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Preface

GENOMICS: THE LINK BETWEEN GENETICS

AND PHYSIOLOGY

Access to the interpretation of entire-genome sequence information for numerous

organisms representing Archaea, Eubacteria, Cyanobacteria, and a number of
Eukaryote model organisms such as Saccharomyces cerevisiae (baker’s yeast),
Caenorabiditis elegans (worm), Drosophila melanogaster (fruit fly), and Arabi-
dopsis thaliana (mustard cress) have brought fundamental changes to biology—
the comprehension of life as a whole instead of its smallest model unit. Additional
genomes are currently in large-scale sequencing pipelines, and projections are
that in the next few years, a wealth of whole-genome sequences spanning most
taxonomic boundaries of living organisms will become publicly available. Thus,
what not long ago appeared to be fiction or wishful thinking is quickly moving
into the realm of reality, making it possible to study entire genomes. Scientists
will be able to determine structural organization, monitor expression of the ge-
netic code, and distinguish biochemical function, cellular components, and struc-
tural components defining the ‘‘wholesome’’ phenotype—the functioning not
only of a living cell but also of the integration of all cells in an organism.
Often, the reconstruction of entire metabolic pathways becomes possible
through dynamic retrieval of biochemical functions, stored in the form of the
universal genetic information code, comparative overviews of diverse biological
systems, life histories, morphogenetic processes, and inference with respect to
natural interactions with the environment. This scenario is not typical, however,
because even in prokaryotes and other comparatively lower complexity systems
we are faced with a large number of open questions that often present themselves

iii
iv Preface

through the detection of functionally unknown or novel coding regions. In addi-

tion, examples of functional ambiguity—coding regions for more than one func-
tion, which had held a position of infrequent oddities—are becoming more nu-
merous. As an immediate result of the fast-paced progress that occurred during
the last decade, it became apparent that our knowledge of the complexity and
number of genes was woefully incomplete. With new protein-coding regions ap-
pearing in every organism whose genomic DNA sequence has been determined,
realization came that the function(s) of more than half the genes, encoded by the
Homo sapiens or A. thaliana genome, remain completely unknown. Moreover,
assignments of gene function continue to be missed, although the accuracy of
predictive algorithms continues to improve rapidly, even in cases where estab-
lished biochemical data exist for well-defined coding regions. Some of the com-
plications of annotating well-characterized functions to genes are associated with
nonuniversal biological processes, such as the generation of multiple functionally
diverse proteins through alternate mRNA splicing, single proteins with multiple
functions depending on spatiotemporal expression, and a significant and growing
number of RNAs that are neither protein coding nor ribosomal RNA nor transfer
RNA, yet that seem to have important additional structural or regulatory roles.
Direct interpretation of genome DNA sequence information alone has
brought recognition of how much more complementary gene function analysis
is still needed and simultaneously, has provided new avenues by which to do so.
Genome sequences are catalysts for the generation of hypotheses. Figures 1 and F1
2 illustrate the concepts underlying the term “genomics.” Initially, these concepts F2
were geared toward high-throughput detection of which genes and which tran-
scripts make up the complexity of an organism. In a comparative fashion, patterns
of commonalities and uniqueness were established. In subdisciplines, labeled as
proteomics and metabolomics, the complexity, cellular localization, dynamics of
protein associations, and their interactions with substrates and products are being
determined at the whole-cell level. Thus, all disciplines derived from the original
concept of genomics—considering the genomic information as a whole—are
based on finding commonalities and differences among organisms, spatiotempo-
ral cell stages, physiological conditions, and interactions with the environment
between or within organisms of a given species.
In a way, the underlying idea is similar to the collection of the existing
knowledge that was undertaken some 200 years ago by scholars, encyclopedists
who reasoned that understanding would arise from the compilation and synthesis
of all existing knowledge. Genomics is similar in its attempt to gather data points,
but it is not at all similar to the encyclopedists’ reasoning because, so far, geno-
mics is generating not only the data but also the new tools to understand them.
In this book, we have assembled contributions from dedicated practitioners
who have been responsible for shaping the field of fungal and plant genomics.
The individual chapters are meant to provide technical insight and to highlight
Preface v

Figure 1 Schematic representation of cellular organization. Genes are the genetic infor-
mational units encoding one or more proteins (polypeptide). Proteins are the products of
gene translation. Metabolites are the products of protein function. Components are one or
more proteins or protein products assembled into a subcellular component, a complex or
functional unit that exerts a specific function. Processes are combinations of proteins,
protein products, and cellular components into a network that exerts a new function. En-
gines are biological processes that provide metabolites—carbon, nitrogen, phosphorus,
and sulfur as well as chemical energy required to drive biological processes. Motors are
cellular components that provide energy and motion to protein complexes, cellular compo-
nents, or biological processes (e.g., vesicle movement, polar growth, or chromosome divi-
sion). Machines are cellular components exerting constitutive functions such as transcrip-
tion, translation, and secretion. Factories are cellular components exerting customized
products used by other cellular components or biological processes. Transcriptomics, pro-
teomics, and metabolomics are whole-genome analysis technologies that approach the
transcription, function, and products of the genotype.

how genomics-oriented studies may be used to bring new understanding to estab-

lished models of fungal development, to analyze and solve problems associated
with multiple copies of genes and proteins with seemingly identical functions, and
to exemplify how genomics information may be used for industrial applications.
The sea change in biology has had two foundations. Foremost have been
new technologies that have harnessed computing power in data acquisition and
vi Preface

Figure 2 Components of genomics—the various approaches that use DNA sequence

information to identify protein function in terms of where and when proteins are expressed,
their interacting partners, the phenotype of gene disruption, biochemical function and
three-dimensional structure, the spatial and temporal consequences of protein action, and
the relative importance of gene function for performance of the organism.

integration. These technologies are also a crucial component in every instrument

in the laboratory. Of equal importance has been the bold thought shown by a
number of foresighted individuals whose visionary anticipation of the emerging
computational power allowed them to forge new tools to attack complex prob-
lems. For those involved in experimentation, ‘‘kid in the candy store syndrome’’
best describes the last decade. There is no indication that this state of mind will
end soon.
We wish to thank Joan Bennett (Tulane University) and Anita Lekhwani
(Marcel Dekker, Inc.) for the opportunity and encouragement to produce this
volume, and the National Science Foundation, from which our work has received
funding.

Rolf A. Prade
Hans J. Bohnert
Contents

Preface iii
Contributors ix

Fungi

1. Aspergillus 1
Michael J. Anderson, Jayne L. Brookman, and David W.
Denning

2. Developmental Processes in Filamentous Fungi 41

Reinhard Fischer and Ursula Kües

3. Multiple GATA Transcription Factors Control Distinct

Regulatory Circuits and Cellular Activities in Neurospora 119
George A. Marzluf

4. Molecular Genetics of Metabolite Production by Industrial

Filamentous Fungi 131
Christian P. Kubicek

5. Acquiring a New Viewpoint: Tools for Functional Genomics in

the Filamentous Fungi 163
Todd M. DeZwaan, Keith D. Allen, Matthew M. Tanzer, Kiichi
Adachi, Lakshman Ramamurthy, Sanjoy Mahanty, and Lisbeth
Hamer

vii
viii Contents

Plants

6. Functional and Comparative Genomics of Cyanobacteria 203

Bradley L. Postier and Robert L. Burnap
7. Genomic Analysis of Arabidopsis Gene Expression in Response
to a Systemic Fungicide 237
Huey-wen Chuang, Tzung-Fu Hsieh, Manuel Duval, and Terry
L. Thomas
8. Identification of T-DNA Insertions in Arabidopsis Genes 255
László Szabados and Csaba Koncz
9. Genomic Approaches for Studying Gene Families in Plants 279
Cathal Wilson, Balázs Melikant, and Erwin Heberle-Bors
10. Industrialization of Plant Gene Function Discovery 291
Douglas C. Boyes, Robert Ascenzi, Mulpuri V. Rao, Susanne
Kjemtrup, Andreas Klöti, and Keith R. Davis
11. Functional Genomics of Plant Abiotic Stress Tolerance 315
John C. Cushman

Bioinformatics

12. Using Workflow to Build an Information Management System

for a Geographically Distributed Genome Sequencing Initiative 359
David Hall, John A. Miller, Jonathan Arnold, Krzysztof J.
Kochut, Amit P. Sheth, and Michael J. Weise

Index 373
Contributors

Kiichi Adachi, Ph.D. Mitsui Global Strategic Studies Institute, Tokyo, Japan

Keith D. Allen, Ph.D. Department of Computational Biology, Paradigm Genet-

ics, Inc., Research Triangle Park, North Carolina, U.S.A.

Michael J. Anderson, D.Phil. School of Medicine, University of Manchester,

Manchester, England

Jonathan Arnold, Ph.D. Department of Genetics, University of Georgia, Ath-

ens, Georgia, U.S.A.

Robert Ascenzi, Ph.D. Department of Biochemistry, BASF Plant Science

L.L.C., Research Triangle Park, North Carolina, U.S.A.

Douglas C. Boyes, Ph.D. Department of Agricultural Biotechnology, Paradigm

Genetics, Inc., Research Triangle Park, North Carolina, U.S.A.

Jayne L. Brookman, Ph.D. Research Division of Biochemistry, School of Bio-

logical Sciences, University of Manchester, Manchester, England

Robert L. Burnap, Ph.D. Department of Microbiology and Molecular Genet-

ics, Oklahoma State University, Stillwater, Oklahoma, U.S.A.

Huey-wen Chuang, Ph.D. Department of Biology, Texas A&M University,

College Station, Texas, U.S.A.
ix
x Contributors

John C. Cushman, Ph.D. Department of Biochemistry, University of Nevada–

Reno, Reno, Nevada, U.S.A.

Keith R. Davis, Ph.D. Department of Agricultural Biotechnology, Paradigm

Genetics, Inc., Research Triangle Park, North Carolina, U.S.A.

David W. Denning, F.R.C.P., F.R.C.Path. Section of Infectious Diseases,

School of Medicine, University of Manchester, and Wythenshawe Hospital, Man-
chester, England

Todd M. DeZwaan, Ph.D. Department of Assay Development, Paradigm Ge-

netics, Inc., Research Triangle Park, North Carolina, U.S.A.

Manuel Duval, Ph.D. Department of Biology, Texas A&M University, Col-

lege Station, Texas, U.S.A.

Reinhard Fischer, Ph.D. Department of Biochemistry, Max-Planck-Institute

for Terrestrial Microbiology and Philipps-University of Marburg, Marburg, Ger-
many

David Hall, Ph.D. Research Computer Division, Research Triangle Institute,

Research Triangle Park, North Carolina, U.S.A.

Lisbeth Hamer, Ph.D. Department of Microbiology, North Carolina State Uni-

versity, Raleigh, North Carolina, U.S.A.

Erwin Heberle-Bors, Ph.D. Institute of Microbiology and Genetics, Vienna

Biocenter, University of Vienna, Vienna, Austria

Tzung-Fu Hsieh, Ph.D. Department of Biology, Texas A&M University, Col-

lege Station, Texas, U.S.A.

Susanne Kjemtrup, Ph.D. Department of Agricultural Biotechnology, Para-

digm Genetics, Inc., Research Triangle Park, North Carolina, U.S.A.

Andreas Klöti, Ph.D. Technology Transfer Office, ETH Zurich, Zurich, Swit-
zerland

Krzysztof J. Kochut, Ph.D. Department of Computer Science, University of

Georgia, Athens, Georgia, U.S.A.
Contributors xi

Csaba Koncz, Ph.D. Department of Plant Development Biology, Max-Planck-

Institute for Plant Breeding Research, Cologne, Germany

Christian P. Kubicek, Ph.D. Institute for Chemical Engineering, Vienna Uni-

versity of Technology, Vienna, Austria

Ursula Kües, Ph.D. ETH Zurich, Zurich, Switzerland, and Institute for Forest
Botany, Georg-August-University Göttingen, Göttingen, Germany

Sanjoy Mahanty, Ph.D. Inspire Pharmaceuticals, Inc., Research Triangle Park,

North Carolina, U.S.A.

George A. Marzluf, Ph.D. Department of Biochemistry, The Ohio State Uni-

versity, Columbus, Ohio, U.S.A.

Balázs Melikant, M.Sc. Institute of Microbiology and Genetics, Vienna Bio-

center, University of Vienna, Vienna, Austria

John A. Miller, Ph.D. Department of Computer Science, University of Geor-

gia, Athens, Georgia, U.S.A.

Bradley L. Postier, B.S. Department of Microbiology and Molecular Genetics,

Oklahoma State University, Stillwater, Oklahoma, U.S.A.

Lakshman Ramamurthy, Ph.D. Department of Bioinformatics, Glaxo-

SmithKline, Inc., Research Triangle Park, North Carolina, U.S.A.

Mulpuri V. Rao, Ph.D. Department of Agricultural Biotechnology, Paradigm

Genetics, Inc., Research Triangle Park, North Carolina, U.S.A.

Amit P. Sheth, Ph.D. Department of Computer Science, University of Georgia,

Athens, Georgia, U.S.A.

László Szabados, Ph.D. Institute of Plant Biology, Biological Research Center,

Szeged, Hungary

Matthew M. Tanzer, Ph.D. Department of Biochemistry, Paradigm Genetics,

Inc., Research Triangle Park, North Carolina, U.S.A.

Terry L. Thomas, Ph.D. Department of Biology, Texas A&M University, Col-

lege Station, Texas, U.S.A.
xii Contributors

Michael J. Weise, Ph.D. Life Sciences Scientific Services, Accelys, Inc., San
Diego, California, U.S.A.

Cathal Wilson, Ph.D. Institute of Microbiology and Genetics, Vienna Bio-

center, University of Vienna, Vienna, Austria
1
Aspergillus

Michael J. Anderson and Jayne L. Brookman

University of Manchester, Manchester, England

David W. Denning
University of Manchester and Wythenshawe Hospital,
Manchester, England

I. INTRODUCTION

Aspergillosis, the name given to all diseases caused in animals by fungi in the
genus Aspergillus, includes allergic, superficial, saprophytic, and invasive dis-
ease. More than 180 species of Aspergillus are recognized (1), but only a few
cause disease with any regularity: A. fumigatus, A. flavus, A. terreus, and A. niger
group species. Aspergilli are common saprophytes in the environment, especially
in composting facilities. The pathogenic Aspergilli are found the world over, but
soil isolation rates do increase towards the equator. Most aerobiology studies
have been done in Europe and some have shown a seasonal variation in airborne
Aspergillus counts. Aspergillus species comprise from 1% to 6% of the total air
flora outside and, if speciated, A. fumigatus comprises from 4% to 41% of the
Aspergillus total (2). The usual concentration of Aspergillus conidia in outside
air is 1 to 30 conidia/m3, but can rise to as high as 7 ⫻ 107 /m3 inside a barn,
after hay or straw disturbance. In hospitals, conidia concentrations in air also
vary typically from 0 to 1 ⫻ 102 /m3 with much variation in the same site (3–5).
Inside human dwellings, Aspergillus species may be found in high concentrations
in potted plants (50 conidia/g soil) (6), damp cellars, dusty crawl spaces, and
condiments, especially pepper (106 conidia/g) (2) and ground spices.
Human disease has been increasing over the 5 decades since invasive asper-
gillosis was first described in the immunocompromised patient (7). Invasive as-
pergillosis (IA) is the most common life-threatening invasive mold infection
1
2 Anderson et al.

worldwide. It usually complicates treatments and diseases associated with immu-

nosuppression, including allogeneic bone marrow transplantation, lung and liver
transplantation, the treatment of acute leukemia, late-stage acquired immunode-
ficiency syndrome (AIDS) and a variety of other diseases treated with corticoste-
roids (8). Invasive aspergillosis rarely affects nonimmunocompromised patients.
The incidence of invasive aspergillosis was calculated to have risen 14-fold in
the 15-year period up to the end of 1992, as seen in autopsy data from one major
teaching hospital in Frankfurt, Germany, with 5% of autopsied patients having
invasive aspergillosis in the last year of this survey (9). In a national autopsy
survey in Japan from 1969 to 1994, invasive aspergillosis increased from 0.4%
to 1.3% in all autopsies (10). Another autopsy series in a European teaching
hospital demonstrated a 4% rate of invasive aspergillosis in unselected autopsies
(11). A culture-based population study in the San Francisco area (requiring cases
to have, for example, two culture-positive bronchoscopy specimens or a sterile
site positive by culture, which probably underrepresents invasive aspergillosis
by perhaps 90%) showed increasing rates of disease over the last 25 years (12).
Comparable figures from previous surveys had put the figure at 1.9 cases per
million population in 1970, linearly rising to 12.4 per million in 1992/1993.

II. CLINICAL MANIFESTATIONS OF

ASPERGILLUS INFECTION
A. Allergy
Wheezing in patients exposed to Aspergillus was recognized in the late 1800s,
but was poorly understood. Allergic bronchopulmonary aspergillosis (ABPA),
an extreme form of continuing local allergy to Aspergillus, was first reported in
1952 in 3 patients from the London Chest Hospital (13). Allergic bronchopulmo-
nary aspergillosis complicates asthma and cystic fibrosis (CF), and patients de-
velop exacerbations of the asthma, CF, or both. They are commonly “difficult-
to-control” patients, in the pulmonary sense. Characteristic presentations include
new pulmonary shadows, which resolve with steroids, and coughing up plugs of
material. The diagnosis is made by a combination of criteria of which episodic
wheezing (asthma), transient pulmonary shadows, elevated serum total immuno-
globulin E (IgE) and Aspergillus-specific IgE, positive Aspergillus precipitins
(IgG), and central bronchiectasis are the most important. Central bronchiectasis is
not a useful diagnostic criterion in CF, as it is universally present. Some patients,
especially those with long-standing disease, have barely detectable Aspergillus
antibodies (14). Other criteria that have been used include peripheral eosinophilia
and a positive immediate skin test to Aspergillus. Eventually, patients with un-
treated ABPA develop pulmonary fibrosis. Acute exacerbations are best treated
with systemic corticosteroid therapy. Itraconazole (one of only two licensed oral
Aspergillus 3

antifungal agents available for aspergillosis) has shown benefit in several open
studies (15, 16) and in a recent controlled trial (17).
Mold allergy is common among asthmatics, and this may represent a milder
form of ABPA, although multiple other fungi are implicated. It is estimated that
there are 275 million asthmatics worldwide (18). Of these, 5% have severe
asthma, and there are around 100,000 deaths per year. Recent prevalence data
in a population of asthmatics attending hospital regularly (19) compared the rates
of fungal allergy in those admitted to the hospital at least twice annually with
those not admitted. Skin testing to Aspergillus, Alternaria, Cladosporium, Peni-
cillium, and Candida showed the frequency in the former to be 76%, but only
16% in the latter group. Rates of allergy to house-dust mite antigen were equiva-
lent. Of interest, sensitization to Aspergillus is also common in patients with
chronic granulomatous disease and hyper-IgE syndrome, both diseases usually
being associated with invasive aspergillosis (20).
Allergic sinus disease caused by Aspergillus and other molds has been rec-
ognized in the last 15 or so years (21). The frequency of allergic fungal sinusitis
or “eosinphilic fungal rhinosinusitis” is not known, and agreed-upon criteria for
diagnosis are currently being evolved. Probably many cases of chronic sinusitis
are related in part to fungal allergy, but this is hard to prove, as markers of allergy
in blood are present in fewer than 30% of cases (21).

B. Saprophytic or Chronic Invasive/Necrotizing

Aspergillus Disease
“Aspergilloma” is the term given to the colonization of an intrathoracic cavity
by Aspergillus. A fungus ball is formed when spores are deposited in the cavity
and germinate on the wall, where mycelia and debris attach to create an amor-
phous mass. An aspergilloma may form in any pre-existing lung cavity. There
are many causes of pulmonary cavities including tuberculosis, sarcoidosis, pneu-
moconiosis, histoplasmosis, and bullae. Some idea of the prevalence of aspergil-
loma can be gained from a review of 60,000 chest radiographs: aspergillomata
were identified in 0.01% (22). During an 11-year period, 15 patients with as-
pergilloma were admitted to a Veteran’s Administration Hospital, representing
0.02% of admissions (23). The frequency is much higher in patients with cavities
of 2 cm or more in diameter. For example, in tuberculosis cavities of this size,
15% to 20% of British patients developed an aspergilloma (24). In a series of
patients with pulmonary sarcoidosis, 10 of 19 (53%) patients with cystic paren-
chymal damage had aspergillomas, compared with none of 81 patients with non-
cystic pulmonary sarcoidosis (25). Many patients with aspergillomas have slowly
progressive cavitary damage to the lung, which is termed “chronic necrotizing
pulmonary aspergillosis (CPNA).” In AIDS, for example, progression of asper-
gillomas over time is seen with considerable morbidity and some mortality. This
4 Anderson et al.

progression probably reflects invasion of cavity walls by Aspergillus and is, there-
fore, not strictly saprophytic Aspergillus disease.
The symptomatology of aspergilloma and CPNA is similar and variable in
individual patients over time. Most patients are asymptomatic when an aspergil-
loma first forms. The most common presentation of aspergilloma is coughing up
blood, which is because of new vessel formation in the vicinity of the aspergil-
loma. About 40% of patients are sensitized to Aspergillus and develop wheezing,
weight loss, and malaise with or without fever. The patient is typically in the
fourth to sixth decade of life and, as with all forms of aspergillosis, more men
than women are affected. Chronic necrotizing pulmonary aspergillosis has similar
clinical features, although weight loss is more profound.
Plain radiography of the chest in cases of aspergilloma shows a number of
typical features, including a round solid mass that may be mobile within a cavity,
separated from the wall by a rim of air. In classic aspergillomas, pleural thick-
ening is also present and varies from several millimeters to 2 cm. All patients
with CPNA have radiological evidence of a small or large cavitary lesion in the
lung, usually in one or both upper lobes. Initially, infiltrates are ill-defined areas
of consolidation or small cavities that progress to form well-defined multiple
cavities with thickened walls—the cavities often contain an aspergilloma, debris,
or fluid. Invasion of the pleura may follow.
Aspergillus precipitins are detectable in more than 95% of aspergilloma
patients. The precipitin test may, however, be positive in some patients with
cavities who do not have an overt aspergilloma, and these patients probably have
CNPA. Two-thirds of patients have elevated levels of total IgE and Aspergillus-
specific IgE. Most patients with aspergillomas have positive respiratory cultures
of Aspergillus, usually A. fumigatus, and multiple variants and genotypes may
be recovered. In a retrospective series, 25% of patients with pulmonary asper-
gillomas, 100% with a sinus aspergilloma, and 8% with disseminated aspergillo-
sis had calcium oxalate crystals present in their tissue and, occasionally, renal
oxalosis was observed (26). Aspergillus niger may be a more prolific producer
of oxalate than A. fumigatus (27). Evidence of inflammation, such as the presence
of C-reactive protein, is common in CPNA. We have recently observed mannose-
binding protein deficiency as a probable association with CNPA (28). Pathologi-
cally, aspergillomas have multiple hyphae in cavities without invasion of cavity
walls. In CPNA, hyphae may also be found in abnormal cavities without invading
tissue. Distinguishing between aspergilloma and CNPA is clinically difficult and
depends on progression over time.
Spontaneous resolution of aspergillomas occurs in 10% of cases within 3
years. However, the consequences of this type of Aspergillus infection can be
dramatic. One report focused on the long-term outcome of 23 patients with asper-
gilloma and found that 5 died directly from complications of this infection (respi-
ratory failure or hemoptysis) (29). Many patients with aspergillomas are elderly
Aspergillus 5

and have significant underlying disease, including severe respiratory compromise

in addition to the aspergilloma. Thus, many patients die with an aspergilloma
rather than of it. Treatment is problematic. Responses have been reported to intra-
cavitary amphotericin B and older drugs, oral itraconazole, surgical resection,
and corticosteroids. Bleeding is managed by resection or embolectomy. Likewise,
CNPA runs a slowly progressive course over months or usually years, if not
treated. Itraconazole or intravenous amphotericin B are appropriate for CNPA.
Gamma-interferon has been used successfully in a few patients. Uncontrolled,
cavities expand, reducing pulmonary capacity; then, local pulmonary fibrosis oc-
curs and eventually the patient is left with little functional lung. Sometimes, the
systemic features are more prominent and cachexia, mimicking carcinoma, is the
eventual outcome.

C. Invasive Aspergillosis
The incidence of invasive aspergillosis is variable in different patient groups
(Table 1) and over time in the same institution. It is slightly more common in
men, for reasons that are unexplained. In some studies, it has followed a seasonal
distribution, but this has not been shown in most studies. Specific defects that
put patients at risk include severe neutropenia, corticosteroid therapy (including
transplantation), prior pulmonary damage or infection, neutrophil defects, such

Table 1 Incidence of Invasive Aspergillosis in Different Host

Groups

Range (%)

Heart and lung or lung transplantation 19–26a

Chronic granulomatous disease 25–40b
Acute leukemia 5–24
Allogeneic BMT 4–9
Autologous BMT without growth factors 0.5–6
AIDS 0–12
Liver transplantation 1.5–10
Heart and renal transplantation 0.5–10
Severe combined immunodeficiency 3.5
Burns 1–7
Systemic lupus erythematosus 1
Autologous BMT with growth factors ⬍1
a
Distinguishing colonization from disease is particularly difficult in these patients.
b
Lifetime incidence.
BMT, Bone marrow transplantation.
6 Anderson et al.

as occur in diabetes mellitus, AIDS, chronic granulomatous disease, chronic liver

disease, and because of excess alcohol consumption. Mitochrondrial defects
(Pearson and MELAS syndromes) have recently been associated with invasive
aspergillosis (30, 31). Some patients with no apparent risk factors develop inva-
sive aspergillosis, often after respiratory or other infections.
The majority of patients (80%–90%) develop invasive pulmonary aspergil-
losis (IPA). A few have airways or sinus infection; other manifestations are rare.
Interestingly, acute invasive sinusitis is extremely uncommon in solid organ
transplant patients and occurs mostly in hematology patients or, in a more chronic
form, in those with less extreme immunodeficiency. A classification of invasive
aspergillosis is shown in Table 2. Local extension, as in sinus disease spreading
to the orbit, is distinct from disseminated disease, which refers to distant spread
from the portal of entry, presumably blood-borne.
Clinical manifestations of disease differ according to patient group. About
two-thirds of patients have a fever. Of those with invasive pulmonary aspergillo-
sis, a mild unproductive cough is the first manifestation in about 60% of patients,
about 40% have chest pain, and 25% cough up blood. Most patients have no
symptoms and 10% to 40% manifest no symptoms until a few days before death.

Table 2 Classification of Invasive Aspergillus Infection

1. Infection associated with tissue damage, surgery or foreign body

A. Keratitis and/or endophthalmitis
B. Cutaneous infection, eg, burn wound aspergillosis
C. Operative site infection, eg, prosthetic valve endocarditis, wound infection
after liver transplantation, and subdural empyema
D. Foreign body-associated, eg, Hickman or other IV line-associated and CAPD
catheter
2. Infection in the immunocompromised host
A. Primary cutaneous aspergillosis, esp. in leukemic children
B. Pulmonary aspergillosis
i) acute invasive
ii) chronic necrotizing
C. Airways aspergillosis:
i) obstructing bronchial aspergillosis
ii) invasive Aspergillus tracheobronchitis
iii) ulcerative Aspergillus tracheobronchitis
iv) pseudomembranous Aspergillus tracheobronchitis
D. Rhinosinusitis
E. Disseminated aspergillosis, esp. cerebral aspergillosis

CAPD, Continuous ambulatory peritoneal dialysis.

Aspergillus 7

Early diagnosis depends on appropriate testing, and at present, 30% remain un-
diagnosed and untreated at death.
The approach to diagnosis depends on the type of patient. In neutropenic
patients with leukemia, essentially all are hospitalized, and frequent observation
and testing is possible. In these patients, a combination of galactomannan-antigen
testing (32, 33) and early computed tomographic (CT) scanning of the lungs (34),
followed up by bronchoscopy and surgery, if necessary, is the optimum approach.
These patients also have elevated fibrinogen and often C-reactive protein or other
acute phase response markers circulating in blood, but the sensitivity and speci-
ficity of each are unclear. The same approach is useful for bone marrow (hemato-
poietic stem cell) transplant patients, but as the risk period extends for months,
regular testing is problematic. Polymerase chain reaction (PCR) diagnosis has
been useful in some studies (35), although not all, probably reflecting both techni-
cal and patient differences. In other patients, culture of respiratory secretions
followed by biopsy is the usual approach to diagnosis, with poor results overall.
Generally, better diagnostic approaches are needed, especially in nonhematologi-
cal patients.
The treatment of IA is problematic. There are only two established licensed
drugs useful for its treatment—amphotericin B and itraconazole. The response
rates to lipid-associated amphotericin B are not different from standard amphoter-
icin B, but the therapeutic ratio has improved, certainly with respect to nephrotox-
icity. Randomized studies between oral itraconazole and intravenous amphoteri-
cin B were unsuccessful, so comparative response rates can only be estimates.
The response rate to amphotericin B varies from approximately 1% (cerebral
aspergillosis in immunocompromised patients) to 60% to 80% in heart and renal
transplant patients with pulmonary or cutaneous infection. Collected series sug-
gest an overall response rate of approximately 35% (36–39). Response rates to
itraconazole appear to be approximately 40% to 60%, again with a wide range
in different patient groups and different settings (38, 40). Less data are available
on the efficacy of itraconazole compared with amphotericin B in more immuno-
compromised settings (such as allogeneic bone marrow transplant patients) and
persistently neutropenic patients, although the drug may be effective in these
settings. It is well known that the response rates to amphotericin B do not exceed
15% in these difficult situations. Patients who fail amphotericin B may respond
to itraconazole, and the converse is also true. Acute invasive Aspergillus sinusitis
appears to respond better to amphotericin B than azoles. Azole resistance has
been documented in A. fumigatus (41, 42). Aspergillus terreus is innately resistant
to amphotericin B (41), and amphotericin B resistance has also been reported in
some isolates of A. flavus and A. fumigatus (41).
The newer drugs being tested include voriconazole, posaconazole, ravu-
conazole, and three candins, caspofungin, micafungin and anidulafungin. All the
8 Anderson et al.

azoles have the same mode of action as itraconazole, but may be slightly more
active, especially posaconazole. Voriconazole is more active than amphotericin
B in a recently completed comparative study (42a) and was licensed for the treat-
ment of IA in 2002. The candins are noncompetitive inhibitors of glucan synthase,
but do not appear to be fungicidal. They have been shown to be active in mice
and patients.

III. VIRULENCE AND PATHOGENICITY

Certain species of the genus Aspergillus are remarkable opportunistic animal

pathogens. As detailed above, the diversity of diseases produced by A. fumigatus
alone is very broad and even broader if the other pathogenic Aspergilli are also
considered. For instance, sinusitis is often caused by A. flavus and otitis externa
is most often caused by A. niger. Aspergillus terreus causes disseminated asper-
gillosis in German Shepherd dogs. Aspergillus clavatus and Aspergillus versi-
color are thought to be significant allergens and producers of unusual mycotoxins.
Where these have been studied, differences between the pathogenic and non-
pathogenic species of Aspergillus will be highlighted, especially between A. fumi-
gatus and the model organism, A. nidulans. These differences will be important to
bear in mind when investigating pathogenicity and in comparisons of the species’
genomes.
Few data support the hypothesis that different strains of A. fumigatus are
more or less pathogenic, partly because these depend on the definition of pathoge-
nicity. Fiftyfold differences in inocula able to cause lethal infection have been
documented between isolates from patients (43). Significant differences in viru-
lence between environmental and clinical isolates also have been shown, although
the differences between the clinical isolates were not tested and so the data proba-
bly just reflected the natural level of variation in the population (44). Molecular
typing of large numbers of isolates does not reveal any grouping of the clinical
isolates into types or clades as would be expected with an opportunistic pathogen
(45, 46). These data could be the consequence of grouping together all the clinical
isolates, despite the fact that patients with different forms of aspergillosis were
included.
It is highly probable that different mechanisms of pathogenesis operate in
the different clinical situations. However, in this section, only clinical and biolog-
ical factors relevant to pathogenesis will be considered for IA.

A. Invasive Aspergillosis
The hallmark of IA is tissue invasion and damage by Aspergillus. The vast major-
ity of cases occur in immunocompromised patients, but occasional cases in “nor-
Aspergillus 9

mal” people also occur. Tissue damage, such as that caused by trauma or burns,
may be followed by IA. Given this, A. fumigatus is best considered an opportunis-
tic pathogen, but it can also be a rare primary pathogen. It may also be a primary
pathogen in other vertebrates and occasionally in invertebrates as well. These
observations lead to a key question:
Are the pathogenicity factors, which must exist in A. fumigatus to cause dis-
ease in the normal host, also operative in the immunocompromised patient?

If A. fumigatus were a true primary pathogen, then it would have evolved along
with its host(s) and would express factors specifically involved in the infection
process that would enable it to avoid any host responses, invade tissue, adhere
to certain cell types, etc. However, because A. fumigatus is such a rare pathogen
of normal hosts and the epidemiological data suggest that all environmental iso-
lates are pathogenic, then essentially it is an opportunistic pathogen. Therefore,
the different factors that are required for growth in the host (pathogenicity factors)
will presumably operate in any opportunistic situation regardless of the immune
status of the patient. However, it might be possible, because of natural variation
in the population, that more aggressive strains do exist and that only these strains
are able to infect less immunocompromised people. It would be interesting to
study the epidemiology and population genetics of such isolates and to test their
virulence in animal models.
Invasive aspergillosis occurs in many different immunocompromised con-
texts, and the manifestations of infection may be different. For example, slowly
progressive pneumonia with invasion of the chest wall is common in chronic
granulomatous disease. This pattern of disease contrasts with focal pulmonary
infection in neutropenic patients with leukemia, in which nodules or consolida-
tion are common. Such lesions very rarely even cross the pleura and never invade
the chest wall. This raises another key question:
Is it the host response, is it Aspergillus-derived pathogenicity factors, or is
it the interaction of the two that determines the manifestations of disease?

In the same patient group with essentially equivalent degrees of immunosuppres-

sion, the pace of progression and manifestation of IA is variable. For example,
in allogeneic bone marrow transplant patients, about 40% get cerebral aspergillo-
sis, and 60% do not. In heart transplant recipients, about one-third develop bilat-
eral pulmonary aspergillosis, one-third disseminated disease, and one-third small
pulmonary nodules. What factors determine the patterns of disease in different
patients? Is it host genetic factors, isolate to isolate variation (with variability in
pathogenicity factors), or both? The application of genome technologies derived
from the human, A. fumigatus, and other fungal genome sequences, as well as
epidemiological studies of patients and A. fumigatus isolates, should begin to
address such complex issues. In fact, it could be argued that such studies of an
10 Anderson et al.

opportunistic pathogen with so many manifestations of disease is only possible

in this postgenomic era.
Another issue that requires consideration in any discussion of pathogenicity
is whether an essential growth factor (such as para-aminobenzoic acid [47] or
uridine and uracil [48]) can be legitimately termed a “pathogenicity factor.” Dele-
tion of any key enzyme necessary for growth in the mammalian host, which
results in an avirulent mutant in a model system, is an important observation but
does little to explain pathogenesis, as it indicates essentially the prototrophic
requirement for survival in man. An extension of this argument is that the demon-
stration of the importance of any pathogenicity factor should include information
on growth rate. Experiments conducted years ago concluded that reduced growth
rate was associated with reduced virulence (49). However, no group has systemat-
ically studied growth rate in the host, and none in parallel with a demonstration
of virulence. Clearly, the ability to grow at 37° C is critical to infection and only
about 13 Aspergillus species are able to do so with any vigor (1). In addition,
the small diameter (2–3 µm) of A. fumigatus conidia enables penetration, deep
into the lung alveoli.
Infection by any micro-organism requires several steps. Pathogenicity
should, therefore, be considered in the same way.

B. Adherence
The organism has to evade the surface defenses in the respiratory tract and the
mucous ladder long enough to germinate. Normally, conidia would be removed
from the lung by ciliated movement long before germination occurs. However,
if this process is defective, fungal products, including gliotoxin, may slow ciliary
beating and damage epithelia locally (50). Then attachment to epithethal cells,
the basement membrane, or both will occur. Adhesion may be to carbohydrate
or protein molecules. Aspergillus fumigatus conidia have been shown to bind to
laminin (51), which is a major noncollagenous extracellular matrix protein. Co-
nidial binding was mainly to the P1 fragment. After binding, production of elas-
tase degraded laminin in situ. The external rodlet layer of conidia bound most
strongly and the number of binding sites decreased as the conidia swelled with
a concomitant loss of adherence (52). The laminin ligand was identified as a 72
kDa cell wall glycoprotein. The binding of spores to laminin and epithelial cells
has been shown to be inhibited by surfactant protein D enriched-broncho-alveolar
lavage and enhanced by diffusates from the spore surface (53).
Avid binding of fibrinogen to conidia, conidial heads, and hyphae has been
shown in various pathogenic Aspergilli (54). Binding to individual conidia varied
substantially and sometimes was absent. Conidia seemed to bind more fibrinogen
than hyphae. Aspergillus niger spores bound the most fibrinogen molecules, fol-
lowed by A. fumigatus and A. flavus, and then A. terreus. Nonpathogenic species,
Aspergillus 11

such as A. nidulans and A. candidus, bound relatively little fibrinogen. Conidia

have been shown to bind to the D domain of fibrinogen (55). There are an average
of 1,200 sites of a proteinaceous nature on a conidium, and the number of sites
decreased during germination. Recent data have suggested that negatively
charged carbohydrates, such as sialic acids, on the surface of conidia bind to the
glycosaminoglycan-binding domain of fibronectin (56, 57). It was also demon-
strated that A. fumigatus conidia bound significantly better to fibronectin than
those of A. flavus, A. wentii, and A. ornatus (56), which reflected the relative
sialic acid densities (57). Conidia have been shown to bind to pulmonary surfac-
tant proteins through their carbohydrate recognition domains (58, 59). In addition,
lactoferrin has been shown to bind both resting and swollen conidia (60). This
may have implications for iron availability and, thus, for growth of the organism
or for phagocyte killing of conidia.
Hydrophobins are small, secreted, moderately hydrophobic proteins that
contain eight cysteine residues. They are structural proteins that make cell walls
hydrophobic and are present in the outer spore wall. One hydrophobin, RodA,
is the major component of the rodlet layer of A. fumigatus and A. nidulans co-
nidia, and disruption of the gene results in conidia lacking this layer (61–63).
RodA⫺ mutant colonies are more readily wettable with water, and conidia are
not dispersed into the air on shaking. The mutant conidia still aggregate readily,
and those of A. fumigatus do not have altered binding to pneumocytes, lami-
nin, and fibrinogen (63). The spores do, however, adhere less to collagen (63).
A detailed physicochemical comparison of A. fumigatus and A. nidulans rodA⫺
spores has shown differences between the two species (64). Although the rodA⫺
spores of both species were significantly less hydrophobic, removal of the rodlet
layer reduced the hydrophobicity of A. fumigatus conidia less than those of A.
nidulans and, for instance, no difference in adhesion to latex spheres was ob-
served between wild-type and mutant A. fumigatus spores in contrast to a sig-
nificant difference between those of A. nidulans (64). These data suggest that,
although A. fumigatus and A. nidulans have an outer spore rodlet layer composed
mainly of RodA, differences must exist in other components of this layer.

C. Evasion of Phagocytosis by Conidia

The first immunological line of defense against Aspergillus is macrophage func-
tion—both resident and monocyte-derived macrophages. These cells are capa-
ble of ingesting and killing spores by an opsonin-independent nonoxidative
method (65). Surfactant enhances phagocytosis of conidia. Macrophage killing
is depressed by glucocorticoids—not through a T-cell-related mechanism, but
possibly by causing failure of phagolysosomal fusion and affecting cytokine
production. Granulocyte-macrophage colony stimulating factor reverses steroid
depression in vitro (66), whereas gamma-interferon has been shown not to en-
12 Anderson et al.

hance macrophage defense. Other factors that negatively affect resident macro-
phage function include total body irradiation and some forms of chemotherapy.
Attachment of immune effector cells to A. fumigatus conidia has been stud-
ied and various receptors identified. A mannosyl-fucosyl-binding receptor is pres-
ent on macrophages, which in mice is calcium dependent but is not in humans
(67, 68). Another receptor, recognizing chitin components, is also likely given
the pattern of inhibition seen (67). Beta-glucan inhibited conidial binding to hu-
man (but not murine) monocytes by 50% to 85% (68). Further work showed that
oligosaccharides with either a β-l,3 or a β-1,4 linkage inhibited binding (68).
Aspergillus conidia also bind to type II pneumocytes, probably through a lacto-
sylceramide receptor (69). Finally, activated lymphocytes have been shown to
reduce the adherence of germinating conidia to a surface (70).
Complement is also capable of binding Aspergillus conidia and hyphae.
Binding to conidia by C3 is relatively rapid but weak in comparison to binding
to swollen conidia or hyphae (71). More molecules of complement bound to “less
pathogenic” species, such as A. glaucus, A. niger, A. nidulans, A. ochraceus, A.
terreus, A. versicolor, and A. wentii, compared with A. fumigatus and A. flavus
(72). Such differences between species could be part of the explanation for the
relative frequency of infection. Isolate to isolate variation was also observed (72).
A 54–58 kDa surface protein that binds C3 has been identified (73).
The blue-green pigment of the A. fumigatus conidium has been shown to
be the product of a single biosynthetic pathway. At least seven genes have been
identified that are involved in the conversion of acetate to a melanin. Six of these
genes are clustered within a 19-kb region of the genome (74). Mutation in the
first gene of the pathway (pksP), which codes for a polyketide synthase, results
in white conidia (75, 76); mutation of the second gene in the pathway results in
yellow conidia (77); mutation of the third and fourth genes in the pathway results
in reddish-pink conidia (74, 78), and mutation in later genes results in brown
conidia (74). Parts of the biosynthetic pathway have been elucidated and have
been shown to be the same as parts of the 1,8 dihydroxynaphthalene (DHN)-
melanin pigment pathway present in the black and brown-spored fungal species,
Magnaporthe grisea and Colletotrichum lagenarium and so, like these two spe-
cies, A. fumigatus produces a DHN-melanin. The first step, however, has been
shown to be the same as that in A. nidulans. This step involves a polyketide
synthase that converts acetyl CoA and malonyl CoA to the yellow naphthopyrone
YWA1 (77). The two pathways diverge after this, and it has been shown that
the A. nidulans pathway does not involve a DHN intermediate (79). As with A.
fumigatus, mutation of the A. nidulans polyketide synthase gene (wA) results in
white conidia. These white conidia, however, differ from those of A. fumigatus
pksP⫺ mutants, as they exhibit the ornamentation characteristic of wild-type co-
nidia, whereas A. fumigatus white conidia are smooth (80). The pigments of both
A. fumigatus and A. nidulans conidia have been shown to quench reactive oxygen
Aspergillus 13

species produced by phagocytes, to inhibit the stimulation of phagocytes (80)

and, in A. fumigatus, to inhibit C3 binding and subsequent phagocytosis (76,
78). Aspergillus fumigatus mutants with white conidia have significantly reduced
virulence in immunocompetent murine models (76, 81), and mutants with yellow
and reddish-pink conidia are also significantly less virulent (44). Interestingly,
though, a white-spored clinical isolate of A. fumigatus has been reported (82).
These data suggest that pigmented conidia are important for survival in the
host, but that the exact nature of the pigment is not so crucial. For instance, as with
A. nidulans, the production of the green pigments of A. flavus and A. parasiticus is
not prevented by inhibitors of DHN-melanin biosynthesis (83).

D. Germination
Once adherent, conidia will germinate and enter logarithmic phase growth. Two
studies have shown that tissue extracts can decrease the germination time of A.
fumigatus conidia (84, 85). Data from our unpublished experiments have shown
that a complex medium containing lecithin can decrease the time when germ
tubes are first observed by 30 minutes. Hydrocortisone, however, has no effect
on time to germination (86). A few studies of A. fumigatus have investigated the
changes in morphology that occur on germination. During the first few hours
after addition to a suitable medium, the conidia undergo a phase of isotropic
growth (swelling). Major changes have been observed in the cell wall during
this phase with the disintegration of an outer convoluted layer and associated
carbohydrates, and the unmasking or synthesis of other carbohydrates (87). These
changes result in an increased hydrophobicity and cause the conidia to stick to-
gether (agglutinate). Inhibition of protein synthesis was shown to reduce aggluti-
nation, but not swelling. After this phase of isotropic growth, polar growth occurs
with the production of a germ tube. Factor(s), possibly of a proteinaceous nature,
released by leukocytes during the inflammmatory process have been shown to
limit germ tube formation (88).
In a comparative study, definite differences were observed in morphologi-
cal events during germination between a strain of A. fumigatus and a strain of
A. nidulans (89). For instance, after the first mitosis, more than 90% of A. fumiga-
tus cells had established polar growth, whereas more than 90% of A. nidulans
cells were polar only after the second mitosis. By the fifth mitosis, just 19% of
A. fumigatus cells possessed a second germ tube, whereas 98% of A. nidulans
cells possessed a second germ tube. Septum formation was never observed in A.
fumigatus germlings with fewer than 16 nuclei, and of these germlings, 21% had
a septum. In A. nidulans, septum formation was observed in germlings with 8
nuclei, and 55% of those germlings with 16 nuclei had a septum. The first septum
formed in A. fumigatus is not laid down at the base of the germ tube, as is the
case with A. nidulans, but can be found anywhere within about 20 µm of the
14 Anderson et al.

base. Finally, this study showed that approximately 4% to 5% of the cells of both
species are in mitosis at any time.
Germination in A. fumigatus has also been investigated at the molecular
level. Glucan synthase activity is important for germ tube formation and hyphal
growth, because treatment with an inhibitor of glucan synthase resulted in pro-
found morphological changes (90). The glucan synthase complex, along with
newly synthesized β-1,3 glucans, is located at the apex of the germ tube (91).
Using antibodies to mammalian myosin, an 180 kDa immuno-analogue has been
detected in resting and swollen conidia, and in germ tubes (92). A role for myosin
in conidial swelling and germination is indicated by the fact that BDM, a potent
general inhibitor of myosin ATPase, significantly affected swelling and abolished
germination. The involvement of actin was also investigated in the same study
and a 43 kDa immuno-analogue was similarly detected in resting and swollen
conidia, and in germ tubes. An actin inhibitor, cytochalasin B, had no effect on
swelling, but partially inhibited germination. These data suggest that actin might
be required for apical growth, as has been previously described for A. nidulans
(93).

E. Growth Rate and Morphology

Factors affecting growth rate and patterns of growth could determine pathogenic-
ity, but are more likely to influence the manifestation of disease. Such factors
would include the intrinsic growth rate of any given isolate, physical barriers to
hyphal extension, immune attack, hormonal effects, nutrient supply including
oxygen, and antifungal therapy. The three-dimensional pattern of growth in the
human host may differ between isolates, and the extent of tissue invaded will be
determined by this. One aspect of this is branching frequency, which has not been
extensively studied to date in pathogenic Aspergilli. One of the chitin synthases of
A. fumigatus (ChsG) has been shown to be important in determining branching
frequency (94). Disruption of the chsG gene results in a mutant that, although
its growth rate is unaltered, produces denser colonies with almost twice as many
hyphal tips as normal. This mutant behaves differently in a murine model with
a delay in the development of respiratory illness and a statistically significant
reduction in mortality. The mutant is capable of invasive growth, but the colonies
within the lung tissue are smaller and more highly branched.
The specific growth rates of Aspergillus species are approximately the same
and, for instance, have been measured in the range of 0.43 to 0.48 h⫺1 on modified
Vogel’s medium at 32° C (86). This equates to a hyphal extension rate of 1–2
mm/hr. Addition of hydrocortisone in physiological concentrations increased the
specific growth rate by about 40% for A. fumigatus, about 30% for A. flavus (86),
and not at all for A. niger, A. oryzae, and A. nidulans (86, Anderson, unpublished
data). Other human (sex) hormones had no effect.
Aspergillus 15

Immune attack on Aspergillus hyphae appears to be mediated by comple-

ment (ineffectually) (71), phagocytic cells (especially neutrophils) (65, 95, 96)
and platelets (97). Given that those patients whose primary immune defect is
phagocytic cell dysfunction (as opposed to profound neutropenia), have more
slowly progressive IA, it is likely that the in vivo growth rate of Aspergillus
is substantially reduced by phagocytic attack. Such effects have not been mea-
sured.
Aspergilli are capable of growth at oxygen concentrations as low as 0.1%
(98). Rare instances of isolates being grown in anaerobic conditions have been
described. The invasion of devitalized tissue after trauma and the persistence of
the organism in infarcted lung, as a result of IA, requires such micro-aerophilic
growth. In addition, the action of amphotericin B on Aspergillus may be different
(ie, protective) at very low oxygen tensions (41).

F. Tissue Invasion
It is not clear that Aspergillus respects physical barriers to growth. However,
demarcation of infection by the pleura is common in neutropenic patients, and
this may constitute a physical barrier for the organism in the absence of phagocyte
attack. This hypothesis remains to be proven. Once germinated, hyphae penetrate
epithelial tissue. It is not known how this occurs. Does Aspergillus cause separa-
tion of epithelial cells by disrupting tight intercellular junctions? Do conidia get
ingested by epithelial cells, germinate within the vacuole, and then penetrate the
lung interstitium from there? Do germinating conidia damage and kill epithelial
cells, leaving a gap for hyphal penetration? Elongating hyphae have been demon-
strated in tissue culture to penetrate pneumocytes (69).
In many, but not all, instances of clinical disease, hyphae penetrate blood
vessels, particularly small arteries, leading to distal (hemorrhagic) infarction.
Vessel wall penetration is not the result of elastic degradation, but is rapid and
effected simply by turgor pressure (99). Hyphae grow within the lumen of the
vessel, leading to further thrombosis. These pathological features are similar to
icthyma gangrenosum and pneumonia caused by Pseudomonas aeruginosa, to
clostridial infection and to mucormycosis. Common pathogenetic factors are
likely, as the pathology is so similar. Phospholipases may be responsible, at least
in part, for this pathology (100), by inference from established pathogenetic
mechanisms in clostridial infection.
Some patients with IA do not have evidence of vessel invasion. Vessel
invasion appears to be more common in neutropenic patients, but is not universal.
This variation in pathology attests to both organism and host related factors being
responsible for pathological appearances. Those patients without vessel invasion
tend to have nodules with surrounding acute or chronic inflammation, sometimes
with Langerhans’ giant cells (multinucleated macrophages). This pathology is
16 Anderson et al.

much more frequent in patients with milder forms of immunosuppression, often

with phagocyte dysfunction. Even in those patients with the most severe immuno-
suppression, the prognosis seems to be better, all things considered, when there
is nodular disease without vessel invasion. Thus certain pathologies appear to be
associated with different radiological appearances and outcomes, for unexplained
reasons. One possible candidate for inducing granuloma formation is phthioic
acid, which is also produced by Mycobacterium tuberculosis. Phthioic acid is
produced by a minority of A. fumigatus isolates (101). A knock-out murine model
of chronic granulomatous disease demonstrated exuberant granulomatous re-
sponses in the lungs in response to killed Aspergillus hyphae—possibly an exag-
gerated “foreign body” reaction (102).

G. Dissemination
In a minority of cases, IA is a disseminated infection, with involvement of brain,
skin, kidneys, heart valve, and other organs. Dissemination occurs through the
bloodstream. The viable hyphal fragments (no conidia are produced in tissue)
presumably travel in the bloodstream, and two clinical observations are pertinent
to evaluating this. First, positive blood cultures of Aspergillus do occur, but are
uncommon (103, 104). Secondly, detection of Aspergillus DNA in blood occurs
almost universally in allogeneic bone marrow transplant recipients with IA (35),
and such patients have a high dissemination rate. Hence, “DNA-emia” is com-
mon, but fungemia is not. Patients who have had splenectomies may be more
likely to disseminate and have multiple sites of infection, and so the spleen
may filter out some hyphal fragments without becoming infected. Intermittent
fungemia is, therefore, likely.

IV. TOOLS FOR GENETIC ANALYSIS

A. Transformation
To manipulate the genomes of Aspergillus species in a targeted way, transforma-
tion systems suitable for use in these organisms were required. Historically, the
model fungus A. nidulans has been the subject of many genetic studies. The genes
and approaches for mutant selection and transformation procedures in A. nidulans
have since been used in other Aspergilli, such as A. niger and A. fumigatus (105).
Enzymatic removal of the fungal cell wall from recently germinated spores is used
to produce protoplasts before DNA can be taken up into the fungal cells using
polyethylene glycol (PEG) fusion/heat shock-type methodologies. The first such
use of transformation to disrupt a gene in A. fumigatus was reported in 1992 (106).
Another approach, which is technically simpler, is the use of electroporation; this
has been carried out successfully in A. fumigatus with swollen spores (107, 108).
Aspergillus 17

In general, either a dominant marker for antibiotic resistance, such as hy-

gromycin resistance, or complementation of an auxotrophic mutation, such as
pyrGI (108), is used for selection of the transformants. We have encountered
problems with the use of antibiotic markers in clinical isolates of A. fumigatus.
Sensitivity to individual antibiotics can vary greatly with different strains, and
we have commonly observed false positives during culture. These positives are
resistant to the antibiotic in comparison with the parental strain, but do not contain
an antibiotic resistance marker gene. Furthermore, unlike true transformants, the
resistance is lost almost immediately when selection is removed, suggesting a
possible up-regulation of efflux pumps in these A. fumigatus strains. This phe-
nomenon is likely to be a strain variation effect as other workers find the hygro-
mycin marker to be very useful. Indeed, nearly all reports of gene replacements
or disruptions in A. fumigatus have mainly used hygromycin resistance and, occa-
sionally, phleomycin resistance as the selectable marker. These studies have prin-
cipally been carried out to investigate whether potential pathogenicity factors
actually have an effect on pathogenicity, by using the mutants in animal models
of infection. Only three studies have been reported that have sequentially dis-
rupted two genes, in all cases using both hygromycin and phleomycin resistance
as the selectable markers (94, 109, 110).
A common target for an auxotrophic marker in filamentous fungi and yeasts
is the pyrG gene; this encodes the enzyme orotidine 5′-monophosphate decarbox-
ylase. Mutations in this gene result in strains auxotrophic for uridine or uracil.
This system is particularly attractive for use in molecular genetics, as the URA⫹
phenotype can also be selected against with the toxic analogue, 5-fluoro-orotic
acid (111). As a consequence, URA3 or pyrG blaster cassettes have been devel-
oped for several fungal species, which permits the sequential disruption of genes.
A pyrG blaster consisting of the A. niger pyrG gene flanked by a direct repeat
that encodes the neomycin phosphotransferase of transposon Tn5 has been con-
structed for use in A. fumigatus and other Aspergillus species (112). Promoters
from A. niger and A. nidulans function in A. fumigatus, presumably because of
the close evolutionary relationship of the species, and so heterologous genes from
other Aspergilli will complement auxotrophies in A. fumigatus and vice-versa,
although the complementation is not always thought to be as efficient as with
the homologous form of the gene.
Weidner et al. (108) have developed a homologous transformation system
for A. fumigatus that permits complementation of pyrG mutations using its own
pyrG gene. This system has been adapted by introducing a mutation into the
cloned pyrG gene (108). An integrative vector using this mutant allele in combi-
nation with promoter-reporter gene fusions can be constructed and used to select
prototrophic transformants from a strain containing a different mutant allele of
pyrG. Some of the transformants will contain an integration at the pyrG locus,
and these transformants can be used to study gene expression in A. fumigatus.
18 Anderson et al.

An example of a gene reporter study in A. fumigatus involved the fusion of the

alp gene promoter to the lacZ reporter gene and integration at the alp locus (110).
Expression of the gene, which encodes an extracellular alkaline protease, was
demonstrated during colonization of the lung in a murine model.
Transformation in Aspergillus species is generally achieved through inte-
gration of the transforming DNA into the genome. Autonomously replicating
plasmids have been described in A. nidulans, although they are not yet widely
used. Further development of these systems would provide extremely useful tools
for the Aspergilli (e.g., see 113). Recently, May and coworkers described the
use of an autonomously replicating plasmid-based genomic library in the model
organism A. nidulans. They have used this library to correlate gene dosage with
drug resistance (114) and to characterize cell-signaling pathways (115). They
also showed that these plasmids may have some utility in A. fumigatus (114).

B. Complementation of Mutations
Banks of A. fumigatus mutants have been generated through the traditional meth-
ods of UV (44, 81) or chemical mutagenesis (48, 78, 116) and screened using
easy-to-score phenotypes such as spore color (44, 78, 81, 116), colony morphol-
ogy (116), or auxotrophy (48). Two studies have performed screens for comple-
mentation of spore color mutations by transforming the A. fumigatus mutants
with a cosmid library using the selectable marker hygromycin resistance. Blue-
green wild-type spores were obtained when an integrated cosmid clone comple-
mented the mutations that resulted in either white conidia (75) or reddish-pink
conidia (78) and enabled the investigators to identify the mutated gene.
Complementation of mutations can also be used to establish if genes are
orthologues. A few studies have used either A. fumigatus or A. nidulans genes
to complement a mutation in the other species (62, 108, 117). For instance, a
hydrophobin gene from A. fumigatus, whose product is 86% identical to the RodA
protein of A. nidulans, was shown to complement the rodletless phenotype in
A. nidulans (62, 117). A homologous gene present in A. fumigatus, which was
identified using an A. nidulans gene-specific probe, was able to complement a
wA mutation in A. nidulans and, therefore, presumably encodes the orthologous
polyketide synthase (117). The reverse complementation was also demonstrated
to work where the wA gene of A. nidulans complemented a white conidial muta-
tion in A. fumigatus (117).

C. Parasexual Analysis
Aspergillus fumigatus, A. flavus, and A. niger are haploid organisms that do not
show a natural sexual cycle. Aspergillus nidulans differs in this respect and under-
goes a sexual cycle producing diploids under natural conditions at a low but
Aspergillus 19

measurable rate. A sexual cross is required to generate linkage data between

genetic loci. Genetic analyses such as these were made easier in A. nidulans and
in other Aspergilli by the discovery of the parasexual cycle in A. niger by Ponte-
corvo and in A. nidulans by Roper, both in 1952 (117a,b).
In the parasexual cycle, two fungal strains, regardless of compatability, can
be induced to form heterokaryons when grown in close proximity. The haploid
nuclei will then fuse to form a diploid nucleus that can be maintained in a stable
form. This diploid can be returned to the haploid state by treatment with destabi-
lizing agents, such as fluorophenylalanine or benomyl. Selection of the haploid
and diploid forms usually relies on the use of spore color mutants or mutants in
metabolic processes, such as nitrate assimilation.
The long history and relative ease, compared with other Aspergillus species,
of genetic study in A. nidulans means that many mutants are available and that
mutant alleles have been assigned to chromosomes by linkage studies. The situa-
tion in A. fumigatus is nowhere as advanced in the numbers of mutants available
and no linkage data are known. Spore color mutants have been reported (44, 74,
76, 78, 81, 116) and data have been published on the generation of diploids using
the parasexual cycle (116, 118, 119, Brookman, unpublished data). The use of
such diploids will enable heterozygotes to be produced with one copy of a gene
disrupted. If the gene is essential, then the haploid progeny produced after hap-
loidization will not be viable, whereas nonessential disruptions will produce via-
ble progeny. Haploid progeny generated from the nondisrupted allele will be
viable in all cases, but can be distinguished from the disrupted progeny by the
absence of the selectable marker (e.g., pyrG or hygromycin resistance) (119).

D. Molecular Mutagenesis
Genome-wide molecular mutagenesis approaches are required to assist in the
understanding of important biological questions in A. fumigatus, such as the iden-
tification of pathogenicity factors required in the infection process. A number of
approaches have been suggested for use with pathogens, and these are discussed
below.
Brown et al. (107) have developed a restriction enzyme-mediated integra-
tion (REMI) approach for use in A. fumigatus. Transformation by PEG-fusion
in the presence of XhoI or KpnI resulted in a high level (⬎75%) of transformants
with a single-copy integration into the A. fumigatus genome. Electroporation of
spores without the addition of restriction enzyme resulted in up to 10 times higher
transformation frequencies than were obtained with protoplasts and between 53%
and 73% of the transformants had single copy integrations (107). Production of
libraries of such mutant strains could then be used in the investigation of gene
function as has been described in plant fungal pathogens (120, 121). The gene
responsible for the altered phenotype observed can be identified using plasmid
20 Anderson et al.

rescue or inverse polymerase chain reaction (PCR). In addition, the use of in-
sertional mutagenesis in combination with a diploid strain will enable screens to
be made for essential genes (119). Recently, de Souza et al. (122) have used a
REMI system to investigate gene systems responsible for multidrug resistance
in A. nidulans. They were able to characterize these mutants further than is cur-
rently possible with A. fumigatus by performing genetic crosses with other drug-
resistant strains. They were able to identify 4 new REMI-generated mutant loci
from a total of 12 different complementation groups for the multidrug resistance/
sensitivity phenotype in A. nidulans.
Signature-tagged mutagenesis (STM) has been used for genome-wide
searches for virulence factors in pathogenic bacteria (123). This methodology
produces insertional mutants with tagged sequences to allow monitoring of entire
populations. This can be useful for studying changes in a population during infec-
tion of an animal model and can identify organisms with altered pathogenic phe-
notypes. The genetic inactivation giving rise to the changed phenotype can be
determined through the use of the signature tag on the insertion.
An STM screen has been carried out in A. fumigatus (47). Two signature-
tagged mutant libraries, one made using REMI protoplast transformation and the
other using non-REMI electroporation, were screened in a murine model of IPA.
Pools of up to 96 mutants were screened at a time. After two rounds of STM,
35 out of 4648 (0.8%) mutant strains failed to give strong hybridization signals
from the fungi recovered from murine lungs. These strains were tested against
a reference strain by infection in which the inoculum consisted of approximately
50% of each strain. The 2 strains with the lowest recovery rates from the lung
were investigated further. Plasmid rescue was used to identify where the inser-
tional mutagenesis had occurred. There were no DNA or protein database hits
to one of the sequences, but the other sequence was identified as the promoter
of the para-aminobenzoate synthase gene. A mutant defective in the synthesis of
an essential growth factor might be expected to have an attenuated virulence,
although not all such auxotrophic mutants present in the STM libraries were
identified by the screen as having a reduced virulence (0.25% of the strains were
found to be auxotrophs or have abnormal morphology). Another important obser-
vation the authors made about these screens was that, although several strains
were repeatedly identified as attenuated in pools of 96 strains, they were not
effectively outcompeted when present as 50% of the inoculum. This contrasts
with the virulence determinants of bacterial pathogens that have specifically
evolved to play an important role for survival in the host. The degree of attenua-
tion of the corresponding mutant strains of bacteria is, therefore, more likely to be
independent of the inoculum. In contrast, because A. fumigatus is an opportunistic
pathogen where many factors play a more nutritional or physiological role for
survival in the host, a slight reduction in the fitness of a mutant will probably
Aspergillus 21

result in it being outcompeted in a mutant pool, but not when there is just one
other strain present.

E. Additional Genetic Tools Required

Development of additional tools will assist in the genetic analysis of function
and pathogenicity in A. fumigatus. Steps have already been taken in setting up
some of these. For instance, insertional mutagenesis using a transposon might
result in a bank of mutants containing only single insertions rather than the vari-
ous possibilities that can result when using transformation-mediated insertional
mutagenesis (tandem insertions, multiple integrations, and DNA rearrange-
ments). Nicosia et al. (124) have recently described the transposition of a Fusa-
rium oxysporum transposon, impala, in A. nidulans. These authors report the
failure of a transposon trap in this organism to isolate active homologous transpo-
sons. We have had a similar lack of success in A. fumigatus despite exhaustive
efforts. The extension of the approach of heterologous transposition into A. fumi-
gatus and, if successful, development of an active transposon-tagging system
would be most beneficial for the elucidation of genes responsible for important
phenotypes, such as resistance to host defenses and antifungal drug resistance.
It would be desirable, also, to have a systematic procedure for knocking
out genes. It will not be possible to use a system similar to that developed for
Saccharomyces cerevisiae, in which short-flanking (⬍50 bp) or long-flanking (a
few 100 bp) homologous regions are added to either end of a selection cassette
using PCR (125, 126), as recombination in filamentous fungi requires longer
regions of homology. Generally, the longer the extent of homologous overlap,
the more efficient is the recombination. A system based on in vitro transposition
has been developed in Magnaporthe grisea for mutating genes that should be
applicable to other filamentous fungi (127). Individual cosmid bacterial artificial
chromosome (BAC) clones or entire libraries can be mutagenized in vitro using
a transposon carrying a selectable marker. Sequence analysis of the transposon
insertion sites will permit selection of the required insertional mutation, and the
entire cosmid/BAC insert can be used in a transformation to efficiently generate
homologous recombinants.
It will also be necessary to develop regulatable titratable promoters for use
in A. fumigatus to assist in the study of essential genes. Promoters, such as alcA,
which is repressed by glucose, have been used in other Aspergillus species. These
are generally very strong promoters, and the overexpression of a gene product
in a pathogenic species could cause problems with genetically modified organism
regulations. Such problems might be circumvented by using a disabled strain of
A. fumigatus, which has, for instance, been made auxotrophic for essential factors
required for growth in the host.
22 Anderson et al.

V. TOOLS FOR PHENOTYPIC ANALYSIS

A. Growth Rate
Measurement of growth rate in Aspergillus is problematic, especially in the ani-
mal host. In vitro, the traditional approach has been to determine dry weight,
after broth cultures. This method is useful for a large mass of Aspergillus, but
inaccurate for small quantities. Measurement of growth rate directly in liquid
cultures is hindered by the clumping of the hydrophobic spores, which results in
pellet formation. Pellet formation can be reduced sufficiently to use optical den-
sity readings to generate growth curves by adding the polymer Junlon (128, A.
Mousavi, personal communication). It is possible to obtain filamentous growth
by growing Aspergillus in a thin liquid layer (129), and this behavior has been
exploited in minimum inhibitory concentration (MIC) testing in microtiter plates.
Reproducible growth curves have been obtained for A. fumigatus by using micro-
titer plates in a plate reader and measuring optical density automatically (130).
Colony diameter measurement on agar is not useful, as it does not reflect
branching frequency. However, hyphal extension rates on agar can be measured
microscopically, using video capture, and this provides a highly accurate means
of determining specific growth rate (86).
Measurement of cell wall mass can be done by assaying chitin directly (as
has been performed in host tissues) (131–134) or by measuring incorporation of
radiolabeled glucosamine, a precursor of chitin (86,94). In many animal model
systems, colony forming units (cfu) are measured, usually as an indicator of anti-
fungal drug activity (43 and others). There is some uncertainty about how accu-
rate such cfu measurements are, as ungerminated conidia and hyphal fragments
may yield individual colonies. Small differences (e.g., ⬃ one log) are probably
not significant. It should be possible to measure indirectly the amount of actively
respiring fungus in infected tissue using molecular techniques, such as quantita-
tive reverse-transcriptase PCR. The relative amounts of a fungal and a mamma-
lian housekeeping gene should provide a measure of the progress of infection in
the host.

B. Animal Models
Multiple different animal models have been used in the investigation of the patho-
genesis of IA and other forms of aspergillosis. In many instances, animal models
have been a critically important component of the evaluation of new antifungal
drugs.
The pulmonary route of infection is the most common in humans, but most
murine models have used intravenous inoculation. Such infections lead to a dis-
seminated infection primarily involving the kidneys, liver, brain, and to a lesser
extent, the lung (135, 136). Pulmonary models usually result in very severe dis-
Aspergillus 23

ease and no dissemination to other organs (137). Mice, like most animals, includ-
ing humans, are intrinsically resistant to Aspergillus. Large inocula are required
to establish infection by the intravenous route and no inoculum is high enough
to infect normal mice by the pulmonary route. For this reason, mice (and most
other animals) need to be immunosuppressed to establish infection. This is typi-
cally done with chemotherapy agents that induce neutropenia, such as cyclophos-
phamide or corticosteroids (138). From an immunological perspective, these
modes of immunosuppression are very different and need to be considered sepa-
rately (136). The models that have been established in mice are usually rapidly
fatal (3–7 days). Approaches to reducing suffering in mice include timed sacri-
fice, sacrifice of mice that are obviously very ill, and determination of a tempera-
ture threshold that predicts death (Denning, unpublished data).
From the perspective of assessing pathogenicity determinants, the “whole
mouse” mortality assay has been used most commonly. Substantial differences
in the virulence of different isolates of A. fumigatus can be discerned (43, 44),
but mutant strains containing individual or even double gene disruptions have
not yielded any meaningful differences in mortality (139). The exceptions to
this have been the demonstration of reduced or nonvirulent auxotrophic mutants,
defective in their ability to synthesize para-aminobenzoic acid, pyrimidine, and
lysine, or to regulate nitrogen metabolism (47, 48, 140, 141), and that pigmentless
mutants are more readily killed and therefore less virulent (76, 81). However,
these observations, although important, do not explain the differences in virulence
between isolates.
The study of immune mechanisms of both allergic and invasive disease
has been done in different strains of mice and in mutant (knock-out) mice. For
example, cytokine concentrations have been measured in the pulmonary fluids
of mice after challenge (142). The administration of cytokine inhibitors has also
been revealing (143, 144). The role of interleukin-10 (IL-10) in IA has been
studied in IL-10 gene knock-out mice (145). Allergic responses have been studied
in several gene knock-out mice, including C–C chemokine receptor 1, chemo-
attractant protein-1 receptor, and interleukin-4 (146–148). A recent development
has used knock-out mice deficient in one or more genes critical for the oxygen-
dependent respiratory burst, which mimic some phenotypes of chronic granuto-
matous disease (102, 149). Remarkably, the inoculum used in these models is
several logs lower than in neutropenic or corticosteroid-treated mice and can be
modulated to produce acute or chronic infection (102). These mutant murine
models will be valuable for studying many aspects of the interaction of host with
Aspergillus, such as surfactant, cytokine, and mannose-binding protein polymor-
phisms.
Species other than mice used in models include the rat, rabbit, guinea pig,
chick, turkey poult, and pigeon. Reasons for these choices are summarized as
follows. Rats have been used because direct tracheal inoculation is possible, thus
24 Anderson et al.

standardizing the inoculum to the lung. Rats have, in addition, been used for
delivering aerosolized antifungal drug therapy (150, 151). Rabbits, as larger spe-
cies, allow multiple sequential blood samples to be taken. This has facilitated
pharmacokinetic and pharmacodynamic studies (152) and temporal studies of
antigenemia (38). Scanning of the lungs with CT or magnetic resonance has also
been pioneered to follow the course of the infection (153). Guinea pig models
have been used in antifungal drug studies to facilitate better drug exposure, espe-
cially with the azoles that are metabolized very quickly in mice and rats. This
has been particularly useful for the comparative evaluation of voriconazole (154).
Turkey poults have been used to study pathogenesis and immunological protec-
tion (155).

VI. ASPERGILLUS GENOMICS

A. The Aspergillus fumigatus Genome Sequencing Project
An international project has been initiated to sequence the genome of A. fumiga-
tus. A pilot project was started at the Wellcome Trust Sanger Institute and the
University of Manchester in July 1999, with funding from the Wellcome Trust,
to make a BAC library, map the clones into contigs, and sequence at least one
megabase (Mb) of DNA.
A clinical isolate (AF293) from the collection of Denning was selected as
the strain to be sequenced (156). As the primary purpose of the sequencing effort
is to assist in the understanding of pathogenicity, it was essential to select an
isolate of proven and known pathogenicity. Unfortunately, the details for almost
all isolates in culture collections are paltry and insufficient to be confident about
the pathological features of a given infection. It was also essential to select an
isolate grown from a sterile source rather than respiratory fluids to ensure that
it was not a colonizing isolate. Certain features of pathogenicity were important
to “capture,” including vascular invasion and lung infarction. This isolate was
grown from the lung of a 57-year-old woman from Shrewsbury, UK, with aplastic
anemia who died of undiagnosed invasive pulmonary aspergillosis. The aplastic
anemia may have been caused by gold therapy for rheumatoid arthritis. Her lung
revealed hemorrhagic nodules at autopsy, which indicates that there was vascular
invasion and infarction. The isolate is susceptible to itraconazole and amphoteri-
cin B, and has been shown to be virulent in two murine models. The isolate was
confirmed as A. fumigatus Fresenius by both traditional taxonomy and molecular
phylogeny. The isolate has been deposited in the National Collection of Patho-
genic Fungi (UK), the Centraal Bureau voor Schimmelcultures (Netherlands),
and the Fungal Genetics Stock Center (USA). Resolution has not been optimal,
but electrophoretic karyotyping has determined that AF293 possibly contains
eight chromosomes (Anderson, unpublished data and J.-A. van Burik, personal
Aspergillus 25

communication). There are two small chromosomes of 1.7 and 1.8 Mb (chromo-
somes 8 and 7). Chromosomes 6, 5, and 4 are all around 3.5 Mb and form a “blob”
on pulsed-field gels. Chromosome 3 is 4.0 to 4.3 Mb in size and chromosomes 2
and 1 are both 5.0 to 5.3 Mb.
Three BAC libraries have been used for the physical mapping stage of the
project. Two were constructed at the Sanger Institute (Quail and Woodman) as
part of the pilot project, using chromosomal DNA provided by the University of
Manchester (Anderson). The vector used was pBACe3.6; one library (B28) was
made using Sau3AI partially digested restriction enzyme fragments and the other
(B46) using EcoRI. The B28 library has an average insert size of 80 kb with a
range from 15 to 160 kb. Random clones (36 ⫻ 96) have been picked into repli-
cate microtiter plates and gridded onto membranes from a total of about 24,000,
and paired end-sequences have been generated. The B46 library has an average
insert size of 75 to 80 kb and a titer of 100,000. The third library was constructed
at The Centre d’Etude du Polymorphisme Humain (Billaut, France) using DNA
provided by the Institut Pasteur (Paris). The vector used was pBeloBac11 and
the enzyme HindIII. The mean insert size is 77 kb, with a range from 25 to 190
kb. Paired end-sequences have been generated from 90 ⫻ 96 clones at the Institut
Pasteur (Glaser and Kunst).
As library B28 was likely to have a more random clone distribution as the
consequence of using a 4-base-pair-specific restriction enzyme, this library was
used as the primary library for the physical mapping. The clones were digested
with the restriction enzyme PstI, which gave the optimal number of fragments
to enable fingerprint contigs to be built (20–50 per clone). In addition, 36 ⫻ 96
clones have also been fingerprinted from the two other BAC libraries. Fingerprint
contigs were built up using the fingerprinted contigs (FPC) software developed
at the Sanger Institute. A contig centered around the niaD locus has been se-
quenced as part of the pilot project. This contig was extended using the end-
sequencing and fingerprinting data until it was more than 900 kb in size.
Five sequencing centers are involved in sequencing the genome of A. fumi-
gatus. The Institute for Genomic Research (TIGR) (USA) (Nierman and Feld-
blyum) has been awarded a grant by the National Institutes of Health (NIH) (in
collaboration with the University of Manchester) under the National Institute of
Allergy and Infectious Diseases (NIAID)-funding mechanism for sequencing
small genomes. Continuation of the sequencing at the Sanger Institute (Hall and
Barrell) has been funded by an additional grant from the Wellcome Trust. The
Spanish government, through its research agency Fondo de Investigaciones Sani-
torias (FIS), has awarded a grant to three collaborating centers: Salamanca Univer-
sity (Sánchez-Pérez, Vazquez, and del Rey), Complutense University (Arroyo and
Nombela), and the Centro de Investigaciones Biológicas (Peñalva and Rodriguez).
The sequencing phase of the project will involve a whole genome shotgun
approach followed by gap closure and annotation. Shotgun sequencing will be
26 Anderson et al.

done at the Sanger Institute and TIGR to a genome coverage of 10 times, and was
initiated in April 2001. The sequence will be completed by closing sequencing and
physical gaps. The intention is to generate as complete a sequence as possible
(excluding the ribosomal DNA repeat region, centromeres, and telomeres), and
both the Sanger Institute and TIGR will annotate this complete sequence.
The genome sequencing project is being coordinated by a committee, which
includes Dr. Denning (chair), Dr. Nieman (TIGR), Dr. Hall (Sanger), Professor
Bennett (Tulane), Dr. Latgé (Pasteur), Professor Turner (Sheffield), Dr. Dixon
(NIH), and Dr. Anderson (secretary). Progress about the project is available at
the Sanger, TIGR, and Aspergillus websites.

B. Comparative Genomics
When the complete genomic sequence of A. fumigatus has been generated, and
even when whole genome shotgun assemblies are released, it will be possible to
compare this genome with the sequences available for other Aspergillus and fun-
gal species. Researchers will be able to carry out many different types of analyses
from metabolic, regulatory, evolutionary viewpoints, among others, and so only
some of the possibilities within the Aspergilli are discussed here.
Because the species within the genus Aspergillus have recently evolved
from each other, it is expected that there will be much conservation in the organi-
zation of their genomes. Already, for instance, the evidence from genetics and
electrophoretic karyotyping suggests that most species have eight chromosomes.
Therefore, one of the first questions that will be resolved from the sequencing
of A. fumigatus is how many chromosomes it also has. There should, in addition,
be a high level of conservation in gene order between species. It will be possible
to make a direct comparison with the sequenced cosmids from chromosome VIII
of A. nidulans, but the sequence of chromosome IV and the whole genome shot-
gun of A. nidulans will be of only limited use in comparing gene order unless
the contigs are bigger than the average gene. However, extensive genetic and
physical maps are available for A. nidulans, and so it should be possible to carry
out comparisons at this level. The only preliminary evidence that there will be
conservation in chromosome structure is that the nitrate-assimilation gene cluster
has been mapped to the largest chromosomes in both species (157, 158).
A direct comparison of the gene complements in various Aspergillus spe-
cies will be of greatest interest for many researchers, and here, even information
from partial whole genome shotgun and EST sequencing will be valuable. The
total number of genes is not expected to vary much between species, but the
differences will be most revealing. It will be interesting to see which additional
genes are present in the more pathogenic species, such as A. fumigatus. For exam-
ple, it has been shown that the gene, which encodes the cytotoxic ribonuclease
mitogillin, is not present in A. flavus, A. niger, or A. nidulans (159).
Aspergillus 27

Many differences between the species might be expected to occur in the

so-called “dispensible” metabolic pathways (160), which includes biosynthetic
pathways involved in the production of secondary metabolites (e.g., 74, 161–
163). These dispensable metabolic pathways are often arranged into gene clusters,
where the genes are coregulated and contain definable cis-acting elements in their
promoters. It is possible, perhaps, when these genes are present in clusters, that
they are more readily gained or lost. It will initially be easy to see if A. fumigatus
contains pathways or portions of pathways that have been studied in other Asper-
gilli: an obvious example would be the aflatoxin biosynthetic pathway, as A.
fumigatus produces an intermediate in this pathway (sterigmatocystin). An exam-
ple of a difference between A. fumigatus and A. nidulans in a disposable meta-
bolic pathway is the conidial pigment biosynthetic pathway. The first enzyme in
the pathway is the same in both species; however, after that the genes and en-
zymes are different. The two genes in A. nidulans, wA and yA, are located on
different chromosomes, whereas most of the genes for pigment synthesis in A.
fumigatus are located in a 19-kb cluster. Interestingly, the wA orthologue is lo-
cated at one end of this cluster and the largest intergenic distance (1.1 kb) in the
cluster lies between it and the next gene (74).
Another area where differences have been reported between Aspergillus
species is in the number of copies of a protein family. For instance, five chitin
synthases have been detected in A. nidulans, whereas seven genes have been
detected in A. fumigatus (164). Two extracellular metalloproteinases have been
reported in A. fumigatus, but only one of these is present in A. flavus (165, 166).
One obvious difference between A. fumigatus and A. nidulans is the lack
of a detectable sexual cycle in A. fumigatus, and it will be interesting to see if
this is due to missing or nonfunctional genes. It had been assumed that the yeast
Candida albicans was an asexual organism, but a mating-type-like locus was
identified in the whole genome shotgun sequence, and subsequent experiments
have demonstrated that C. albicans is able to undergo mating (167, 168). Addi-
tional analysis of the C. albicans sequence, comparing it with S. cerevisiae, indi-
cated that C. albicans should be able to undergo meiosis as well (169).
It will, of course, be possible to use the gene micro-arrays that will be
produced as a consequence of the A. fumigatus genome sequencing project, to
carry out cross-species comparisons and screen for those genes that are unique
to A. fumigatus. Electrophoretic karyotyping has shown that there is considerable
variation in chromosome size between isolates of A. fumigatus (Anderson, unpub-
lished data), and so these microarrays could also be used to check the gene com-
plements of isolates as well.
Even though relatively little research has been carried out on the genes
and proteins of A. fumigatus, there has been much research published on other
Aspergillus species, especially A. nidulans. Therefore, it will be possible to iden-
tify orthologues in other species and start with the premise that the protein per-
28 Anderson et al.

forms the same role in A. fumigatus. This has generally been shown to be the
case with the orthologous genes that have been studied, such as the hydrophobin
RodA (62), the polyketide synthase involved in pigment biosynthesis (77), the
pyrG gene (108, 112), the pdmA gene (114), and the brlA fluG, flbA, and trpC
genes (117). One notable exception is the A. fumigatus chitin synthase, ChsG
and its orthologue in A. nidulans, ChsB. Both chitin synthases are class III en-
zymes, and they are 90% identical at the protein level and 76% identical at the
DNA level over the coding region. At the level of gene structure, there is complete
conservation in the positions of the start and stop codons and the splice junctions
(except for the 5′ splice site of intron 1), and the exon sizes vary by multiples
of 3 (A. fumigatus/A. nidulans exon 1: 84/93; exon 2: 475/481; exon 3: 362/
362; exon 4: 847/847; exon 5: 830/830; exon 6: 138/138). Both enzymes have
been shown to have important roles in hyphal tip growth, but the severity of the
phenotype in the disrupted mutants differed. The A. fumigatus chsG⫺ mutant was
more highly branched than normal, and its conidia failed to mature properly (94),
whereas the phenotype of the A. nidulans chsB⫺ mutant was more severe, with
the highly branched hyphae also displaying enlarged tips with bulges along their
lengths; in addition, no conidiophores or conidia were formed (170, 171). The
differences in phenotype is not the consequence of A. fumigatus containing an
additional class III enzyme, ChsC, because a mutant disrupted in both genes
displays the same phenotype as a chsG⫺ mutant (94). It is still possible, however,
that functional redundancy exists, and that one of the other chitin synthases in
A. fumigatus is important for some of the roles carried out only by ChsB in A.
nidulans. It will, therefore, require complementation experiments to prove for-
mally that these two enzymes can perform the same functions in both organisms.

VII. FUTURE APPROACHES TO UNDERSTANDING

PATHOGENICITY

It is highly likely that there are multiple pathogenicity determinants, some opera-
tive in certain host settings and others in other settings. So the ability of Aspergil-
lus to invade the cornea of the eye will likely depend on different organism-
related factors than those required for the invasion of a burn wound or a lung in
a neutropenic patient. Given this, highly specialized model systems will need to
be developed to dissect out the detailed mechanisms involved. A transcriptome
analysis of Aspergillus will be of little use if very broad questions are asked,
such as which genes are involved in pulmonary infection and RNA is derived
from a rapidly lethal murine model, two or more days after infection. If, on the
other hand, RNA is derived from each and every step of the infection process,
then the data derived will be much more useful. Knock-outs of genes thought to
be important can then be produced and studied in each stage of the infection
Aspergillus 29

process. The use of different models, with different wild-type A. fumigatus iso-
lates may also be revealing, in that alternative pathogenetic mechanisms may be
revealed. Multiple knock-outs, with phenotypic correlates, could start to unravel
the complexities of why Aspergillus is such a common allergic and opportunistic
pathogen, and yet can also cause disease in normal hosts.

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Exploring the Variety of Random
Documents with Different Content
"And some brandy," said Fischer. "This scratch is deucedly painful."
There was a moment's silence. Then Chérie, taking a step towards
the door, said, "I will fetch some brandy."
"I'll come too," said Mireille.
"No, no, no, no," cried Von Wedel, catching hold of them each by
one arm. "You two want to run away. I know your tricks! No. The
vixen stays here; and the angel"—bending to gaze into Chérie's face
—"comes with me and shows me where the brandy is kept."
"She shan't! she shan't!" screamed Mireille, clinging to Chérie's arm.
"Donner und Blitz!" exclaimed Von Wedel, "what a little demon. You
just catch hold of her, Glotz, and keep her quiet."
Glotz, who had been sitting at the table eating silently, rose and
dried his mouth on one of the beflowered tissue-paper serviettes. "I
know where the cellar is," said he, "I saw it on my round with the
Herr Kapitän. If the Herr Kapitän permits, I will fetch the brandy
myself." And he left the room quickly, paying no heed to Von Wedel's
murmured remark that he was a confounded interfering head of a
sheep.
Louise had burst into tears when Von Wedel had told Glotz to hold
Mireille, and although the captain patted her hand and told her not
to cry she went on weeping bitterly while she bandaged his arm.
Von Wedel looked at her a moment and then turned to Chérie.
"What relation are you to that weeping Niobe? I forget."
"Sister-in-law," murmured Chérie inaudibly.
"What? Speak louder. I can't hear," said Von Wedel, seating himself
on a corner of the table and lighting one of Dr. Brandès's cigars.
"Sister-in-law," repeated Chérie faintly.
"Sister-in-law? Good." He puffed at the cigar. "And I'll be your
brother-in-law, shall I? Ah, here is the wine!" he exclaimed as the
door was thrown open.
But it was not the wine. It was another officer, dressed like the
others in a grey uniform bereft of all insignia; he was very red and
covered with dust and mud. He saluted the captain and nodded to
the lieutenant, loosened his belt and flung his grey helmet on the
piano where the others lay.
"Ah, Feldmann," cried Captain Fischer. "What have you done?"
"My duty," said the new-comer in a curious hoarse voice.
"Der Pfarrer?" ... questioned Von Wedel.
The man nodded and made a grimace. "And that idiot of a scout-boy
too. It was he who fired at you," he said turning to Fischer.
"It was not," said the captain. "It was an old man, from a window.
Near the church."
"Oh well, I didn't see any old man," said Captain Feldmann. "And
these civilians must be taught their lesson.... What have we here?"
he added, surveying the table. "I am famished." And he took two or
three sandwiches, placed them one on the other and ate them.
"Beastly hole, this," he remarked, with his mouth full. "We needn't
have come here at all."
"Oh yes, we need," declared Fischer very sternly.
"Well, we won't discuss that," said Feldmann. "And anyhow we are
going on in the morning. I should like something to drink."
Chérie had flushed to the roots of her hair. She had grasped the one
thing only—they were going on in the morning! At any cost she must
tell Louise that wonderful news. And she did so rapidly, in low tones,
in Flemish.
Louise, who had finished bandaging the officer's wounded arm,
burst into tears again; this time they were tears of joy.
"What are these women?" inquired Feldmann, glancing around with
his mouth full. "They look like ballet-dancers."
"That one," said Von Wedel, with a coarse laugh, pointing at Louise,
"is the weeping Niobe; and that" indicating Mireille—"is the demon
child. And this"—taking Chérie's wrist and drawing her towards him
—"is my sister-in-law and an angel."
"And this is Veuve Clicquot '85," said Glotz entering with some
bottles in his hand and stepping as if casually between Chérie and
her tormentor.
The men turned all their attention to the wines, and sent Glotz to
the cellar three or four times to fetch some more.
They wanted Martel; they wanted Kirsch; they wanted Pernod. Then
they wanted more champagne. Then they wanted more sandwiches,
which Louise went to make. Then they wanted coffee, which
Feldmann insisted upon making himself on a spirit-lamp. They set
fire to the tablecloth and to the tissue-paper serviettes, which they
threw down and stamped out on the carpet.
Von Wedel sat down at the piano and sang "Traum durch die
Dämmerung," and Feldmann wailed a chorus. Then Feldmann
recited a poem. He was very tipsy and had to put one arm around
Glotz's neck and lean heavily on Glotz's shoulder in order to be able
to stand up and gesticulate.

"Liebe Mutter, der Mann mit dem Kocks ist da!"

"Schweig still, mein Junge, das weiss ich ja.
"Hab'ich kein Geld, hast du kein Geld,
"Wer hat denn den Mann mit dem Kocks bestellt?"

Great laughter and applause from Captain Fischer and Von Wedel
greeted this; only Glotz remained impassive; with Feldmann's arm
around his neck, his chubby countenance unmoved, his expression
vacant.
For some time they paid no heed to the three women clustered
together in the furthest corner of the room, except to stretch out a
detaining hand whenever they tried to move towards the door.
"No," declared Von Wedel, leering at them through his light, vague
eyes. "No. You don't leave this room. Not all three together. Only
one at a time; then we're sure she'll come back."
So they clung together with pale bewildered faces, whispering to
each other every now and then the comforting words, "They will go
away in the morning."
But the morning was not yet.
When Captain Fischer suggested that it was time to go to bed, the
others called him an old screech-owl; whereupon Captain Fischer
explained to them at great length that military discipline did not
permit them to call him a screech-owl. And he called Louise to
witness that he had been called a screech-owl.
But now Feldmann was singing "Gaudeamus igitur," so the captain
joined in too.
"Come along," said Von Wedel, lurching towards Chérie with two
glasses in his hand; "come, turtle-dove, Brüdershaft trinken!" He
forced one of the glasses into her hand. "You must drink the pledge
of brotherhood with us. Like this"—and he made her stand face to
face with him, pushing his left arm through hers and raising his glass
in his right hand.
Chérie shrank back, seeking refuge behind Louise. But he dragged
her forward and caught her by the arm again.
"Obedience!" he roared, scowling at her. "Now sing; 'Lebe, liebe,
trinke, schwärme'—and when I get to the words 'froh mit mir,' we
clink our glasses together."
"Please not! please not!" implored Chérie.
"Froh mit mir"—repeated he, glaring at her through his heavy lids.
And he sang:

Lebe, liebe, trinke schwärme

Und erfreue dich mit mir.
Härme dich wenn ich mich härme
Und sei weider
froh
mit
mir!

At the last three words he clinked his glass against Chérie's. "Drink!"
he commanded in a terrible voice. "If you do not drink, it is an insult
which must be punished."
With a sob Chérie raised the glass to her lips.
Louise was wringing her hands. "The brute! the brute!" she cried,
while Mireille holding her mother's skirts stared wide-eyed at the
scene.
Captain Fischer looked across at Louise. "My Samaritan," ... he
mumbled. "My sister of mercy...." He rose and approached her with
a stupefied smile.
Mireille rushed at him like a little fury. "Go away," she screamed, "go
away!"
The Herr Kapitän took her not unkindly by the shoulders. "Little girls
should be in bed," he said thickly. "My little girls are in bed long
ago."
Louise clasped her hands. "I beg you, sir, have pity on us; let us go
away.... The house is yours, but let us go away."
"Where do you want to go?" he asked dully.
"To our rooms," said Louise.
"You have no rooms; they are ours," he said, and bending forward
he widened his eyes at her significantly.
Louise looked about her like a trapped animal. She saw Von Wedel
and Feldmann who had Chérie between them and were forcing her
to drink out of their glasses; she saw Glotz seated on the piano-stool
looking on with fat, impassive face; she saw the man before her
bending forward and leering suggestively, so close that she could
feel his hot, acrid breath on her face. The enemy! The man with
mud and blood on his feet ... he was putting out his hand and
touching her——
She fell on her knees and dragged Mireille down beside her! she
lifted up her hands and raised her weeping face to him. "Your
children ... you have children at home ... your little girls are in bed
and asleep ... they are safe ... safe, locked in their house.... As God
may guard them for you, oh protect us! spare us! Take care of us!...
Be kind—be kind!" She dropped forward with her head on his feet—
on Claude's slippers—and little Mireille with quick tears rolling down
her face looked up at him and touched his sleeve with a trembling
hand.
He looked down and frowned. His mouth worked. Yes. He had three
yellow-headed little girls in Stuttgart. It was good that they were in
Stuttgart and not in Belgium. But they were little German girls, while
these were enemies. These were belligerents. Civilians if you will,
but still belligerents....
He looked down at the woman's bowed head and fragile heaving
shoulders, and he looked at the white, frightened child-face lifted to
his. "Belligerents" ... he growled, and cleared his throat and
frowned. Then his chin quivered. "Get away," he said thickly. "Get
away, both of you. Quick. Hide in the cellar—no—not in the cellar, in
the stable—in the garden—anywhere. Don't go in the streets. The
streets are full of drunken soldiers. Go."
Louise kissed his feet, kissed Claude's slippers, and wept, while
Mireille smiled up at him with the smile of a seraph, and thanked
and thanked him, not knowing what she thanked him for.
"But—what of Chérie?" gasped Louise, looking round at the
frightened wild-rose figure in its white dress, trembling and weeping
between the two ribald men.
"You shall take her with you," said Fischer, and he went resolutely
across the room and took Chérie by the arm.
"What? What? You old reprobate," roared Feldmann, digging him in
the ribs, with peals of coarse laughter. "You have two of them! What
more do you want, you hedgehog, you? Leave this one alone."
"You leave her alone, too. I order her to go away." Fischer frowned
and cleared his throat and tried to draw Chérie from Feldmann's and
Von Wedel's grasp.
"What do you mean?" asked Von Wedel, going close up to Fischer
and looking him up and down with provocative and menacing air.
"I mean that you leave her alone," puffed the captain. "Those are
my orders, Lieutenant—and if they are not obeyed you shall answer
for it."
"You old woman! you old head of a sheep," shouted Von Wedel;
"answer for it, shall I? You are drunk; and I'm drunk; and I don't
care a snap about your orders." And dragging Chérie's arm from
Fischer's grasp he pushed him back and glowered at him.
"Your orders ..." stuttered the intoxicated Feldmann, placing his
hand on Fischer's shoulder to steady himself, "your orders ... direct
contradiction with other orders ... higher orders ..." He wagged his
head at Fischer. "The German seal must be set upon the enemy's
country.... Go away. Don't be a screeching owl."
"And don't be a head of a sheep," added Von Wedel. "Vae victis! If it
isn't you, it'll be somebody else. It'll be old Glotz—look at him ...
sitting there, all agog, arrectis auribus! Or it will be our drunken men
downstairs. Just listen to them!..."
The drunken men downstairs were roaring "Die Wacht am Rhein."
Von Wedel's argument seemed to carry conviction.
"Vae victis!" sighed Fischer, swallowing another glass of brandy and
looking across the room at the trembling Louise. "If it isn't I ... then
Glotz ... or somebody else ... drunken soldiers...."
He went unsteadily towards Louise, who stood clutching at the
locked door. "Woe to the vanquished, my poor woman ... seal of
Germany ... higher orders.... Why should I be a head of a sheep?..."
BOOK II
 CHAPTER VI
It is pleasant to sit in a quiet English garden on a mild September
afternoon, sipping tea and talking about the war and weather, while
venturesome sparrows hop on the velvety lawn and a light breeze
dances over the flower-beds stealing the breath of the mignonette to
carry back at nightfall to the sea.
Thus mused the gentle sisters, Miss Jane and Julia Cony, as they
gazed round with serene and satisfied blue eyes on the lawn, the
sparrows, the silver tea-set, the buttered toast, and their best friend,
Miss Lorena Marshall, who had dropped in to have tea with them
and whose gentle brown eyes now smiled back into theirs with the
self-same serenity and satisfaction. All three had youthful faces
under their soft white hair; all three had tender hearts in their
somewhat rigid breasts; all three had walked slender and tall
through an unblemished life of undeviating conventionality. They
were sublimely guileless, divinely charitable and inflexibly austere.
"It is pleasant indeed," repeated Julia in her rather querulous treble
voice. Julia had been delicate in her teens and still retained some of
the capricious ways of the petted child. She was the youngest, too—
scarcely forty-five—and was considered very modern by her sister
and her friend. "Of course the Continent is all very well in its way,"
she went on. "Switzerland in summer, and Monte Carlo in winter
——"
"Oh, Julia," interrupted Miss Jane quickly, "why do you talk about
Monte Carlo? We only stayed there forty-five minutes."
"Well, I'm sure I wish we could have stayed there longer," laughed
the naughty Julia. "The sea was a dream, and the women's clothes
were revelations. But, as I was saying, England is, after all——"
We all know what England is, after all. Still, it is always good to say
it and to hear it said. Thus, in the enumeration of England's
advantages and privileges a restful hour passed, until the neat maid,
Barratt, came to announce the arrival of other visitors. Mrs.
Mulholland and her daughter Kitty had driven round from Widford
and came rustling across the lawn in beflowered hats and lace veils.
Fresh tea was made for them and they brought a new note into the
conversation.
"Are you not thinking of taking a refugee?" asked Mrs. Mulholland.
"The Davidsons have got one."
"The Davidsons have got one?" exclaimed Miss Marshall.
"The Davidsons have got one?" echoed Miss Jane and Miss Julia
Corry.
"Yes, indeed," said Mrs. Mulholland somewhat acidly. "And I am sure
if they can have one in their small house, you can; and we can."
"Refugees are all the rage just now," remarked Kitty. "Everybody
who is anybody has them."
"Yes, but the Davidsons ..." said Miss Marshall. "Surely they cannot
afford it."
"They have dismissed their maid," explained Mrs. Mulholland, "and
this poor Belgian woman has to do all their housework."
"Yes; and Molly Davidson says that she is really a countess," added
Kitty, "and that she makes the beds very badly."
"Poor soul!" said Miss Jane.
"I certainly think," continued Mrs. Mulholland, "that the Davidsons of
all people should not be putting on side with a foreign countess to
make their beds for them, while others who have good houses and
decent incomes simply look on. In fact," she added, "I have already
written to the Committee in Kingsway offering hospitality to a family
of two or three."
"That is very generous of you," said Miss Jane; and Miss Julia shyly
patted the complacent white-gloved hands reposing in Mrs.
Mulholland's lap.
"We had not thought of it ourselves, so far," said Miss Jane. "But if it
is our duty to help these unfortunates, we shall certainly do so."
"Of course you will. You are such angels," exclaimed the impulsive
Kitty, throwing a muscular arm around Miss Jane's prim shoulders
and kissing her cheek. And Miss Jane liked it.
"How does one set about it?" asked Miss Marshall; "I might find
room for one, too. In fact I should rather like it. The evenings are so
lonely and I used to love to speak French."
Mrs. Mulholland, to whom she had turned, did not answer at once.
Then she replied drily: "You can write to the Refugee Committee or
the Belgian Consulate. The Davidsons got theirs from the Woman's
Suffrage League."
Then there was a brief pause.
"But I hear that the committee is frightfully particular," she went on.
"They don't send them just to any one who asks. One must give all
sorts of references. In fact," she added, with a chilly little laugh, "it
is almost as if one were asking for a situation oneself. They want to
know all about you."
There was another brief silence, and then Mrs. Mulholland and Kitty
took their leave.
To Miss Julia, who accompanied them to the gate, Mrs. Mulholland
remarked, "The idea! Miss Marshall wanting a refugee! With her
past!"
"What past?" inquired Miss Julia, wide-eyed and wondering.
"Oh," snapped Mrs. Mulholland, tossing her head, and the white lace
veil floating round her sailor-hat waved playfully in the breeze,
"when people live abroad so long, there is always something behind
it."
She stepped into her motor, followed by the pink-faced, smiling Kitty,
and they drove away to pay some other calls.
Miss Julia returned to the lawn with a puckered brow and a
perturbed heart. Neither she nor her sister had ever thought of Miss
Lorena Marshall's past; Miss Marshall did not convey the impression
of having a past—especially not a foreign past, which was associated
in Jessie's mind with ideas of the Moulin Rouge and Bal Tabarin. The
neat black hat sitting firmly on Miss Marshall's smooth pepper-and-
salt hair could never be a descendant of those naughty French petits
bonnets which are flung over the mills in moments of youthful folly.
Her sensible square-toed boots firmly repelled the idea that the feet
they encased could ever have danced adown the flowery slopes of
sin.
"I do not believe a word of it," said Miss Julia to herself, and later on
to her sister. Miss Jane was indignant at the suggestion. "This village
is a hotbed of cats," she said cryptically; and when the vicar looked
in after dinner to discuss arrangements for a Church concert they
confided in him and asked his opinion. Had he known Miss Lorena
Marshall before she came to Maylands? Did he think she had a past
—a Continental past?
The vicar thought the suggestions ridiculous and uncharitable.
"Of course," said Miss Jane, toying with her favourite angora cat's
ear as he lay purring comfortably in her lap, "we are narrow-minded
old maids." The vicar made a deprecating gesture. "Yes, yes, we are.
And we like to be sure that our friendships are not misplaced."
"We are narrow-minded old maids," echoed Miss Julia. The two Miss
Corrys always said that, partly in order to be contradicted and partly
in that curious spirit of humility which in the English heart so closely
borders on pride. For is not the acknowledgment of a certain kind of
inferiority a sign of unmistakable superiority?
When we say we are a humdrum nation, when we say we are a dull
and slow and stodgy nation, do we not in our heart of hearts think
that it would be a good thing if other nations took an example from
our very faults?
Even so when Miss Corry said, "We are narrow-minded old maids"—
she felt with a little twinge of remorse that the statement was not
altogether sincere. Did she really, in her heart of hearts, think it
narrow-minded to abhor vulgarity, to shun coarseness, to shrink
from all that might be considered indecorous or unseemly? Then
surely to be narrow-minded was better than to be broad-minded,
and she for one would certainly refuse to change her views. Was
narrow-mindedness mindedness nowadays not almost a synonym for
pure-mindedness?
And—"old maids"! Did she really consider herself and her younger
sister old maids? Had they—just because they had chosen to remain
unmarried—any of the crotchety notions, the fantastic, ineradicable
habits that old maids usually get into? Did they go about with a
parrot on their shoulder like Miss Davis? Or dose themselves all day
with patent medicines, like the Honourable Harriet Fyle? Did they
fret and fuss over their food, or live in constant terror of draughts
and burglars? Certainly not. And—come now—did they really feel a
day older than when they were twenty-two and twenty-five
respectively? Or did they look any older?—except for their hair
which, had they chosen, they could easily have touched up with
henné or Inecto? Were they not able to do anything, to go
anywhere? Were their hearts not as young, and fresh, and ready for
love if it happened to come their way, as Kitty Mulholland's or Dolly
Davidson's? Did not their elder brothers—the parson and the Judge
—always speak of them still as "the girls"?
No. Miss Jane and Miss Julia Corry were not quite sincere when they
called themselves "narrow-minded old maids," and accordingly they
had qualms and conscience-pricks when they did so.

A week later the two sisters returned Mrs. Mulholland's call. They
fluttered into the large drawing room full of the subdued murmur of
many voices, and were greeted absent-mindedly by the busy hostess
and effusively by Kitty. The Davidsons were there, quite unsuitably
attired (remarked Miss Jane to Miss Julia; nobody wore satin at tea),
and they were explaining volubly to a group of ladies how it
happened that their Belgian countess-refugee had suddenly left
them.
"First of all, she was not a countess at all," explained Dolly Davidson.
"And she was not even a Belgian," Mrs. Davidson added, in
aggrieved tones. "I cannot understand the W.S.L. sending her to us.
Why she confessed before she went away that she was a variety
artist from Linz and could only speak German and Czech. We always
thought the language she spoke was Flemish. It has been a most
unpleasant affair."
Every one was tacitly delighted. Mrs. Davidson had been giving
herself such airs of importance with her countess, and now it turned
out that she had been playing Lady Bountiful to an alien enemy from
a Bohemian Café Chantant. One would have to be super-human not
to rejoice. "How did you get rid of her?" asked one of the ladies,
discreetly repressing her smiles.
"A villainous-looking man came to fetch her, late in the evening,"
said poor Mrs. Davidson, blushing. "They made a frightful noise in
the hall, quarrelling or something."
"Then they both went upstairs," piped up Dolly Davidson; and
pointing to her brother, a lumpish youth who at that moment had his
mouth full of cake. "We sent Reggy upstairs to tell them to go away
at once. But Reggy only looked through the keyhole and wouldn't
come down again until mother fetched him."
"It isn't true," mumbled Reggy.
"Finally we had to send for the police," said Mrs. Davidson, with
tears of mortification in her eyes.
Mrs. Mulholland confessed that she felt rather nervous about her
own refugees who were expected at any moment. "I wish I could
countermand them," she said; but her sympathizing friends all
agreed that having asked for them she must keep them when they
came.
They arrived the following day—an uninteresting woman, with two
torpid boys and a thin girl of fifteen.
The boys ate a great deal, and the girl was uncannily intelligent.
Since landing in England they had had it drummed into them that
they were heroes; they had been acclaimed with their compatriots
as the saviours of Europe; they had had speeches made to them
apprising them of the fact that the gratitude of all the world could
never repay the debt that civilization owed them. They therefore
accepted as their due the attentions and kindness shown them. They
ate jam at all their meals and asked for butter with their dinner; they
drank red wine and put a great deal of sugar in it; they complained
that the coffee was not good. They borrowed Mrs. Mulholland's seal-
skin coat and Kitty's silk scarves when they felt chilly, and they sat in
the drawing-room writing letters or looking at illustrated papers all
day long. They spoke French in undertones among themselves and
accepted everything that was provided for them without any undue
display of gratitude. Had they not saved Europe? Would Mrs.
Mulholland still have a seal-skin coat to her back but for Belgium?
Had it not been for King Albert, would not the Uhlans and the
Death's Head hussars be sprawling on the Mulholland sofa, eating
the Mulholland jam, criticizing the Mulholland coffee? Comment
donc!
And had they not themselves, in order to save Europe, given up their
home and their business—a stuffy little restaurant (Au Boeuf à la
Mode, Épicerie, Commestibles) down a dingy Brussels street?
The restaurant soon became a Grand Hotel in their fond
reminiscences. Le souvenir, cet embellisseur, swept the sardine-tins,
the candles, the lemons, and the flies from its windows, built up a
colonnaded front, added three or four stories and filled them with
rich and titled guests.
"What was the name of your hotel?" inquired Mrs. Mulholland. "We
stopped in Brussels once on our way to Spa, and I remember that
we stayed in a most excellent hotel—The Britannique, or The
Metropole, or something."
"Tell them," said Mme. Pitou to her daughter Toinon who acted as
interpreter,—"tell them the name of our hotel—in English."
"Restaurant to the Fashionable Beef," said Mademoiselle Pitou; and
Madame Pitou sighed and shook her head despondently. "Hotel," she
corrected, "not Restaurant. 'Hotel to the Fashionable Beef.' Toinon,"
she added, "do ask these people to give us potage aux poireaux this
evening, for I cannot and will not eat that black broth of false turtle
any more."
 CHAPTER VII
The craze for refugees cooled slightly in the neighbourhood after
that. The first rush of enthusiastic generosity abated, and when
friends met at knitting-parties and compared refugees there was a
certain ægritude on the part of those who had them, and a certain
smiling superiority on the part of those who had not. They were
spoken of as if they were a disease, like measles or mumps.
"I hear that Lady Osmond has them," said Mrs. Mellon.
"Has she really?"
"Yes. And poor Mrs. Whitaker, too."
"Mrs. Whitaker? You don't say so."
"Yes, indeed. Mrs. Whitaker has them. And she feels it badly."
"I will run over to see her," said the sympathetic Mrs. Mulholland. "I
am so fond of the dear soul."
But that very afternoon Mrs. Whitaker herself called on Mrs.
Mulholland, at Park House.
"How do you do, my poor dear Theresa?" began Mrs. Mulholland,
taking Mrs. Whitaker's hand and pressing it. "I hear——"
"Yes, yes," said Mrs. Whitaker rather fretfully, drawing her hand
away. "Of course you have heard that I have them." There was a
brief silence. "I must confess I did not expect quite such dreary
ones."
"Dreary, are they?" exclaimed Mrs. Mulholland. "Is that all?"
"It's bad enough," sighed Mrs. Whitaker. "You have no idea what
they are like. Three creatures that look as if they had stepped out of
a nightmare."
But Mrs. Mulholland overflowed with her own grievances. "Do they
borrow your clothes and use all your letter-paper and order your
dinners?" asked Mrs. Mulholland, quivering with indignation. Her
cook had just given notice on account of Madame Pitou going into
the kitchen and making herself a timbale de riz aux champignons.
"No. They don't do that. But they sit about and never speak and look
like ghosts," said Mrs. Whitaker. "When you have time you might
drop in and see them."
"I think I'll run over with you now," said Mrs. Mulholland; "though I
don't for a moment believe they can be as bad as mine."
She put on her garden-hat and her macintosh, told Kitty not to let
the Pitous do any cooking in the drawing-room, and went out with
Mrs. Whitaker. They took the short cut across the fields to Acacia
Lodge.
"What language do they speak?" asked Mrs. Mulholland, as she
proceeded with Mrs. Whitaker through the green garden-gate and
down the drive.
"They never speak at all," replied Mrs. Whitaker; "and I must say I
had looked forward to a little French conversation for Eva and Tom.
That is really what I got them for."
They walked on under the chestnut-trees towards the house. Eva in
trim tennis attire and George in khaki came to meet them, running
across the lawn.
"I've beaten George by six four," cried Eva, waving her racket.
"That's because I let you," said her brother, shaking hands with Mrs.
Mulholland and allowing his mother to pat his brown cheek.
"Handsome lad," murmured Mrs. Mulholland, and wished she had
brought Kitty with her, even though the Pitous should profit by her
absence to prepare their tête-de-veau en poulette on the drawing-
room fire. "Where are ... they?" she added, dropping her voice and
looking round.
"I don't know," said Eva. "I have not seen them all the afternoon."
"I have," said George. "They are in the shrubbery."
"You might call them, dear boy," said his fond mother.
"Not I," said George.
"I will," said Eva, and ran down the flower-bordered path swinging
her racket.
"Sweet girl," said Mrs. Mulholland, following Eva's slim silhouette
with benevolent eyes, and then gazing even more benevolently at
George Whitaker's stalwart figure. "She and my Kitty should really
see something more of each other."
Mrs. Whitaker threw a penetrating glance at her friend's profile.
"Schemer," she murmured to herself. "Certainly," she said aloud. "As
soon as George goes to Aldershot I hope your dear daughter will
often come here."
"Cat," reflected Mrs. Mulholland. And aloud she said, "How delightful
for both the dear girls!"
George had sauntered with his long khaki limbs towards the
shrubbery, but Eva reappeared alone.
"They won't come," she said.
"What!" exclaimed Mrs. Mulholland.
"Why not?" asked Mrs. Whitaker.
"They don't want to," said Eva. "The tall one shook her head and
said, 'Merci.'"
"I am not surprised," laughed George, "considering they have been
exhibited to half the county within the last three days."
"I'll fetch them myself," said Mrs. Whitaker sternly. Then she turned
to her son. "George, you who are half a Frenchman after your visit
to Montreux, do tell me—how do I say in French, 'I desire you all
three to come and be introduced to a very dear friend of mine?'"
There was a brief silence; then George translated. "Venny," he said.
"Is that all?"
"Yes," said George.
His mother was about to go when Mrs. Mulholland suggested: "Had
we not both of us better take a turn round the garden, and casually
saunter into the shrubbery?"
"Perhaps so," said Mrs. Whitaker.
And so they did. George followed them slowly, with Eva hanging on
his arm. She was very fond and proud of her soldier brother.
They entered the shrubbery and saw seated upon a bench three
figures dressed in black, who rose to their feet at their hostess's
approach.
"Goodness gracious! how uncanny they look!" whispered Mrs.
Mulholland, and added, with a smile of half-incredulous pleasure, "I
believe they really are worse than mine."
The three black figures stood silent and motionless, and Mrs.
Mulholland found herself gazing as if fascinated into the depths of
three pairs of startled, almost hallucinated eyes, fixed gloomily upon
her.
Mrs. Whitaker addressed them in English, speaking very loud with an
idea of making them understand her better. They seemed not to
hear, they certainly made no attempt to answer her amiable
platitudes.
Mrs. Mulholland, moved to something like pity by their stricken
appearance, put out her hand saying, "How do you do?" and two of
them laid their limp fingers in hers—the third, whom she now
noticed was a child although she wore a long black skirt, neither
stirred nor removed her stony gaze from her face. There was an
embarrassing pause. Then Mrs. Mulholland asked with a bright
society smile—
"How do you like England?"
No answer.
"George, dear, ask them in French," said his mother.
George stepped forward blushing through his tan. "Um ... er ..." he
cleared his throat. "S'il vous plaît Londres?" he inquired timidly.
He addressed the tallest, but she gazed at him vacantly, not
understanding. The little girl stood next to her—the large tragic eyes
in her small pale face still fixed on the unknown countenance of Mrs.
Mulholland. She conveyed the impression that she had not heard
any one speak.
George, blushing deeper, turned towards the third ghost standing
before him, coughed again and repeated his question, "S'il vous plaît
Londres?"
Then a strange thing happened. The third ghost smiled. It was a real
smile, a gleaming smile, a smile with dimples. The ghost was
suddenly transformed into a girl. "Merci. L'Angleterre nous plaît
beaucoup." That was in order not to hurt the "half Frenchman's"
feelings. Then she added in English, "London is very nice."
"Oh," snapped the astonished Mrs. Whitaker, "you speak English?"
and her tone conveyed the impression that something belonging
exclusively to her had been taken and used without her permission.
"A little," was the murmured reply. The smile had quickly died away;
the dimples had vanished. Under Mrs. Whitaker's scrutiny the girl
faded into a ghost again. The two ladies nodded and moved away.
George and Eva, after a moment's hesitation and embarrassment,
followed them.
"What strange, underhand behaviour!" commented Mrs. Whitaker;
"never to have told me she understood English until today."
"I suppose they were trying to find out all your family concerns,"
said Mrs. Mulholland.
A word that sounded like "Bosh" proceeded from George, who had
turned his back and was walking into the house.
"I think they were just dazed," explained Eva. "They look almost as
if they were walking in their sleep. I never even noticed until today
that they were all so young. Why, the little one is a mere kiddy;" she
twisted round on her heel. "I think I shall go back and talk to them,"
she added.
"No," said her mother. "You will stay here."
That evening when Mr. Whitaker came back from the City his
daughter had much to tell him, and even the somewhat supercilious
George took an interest and joined in the conversation.
"The ghosts have spoken, papa!" cried Eva, dancing round him in
the hall. Then as soon as he was in the drawing-room she made him
sit down in his armchair and kissed him on the top of his benevolent
bald head. "And—do you know?—they are really not ghosts at all;
are they, mother?"
Mrs. Whitaker did not look up from her knitting. But her husband
spoke.
"They are the wife, the sister, and the daughter of a doctor," he said.
"At the Belgian Consulate I was told they were quite decent people.
My dear Theresa," he added, looking at his wife, "I think we ought
to have asked them to take their meals with us."
"I did so," said Mrs. Whitaker, with some asperity. "I did so, although
they do look like scarecrows. But they say they prefer having their
meals by themselves."
"Then you must respect their wishes," said Mr. Whitaker, opening a
commercial review.
"Just fancy, Pops," said Eva, perching herself on the arm of her
father's chair, "the youngest one—the poor little creature with the
uncanny eyes—is deaf and dumb."
"How sad!" said her father, caressing his daughter's soft hair.
"Did her mother tell you so?" asked Mrs. Whitaker, looking up from
the grey scarf she was knitting.
"No, not her mother," explained Eva; "the other one told me. The
one with the dimples, who speaks English. She is sweet!" cried the
impulsive Eva, and her father patted her hair again and smiled.
"Her name is Sherry," remarked George.
"Oh, George, you silly," exclaimed Eva. "You mean Chérie."
"How do you know her name?" snapped Mrs. Whitaker, laying down
her knitting in her lap and fixing stern inquisitorial eyes upon her
son.
"She told me," said George, with a nonchalant air.
"She told you!" said his mother. "I never knew you had any
conversation with those women."
"It wasn't conversation," said George. "I met her in the garden and I
stopped her and said, 'What is your name?' and she answered,
'Sherry.' That's all."
"Queer name," said his father.
"My dear Anselm, that is really not the point—" began Mrs. Whitaker,
but the dressing-gong sounded and they all promptly dispersed to
their rooms, so Anselm never knew what the point really was.
After dinner Eva, as usual, went to the piano, opened it and lit the
candles, while her father sat in the dining-room with the folding-
doors thrown wide open, as he declared he could not enjoy his port
or his pipe without Eva's music.
"What shall it be tonight, Paterkins?" Eva called out in her birdlike
voice. "Rachmaninoff?"
"No. The thing you played yesterday," said her father, settling
himself comfortably in his armchair, while the neat maid quietly
cleared the table.
"Why, that was Rachmaninoff, my angel-dad," laughed Eva, and
twisted the music-stool to suit her height.
George came close to her and bending down said something in an
undertone.
"Good idea," said Eva. "Ask the mater."
"You ask her," said George, sauntering into the adjoining room,
where he sat down beside his father and lit a cigarette.
Eva went to her mother, and coaxed her into consenting to what she
asked. Then she ran out of the room and reappeared soon after,
bringing with her the three figures in black. As they hesitated on the
threshold, she slipped her arm through the arm of the reluctant
"Sherry" and drew her forward. "Do come!—Venny!" she said, and
the three entered the room.
They were quite like ghosts again, with pale faces and staring eyes
and the rigid gait of sleep-walkers.
They sat down silently in a row near the wall, and Eva went to the
piano and played. She played the Rachmaninoff "Prelude," and when
she had finished they neither moved nor spoke. She wandered off
into the gentle sadness of Godard's "Barcarole," and the three
ghosts sat motionless. Schumann's "Carnaval" did not cheer them,
nor did the "Moonlight Sonata" move them. When Eva at last closed
the piano they rose, and the two eldest, having silently bowed their
thanks, they left the room, conducting between them the little one,
whose pallor seemed more spectral and whose silence seemed even
deeper than theirs.
"Poor souls! poor souls!" growled Mr. Whitaker, clearing his throat
and knitting his brows. "Theresa, my dear," to his wife, "see that
they lack for nothing. And I hope the children are always very kind
and considerate in their behaviour to them. George," he added,
turning what he believed to be a beetling brow upon his handsome
son, "I noticed that you stared at them. Do not do so again. Grief is
sensitive and prefers to remain unnoticed."
George mumbled that he hadn't stared and marched out of the
room. Eva put her arms round her father's neck and pressed on his
cheek the loud, childish kisses that he loved.
"May I go and talk to them a little?" she asked, in a coaxing whisper.
"Of course you may," said her father, and Eva ran out quickly, just as
her mother looked up to say, "What is it?"
"I have sent Eva to talk to those unhappy creatures," said Mr.
Whitaker. "We must try and cheer them a little. It is nothing less
than a duty. Poor souls!" he repeated, "I have never seen anything
so dismal."
"I think we fulfil our duty in providing them with shelter and food,"
said Mrs. Whitaker.
"You think nothing of the kind, Theresa," said Mr. Whitaker.
"I do," asserted his wife. "And as for Eva, she is already inclined to
be exaggeratedly sentimental in regard to these people. She is
constantly running after them with flowers and cups of tea."
"Nice child," said her father, with a little tightening in his throat.
"She is not a child, Anselm. She is nineteen. And I do not wish her
to have anything to do with those women."
"Theresa?" said her husband, in a high questioning voice. "Theresa.
Come here."
Mrs. Whitaker did not move. "Come here," he repeated in the
threatening and terrible tone that he sometimes used to the children
and to his old retriever Raven—a tone which frightened neither child
nor beast. "Come here."
Mrs. Whitaker approached. "Sit down," he said, indicating a footstool
in front of him; and Mrs. Whitaker obeyed. "Now, wife," he said, "are
you growing hard and sour in your old age? Are you?"
"Yes," said Mrs. Whitaker. "I am."
"Ah," said Mr. Whitaker, "that's right. I knew you weren't." And he
laughed, and patted her cheek.
This was not the answer Mrs. Whitaker was prepared for and she
had nothing ready to say. So the wily Mr. Whitaker went on, "I have
noticed lately in you certain assumed asperities, a certain simulated
acrimony.... Now, Theresa, tell me; what does this make-believe bad
temper mean?"
Mrs. Whitaker felt that she could weep with rage. What is the good
of having a bad temper when it is not believed in? Of what use is it
to be sore and sour, to feel bitter and hard, in the face of smiling
incredulity?
"With other people, my dear," continued Mr. Whitaker, "you may
pretend that you are disagreeable and irascible, but not with me. I
know better."
This simple strategy had proved perfectly successful for twenty years
and it answered today, as it always did.
"I am disagreeable, I am irascible, I am bitter, and hard, and cross,"
said Mrs. Whitaker, whereupon Mr. Whitaker closed his eyes, smiled
and shook his head.
"Don't keep on shaking your head like a Chinese toy," she added.
"Anselm, you really are the stupidest man I have ever seen." And
then she laughed. "It is dreadful," she added, putting aside the hand
he had laid on her shoulder, "not to be believed when one is cross,
not to be feared when one is angry. It makes one feel so helpless."
"You may be helpless," he said; "womanly women mostly are. But
you are never cross and you are never angry. So don't pretend to
be."
Now Mrs. Whitaker was tall and large and square; she was strong-
minded and strong-featured; she was what you would call a
"capable woman"—and none but her own inmost soul knew the
melting joy that overcame her at being told that she was helpless.
She raised her hand to the hand that lay on her shoulder again, and
patted it. She bent her head sideways and laid her cheek upon it.
"Now, what's the trouble?" said her husband.
"The trouble ... I can hardly express it," she spoke hesitantly, "either
to myself or to you. Anselm!" she turned her eyes to him suddenly,
the eyes full of blueness and temper and courage he had fallen in
love with in Dublin long ago. "I hate those three miserable women,"
she said. "I hate them."
"What!" cried her husband, drawing his hand away from hers.
"I fear them, and I hate them!" she repeated.
"What have they done?"
"They have done nothing," said his wife, with drooping head and
downcast eyes. "But I cannot help it. I hate and fear them ... for the
children's sake."
"What do you mean?" Mr. Whitaker was sitting very straight. The
thin soft hair still crowning his brow was ruffled.
"The mystery that surrounds them frightens me," said Mrs. Whitaker.
"I don't know where they come from, what they have seen, what
they have lived through. I should like to be kind to them, I should
like to encourage the children to cheer them and speak to them. But
there is something ... something in their eyes that repels me,
something that makes me want to draw Eva away from them. I
cannot express it. I don't know what it is."
There was a brief silence. Then her husband spoke. "A woman's
instinct in these things is right, I suppose. But to me it sounds
uncharitable and cruel."
Mrs. Whitaker rose to her feet, her face flushing painfully. "Are we
called upon to sacrifice our daughter's purity of mind, her ignorance
of evil, to these strangers? Is it our duty to encourage an intercourse
which will tear the veil of innocence from her eyes?"
"I am afraid so," said Mr. Whitaker gravely. "How can our daughter
have pity on human suffering while she does not know its meaning?
True charity, Theresa, cannot be blind; compassion must know the
ills it tries to heal. My dear, we are face to face with one of the
problems—one of the minor problems perhaps, but still a very real
problem—which this ghastly war has raised. Think for a moment,
Theresa; how can our girls, who are called upon to nurse the
wounded in body, and comfort the stricken in soul, live in the midst
of puerile ignorance any longer? Painful though it may be, the veil
you speak of, the white veil that hides from a maiden's eyes the sins
and sorrows of life, must be rent asunder."
"It is cruel! it is cruel!" cried the mother.
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