Available online at www.sciencedirect.com
Role of Pseudomonas aeruginosa type III effectors in disease
Joanne Engel and Priya Balachandran
Pseudomonas aeruginosa uses a type III secretion system
(T3SS) to directly inject four known effectors into host cells.
ExoU is a potent cytotoxin with phospholipase A2 activity that
causes rapid necrotic death in many cell types. The biological
function of ExoY, an adenylate cyclase, remains incompletely
defined. ExoS and ExoT are closely related bifunctional
proteins with N-terminal GTPase activating protein (GAP)
activity toward Rho family proteins and C-terminal
ADP ribosylase (ADPRT) activity toward distinct and
non-overlapping set of targets. While almost no strain encodes
or secretes all four effectors, the commonly found
combinations of ExoU/ExoT or ExoS/ExoT provides redundant
and failsafe mechanisms to cause mucosal barrier injury, inhibit
many arms of the innate immune response, and prevent wound
Departments of Microbiology/Immunology and Medicine, Microbial
Pathogenesis and Host Defense Program, Box 0654 UCSF, 513
Parnassus Ave, San Francisco, CA 94143, United States
Corresponding author: Engel, Joanne (firstname.lastname@example.org)
Current Opinion in Microbiology 2009, 12:61–66
This review comes from a themed issue on
Host-microbe interactions: bacteria
Edited by Brendan Kenny and Raphael Valdivia
Available online 23rd January 2009
1369-5274/$ – see front matter
# 2008 Elsevier Ltd. All rights reserved.
Pseudomonas aeruginosa, a ubiquitous Gram negative
pathogen widespread throughout the environment,
is a leading cause of opportunistic infections in
humans . In normal hosts, with an intact epithelial
barrier, P. aeruginosa rarely causes disease. However, in
the setting of epithelial damage, as is seen in immu-
nocompromised and/or hospitalized patients, P. aerugi-
nosa is a common cause of nosocomial infections. Most
of these are acute infections, including sepsis, venti-
lator-associated pneumonia, and infections in post-
operative wound and burn patients. P. aeruginosa also
chronically colonizes Cystic Fibrosis (CF) patients,
leading to severe pulmonary damage and death.
mortality remains as high as 40% in acute infections,
P. aeruginosa has a large armamentarium of secreted
virulence factors that rely on specialized export systems,
type VI secretion systems [2–4]. The type III secretion
system (T3SS), a contact-dependent sec-independent
protein secretion pathway that forms a conduit for the
translocation of bacterial effectors into the host cell, is
thought to play a key role in the pathogenesis of acute P.
aeruginosa infections. The T3SS of P. aeruginosa contrib-
utes to epithelial cell and macrophage damage in vitro, in
animal models of disease, and in human infections .
This review summarizes exciting progress in recent years
in understanding the effectors and in placing these find-
ings in the context of the pathogenesis of this important
opportunistic pathogen of humans.
Four T3SS effectors have been identified in P.
In contrast to some organisms that encode a multitude of
effectors, only four T3SS effector molecules have been
identified in P. aeruginosa so far: ExoU, ExoS, ExoT, and
ExoY (Figure 1). Additional T3SS effectors may be
revealed as more strains are sequenced and further ana-
lyzed. ExoT and ExoY are encoded by almost all strains,
though not all strains produce functional ExoY due to the
presence of frameshift mutations. ExoS and ExoU are
variably encoded genes and are almost never found in the
same strain . Although the T3SS system and its associ-
ated effectors were probably acquired by horizontal trans-
mission, only ExoU and ExoS bear characteristic
hallmarks of recent acquisition. ExoS is flanked by 10
base pair repeats that are only present as a single copy in
non-ExoS-encoding strains, consistent with horizontal
gene transfer. Interestingly, ExoS is more similar than
ExoT to other bacterially encoded ADPRTs, suggesting
In this scenario, ExoS would have then undergone a
deletion event in some strains. Four distinct configur-
ations of the genome region containing ExoU have been
identified by comparative sequencing, though no allelic
variation was observed in the coding region of ExoU,
consistent with relatively recent dissemination of this
gene in natural populations. Further analysis suggests
that this gene was probably acquired by transposition
onto a transmissible plasmid followed by the transfer
of the plasmid onto different strains and subsequent
integration into the tRNAlysgene, where it acquired
insertion sequences and underwent deletions and re-
Most strains examined so far can be divided into two
groups. ExoU and ExoT producing strains (including
Current Opinion in Microbiology 2009, 12:61–66
PA103 and PA14) cause rapid necrotic host cell death and
are poorly internalized. By contrast, ExoS and ExoT
producing strains (as exemplified by PAK and PA01)
are more efficiently internalized and result in caspase-
dependent host cell death with a slower time course.
Interestingly, all four T3SS effectors require a host co-
factor for activity; this attribute may prevent them from
damaging bacteria before translocation. Both genotypes
(ExoS/ExoT and ExoU/ExoT) are associated with acute
infections in humans, though ExoU-producing strains are
under-represented in persistently infected CF patients
. The expression of the T3SS is downregulated in CF
isolates from chronically colonized CF patients, consist-
ent with the notion that bacterial persistence requires the
downregulation of many virulence factors .
ExoU, a potent phospholipase
ExoU possesses phospholipase A2-like activity with
broad substrate specificity [9??]. The chaperone for
dismutase (SOD) has been reported to function as the
co-factor, though the enzymatic activity of SOD was not
required . Interestingly, upon translocation into the
host, ExoU rapidly associates with the membrane, which
may bring ExoU in close proximity to its substrate phos-
pholipids [11,12]. ExoU has also been reported to be
ubiquinated. This post-translational modification does
not alter the degradation of ExoU, and its biological
significance is as yet unclear .
ExoU production and activity are required for virulence
in a mouse model of acute pneumonia and are associated
with up to 90% of cases of severe disease in human
infections . In addition to its cytotoxic effects, ExoU
triggers an arachidonic acid-dependent inflammatory cas-
cade in vivo and induces expression of inflammatory
genes [13–15]. ExoU is predicted to cleave surfactant,
a key component of alveoli . In human infections and
in mouse models of pneumonia, ExoU may specifically
target and kill neutrophils, leading to localized areas of
immunosuppression that render the host susceptible to
secondary infections, including viral infections [17?,18??].
Host-microbe interactions: bacteria
The T3SS effectors of P. aeruginosa have diverse and pleiotropic effects on host cell function. The light blue cells represent the polarized epithelial
barrier; the dark blue cell represents an injured, depolarized cell at the epithelial barrier. A typical neutrophil is shown in green and a macrophage is
shown in orange. The effects of the T3SS effectors are diagrammed. ExoS induces apoptosis, inhibits cell migration, disrupts tight junctions, and
disrupts the actin cytoskeleton in epithelial cells (and probably endothelial cells). ExoT inhibits cell division and cell migration, disrupts focal adhesions,
and can induce cell death. Both ExoS and ExoT inhibit bacterial uptake into epithelial and phagocytic cells. They may inhibit neutrophil and
macrophage function as well. ExoS may also inhibit vesicular trafficking (not pictured). ExoU possesses phospholipase A2 activity, leading to rapid cell
death in many cell types. Finally, ExoY is an adenylate cyclase that may disrupt the actin cytoskeleton. Together, these T3SS effectors efficiently inhibit
wound repair and the host innate immune response, perpetuate tissue injury, and render the host susceptible to further colonization and injury by P.
aeruginosa and other pathogens.
Current Opinion in Microbiology 2009, 12:61–66 www.sciencedirect.com
ExoU has recently been shown to inhibit caspase-1-
mediated pro-inflammatory cytokine production [19?].
The extensive tissue destruction induced by ExoU com-
bined with the modulation of the host inflammatory
response, particularly its ability to induce localized
immunosuppression, probablyexplains its prominent role
in the pathogenesis of severe acute P. aeruginosa infec-
tions. However, the prevalence of ExoU-producing
strains in the natural environment may reflect its role
in avoiding natural predators, such as water and soil
ExoY, a host factor-dependent adenylate
ExoY is a host factor-dependent adenylate cyclase
initially identified by a proteomic analysis of T3SS
proteins. It requires an as yet to be identified host cell
co-factor for activity. Its role in virulence remains uncer-
with tissue culture cells  and is toxic when expressed
in yeast [21?].
ExoS and ExoT are closely related bifunctional
proteins with GAP and ADPRT activity
ExoT and ExoS are closely related bifunctional proteins
that possess two distinct enzymatic activities that work
redundantly to disrupt the actin cytoskeleton, resulting in
profound effects on host cellular processes . It is
unknown whether there is additional evolutionary benefit
to encoding both enzymatic activities on a single protein.
The N-terminus of ExoS (as well as ExoT) contains a
membrane localization domain (MLD). ExoS is initially
delivered to the plasma membrane in a cholesterol-de-
pendent but clathrin-independent, caveolin-indepen-
dent, Dynamin II-independent, and Arf6-independent
manner. ExoS subsequently localizes to the perigolgi
region, utilizing a microtubule-dependent but actin-inde-
pendent pathway that involves Rab 5-positive, Rab 9-
positive, and Rab 6-positive endosomes, even though the
ADPRT-activityofExoScaninhibitRab 5function [23?].
The MLD may regulate substrate specificity; it is
required for ExoS to efficiently ADP ribosylate mem-
brane-localized Ras and for both ExoS and ExoT Rho-
GAP activity toward Rho family GTPases [24?,25,26??].
The FAS binding domain of ExoS, required for the
ADPRT activity but not the GAP activity, is unusual
[27?]. While 14-3-3 proteins typically bind to a phospho-
serine-containing motif, the ExoS and ExoT-FAS bind-
ing motif binds 14-3-3 in a novel, phosphorylation-
independent manner that relies on hydrophobic inter-
actions. Key mutations in the ExoS 14-3-3 binding
domain affect its ability to cause death in a mouse model
of pneumonia. Competition for 14-3-3 proteins could be
an additional mechanism by which ExoS and ExoT
disrupt normal host signaling pathways.
The N-terminal domains of ExoS and ExoT exhibit GAP
activity toward Rho, Rac, and Cdc42. These toxins are
members of a growing family of bacterially encoded GAP
proteins that arose by convergent evolution and that now
include Yersinia YopE, Salmonella SptP, and Aeromonas
AeroT . As would be predicted of proteins with GAP
activity, in cell-based assays, the GAP domains of ExoS
and ExoT disrupt the actin cytoskeleton, inhibit bacterial
internalization into epithelial cells and macrophages,
induce host cell rounding, and prevent wound healing
. More recently, the GAP activity of ExoT has been
shown to inhibit early stages of cytokinesis, probably by
inactivating Rho .
The C-terminal domains of ExoS and ExoT encode
biglutamic acid ADPRTs that differ from those of other
bacteria with respect to the number of target proteins, the
ability to ADP-ribosylate target proteins at more than one
arginine residue, and the requirement for a eukaryotic co-
factor (FAS) for enzymatic activity. While the ADPRT
domains of ExoS and ExoT are highly homologous and
both require FAS for activity, their targets are very
has defined regions that are responsible for their differ-
ences in substrate specificity .
In vitro and in vivo studies suggest that the major targets
of the ExoS ADPRT domain of ExoS include Ras, Rac,
Ral, Rap, various Rabs (3-8, and 11 in vitro and 5-8 and 11
in vivo), Rac, Cdc42, cyclophilin, vimentin, Hsp27, and
ezrin/radixin/moesin proteins [22,30,31]. The ADPRT
activity of ExoS inhibits clathrin-coated vesicle-depend-
ent transport, including Rab-5-dependent endocytosis of
epidermal growth factor receptor [23?]. ADP ribosylation
of moesin by ExoS prevents phosphorylation by protein
kinase C, providing the first example of a bacterial toxin
that affects phosphorylation of a mammalian protein .
There are many physiological consequences of ExoS-
mediated ADP ribosylation of host proteins. ExoS
ADPRT activity is cytotoxic through a Fas receptor/
caspase-8 independent pathway , possibly by stimu-
lating c-JUN NH2-terminal kinase (JNK) phosphoryl-
ation while inhibiting ERK1/2 and p38 phosphorylation
. It has also been shown recently that the ADPRT
domain of ExoS disrupts tight junctions and probably
contributes to the ability of an ExoT/ExoY/ExoS produ-
cing strain to alter the subcellular localization of ZO-1,
occluding, and ezrin in human airway cells [34?].
Initially, it was thought that the ADPRT domain of ExoT
was catalytically inactive, as it had very little enzymatic
activity toward a synthetic substrate in vitro. However,
mutations in the GAP or ADPRT domain suggested that
the ADPRT domain affected cell migration, focal
adhesion formation, wound healing, and cell rounding.
In vivo substrates of ExoT identified by radiolabelling
Role of Pseudomonas aeruginosa type III effectors in disease Engel and Balachandran63
Current Opinion in Microbiology 2009, 12:61–66
permeabilized cells with
CrkII, phosphoglycerate kinase, and ExoT itself .
32P-NAD include CrkI and
ADPRT domain of ExoT explains the observed ability of
the ExoT ADPRT domain to inhibit focal adhesions, cell
migration, wound healing, and bacterial uptake [36,37].
The Crk family of proteins are widely expressed adaptor
proteins that lack enzymatic activity, but consist of an N-
terminal SH2 domain and 1-2 SH3 domains, providing a
platform to recruit and assemble multi-protein complexes
including Rac, p180 Dock (a Rac GEF), and Abl kinase.
Crk family proteins are involved in many cellular pro-
cesses including focal adhesions, epithelial to mesench-
ymal transition,cell migration,
apoptotic programmed cell death. ExoT ADP-ribosylates
CrkI and CrkII isoforms on a crucial arginine in the SH2
domain and interferes with their ability to be phosphory-
lated in cell-based assays . Using ExoT as a cell
biology ‘probe’, it was recentlydiscovered that abscission,
the final stage of cytokinesis in which the two daughter
cells separate, requires Crk and other focal adhesion
components, and that ExoT inhibits cell division .
In addition, by interfering with Crk function, ExoT
induces apoptosis [38??].
Little is known about the degradation of T3SS effectors
following infection into the host cytosol. Recent in vitro
and in vivo studies reveal that the association of the
ADPRT domain of ExoT with Crk also brings it in
contact with a host ubiquitin ligase, Cbl-b [39??], which
targets ExoT for polyubiquitination and proteasomal
degradation. Consistent with these in vitro observations,
Cbl-b deficient mice showed higher susceptibility to in-
fection by ExoT containing P. aeruginosa in murine
pneumonia and systemic infection models. Bone marrow
transfer experiments suggest that Cbl-b in both hemato-
poietic and non-hematopoietic tissues is important in
(Lemus A, Balachandran P, Engel J, unpublished data).
These studies provide the first example of a mammalian
gene product that is specifically required for in vivo
resistance to disease mediated by a T3SS effector and
demonstrate that proteasome-mediated degradation of
T3SS effectors may play a role in altering the course of
The T3SS itself may contribute to virulence
While much of the T3SS-mediated damage is probably
caused by the translocated effectors, there is increasing
evidence that insertion of the needle complex itself can
contribute to host cell injury, possibly by allowing ion
influx and/or by activating the innate immune response
though activation of caspase-1-dependent cleavage of IL-
1B . In the case of P. aeruginosa, this intracellular
signaling goes through Ipaf [41,42?] and is inhibited by
ExoU [19?] and possibly by ExoS ADPRT activity [43?].
ExoT can partly protect against the needle-mediated
Utilizing the T3SS for diagnostics and
As the presence of an active T3SS correlates with worse
outcome in clinical infections, future clinical strategies
may be aimed at identifying subsets of patients colonized
or infected with T3SS-active strains of P. aeruginosa and
example, in one recent study, the presence of T3SS-
active strains correlated with higher bacterial burden
and a greater risk of death in mechanically ventilated
patients [45?]. Chemical inhibitors of ExoS [21?], ExoU
[46?], and the T3SS apparatus itself [47?], have been
identified and could serve as useful adjuncts to antimi-
The T3SS is also a potential vaccine candidate. In mouse
models of acute and chronic pneumonia, vaccination
against PcrV (a component of the translocation complex)
orpassive transferof anti-PcrV antibodies,protectsagainst
PA-induced damage [48,49]. Although the T3SS is down-
regulated in CF patient isolates, CF patients make anti-
bodies against the T3SS, suggesting a role for the T3SS in
the pathogenesis of CF lung disease . Prevention of
colonization with P. aeruginosa isolates with an active
TTSS may allow for early therapeutic intervention and
improved clinical outcome for patients with CF.
While few in number, the four known T3SS effectors of
P. aeruginosa have failsafe, redundant, and profound
effects on the host cell that contribute to its virulence
in the context of acute infections and provide attractive
targets for new diagnostic and therapeutic strategies.
ExoU, ExoS and ExoT, and perhaps ExoY, are capable
of disrupting the epithelial barrier and of inhibiting
wound repair by inhibiting cell migration and cell pro-
liferation, inducing apoptosis, and disrupting tight junc-
tions at multiple steps. The potent cytotoxic activities of
ExoU result in rapid necrosis of the epithelial barrier. By
preventing wound healing, P. aeruginosa perpetuates the
injured state, resulting in further damage and dissemina-
tion to distant organs. In addition, these effectors would
be predicted to interfere with macrophage and neutrophil
function and migration. Their effect on cytokine pro-
duction is just beginning to be explored. As humans are
accidental hosts, it is likely that these toxins are also
jungle of microbial predators in its natural environment.
I thank Dr Alan Hauser and members of the Engel lab for their suggestions.
Because of space constraints, I apologize to the investigators whose work I
was unable to cite. Work in my laboratory is supported by grants from the
National Institutes of Health (AI42806, AI065902).
Host-microbe interactions: bacteria
Current Opinion in Microbiology 2009, 12:61–66 www.sciencedirect.com
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
? of special interest
?? of outstanding interest
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