Nature Reviews Microbiology | AOP, published online 15 December 2014; doi:10.

1038/nrmicro3393

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Bacteria, the endoplasmic reticulum

and the unfolded protein response:
friends or foes?
Jean Celli1 and Rene M.Tsolis2

Abstract | The unfolded protein response (UPR) is a cytoprotective response that is aimed at
restoring cellular homeostasis following physiological stress exerted on the endoplasmic
reticulum (ER), which also invokes innate immune signalling in response to invading
microorganisms. Although it has been known for some time that the UPR is modulated by
various viruses, recent evidence indicates that it also has multiple roles during bacterial
infections. In this Review, we describe how bacteria interact with the ER, including how
bacteria induce the UPR, how subversion of the UPR promotes bacterial proliferation and
how the UPR contributes to innate immune responses against invading bacteria.
Lysosomes
Membrane-enclosed
organelles of eukaryotic cells
that contain acid hydrolases
capable of degrading biological
polymers.

Secretory pathway
A series of intracellular steps
that eukaryotic cells use to
transport proteins to specific
cellular locations and also for
extracellular secretion, which
involves the endoplasmic
reticulum and Golgi apparatus.

Paul G.Allen School for

Global Animal Health,
Collegeof Veterinary
Medicine, Washington State
University, Pullman,
Washington 99164, USA.
2
Department of Medical
Microbiology and
Immunology, University of
California Davis School
ofMedicine, Davis,
California95616, USA.
e-mails:
jcelli@vetmed.wsu.edu;
rmtsolis@ucdavis.edu
doi:10.1038/nrmicro3393
Published online
15 December 2014
1

Bacterial pathogens that have an intracellular life cycle

have devised various strategies to subvert specific
compartments within host cells and generate niches
that ensure their survival, persistence and proliferation. Bacterial entry into eukaryotic cells generally
results in bacteria residing within phagosomes, which
are intracellular compartments that are dedicated to
innate immune detection and degradation of incoming
microorganisms, leading to antigen presentation and
the development of adaptive immunity. Despite these
immune processes, bacteria that are entrapped within
phagosomes can survive using various means, including interference with phagosomal maturation to impair
fusion with lysosomes, phagosomal disruption and
release into the cytosol. Bacteria can also transform the
original phagosome into a unique vacuole that acquires
functional properties of less bactericidal intracellular
compartments; for example, bacteria can modulate the
phagosome to interact with the endoplasmic reticulum
(ER), which is a large membrane-bound organelle that
ensures the biosynthesis of proteins, carbohydrates
and lipids, and coordinates their transport along the
secretory pathway. The ER delivers these components
to their destination compartments, which include the
ER itself, the Golgi apparatus, the plasma membrane,
the extracellular milieu or the endocytic pathway and
autophagic pathway. Given its biosynthetic functions
and role along the secretory pathway, the ER is a nutrient-rich intracellular location that is presumably devoid
of bactericidal effectors, such as antimicrobial peptides or hydrolytic enzymes, which makes it a suitable

niche for the survival, persistence and proliferation of

intracellular bacteria.
The ER has crucial roles in cellular homeostasis by
controlling the processing and folding of secretory and
membrane proteins. When protein folding requirements
exceed the ER processing capacity, unfolded proteins
accumulate, induce ER stress and trigger the unfolded
protein response (UPR), which is an evolutionarily conserved cytoprotective signalling pathway. By inhibiting
mRNA translation and by increasing the ER protein
folding capacity and ERassociated degradation (ERAD),
the UPR can relieve physiological stress on the ER and
maintain cellular homeostasis1. Failure to restore ER
functions results in programmed cell death. In addition,
the UPR triggers signal transduction events associated
with innate immunity and host defence, linking this
physiological response to the detection of intracellular
pathogens2.
Viral infections have long been known to exert stress
on the ER and to induce the UPR owing to the demand
that these infections place on protein synthesis, and
several viruses modulate the UPR to ensure viral protein production, replication and cell survival3 (BOX1).
Similarly, bacterial proliferation in the ER probably
results in ER stress and the induction of the UPR. In
agreement with this scenario, recent evidence indicates
important roles of the UPR in either promoting or counteracting intracellular proliferation of bacterial pathogens that subvert ER functions and in sensing the effects
of bacterial protein delivery into cells. In this Review,
we present recent findings that support the UPR as a

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Box 1 | Viruses and the unfolded protein response
Viral replication co-opts endoplasmic reticulum (ER) functions to produce viral
glycoproteins, which leads to induction of the unfolded protein response (UPR)109.
Asthe downstream effects of UPR activation, including translational attenuation,
ER-associated degradation (ERAD) and cell death, can inhibit viral protein production,
viruses can manipulate and avoid the UPR to replicate successfully. Although the exact
mechanisms that are involved are unknown for most viruses, some of the viral proteins
that are involved have been identified; for example, cytomegaloviruses (CMVs) can
both induce and modulate the UPR110112. Induction of the UPR by human CMV (HCMV)
protein US11 has been proposed to lead to degradation of major histocompatibility
complex (MHC) classI proteins, which is a mechanism that may promote chronic
infection112. Furthermore, HCMV protein UL50 and its mouse CMV homologue M50 can
both downregulate inositol-requiring enzyme 1 (IRE1) to suppress the UPR111. Hepatitis
C virus also downregulates the IRE1X-box-binding protein 1 (XBP1) pathway to
promote viral replication113,114. Rotavirus can modulate the UPR in two ways: its NSP3
protein blocks translation of UPR target gene transcripts115, and cellular infection leads
to sequestration of UPR signalling components, such as protein kinaseR (PKR)-like ER
kinase (PERK) and C/EBP-homologous protein (CHOP), in viral inclusion bodies116.
Asbacteria that replicate within the ER encounter a similar environment, they may
share some of these strategies to modulate the UPR; however, additional studies are
needed to test this idea. Furthermore, similar to what has been proposed for Brucella
abortus infection62, stimulation of the UPR during viral infection may function as a
pathogen-associated pattern that activates antiviral innate immunity117.

Endocytic pathway
An array of intracellular
membrane-bound
compartments (including early
endosomes, late endosomes
and lysosomes) that internalize
molecules at the plasma
membrane and sort them for
either degradation or recycling
back to the plasma membrane.

Autophagic pathway
A series of processes of
recognition, capture and
degradation of cytosolic
content, such as protein
aggregates, damaged
organelles and invading
microbes, which occur
via engulfment into
double-membrane bound
autophagosomes and
maturation along the endocytic
pathway into degradative
autolysosomes.

ERassociated degradation
(ERAD). An essential
component of endoplasmic
reticulum (ER) quality control,
the ERAD is a process of
removal of misfolded or
misassembled proteins in the
lumen or membranes of the ER
via polyubiquitination and
targeting to the proteasome
for degradation.

Programmed cell death

A genetically regulated process
of cellular suicide that involves
specialized intracellular
machineries and enables the
elimination of specific cells.

key component in the crosstalk between the ER, intracellular bacteria and their pathogenic activities, and we
discuss how the UPR may contribute to inflammatory
and immune responses against intracellular bacteria.

The UPR: components and functions

Nearly one-third of the eukaryotic proteome is synthesized in the ER and enters the secretory pathway.
In the lumen of the ER, proteins fold into their correct
conformation with the assistance of the protein glycosylation machinery and dedicated chaperones, such as
the binding immunoglobulin protein (BiP; also known
as GRP78), calnexin and calreticulin4. Under certain
stress conditions, such as elevated synthesis of secretory proteins, and perturbations in calcium homeostasis and redox balance, the abundance of misfolded
proteins exceeds the capacity of the protein folding
machinery. In response to these perturbations, three
sensors that are located in the ER membraneinositolrequiring enzyme 1 (IRE1; also known as ERN1), doublestranded RNA-dependent protein kinaseR (PKR)-like
ER kinase (PERK) and activating transcription factor 6
(ATF6)induce the UPR1,4,5 (FIG.1). Each of these proteins has an ER-targeting transmembrane domain that
links a cytosolic effector domain and a domain that is
localized in the lumen of the ER. Although the precise
molecular mechanisms that lead to activation of these ER
sensors are still unclear, experimental evidence supports
two different inputs to this process. The first is direct
binding of unfolded proteins to the luminal domain.
This interaction has been demonstrated for yeast IRE1,
for which the dimerized protein was shown to form a
polypeptide-binding groove6, and a similar structural
feature has been identified in PERK1. A second input
is binding of the luminal domains of IRE1, PERK and
ATF6 by the ER chaperone BiP. When the capacity of the
cell for protein folding is exceeded, BiP is recruited away

from IRE1, PERK and ATF6 to assist in refolding proteins within the ER, which correlates with activation of
IRE1, PERK and ATF6. Additional signals that emanate
from the ER membrane, as well as the cytosol, have also
been proposed to activate the UPR1. Activation of IRE1,
PERK and ATF6 initiates signalling pathways of the UPR
to restore homeostasis in the ER, by increased production of chaperones to assist in protein folding, arrested
translation of proteins not involved in resolving ER stress,
and degradation of terminally misfolded proteins via the
ERAD pathway, which coordinates their retrotranslocation
through the ER membrane, polyubiquitination and targeting
to the proteasome7 (FIG.1).
IRE1 is a multifunctional protein that has kinase
and endonuclease activities. The activity of IRE1 is
downregulated by binding of BiP810 and is stimulated
by the direct binding of unfolded proteins2,6. Following
activation, IRE1 aggregates and autophosphorylates,
activating its endonuclease activity 11. The site-specific
endonuclease activity of IRE1 splices the mRNA that
encodes the transcription factor X-box-binding protein
1 (XBP1), which results in the translation of an active
protein12. Production of XBP1 directs the expression of
ER-resident molecular chaperones and protein-folding
enzymes13. In addition to splicing the Xbp1 transcript,
IRE1 has a non-specific endonuclease activity that is
responsible for rapid degradation of ER membraneassociated mRNA by a process known as regulated
IRE1dependent decay (RIDD) 14,15. This response
reduces the amount of protein that enters the secretory
pathway (FIG.1).
Similar to IRE1, PERK is also an ER stress-activated
kinase. Following activation by ER stress, PERK homo
dimerizes and transphosphorylates, which is accompanied by the dissociation of BiP. Activated PERK can
then phosphorylate its target, the eukaryotic translation
initiation factor- (eIF2)8,16. Phosphorylation of eIF2
inhibits synthesis of secretory proteins by inhibiting the
assembly of the 80S ribosome, thereby promoting cellular survival during ER stress17. At the same time, phosphorylated eIF2 directs translation of the mRNA that
encodes the transcription factor ATF4, which induces
expression of UPR target genes that are involved in
amino acid transport and oxidative stress resistance17.
Under conditions of prolonged or severe ER stress that
the UPR cannot resolve, ATF4 also increases the levels
of the transcription factor C/EBP-homologous protein
(CHOP), which upregulates genes that are involved in
the induction of apoptotic cell death18 (FIG.1).
The third branch of the UPR is initiated by ATF6.
Activation of ATF6 results in its translocation to the Golgi
apparatus, where it is cleaved by the Golgi-resident proteases site 1 protease (S1P) and S2P19. The active amino
terminus of ATF6 is translocated to the nucleus, where it
binds the ER stress response element upstream of a subset of UPR genes to activate their transcription20. The set
of ATF6dependent genes partially overlaps with genes
induced by IRE1 and PERK and includes genes with functions in protein folding, protein transport and lipid biosynthesis20,21. In addition, ATF6 can homodimerize with
XBP1 to activate genes that are involved inERAD22 (FIG.1).

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Unfolded proteins

BiP

ER
Cytosol

PERK

BiP
P

BiP
P

IRE1

ATF6

elF2
Golgi

elF2
P
ER-associated
mRNA turnover
(RIDD)

80S ribosome
Translation
attenuation

Xbp1 splicing

S1P
S2P

Xbp1

Targeted
translation

ATF4

XBP1

ATF6

ATF4

XBP1

ATF6

Nucleus

CHOP (apoptosis),
ER chaperones, oxidative
stress resistance and
amino acid metabolism

Chaperones
and ERAD

ERAD and
ER chaperones

UPR transcriptional programme

Retrotranslocation
The process by which the
transport of misfolded proteins
are transported from the
endoplasmic reticulum lumen
to the cytosol for degradation
by the proteasome.

Polyubiquitination
A post-translational
modification of proteins that
includes the covalent addition
of chains of ubiquitin
monomers that function as
signals for proteasomal
degradation of misfolded
proteins.

Proteasome
A large protein complex that
degrades unneeded or
misfolded proteins that
have been tagged via
polyubiquitination.

Figure 1 | Cellular responses to endoplasmic reticulum stress. In response to an increaseNature

in unfolded
proteins in the
Reviews | Microbiology
endoplasmic reticulum (ER) lumen, three sensors that are located in the ER membrane protein kinase R (PKR)-like ER
kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) activate the unfolded
protein response (UPR). Under homeostatic conditions, PERK, IRE1 and ATF6 are bound by binding immunoglobulin protein
(BiP), which suppresses their activity. In response to ER stress, BiP is recruited away from PERK, IRE1 and ATF6 to promote
protein folding, which leads to the activation of these ER stress sensors. PERK is a kinase that auto-activates, dimerizes
and phosphorylates its target, eukaryotic translation initiation factor- (eIF2). eIF2 prevents the assembly of functional
80S ribosomes, and directs the selective translation of the transcription factor ATF4, which activates transcription of
genes involved in the UPR, including the transcription factor C/EBP-homologous protein (CHOP), which is involved in the
induction of apoptotic cell death. IRE1 is a bifunctional enzyme that has kinase and endonuclease activity. Inresponse to
ER stress, IRE1 autophosphorylates via its kinase domain, which leads to dimerization and activation of its endonuclease
activity. The endonuclease function of IRE1 degrades mRNA in the ER (in a process termed regulated IRE1dependent
decay (RIDD)) to decrease protein biosynthesis. IRE1 also splices the transcript that encodes X-box-binding protein 1
(XBP1), which is a second transcription factor that directs the expression of genes involved in restoring cellular
homeostasis. ATF6 is a transcription factor that is anchored in the ER membrane, but in response to ER stress, it
translocates to the Golgi apparatus, where the transcription factor domain is released from the membrane by sequential
action of site 1 protease (S1P) and S2P, which enables its translocation to the nucleus to activate transcription of UPR
target genes. ATF4, XBP1 and ATF6 upregulate the transcription of chaperones, which are components of the
ER-associated degradation (ERAD) pathway and factors that are involved in autophagy and apoptosis that function to
restore cellular homeostasis, or if the disruption to ER function cannot be resolved, initiate programmed cell death.

One of the cellular responses downstream of the

UPR is the induction of autophagy. Induction of the UPR
increases membrane lipid biogenesis and triggers ER
expansion via the XBP1 and ATF6 pathways as a means
to enhance ER function2325. ER expansion is regulated by
selective autophagy of the ER (so-called reticulophagy
or ERphagy), which promotes the restoration of cellular
homeostasis following resolution of ER stress. Although
induction of autophagy has been shown to promote the
survival of cancer cells26, the effect of ER stress-induced
autophagy on cell survival seems to be context dependent,

as ER stress-induced autophagy led to death of primary

cells27. Under conditions of strong or sustained ER
stress, a failure of the cell to restore homeostasis triggers
apoptotic cell death. Although the precise pathways of
apoptosis that are induced by ER stress are not known,
the PERKeIF2ATF4CHOP pathway has an important role, by reversing translational arrest, increasing
generation of reactive oxygen species and promoting
calcium efflux from the ER. Together, these signals lead
to cytochromec release from the mitochondria and loss
of membrane potential, which results in apoptosis18.

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L. pneumophila
Brucella

RicA

GDPRAB2
?

EE
BCV

GTPRAB2

LE

Golgi
GDPRAB1
DrrA

S. negevensis
Chlamydia spp.

Lys

LepB
ERES

GTPRAB1
GTPARF1
RalF
GDPARF1

Nucleus
LCV

Vacuole

ER

Figure 2 | Trafficking and biogenesis of endoplasmic reticulum-associated replicative organelles by bacterial

Nature Reviews
| Microbiology
pathogens. Legionella pneumophila recruits early secretory vesicles to its plasma membrane-derived
phagosome
via
delivery of the Dot/Icm type IV secretion system (T4SS) effectors DrrA, LepB and RalF. These effectors modulate activities
of the small GTPases RAB1 and ARF1 on the Legionella phagosome (see inset) to bypass the endocytic pathway, and to
promote fusion of the Legionella phagosome with the endoplasmic reticulum (ER) and biogenesis of an ER-derived
replicative vacuole, termed the Legionella-containing vacuole (LCV). Brucella spp. reside within a vacuole, termed the
Brucella-containing vacuole (BCV), which traffics along the endocytic pathway, interacting with early endosomes (EEs),
late endosomes (LEs) and lysosomes (Lys). The BCV is then redirected to ER exit sites (ERESs) and fuses with the ER via
the action of VirB type IV effector proteins and the small GTPases SAR1 and RAB2. The Brucella effector RicA binds
GDP-bound RAB2 in an unknown manner and is required for accumulation of activated, GTP-bound RAB2 on BCVs
(seeinset). Chlamydia spp. and Simkania negevensis control the traffic and maturation of their vacuole into a large
inclusion that physically interacts with the ER at specific contact points (termed synapses).

In addition to autophagy, UPR signalling also interacts with innate immune signalling pathways that lead
to inflammation, as activation of IRE1 by ER stress can
activate the mitogen-activated protein kinase (MAPK)
JNK, which is a key player in the response to inflammatory stimuli28 (see below). Therefore, although residing in
the ER might promote the survival of intracellular bacteria, it is possible that UPR induction might function as
an innate immune mechanism against invading bacteria.

The ER: a safe niche for intracellular bacteria

Although the ER is thought to provide a hospitable,
nutrient-rich organelle devoid of bactericidal functions,
only a few bacterial pathogens take advantage of this
environment to ensure their intracellular survival and
proliferation. The first evidence of bacterial interactions
with the ER came from the observations that Legionella
pneumophila and Brucella spp. occupy ribosome-studded
intracellular vacuoles2931, which were confirmed to be
derived from the ER at both ultrastructural and functional levels3236. Since these seminal demonstrations,

Legionella longbeachae37, Chlamydia trachomatis 38,39 and

the Chlamydia-related bacterium Simkania negevensis 40
have also been shown to survive and replicate in
organelles that closely interact with theER.
Bacterial trafficking to the ER. The trafficking events
leading to the biogenesis of ER-derived organelles containing L.pneumophila and Brucella spp. have been well
characterized. Both invoke subversion of early secretory vesicles but involve distinct mechanisms (FIG.2).
Following phagocytosis by macrophages, plasma membrane-derived Legionella-containing vacuoles (LCVs)
avoid phagosomal maturation along the endocytic pathway. Instead, LCVs intercept early secretory vesicles trafficking between the ER and the Golgi apparatus, which
cover and subsequently fuse with LCVs, transforming
them into vacuoles that have functional features of early
secretory compartments34,36,41. These events eventually enable LCVs to fuse with ER membranes34,36,41 and
become replication-permissive organelles that expand
following bacterial proliferation (FIG.2).

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Box 2 | TypeIV secretion systems in pathogenic bacteria
TypeIV secretion systems (T4SSs) are multiprotein complexes organized in
ATP-powered, membrane-spanning machineries that deliver proteins, proteinprotein
complexes or nucleoprotein complexes into either bacterial or eukaryotic cells. T4SSs
are expressed in both Gram-negative and Gram-positive bacteria and fulfil a range of
functions, including DNA transfer and effector protein delivery into target cells,
therefore, playing important parts in transfer of antibiotic resistance traits via bacterial
conjugation, or in virulence mechanisms of pathogenic bacteria118. On the basis of
phylogenetic relationships with the T4SS harboured by the canonical conjugative
plasmids, T4SSs have been classified in subfamilies (typeIVA and typeIVB) that are
represented in pathogenic bacteria by the VirB and Dot/Icm systems, respectively
(reviewed in REF.119). Many pathogenic bacteria express T4SSs to mediate their
interactions with host cells. These include Agrobacterium tumefaciens, Helicobacter
pylori, Bordetella pertussis, Rickettsia spp., Brucella spp. and Bartonella spp., which
encode typeIVA VirB T4SSs that deliver DNAprotein complexes, multisubunit protein
toxins and proteins into eukaryotic cells, and the intracellular pathogens Legionella
pneumophila and Coxiella burnetii, which express typeIVB Dot/Icm T4SSs that deliver
effector proteins into target cells120. T4SSs such as the Brucella spp. VirB apparatus121
and the Legionella spp. and Coxiella spp. Dot/Icm systems122 are essential to the
survival and proliferation of these intracellular pathogens, as they translocate into host
cells a range of effector molecules that take control of host functions associated with
the biogenesis and trafficking of the bacterial vacuole. As such, they constitute key
virulence factors of many pathogenic bacteria.

SNARE proteins
(Soluble N-ethylmaleimidesensitive factor attachment
protein receptor proteins).
Proteins that are involved in
mediating intracellular
membrane vesicle fusion in
eukaryotic cells.

Similarly, Brucella spp. reside within a Brucellacontaining vacuole (BCV) following phagocytic uptake
or entry within non-professional phagocytes. However,
unlike LCVs, BCVs first undergo maturation along
the endocytic pathway to become endosomal BCVs
(eBCVs)42 and partially acquire phagolysosomal properties33,4345. This trafficking step is followed by interactions of BCVs with ER exit sites (ERESs)43,45, which are
sub-compartments of the ER dedicated to formation and
budding of secretory vesicles before their transport to
the Golgi apparatus. Sustained BCVERES interactions
lead to biogenesis of ER-derived vacuoles named replicative BCVs (rBCVs) through progressive exchange
of endocytic membranes for ER-derived membranes43.
rBCVs support bacterial replication, which is accompanied by further ER membrane accretion and a dramatic
reorganization of the ER into rBCVs when bacterial
proliferation is extensive35 (FIG.2).
The obligate intracellular bacteria Chlamydia spp.
resides within a large vacuole the Chlamydia inclusion
which intercepts exocytic traffic between the Golgi
apparatus and the plasma membrane to acquire sphingolipids that are necessary for inclusion biogenesis and
bacterial growth46,47. Interactions between the Chlamydia
inclusion and the ER have recently been revealed in the
form of points of contact, known as synapses, between
these compartments38,39. These synapses may deliver
ER-derived material to the inclusion39 and are important for inclusion biogenesis and bacterial growth38,39.
Similarly, the Chlamydia-like organism S.negevensis
generates a large networked vacuole that also harbours
contact sites with the ER40, which suggests a common
strategy of ER subversion among Chlamydiales (FIG.2).
Mechanisms of biogenesis of ER-derived bacterial vacuoles.
Although the mechanisms underlying Chlamydia
spp. inclusion interactions with the ER remain to be

characterized, extensive studies of the biogenesis of

L.pneumophila and Brucella abortus ER-derived replicative organelles have provided useful insights into how
these pathogens reach the secretory compartment and
establish residency in the ER. Both pathogens express
a typeIV secretion system (T4SS), the Legionella Dot/
Icm and Brucella VirB T4SS (BOX2), which directs the
intracellular fate of the bacteria by delivering effector
proteins into infected cells that modulate host cell pathways. Concomitant with uptake, L.pneumophila delivers the Dot/Icm effector proteins DrrA (also known as
SidM), LepB and RalF, which function in a coordinated
manner to mediate targeting of LCVs to the ER via the
modulation of small GTPases that normally control
early secretory vesicle transport (FIG.2). DrrA functions
as a GDPGTP exchange factor (GEF) that recruits and
activates RAB1 on plasma membrane-derived LCVs
to promote recruitment of early secretory vesicles48,49.
This process is negatively regulated by LepB, which is
a GTPase-activating protein (GAP) that deactivates
RAB1 (REF.50). Fusion of secretory vesicles with LCVs
involves an unusual DrrA-dependent pairing of plasma
membrane and ER-derived vesicle SNARE proteins51,52,
which enables direct fusion between a plasma membrane-derived vacuole and early secretory vesicles,
thereby mediating LCV bypass of the endocytic pathway.
Subsequent to LCV fusion with secretory vesicles, RalF
functions as a GEF for ARF1 and promotes its recruitment to LCVs and fusion with ER membranes to generate an ER-derived Legionella spp. replication-permissive
organelle41,53,54. Hence, this pathogen uses an array of
effector proteins that co-opt membrane trafficking processes between secretory compartments to promote its
residence within an ER-derived organelle.
The role of the Brucella VirB T4SS in conversion of
the BCV into an ER-derived organelle has been established on the basis of the inability of virB mutants to
undergo sustained interactions with ERESs and to fuse
with the ER, which results in failure to replicate and killing within eBCVs35,43,55. Through acidification and, presumably, through additional intravacuolar cues, eBCVs
signal intracellular Brucella spp. to induce expression of
the VirB apparatus44,45,56. Unlike Legionella spp., very little
is known about the effector proteins that the Brucella
VirB T4SS delivers during infection. The recent identification of 14 Brucella spp. proteins that are delivered
into host cells by the VirB apparatus5761 is an important
step towards a molecular understanding of VirB functions, but it has yet to yield information about whether
these effectors mediate rBCV biogenesis. Several VirB
effectorssuch as VceC, BspA, BspB, BspC, BspD and
BspFtarget compartments of the secretory pathway
when ectopically expressed in mammalian cells60,62,
which suggests that they may modulate specific functions associated with secretory transport. Accordingly,
expression of BspA, BspB and BspF impairs secretory trafficking during Brucella spp. infection60, which
indicates that the bacterium uses typeIV secretion to
modulate functions of the secretory pathway. Indeed,
Brucella spp. require functional ERESs and the small
GTPase SAR1 which controls coatomer protein

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Box 3 | Toxoplasma gondii and the unfolded protein response
The apicomplexan protozoan parasite Toxoplasma gondii occupies a parasitophorous
vacuole that forms close interactions with the endoplasmic reticulum (ER)123,124, which
suggests that it may influence functions of this compartment. Consistent with this,
recent evidence indicates that apoptosis of T.gondii-infected cells involves activation
of ER stress pathways125,126, via induction of C/EBP-homologous protein (CHOP)
caspase12JNK pathway, which highlights a role for unfolded protein response (UPR)
induction during T.gondii infection. Whether the UPR is actively modulated by T.gondii
or an indirect response to cellular effects caused by the parasite needs further
clarification, but the demonstration that the T.gondii rhoptry protein ROP18 destabilizes
the UPR transducer activating transcription factor 6 (ATF6) to promote its
pathogenesis127 strongly suggests that this pathogen manipulates UPR signalling to
its benefit.

complex II (COPII)-dependent vesicular budding from

ERESs63,64 to generate rBCVs43. Biogenesis of rBCVs
also requires the small GTPase RAB2, to which the
Brucella spp. effector RicA binds58,65. RicA specifically
interacts with the GDP-bound form of RAB2 but does
not show any GEF activity 58; therefore, how this interaction modulates RAB2 function remains unclear. Unlike
LCVs, ARF1dependent vesicular trafficking between
ERESs and the Golgi apparatus is not required for rBCV
biogenesis43, which highlights mechanistic differences in
how L.pneumophila and Brucella spp. reach theER.

Bacteria and the UPR

Although many of the mechanistic underpinnings of bacterial interactions with the ER remain to be understood,
Legionella spp. and Brucella spp. are model organisms
to study the bacterial exploitation of this compartment
and its consequences. The rare use of the ER by bacterial
pathogens for survival and replication raises the question
of its permissiveness to infection. Much like many viruses
need to modulate the UPR to ensure their replication
while overwhelming the biosynthetic capacity of the ER
(BOX1) and protozoan parasites modulate UPR pathways
to promote their pathogenesis (BOX3), bacteria that proliferate within the ER must be able to deal with the consequences. Although these consequences are still unclear,
bacterial replication within the ER elicits the UPR, which
could trigger sensing of bacterial pathogenic activities.
Bacterial induction of the UPR. Several examples support the idea that bacterial infections trigger ER stress
and can be sensed via the UPR. The UPR is induced
in macrophage-rich granulomatous lesions of mouse
lungs that are infected with virulent Mycobacterium
tuberculosis, in which apoptotic events are detectable66
(TABLE1). Induction of the UPR has also been correlated
with Helicobacter pylori-induced gastric carcinogenesis67
and occurs in gastric epithelial cells via the action of the
vacuolating cytotoxin VacA68. VacA intoxication activates
PERK and eIF2, which results in CHOP induction,
mitochondrial dysfunction and apoptosis68 (TABLE1). This
highlights the role of ER stress in responses to infections
and toxin activities.
Similarly, invitro models of infection have revealed
induction of the UPR in macrophages and epithelial
cells that are infected with either Brucella melitensis and

B.abortus 62,69 or with Listeria monocytogenes 70 (TABLE1).

The first demonstration of a role of components of the
UPR in the Brucella spp. intracellular life cycle was the
discovery that IRE1 is necessary for the intracellular
growth of B.abortus, using an RNA interference (RNAi)
screen for ER-associated factors that are required for
bacterial replication in Drosophila melanogaster S2
cells71. IRE1 dependency of B.abortus replication, which
has been confirmed in mammalian cells, suggests that
the UPR is beneficial to the infection. However, depletion of neither ATF6 nor PERK reproduced the effect of
IRE1 depletion71, which made a role for a canonical UPR
activation in B.abortus replication less substantiated.
Direct evidence that B.melitensis infection of mouse
macrophages invitro and invivo leads to the induction of the three major signalling pathways of the UPR
recently established that B.melitensis infection induces
physiological ER stress69. However, whether bacterial
residence and proliferation in the ER is required for
induction of the UPR was not addressed in this study,
and the fact that a VirB-deficient mutant (which does
not reach the ER or replicate) also induced UPR signalling 69 suggests that this response may not be related to
bacterial replication in the ER. It will also be important
to address whether the three main UPR regulators are
required for B.melitensis-induced UPR, or whether
only IRE1dependent signalling contributes to bacterial replication. Counteracting the UPR response
with the pharmacological chaperone tauroursodeoxycholic acid (TUDCA) reduces intracellular growth of
B.melitensis, which suggests that the induction of the
UPR promotes bacterial intracellular growth69. However,
obtaining more direct evidence will be important to
further substantiate this beneficial role of theUPR.
Induction of the UPR by L.monocytogenes occurs
via extracellular secretion of its cytolysin listeriolysin O
(LLO), which induces all branches of the UPR and leads
to ER stress-specific apoptosis70 (TABLE1). The molecular mechanisms by which this toxin functions remain
unclear but may be related to the ability of LLO to alter
intracellular calcium homeostasis via its pore-forming
activity 72, as calcium imbalance causes ER stress.
Nonetheless, evidence that LLO treatment of mammalian
cells itself triggers the UPR indicates that the UPR may be
induced via external stimuli and may not be restricted to
ER-dwelling pathogens.
Bacterial subversion of the UPR. Although the occurrence of UPR induction following bacterial infection
is now supported by several examples, indications that
bacteria can modulate this response are less common but
are starting to emerge. L.pneumophila recruits components of the ERAD on its vacuole to mediate turnover
of bacterial effectors on the vacuolar surface54 and the
proteasome to generate amino acids that are necessary
for its intracellular growth73, but whether L.pneumophila
modulates the UPR is undocumented. By contrast,
S.negevensis generates a replicative vacuole that is associated with the induction of ER stress, which the bacterium
subsequently downregulates40. In addition, S.negevensis
prevents the induction of ER stress following treatment

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Table 1 | Bacteria and bacterial products that modulate the unfolded protein response
Bacteria

Toxin or effector

Effect

Mode of action

Refs

Brucella abortus

VceC

Induces the UPR

BiP binding

62

BspC, BspG, BspH, BspI and BspK

Induces the UPR

N.D.

60

Brucella melitensis

TcpB

Induces the UPR

N.D.

Escherichia coli (Shiga

toxin-producing)

Subtilase (SubAB)

Activates IRE1, ATF6 and PERK

BiP degradation

Shiga toxin 1 (Stx1A)

Activates IRE1

IRE1 binding?

Helicobacter pylori

VacA

Induces the UPR

PERK activation

Listeria monocytogenes

Listeriolysin O

Induces the UPR

Intracellular calcium imbalance?

70

Mycobacterium tuberculosis

N.D.

Induces ER stress

N.D.

66

Simkania negevensis

N.D.

Inhibits the UPR

N.D.

40

Vibrio cholerae

Cholera toxin (CTA)

Activates IRE1

IRE1 binding

Various

Pore-forming toxins

Activates IRE1 and ATF6

p38 MAPK pathway activation

69
7577
103
67,68

2
74

ATF6, activating transcription factor 6; BiP, binding immunoglobulin protein; ER, endoplasmic reticulum; IRE1, inositol-requiring enzyme 1; MAPK,
mitogen-activated protein kinase; N.D., not determined; PERK, protein kinase R (PKR)-like ER kinase; UPR, unfolded protein response.

with tunicamycin or thapsigargin, as measured by

decreased induction of BiP, impaired nuclear translocation of CHOP and reduced activation of eIF240. Both
compounds induce the UPR by perturbing distinct ER
functions tunicamycin inhibits N-linked glycosylation
in the ER and affects protein folding, whereas thapsigargin blocks the sarco-ER calcium ATPases (SERCAs),
leading to depletion of ER calcium storeswhich indicates that S.negevensis probably interferes with common
UPR signalling pathways via mechanisms that are yet
to be determined. Interestingly, high concentrations of
these UPR inducers can overcome S.negevensis-mediated
inhibition of the UPR and affect bacterial growth40,
which suggests that the bacterium actively modulates this
response to benefit its intracellular proliferation.
Additional evidence that bacteria modulate the UPR
originates from the study of bacterial toxins (TABLE1).
Similar to the effect of LLO during L.monocytogenes
infections70, pore-forming toxin (PFT) intoxication of cells
leads to induction of the UPR via the p38 MAPK pathway.
Pore formation probably triggers p38 MAPK activation,
which, in turn, induces the UPR via the IRE1XBP1 and
ATF6 pathways, but not via the PERK pathway, which
suggests that the induction of the UPR by PFTs is specific
and distinct from that induced by unfolded proteins74.
UPR induction is protective against PFTs74, which indicates that the UPR functions as a defence mechanism
against cellular injury that is caused by bacterial toxins.
Studies of the mode of action of the AB5 cytotoxin family subtilase SubAB from Shiga-toxigenic Escherichia coli
(STEC) have shown that SubAB specifically cleaves the ER
chaperone BiP75,76, owing to unique structural features of
its active site. BiP cleavage triggers the IRE1, PERK and
ATF6 signalling branches of the UPR to induce transient
ER stress and causes cell cycle arrest75,77 (FIG.3). Cell cycle
arrest at the G0 or G1 phase is thought to be a consequence
of decreased cyclin D1, which occurs as a result of both
inhibition of translation and proteasomal degradation75.
This exemplifies the ability of pathogenic bacteria to
induce the UPR, via delivery of specific proteins that have
enzymatic activities directed towards host cell factors.

The observation that induction of the UPR following B.melitensis infection promotes intracellular growth
also suggests that the UPR might be targeted by specific
bacterial mechanisms to promote survival. Induction of
the UPR by B.melitensis depends on the protein TcpB
(Toll/interleukin 1 (IL1)-like receptor (TIR) domaincontaining protein; also called Brucella TIR protein A
(BtpA))61, as a mutant lacking tcpB failed to induce the
UPR and purified TcpB was sufficient to cause induction
of the UPR69 (TABLE1). TcpB has been shown to antagonize
Toll-like receptor2 (TLR2) and TLR4 signalling, via its
interaction with myeloid differentiation primary response
protein88 (MYD88) and the TIR domain-containing
adapter protein MAL (also known as TIRAP) and inhibition of nuclear factor-B (NF-B) activation7883, and
also stabilizes microtubules80, which potentially affects
ER morphology. It will therefore be interesting to examine whether TcpB-mediated UPR induction results from
any of these activities or whether its UPR-inducing effect
reflects yet another function. The recent demonstration
that TcpB is translocated into host cells by B.abortus,
possibly in a VirB-dependent manner 61, emphasizes the
concept that bacterial pathogens may trigger ER stress
via the delivery of effectors or toxins, but also raises
the question of VirB dependency of UPR induction by
Brucella spp.; if TcpB is a VirB effector, one would expect
that a VirB-deficient strain also fails to induce the UPR.
Hence, further studies are needed to clarify these aspects
of induction of the UPR by Brucellaspp..
However, induction of the UPR by Brucella spp. may
result from more complex interactions with host cells
involving several effectors; for example, the VirB T4SS
effector VceC induces IRE1dependent signalling, which
leads to induction of the pro-inflammatory cytokines
tumour necrosis factor (TNF) and IL6 (REF.62). VceCmediated stimulation of UPR signalling possibly results
from its interactions with, and possible sequestration
of, the ER chaperone BiP, which could alter its ability to
downregulate IRE1 activation9,10 (FIG.3). In addition, individual expression of various Brucella spp. effectors (BspC,
BspG, BspH, BspI and BspK) also leads to induction of

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Stx1A CTA

ER
VceC

PERK

SubAB

BiP

BiP

BiP

VceC
P

IRE1
P

Cytosol
elF2

ATF6
P

TRAF2

elF2
P

Xbp1 splicing

JNK

Golgi

Xbp1
ER-associated
mRNA turnover
(RIDD)

IKK

XBP1

RIG-I

S1P
S2P

MAVS
NF-B

ATF4

IB

AP-1

NF-B

ATF6

IRF3
Nucleus
ATF4

NF-B

CHOP (apoptosis)

XBP1

Pro-inammatory genes
AP-1

Pro-inammatory genes

IRF3
Type I interferons

Type I interferons
and chaperones
ATF6
Chaperones

Figure 3 | Induction of the unfolded protein response and inflammation by bacterial virulence
factors.|Several
toxins
Nature Reviews
Microbiology
and type IV secretion system (T4SS) effectors have been implicated in the activation of the unfolded protein response
(UPR) and subsequent inflammation. The Brucella abortus T4SS effector VceC targets the endoplasmic reticulum (ER) and
binds to binding immunoglobulin protein (BiP), which may inhibit its interaction with protein kinase R (PKR)-like ER kinase
(PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6), thereby promoting the activation
of the UPR. The A subunit of the Vibrio cholerae toxin (CTA) and the A subunit of the Shiga toxin (Stx1A) of Shiga
toxin-producing Escherichia coli (STEC) bind IRE1 and activate its endonuclease activity. The subtilase cytotoxin (SubAB)
of STEC also localizes to the ER and can cleave BiP, which leads to activation of PERK, IRE1 and ATF6. Activation of PERK
leads to the phosphorylation of the eukaryotic translation initiation factor 2 (eIF2) and results in the translation of the
transcription factor ATF4, which activates transcription of genes involved in the UPR. Activated IRE1 interacts with the
tumour necrosis factor (TNF) receptor-associated factor 2 (TRAF2), which leads to the production of pro-inflammatory
cytokines. Activated IRE1 forms a complex with TRAF2, which activates JNK, an upstream signalling molecule that leads
to activation of the transcription factor activator protein 1 (AP1). Activation of nuclear factor-B (NF-B) occurs by the
recruitment of inhibitor of NF-B (IB) kinase (IKK) by TRAF2. IRE1dependent mRNA decay (RIDD) generates
immunostimulatory mRNA fragments that are recognized by the innate immune receptors retinoic acid-inducible protein
I (RIG-I) and mitochondrial antiviral signalling protein (MAVS) to activate NF-B and interferon-regulatory factor 3 (IRF3),
which drives expression of pro-inflammatory cytokines and typeI interferons. IRE1mediated activation of X-box-binding
protein 1 (XBP1) also promotes the transcription of typeI interferons. In the context of cellular intoxication by SubAB,
ATF6also has a role in the activation of NF-B. ATF6 is a transcription factor that is anchored in the ER membrane, but in
response to ER stress, it translocates to the Golgi apparatus, where the transcription factor domain is released from the
membrane by sequential action of site 1 protease (S1P) and S2P, which enables its translocation to the nucleus to activate
transcription of UPR target genes, but the precise mechanism for ATF6 translocation following STEC infection is not known.

ER stress in HeLa cells, regardless of whether these proteins target the secretory compartment 60. Interestingly,
all of these effectors induced BiP overexpression, but not
CHOP overexpression60, which suggests that they modulate the IRE1 and ATF6 pathways, but not the PERK
pathway, potentially avoiding inhibition of translation,
cell cycle arrest and cell death. Hence, our current knowledge of induction of the UPR by Brucella spp. suggests
that a multifactorial process enables the bacterium to
finely tune this pathway for its own benefit.

The UPR and innate immune signalling

The co-occurrence of ER stress and inflammation in
many chronic pathologies, including neurodegenerative diseases, diabetes, obesity and inflammatory bowel
disease, suggests that there are interactions between the
UPR and inflammation84. Indeed, pro-inflammatory
cytokines can promote ER stress signalling via oxidative stress, which can alter redox homeostasis in the ER
and lead to protein misfolding. In addition, nitric oxide
that is produced during inflammation can perturb ER

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Thioredoxin-interacting
protein
(TXNIP). A protein that is
induced via protein kinaseR
(PKR)-like ER kinase (PERK)
and inositol-requiring
enzyme1 (IRE1), and that
activates inflammatory
processes and cell death
following unabated
endoplasmic reticulum stress.

Hepcidin
A hormone that regulates iron
homeostasis in mammals; it
has a role in innate immunity
as it limits iron availability to
bacteria.

function via S-nitrosylation of protein disulphide isomerase (PDI), which inhibits its ability to catalyse disulphide
bond formation during protein folding 85. Furthermore,
enhancement of pro-inflammatory cytokine responses by
the IRE1XBP1 pathway in response to TLR2 and TLR4
ligands suggests that there are additional points of intersection between innate immune and ER stress signalling
pathways86.
Conversely, ER stress can activate pro-inflammatory
signalling pathways via multiple mechanisms (FIG.3).
Overexpression of viral proteins in the ER has long been
known to activate the NF-B and activator protein 1
(AP1) transcription factors, which induce expression of
pro-inflammatory cytokines, such as IL6 (REFS87,88).
One of the pathways that links ER stress to pro-inflammatory cytokine expression is dependent on the kinase
activity of IRE1. Activated IRE1 forms a complex with
TNF receptor-associated factor 2 (TRAF2), which activates JNK an upstream signalling molecule that leads
to AP1 activation28. In the TNF receptor signalling pathway, activated TRAF2 can also recruit inhibitor of NK-B
(IB) kinases, leading to their activation, which suggests
that recruitment of TRAF2 to the ER membrane by IRE1
activation during the UPR may also promote activation of NF-B. RIDD can also activate innate immune
responses via generation of immunostimulatory mRNA
fragments in the cytosol2,15. Similar to fragments that are
generated by the antiviral endoribonuclease RNase L89,90,
the mRNA fragments generated by RIDD can be detected
by the innate immune receptor retinoic acid-inducible
protein I (RIG-I) to activate NF-B and interferonregulatory factor 3 (IRF3), which drives expression of
pro-inflammatory cytokines and interferon- (IFN)2,89,91.
The endonuclease activity of IRE1 also activates XBP1,
which has been shown to promote transcription of IFN
via binding of an upstream enhancer, as well as promoting transcription of IL6 and TNF86,92. In addition
to the NF-B pathway, under conditions of irremediable ER stress, IRE1 can also activate NLRP3 (NOD-,
LRR- and pyrin domain-containing 3) inflammasomes
in response to redox stress via thioredoxin-interacting
protein (TXNIP), which leads to activation of IL1 and
pyroptotic cell death9395.
The PERKeIF2ATF4CHOP branch of the UPR,
which mediates inhibition of translation, interacts with
innate immune signalling via activation of NF-B. AsIB
has a short half-life, translational inhibition by this pathway results in a more rapid turnover of IB and, consequently, activation of NF-B (FIG.3). In the context of
cellular intoxication by SubAB, ATF6 also contributes
to the activation of NF-B; however, the precise mechanism for this is unknown96. An ATF6like transcription
factor, cyclic AMP response element-binding protein H
(CREBH), is activated by ER stress in a similar manner
to ATF6, requiring cleavage by S1P and S2P, and has a
role in innate immunity by activating hepcidin expression,
thereby limiting iron availability to infecting bacteria97,98.
It is therefore likely that, depending on the cellular and
tissue context of infection, different combinations of
these pathways may have a role in the induction of innate
immune responses by ERstress.

Studies of viral infection have shown that hijacking

of ER functions for production of viral proteins can trigger innate immune responses; however, several viruses
can exploit the resulting UPR to promote their replication3(BOX1). Therefore, it is conceivable that subversion
of ER function for intracellular replication of bacterial
pathogens, such as Legionella spp., Brucella spp., Simkania
spp. or Chlamydia spp. may also activate a subset of these
surveillance pathways.
Brucella spp. are excellent models to study the intersection of the UPR and innate immunity as they signal
only weakly through TLRs, such as TLR2 and TLR4
(REFS99,100). Furthermore, in a mouse model of infection, innate immune responses to Brucella spp. are highly
dependent on function of the VirB T4SS101. In infected
macrophages, the IRE1 branch of the UPR seems to have
a key role in the innate immune response to the T4SS, as
silencing of IRE1 dampened the production of inflammatory cytokines by infected cells. VceC, which is a
T4SS effector protein that localizes to the ER, can activate IRE1dependent secretion of IL6 and TNF62. As
described above, the newly identified T4SS substrates
BspC, BspG, BspH, BspI and BspK can also activate the
IRE1 pathway in HeLa cells; therefore, it will be interesting
to determine whether they synergize with VceC in inducing expression of pro-inflammatory cytokines during
infection60. Furthermore, it is not known how BtpA, which
has recently been shown to induce all three branches
of the UPR, influences UPR-induced inflammation69.
However, induction of UPR-dependent inflammation
during Brucella spp. infection may underlie some similarities in the host response between Brucella spp. and
viral infections with regard to their gene expression
profiles invivo101,102.
Bacterial toxins can also induce UPR-dependent
inflammation (TABLE1). Some toxins, such as Shiga toxin
and cholera toxin, reach their cytosolic targets via cellular uptake and retrotranslocation to the ER, where
they fold before reaching their targets in the cell cytosol.
Localization of an enzymatically inactive A subunit of
cholera toxin (CTA) to the ER was recently found to activate NF-B via an IRE1dependent pathway in intestinal
epithelial cells, via binding of unfolded CTA to IRE1 (REF2)
(FIG.3). Interestingly, the pathway that leads to NF-B activation by CTA involves activation of RIDD and sensing
of the resulting mRNA fragments via a RIG-Imitochondrial antiviral signalling protein (MAVS)-dependent pathway. The A subunit of Shiga toxin (Stx1A), which triggers
death of intoxicated cells via the UPR103,104, is also able to
activate IRE1 and induce NF-B activation, probably via
a similar mechanism to that identified for CTA2 (FIG.3).
Notably, activation of immune cells, particularly Tcells,
by Shiga toxins has been postulated to play a part in disease progression of haemolytic uremic syndrome caused
by STEC, which raises the possibility of a connection
between activation of the UPR in intoxicated cells and systemic inflammatory pathology 105. Interestingly, although
cleavage of BiP by SubAB of STEC leads to induction of
the three branches of the UPR and, consequently, to a
transient activation of NF-B96,106, pre-stimulation of cells
with SubAB at sub-cytotoxic doses was shown to blunt the

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REVIEWS
induction NF-B activation by heterologous stimuli, such
as TLR4 agonists107. Therefore, the effect of this toxin on
inflammatory responses during an infection may depend
on the toxindose.
The above examples show that eliciting the UPR
functions as a pathogen-associated molecular pattern of
infection initiates innate immune signalling in response
to pathogens that target the ER directly and pathogens
that, via the disruption of other cellular processes, perturb
ER function. Furthermore, in the context of additional
infection-related signals, including TLR ligands86 and
pro-inflammatory cytokines5, cellular surveillance of ER
function is heightened to generate a more rapid innate
immune response to ERstress.

The UPR: friend or foe?

Although several bacterial pathogens can induce
the UPR during infection, it is not clear in each case
whether this response benefits the host or the pathogen. Modulation of ER function by intracellular bacteria
can promote infection by providing a replicative niche,
but at the same time, the resulting disruption of the
secretory pathway can aid the innate immune system
in recognizing intracellular infection and in mounting
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an appropriate defence. However, considering the more

rapid evolution of bacterial pathogens compared with
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Acknowledgements

This work was supported by Washington State University

funds and Public Health Service (PHS) grant AI112649 to
J.C., and PHS grants AIAI050553, AI097107 and AI090387
to R.M.T.

Competing interests statement

The authors declare no competing interests.

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