acetyl neuraminic acid, and the effect of host
lactate are described. This investigation revealed a new bacterial component im-
portant in pathogenicity, the host factors responsible for its production and the
metabolism involved.

K e y w o r d s : bacteria; pathogens; in vivo; gonococci; sialylation; lipopolysaccharide

1. INTRODUCTION

Pathogenicity (virulence) is the capacity to cause disease. Bacterial

pathogens form only a small part of the bacterial world but they attract the
most attention. They have unique biological properties, which enable them
to enter the tissues of man or animals and cause sickness and sometimes
death. These biological properties are indicated by the progression of the
disease process. The skin is a formidable barrier against bacterial attack. It
can be breached by gross trauma or vector bite and a few pathogens, e.g.
staphylococci are able to exploit small abrasions to cause skin infections.
However, the usual route of entry for most pathogens is not through the
skin but over the internal surfaces of the respiratory, alimentary or urogen-
ital tracts. Initially, only a small number of bacteria are deposited on these
mucous surfaces, so the first requirement for pathogenicity is survival and
growth on them. Survival entails competition with commensals that nor-
mally inhabit these surfaces, penetration of mucus that covers them and
adherence to epithelial cells. Next, most pathogens need to penetrate into
the tissues to be effective, although some, like Vibrio cholerae, can cause dis-
ease while remaining on the mucous surface. Penetration may be achieved
by entry into and egestion from epithelial cells, by passage between them
or destruction of them. The third requirement is the ability to grow and
multiply in the environment of host tissues, otherwise the pathogen can-
not cause harm. On the mucous surfaces and within the tissues, pathogens
have to contend with antibacterial substances in body fluids and within
the cells they infect. Also, there are polymorphonuclear (PMN) phagocytes
and macrophages that can ingest and kill them. These defence mechanisms,
which act against any invading pathogen, are present at the site of infec-
tion and, within a few hours, are reinforced by the inflammatory response.
Clearly, ability to withstand these host defences is the fourth requirement
 Questions about the Behaviour of Bacterial Pathogens in Vivo 5

for pathogenicity. This quality is needed again as bacteria spread through

the lymphatic system into the bloodstream, where many macrophages line
the vessels of the lymph nodes, spleen and liver. A few days after initial
infection, the pathogen faces an even greater obstacle, the specific immune
response. To survive, it must either suppress or circumvent antibody- and
cell-mediated immunity. Finally, entry and growth in the host and inhibi-
tion of host defence are not enough. To be pathogenic, the bacteria must
damage the host, thus causing disease. This can be achieved directly ei-
ther by production of toxins or lysis of host cells by intracellular bacteria.
It can also be accomplished indirectly by stimulation of host cytokines or
immunopat hology.
To summarize, for the many pathogens that do not enter the host by
direct penetration of skin, there are five essential biological requirements
for pathogenicity: colonize mucous surfaces; penetrate them; grow in the
tissues; inhibit host defence; and damage the host. The cardinal fact about
pathogenicity is that it is multifactorial. Many genes are involved. Their
products, the determinants of pathogenicity (virulence determinants), are
the molecular bases for the five essential biological properties (Smith 1995).
The goal of studies on bacterial pathogens is to recognize these determi-
nants, to identify them and to relate their structure to function.
In the past, most of our knowledge about these determinants has come
from experiments with bacteria grown in culture. Almost all pathogens have
been investigated and many determinants have been identified. Examples
are the pili of gonococci, which aid mucosal colonization; the Ipa proteins
of shigellae, which are involved in invasion of colonic epithelium; the ente-
rochelin of Esherichia coli, which aids acquisition of iron for growth; the
capsular polysaccharides of pneumococci that interfere with host defence;
and the lethal toxin of diphtheria bacilli (Smith 1995; Finlay &c Falkow
1997).
Now, the situation has changed. There is a burgeoning interest in the
activities of bacteria within the infected host. Environmental conditions
in vivo (osmolarity, pH, Eh, and nutrient and substrate availability) differ
from those in laboratory cultures. They are more complex. Pathogens may
be in films on mucosal surfaces or free in body fluids, cell cytoplasm or cell
vacuoles; in all cases, the environment is neither simple nor defined. Also,
the conditions change during the course of infection due to inflammation,
tissue breakdown and spread from one anatomical site to another. Within
6 H. Smith

cells, the environment can alter when bacteria invade due to changes in host
gene expression. Since environmental conditions affect bacterial growth,
metabolism and regulation of gene expression (Busby et al. 1998; Marshall
et al, this issue) we should expect bacteria taken from infected animals to
be different, in some respects, from those grown in vitro, a fact now well
established for many pathogenic species (Smith 1990, 1996).
Clearly, the activities of bacteria in vivo must be explored for a fuller
appreciation of pathogenicity. There are three aspects to the full picture.
First, there are observations on the bacteria themselves and identification
of virulence determinants formed by them at different stages of infection.
Second, there is recognition of host factors that affect bacterial behaviour
and production of virulence determinants. Third, there is investigation of
the underlying metabolic interactions between bacterial and host factors.
The first aspect is relatively easy to accomplish and recently new methods
for doing so have been devised. The second is not easy to achieve because
of the complexity of the environment in vivo and the fact that it changes
as infection proceeds. Nevertheless, there is relevant information in the
literature and some new methods have been evolved. The final aspect is
very difficult to study effectively in vivo; some progress might be made if
events in vivo can be simulated in vitro. It is not surprising that the first
aspect receives most attention. Indeed, one can understand an attitude to
concentrate solely on bacterial properties because the environment in vivo
and its influence are too complex to analyse properly. However, this leaves
part of the story untold.
The new methods for studying bacterial pathogens in vivo are listed in
Table 1. Some require the pathogen to have robust genetics that are easily
manipulated, which is not always the case, e.g. for Campylobacter jejuni.
The objectives of this Discussion Meeting were to describe these methods
and to show how they, and a recent surge in conventional studies, are ad-
vancing knowledge. This paper sets the scene by posing some questions and
suggesting, with examples, methods whereby answers may be forthcoming.

2. QUESTIONS A B O U T T H E D E T E R M I N A N T S OF
B A C T E R I A L G R O W T H IN VIVO

The multifactorial nature of pathogenicity means that the determinants of

all five requirements are essential for its manifestation. However, growth
 Questions about the Behaviour of Bacterial Pathogens in Vivo 7

Table 1. New methods for studying bacterial behaviour in vivo.

Function Method Principle Reference

Following Confocal laser Possible to examine a Richter-Dahlfors et

infection in scanning few bacteria in al. 1997
animals microscopy (CLSM) thick slides
Fluorescence- Rapid identification Valdivia & Falkow
activated cell of host cells 1997a
sorting (FACS) containing
fluorescent bacteria
Laser microprobe Measuring viability of Haas et al. 1993;
mass spectrometry bacteria in biopsies Seydel et al. 1992
by N a + / K + ratios
Photonic and radio Pathogens made Contag et al. 1995;
detection of bioluminescent by a Perin et al. 1997
pathogens in vivo luciferase or
radiolabeled by
technetium-99 m

Measuring Quantitative Measurement of Akins et al. 1995;

environmental fluorescence fluorescing dyes Aranda et al 1992
parameters in microscopy use of that react to Garcia-del Portillo
vivo reporter genes environmental et al. 1992; Pollack
factors 1986
LacZ fusions to
genes that respond
to certain levels of
compounds in the
environment
X-ray microanalytical Morgan 1985;
electron microscopy Spencer et al. 1990

Detection of in vivo expression Genes expressed in Camilli et al. 1994;

genes technology (IVET) vivo provide Camilli &
expressed in promoters for Mekalanos 1995;
vivo various reporting Heithoff et al. 1997;
systems Lowe et al. 1998;
Mahan et al. 1994,
1995; Wang et al.
1996a, b; Young &
Miller 1997
Differential Promoters of genes Valdivia & Falkow
fluorescence expressed in vivo 1996, 1997a
induction (DFI) drive expressions of
green fluorescent
protein
8 H. Smith

Table 1. (Continued)

Function Method Principle Reference

Differential display of cDNAs prepared from Abu-Kwaik &

cDNAs mRNAs of genes Pedersen 1996;
expressed in vivo Plum&
and compared with Clark-Curtiss 1994
cDNAs from
organisms in vitro
Reaction with Reaction of products Akins et al. 1995; Suk
antibodies produced of gene libraries et al. 1995; Wallich
by infection with antibodies et al. 1995
evoked by infection
compared to those
of antibodies formed
against killed
bacteria
Labelling proteins Diaminopimelate used Burns-Keliher et al.
with by bacteria and not 1997
diaminopimelate by host cells

Direct Signature-tagged Non-recovery from Hensel et al. 1995;

identification mutagenesis (STM) animals after inocu- Chiang &
of virulence lating individually Mekalanos 1998;
genes tagged insertion Coulter et al. 1998;
mutants indicates Mei et al. 1997;
genes required for Shea et al. 1996
infection
Virulence Complementation of Collins 1996;
complementation avirulent strains by Pascopella et al.
gene libraries from 1994
virulent strains

Global analysis Complete genome Aids the identification Strauss & Falkow
of potential sequencing of new genes 1997; Tang &
gene expressed in vivo Holden 1999
expression
Chip technologies Microarrays of probes De Saizieu et al. 1998;
allow monitoring of Lockhardt et al.
expression of many 1996; Ramsay 1998;
genes in parallel Schena et al. 1996

holds the primary position because without it other determinants would

not be formed. This is the first reason for dealing with growth separately.
The second is that the new methods for recognizing genes expressed in vivo
 Questions about the Behaviour of Bacterial Pathogens in Vivo 9

(Table 1) are underlining its importance. Many of the genes detected are
involved in the acquisition of nutrients and their metabolism, e.g. members
of the 100 or more genes demonstrated by in vivo expression technology
(IVET) for infections of Salmonella typhimuriun in mice and macrophages
(Heithoff et al. 1997). Finally, compared with other aspects of virulence,
growth and metabolism have been neglected because it is difficult to do
meaningful experiments. A discussion of these difficulties and possible ways
of solving them could encourage more work in the area.
Perhaps the first point to emphasize is that it is not just growth, but
rate of growth, that is important in pathogenicity. On epithelial surfaces,
receptors for some bacterial adhesins are present in mucus and could delay
contact between the pathogen and the surface. Their influence can be over-
whelmed by rapid and substantial bacterial growth in mucus (McCormick
et al. 1988; Mantle & Rombough 1993). At primary lodgement, the few
bacterial invaders must multiply rapidly to replace losses inflicted by the
powerful host defences of the inflammatory response. In acute disease, rapid
growth of the pathogen in tissues is needed to produce harmful effects before
a protective immune response is mounted. In chronic disease, slow growth
at all stages may lead to less stimulation of immune responses. To form
carrier states, a resistant stationary phase of the pathogen (Kolter et al.
1993; Kolter 1999) may be an advantage. These different growth rates will
be determined by prevailing environmental conditions, which will also in-
fluence the size of pathogen populations that can be sustained by different
tissues.
The important questions regarding bacterial growth in vivo are as fol-
lows. What is the pattern of a developing infection and growth rates and
population sizes at different stages? What nutrients become available or
depleted as infection proceeds and how do they relate to growth rates and
population size? How are the nutrients metabolized and by what bacterial
determinants? How do bacteria handle nutrient deficiences and antagonis-
tic biochemical conditions? They are discussed in two sections, bacterial
activities and host factors.

(a) Bacterial activities

The classical method of following pathogenesis is to take samples of body

fluids and tissues during the course of infection, either from live animals
10 H. Smith

or those killed at intervals, and then to examine them outside the host by
in vitro methods. The latter include total and viable counts, and light and
electron microscopy. Recently, confocal laser scanning microscopy (CLSM),
fluorescence-activated cell sorting (FACS), and laser microprobe mass spec-
trometry (Table 1) have added new dimensions to these classical methods.
For example, CLSM of immunostained sections of livers of mice infected
with realistically small doses of S. typhimurium showed that the pathogen
resides intracellularly in macrophages and is cytotoxic to them (Richter-
Dahlfors et al. 1997), as occurs with cultured macrophages (Chen et al.
1996). Hence, efforts to identify the molecular determinants of cell cul-
ture cytotoxicity are now relevant to behaviour in vivo. The major advance
has been to use non-invasive methods such as photonic imaging and ra-
diolabelling (Table 1) for following the pattern of infection. For example,
photonic imaging provided a surprise about salmonellosis of mice; after oral
infection, the organisms were concentrated in the caecum rather than the
ileum (Contag et al. 1995).
Increases or decreases in bacterial populations have been measured by
counting bacteria in blood, lymph glands, spleen, liver and other relevant
tissues, e.g. Peyer's patches (Curtiss et al. 1988; O'Callaghan et al. 1988).
Now, the new non-invasive methods can be used for evaluating population
changes in different tissues.
In considering growth rates in vivo, it should be remembered that pop-
ulation size is the result of bacterial growth and destruction or removal by
the host. If a population increases rapidly, there is no doubt that bacteria
are multiplying rapidly. But, the precise growth rate is unknown because,
although dominated by the growing pathogen, host defence will have some
effect. When the population increases slowly, or even decreases, as happens
early and late in the disease process, multiplication rates are not clear. A
rapid growth rate may be masked by an equally quick destruction by the
host. Certainly, a stationary population does not necessarily mean that the
pathogen has stopped growing.
Methods for measuring doubling times in tissues are available. The first
methods relied on a genetic marker distributing to only one of two daugh-
ter cells in each succeeding generation. The proportion of the bacterial
population carrying non-replicating markers, examined at intervals during
infection, revealed the number of preceding generations (Maw & Meynell
1968; Hormaeche 1980). The method was used for infections of E. coli and
 Questions about the Behaviour of Bacterial Pathogens in Vivo 11

S. typhimurium in mice but its scope was limited by the need for a non-
replicating marker. The next method, used for mice infected with E. coli
and Pseudomonas aeruginosa (Hooke et al. 1985; Sordelli et al. 1988), could
have wide application. Growth rates were calculated from increases in ratios
of wild-type organisms (which multiply in vivo) to temperature-sensitive
mutants (which should not multiply in vivo) during the course of infection.
Unfortunately, this method has not been exploited. Recently, a combina-
tion of the two methods has been used to compare growth rates in mice of
virulent and attenuated strains of S. typhimurium (Gulig & Doyle 1993).
The marker, inherited by only one of the progeny on division in vivo, was
the temperature-sensitive Cm r plasmid pHSG 422, which is maintained on
replication at 30°C but not at 37°C. Overall, these methods indicated that
growth in vivo was slower than in vitro in some cases (Maw &; Meynell
1968; Hormaeche 1980) and similar in others (Hooke et al. 1985; Sordelli
et al. 1985). But, their use has been limited. Since the 1960s, when the
subject was first raised, only three pathogens, E. coli, S. typhimurium and
P. aeruginosa, have been examined.
In view of the importance of growth rates in pathogenicity, it would be
a great advance if an easily used, non-invasive method for measuring them
in vivo could be devised. If a method became available, particular attention
should be given to growth rates in the early, crucial stage of infection, which
are obscured by the bactericidal effects of host defences. Also, the possible
occurrence of non-growing bacterial populations in persistent infection and
carrier states should be investigated, in view of the knowledge accumulating
about stationary bacterial populations in vitro (Kolter et al. 1993; Kolter
1999).

(b) Host factors

The first method to recognize nutrients that might determine growth in

vivo relies on the fact that most key nutrients, e.g. iron, will also be neces-
sary for growth in vitro. Hence, the first step is to grow the pathogen in a
defined medium and observe the effects of deleting specific nutrients. Then,
the presence in vivo of the identified nutrients can be ascertained. Much in-
formation on sugars, aliphatic-, hydroxy- and long-chain fatty acids, amino
acids, purines, pyrimidines, vitamins and metal ions in blood, body fluids,
neutrophils, macrophages and other tissues is known from physiological,
12 H. Smith

pathological and pharmacological studies (Lentner 1981, 1984). Also, tis-

sue samples can be analysed by established biochemical methods.
Some key nutrients may not be revealed by these studies in vitro. Ery-
thritol is used preferentially by Brucella abortus in a medium containing
glucose (Anderson & Smith 1965). It is concentrated in the placenta, foetal
fluids and chorions of pregnant cattle, and during brucellosis promotes in-
fection of these tissues leading to abortion (Keppie et al. 1965; Smith et al.
1962; Williams et al. 1964). Its importance in the metabolism of B. abortus
was discovered by noting growth stimulation when foetal fluids or placental
extracts were added to cultures in vitro and then purifying the stimulant.
This procedure could be applied to other pathogens, particularly those that
show tissue tropism in disease.
Auxotrophic mutants of pathogens can be used to check the availability
of specific nutrients in vivo. For example, auxotrophs unable to synthesize
p-aminobenzoic acid, purines, thymine and histidine have been prepared
from Salmonella typhi, S. typhimurium and Shigella flexneri (Ahmed et al.
1990; Curtiss et al. 1988; Fields et al. 1986; Karnell et al. 1993; Leuing &
Finlay 1991; Levine et al. 1987; O'Callaghan et al. 1988). They have low
virulence for mice, rabbits, monkeys or man due to impaired growth in
vivo, indicating that p-aminobenzoic acid, purines, thymine or histidine are
absent or scarce in these animals. Similar auxotrophs for other nutrients
could be prepared from different pathogens. The absence or presence of
the specific nutrients would be indicated by comparing their multiplication
rates in vivo with those of wild-types. This method reveals deficiencies in
nutrients that may be required by other pathogens. It does not provide
information on nutrients that the wild-type uses in vivo to synthesize the
particular metobolite required by the auxotroph.
Another method for recognizing key nutrients of growth in vivo covers
the possibility that they may not be the same as those needed for growth
in vitro. Bacteria grown in vivo can be investigated by the new methods
(Table 1) for genes that code for enzymes involved in acquiring and metab-
olizing nutrients. The nature of these enzymes will indicate nutrients used
in vivo. In a signature-tagged mutagenesis (STM) study of staphylococci in
infected mice, a prominent identified virulence gene (i.e. one which when
mutated results in reduced virulence) coded for a proline permease, indi-
cating that scavenging for proline is essential for virulence (Schwan et al.
1998).
 Questions about the Behaviour of Bacterial Pathogens in Vivo 13

After key nutrients have been identified their concentrations in the tis-
sues can be obtained from the literature (Lentner 1981, 1984) or measured
by appropriate methods. The latter is relatively easy for normal uninfected
tissues but monitoring nutrient concentration in infected tissues and their
changes as disease progresses is extremely difficult, even if good animal
models are available.
Measuring environmental parameters within infected cells is not easy
but some progress has been made using new methods. Quantitative flu-
orescence microscopy (Table 1) has been used to measure intraphagoso-
mal pH in macrophages (Aranda et al. 1992) and LacZ reporter genes
(Table 1) have been used to indicate Ca 2 + , Fe 2 + and Mg 2 + levels
in tissue culture cells (Pollack et al. 1986; Garcia-del Portillo et al.
1992).
The relevance of identified nutrients to infection in vivo can be tested by
two methods. The virulence of the pathogen may be enhanced by injecting
additional nutrient. Also, reduced virulence of mutants unable to use it
could be investigated. If it is irreplaceable, e.g. iron, these mutations will
usually be lethal. If, however, the nutrient is preferred but replaceable by
another, e.g. erythritol by glucose for B. abortus (Anderson & Smith 1965),
then the required mutant might be obtained.
Some biochemical conditions existing in vivo, which might have adverse
effects on growth, e.g. high osmolarity, low pH and anaerobic conditions,
can be identified and quantified by reference to the literature or by analysis.
Antagonistic influences may also be indicated by the functions of genes
whose expression in vivo is detected by the new methods (Table 1), e.g.
those that deal with variations in osmolarity.
The final questions posed at the beginning of this section — How are the
nutrients metabolized and by what bacterial determinants? How do bac-
teria handle nutrient deficiences and antagonistic biochemical conditions?
— are extremely difficult to answer. Trying to investigate the metabolism
of pathogens growing in infected tissues is well nigh impossible. If some
aspect of growth in vivo, such as doubling time or population size, could be
simulated in vitro by culturing the pathogen in a medium to which nutri-
ents known to be important for growth in vivo are added at the appropriate
concentrations, some meaningful observations could be made by established
methods of bacterial physiology. Similarly, the effects of adverse conditions
could be investigated provided the phenomenon in vivo could be simulated
14 H. Smith

in vitro. In both cases, the enzymic products of genes shown to be expressed

in vivo by the new methods (Table 1) should be kept in mind. Also, the
complete genomes of pathogens will reveal their overall metabolic potential,
e.g. the presence, absence or incompleteness of a citric acid cycle (Huynen
et al. 1999).
In view of the complexity of the experimental systems, it is hardly sur-
prising that there has been little progress in answering questions on the
nutrients and metabolism that underpin bacterial growth in vivo. How-
ever, there are a few examples showing that answers can be obtained. The
best is the acquisition of iron by pathogens in vivo. Iron is an essential
nutrient for all bacteria. First, it was shown that availability of iron in
vivo is restricted by chelation to host transferrin and lactoferrin, and that
injection of iron salts enhanced virulence of many pathogens in various
animal models (Bullen 1981). Then, molecular studies were conducted in
vitro under iron-limiting conditions. These showed that different pathogens
adopt numerous strategies to overcome iron restriction (Weinberg 1995).
In some cases, siderophores are excreted, which chelate iron and return
to bacteria via specially induced cell-surface protein receptors (Brown &
Williams 1985; Weinberg 1995). After internalization, the siderophores give
up their iron under the influence of reductases (Halle k. Meyer 1992). In
other cases, transferrin-bearing iron interacts with cell-wall protein recep-
tors and iron is delivered into the bacteria (Cornelissen et al. 1992; Anderson
et al. 1994). In both cases, the cell-wall receptors were shown to be present
on bacteria in patients or infected animals (Brown & Williams 1985; Cor-
nelissen et al. 1992; Smith 1990, 1996). Finally, mutants deficient in the
determinants of iron acquisition were shown to be attenuated in virulence
tests, e.g. a gonococcal mutant deficient in the transferrin receptor was
less infective for human volunteers than the wild-type (Cornelissen et al.
1997).
Two other examples relate to tissue localization by pathogens. Urea is
a growth stimulant for Proteus mirabilis, which causes severe kidney infec-
tions (Braude & Siemienski 1960). The fact that this growth stimulation
contributes to localization in the kidney was supported by the impaired
growth in the kidneys of mice of a urease-deficient mutant (MacLaren 1968).
Also, the following evidence supports the role of erythritol in stimulating
massive growth by B. abortus in foetal tissues of cattle, resulting in abor-
tion (Keppie et al. 1965; Smith et al. 1965; Williams et al. 1964). Injections
 Questions about the Behaviour of Bacterial Pathogens in Vivo 15

of erythritol enhanced infection of B. abortus in newborn calves. Erythritol

analogues inhibited growth of B. abortus in vitro and in vivo. A strain (S19)
of B. abortus unable to use erythritol did not cause abortion.
Finally, there are results emerging from use of the new methods of
detecting gene expression in vivo. Two previously unrecognized genes
of E. coli, guaA and argC, induced in urine appear important in
uropathogenesis (Russo et al. 1996). Urine contains no guanine and only
low levels of arginine and the induced genes allow E. coli to synthesize
them. Deletion mutants do not grow in urine and in mice are less viru-
lent than the wild-type. In addition to these genes, the osmoregulatory
transporter ProP, coupled with osmoprotective betaine, allow E. coli to
grow in human urine and to colonize the urinary tract of mice (Culham
et al. 1998). Turning to V. cholerae, STM identified an attenuated bi-
otin auxotroph from the intestine of infected mice (Chiang & Mekalanos
et al. 1998). This suggested that biotin synthesis is a virulence attribute
and that there was little available biotin in the infant mouse intestine, a
fact supported by enhanced colonization when biotin was added to the
inoculum.

3. QUESTIONS A B O U T P R O D U C T I O N IN VIVO OF
D E T E R M I N A N T S OF M U C O S A L COLONIZATION,
PENETRATION, INTERFERENCE WITH HOST
D E F E N C E A N D D A M A G E TO T H E H O S T

Far more is known about these determinants than those responsible for
growth.

(a) Bacterial activities

The fact that environmental conditions in vivo differ from those in vitro
and change as infection proceeds has the following implications. First, some
putative virulence determinants indicated by experiments in vitro may not
be formed in vivo, and even if they are, they may not be necessary for
virulence. Second, some determinants formed in vivo may not be produced
in vitro. Third, the complement of determinants may change as infection
proceeds and different anatomical sites are affected. The questions relate
to the validity of these implications.
16 H. Smith

(i) Confirming production in vivo and relevance to virulence of putative

determinants recognized in vitro
It is now standard practice in most studies of pathogenicity to con-
firm the production in vivo of putative determinants. Bacteria harvested
directly from patients or infected animals can be examined by conventional
methods (Smith 1990, 1996). The profiles of homogenates run on sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) can be ex-
amined for bands corresponding to those of purified putative determinants.
If the latter are antigenic, specific antisera can be used to immunoblot the
profiles and for fluorescence microscopy of bacteria in tissue sections. Also,
convalescent sera from patients or animals can be examined for appropri-
ate antibodies by using them to immunoblot SDSPAGE profiles of in vivo-
and in vitro-giown organisms. In addition, the new methods for detecting
gene expression in vivo (Table 1) can confirm production in vivo of pu-
tative determinants, e.g. tcp (the toxin-coregulated pilus) of V. cholerae
by STM (Chiang & Mekalanos 1998). Usually the putative determinant
is produced in vivo but not always. For example, capsular polysaccharide
type 5 is formed by Staphylococcus aureus in vitro in aerated cultures but
minimally in the lungs and nasal polyps of cystic fibrosis patients (Herbert
et al. 1997). It is also usual to prove relevance to virulence of the puta-
tive determinant by showing that deficient mutants are less virulent than
wild-type strains in animal models of infection, followed by complement-
ing the deficient gene (Falkow 1988). In most cases, relevance to virulence
is proven, but not always, even when the putative determinant is present
in vivo. The 17 kDa product of the ail gene of Yersinia enterocolitica is
formed in Peyer's patches of mice (Wachtel & Miller 1995) but experi-
ments with an aiZ-deficient mutant indicated that the gene is not required,
either for primary invasion or to establish systemic infection. In cell cul-
tures, Opa proteins are determinants of cell invasion by gonococci grown in
vitro (Dehio et al. 1998). Gonococcal strain FA1090 produces at least eight
antigenically distinct Opa proteins. When an Opa-negative variant was in-
oculated into volunteers, Opa proteins were expressed in a large proportion
of the re-isolates. The predominant Opa variants differed between subjects.
Hence, Opa proteins are formed in vivo. However, one variant expressing
Opa protein F, which was highly represented in the re-isolates, was no more
infective for volunteers than an Opa-negative variant (Jerse et al. 1994).
 Questions about the Behaviour of Bacterial Pathogens in Vivo 17

Another encouraging trend is that results of cell culture tests for viru-
lence determinants are being viewed in relation to the pathology of disease.
In the forefront of such studies is penetration of the mucosa by intestinal
pathogens for which the determinants of invasion of epithelial cell lines are
known. However, in vivo these pathogens do not usually invade epithelial
cells directly. They penetrate the mucosa via M cells present in Peyer's
patches and elsewhere (Jepson & Clark 1998). Now, the determinants of
invasion of cell lines are being investigated for a role in this process. Pioneer-
ing studies were done on S. flexneri (Sansonetti et al. 1999). Experiments
with Hela cells showed that entry, intracellular movement and transfer be-
tween cells were determined by plasmid gene products IpaB, IpaC, IpaD
and TCSD- Then, experiments with confluent polarized colonic epithelial
cell lines, e.g. CaCo-2 cells showed that shigellae could not penetrate the
intact brush border that exists in vivo and is absent from Hela cells. In
vivo, shigellae are ingested by M cells and delivered to macrophages in the
lamina propria. IpaB causes apoptic death of the macrophages and IL-1/3
is released. Inflammation follows with disruption of the epithelial cells so
that shigellae can invade the sides and bases of these cells, using the deter-
minants recognized in the Hela cell studies. In vivo, S. typhimurium causes
membrane ruffling of M cells, then is internalized leading to cell destruc-
tion as for tissue culture cells (Jones et al. 1994; Jepson & Clark 1998).
Mutants deficient in salmonella pathogenicity island SPIl genes, and thus
unable to enter tissue culture cells, caused membrane ruffling and entered
M cells but to a lesser degree than the wild-type (Clark et al. 1996; Jepson
& Clark 1998). Also, they killed mice after oral inoculation but the LD50S
were higher than for the wild-type. Hence, the determinants of invasion
of cell lines by S. typhimurium have some role in invasion via the M-cell
system in vivo but unknown determinants are also involved.

(ii) Examining bacteria grown in vivo for hitherto unknown virulence

determinants
The fact that some virulence determinants may not be formed in vitro is
generally accepted. Increasingly, the genes and their products in bacteria
harvested from patients and infected animals are being compared with those
of bacteria grown in vitro to reveal differences that may be biologically im-
portant. Also, for intracellular pathogens, organisms grown in macrophages
18 H. Smith

are compared with those from cultures. Such studies are aimed at recog-
nizing potential diagnostic aids and immunizing antigens as well as vir-
ulence determinants. Over the past ten years, conventional studies using
SDS-PAGE have revealed many hitherto unknown bacterial components of
numerous different pathogens (Smith 1990, 1996). Now, the new methods
for detecting genes expressed in vivo (Table 1) are recognizing new genes
of potential importance.
Having recognized a previously unknown bacterial component, the next
step is to prove that it is a virulence determinant by biological tests re-
lated to pathogenicity and virulence tests on appropriate mutants. Unfor-
tunately, in conventional studies, this follow-up has not been as popular
as the original demonstration of a new component. For example, in the in-
testinal lumen of mice, Y. enterocolitica produced a plasmid-encoded outer
membrane protein (23 kDa), which had not been seen in culture. On inva-
sion of Peyer's patches, this protein and two further proteins (210 and 240
kDA) were formed (Nauman et al. 1991). There the matter remains. The
functions of these novel proteins in intestinal invasion have not been inves-
tigated. Follow-up on hitherto unknown genes revealed by the new methods
has been much better. In many cases, virulence tests have been done on ap-
propriate mutants and, for STM, the method itself detects only virulence
genes. As a result, many new virulence genes have been recognized, e.g.
by IVET and STM for V. cholerae (Camilli & Mekalanos 1995; Chiang &
Mekalanos 1998), S. typhimurium (Hensel et al. 1995; Mahan et al. 1993),
Y. enterocolitica (Young &: Miller 1997) and S. aureus (Lowe et al. 1998;
Mei et al. 1997).

(hi) Identifying virulence determinants as infection proceeds, together

with the regulatory processes involved
There are some indications that the requirements for virulence determi-
nants change as infection proceeds. For example, in experiments on gono-
coccal infection in volunteers (see §4), there was an indication that gono-
cocci lacking a sialylated lipopolysaccharide (LPS) are optimal for initial
invasion of epithelial cells but that later, to cope with host-defence mech-
anisms, production of sialylated LPS is an advantage. However, proof of
specific changes in virulence-determinant complements at different stages
of infection in animals has not been obtained. In studies with macrophages,
 Questions about the Behaviour of Bacterial Pathogens in Vivo 19

switch-on of genes at different times during an infection has been de-

tected. Differential fluorescence induction (DFI) (Table 1) distinguished
two classes of macrophage-induced genes, one induced within an hour of
S. typhimurium entering and the other after four hours (Valdivia & Falkow
19976).
Some observations have been made on the operation in vivo of regulatory-
systems detected in vitro. Two examples are discussed. The ToxR/ToxS vir-
ulence regulon of V. cholerae comprises over 20 genes involved in coloniza-
tion (e.g. tcp genes which code for toxin-coregulated pili) and production
of cholera toxin (Skorupski & Taylor 1997; DiRita et al., this volume). It
depends on a transcriptional activator ToxR, ToxS (a stabilizer of ToxR)
and ToxT, another transcriptional activator that is positively regulated by
ToxR. The regulon is modulated in vitro by temperature, osmolarity, pH,
oxygen status and availability of amino acids. The PhoP/PhoQ regulator
of S. typhimurium is a two-component regulatory system. PhoQ is the sen-
sor and PhoP is the transcriptional activator that is phosphorylated by
PhoQ. It controls expression of more than 40 genes, including those needed
to resist killing within phagocytes and those involved in lipid A synthe-
sis (Garcia Vescovi et al. 1994; Guo et al. 1997). In vitro, it is affected
by pH, oxygen tension, carbon and nitrogen starvation and phosphate and
magnesium concentrations (Garcia Vescovi et al. 1996).
In considering whether these and other regulons operate in vivo, it
should be remembered that production in vivo of a virulence determinant
whose formation in vitro is controlled by a certain regulator does not nec-
essarily mean that control in vivo is effected by the same regulator. To
confirm that the regulator is involved, expression of its genes should be de-
tected in vivo; deletion of the genes should reduce virulence; and relevant
environmental parameters should be present at the level at which they are
effective in vivo.
The IVET method did not detect expression of the cholera toxin gene
nor other ToxR/ToxS regulated genes in the intestines of infected mice
(Camilli & Mekalanos 1995); but STM in the same model detected mu-
tants with insertions in tcp and toxT (Chiang &: Mekalanos 1995). IVET
showed that the genes of the PhoP/PhoQ system were expressed in mice
infected with S. typhimurium (Heithoff et al. 1997; Conner et al. 1998).
However, in infected macrophages DFI and IVET identified two and one
gene, respectively, which were not controlled by PhoP/PhoQ (Valdivia &
20 H. Smith

Falkow 1997a; Heithoff et al. 1999), thus indicating that this system is
not the only one operating. With regard to virulence tests on mutants, a
ToxR/ToxS-deficient mutant of V. cholerae produced less colonization and
diarrhoea in volunteers than did the wild-type (Herrington et al. 1988), and
PhoP/PhoQ-deficient mutants were avirulent for mice (Miller et al. 1989;
Garcia Vescovi et al. 1996). The influence of environmental parameters is
discussed later.
Recently, two new regulatory systems have been described, quorum
sensing and type III secretion (Williams et al., this issue; Cornells, this
issue). They are summarized here, in order to ask some questions about
their operation in vivo. In quorum sensing, transcriptional activators of
virulence-determinant production only go into operation when a signifi-
cant cell population has been attained (Guangyong et al. 1995; Winson et
al. 1995). The cell-density dependency reflects a cell-to-cell communication
system based on accumulation of signal molecules to a threshold concentra-
tion. The signalling molecules for P. aeruginosa are N-(3-oxododecanoyl)-
L-homoserine lactone and N-butanoyl-L-homoserine lactone, which switch
on two transcriptional activators, LasR (regulates expression of the elas-
tase LasB) and RhiR (regulates expression of rhamnolipid), respectively
(Lafiti et al. 1996; Pesci & Iglewski 1997). Together they regulate virulence
determinants, secondary metabolites and survival in the stationary phase.
The signalling molecule for S. aureus is an octapeptide which activates the
accessary gene regulator (Agr), which positively regulates production of ex-
tracellular toxins (e.g. a-toxin, /3-toxin and toxic shock syndrome toxin 1)
and fibronectin-binding proteins (Guangyong et al. 1995).
In vivo, quorum sensing cannot operate early in infection because the
numbers of pathogens are too small. To be certain that it operates later,
evidence is needed for expression of relevant genes, reduced virulence if
these genes are mutated, attainment of requisite population densities and
detection of signalling molecules. Some of this evidence is emerging. Ex-
amination of RNAs from P. aeruginosa in the sputum of cystic fibrosis
patients indicated that lasR transcription occurs and may coordinately
regulate virulence genes lasA, lasB and toxA (Storey et al. 1988). Also,
ZasiZ-deficient mutants of P. aeruginosa were less able than the wild-type
to produce pneumonia in neonatal mice (Tang et al. 1996). They were, how-
ever, equally able to infect the corneas of mice (Preston et al. 1997). IVET
demonstrated the expression of the agrA gene of S. aureus in mice (Lowe
 Questions about the Behaviour of Bacterial Pathogens in Vivo 21

et al. 1998) and staphylococcal mutants defective in agrA were of reduced

virulence (Gillaspy et al. 1995).
Type III secretion systems respond when bacteria contact eukaryotic
cells. They induce secretion of virulence determinants from the bacterial cell
and deliver them into host cells (Hueck 1998). A good example is the Yop
protein system of Yersinia spp. (Cornells 1998; Cornells et al. 1998). The
Yop virulon is encoded by a 70 kb plasmid, pYV. It consists of (i) a secretion
apparatus Ysc comprising over 20 proteins, (ii) a delivery (to host cells)
system consisting of YopB, YopD, LcrV and YopQ/YopK, (iii) a control
element, YopN, TycA and LcrG, and (iv) a set of effector proteins that harm
phagocytes, YopE, YopH, YpkA/YopO, YopM and YopT. Transcription of
genes is influenced by temperature changes and cell contact.
S. typhimurium has two type III secretion systems comprising many
genes dealing with secretion, delivery, regulation and production of effects
on host cells. One deals with export and translocation to host cells of in-
vasion proteins that are responsible for membrane ruffling and entry of
epithelial cells in culture (Galan 1996; Hueck 1998). The other is involved
with virulence in mice and proliferation in macrophages (Shea et al. 1996;
Hensel et al. 1998).
In proving operation in vivo of type III secretion systems, it will not
be easy to show that they are switched on by contact with relevant host
cells in tissue sections or biopsies as has been demonstrated for contact
with tissue culture cells (Petterson et al. 1996). However, the new methods
such as CLSM may help in this respect. It will be easier to demonstrate
the expression of the regulatory genes of these complex systems in vivo
and reduction of virulence when these genes are mutated. Indeed, this has
been done for the second type III secretion system of S. typhimurium. The
system was discovered by STM showing expression of relevant genes in vivo.
Mutation of one regulatory gene, P3F4, resulted in loss of virulence (Shea
et al. 1996).

(b) Host factors

First, as for growth, factors in the environment that could affect production
of virulence determinants (osmolarity, pH, Eh and metabolites) should be
recognized and their levels or concentrations in vivo determined. Then, at-
tempts should be made to mimic in vitro the conditions in vivo and observe
22 H. Smith

the effect on virulence-determinant production and its regulation. Although

receiving far less attention than bacterial activities, such experiments are
beginning. Six invasion genes of S. typhimurium were maximally expressed
in vitro at an oxygen tension, osmolarity and pH likely to exist in the ileum
(Bajaj et al. 1996). Conditions designed to mimic those of the intestine
showed that two type III secretion genes of S. typhi, invG and prgH were
induced by high osmolarity, anaerobic conditions and pH 6.5, and strongly
repressed at pH 5.0 (Leclerc et al. 1998). When S. typhi was grown at an os-
molarity (300 mM NaCl) similar to that of the human intestine, production
of flagellin and salmonella invasion proteins increased, and that of the sur-
face Vi antigen, which prevents their secretion, was depressed (Arricau et
al. 1998). This would facilitate contact with and entry into epithelial cells.
The position was reversed at an osmolarity (150 mM NaCl) similar to that
within the tissues (Arricau et al. 1998). The Yst regulated toxin of Y. en-
terocolitica is produced at 37°C in vivo but not in culture media unless
the temperature is below 30°C. However, if the osmolarity and pH of the
medium are adjusted to values normally present in the ileum, toxin produc-
tion at 37° C occurs (Mikulskis et al. 1994). Similarly, in vitro expression
of the invasin gene inv by Y. enterocolitica is depressed at 37° C compared
with that at lower temperatures, but it increases significantly if the pH is
less than 7 (which occurs in the stomach) and when concentrations of N a +
increase (which occurs near the enterocyte brush border) (Pepe et al. 1994).
The level of inv expression in the mouse intestine is comparable to that at
23°C in vitro.
In some cases the environmental conditions that affect regulons in
vitro have been shown to exist in vivo. The PhoP/PhoQ system of S. ty-
phimurium appears to be controlled in vivo by Mg 2 + as it is in vitro. During
infection, S. typhimurium resides in phagosomes where the Mg 2 + concen-
tration (estimated 50-100 /xM) is permissive for phoP/phoQ expression,
unlike the high concentrations (0.5-1.0 mM) found in cytosols and body
fluids (Garcia Vescovi et al. 1996). Also, a phoQ mutant that was less re-
sponsive to Mg 2 + was of attenuated virulence for mice (Garcia Vescovi et al.
1996). In contrast, the position on the ToxR/ToxS system is not as clear.
Classic strains of V. cholerae form toxin maximally in vitro at low tem-
peratures and under aerobic conditions at low pH, whereas in the human
intestine, toxin production occurs at 37°C and under anaerobic conditions
at high pH. The actual environmental control of the ToxR/ToxS system in
 Questions about the Behaviour of Bacterial Pathogens in Vivo 23

vivo is still under investigation (Skorupski et al. 1997; DiRita et al., this
volume).

4. SIALYLATION OF G O N O C O C C A L LPS B Y H O S T
C M P - N A N A A N D E F F E C T OF LACTATE: A
P A R A D I G M FOR INVESTIGATION OF B E H A V I O U R
IN VIVO

Sialylation of gonococcal LPS by host-derived cytidine 5'-monophospho-N-

acetyl neuraminic acid (CMP-NANA) and lactate has a major influence
on many aspects of gonococcal pathogenicity. This was revealed by investi-
gating the cause of a biological property of gonococci in urethral exudates
which was lost on subculture in vitro. This work shows how bacterial ac-
tivities in vivo, relevant host factors and the metabolism concerned can be
identified. References to papers up to 1995 are given in Smith et al. (1995).

(a) Sialylation of LPS by host CMP-NANA affects

pathogenicity

Gonococci in urethral exudates are resistant to complement-mediated

killing by fresh human serum. In most cases, resistance is lost on one subcul-
ture in vitro but it can be restored by incubation with blood cell extracts.
Fractionation showed that the resistance inducing activity is due to CMP-
NANA. After growing gonococci with CMP- 14 CNANA, autoradiography
of LPS bands separated by SDS-PAGE showed that some, but not all,
LPS components are sialylated. One sialylated component of 4.5 kDa is
conserved in many strains and its side chain is Gal/?l-4GlcNac/?l-Gal/3l-
4Glc. The sialylated LPS forms an irregular surface coat, which is seen
on gonococci in urethral exudates whose LPS was shown to be sialylated.
A previously unknown gonococcal sialyltransferase was demonstrated in
gonococcal extracts. LPS sialylation is responsible for serum resistance since
conversion to resistance accompanies sialylation by CMP-NANA and rever-
sion to sensitivity occurs when sialyl groups are removed by neuraminidase.
The crucial importance of LPS sialylation in serum resistance of gonococci
is now generally accepted (Vogel & Frosch 1999). Sialylation of LPS also
interferes with the following host-defence mechanisms: absorption of com-
plement component C3; ingestion and killing by PMN phagocytes; killing
24 H. Smith

by antisera against gonococcal proteins; and stimulation of the immune re-

sponse. On the other hand, sialylation prevents invasion of epithelial cells.
Observations on volunteers were consistent with these results. When they
were inoculated with a strain whose LPS could not be sialylated, a variant
was selected in vivo whose LPS could be sialylated (Schneider et al. 1991).
This variant, recovered from the volunteers, was more virulent than the
parent strain provided it was inoculated after being grown in a medium
without CMP-NANA (i.e. its LPS was not sialylated so that it could in-
vade epithelial cells) (Schneider et al. 1995, 1996). If the inoculum was
grown with CMP-NANA, it did not infect volunteers so well, consistent
with inhibition by LPS sialylation of the ability to invade epithelial cells
(Schneider et al. 1996).
A sialyltransferase-deficient mutant, in contrast to its parent strain, did
not become serum resistant when incubated with either CMP-NANA or
blood cell extracts (Bramley et al. 1996). Hence, the latter do not con-
tain a mechanism for sialylating gonococcal LPS, which is independent of
CMP-NANA. Also, unlike the wild-type, incubation of the mutant with
CMP-NANA did not increase resistance to ingestion and killing by PMN
phagocytes, killing by antiserum to porin I and human complement, bind-
ing of C3 of complement, and invasion of epithelial cells (Gill et al. 1996).
The mutant was unable to sialylate any of its LPS components, which were
shown by mass spectrometry to be similar to those of the parent strain.
They included components sialylatable by the sialyltransferase from the
parent strain, such as the 4.5 kDa conserved component mentioned above
(Crooke et al. 1998). Hence, loss of ability of the mutant to be converted
by CMP-NANA to resistance to serum killing, and all the other facets of
pathogenicity mentioned above, is attributed to loss of sialyltransferase ac-
tivity rather than inability to synthesize the LPS substrate for sialylation.
Final confirmation would have come from complementation of the mutant
by the gene for the sialyltransferase which has been cloned and sequenced
(Gilbert et al. 1996). This could not be achieved (Crooke et al. 1998) so it
seems that multiple genetic loci may be essential for LPS sialylation. The
mutant has not been examined in human volunteers. A sialyltransferase-
negative mutant of gonococcal strain FA 1090 was as infective as the wild-
type for volunteers (Cannon et al. 1998), but this is not unexpected because
this strain is fully resistant to serum killing (Cohen et al. 1994) and does
not require LPS sialylation to make it so.
 Questions about the Behaviour of Bacterial Pathogens in Vivo 25

(b) Host lactate enhances LPS sialylation, and LPS and

protein synthesis

Another blood cell factor, which enhances the ability of CMP-NANA to

sialylate gonococcal LPS and to induce serum resistance, has been identi-
fied as lactate (Parsons et al. 1996a). The enhancement occurs with minute
quantities of lactate in a defined medium containing high concentrations of
glucose. The action of lactate is separate from that of CMP-NANA because
enhanced sialylation occurred when gonococci were pretreated with lactate
(Parsons et al. 19966). Lactate did not increase the gonococcal content of
sialyltransferase (Gao et al. 1998). On the other hand, there was a marked
increase in LPS synthesis (10-20%), which could explain the enhancement
of sialylation because additional receptors for sialyl groups are provided.
The increase in LPS synthesis was paralleled by increases in protein syn-
thesis and ribose content, presumably reflecting additional ribosome pro-
duction (Gao et al. 1998).

(c) Metabolic effects of lactate on gonococci growing in a

medium containing glucose, as occurs in vivo

The increases in LPS, protein and ribose synthesis, first noticed under the
conditions for detecting enhancement of sialylation by lactate, also occurred
when both glucose and lactate concentrations in the defined medium were
adjusted to levels akin to those occurring in vivo (Gao et al. 1998). Hence,
there appears to be a general stimulation of gonococcal metabolism when
lactate is added to media containing glucose, and there is other evidence.
Lactate increased oxygen consumption by gonococci in a solution containing
glucose (Britigan et al. 1988). Also, in the above-defined medium, growth
rate was faster and lactate was metabolized side-by-side with glucose and
more rapidly (Regan et al. 1999). When gonococci were grown with 1 4 C-
labelled lactate in this medium (Yates et al. 1999), tricene SDS-PAGE on
homogenates showed that lactate is not a general carbon source. Label was
concentrated in a low Mr component, LPS and a few proteins. N-terminal
sequencing of the three most heavily labelled proteins showed one (M r ca.
58 kDa) to be the chaperone, GroEl and another (M r ca. 35 kDa) porin
IB. Nuclear magnetic resonance after 13 C labelling, and thin layer chro-
matography following 14 C labelling (Yates et al. 1999), showed the low M r
26 H. Smith

component to be lipid. Gonococcal membrane lipids consist mainly of phos-

phatidyl ethanolamine and glycerol esterified to palmitic, myristic, a 16:1
and a 18:1 acid (Sud & Feingold 1975). In the glucose-containing medium,
the carbon atoms from the 13 C lactate were incorporated specifically into
the fatty acid portions, in contrast to both glycerol and fatty acid moeities
when 13 C glucose was used without lactate present. The location in the
LPS of the 14 C label from the lactate is not yet known but the fatty acid
residues seem likely.
The incorporation of lactate carbon into GroEl is interesting in two
respects. GroEl would ensure correct folding of the products of the large
increase in protein synthesis (Gao et al. 1998). Also, it could contribute
to the inflammation seen in gonorrhoea, since it is a potent stimulator of
relevant cytokines (Coates & Henderson 1998); and it is significant that
patients have high levels of antibody to GroEl (Demarco de Hormaeche et
al. 1991). Porin IB plays a major metabolic role, membrane transport, in
gonococci (Gotschlich et al. 1987) and can contribute to pathogenicity by
inserting into host-cell membranes (Bjerknes et al. 1995). Stimulation of
lipid formation would aid membrane synthesis and therefore metabolism
and growth. It is fascinating that gonococci have adapted to use lactate
and glucose, which are ubiquitous in vivo, to produce a vibrant metabolism
and a large content of virulence determinants such as LPS and GroEl. The
marked effects of minute amounts of lactate on LPS, protein and ribose
synthesis in a medium containing large quantities of glucose suggests that
lactate may have a signalling as well as a metabolic role.

(d) Extension of the work to meningococci

The work on gonococci stimulated similar investigations on meningococci.

Some strains contain LPS components that are endogenously sialylated
(serogroups B, C, W and Y) and others have components that can be sia-
lylated exogenously by host CMP-NANA (groups A and 29E) (Smith et al.
1995). An LPS sialyltransferase is present (Smith et al. 1995). LPS sialyla-
tion affects facets of pathogenicity but not as markedly as for gonococci be-
cause capsular polysaccharide is the more powerful virulence determinant.
It is sometimes difficult to distinguish between their respective roles. LPS
sialylation inhibits meningococcal invasion of epithelial cell lines, endothe-
lial cells and mono- and PMN phagocytes (McNeil & Virgi 1997; Virgi et al.
 Questions about the Behaviour of Bacterial Pathogens in Vivo 27

1993). Also, it interferes with opsonophagocytosis of some strains (Smith et

al. 1995). T h e position regarding serum resistance is equivocal; some papers
indicate t h a t L P S sialylation is important (Esterbrook et al. 1997; Kahler
et al. 1998) and others t h a t it is less so (Vogel et al. 1997; Vogel & Prosch
1999). In a n outbreak of group B meningitis, a n immunotype capable of
L P S sialylation was associated with invasive disease and a n immunotype
incapable of L P S sialylation with the carrier state (Smith et al. 1995). T h e
effect of lactate on meningococci has not yet been investigated.

5. CONCLUDING REMARKS

I hope this paper has m a d e clear t h e pertinent questions about the be-
haviour of bacterial pathogens in vivo; a n d has indicated how they might
be answered, despite difficulties in some areas, by a combination of conven-
tional and newly devised methods.
I a m indebted to J. A. Cole, M. J. Gill, N. J. Parsons C. W . P e n n a n d
E. Yates for critical reading of t h e manuscript.

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Discovering Diverse Content Through
Random Scribd Documents
Baxter and compel him to rest in England without forcing me to break my
vows."

"Your vows, your precious vows, always your vows!" she cried, in
anger and great contempt.

"Yes," he retorted instantly, "my vows, always my vows. They are

precious to me indeed, and I will beg you not to speak of them lightly."

She faced him with increasing anger. But, before she could speak,
Antonio suddenly repented himself of his sharpness.

"Isabel," he said, in quieter tones. "Think. You despise me for keeping

my vows. But suppose I had vowed my vows to you. And suppose I should
break them, for some other woman. What then?"

"I would kill her. And you too."

For a moment her wrathful excitement hindered her logical perceptions;

but as soon as she recognized his meaning she cried:

"It's different, all different! I'm real; your Bride isn't. Besides, She has
deserted you. She's run away, or She's dead. You are free."

"No, Isabel," he said. "Think again. Suppose to-day I should vow my

vow to you. Suppose your father, or someone else, should pluck you
suddenly from my side so that I could never find you again. Nay, more.
Suppose you were untrue to me and that you abandoned me. Would you
have me say: 'She has gone. I shall never see her again. To-morrow I will
seek another bride?' No, Isabel, no. If you say Yes, I shan't believe it. I
know your soul too well. Even if you broke yours, my vow would still be
there, and you would despise me for not keeping it. Am I right or wrong?"

He had unguardedly lowered his tones to a perilous tenderness, and he

was unconsciously gazing at her with the gaze she could never resist. Her
lips lost their hardness and began to tremble, and her eyelids drooped over
her eyes.
 Antonio involuntarily recoiled from the danger. He knew in an instant
that his fate was quivering in the balance. His heart had bled at every harsh
word he spoke to her; and he knew that to sweep away the last shaken ruins
of his defenses, she needed only to throw herself weeping into his arms. He
knew that if she should once sob out, "Antonio, Antonio, don't send me
away," his doom would then and there be sealed.

All this Antonio knew. But Isabel did not know it. His sudden
movement of recoil stung her back into anger.

"Are you right or wrong?" she echoed bitterly. "You're right, of course.
You always are. Even when you're wrong fifty times over, you can argue
yourself into the right. I call it cowardly."

He exhaled a deep breath. The peril was past. Her scorn he could
withstand.

"I have come to the end," she cried. "The very end. Listen. You are
blighting my life, but I won't let you blight your own. Mark me well. This
place is mine. These lands are mine. I have the right to go to-night and to
set the whole abbey ablaze; and where will your work be then?"

The threat did not alarm him; but the cruelty of it, coming from such
lips as hers, cut him to the marrow. He was on the point of retorting that the
place was not hers at all, and that her father had deceived her on a wretched
point of money. But her anguish was bitter enough without this new
mortification; so he held his peace.

"I can make a bonfire of it this minute," she went on passionately. "I
hate it. How I should love to see it blaze! But I won't. And I won't sell this
place. And when I've left it on Thursday, I'll never come back till you seek
me on your knees. Never!"

Still Antonio held his peace. Isabel picked up her little bag. But she did
not turn immediately towards home. She stood awaiting his final word.
When it failed to come her indignation rose to its climax.
 "No!" she cried. "I've altered my mind. I will come back. I foresee the
end. You will never seek me. You hate me. But I will come back. You'll go
on slaving, slaving, starving, starving, praying, praying, and breaking hearts
in the name of God. But I will come back. You'll succeed. You'll regain the
abbey. You'll fill it with monks. But remember. I will come back. On the
day of your triumph, I will be there. It isn't only you Southern people who
love revenge. I will be there. I will come back!"

Antonio had been silently praying for sudden grace in his own dire
need; but he ceased to pray for himself and prayed with all his soul for her.
She turned to go.

They stood facing one another as they had stood so often during these
two bitter days of their ordeal. Try as he would the monk could not conceal
his agony of holy love; and under the spell of his gaze the devil of
revengeful hate which had entered into Isabel rent her poor heart and fled
away. They looked at each other a long time. Then, in a breaking voice, she
said softly:

"Antonio. I don't hate you. I love you. This is the very last time. Do you
send Isabel away? Is it true that I must go?"

With a sharp moan of anguish and with hands thrust out for mercy he
gave his answer.

"For the love of Jesus Christ," he cried. "Go! And may the merciful God
help us both!"

He closed his eyes in desperate prayer. But God and the Virgin Mother
and the whole company of heaven seemed to have forsaken him. No light
shown, no supernal fortitude came down. Instead of a vision of ministering
angels, his mind's eyes saw only Isabel. Isabel, standing there. Isabel,
weeping. Isabel, wounded to death by his cruel sword. Isabel, hoping
against hope for his mercy. Isabel, his Isabel, rarer than gold, lovelier than
the dawn, purer than snow, waiting to dart like a bird into the nest of his
love.
 He could fight no longer. Stepping one staggering step forward he held
out his arms and opened his eyes.

She had vanished.

A moment later he caught sight of her pressing up the path above him.
She was going swiftly, looking neither to the right hand nor to the left. Now
and again a ray of the sinking sun shone upon her hair, till she seemed a
queen crowned or a saint glorified.

With all his heart Antonio yearned to leap after her, to capture her like a
shy creature of the woods, and to bear her back in triumph, seated on his
shoulder as she had sat after the thunderstorm. But his limbs refused to
obey. His feet seemed to have been rooted for centuries in the granite. He
could not move an inch.

Two cypresses, which they had often halted to admire, hid her from his
sight. A groan, which he could not stifle, broke from the monk. There was
one more point in the path, one only, where she could reappear. Would she
turn round? Would she look back? As he waited, red-hot pincers seemed to
be working and worming within him as if they would have his heart out of
his body. He felt as if he were bleeding at every pore.

She reappeared. She did not turn round. She did not look back. She was
gone.
 BOOK VI

"ITE, MISSA EST"

Having charged José to place himself at the disposal of Mrs. Baxter,

Antonio took the road for Villa Branca about an hour after sunrise. Utter
weariness had brought a few hours' sleep to his eyelids; but he felt unrested
and unrefreshed. By the time he reached Santa Iria fatigue compelled him to
hire a horse.

While his mount was a-saddling the monk sat musing outside the wine-
shop. What was Isabel doing? Of what was she thinking? Had she slept?
Was she truly hating him at last? Would she come once more to the
cascade?

In answer to this last question he could hardly restrain himself from

leaping on the half-ready horse and galloping off like a whirlwind to her
presence. At the moment of his leaving the farm-house, two hours before,
this all-day expedition to Villa Branca had seemed the height of prudence;
but he suddenly saw it as the depth of cowardice and brutality. She would
come to the cascade, in vain; and, later on, she would learn from José's lips
how he had turned tail and run away. Antonio cringed and burned. A
moment later, however, he knew that he had done right. She would not be at
the cascade.

"To-morrow," he said to himself, with a dull pain gnawing in his cold

and heavy heart, "I shall see her for the last time. She will make no sign.
She will say good-bye as if there has been nothing between us. Blessed
Mother of God, help us to the end!"

He took out Sir Percy's letter and perused it once more to distract his
thoughts. He read:

Dear Senhor da Rocha,—

A post just to hand apprises me of your gentility to my daughter and her

governess. The fact that I fully expected such courteous behavior on your
part does not diminish my gratitude in respect of it; and I beg you to believe
in the sincerity of my regret that I shall be unable to present my
acknowledgments in person.

I indulge the hope that a proposal which I am about to make may not be
unacceptable to you. From our mutual friend Mr. Austin Crowberry I learn
that you wished to purchase the abbey domain, but that your offers were
unacceptable to the Minister of Finances.

I have paid a deposit of £500 to the chief of the Fazenda at Villa

Branca, and am engaged to pay £300 on New Year's Day and the balance
(£2500) in five half-yearly instalments. As I have become closely associated
with an enterprise which will involve my residing alternately in Lisbon and
London, I should find it convenient to transfer to yourself my whole bargain
as regards the abbey. That is to say, I forfeit the £500 already paid and
leave you to find £2800 on the dates above referred to. I also ask your
acceptance of the larger articles of English furniture recently placed by me
in the guest-house, and I have instructed Jackson, my man, to bring away
personal luggage only.

As my movements are erratic, perhaps you will indulge me by

completing the business with my agents, Messrs. Lemos Monteiro and
Smithson, Rua do Carmo, Lisbon, who have written to Villa Branca
preparing the officials for your visit. Failing your approval I will make
other arrangements; but, meanwhile, I beg that you will add to your
unfailing kindness by taking care of the keys, and that you will believe me to
be

Your obliged and obedient servant,

Percival Kaye-Templeman.

Once in the saddle, with the well-beloved music of horse-hoofs in his

ears, Antonio found it easier to abstract his mind from bitter thoughts. He
applied his whole brain to problems of finance. Two thousand five hundred
pounds in two years and a half. At first it had staggered him; but he was
going to take the risk. His own and José's hard cash hoardings would pay
the New Year's Day instalment nearly twice over. By mortgaging the farm
and the sea-sand vineyards, and by pledging his personal credit he could
pay the July five hundred and keep two or three hundred towards the
instalment due the following January, making up the balance from the year's
wine-sales. Fifteen hundred pounds would remain payable; and this sum he
hoped to raise in due course by a bold stroke involving a mortgage on the
abbey itself.

The chief of the Fazenda received his visitor effusively. This time the
monk was not required to lean against a pile of stolen books. He sat in the
chief's own chair and was offered wine of the chief's own stealing. As three
hundred pounds of Isabel's money had stuck to the chief's fingers the great
man was more than willing to accept Antonio in Sir Percy's place; for he
had just learned that the Englishman would be unable to meet his
obligations, and he was mortally afraid of a reopening of the transaction in
Lisbon. He even threw out mysterious hints as to further concessions which
might be arranged. Antonio listened attentively. His conscience allowed him
to plan the outwitting of the Portuguese Government as regards money
which was not honestly theirs. But as soon as he perceived that the official
was bent on more pickings for himself the monk became obtuse. He was
not willing to assist any man in the work of more completely damning his
soul; and, although Antonio clearly foresaw that he was making an enemy
and preparing sore troubles for himself in the future, he steadfastly held out
against temptation.
 The autumn day was drawing to its twilight when Antonio, having given
up his horse at Santa Iria, trudged up the path to his own door. Half the way
home Isabel had queened his whole mind. On leaving Villa Branca he had
sought to preoccupy himself with the most complicated arithmetic; but,
little by little, Isabel had reclaimed her empire. As he mounted the doorstep
his heart thumped heavily. Had she written? Had she sent a message by
José? Or, most terrible and beautiful possibility of all, would he find her
sitting in the house, as in her rightful place?

He entered. There was no Isabel enlightening the dim and cheerless

room. He hurried to the table whereon, José was accustomed to leave the
letters. There was nothing. His heart chilled and shrank. Still, there was to-
morrow. Yes. He was certain to see her to-morrow.

José stamped in noisily and handed Antonio two keys.

"They have gone," he said.

So sharp a blade of anguish pierced his soul that Antonio let the keys
fall on the brick floor.

"Gone?" he echoed. "Who? When? Why? Where?"

"The English senhoras," answered José. "They started about three

o'clock, to Lisbon."

Antonio sank down upon a coffer. He had used up the last of his
strength in tramping from Santa Iria, and he had eaten nothing all day.

"I don't understand it very well," continued José. "I reached the guest-
house at half-past eight. I thought they weren't to leave until to-morrow. I
worked under the Senhor Jaxo. He didn't hurry himself at all. Joanninha
brought us cold meat and white bread and strong wine. Joanninha is the
cook. She has the longest tongue, your Worship, in Portugal. She made me
angry, talking about your Worship."

"About me? How?" asked Antonio. He felt sick and faint.

 "She heard me say that your Worship would attend the senhoras to-
morrow morning. She said: 'Where is his Excellency to-day? I suppose he's
gone to see Senhor Jorge's Margarida.' I said: 'No, his Worship has
something better to do. He has gone to Villa Branca to mind his own
business, and it would be a good thing if everybody else would do the
same.' There was an English servant in the room, called Ficha. She's maid
to the Senhorita Isabel. Joanninha translated to her what I'd said, and they
both laughed, and I was very angry."

"What has this to do with the senhoras going away in such a hurry?"
asked Antonio. But, even as he finished putting the question, his own fears
supplied the answer.

"It's nothing to do with the senhoras hurrying away at all," said José
humbly. "I beg your Worship's pardon for repeating such nonsense. All I
know is that some bells rang and the Senhor Jaxo went out, and when he
came back he was in a great rage. Joanninha told me that the Senhorita
Isabel had decided to go to her illustrious father at once, and that nobody
dared oppose her."

"Did you see the senhoras? Were they well?"

"I think they were well, because I heard them quarreling," José
answered. "The dark senhora, the old one, has a temper that made me
tremble, your Worship. They went away, the senhoras and the servants in
two old shut-up carriages, but they are going to hire a better carriage on the
way. I saw the old senhora, when she handed me the keys. She sent you a
long message, but I don't think Joanninha could translate it properly. So I
asked would she write, but she didn't. They locked all up and gave me the
keys. Then they went away. They didn't say when they will come back. I
think, your Worship, that they are all mad."

"José," said his master, after a long silence, "I have eaten nothing all
day. Let me break my fast. Afterwards I have something to tell you. Prepare
me what you can while I change my clothes."

He climbed the steep and narrow stairs painfully. His cold tub revived
him, and his old clothes gave him ease. But, as he lifted his worn cloak
from its hook, the wound in his heart burst open afresh. He remembered
how often Isabel had sat, in all her daintiness, upon that same cloak's clean
but rusty folds; and how, on her own confession, she had "cried and cried
and cried like a baby" at the sight of its threadbareness.

By the time he descended José had grilled two small trout and was
placing a bottle of good white wine upon the table. Antonio's heart was
wrung anew at the thought of the simple fellow's unfailing devotion. Isabel
had come and had gone; but José remained, loving and serving his strange
master with a dumb love passing the love of women. The monk forced his
faithful disciple to sit down at table with him and to take his fair share of
the dainty fish and the animating wine. When they had finished eating and
drinking he said:

"José, I have been a good deal in and about the guest-house and the
abbey since we saved the azulejos, and many strange things have happened.
The end of it all is this. Here are the keys of the guest-house. Upstairs, in
the green box, I have all the keys of the abbey. To-day, as you know, I have
been to Villa Branca. We are in legal possession of the abbey domain, and
everything in it. Within three years we must raise three thousand pounds.
With God's help it can be done. The English people will never come back."

He closed his eyes wearily. When he half-opened them he saw José by

the light of the one candle, bowing his head and silently repeating thankful
prayers. The monk quailed. For himself, as well as for José, this ought to be
a night of praise and rejoicing. Yet Antonio found it the darkest hour of his
life. The abbey keys seemed no more than a few bits of metal. Or, if they
were more than bits of metal, they were the keys of a prison, the keys which
were locking Isabel outside his life.

He took his candle and went to bed. But, despite his weariness, he could
not sleep. Where was she? In what rough inn, amidst what discomforts and
indignities, was she lying? If he jumped up at once and tramped southward
until he could find a horse, when would he overtake her? To-morrow, he
calculated, about noon. He imagined himself thundering after her chariot,
like a highwayman in a picture. He pictured her pretty alarm, her radiant
joy, her gracious forgiveness, their ecstasy of reunion.
 Suddenly the monk remembered with a shock that he had not said all his
Office. Busy or idle, sick or well, glad or sad, he had never failed to recite it
before. He still had None, Vespers, and Compline to say. Lighting the
candle and opening his breviary he began to repeat the holy words. But he
had not uttered half a dozen sentences before he shut the book with a snap.

Half an hour later he arose, put together all the keys, and went down
stairs. The new moon had not set, and its brightness lured him forth from
his narrow room into the peace of the night. As a matter of course he took
the path to the abbey.

Although the ruts of wheels, her wheels, made him shiver he did not
turn back. He opened the chapel with the long key she had so often handled,
and sitting down in his old stall, he tried to say the rest of None; but a white
form, her form, hindered him, and a soft, glad voice, her voice, cried:
"Antonio, Antonio, Antonio—what a beautiful name!" He groped his way to
his own cell, and he could almost see and hear her opening his cupboards.
He hastened through the cloisters and escaped into the wood by the secret
door.

Some dead leaves fled before him, their tripping sound was no lighter
than the fall of her elfin feet. The moon suddenly peeped at him through a
clearing; and he saw her moon-white shoulders. The chirrup of a brimming
brook struck upon his ear; and he seemed to be carrying her once more in
his arms, while she murmured: "Listen, Antonio, all the world is singing."

He knew that the guest-house must tear his wound wide open, and that
he ought to hurry home to the farm; but an irresistible influence drew him
on. He reached the broad path. He stood under the casement whence she
had flung the white rose. It was still ajar.

He turned the key in the lock and entered the ghostly and silent house.
There was enough moonlight in the salon to show him the blue ottoman
whereon she had so often sat. He hurried out of the room with a heart ready
to burst.

At the foot of the stairs he paused. They led to her chamber. Could he
bear to cross its threshold, to lean out of the window as she had leaned out
after the thunder, and to look at the bed where she had lain sobbing for his
sake? He knew he could not bear it. But his intellect had ceased to govern
him and he ascended the stairs.

A broad moonbeam lit up every corner of her chamber. Like a man

dazed he lurched to the window. There were the roses and there were the
thorns. He turned to gaze at her couch. The fine linen had been taken away;
but there was the place where she had lain, there was the pillow which her
golden head had pressed. What had her last night been? Had she hated him
or did she love him still? Had she cursed God or had she prayed?

For a moment his mind turned the question over in a numb, impersonal
way. Then he came back with a rush to himself and, in a single moment, his
chalice of agony welled up and brimmed over. He flung himself down on
his knees and stretched out desperate hands and hungry arms across the
narrow bed.

Although long minutes passed his dry-eyed, stony anguish remained.

But at last his inward, spiritual man spoke. Was he committing a grievous
sin? Was he breaking, in spirit, a vow which he was only keeping in the
letter? Had he forsaken the Creator for a creature?

Slowly, but very surely, his conscience framed the answer. No, he had
not sinned. In all his desire of her there was still nothing of the carnal mind.
He was racked and scorched by anguish, not because he had lost her love,
but because he had been forced to break her heart by refusing her his own.
She was a child, a poor lonely child with neither man nor woman to love
her, nor any God to console her; and he, Antonio, had flung her back into a
still blacker frost and sharper famine, to pine and wither without love and
without faith. Yet, in all this, he had simply obeyed God. He had obeyed the
God who commanded Abram to offer up Isaac, the God who "spared not
His own Son, but freely gave Him up for us all."

The moonbeam softly faded from the chamber. But Antonio did not
move. His weary limbs and exhausted brain could resist no longer; and, still
kneeling against her pillow with his arms outstretched across her bed, he
fell asleep.
 II

When the monk awoke day was dawning. For a while memory failed
him. But as soon as he understood that he was in Isabel's room he leaped up
and hastened downstairs.

He knew that he ought to go straight home. But his feet, despite their
soreness, turned towards the stepping-stones. He retraced the path by which
she had left him, hardly thirty-six hours before. Past the cypresses, through
the mimosas, he went; and before the sun rose he was standing in the icy
spray of the thunderous waterfall. He longed to plunge into the crystal pool;
but her invisible presence abashed him, and with an ever-sharpening pain he
hurried away.

As he regained the farm, he found José burning some dead leaves. Why
could he not tear down these clinging memories of Isabel from his heart, as
José could tear down ivies from the trees, and fling them a-top of the
glowing, fuming pyre? The gust of pale, acrid smoke which nipped his
nostrils was bitter-sweet.

After a dip in the brook he drank some of the sham coffee and forced
down a hunk of coarse bread. But when he faced his routine he found that
he could neither work nor pray. The black and red letters in his breviary
danced impishly before his eyes; and when he took up a pen to write out
some accounts he marked the paper with more blots than figures. Both door
and window were wide open to the morning breeze; yet the room suffocated
him.

At last a plan formed in Antonio's brain and he did not delay its
execution. Stuffing a piece of bread in his pocket he sought out José and
said:
 "To-morrow my hard work will begin. To-day I am going to Navares.
After to-night I will not leave you so much alone."

He set out, striding northward with long strides. Every stride was a
symbol of his renunciation; for he knew that by this time Isabel would have
left her inn and that every moment was taking her farther southward to
Lisbon. On he pressed. As landmark after landmark came in sight a flood of
old memories diluted his bitter potion of new-brewed sorrow. He lived over
again the afternoon of his dusty march from the monastery amid a throng of
monks and soldiers and the evening of his solitary return. But not for long.
An hour before the white houses of Navares shone in the morning sun
Isabel had once more become the sole tenant of his mind.

The doors of the Navares' corn-factor's granary, where the monks had
held their council, were wide open; but Antonio did not pause to look
inside. As on the night of his flight, he hurried through the town and only
rested when he came to the knoll where he had bivouacked twice before.
Thence, after munching a little bread, he took the short cut through the
maize-fields to the village of the old cura; for the old cura's grave was the
goal of his hasty pilgrimage.

By an irony of fate a rustic wedding had drawn the whole population to

the church and churchyard. Their mirth so mocked the pilgrim's mood that
he had a mind to go away. But he mixed with the throngs until his
resentment at their gaiety was turned to thankfulness for the excess of
human joy over human sorrow. At last a horn was blown from the door of a
neighboring barn, and the crowd swept out of the churchyard like
stampeding buffaloes.

The plain grave of the old cura lay in a sheltered corner on the north
side of the chancel. Pious hands had brightened it with a yellow and purple
nosegay that very morning. Antonio did not kneel down. He simply
uncovered his head and strove to pray. For five minutes it was like chewing
chaff. Some devil whispered in the monk's ear that his errand was not only
silly, but in doubtful taste. The old cura was a saint, no doubt; but what had
so rough a diamond to do with so soft and lustrous and exquisite a pearl as
Isabel? Thus spake the devil, but Antonio refused his ear. Knowing that
prayer comes with praying, he prayed on.
 Not until he had replaced his hat on his head and was about to go were
his prayers answered. But when the answer came, it was an answer indeed.
It almost struck him down, like the great light which struck down Saul on
the way to Damascus, and he was forced to lean against the church wall. It
was an answer which both healed the worst of his grief and showed him the
most of his duty in a single flash. It thrust into his hand a golden key to the
whole mystery of Isabel, past and future.

Like a man whose shoulders have suddenly been eased of a burden he

swung out homewards, holding his head high. Without knowing it, he
talked to himself aloud, uttering broken phrases of hope and thankfulness.
Yes, he had found the key, the master-key to all that had happened. As he
strode along he recalled his association with Isabel from the beginning, and
there was no lock his key did not fit.

Even the problem which had tried his faith most sorely was solved. In
confiding to him her story of the mysterious influence which he had begun
to exercise over her, four years before she saw his face, Isabel had declared
that their lives were interfused in an irresistible destiny. She had spoken of
this as a fact more undeniable than the sun and moon. She evidently
believed with her whole soul that God's hand had brought them together.
Yet Antonio, all through her pleading, had remained more persuaded than
ever that the selfsame God had called him to the celibate life. And the
apparent impossibility of reconciling these two equally clear, equally honest
convictions had kindled a fiery ordeal for the monk's faith. The only way
out of it seemed to be that all inward illumination was a delusion—totum
corpus tenebrosum, "the whole body full of darkness"—and that perhaps
there was no Divine Enlightener at all. But this wonderful new thought
which had come to him at the old cura's grave explained everything. He
thrust it into the most complicated wards of his spiritual doubts, and it
turned as smoothly as the damascened key was wont to turn in the lock of
the chapel. The doors of Isabel's soul rolled open before his eyes, and a
bright light shone into the furthest cranny.

As for his duty to her in the present and in the future, he understood it
no less certainly than he understood her chaste love for him in the past.
And, as soon as this duty was plain, he made haste to begin doing it; for it
was a duty of prayer, of specific, faithful, heroic, loving, unceasing prayer.
He prayed as he walked, with increasing exultation.

So rapt was he by his holy work that Antonio hardly noticed the
difference between the dusty, lonely road and the cobbled streets of noisy
Navares. He pressed southward without a pause. Was he not going home?
After a day and a night of banishment had not the farm once more become
the tranquil home of his body, and had not the chapel once more become the
rapturous home of his soul? He strode the last long league of his homeward
journey as if it had been the first; and when he met José at the gate his face
was shining like an angel's.

True to his word, Antonio rose early the next morning and threw
himself body and soul into hard work. Now that the abbey domain had
come under his care, there were hundreds of things to be done. As the sunny
and well-drained slopes were exceptionally suitable for the culture of a
profitable amber-colored wine, Antonio decided to double the area of the
monk's old vineyard immediately. In order to effect this extension and to
repair the damage done by seven years' neglect, it became necessary to
engage nearly a score of helpers, half a dozen of whom would have to be
retained in permanent employ. José, with one resident laborer, continued to
live at the farm, while the monk quietly resumed occupation of his own cell
in the monastery.

On Mondays, Wednesdays, and Fridays, Antonio dined at the farm with

José; and on Tuesdays, Thursdays, and Saturdays, José dined with his
master beside the stream in the monastery kitchen. At these week-night
meals, the conversation was usually a review of the day's operations and a
debate as to the work of the morrow; but on Sundays, when dinner was
eaten ceremoniously in the guest-house, such topics were not mentioned,
and the talk was of the great world's doings as chronicled in Antonio's
English paper, of Portugal's troubles, and, above all, of churchly and holy
things.

Not only during these Sunday talks, but also throughout their work-a-
day intercourse, José was conscious of a change in Antonio. Hitherto, the
monk had simply accepted the shaggy fellow's dumb affection; but, after
the day of his visit to the old cura's grave, he began to show that he requited
it as well. The last remains of his aloofness vanished, his speech grew
gentler, and he became more watchful of José's health and comfort. Nor was
the monk's manner changed towards José alone. In all things and to all
persons he was more tender and less cold.

On the long winter evenings the two men busied themselves with blue
pigments and white glazes, until they succeeded in fabricating tolerable
copies of the two broken azulejos. When this was achieved, they began a
series of experiments, with a view to distilling a new liqueur from
eucalyptus. By rashly gulping down a mouthful of the first pint, José almost
burned out his tongue. Nevertheless, they persevered; and, in the long run,
the monkish talent for cordial-making enabled Antonio to mollify the
harshness of the fiery elixir, and to render it palatable. In January they
shipped samples to agents in fever-cursed regions of Spanish America, and
offered to supply the liqueur in bulk at a high price.

Meanwhile, Antonio was waxing stronger in faith, and hope, and love.
Every day he recited the whole of his Office in his old stall, sometimes with
José's assistance, sometimes alone. He began also to hear Mass in the
village church every Wednesday and Friday, and to say the whole rosary
every Sunday afternoon. In meditating on the fifteen Mysteries, he
habitually applied them to the case of Isabel; and, somehow, these thinkings
never became trite or stale. In pursuance of his plan for Isabel's well-being,
he redoubled his prayers, and offered half his Mass-hearings and
communions with the same intention.

The winter passed and the spring came; and still he had not heard a
word from her or about her. Sometimes a memory of her would suddenly
overwhelm him. When he dined at the farm with José there seemed to be
always three persons, not two, at the table. He felt that she was sitting at his
right hand, where she had sat when he gave her the painted bowl; and so
strong was his sense of her presence that he would often halt in the midst of
a sentence, as if to ask her pardon for the dryness of the talk. After the
morrow of her flight, he never visited the stepping-stones, although he
repeatedly gave José minute instructions for the conserving of the pool's
beauties. As for Isabel's chamber, he locked it up, and never re-entered it.
Yet, in spite of this reverence for everything she had touched, he never
moped or repined. He confided Isabel, as he had confided the fate of the
abbey, to the might and love of God.

When July came, he made a novena in honor of Saint Isabel, the holy
queen of Portugal, whose silver shrine was the glory of the Poor Clare's
great convent opposite Coimbra, on the heights above the Mondego. And in
August he received a long letter from young Crowberry. Seven of its eight
pages were concerned with England's theological and ecclesiastical affairs:
but in the midst of the page devoted to personal matters, the young man had
written:

Of course, you know that Isabel has taken her father to live at
Weymouth. I never see them; but I hear they are both well, and that Sir
Percy has become quite reasonable and docile. Have they told you how she
put her foot down and sent away that Excellent Creature, Mrs. Baxter? If
she hadn't pulled up Sir Percy I'm told he would have died. Now, what did
you really and truly think of Isabel? Did you see much of her, or did she
sulk? Tell me when you write.

Antonio wrote a long letter in reply; but he did not tell young
Crowberry what he really and truly thought about Isabel, nor did he so
much as mention her name. His novena was answered. It was enough for
him to know that Sir Percy lived, and that she was well.

The grape-harvest in September was a good one, and it was only by

cutting an hour from his sleep-time that the monk could fill full his
appointed measures of work and prayer. Then came October, with its
vintage of memories. On the anniversary of Senhor Jorge's serao Antonio
could be serene; for Margarida had just been happily married to a handsome
and honorable young man of Leiria, the son of a prosperous builder. But
with the approach of the anniversary of his first meeting with Isabel he
grew troubled; and, to divert his thoughts, he departed hurriedly for Lisbon,
where he had business to transact with the shippers of his wines and
cordials. In Lisbon he learned that a journey to England would be to his
advantage. But England meant Isabel; so, on the anniversary of her flight
from the guest-house, he turned his back on the capital and hastened home.

By mortgaging his farm the monk succeeded in paying the third

instalment of the abbey's price. He faced the New Year with less than
twenty pounds of ready money, and with the obligation to find five hundred
by the first of July. A request for a more flexible arrangement was flung
back at him by the Fazenda official with vindictive contempt. As the spring
advanced, Antonio laid his plan for the immediate outright purchase of the
abbey on a fifteen hundred pound mortgage before four separate persons;
but without exception they either could not or would not entertain it. In
these circumstances he felt bound to cut down his gifts to village charities
and his bounties to the hangers-on of the countryside. As a result, José came
home one day with a black eye, received while he was punishing three
village loafers for calling the Senhor da Rocha a skin-flint and a miser.

By May-day Antonio's sales of stock and the pledging of his credit had
brought him in only three hundred pounds, and there was nothing left that
he could pawn without crippling himself hopelessly in the near future. But
he was not cast down. He was doing his utmost, and he calmly left the rest
with God.
 III

Very early one morning, at the end of May, Antonio heard light
footsteps passing his cell. Although he sprang up immediately from bed he
could not open his door in time to see the intruder's face or form. He caught
no more than half a moment's glimpse of a slender and darkly garbed figure
disappearing round the angle of the corridor.

Having scrambled into his clothes, he started in pursuit. The light tap-
tap of shod feet on the stones told him that his visitor was making for the
chapel. The monk, who was barefooted, followed noiselessly.

Peeping into the chapel through the little door amid the azulejos,
Antonio saw a tall spare man kneeling before the altar. Even if his back had
not been turned to Antonio it would have been impossible to see his face,
because he was hiding it in his hands. The stranger wore a long black cloak,
uncomfortably thick and heavy for the torrid Portuguese summer. But it was
plain that he did not find it too warm. With long, thin, death-pale hands, he
drew its folds more closely round his body; and, as he did so, the familiar
movement revealed his identity to Antonio.

It was Father Sebastian.

Antonio hurried forward and knelt at his side. But Sebastian did not
move, nor did he cease praying for four or five minutes; and when at last he
turned towards Antonio it was without the slightest sign of surprise. Rising
painfully, he left the altar and made a gesture, inviting Antonio to follow
him.

As Sebastian had stood next to Antonio in juniority among the choir-

monks, the stalls of the two men were side by side. Sebastian sat down in
his old place and Antonio did likewise. The chapel was dim; but the
younger man could see that the elder's body had wasted almost to a
skeleton. Yet there was nothing repellent about him. The bloom on his
cheeks and the fire in his eyes had the solemn beauty of a sunset in an
autumnal forest. When he began to speak his voice was so soft and sweet
that it seemed to come from some far-off holy height.

"To-day, Father Antonio," he said, "completes the ninth year since you
sat on the cloister roof and heard the hoofs of the horsemen who had come
to thrust us from this house. And, this morning, it is just nine years since
you were raised to the priesthood. I asked our Lord to give me strength for
the journey, so that I might spend this anniversary with you. He has heard
me."

"Who told you that I was here?" Antonio asked.

Sebastian did not reply. But there was that in his eyes which gave
Antonio a sufficient answer. Here was a saint who walked in the light.

"Nine years," mused Sebastian aloud. "And you have not yet said your
first Mass."

"No," replied Antonio. "But God is good. Every year He enables me to

send a little cask of wine for the altar to a poor church in England. Six days
a week I work amid wine; and is not wine the matter of His great
Sacrament? It consoles me to know that although I cannot say Mass, I can
serve His table. Although I cannot, like Mary, his mother, bear Him in my
hands, I can be like those other Marys at the sepulcher. Emerunt aromata ut
venientes ungerent Jesum: 'They brought sweet spices that they might
anoint Jesus.'"

"He is not a God of the dead, but of the living," said Sebastian, in sweet,
far-off tones. "We do not offer a dead Christ. Say rather that you are like
that favored unknown to whom He sent two disciples saying, Ubi est
diversorium ubi pascha cum discipulis meis manducem: 'Where is the
guest-chamber where I may eat the Passover with My disciples?' But come.
Our time together is short, and there is much to say. First of all, I have
brought your breviary which you charged me to keep."

He pointed to a package lying on the Prior's seat. Antonio rose and took
it with joyful gratitude. When he returned to his stall he said:
 "Suffer my questions first. Whence do you come? Where have you lived
these nine long years?"

"For a few months I was with the English fathers in Lisbon," Sebastian
answered. "They were kind; but when it became plain that the Portuguese
Benedictine congregation must come to an end, I crossed Spain and sought
asylum at the Montserrat, where men used to believe the Holy Grail was
treasured. There was much work for me to do there in the School of Music;
and I found strength to do it, for we lived like eagles high up in the pure air,
three thousand feet above the sea. But Madrid followed the example of
Lisbon. Greedy eyes were cast on our possessions. They accused us of
being Carlists, just as in Portugal they accused us of being Miguelistas: and
only eighteen months after leaving this abbey, I was again an exile. Since
then I have dwelt in three religious houses; and every one of them has been
suppressed."

"Can it be," asked Antonio uneasily, "that the Orders are themselves to
blame, as men say? Here we dwelt in simplicity and piety, living by our
own labor and feeding the poor. But was this house an exception? Had the
majority of other monks indeed sunk into gluttony and sloth?"

"In every monastery from which I have been driven," said Sebastian,
"our evictors poured regrets and compliments upon us. It was always the
misdeeds of 'others,' for which we had to suffer. But whenever I questioned
an exiled community, I found they had received the same compliments.
Those mysterious 'others' have still to be found. According to the statesmen,
all religious houses individually are fountains of light and blessing to their
neighbors; but collectively they are a dark curse on the nations."

"Unbelieving men are determined to mulct us of all we have," said

Antonio, "and therefore they must needs invent crimes to suit our
punishment. They hang us first and indict us afterwards."

"They oppress us," agreed Sebastian, "in the great and sacred name of
liberty. But the avarice of godless men is the mainspring of it all. I have
seen five houses confiscated 'for the good of the People'; and in not one
case have the People received a third of the plunder. But enough of this. Tell
me your own story."
 "Where is the Prior?"

"He is dead. He died in Belgium."

"The Cellarer?"

"He is dead. He died in Brazil."

"Father Isidoro?"

"He is dead. He died in Spain."

With a sinking heart, Antonio named the choir-monks one by one; and,
after each name, Sebastian answered: "He is dead." Father Sebastian
believed that Brother Cypriano was still alive; but, of the Fathers, only he
and Antonio were left.

"Requiem aeternam dona eis, Domine," murmured Antonio.

"Et lux perpetua luceat eis," Sebastian responded.

"To-morrow morning," added Sebastian, "will be the anniversary of the

Abbot's last Mass. If our Lord will give me strength I shall say Mass at this
altar once more."

After a pause, Antonio began to relate his history from the moment of
his quitting the council at Navares. Every fact that threw light on his
operations for regaining the abbey he stated with precision. But he did not
mention Margarida, and he referred to Isabel only as Sir Percy's moneyed
daughter. When he had finished, Sebastian looked at him with steadfast
pitiful eyes and said:

"These have been great sacrifices and cruel hardships for the sake of our
Lord, and they will not be in vain. But you have not told me all. My brother,
I feel that you have kept silence concerning your most costly sacrifice, your
bitterest ordeal. Why not tell me all?"

Antonio's pride rebelled. The desire to ease his heart by pouring out its
hoard of solitary grief was strong; but his gentleman's instincts of reticence
were stronger. For some time he remained silent. But an inward voice
sternly bade him speak; and he spoke.

He told the short tale of Margarida. Then he unfolded the whole case of
Isabel, glossing over nothing. He scrupulously added an account of his
actions and feelings on the night and morrow of her flight. When he had
finished he sat with bowed head and waited for Sebastian's judgment. But
Sebastian remained silent.

"You do not speak," said Antonio. "Perhaps I have given you the
impression that my ordeal was carnal, and that this English maiden was a
direct emissary of Satan. If you think so, I have spoken blunderingly
indeed."

"Satan exists and he is busy enough," returned Sebastian. "But in trying

to find the cause of any strange thing that happens I have learned to think of
Satan last. Nearly all our temptations arise from our own self-love and
carelessness. Many other temptations are God's provings and perfectings of
our spiritual mettle. Satan is not omnipresent and his angels are only a
shrunken legion. But have patience. Let me think."

He resumed his meditation. At last he turned and said:

"This was not a temptation from the devil. Neither did it spring from
corruptness in your heart or in hers. I am persuaded that our Lord's work is
somehow in it all. Perhaps you will never know in this world what work it
is; but that is not your affair."

"Sometimes," said Antonio slowly, "it troubles my conscience. As I told

you just now, I didn't hold out to the very end. I gave way within my heart;
but when I opened my eyes she had vanished."

"You do wrong to be troubled," said Sebastian. "You held out to the

bitter end of the trial God had appointed you. When you told this Isabel
finally to go, she went. That was the end. All that happened afterwards was
mere reaction."
 "The next day," persisted Antonio, "I did not say my Office. My heart
bled for her as it never bled for the Abbot, or for you, Sebastian, or for this
place."

"It bled for her, not for yourself," Sebastian explained. "In profane love,
the lover who thinks he is grieving for the beloved is only grieving over his
own loss of her, over his own short bereavement, or over his own
humiliation and discomfiture. With you, Antonio, it was not so. You did not
wish to take; you wished to give."

"Do not make me out a saint when I know I am a sinner," said Antonio,
almost sharply. "If she had been old, and tart, and ugly, would my heart
have bled for her all the same?"

"Perhaps not," Sebastian retorted. "But, if she had been old and ugly,
neither would there have been much virtue in giving her up. Do not
complain of her beauty. You had heroic work to do, and her beauty helped
you to do it better. In England there are Puritans who would say that these
azulejos and these gilded carvings must hinder us from doing the Work of
God."

"I do not follow you," said Antonio.

"Tell me," Sebastian asked abruptly, "how you stand with the payments
you have bound yourself to make."

Antonio drew from his breast an account over which he had pored and
pored for a month without making the adverse balance a vintem less.
Sebastian conned it attentively from beginning to end. Then he said:

"Follow me to my old cell and bring me paper and ink."

He rose with so much difficulty that Antonio had to support him; but
once fairly on his feet he moved quickly over the pavement. At the door of
the cell Antonio left him; but before he had finished cutting a new quill and
replenishing the sand-sprinkler in his own room, Sebastian rejoined him.
Sitting down painfully at the tiny table he swiftly wrote a very short letter.
Without reading it over he folded it, sealed it with a small brass seal which
he drew from his pocket, and addressed it to a Spanish nobleman in a small
town of the Asturias.

"Let this be despatched at once," he said. "There is no time to lose."

"A post leaves Navares in three days," replied Antonio. "José shall take
the letter there this morning."

"It is well," said Sebastian. "And when this José returns, let me see him
as soon as he is rested."

The cell was brighter than the chapel, and Antonio perceived that his
friend was become almost as insubstantial as a ghost. He called to mind a
passage from a new English poet about a man who, having wasted to a
shadow, was ready to be resumed into the Great Shadow, the shadow and
blackness of death. But Sebastian seemed rather to be a pure white flame,
waiting to be drawn into the Great Light.

"You have not broken your fast," cried Antonio in shame and alarm.
"You must eat. I have good wine. You must rest. You must sleep. When the
heat is over we will talk again, and you shall see José."

"It has been meat and drink and rest and sleep to see you again, Father
Antonio, and to hear what you have told me," the other answered. "But you
are right. I must sleep. I will obey your orders."

At breakfast Sebastian ate and drank nothing save an ounce or two of

bread and an egg beaten up in white wine. When the meal was over he
declined Antonio's pressing offer of a comfortable bed from the guest-
house, and lay down on the straw mattress in his own cell. There he soon
fell into so profound a sleep that he did not hear Antonio drenching the
window with bucketful after bucketful of water to counteract the blazing
heat.

At night José, wearing his best coat and his most diffident manner,
dined with the two monks in a corner of the refectory. Sebastian, with bright
eyes and glowing cheeks, did most of the talking. He praised the wine and
the food, although he touched little of either; and throughout the repast he
was full of an eager cheerfulness such as Antonio had never seen in him
before. After dinner he drew from José an exact account of his mental and
spiritual state: for Antonio had told him of the poor fellow's desire to
become a monk.

"José," he demanded, at the end of his questioning, "You have learnt

Latin. Can you translate, Irascimini: et nolite peccare?"

"I can, Father," answered José proudly. "It means, 'Be angry and sin
not.'"

"So it does. You did well to be angry with the greedy and lazy good-for-
nothings who spake evil of Father Antonio. But you did ill to thrash them
and to come home with that black eye. Go on being angry with sin; but
learn to love sinners."

"Can't I be a monk, Father? May I not have the habit?" pleaded José, in
consternation. "I am glad I thrashed them; but I'm sure I shan't need to
thrash them again."

"The habit is a comfort and a help," Sebastian replied, "but we must not
give it you to-night. Live as you have been living, in the love of our Lord
and in obedience to Father Antonio. For the present you can wear no habit
more acceptable to God than the coat in which you do your daily duty about
the farm. Do not hang your head. I foresee that an abbot will once more rule
within these walls, and that you, José will die as one of his family. Have
patience."

A sudden change came over Sebastian as he ceased speaking. The hectic

bloom faded from his cheeks, and the heavy lids drooped over his
preternaturally bright eyes. A moment later he sank forward against the
table. Antonio and José sprang at once to his help. He had swooned. They
made haste to bear him bodily to his cell. It was an easy task; for beyond
the weight of his cloak there seemed to be hardly anything to carry. After
they had laid him on his bed and dashed water from the torrent in his face,
he revived and said faintly:
 "Thanks, thanks, thanks, I am well. Leave me. I shall say mass to-
morrow at five o'clock. Leave me."

He fell into another unnatural sleep. But Antonio did not leave him. All
through the short warm night he watched and prayed. At last the dull chant
of the Atlantic was drowned under the glittering trills of near blackbirds.
Day dawned. The sun rose above Sebastian's Spain; and the sleeper awoke.

He answered the traditional Benedicamus Domino with so ringing a Deo

gratias that Antonio thought a miracle had happened. Sebastian looked
stronger and healthier than ever before. Even José, who had been sleeping
heavily on the corridor floor, was aroused by Sebastian's two words.

They repaired to the chapel. There Father Sebastian heard the

confessions of his two companions. Without delay he proceeded to the
sacristy. Antonio followed him and began to lift from its drawer one of the
less costly vestments which had never been taken away. It was green and
gold, as appointed in the Ordo for that day. But Sebastian, having bidden
him replace it, drew forth a black chasuble, simply embroidered with a
plain white cross. Antonio felt justly rebuked. When the Abbot was dead,
and the Prior and all the fathers save two, surely it was meet that the
survivor's Mass should be a Mass of requiem.

From his pocket-case, Sebastian took the unconsecrated wafers which

he had brought from Lisbon. He finished his vesting and preparation and
they re-entered the chapel. José was kneeling devoutly on the lowest step of
the sanctuary. Outside, hundreds of birds were in full boisterous song.

Father Sebastian went to the foot of the altar and began to say Mass. He
uttered the words quickly and clearly, and made the genuflections without
difficulty. Indeed, Antonio, as he poured water over the white and fleshless
fingers at the psalm Lavabo, marveled more than ever at the miracle of his
friend's sudden strength. At the commemoration of the dead, the intensity of
Sebastian's recollection seemed to make the whole chapel thrill and throb,
like a bed of reeds in a wind.

After he had given the most holy Body to Antonio and to José,
Sebastian concluded the Mass and returned to the sacristy with a firm tread.
He laid aside the sacred vestments and came back to his old stall in order to
make his thanksgiving. Antonio, also in his old stall, knelt at Sebastian's
side.

The ascending sun cleared the top of the hill and shone into the chapel.
The diadem of the Holy Child blazed with glory. In all the trees happy birds
redoubled their songs.

Half an hour passed. José, arising noisily, made Antonio open his eyes.
But Father Sebastian knelt without moving against the sloping book-board.
José clattered out. Still Father Sebastian did not move. Antonio waited,
revering his friend's ecstasy of communion with his Lord. He waited long.
But meanwhile a broad sunbeam had been working westward; and at last it
poured its burning gold upon the bended head.

Antonio was stepping softly forward to screen his friend from the fierce
ray when a sudden instinct bade him kneel down and look into Sebastian's
face. But Sebastian's wide-open, rapturous eyes did not gaze into Antonio's;
nor were they beholding any earthly thing. So beautiful was the sight that
Antonio's exclamation was more a shout of joy than a cry of fear. Into his
mind there rushed the words of Isaias which had been Sebastian's favorite
scripture in the old days, Regem in decore suo videbunt oculi ejus: "His
eyes shall see the King in His beauty; they shall behold the land which is
very far off."

Antonio and José buried the body of Sebastian that night on the sunny
side of the cloister, between the third and fourth pillars, just under the tile-
picture of Enos, with its legend, Ambulavit cum Deo et non apparuit, quia
tulit eum Deus: "He walked with God and was no more seen, for God took
him."
 IV

On the feast of Saints Peter and Paul, just thirty days after Sebastian's
death, Antonio heard Mass in the village church. Forty-eight hours were left
to him before his payment to the Villa Branca Fazenda became due. In the
strong-box at home he had only three hundred and twelve pounds towards
his debt of five hundred. Nothing had been received from Sebastian's friend
in Spain, although sufficient time had elapsed for a reply to reach the farm.
Nevertheless, Antonio rose from his knees at the end of Mass and took his
way homeward with a serene spirit.

From the point where he and José had seen the ruts of young
Crowberry's wheels nearly two years before, the monk heard thumping
hoofs. He gazed down the road and saw an advancing cloud of dust. A few
moments later he made out the milk-white Branco which had succeeded
coal-black Negro as the Navares' post-horse. Thomé, the postman, drew
rein and handed Antonio two letters.

The first was from young Crowberry. It ran:

Dear Friend da Rocha.

You will be sorry to hear that my father died last week, suddenly. I know
you will pray for him; and I hope you will pray for me too.

Strange to say, Sir Percy also passed away last week, two days after my
father. I saw it in the papers, but I know no details. At Christmas my father
saw him at Weymouth, and he seemed well.

As our affairs are tangled, I have much to do. Write to me soon. My

thoughts turn to you very often nowadays. Tell me how you do, all round. I
remain, your sincere friend. Edward Crowberry.