© 2003 Nature PublishingGroup
Infectious diseases are more medically relevant than
ever.More than a third ofthe annual worldwide deaths
are the result of infectious diseases,such as malaria,
tuberculosis and human immunodeficiency virus
(HIV)/AIDS (see online link to the World Health
Report 2002). There is an urgent need for animal
models in the field of infectious diseases.In order to
develop new therapies and vaccines,we need to have a
detailed understanding ofthe events that are triggered
in a host after bacterial,viral or parasitic infection.
There is no substitute for using animal studies if we
want to understand in detail the dynamics of
host–pathogen interactions or the complex interac-
tions between the different cell types and organs that
are involved in the host response to a pathogen.
Mice are an ideal organism in which to understand
human infectious diseases.Despite some differences,
the immune systems of mice and humans are similar
and they can often be challenged with the same,or
similar,pathogens1.In this review,we argue that the
mouse is an excellent model for the study of human
infectious disease.To illustrate this,we focus specifi-
cally on bacterial infections and provide examples of
studies using one of two genetic approaches that have
been applied to the field of infectious disease: for-
ward genetic screens and targeted gene knockouts.
Forward genetic screens in mice have been used to
identify and characterize genes that have a role in the
susceptibility or resistance to infection.Targeted gene
knockouts in mice have been used to characterize the
mechanism of gene action during infectious
processes.In all cases, we emphasize how the tools
that are available for the mouse geneticist can be used
to understand infection and immunity and discuss
how applicable the results from mouse studies are to
the understanding of human disease.Finally,we dis-
cuss the future potential for using mice to understand
disease and,in particular,to follow the dynamics of
bacterial infection in vivo.
Forward genetic screens
Naturally occurring mutations that affect the mouse
immune system and segregating in a Mendelian
manner have been investigated since the late 1930s —
long before mouse genes could be experimentally
targeted2,3. Most mouse mutants with a defective
immune system have a broad vulnerability to infection
and show signs ofimmunodeficiency.In this section,
we give a few key examples offorward genetic studies
and describe how they have enabled the identification
and characterization ofgenes that determine resistance
or susceptibility to bacterial infection.
Cloning Slc11a1 (Nramp1). More than 20 years ago,
mutations that confer resistance to three intracellular
pathogens (Mycobacterium bovis BCG, Salmonella
typhimuriumand Leishmania donovani) were mapped to
the same position on mouse chromosome 1 (REFS 4–7).It
was soon realized that a single gene might confer resis-
tance to all three pathogens and the locus was,therefore,
called Bcg/Ity/Lsh (REFS 5,7).Additional in vivoand in vitro
studies showed that Bcg/Ity/Lshmediates resistance in the
MICE,MICROBES AND MODELS OF
Jan Buer*‡and Rudi Balling*
We urgently need animal models to study infectious disease. Mice are susceptible to a similar
range of microbial infections as humans. Marked differences between inbred strains of mice in
their response to pathogen infection can be exploited to analyse the genetic basis of infections. In
addition, the genetic tools that are available in the laboratory mouse, and new techniques to
monitor the expression of bacterial genes in vivo, make it the principal experimental animal model
for studying mechanisms of infection and immunity.
NATURE REVIEWS |GENETICS
VOLUME 4 |MARCH 2003 |1 9 5
*German Research Centre
for Biotechnology (GBF),
Mascheroder Weg 1,
Hannover Medical School,
Correspondence to R.B.
R E V I E W S
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expressed in the PHAGOLYSOSOMESofmacrophages,where it
transports divalent metal cations against a proton gradi-
ent. Recent data suggests that Nramp1 has many
pleiotropic effects on macrophage activation13.
Nonetheless,its expression in the phagolysosome sup-
ports the idea that Nramp1 is a host susceptibility factor,
because macrophages are important in the immune
response to infections by intracellular pathogens (BOX 1a).
initial phase ofinfection,controlling intracellular micro-
bial replication in MACROPHAGES8.A positional cloning
approach based on high resolution genetic9,10,physical11
and transcriptional12mapping was used to identify the
locus.The gene — originally called natural resistance-
associated macrophage-protein 1(Nramp1) — encodes a
90–100 kDa integral membrane phosphoglycoprotein
with 12 transmembrane domains. It is exclusively
Phagocytic cells that respond to
non-selfmaterial by releasing
substances that stimulate other
cells ofthe immune system and
are involved in antigen
An organelle in a phagocytic cell
that is formed by fusion ofan
ingested particle with a lysosome
containing hydrolytic enzymes
that digest the particle.
Antigen-specific cells of the
The process by which factors of
the immune-serum coat bacteria
A large group ofinteracting
plasma proteins that act together
to attack extracellular pathogens.
A collective term for parasitic
tapeworms and roundworms.
Box 1 |Immunity to infection
The immune system of vertebrates provides protection from a wide range of pathogens.There are two components
in this system:a nonspecific,constitutive (innate) set of defences which acts immediately against microbe and a
specific,inducible (adaptive) reaction,which accounts for the generation of immunological memory.
Both systems consist ofcellular and soluble (humoral) components and communicate through a network ofsoluble
regulatory substances (cytokines,chemokines and complement factors) and corresponding receptors.
Macrophages (and neutrophils) engulfand destroy microbes after first encounter and — together with natural
killer (NK) cells — secrete cytokines that orchestrate innate and adaptive immunity (panel a).The dendritic cells
(DCs) — specialized relatives ofmacrophages — present antigens to LYMPHOCYTES(white blood cells) to stimulate
adaptive immunity (panel b).
Innate immune recognition is based on the detection ofconstitutive and conserved microbial products (pathogen-
associated molecular patterns,PAMPs) — including lipopolysaccharide (LPS) — through pattern recognition receptors
(PRRs),including Toll-like receptors (TLRs).Binding to TLRs activates macrophages and DCs,causing them to release
pro-inflammatory cytokines and chemokines and triggering functional maturation ofDCs by upregulating the
receptors CD80 and CD86.This leads to the initiation ofantigen-specific adaptive immune responses and the functional
differentiation ofT cells.Innate immunity therefore acts to focus and control acquired immunity.
Acquired immunity is mediated by lymphocytes,which have evolved to express an enormous array ofrecombinant
receptors for antigen capable ofrecognizing any pathogen that the host might ever encounter.Antibodies produced by
B lymphocytes (humoral immunity) and T-cell receptors expressed on the cell surface ofT lymphocytes (cell-mediated
immunity) recognize and bind to specific antigens,resulting in a variety ofresponses that depend on the nature ofthe
target.Antibodies can neutralize bacterial toxins and OPSONIZEbacteria.
Cytotoxic T cells kill those cells that display foreign antigens on their cell surface that are presented by major
histocompatibility complex (MHC) class I molecules.CD4+T-Helper cells (TH1 and TH2) recognize foreign antigens
presented by MHC class II molecules.TH1 cells produce interferon-γ (IFN-γ),whereas TH2 cells preferentially secrete
IL-4,IL-5 and IL-10.TH1 cells have an important role in the initiation of the cell-mediated immune response
against intracellular pathogens,due to secretion of IFN-γ which activates macrophages.In the mouse,IFN-γ also
stimulates the production of IgG2a and IgG3 antibody subclasses that contribute to anti-microbial immunity by
virtue of their COMPLEMENT binding and opsonizing activities.TH2 cells control activation and differentiation ofB cells.
IL-4 controls IgE antibody production which has a central role in the immune defence against HELMINTHS.It also
stimulates the production ofIgG1 in the mouse that neutralizes antigens.Regulatory T cells (mainly ofthe CD4+CD25+
phenotype) are involved in downregulating the immune response.An adaptive response also generates a memory,
which allows a more rapid response on a second exposure to the pathogen (or antigen).Figure modified with
permission from REF.30© (2001) Nature Publishing Group.
IL-8 and other chemokines
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R E V I E W S
the permissive A/J and the highly restrictive C57BL/6J
mouse strains was mapped to a single locus on chro-
mosome 13 called Lgn1.This region contains multiple
copies of the gene baculoviral inhibitor of apoptosis
protein repeat-containing 1 (Birc1, also called
Naip)22,26,27.Genomic bacterial artificial chromosome
(BAC) clones from the crucial interval were transferred
into transgenic mice in an attempt to functionally com-
plement the Lgn1-associated susceptibility ofA/J mice
to L.pneumophila.Birc1e(also called Naip5) is the only
known full-length transcript encoded in the region that
is capable offull or partial complementation28,29.
The mechanism ofBirc1eaction is not yet known;it
encodes a protein that is homologous to plant innate
immunity proteins and has been implicated in signalling
pathways related to apoptosis regulation.It is possible that
in normal mouse macrophages,constitutive or inducible
expression of Birc1emight prevent the induction of
apoptosis.Whereas,in the A/J mouse strains the Birc1e-
mediated inhibition ofL.pneumophilareplication might
be disrupted through mutation.However,one might also
argue thatBirc1eregulates Legionellapermissiveness by
detecting the presence ofLegionella-secreted virulence
factors and initiating an unidentified cellular response
that prevents the bacterium from gaining access to the
replicative phagosome29.Future work in mice to resolve
the remaining questions of the function of Birc1ein
Legionellapathogenesis will provide important informa-
tion to distinguish between these and other possibilities
and to enhance our understanding of Legionnaire’s
disease in humans.
Cloning Tlr4. Nramp1 and Birc1ewere cloned from
mice strains with altered susceptibility to pathogens
and gave an indication as to which loci might affect
human responses to infectious disease.By contrast,the
human Toll-like receptor 4 (TLR4) was identified
before its mouse orthologue.However,studies in mice
have been crucial in determining the mechanism ofthe
TLRs in host defence30.
The first identified member of the Toll family,
DrosophilaToll,was discovered as a maternal-effect
gene that functions in a pathway that controls
dorsoventral axis formation in fruitfly embryos31and,
later,was shown to have a key role in the antifungal
immunity of Drosophilaadults32.In mammals,TLRs
have evolved to recognize products unique to micro-
bial metabolism — such as LIPOPOLYSACCHARIDE (LPS),
peptidoglycan and unmethylated CpG islands — and,
in response, to induce innate immunity (BOX 1).
Human TLR4was the first characterized mammalian
Toll(REF.33).It is expressed in a variety ofcell types,most
predominantly in the cells of the immune system,
including macrophages and dendritic cells (DCs) (BOX 1).
It was studies in mouse that led to the realization that
TLR4encodes the signal-transducing receptor for LPS.
The Lpsmouse gene was cloned from the LPS-non-
responsive C3H/HeJ mouse strain and was found to be
Tlr4;this was confirmed in Tlr4knockout mice34,35.
Once the mouse Tlr4gene had been identified its mode
ofaction could be studied further.
Sequence analysis ofinbred strains showed that suscepti-
bility was associated with a glycin-to-aspartate (G169D)
substitution in the TM4 domain ofthe Nramp1 protein.
Nramp1knockout mice are susceptible to infection with
and L.donovani,but can be rescued ifthe resistant G169
allele is introduced transgenically.This confirms that
Nramp1 — now renamed solute carrier family 11a
member 1 (Slc11a1) — is indeed Bcg/Ity/Lsh14,15.
Human SLC11A1 is linked to several infectious
diseases, including leprosy and HIV, as well as to
autoimmune diseases such as rheumatoid arthritis13.
AlthoughNramp1knockout mice are susceptible to
infection by a number ofMycobacteriumspecies,this
gene does not seem to function in conferring resistance
in mice or humans toMycobacterium tuberculosis —
the tuberculosis-causing bacterium16. Studies of
Nramp1 in host defence against clinically relevant
strains ofM.tuberculosisshowed no difference in sur-
vival between Nramp1–/–and wild-type mice17.In addi-
tion,no association with the Nramp1locus was found
in a genetic linkage study for M.tuberculosissuscepti-
bility in inbred strains of mice18.The above findings
show how the role of an individual gene can change,
depending on the specific mechanisms of bacterial
pathogenesis and the host genetic background.
Cloning Birc1e (Naip5).The study of mouse strains
with an increased susceptibility toLegionnaires’ disease
represents another example ofhow phenotypic differ-
ences in mouse models ofinfectious disease contribute
to a better understanding ofhost susceptibility.In this
case,it has not yet been shown whether the conclusions
drawn in mice are applicable to humans.
Legionella pneumophila — the causative agent of
Legionnaires’ disease — was first discovered in 1976 by
scientists at the Centers for Disease Control and
Prevention who were investigating an epidemic ofsevere
pneumonia among attendees at an American Legion
convention in Philadelphia.It infected 182 individuals,
of whom 29 died. L. pneumophila is an intracellular
FACULTATIVE PARASITE that exploits a poorly understood
process in host macrophages19.The ability ofLegionella
to cause disease is intimately linked to its ability to
replicate in macrophages by first creating a specialized
vacuole that is morphologically similar to the endoplas-
mic reticulum (ER) ofits host.It is only within the past
few years that clues — some from studies ofLegionella-
sensitive mice strains — have begun to emerge to explain
how L.pneumophilagains control ofthe PHAGOSOMEfrom
the host phagocyte.Successful intracellular survival and
replication ofL.pneumophilaseems to involve modula-
tion ofhost macrophage apoptosis20,21.Determining how
Legionella exploits normal cellular processes during
infection will help us to understand Legionnaire’s disease
and to identify macrophage defence mechanisms that
are important in other infectious diseases.
Most inbred mouse strains are highly resistant to
L.pneumophiladisease,and their macrophages support
little or no growth of the microorganisms, with the
exception ofthe A/J strain22–25.The difference between
A parasite that is able to survive
away from its host.
A membrane-bound vesicle that
contains microbes or particulate
material from the extracellular
(LPS).A complex glycolipid
found on the surface ofGram-
negative bacteria that is a
powerful inflammatory stimulus.
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R E V I E W S
molecules (FIG.1).LPS is first bound to a serum pro-
tein — LPS-binding protein (LBP) — which func-
tions by transferring LPS monomers to Cd14 (REF.40).
Cd14 is a high-affinity LPS receptor that can either be
secreted into serum,or expressed as a glycophospho-
inositol (GPI)-linked protein on the surface of
macrophages40.Cd14-deficient mice have a profound
defect in responsiveness to LPS,showing the impor-
tance of Cd14 in LPS recognition41.Another compo-
nent of the LPS receptor complex is MD-2 (REF.42).
MD-2 is a small protein that lacks a transmembrane
region and is expressed on the cell surface in associa-
tion with the ectodomain of Tlr4 (REF.42).Although
its precise function is not known,MD-2 is required
for LPS recognition by Tlr4 (REF.43).The molecular
mechanism of Tlr-mediated recognition is one of the
most challenging issues in Toll biology.Several lines
of evidence indicate that Tlr4 might,in fact,interact
with LPS directly44,45; however, this interaction is
clearly aided by Cd14 and MD-2 (REF.46).
The identification and functional characterization
ofTlr4 in the mouse has brought our understanding of
the innate immune system to a new level.The role of
the TLRs in host defence is fundamental and it is likely
that their function affects most aspects of the mam-
malian immune system.Loss-of-function mutations in
TLRs are likely to result in immunodeficiencies,
whereas gain-of-function mutations might predispose
an individual to inflammatory or autoimmune disor-
ders. The importance of the TLRs in the control of
adaptive immune responses also makes them crucial
targets for immune intervention.The mouse model
system will provide all the essential tools needed to
address these issues in the future.
Targeted gene knockouts
In the previous section we showed the power ofusing
forward genetic screens for mice with altered suscepti-
bility to bacterial infections.This enabled the identifica-
tion ofloci that are involved in the infectious process.
Now,we describe how reverse genetics approaches — in
the form oftargeted gene knockouts — have led to the
successful genetic dissection of molecular pathways
involved in defence against microbial pathogens.
Although this review focuses mainly on bacterial infec-
tions,knockout mice have also been used as model sys-
tems to test susceptibility to other pathogens,including
viruses,fungi and parasites.
There are now many hundreds of knockout mice
in which different genes have been inactivated. In
investigations of infectious disease,the main studies
with knockout mice have focussed on genes that
encode CYTOKINESand their receptors.From these stud-
ies,the high level of complexity and the considerable
redundancy of the immune system became evident.
Many of these cytokine-pathway knockouts result in
mice with altered susceptibility to bacterial infection
(TABLE 1) (REFS 47–71).Cytokines are a key part of the
immune response,enhancing the bactericidal capacity
of phagocytes,recruiting additional innate cell popu-
lations to sites of infection,inducing dendritic-cell
Variation in the inflammatory response in inbred
strains ofmice after challenge with LPS was noted for
nearly 40 years36.In 1968,Sultzer showed that inbred
C3H/HeJ mice were innately resistant to lethal chal-
lenge with LPS and generally hyporesponsive to LPS
in vitro37.Initial genetic analysis showed that the pheno-
type is under the control of a single locus with two
allelic forms:the LPS responsive allele Lpsn(normal,
wild-type) and the hyporesponsive allele Lpsd
(defective,C3H/HeJ) (REF.38).The Lpsdallele impairs
LPS-mediated macrophage activation and survival
of infection with gram-negative bacteria such as
S. typhimuriumand Klebsiella pneumoniae. Indeed,
C3H/HeJ mice are extremely susceptible to
S.typhimurium infection39.Subsequent genetic map-
ping narrowed the position of the Lpsgene down to a
2–3 Mb interval on mouse chromosome 4 and led to
the independent cloning of Tlr4 by the groups of
Beutler34and Malo35. Subsequently, at least 10
Tlrgenes have been identified in mice and humans.
C3H/HeJ mice are unresponsive to LPS due to a
point mutation in the cytoplasmic Toll/interleukin-1
receptor (TIR) domain of Tlr4, which prevents
downstream signalling30,34,35.Recognition of LPS by
Tlr4 is complex and requires several accessory
A group ofproteins that form a
dynamic network ofintercellular
messenger molecules that
regulate various aspects of
immune response to infection.
DC - maturation
Figure 1 |Toll-like receptor (TLR4) signalling. TLR4detects
the presence of gram-negative bacteria through lipopoly-
saccharide (LPS) presence. The extracellular domain of TLR4
associates with CD14 and MD-2, whereas the cytoplasmic
Toll/IL-1 receptor (TIR) domain (red) forms a signalling complex
with the two TIR domain-containing signalling adaptors MyD88
and MyD88-adaptor-like/TIR-domain-containing adaptor protein
(MAL/TIRAP) and IL-1R-associated kinase (IRAK). MyD88 and
IRAK are shared by several members of the Toll-like receptor
family. Responses that might be specific to TLR4signalling are
mediated by TIRAP/MAL, including dsRNA-binding protein
kinase (PKR) and IRAK2 activation. The set of genes that is finally
induced might be tailored for the subsequent elimination of the
pathogen being recognized. Modified with permission from
REF.30 © (2001) Nature Publishing Group.
© 2003 Nature PublishingGroup
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been reported in the human IFNGR1gene and are associ-
ated with susceptibility to severe mycobacterial infections
that arenot normally virulent in immuncompetent
patients76,77.Complete deficiency ofIFNGR1 results in
early childhood lethality caused by vaccinations with
M.bovisBCG or fatal infections with environmental
non-tuberculous mycobacteria (NTM).The phenotypes
observed in Ifngand Ifngrknockout mice,and the clin-
ical phenotypes of patients with null mutations in
IFNGR1 and IFNGR2,show that IFN-γ signalling is
important in the development ofmature bactericidal
GRANULOMAS, which are needed to control bacterial
replication in infected organs and confer protective
immunity.So,even though the spectrum ofpathogens
that cause infections in gene-deficient patients and
mice seem to be different, knockout mice can be
important animal models for the study ofhost-defence
mechanisms,and the differences can be informative in
understanding the biology ofinfectious disease.
Tumour-necrosis factor-α.TNF-αactivates antibacterial
effector systems in the mouse and mediates septic shock
in patients 78.Knockout mice have been generated for
Tnfand for both receptor genes (Tnfrp55and Tnfrp75).
Tnf-deficient mice are extremely susceptible to infec-
tions with the human pathogens M.tuberculosisand
L.monocytogenesand are therefore an important model
system to study the proinflammatory effects ofTNF-αin
human infectious disease78.Tnfrp55-deficient mice have
also been infected with a broad spectrum ofbacterial
pathogens and are highly susceptible to infections
with M. tuberculosis, M. avium, M. bovis BCG,
S. typhimurium, Streptococcus pneumoniae and
L.monocytogenes48,75,79–81.The resistance of Tnfrp75
receptor knockout mice is more similar to wild-type
mice,which is probably due to the independent signal-
transduction pathways ofboth receptors as indicated by
the lack of sequence homology in the intracellular
domains of the Tnfrp55 and Tnfrp75 signalling
proteins82.The antibacterial effects ofTnf-αare mostly
mediated by Tnfrp55 (REFS 56,80).However,deletion of
Tnfrp75has shown that this receptor is important in
low-dose TNF-induced lethality83.
In the pathogenesis ofmycobacterial infections,the
formation of granulomas is a hallmark of a
coordinated host defence.Mycobacteria and mycobacte-
ria-infected cells are physically sequestered from the sur-
rounding tissue by activated T cells and macro-phages,
which form a highly organized epithelial structure
around infected cells. A recent study investigated
M. avium infections and granuloma formation in
Tnfrp55knockout mice79.They reported that the prema-
ture death observed in these mice after M.aviuminfection
is not a function ofbacterial replication in affected organs
nor a function ofthe virulence ofthe M.aviumsubstrain
used.Before M.avium-infected Tnfrp55knockout mice
die,their granulomas undergo acute disintegration,with
persistent granulomatous lesions and extensive tissue
necrosis in all infected tissues.T cells and interleukin-12
(IL-12) are crucially involved in this inflammatory granu-
loma disintegration84 .Depleting T cells that express the
maturation and directing the ensuing specific
immune response to the invading microbes (BOX 1).
Here,we concentrate in more detail on two cytokine
pathways ofhost immune response — the interferon-γ
(IFN-γ) and the tumour necrosis factor-α (TNF-α)
signalling pathways — which have been extensively
studied in infection experiments in the mouse.IFN-γ
pathway knockout mice provide important hints for
the investigation of genetic-based susceptibility to
bacterial infections in humans.In addition,we show
the pleiotropic function of interleukin-10 (Il-10) in
the control of pro-inflammatory cytokine networks,
which is just beginning to be unravelled in the mouse.
Due to the promising data that were obtained from
studies in knockout mice, IL-10 is, at present, a
key target of therapeutic intervention in human
Interferon-γ.IFN-γis a pleiotropic cytokine that has a
role in promoting innate and adaptive immunity.IFN-γ
interacts with a ubiquitously expressed receptor that
consists oftwo subunits:a 90-kDa α-chain (encoded by
Ifngr1) that is involved in ligand binding and receptor
signalling,and a 62-kDa β-chain (encoded by Ifngr2)
that is also necessary for signal transduction.Mice with
null mutations in the genes that encode IFN-γand both
receptor subunits have been generated and are extremely
susceptible to infection with Listeria monocytogenes,
S.typhimuriumand M.tuberculosis (REF.75).Taking into
account the variety ofhost-defence mechanisms that are
activated on IFN-γstimulation,the broad susceptibility
against many bacterial pathogens in IFN-γ pathway
knockout mice was expected.However,it is quite surpris-
ing that null mutations in the homologous genes in
humans lead to a selective susceptibility to infections with
atypical mycobacteria that normally do not cause dis-
ease16.Dominant-negative and recessive mutations have
A site ofchronic inflammation
that is usually triggered by
persistent infectious agents,such
Table 1 |Cytokine-deficient mice
Cytokine knockout Selected bacterial infections studiedRole in
Tumour necrosis factor-α
Listeria monocytogenes47, Mycobacterium PI
tuberculosis48, Salmonella typhimurium49,
S. typhimurium49, S. aureus52
M. tuberculosis51, L. monocytogenes55,
Escherichia coli 54
L. monocytogenes58, M. tuberculosis59,
M. tuberculosis61, Helicobacter pylori50
S. typhimurium64, L. monocytogenes65,
M. tuberculosis66, Helicobacter pylori50,
L. monocytogenes57, M. tuberculosis68
M. tuberculosis69, S. aureus70
M. tuberculosis71, L. monocytogenes58
Interleukin-1α and β
This list is not exhaustive and does not include of the use of cytokine knockout mice for the study of
viral and parasitic infections. PI, pro-inflammatory effects; TH1, TH1-mediated immunity; TH2, TH2-
mediated immunity; AI, anti-inflammatory effects.
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gene-targeted mouse models will address the relevance
ofT-cell homeostasis in other granuloma-developing
infectious diseases,providing new insights into infectious
Interleukin-10.IL-10 is one of the key regulators of
homeostasis in the anti-infective immune response.
The central and necessary role of IL-10 in protecting
against severe inflammatory pathology has been clearly
shown in many models ofexperimental infection with
intracellular pathogens using Il-10–/–mice85–87.From
these experiments, a clear picture is emerging that
IL-10 is involved in limiting inflammation as well as
being involved in the host response against viral and
IL-10 might have a role in tolerance and in protec-
tion against autoimmunity86. In this context,
CD4+CD25+regulatory T cells that produce high
levels of IL-10 have been described that can inhibit
the proliferation of NAIVE CD4+T cells and/or the
induction of pathology by mucosal antigens,such as
inflammatory bowel disease85,89,90.IL-10,as well as IL-4
and transforming growth factor (TGF)-β, might have
an essential role in tolerance to self antigens and to
mucosal antigens.However,Tgfb1–/–mice develop a
multiorgan auto-immune syndrome91,whereas Il-4
and Il-10-deficient mice do not.Il-10-deficient mice
spontaneously develop ENTEROCOLITISwhich seems to
be due to a defect in Il-10-producing regulatory
T cells that modulate responsiveness to intestinal
flora92.Il-10–/–mice bred under germ-free conditions
do not develop colitis.It is important to note that such a
lost oftolerance to selfflora is also a feature ofhuman
inflammatory bowel disease74.
The impact gained from the study of mouse mod-
els of inflammation on the understanding of human
inflammatory bowel disease is difficult to exaggerate.
Research on the Il-10–/–mice and other related mouse
models of inflammation has provided the framework
for research on new treatments of the disease.
A recent experiment has used the commensal
bacterium Lactococcus lactis,which is engineered to pro-
duce the anti-inflammatory cytokine Il-10 (REFS 72,73).
Strikingly,in Il-10–/–mice the administration of this
engineered bacterium prevented animals from devel-
oping colitis.This finding holds enormous promise for
the use ofour commensal bacteria to control cytokine
networks in patients with inflammatory bowel disease.
markers CD4 and CD8,by injectingcell-specific antibod-
ies into Tnfrp55knockout mice,prolonged the survival
time ofthe gene-deficient mice to the survival time of
wild-type mice.Therefore,TNF-αapparently controls
apoptosis ofT cells in a granuloma,regulating the num-
ber ofT cells that are present in this structure.T cells
promote inflammatory responses through secretion of
IFN-γ (REFS 66,84).This process might be fatal for tissue
destruction ifit becomes hyperinflammatory.
Disintegration of granuloma is also crucial for the
outcome of human tuberculosis.The use of Tnfrp55
knockout mice has been successful in uncovering the role
ofTNF-αsignalling in the maintenance ofgranulomas
— that is, in M. aviuminfections. Future studies in
NAIVE T CELL
A cell that has not yet
Inflammation ofthe small
intestine and the colon.
1 mm10 µm 1 µm
Figure 2 |The infected host — a complex and dynamic environment. In the mouse, a variety
of host-defence mechanisms control and effectively prevent invasive microbial disease. However ,
bacterial pathogens can overcome protective host barriers and, in selected cases, take advantage
of innate host responses. a | Dynamics of bacterial infection by video confocal microscopy in vivo.
Tissue section of an infected mouse spleen. The spleen consists of red pulp, which is the site of red
blood cell destruction, interspersed with lymphoid white pulp. Antigens introduced directly into the
bloodstream are picked up by antigen-presenting cells in the spleen, and lymphoid-cell sensitization
then occurs in the white pulp. Left, longitudinal section; right, cross section; b,c | Four-colour
confocal micrograph illustrating that infected ERT-9 positive macrophages (green) interact through
MOMA-1 positive macrophages (red) with B-cells (blue). Listeria monocytogenes (turquoise, arrow).
Visualization through cell-type-specific antibody staining. d | Field emission scanning electron
micrograph of Streptococcus pyogenes strain A20 infected C3H/HeN mouse. The red pulp area of
mouse spleen is highly infected after 48 hours of infection with 105Streptococcus pyogenes
(orange-red, arrow). No bacteria were detectable in the adjacent blood vessel (asterisk).e |
Transmission electron micrograph of L. monocytogenes infected mouse spleen three days after
infection. After invasion of the cells, Listeriaescape from the phagosome and start to move in the
cytoplasm with the aid of an actin tail formed from the recruitment of cellular actin. The micrograph
shows tail formation in vivoin infected mouse spleen. (Scale bars are approximate).
Box 2 |New methods for studying bacterial pathogens in vivo
Gene-expression profiling in vivo
Substractive hybridization | Differential display | DNA microarrays
Techniques for measuring gene expression in vivo
Microscopic approaches,image capture and digital analysis | Confocal microscopy | Image deconvolution | Multiphoton
microscopy | Flow cytometry
Reporter systems for the indirect monitoring ofbacterial mRNA levels in vivo
β-galactosidase | Luciferase | Green fluorescent protein
Identification ofinvivo-induced genes
In vivoexpression technology (IVET) | Differential fluorescence induction (DFI) | Signature-tagged mutagenesis (STM)
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Nobody will doubt that during the last century many,if
not most,ofthe key discoveries in the field ofinfection
and immunity relied on experiments with mice.But
what about the future? With the complete sequence of
the human genome,and many other vertebrate genomes
at hand,is there an alternative to Mus musculusand its
close relatives? If not, what can immunologists and
medical microbiologists expect from the field ofmouse
genetics and genomics in the years to come? What are
the tools that mouse geneticists should develop to help
in providing answers to the most pressing questions in
infectious disease research? More than 30,000 mouse
and human genes need to be annotated.In practice,
this translates to a genetic analysis of gene function.
Both loss-of-function and gain-of-function mutants
have to be generated and analysed to fully understand
the phenotypic consequences ofgenetic perturbation.
Techniques for monitoring bacterial gene expression.
The identification and characterization of bacterial
infectious disease loci cannot be done without in vivo
infection experiments combined with a sophisticated
phenotypic analysis.We therefore describe some ofthe
tools that are available at present for the analysis ofbac-
terial gene expression in vivo,their limitations and some
promising new approaches (BOX 2).Most ofthese assays
are relatively mouse specific:needless to say,we cannot
monitor virulence gene expression by reporter genes in
patients.However,it is thought that they will provide
new information relevant to human infectious disease
in the future.
The complexities ofbacterial gene expression during
mammalian infection cannot be addressed by in vitro
experiments.We know that the infected host (FIG. 2)
represents a complex and dynamic environment,which
is modified during the infection process,presenting a
variety ofstimuli to which the pathogen must respond in
order to survive. This response involves hundreds
ofbacterial in vivo-induced (ivi) genes that have recently
been identified in cell culture and mouse models using
several technologies described below, including
in vivo expression technology (IVET),differential fluo-
rescence induction (DFI), SIGNATURE-TAGGED MUTAGENESIS
(STM),subtractive hybridization,DIFFERENTIAL DISPLAY and
Identification ofin vivo-induced genes.Reporter genes
have been used as the basis for selection procedures
designed to isolate bacterial ivi genes expressed in the
mouse.Analysis of these genes has elucidated several
aspects of in vivo regulation of virulence gene expres-
sion and pathogen adaptation95,96.
The genetic approach ofin vivo expression technology
(IVET) has been used to identify hundreds of genes
that are induced in the host during infection (FIG. 3)
(REFS 95,97,98).Random chromosomal fragments from a
bacterial pathogen are inserted upstream ofa selectable
gene,the expression ofwhich is required for the survival
ofthe specific bacterial strain that is used while it is in the
mouse (FIG.3a).Selectable markers include genes that can
A technique for detecting genes
that are required for survival and
growth in vivothat uses
modified transposons to allow
high-throughput screening of
randomly generated mutants.
A technique for detecting those
genes that are expressed only
under specific conditions;it
involves isolation and
comparison ofmRNA from two
or more populations.
An array ofPCR products or
to either genomic or cDNA
sequences) deposited on solid
glass slides that can be used to
identify patterns ofgene
A mutation that affects the
ability ofan organism to make a
particular molecule that is
essential for growth.
of pathogen DNA
b Antibiotic resistance
Complementation of (pur A)
Screen for absence of
lacZY expression in vitro
Figure 3 | In vivo expression technology approaches.
IVET is a promoter trap technology that has been developed
to select for bacterial genes that are specifically induced when
bacteria infect a host organism. A | IVET vectors contain a
random fragment of the chromosome of the pathogen (red)
and a promoter-less gene that encodes a selective marker
that is required for survival (burgundy). Random integration of
the IVET vector into the pathogen chromosome is performed
by insertion–duplication mutagenesis to create a pool of
recombinant pathogens (this means that the gene in which
the vector has inserted by homologous recombination is not
disrupted). Recombinants can be selected using antibiotic
resistance, as an additional marker is also on the integrated
IVET construct (not shown). Pooled clones are then
inoculated into the mouse. B | The two main types of IVET
promoter trap strategies are (a) the complementation of
auxotrophic mutation and (b) the expression of antibiotic
resistance. Only those bacteria that contain the selective
marker fused to a gene that is transcriptionally active in the
host are able to survive. After a suitable infection period,
bacteria that express the marker are isolated from the spleen
or other organs. The inclusion of lacZY gene (blue) allows
post-selection screening for promoters that are only active in
vivo. Reproduced with permission from REF.111© (2002)
Cambridge University Press.
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in vivo.Subsequent cell infection and bacteria sorting
is used to enrich the in vivo-induced population.
DFI was first applied to S.typhimurium to identify
genes induced in mouse macrophages102.A similar
GFP-based approach has been used more recently to
identify mouse macrophage-induced genes in
The IVET and DFI approaches enable the identifi-
cation of ivi genes but do not address their function
in virulence.The complementary approach of STM
— based on transposon mutagenesis — also enables
the identification of genes that are required for
in vivo survival97,104.The three approaches of IVET,
DFI and STM can be used to recognize different
classes of ivi or virulence-associated genes by testing
the same gene library or mutant pool in different ani-
mal models. For example, STM has been used to
screen for signature-tagged mutants of Staphylococcus
aureus in mouse models for abscess,bacteraemia and
A lack ofgenetic tools has prevented the application
of reporter gene technology to many bacterial
pathogens,and has led to the development ofalternative
techniques for the identification ofivigenes,including
subtractive hybridization and differential display.DNA
microarrays have also been used to identify the bacterial
virulence genes that promote colonization and host
damage (TABLE 2).The approach is based on the assump-
tion that virulence-associated genes are likely to be
co-regulated.Whole-genome expression profiles are
complement AUXOTROPHIC MUTATIONSor that confer antibi-
otic resistance (FIG. 3b). If the DNA fragment that is
inserted upstream ofthe selectable marker is active in the
host,the marker gene is transcribed and live bacteria are
recovered.The DNA fragment can then be sequenced
and the genes identified.The IVET construct includes the
lacZ gene as an operon fusion with the sequence ofinter-
est and the marker gene.After in vivopassage,blue–white
screening ofbacterial clones plated on the chromogenic
β-galactosidase substrate X-Gal enables the identification
ofclones with promoters that are also active in vitroand
that,therefore,can be ignored.An improved recombinase
fusion approach (RIVET) has been used to adapt IVET
for the identification ofgenes that are expressed tran-
siently during infection,which should allow the identifi-
cation oforgan-specific ivi genes99,100.
Differential fluorescence induction (DFI) is an
alternative method for the positive selection of ivi
genes during bacterial infection of cultured mouse
cells101.DFI involves the insertion ofrandom genomic
DNA upstream ofa green-fluorescence-protein (GFP)
reporter gene on a plasmid,followed by introduction
to the bacterial pathogen.Bacteria harbouring GFP
fusions are pooled and used for infection of cultured
mammalian cells. FLUORESCENCE ACTIVATED CELL SORTING
(FACS) is used to isolate GFP-expressing bacteria that
are released from lysed mouse cells.These bacteria are
cultivated on laboratory medium and a second FACS
step is used to isolate bacteria with low in vitro fluores-
cence,which carry ivi fusions that are only induced
(FACS).A method in which
dissociated and individual
living-cells are sorted,in a liquid
stream,according to the
intensity offluorescence that
they emit as they pass through a
Table 2 |Overview of microbial DNA microarrays
Number of ORFs
15×25mer oligos (sense)
15×25mer oligos (antisense)
Z2491; MC58Neisseria meningitidis
Probe type cDNAs are PCR amplified and cleaned inserts. 50mer oligos (oligonucleotides) correspond to a particular open reading frame (ORF) . Oligomatrixes
(15×25 or 13×25) are different oligos all corresponding to one particular ORF. A, Affymetrix; E, Eurogentec; SG, Sigma Genosys. ‘Pathogene’ refers to sequences
isolated from pathogenic E. coli.
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genes of the host.The mouse will be essential in our
attempts to dissect the molecular and cellular mecha-
nisms ofhost-pathogen interactions in the future.The
reason for this optimism is based on the excellent genetic
toolbox that we have available for the mouse,including
conditional gene targeting,tissue specific knock-ins or
knockouts and the combination ofgenome manipula-
tion with sophisticated new reporter assays to follow
infections in an organism.Furthermore,non-invasive
imaging methods,such as whole body nuclear magnetic
resonance (NMR) and two-photon electron microscopy,
are under development and will revolutionize our insight
into the dynamics ofinfection processes.
Future analysis of the complexity of infectious
diseases needs to integrate aspects of population
genetics.We need to know the frequency ofthe alleles
that influence infection susceptibility among those
infected with a pathogen.We also need to know the
genetic variability among the genomes of pathogens.
Rapid progress is already being made in the identifica-
tion ofallelic variation in humans and in the detection
of positive or negative selection at particular loci.A
comparative sequence analysis between the mouse and
the human genome is becoming a key tool to obtain
insight into the evolution ofboth individual gene loci
and the overall structure and evolution ofgenomes108.
The potential ofthe mouse as a model to study the
evolution ofpathogens in the context ofco-evolution
with their host is tremendous but has not yet been
tapped.We know little about the natural pathogens of
mice and even less about their specific interactions in
different genetic backgrounds.Recently,the genome
of the human parasite Plasmodium falciparumwas
published109at the same time as the sequence of
Plasmodium yoelii,one of four species causing rodent
malaria in African wild-thicket-rats110.This strain has
been adapted to grow in laboratory miceand opens
up the potential for comparing modes of infection
between mouse and human malarial parasites.This
raises the possibility of a more sophisticated mouse
model of malaria.Comparative genomics has been
one of the most powerful tools in genome research in
the past years.The era of‘comparative immunity’ has
just begun,but promises to be ofsimilar importance.
measured under a large number ofin vitroand in vivo
conditions and then ‘clustering’ algorithms are used
to identify the subsets of genes that are co-regulated.
Until now,most microarray studies that have investi-
gated host–pathogen interactions have only refined
hypotheses about gene functions.Future studies for ivi
gene analysis should combine gene-profiling data with
experiments on mouse models.
In this review,we have discussed the power of using
mice in the study of bacterial infections. The
approaches outlined are widely applicable to the
study of other infectious diseases,including viral and
parasitic infections.Despite the widespread use of the
mouse in research on infection and immunity,it is
clear that for many human infectious diseases we do
not have suitable animal models available and it will
be necessary to systematically develop new mouse
models for infectious diseases.Taking advantage of
the whole range of different inbred mouse strains will
help in this task. At this point, only a few inbred
mouse strains have been systematically screened for
phenotypic differences.The Phenome project (see
online link) — coordinated by the Jackson laboratory
— addresses this point and tries to collect phenotypic
information for more than 30 defined inbred mouse
strains.Bacterial,viral or parasitic infection challenges
are not yet included in the phenome project.Detailed
knowledge about the influence ofgenetic background
on certain phenotypes,such as immunity to pathogen
exposure,will be extremely important for selecting
the best suited mouse strain for experimental pur-
poses. Mice are also good models because genome
manipulation is possible and human genes can be
ectopically expressed in transgenic mice,allowing the
traits that influence susceptibility to infections to be
The complexity ofinteracting genomes.One ofthe most
intriguing aspects ofinfectious diseases is the fact that we
are dealing with the process of interacting genomes.
Genes that determine the virulence ofa pathogen operate
in a genetic environment ofsusceptibility or resistance
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The authors thank M. Probst-Kepper for stimulating discussions,
M. Rohde, K. E. J . Dittmar and S. Weiss for the figure on the
infected host, and J . Lauber, D. Bruder and R. Geffers for help in
preparing the manuscript. J .B. would like to thank D. Bitter-
Suermann, J . Wehland and H. von Boehmer for ongoing support
and encouragement. The authors are supported by the Deutsche
Forschungsgemeinschaft and the German Federal Ministry of
Education and Research.
The following terms in this article are linked online to:
Birc1e| Cd14 | IFN-γ | IFNGR1 | IFNGR2 | IL-4 | Il-10 |Lgn1 | Lps |
Nramp1 | TLR4 | TNF-α | Tnfrp55 |Tnfrp75 | Toll
Phenome project: http://www.phenome.org
Sigma Genosys: http://www.sigma-genosys.com
World Health Report 2002: http://www.who.int/whr/2002/en
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