Ubiquitin and ubiquitin-modified proteins activate the Pseudomonas aeruginosa T3SS cytotoxin, ExoU

Article (PDF Available)inMolecular Microbiology 82(6):1454-67 · December 2011with28 Reads
DOI: 10.1111/j.1365-2958.2011.07904.x · Source: PubMed
Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that possesses a type III secretion system (T3SS) critical for evading innate immunity and establishing acute infections in compromised patients. Our research has focused on the structure-activity relationships of ExoU, the most toxic and destructive type III effector produced by P. aeruginosa. ExoU possesses phospholipase activity, which is detectable in vitro only when a eukaryotic cofactor is provided with membrane substrates. We report here that a subpopulation of ubiquitylated yeast SOD1 and other ubiquitylated mammalian proteins activate ExoU. Phospholipase activity was detected using purified ubiquitin of various chain lengths and linkage types; however, free monoubiquitin is sufficient in a genetically engineered dual expression system. The use of ubiquitin by a bacterial enzyme as an activator is unprecedented and represents a new aspect in the manipulation of the eukaryotic ubiquitin system to facilitate bacterial replication and dissemination.
Ubiquitin and ubiquitin-modified proteins activate the
Pseudomonas aeruginosa
T3SS cytotoxin, ExoUmmi_7904 1454..1467
David M. Anderson,
Katherine M. Schmalzer,
Hiromi Sato,
Monika Casey,
Scott S. Terhune,
Arthur L. Haas,
Jimmy B. Feix
Dara W. Frank
Departments of
Microbiology and Molecular Genetics
Center for Infectious Disease
Research, and
Biotechnology and Bioengineering
Center, Medical College of Wisconsin, Milwaukee, WI
53226, USA.
Department of Biochemistry and Molecular Biology,
LSU Health Sciences Center School of Medicine, New
Orleans, LA 70112, USA.
Pseudomonas aeruginosa is an opportunistic Gram-
negative pathogen that possesses a type III secretion
system (T3SS) critical for evading innate immunity
and establishing acute infections in compromised
patients. Our research has focused on the structure–
activity relationships of ExoU, the most toxic and
destructive type III effector produced by P. aeruginosa.
ExoU possesses phospholipase activity, which is
detectable in vitro only when a eukaryotic cofactor is
provided with membrane substrates. We report here
that a subpopulation of ubiquitylated yeast SOD1
and other ubiquitylated mammalian proteins activate
ExoU. Phospholipase activity was detected using puri-
fied ubiquitin of various chain lengths and linkage
types; however, free monoubiquitin is sufficient in a
genetically engineered dual expression system. The
use of ubiquitin by a bacterial enzyme as an activator is
unprecedented and represents a new aspect in the
manipulation of the eukaryotic ubiquitin system to
facilitate bacterial replication and dissemination.
Pathogens have evolved a variety of mechanisms to over-
come host barriers to parasitism. Physical barriers include
an intact epithelium, the outward flow of mucosal fluids,
skin keratinization and the synthesis and secretion of
antimicrobial peptides (Collins and Brown, 2010). Other
barriers encode specific systems to recognize and traffic
intracellular invaders through the lysosomal or autophagy
pathways for destruction (Dupont et al., 2010). Pathogens
that have co-evolved with their hosts encode gene prod-
ucts that manipulate the mammalian environment to
enhance replication and spread to the next host.
Microbial interference with the ubiquitylation/
deubiquitylation pathway is now recognized as a major
evasive mechanism (reviewed in Angot et al., 2007; Ryt-
konen et al., 2007; Shames et al., 2009; Collins and
Brown, 2010; Dupont et al., 2010). A variety of bacterial
effector proteins possess deubiquitylation (Orth et al.,
2000; Zhou et al., 2005; Misaghi et al., 2006; Catic et al.,
2007; Rytkonen et al., 2007; Sweet et al., 2007; Ye et al.,
2007; Le Negrate et al., 2008) or E3 ligase activities
(Haraga and Miller, 2006; Janjusevic et al., 2006; Zhang
et al., 2006; Rohde et al., 2007; Kubori et al., 2008). Upon
intracellular delivery, bacterial effectors also serve as ubiq-
uitylation targets (Kubori and Galan, 2003; Schnupf et al.,
2006; Patel et al., 2009) or as scaffolding proteins for
modification complexes (Kim et al., 2005; Nomura et al.,
2006; Angot et al., 2007; Jubelin et al., 2010). Collectively,
these incursions benefit the bacterium by modulating host
signalling pathways, particularly those involved in inflam-
mation, as well as altering host or bacterial protein traffick-
ing and stability.
Pseudomonas aeruginosa is a soil bacterium and sig-
nificant opportunistic pathogen (Pier and Ramphal, 2005).
This organism takes advantage of tissue that is damaged
or hosts with compromised immune systems to establish
a nidus of infection that can spread systemically. It is
particularly problematic in people who suffer neutropenia,
mechanically ventilated patients and individuals with
severe burns. Treatment is difficult because of intrinsic
and acquired resistance to antimicrobial agents (Giama-
rellou, 2000). In contrast to acute infections, P. aeruginosa
can also establish long-term, localized, chronic infections
in cystic fibrosis patients (Hauser et al., 2011). Damage in
this case is not only due to bacterial replication but also to
the host inflammatory response to an invader that cannot
be eliminated.
Pseudomonas aeruginosa is notable for its expression
of many tissue degradative enzymes and toxins that alter
Accepted 24 October, 2011. *For correspondence. E-mail frankd@
mcw.edu; Tel. (+1) 414 955 8766; Fax (+1) 414 955 6535.
authors contributed equally to this work.
Molecular Microbiology (2011) 82(6), 1454–1467 doi:10.1111/j.1365-2958.2011.07904.x
First published online 21 November 2011
© 2011 Blackwell Publishing Ltd
eukaryotic cell physiology. The organism uses a type III
secretion system (T3SS) to deliver or inject at least four
toxins into the cytoplasm of infected cells (Yahr et al.,
1997). Injection of ExoS or ExoT perturbs cellular signalling
and cytoskeletal components (Frithz-Lindsten et al., 1997;
Ganesan et al., 1999). Each enzyme is bifunctional and
contains Rho GAP and ADP-ribosyltransferase domains
(Goehring et al., 1999; Sun and Barbieri, 2003). The intro-
duction of junctional gaps in endothelial tissue is mediated
by the injection of ExoY (Sayner et al., 2004), an adenylyl
cyclase (Yahr et al., 1998). The most cytotoxic component
injected into cells, ExoU, possesses phospholipase A
activity (Sato et al., 2003). ADP-ribosyltransferase, adeny-
lyl cyclase and phospholipase activities are generally not
detectable from purified proteins in vitro unless an eukary-
otic activator/cofactor is present. For ExoS and ExoT the
activators are members of the 14-3-3 family of scaffolding
proteins (Fu et al., 1993). ExoU is activated in the presence
of certain preparations of superoxide dismutase or SOD1
(Sato et al., 2006). The cofactor for ExoY is currently
unknown (Yahr et al., 1998). The interaction of cofactor
proteins with each enzyme is poorly understood and the
development of defined inhibitors will depend on charac-
terizing the mechanisms of activation at the molecular
Our studies have focused on defining the mechanism
of activation of ExoU by SOD1. This protein was origi-
nally identified as an activator of ExoU by using bio-
chemical enrichments of yeast soluble fractions and
proteomic approaches (Sato et al., 2006). Similar to the
14-3-3 activators of ExoS and ExoT, SOD1 is ubiquitous
in eukaryotes, exists in high concentration in the cyto-
plasm and the sequence differences between prokary-
otic and eukaryotic forms potentially accounted for the
specificity of ExoU’s toxicity for eukaryotes (Sato et al.,
2006). Commercially prepared bovine SOD1 (bSOD1) is
also an activator, but the activation activity appears to
depend on the tissue of origin and commercial supplier
(Benson et al., 2010). The amount of bSOD1 or recom-
binant yeast SOD1 (ySOD1) required for activation of
ExoU is relatively high and is not saturable in kinetic
analyses (Benson et al., 2010). These data suggested
that a minor population of SOD1 molecules are respon-
sible for the activation of ExoU.
The aim of this study was to determine the properties of
bSOD1 and ySOD1 mediating the activation of ExoU. Our
analyses indicate that post-translational ubiquitylation of
ySOD1 and monoubiquitin, contained within bSOD1 com-
mercial preparations, is responsible for the activation of
ExoU in vitro. Of the multiple mechanisms that prokaryotic
pathogens use to interact with or modulate the eukaryotic
ubiquitylation system, this is the first report of a T3SS
toxin utilizing ubiquitin as an activator of enzymatic activity
that mediates host cell death.
Activation of ExoU phospholipase activity by peptides
Our first aim was to characterize the minimal region of
superoxide dismutase required to activate ExoU in an in
vitro assay. To test relatively large fragments with potential
overlaps, we used a partial protease digestion approach
and controlled the time of digestion (Koth et al., 2003).
Initial experiments utilized thrombin (2 peptide fragments),
glutamyl endopeptidase (10 fragments), endoproteinase
Lys-C (12 fragments), and trypsin (15 fragments) either
alone or in combinations (data not shown). Bovine liver
SOD1 was denatured to facilitate digestion and the result-
ing peptide fragments were precipitated for testing in a
fluorescence-based in vitro activity assay measuring
cleavage of the phospholipid mimic, PED6 (Benson et al.,
2010). Interestingly, peptide activation of ExoU compa-
rable with non-treated bSOD1 was detectable in all
instances, even samples sequentially digested to comple-
tion with three different proteases (Fig. 1A and B). The
most prevalent products appeared in the 4–8 kDa range,
suggesting that a relatively small fragment possessed the
capability to activate ExoU (approximately 36–72 amino
acids, Fig. 1A).
To more completely digest bSOD1, we used proteinase
K, which possesses 58 predicted cleavage sites within
the protein. Similar to site-specific digestion, full-length
bSOD1 was not detectable on silver stained gels (data not
shown) or by Western blot analysis after the 60 min time
point (Fig. 1C), yet specific activity remained relatively
constant until 75–90 min digestion (Fig. 1D). We postu-
lated that the accumulation of a peptide activator over
time might be demonstrated by an increase in specific
activity but this result was not observed. In fact, digestion
beyond 75 min showed a decrease in specific activity,
indicating that the cofactor was at least partially proteina-
ceous (Sato et al., 2006). Together, these findings sug-
gested that an SOD1 derived peptide or a protease
resistant molecule or both contributed to ExoU activation.
We noted that a peptide corresponding to 6.5 kDa was
not only present in the undigested bSOD1 preparation,
but was also partially retained in the preparation of triply
digested SOD1 (Fig. 1A, lane 11). This peptide was
extracted from the gel and identified by mass spectrom-
etry as ubiquitin (Table S1). To determine if the presence
of ubiquitin was the common factor determining whether
or not different SOD1 preparations activated ExoU, we
examined bovine liver, kidney and red blood cell derived
proteins by Western blot analysis with a monoclonal anti-
body specific to ubiquitin (Fig. S1). Using similar amounts
of SOD1, we detected ubiquitin in the only preparation
capable of activating ExoU, bovine liver SOD1 (Fig. S1
and data not shown). These data suggest that ubiquitin is
a cofactor for ExoU activation.
Ubiquitin activation of ExoU
© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 82, 1454–1467
Ubiquitylated proteins from yeast and mammalian cells
activate ExoU
Commercial preparations of bSOD1 purified from SDS
gels activate ExoU (Sato et al., 2006) suggesting that a
subpopulation of bSOD1 molecules might be modified by
ubiquitin. Western blot analysis of the bSOD1 indicated
that ubiquitin was not covalently associated with liver
SOD1 (Fig. S1). To determine whether the activation
activity associated with recombinant ySOD1 (Sato et al.,
2006) was due to ubiquitylated protein or released
monoubiquitin, ySOD1 was purified by cobalt chromatog-
raphy and subjected to Western blot analysis using
equivalent amounts (4 mg) of monoclonal antibodies to the
Fig. 1. Peptides from a liver bSOD1 preparation activate ExoU in vitro.
A. 10–20% Tris-tricine Coomassie-stained SDS-PAGE gel showing peptides generated using alkylated liver bSOD1. Lanes 1, 6 and 12 are
molecular weight standards; Lane 2, undigested bSOD1; Lane 3, endoproteinase Lys-C (Lys-C) only; Lane 4, Lys-C digestion; Lane 5, Lys-C
and thrombin only; Lane 7, Lys-C + thrombin digestion; Lane 8, Lys-C and glutamyl endopeptidase (Glu-C) only; Lane 9, Lys-C + Glu-C
digestion; Lane 10, Lys-C, Glu-C and thrombin only; Lane 11, Lys-C + Glu-C + thrombin digested bSOD1. Arrows indicate a peptide that is
present in undigested SOD1 and retained in SOD1 digested to completion with endoproteinase Lys-C, thrombin and glutamyl endopeptidase
as outlined.
B. Activity assay using either untreated bSOD1, sequentially and completely digested protein or a protease only control as cofactor material.
‘Triple Digestion’ corresponds to lane 11 in (A).
C. Western blot analysis of bSOD1 from a 1:1000 proteinase K: bSOD1 (w/w) digestion over a 120 min time-course. Anti-SOD1 (1:5000) was
used as the primary antibody in this analysis.
D. Specific phospholipase activity (nmol PED6 cleaved min
rExoU) when proteinase K digested products from denatured bSOD1 are
used to activate rExoU over a digestion time-course (error bars SEM, n = 3). Untreated refers to bSOD1 that is not denatured nor protease
treated and undigested refers to bSOD1 that is denatured but not treated with proteinase K.
D. M. Anderson
et al
© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 82, 1454–1467
hexa-histidine tag or ubiquitin as probes. Anti-histidine tag
antibody recognized a 34 kDa protein when the antigen
was loaded at nanogram levels (Fig. 2A). Anti-ubiquitin
antibodies recognized protein within the same molecular
weight range at levels of 1 mg or higher (Fig. 2A). Detec-
tion of signal with anti-ubiquitin required an exposure time
of 1 h as compared with a few seconds for blots probed
with anti-histidine tag antibody. Monoubiquitin was not
detectable (data not shown). These data support the con-
clusion that a small subpopulation of ubiquitlyated recom-
binant ySOD1 serves as an ExoU cofactor in vitro.
To determine whether ubiquitylated mammalian pro-
teins activate ExoU, an osteosarcoma cell line, U2OS,
was exposed or not exposed to the proteasome inhibitor
MG132. Exposure to MG132 shifts the pool of free ubiq-
uitin to ubiquitin conjugated to proteins. Western blot
analysis of total cell lysates revealed an increase in the
detectable levels of high molecular weight species, con-
sistent with a shift in the accumulation of ubiquitylated
proteins upon treatment with MG132 (Fig. 2B). Immuno-
precipitated material from isotype controls revealed
neither ubiquitylated proteins nor free ubiquitin as
Fig. 2. Ubiquitylated proteins activate ExoU.
A. Histidine tagged recombinant ySOD1 was purified and subjected to a twofold dilution series followed by SDS-polyacrylamide gel
electrophoresis and Western blot analysis. The beginning protein concentration for each blot was 4 mg of purified ySOD1. Left panel, Western
blot of ySOD1 titration probed with antibody to the histidine tag. Right panel, Western blot analysis identical to the left panel except probed
with monoclonal antibody specific for ubiquitin. Equivalent amounts of primary antibody were used for each blot. Twofold more secondary
antibody was used to detect the anti-ubiquitin probe. Exposure times: anti-His, 3 sec; anti-ubiquitin, 1 h.
B. Western blot analysis of ubiquitylated proteins from U2OS cells. Left panel, cultures were subjected to an 8 h treatment with MG132 (10
mM, + lanes) or medium only (- lanes) before harvest. Five percent of each cell lysate (~ 5.0 ¥ 10
cells) was probed with anti-ubiquitin or
antibody to GAPDH (loading control) after electrophoresis (SDS-PAGE, 15%) and Western blot transfer. Right panel, the remaining portion of
each corresponding lysate was subjected to immunoprecipitation with an isotype control or anti-ubiquitin monoclonal antibody and probed in
Western blot analysis of an SDS-PAGE (8% gel) with anti-ubiquitin. Bottom right panel, SDS-PAGE gel (15%) of isotype control or
anti-ubiquitin precipitated material probed with anti-ubiquitin. Free or monoubiquitin is not detectable. A twofold monoubiquitin titration
(100–3.1 ng) was loaded to lanes on the same gel to establish the detection range of the antibody.
C. In vitro assay measuring cleavage of a phospholipid mimic, PED6, using identical amounts of protein shown in each lane for material
immunoprecipitated with anti-ubiquitin or isotype control antibodies. Assay measurements from equivalent amounts of each antibody bound to
Protein G beads exposed to lysis buffer only were subtracted as background. Data are expressed as means SEM, n = 3.
Ubiquitin activation of ExoU
© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 82, 1454–1467
expected. In the absence of MG132 few high molecular
weight ubiquitin conjugates were detectable. Conjugated
protein material increased substantially upon treatment
with MG132 (Fig. 2B). Importantly, in immunoprecipitation
assays ubiquitylated proteins appeared to be enriched
while free ubiquitin was not detectable (Fig. 2B). ExoU
activity assays were performed utilizing the bound mate-
rials as a source of the activator. Beads from the anti-
ubiquitin immunoprecipitated MG132-treated lysates
possessed significant activity (Fig. 2C). Minor activity was
detectable from MG132 untreated cells (Fig. 2C). Overall,
these data support the hypothesis that in addition to free
ubiquitin, ubiquitylated proteins from either yeast or mam-
malian cells serve as cofactors for ExoU.
Various species of ubiquitin but not ubiquitin-like
proteins serve as activators
To address the mechanism of activation of ExoU, it is
important to determine whether different forms of ubiquitin
affect ExoU activity. A PED6 titration was initially con-
ducted in the presence of monoubiquitin or polyubiquitin
to quantify the concentration of substrate required to be
near saturation. For PED6, this concentration was deter-
mined to be 100 mM (data not shown). To determine how
well ubiquitin activated ExoU, each species was titrated
into the assay and phospholipase A
activity was mea-
sured as an increase in cleaved PED6 (fluorescence)
over time (Fig. S2A and B). For most of the ubiquitin
species tested, a lag time in ExoU activation was
observed in the progression curves. This lag in activation
was not dependent on preincubation time and could be
reduced or eliminated as the concentration of activator
was increased. These two points suggest that the struc-
tural ordering of ExoU and the binding to ubiquitin reaches
equilibrium fairly rapidly when sufficient amounts of the
activator are present.
Progression curves were analysed for the rates of enzy-
matic activity after steady-state conditions were reached
and the concentration of activator (nM) as a function of
rate (nmoles PED6 cleaved min
) was plotted (Fig. 3A).
With the exception of monoubiquitin, the enzymatic rate of
ExoU in the presence of all ubiquitin species reached
saturation at concentrations of activator less than 4 mM,
about 2 mM less than the intracellular concentration of
ubiquitin (Haas and Bright, 1987).
Non-linear regression analysis of the Michaelis–Menten
plots (as represented by dashed lines) fit well with calcu-
lated correlation coefficients of 0.89 or greater (Fig. 3A
and Table 1) making it possible to derive kinetic constants
for each ubiquitin species. As the number of ubiquitin
moieties increased for each isoform, the activation con-
stant (K
) corresponding to the K
for ExoU binding to the
ubiquitin, decreased, which is indicative of tighter binding
of rExoU for ubiquitin (Fig. 3B and Table 1). Although
rates of maximal catalysis (V
) were similar, we noted a
disparity in the catalytic efficiency (V
) between the
different forms of ubiquitin. Compared with monoubiquitin,
Fig. 3. Multiple isoforms of ubiquitin activate rExoU.
A. Michaelis–Menten plots representing in vitro phospholipase activity measured from titrations of several ubiquitin species. Nanomoles PED6
cleaved were calculated from steady-state rates of RFU accumulation. Dashed lines represent non-linear regression fit of data. The inset
graph for each ubiquitin species is the double reciprocal plot with linear regression analysis designated as the dashed line.
B. The bar graph shows the affinity of ExoU for different ubiquitin isoforms plotted in terms of 1/K
and chain length (the mean SEM,
*P < 0.01).
D. M. Anderson
et al
© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 82, 1454–1467
activation efficiency of rExoU increases by approximately
10-fold when K48-linked diubiquitin is used as the activa-
tor, and 250- to 500-fold when K48-linked tetraubiquitin,
linear tetraubiquitin or K63-linked octaubiquitin are used
(Fig. 3B and Table 1).
Importantly, ubiquitin-like proteins including ISG15,
FAT10, SUMO-1 and NEDD8 do not activate rExoU at the
highest concentrations tested (10 mM, Fig. S2C and data
not shown). These data suggest that ubiquitin specifically
activates ExoU in vitro. ExoU activation, however, is not
restricted to a specific ubiquitin chain linkage but occurs
with multiple forms. Better activation is observed as the
number of ubiquitin moieties increases up to n = 4
(Fig. 3B).
Recombinant ExoU binds several immobilized
ubiquitin species
To determine if ExoU is able to bind ubiquitin in vitro,a
solid phase binding assay previously established for
ExoU binding to bSOD1 was utilized (Schmalzer et al.,
2010). Recombinant histidine-tagged ExoU was able to
bind both monoubiquitin (Fig. 4A) and K48-linked diubiq-
uitin (data not shown) with similar affinities (K
~ 1.4 nM).
The affinity of rExoU for K48-linked polyubiquitin
~ 0.4 nM, Fig. 4B) is approximately 3.5-fold greater
than monoubiquitin or diubiquitin (Fig. 4C and Table 2).
These data suggest that the association of ExoU with
ubiquitin increases as the number of ubiquitin moieties
increases. Double reciprocal plots (insets for each ubiq-
uitin species) demonstrate that the concentration range
for rExoU is sufficient and the linear regression analysis
suggests that there is good correlation between recombi-
nant protein added and binding (Fig. 4A and B). Histidine-
tagged rPcrV, a protein present at the tip of the type III
needle apparatus, does not bind ubiquitin under these
conditions. These experiments provide evidence that
ExoU associates with ubiquitin and that this association is
enhanced with polyubiquitin species (Fig. 4C).
Monoubiquitin is sufficient for ExoU activity in a
genetically engineered E. coli system
A dual expression bacterial system was constructed in
which both ExoU and ubiquitin were under the control of
tightly repressible promoters (pBAD and T7 polymerase,
Table 1. Activation of ExoU by different isoforms of ubiquitin.
Activator K
Monoubiquitin 17.18 7.58 0.13 0.01 4.6 0.890
K48-linked Diubiquitin 1.19 0.33 0.08 0.01 40 0.988
K48-linked Tetraubiquitin 0.06 0.02 0.10 0.01 1.1 ¥ 10
Linear Tetraubiquitin 0.08 0.02 0.21 0.01 1.5 ¥ 10
K63-linked Octaubiquitin 0.04 0.01 0.15 0.01 2.0 ¥ 10
a. mM, the affinity of rExoU for ubiquitin.
b. nmoles of PED6 cleaved min
c. nmoles of PED6 cleaved min
nmoles ExoU
mM ubiquitin
d. r
is the correlation coefficient listed for the Michaelis–Menten fit for each ubiquitin isoform.
Fig. 4. Recombinant ExoU binds immobilized ubiquitin.
A. The binding of rExoU to immobilized monoubiquitin was determined using a solid-phase binding assay (Schmalzer et al., 2010 and
Experimental procedures). rExoU binding was detected using monoclonal antibody and a fluorescent horse radish peroxidase conjugated
secondary reagent. The data are plotted and analysed by non-linear regression (dashed line). Each point represents three independent
experiments. The closed circles () represent histidine-tagged rExoU and the closed squares (
) represent the negative control protein,
histidine-tagged rPcrV. The inset graph is the double reciprocal plot with linear regression analysis designated as the dashed line.
B. Solid phase binding assay using immobilized K48-linked polyubiquitin.
C. Bar graph shows comparison in affinity (1/K
) of monoubiquitin versus polyubiquitin chains. Data are expressed as the mean SEM, n = 3.
*P < 0.01.
Ubiquitin activation of ExoU
© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 82, 1454–1467
respectively) in E. coli. Western blotting experiments of
bacterial cells demonstrated that ubiquitin, ExoU or a
control protein, PcrV, could be induced independently and
simultaneously, depending on the medium composition
(Fig. 5A and B). A time-course of cell survival after dual
induction (derepression) showed a significant drop in
viability after 1 h in the ExoU-ubiquitin expressing cells,
correlating with the timing of ExoU expression (Fig. 5B and
C). PcrV-ubiquitin expressing cells did not suffer any loss in
viability; in fact, cell numbers, after a 3 h induction,
increased almost sevenfold compared with a nearly 50-fold
decrease in survival of ExoU-ubiquitin induced cells
(Fig. 5C and D). At the final time point, bacterial cells were
harvested by centrifugation, lysed and PED6 substrate
added to the lysate. Only ExoU/ubiquitin-induced lysates
actively cleaved PED6 in vitro without addition of exog-
enous enzyme or activator (Fig. 5E). From these experi-
ments, we concluded that in the presence of monoubiquitin
and ExoU, the bacterial inner membrane serves as an in
vivo substrate for ExoU phospholipase activity.
Table 2. Binding constants for the association of ExoU and different
ubiquitin isoforms.
Immobilized activator K
Monoubiquitin 1.45 0.47 27 430 3 844 0.891
K48-linked Diubiquitin 1.41 0.53 34 025 5 426 0.864
K48-linked Polyubiquitin 0.38 0.08 15 885 1 055 0.959
a. nM units.
b. relative fluorescence units (RFU).
c. r
is the correlation coefficient listed for the non-linear regression
fit for each immobilized ubiquitin isoform.
Fig. 5. Coexpression of ExoU and ubiquitin in E. coli is lethal.
A. Western blot analysis of the independent or simultaneous expression of PcrV and ubiquitin within E. coli cells transformed with two
expression plasmids. The expression of monoubiquitin is induced by the addition of IPTG and the expression of PcrV is induced by the
addition of arabinose to the medium. Each lane is labelled by the number of hours post induction that the sample was harvested for analysis.
B. Western blot analysis of the independent or simultaneous expression of ExoU and monoubiquitin within E. coli cells transformed with similar
expression plasmids as described in A.
C. Cells harbouring inducible ExoU or PcrV and ubiquitin vectors were measured for cell survival after dual expression of both constructs over
a 3 h time-course. Error bars are means SEM (n = 3).
D. Relative survival of each strain at each time point over the induction period in relation to cell number at time zero induction. Error bars are
means SEM (n = 3), *P < 0.0001.
E. Lysates were made using cells harvested from the 3 h time point and one OD
unit was analysed for PED6 cleavage in vitro. Error bars
are for the mean SEM (n = 3).
D. M. Anderson
et al
© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 82, 1454–1467
Permeabilization of bacterial membranes by ExoU in the
presence of monoubiquitin
We next used a microscopic approach to characterize
ExoU phospholipase activity on bacterial membranes.
Cell morphology was first studied using a lipophilic styryl
dye, FM 4-64, for membrane staining. ExoU and monou-
biquitin coexpression resulted in changes in membrane
staining patterns from predominantly rod shapes to punc-
tate, irregular spheres, which accumulated with time
(Fig. 6A). Differential interference contrast (DIC) micro-
scopic images show what appears to be the formation of
coccus-shaped cells and debris after 1 h induction with
the eventual breakdown of any discernable cell structure
at 90 min. PcrV-ubiquitin induced cells, in contrast,
retained a defined rod shape throughout the time-course
(Fig. 6A).
Live cell microscopy was subsequently performed on
immobilized E. coli cells using three fluorescent reagents:
FM 4-64 to stain the membranes, Hoechst stain and
SYTOX green to mark DNA. Intact cells exclude SYTOX
green, but when the membrane barrier is compromised,
the dye flows into the cell, binds to nucleic acids and
green fluorescent signals are amplified. The inclusion of
SYTOX green in the medium confirmed the permeabiliza-
tion of the bacterial membrane as an intense green fluo-
rescence can be seen originating near the septum and
flowing into induced cells (Fig. 6B). A green haze appears
around some cells suggesting that nucleic acids are being
released (Fig. 6B).
Phospholipase-mediated membrane destruction is rapid
Further characterization of ExoU activity aimed to
examine the kinetics of ExoU-mediated destruction of
bacterial membranes. The membrane and DNA staining
system described for Fig. 6 was used to observe SYTOX
Fig. 6. Morphological changes in E. coli cells after induction of ExoU and monoubiquitin.
A. Bacterial cells were induced for expression of ExoU or PcrV with ubiquitin for the indicated times. Cells were stained with a lipophilic dye
FM4-64, washed, and detected by using a filter for FM4-64 or differential interference contrast (DIC). Arrows indicate morphology changes.
B. The coexpression cells were induced for 90 min and stained with nucleic acid stains SYTOX green (membrane impermeable) and
permeable Hoechst 33342 (blue) in the presence of FM4-64 (red). Cells were incubated on poly-lysine coated glass coverslips and washed to
remove unbound cells before image acquisition. Scale bars in overlay images indicate 2 mm.
Ubiquitin activation of ExoU
© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 82, 1454–1467
green infiltration (after a 1 h induction period) into ExoU-
permeabilized cells over a time-course of 30 min. In
agreement with the timing of ExoU expression in previous
experiments, significant SYTOX fluorescence signal was
detectable after 1 h of induction. Once initial punctate
green signals were visible at a pole or septum, approxi-
mately 15 min was required to progress to an intense
green fluorescence throughout the bacterium and then to
loss of nucleic acid and membrane signals in ExoU-
induced cells expressing ubiquitin (Fig. 7). The loss of
fluorescent signals may be related to a release of turgor
pressure and the reduction of intact bacteria to cellular
debris as shown by DIC at 90 min (Fig. 6A). Motion visu-
alization of the occurrence displays a sweeping green
signal moving through doubly induced cells (ExoU and
ubiquitin) as the reporter dye appears to enter through a
predominant site of the membrane (Movie S1). Con-
versely, PcrV and ubiquitin expressing strains retained
membrane staining and excluded SYTOX green over the
entire time-course (Fig. 7 and Movie S2).
ExoU is the most potent type III-secreted toxin synthe-
sized by P. aeruginosa (Lee et al., 2005), but its mecha-
nism of action remained unclear due to limited homology
with other proteins and its potent cytotoxicity in mamma-
lian cells (Sato and Frank, 2004). The use of yeast as a
model system revealed a phenotype of vacuolar fragmen-
tation suggesting membrane destruction or remodelling
(Sato et al., 2003). These data, combined with the inhibi-
tion of cytotoxicity in both mammalian and yeast cells by
phospholipase A
inhibitors, allowed alignment with phos-
pholipases and the identification of a catalytic dyad, S142
and D344 with similarity to the patatin protein family (Phil-
lips et al., 2003; Sato et al., 2003). In vitro phospholipase
activity was detectable only in the presence of other cel-
lular materials indicating the requirement for a eukaryotic
activator (Sato et al., 2003). Affinity methods for identify-
ing this activator generally failed as ExoU appeared to
interact with multiple proteins and the inclusion of deter-
gents or other agents that decrease non-specific protein–
protein interactions inhibited enzyme activity (Sato et al.,
2005; 2006). It was unclear whether this inhibition was
due to the inability of ExoU to associate with phospholipid
substrates or activator or both. Proteomic approaches
and the use of enzymatic activity as a screen resulted in
the discovery of eukaryotic SOD1 as an activator (Sato
et al., 2006). Caveats pertaining to SOD1, however, were
that it had poor specific activity as a cofactor, saturable
kinetics for ExoU could not be obtained in vitro and only
particular commercial preparations of the protein dis-
played activation capabilities (Sato et al., 2006; Benson
et al., 2010). The goal of this study was to determine the
specific properties of yeast and bovine SOD1 that medi-
ated the activation of ExoU. Our results indicate that ExoU
is a unique toxin in that it specifically associates with
ubiquitin and/or ubiquitylated proteins to activate the
enzyme. The mechanism of activation coupled with type
III delivery ensures that eukaryotic cells are specifically
and potently targeted and that the bacterium is protected
from its own enzyme.
In addition to using ubiquitin as an activator, ExoU is
itself ubiquitylated (Stirling et al., 2006). Ubiquitylation of
proteins delivered by type III and type IV secretion
systems has been shown to play critical roles in effector
stability and trafficking (Kubori and Galan, 2003; Schnupf
et al., 2006; Angot et al., 2007; Patel et al., 2009). For
ExoU, two monoubiquitin molecules are added to K178
predominantly via a K63 linkage (Stirling et al., 2006).
Modification appears to have no significant impact on the
half-life of the toxin. In terms of intracellular localization,
ubiquitylated, catalytically inactive ExoU (ExoU-S142A)
as well as ExoUS142A that cannot be ubiquitylated at
K178 (K178R) traffic to the plasma membrane suggesting
that ubiquitin modification is not involved with plasma
membrane localization (Stirling et al., 2006). The obser-
vation of cytotoxicity in the prokaryotic dual expression
system also suggests that eukaryotic proteins, other than
monoubiquitin, are not required for ExoU to traffic to mem-
brane substrates, or compromise membrane integrity.
The discovery of ubiquitin as an activator and the fact
that ExoU is modified by ubiquitin in cells suggests the
hypothesis that ubiquitylated ExoU may self-activate. In
this model, the injection of ExoU would lead to ubiquitlya-
tion at K178, followed by a conformational change of the
molecule. This conformational change might be mediated
by the intramolecular recognition of attached diubiquitin
by another domain within ExoU. Structure–function analy-
ses of ExoU have implicated the importance of C-terminal
residues for phospholipase activity (Finck-Barbançon and
Frank, 2001; Sato et al., 2003; 2005; 2006; Benson et al.,
2010; 2011; Schmalzer et al., 2010). It is also clear from
EPR analyses that ExoU N- and C-terminal residues
change conformation in the presence of ubiquitin
(bSOD1, Benson et al., 2011; data not shown). To account
for these observations, we considered whether the
C-terminus of ExoU might encode an ubiquitin-binding
domain. Using a variety of bioinformatic approaches, no
known ubiquitin binding motifs or domains were identified
(data not shown) in the C-terminus or other regions of
ExoU. Importantly, Stirling et al. showed that ExoU K178R
retained full toxicity, implying that ubiquitylation of ExoU is
not required for phospholipase activity. Finally, rExoU is
produced in bacteria and the in vitro enzyme activity
assay used to measure phospholipase activity lacks ATP
and other enzymes required for ubiquitylation. Whether
intracellular ubiquitylation of ExoU serves to accelerate
D. M. Anderson
et al
© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 82, 1454–1467
Fig. 7. Time-lapse imaging of the
coexpression E. coli strains during
induction. Bacterial cells were induced
for the expression of ExoU or PcrV
with monoubiquitin and stained with
SYTOX green, Hoechst 33342 (blue)
and FM4-64 (red) in the presence of
inducers in a glass-bottom dish. After
1 h post induction, cells were analysed
at 30°C by time-lapse microscopy.
Images were acquired with 10 steps of
a 3 min interval as indicated.
Ubiquitin activation of ExoU
© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 82, 1454–1467
activation is unclear and will require direct testing of
ubiquitin-modified ExoU derivatives.
The activation of ExoU is highly specific to ubiquitin as
ubiquitin-like proteins SUMO-1, ISG15, FAT10 and
NEDD8 (10 mM) do not activate ExoU (data not shown
and Fig. S2C). Although the specificity of activation
relates to ubiquitin, different chain lengths, types of link-
ages or conjugation to other proteins all function to acti-
vate ExoU. Longer chains of ubiquitin, however, have a
greater ability to activate phospholipase activity in vitro.
We postulate that the interaction of ExoU with ubiquitin
may involve multiple sites accounting for the apparent
high affinities measured in the solid phase binding assays
and the absence of an identifiable motif. Polyubiquitin
may act as the best scaffold on which ExoU folds to
produce an active enzyme. Alternatively, cofactor interac-
tion may serve a bifunctional role in this toxin’s activation,
facilitating both a global conformational change (Benson
et al., 2011) as well as contributing to catalysis. Our
kinetic data suggest that a single ubiquitin molecule may
not be able to efficiently accomplish both tasks, as high
concentrations of monoubiquitin are required to reach
saturable kinetics relative to chain-linked counterparts
(Fig. 3 and Table S2). These data indicate that the ExoU–
ubiquitin interaction may define a novel type of binding or
unique motifs.
In summary, we have demonstrated that the
P. aeruginosa phospholipase toxin ExoU is activated by
several ubiquitin isoforms, as well as by ubiquitylated
proteins. This is, to our knowledge, the first report of
ubiquitin serving as an activator for a bacterial toxin. The
exact role of ubiquitin in the activation process is unknown
but is postulated to facilitate a conformational change in
ExoU to allow catalysis. Polyubiquitin molecules associ-
ate with and activate ExoU with the greatest efficiency in
vitro suggesting that either the size of the cofactor or
multiple interaction sites within ExoU are important for
phospholipase activity. The association of ExoU with ubiq-
uitin apparently involves novel structural contacts, as no
recognizable ubiquitin binding motifs were identified
within the ExoU sequence. The toxicity and membrane
degradation observed in an E. coli dual expression
system for ExoU and ubiquitin reinforces the importance
and absolute requirement of a eukaryotic cofactor in regu-
lating the activity of this potent type III effector.
Experimental procedures
Bacterial strains and media
Bacterial strains E. coli DH5a and BL21 (DE3) pJY2 (Enzo
Life Sciences) JN105-ExoU or PcrV pET15b-ubiquitin were
grown in Luria–Bertani (LB) medium at 30°C or 37°C supple-
mented with appropriate antibiotics (ampicillin 100 mgml
gentamicin 10 mgml
; chloramphenicol 30 mgml
). Saccha-
romyces cerevisiae Y258 YJR104C (Open Biosystems) was
maintained in standard defined-ura medium and grown as
per manufacturer’s recommendation (Thermo scientific).
U2OS cells were maintained in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal bovine serum.
Antibodies used were as follows: MAb166 mouse anti-PcrV
(Frank et al., 2002), U29F8 mouse anti-ExoU, rabbit anti-
SOD1 (RDI-Fitzgerald), mouse anti-ubiquitin (Santa Cruz,
sc-271289), mouse anti-His (GE healthcare), goat anti-rabbit
HRP (Sigma), goat anti-mouse HRP (Invitrogen) and mouse
IgG (Santa Cruz, sc-2025). SuperSignal West Pico chemilu-
minescent substrate (Thermo Scientific) was used for
Western blot detection. SUMO-1, FAT10 and all ubiquitin
except K48-linked polyubiquitin (Enzo Life Sciences, BML-
UW0670-0100) were purchased from Boston Biochem.
Bovine ubiquitin was obtained from Sigma and purified to
apparent homogeneity (> 99.9%, Baboshina and Haas,
1996). Recombinant NEDD8 and ISG15 were expressed and
purified as active molecules to apparent homogeneity (> 99.
9%) as described previously (Bohnsack and Haas, 2003 and
Narasimhan et al., 2005). Proteases used for digestion
experiments include: endoproteinase Lys-C (Promega),
glutamyl endopeptidase (Glu-C; Sigma), proteinase K
(Sigma), thrombin (Sigma) and trypsin (Gibco).
Protein purification
Recombinant ExoU was produced and purified as described
in Schmalzer et al. 2010, with the exception that cells were
lysed by passage through a French pressure cell. Recombi-
nant ySOD1 was produced by an overnight galactose induc-
tion (2% final concentration) of cells (final OD
= 2.5–3.0).
Washed cells were suspended in 20 mM Tris-HCl, pH 8.0,
100 mM NaCl, 5 mM b–mercaptoethanol, 1 mM PMSF, pro-
tease inhibitors, DNase and RNase and lysed by bead
beating. Metal affinity chromatography was used for recom-
binant protein purification.
Partial proteolysis
For sequential site-specific protease digestions, bSOD1 was
first denatured and then alkylated in the dark for 30 min at
37°C with 10 mM iodoacetamide. The solution was desalted
and the flowthrough was acetone precipitated. Pellets were
washed with 90% acetone, suspended in buffer and digested
with Endoproteinase Lys-C. The reaction was stopped with
5 mM PMSF, 1% SDS and acetone precipitated. Precipitated
proteins collected by centrifugation were suspended in the
appropriate buffer and digested with glutamyl endopeptidase
following the same procedure. The reaction was stopped as
described, acetone precipitated, and the protein pellet sus-
pended in buffer and digested with thrombin. This reaction
was stopped, acetone precipitated and suspended in 10 mM
, pH 6.3. Single digestions followed similar procedures.
For Proteinase K digestions, bSOD1 was diluted in buffer
before heating to 95°C for 20 min. Aliquots were removed
and placed in stop tubes containing 15 mM PMSF in 10 mM
D. M. Anderson
et al
© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 82, 1454–1467
buffer (controls). Proteinase K was added to the reac-
tion tube and aliquots were removed at their respective time
points and placed into the stop solution and kept on ice until
the last time point. All solutions were acetone precipitated,
washed and suspended in 10 mM KPO
buffer, pH 6.3 for use
in activity assays as described. Supplementary experimental
procedures provide additional detail.
Cell culture and immunoprecipitation
U2OS cells were seeded (7.5 ¥ 10
) into 10 cm dishes to
grow 72 h, 37°C, 5% CO
. At this time (~ 90% confluency)
they were treated with either medium alone or 10 mM MG132
(Sigma) for 8 h. The cells were scraped into PBS, harvested
by centrifugation for 5 min, 2000 g and suspended in NP-40
buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40,
1 mM EDTA) with protease inhibitors (EDTA-free tablet,
Roche) and 5 mM iodoacetamide. Lysates were sonicated
three times with 5 s pulses (Branson Sonifier 150) and placed
on ice 30 min with occasional vortexing. The supernatant was
collected after a 10 min, 16 000 g, 4°C centrifugation step
and placed in a new tube with 5 mg antibody for 12 h, 4°C.
Protein G dynabeads (50 ml, Invitrogen) were then washed in
NP-40 buffer and incubated with the lysate for 1 h at 4°C. The
beads were washed three times with 200 ml PBS and sus-
pended in 50 ml 50 mM MOPS, pH 6.3, 50 mM NaCl. Protein
bound beads were added directly to the in vitro PED6 assay
(30 mM PED6 and 135 nM rExoU) or boiled in SDS loading
buffer for Western blot analysis.
In vitro phospholipase activity assay
The phospholipase A
activity of rExoU was measured using
the established ExoU activity assay previously described
(Benson et al., 2010). Briefly, the assay conditions were opti-
mized such that the reaction included 50 mM MES (pH 6.3),
750 mM monosodium glutamate (MSG; pH 6.3), 100 mM
PED6 and 33.8 nM untagged rExoU in a final volume of 50 ml.
The addition of ubiquitin (in 10 mM KPO
, pH 6.3) was
required for activation of the phospholipase activity of ExoU
and the concentration was dependent on the species used.
Background fluorescence was measured from reactions con-
taining all components except ubiquitin. Fluorescence was
measured every 5 min for no more than 90 min at an excita-
tion wavelength of 488 nm and emission wavelength of
511 nm (495 nm cut-off filter; Spectramax M5 microplate
reader; Molecular Devices). The conversion equation used
was y = 63 654x, where y is RFU and x is nmoles PED6
cleaved (Benson et al., 2010). The data were fit to the
Michaelis–Menten equation and analysed by non-linear
regression using Prism 5.0 (GraphPad Software).
Solid phase binding assay
The interaction of rExoU with several ubiquitin species was
analysed using the previously established solid phase binding
assay with modifications (Schmalzer et al., 2010) related to
the use of ubiquitin isoforms rather than bSOD1. Approxi-
mately 250 ng of the appropriate ubiquitin species was immo-
bilized to the surface of a polyvinyl plate in 10 mM sodium
carbonate pH 9.6 and 0.5% gelatin was used in the blocking
and diluent buffers. The non-specific binding of proteins and
antibodies was evaluated and the data plotted as the
mean standard error of three independent experiments.
The data were fit using non-linear regression and binding
constants were determined using the one-site binding model
in Prism 5.0.
Dual expression studies
Bacterial cells were harvested from plates in LB broth, incu-
bated for 30–45 min at 30°C and adjusted in LB broth with
antibiotics with or without inducers to a starting OD
of 0.25.
Aliquots from cultures (30°C) were removed at the indicated
time points for plating. Colonies visible after overnight growth
(37°C) were counted. After a 3 h induction, cells were har-
vested by centrifugation and suspended to OD
of 0.1 in
assay buffer with DNase, RNase, protease inhibitors and
0.5 mg ml
lysozyme. Further lysis was carried out via soni-
cation and lysates were analysed for phospholipase activity
with PED6 as a substrate. For Western blotting, cells were
grown and induced as described, with addition of 0.5%
glucose medium to control for the addition of arabinose. The
load per lane on SDS-polyacrylamide gels was normalized to
2 ¥ 10
Live cell imaging of coexpression strains
Bacterial cells (initial OD
of 0.4) were grown in the inducing
medium (LB containing 0.1 mM IPTG, 0.5% arabinose, and
appropriate antibiotics) at 30°C for indicated periods. Cells
were collected from 800 ml of culture by centrifugation at
4300 g for 2 min and suspended in 100 ml staining solution for
5–10 min. The staining solution consisted of 1.5 mgml
FM4-64 lipophilic styryl dye in Hank’s balanced salt solution
(HBSS) containing calcium and magnesium in the presence
or absence of 75 nM SYTOX green nucleic acid stain (mem-
brane impermeable) and 20 mgml
Hoechst 33342 nucleic
acid stain (membrane permeable) as indicated (all reagents
from Invitrogen). Stained cells were washed with HBSS twice
before mounting on a glass slide with a coverslip or on a
poly-lysine-coated glass at the bottom of a culture dish for
microscopy. Cells were analysed with a Nikon Eclipse Ti-U
inverted microscope with Chroma Sedat filters using a
100 ¥ oil immersion objective lens (Plan Apo VC with NA
1.40, Nikon). NIS-Elements software (Nikon) was used for
image acquisition.
For time-lapse imaging, cells were stained for 10 min in
100 ml of the staining solution in the presence of inducers
0.1 mM IPTG and 0.5% arabinose on a poly-lysine-coated
glass bottom dish. Unbound cells were removed by washing
with HBSS twice. At the 1 h induction time point, bound cells
were covered with 200 ml staining solution containing induc-
ers for time-lapse microscopy at 30°C. Images were acquired
with 10 steps of a 3 min interval for a total of 30 min.
Statistical analysis
Two-tailed P-values were generated from unpaired t-tests
using Prism 5.0 (GraphPad Software).
Ubiquitin activation of ExoU
© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 82, 1454–1467
This work was supported by the National Institutes of Health
(AI49577 to D.W.F.), the Center for Infectious Disease
Research and the Advancing a Healthier Wisconsin Founda-
tion at the Medical College of Wisconsin.
Angot, A., Vergunst, A., Genin, S., and Peeters, N. (2007)
Exploitation of eukaryotic ubiquitin signaling pathways by
effectors translocated by bacterial type III and type IV
secretion systems. PLoS Pathog 3: e3.
Baboshina, O.V., and Haas, A.L. (1996) Novel multiubiquitin
chain linkages catalyzed by the conjugating enzymes
E2EPF and RAD6 are recognized by 26 S proteasome
subunit 5. J Biol Chem 271: 2823–2831.
Benson, M.A., Schmalzer, K.M., and Frank, D.W. (2010) A
sensitive fluorescence-based assay for the detection of
ExoU-mediated PLA2 activity. Clin Chim Acta 411: 190–
Benson, M.A., Komas, S.M., Schmalzer, K.M., Casey, M.S.,
Frank, D.W., and Feix, J.B. (2011) Induced conformational
changes in the activation of the Pseudomonas aeruginosa
type III toxin, ExoU. Biophys J 100: 1335–1343.
Bohnsack, R.N., and Haas, A.L. (2003) Conservation in the
mechanism of Nedd8 activation by the human AppBp1-
Uba3 heterodimer. J Biol Chem 278: 26823–26830.
Catic, A., Misaghi, S., Korbel, G.A., and Ploegh, H.L. (2007)
ElaD, a deubiquitinating protease expressed by E. coli.
PLoS ONE 2: e381.
Collins, C.A., and Brown, E.J. (2010) Cytosol as battle-
ground: ubiquitin as a weapon for both host and pathogen.
Trends Cell Biol 20: 205–213.
Dupont, N., Temime-Smaali, N., and Lafont, F. (2010) How
ubiquitination and autophagy participate in the regulation of
the cell response to bacterial infection. Biol Cell 102: 621–
Finck-Barbançon, V., and Frank, D.W. (2001) Multiple
domains are required for the toxic activity of Pseudomonas
aeruginosa ExoU. J Bacteriol 183: 4330–4344.
Frank, D.W., Vallis, A., Wiener-Kronish, J.P., Roy-Burman, A.,
Spack, E.G., Mullaney, B.P., et al. (2002) Generation and
characterization of a protective monoclonal antibody to
Pseudomonas aeruginosa PcrV. J Infect Dis 186: 64–73.
Frithz-Lindsten, E., Du, Y., Rosqvist, R., and Forsberg, A.
(1997) Intracellular targeting of exoenzyme S of
Pseudomonas aeruginosa via type III-dependent translo-
cation induces phagocytosis resistance, cytotoxicity and
disruption of actin microfilaments. Mol Microbiol 25: 1125–
Fu, H., Coburn, J., and Collier, R.J. (1993) The eukaryotic
host factor that activates exoenzyme S of Pseudomonas
aeruginosa is a member of the 14-3-3 protein family. Proc
Natl Acad Sci USA 90: 2320–2324.
Ganesan, A.K., Vincent, T.S., Olson, J.C., and Barbieri, J.T.
(1999) Pseudomonas aeruginosa exoenzyme S disrupts
Ras-mediated signal transduction by inhibiting guanine
nucleotide exchange factor-catalyzed nucleotide
exchange. J Biol Chem 274: 21823–21829.
Giamarellou, H. (2000) Therapeutic guidelines for Pseudomo-
nas aeruginosa infections. Int J Antimicrob Agents 16: 103–
Goehring, U.M., Schmidt, G., Pederson, K.J., Aktories, K.,
and Barbieri, J.T. (1999) The N-terminal domain of
Pseudomonas aeruginosa exoenzyme S is a GTPase-
activating protein for Rho GTPases. J Biol Chem 274:
Haas, A.L., and Bright, P.M. (1987) The dynamics of ubiquitin
pools within cultured human lung fibroblasts. J Biol Chem
262: 345–351.
Haraga, A., and Miller, S.I. (2006) A Salmonella type III secre-
tion effector interacts with the mammalian serine/threonine
protein kinase PKN1. Cell Microbiol 5: 837–846.
Hauser, A.R., Jain, M., Bar-Meir, M., and McColley, S.A.
(2011) Clinical significance of microbial infection and
adaptation in cystic fibrosis. Clin Microbiol Rev 24: 29–
Janjusevic, R., Abramovitch, R.B., Martin, G.B., and Steb-
bins, C.E. (2006) A bacterial inhibitor of host programmed
cell death defenses is an E3 ubiquitin ligase. Science 311:
Jubelin, G., Taieb, F., Duda, D.M., Hsu, Y., Samba-Louaka,
A., Nobe, R., et al. (2010) Pathogens bacteria target
NEDD8-conujgated cullins to hijack host-cell signaling
pathways. PLoS Pathog 6: e1001128.
Kim, D.W., Lenzen, G., Page, A.L., Legrain, P., Sansonetti,
P.J., and Parsot, C. (2005) The Shigella flexneri effector
OspG interferes with innate immune responses by target-
ing ubiquitin-conjugating enzymes. Proc Natl Acad Sci
USA 102: 14046–14051.
Koth, C.M., Orlicky, S.M., Larson, S.M., and Edwards, A.M.
(2003) Use of limited proteolysis to identify protein domains
suitable for structural analysis. Meth Enzymol 368: 77–
Kubori, T., and Galan, J.E. (2003) Temporal regulation of
Salmonella virulence effector function by proteosome-
dependent protein degradation. Cell 115: 333–342.
Kubori, T., Hyakutake, A., and Nagai, H. (2008) Legionella
translocates an E3 ubiquitin ligase that has multiple
U-boxes with distinct functions. Mol Microbiol 67: 1307–
Le Negrate, G., Faustin, B., Welsh, K., Loeffler, M., Krajew-
ska, M., Hasegawa, P., et al. (2008) Salmonella secreted
factor L deubiquitinase of Salmonella typhimurium inhibits
NF-kappaB, suppresses IkappaBalpha ubiquitinated and
modulates innate immune responses. J Immunol 180:
Lee, V.T., Smith, R.S., Tummler, B., and Lory, S. (2005)
Activities of Pseudomonas aeruginosa effectors secreted
by the type III secretion system in vitro and during infection.
Infect Immun 73: 1695–1705.
Misaghi, S., Balsara, Z.R., Catic, A., Spooner, E., Ploegh,
H.L., and Starnbach, M.N. (2006) Chlamydia trachomatis-
derived deubiquitinating enzymes in mammalian cells
during infection. Mol Microbiol 61: 142–150.
Narasimhan, J., Wang, M., Fu, Z., Klein, J.M., Haas, A.L., and
Kim, J.J. (2005) Crystal structure of the interferon-induced
ubiquitin-like protein ISG15. J Biol Chem 280: 20365–
Nomura, K., DebRoy, S., Lee, Y.H., Pumplin, N., Jones, J.,
and He, S.Y. (2006) A bacterial virulence protein sup-
D. M. Anderson
et al
© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 82, 1454–1467
presses host innate immunity to cause plant disease.
Science 31: 220–223.
Orth, K., Xu, Z., Mudgett, M.B., Bao, Z.Q., Palmer, L.E.,
Bliska, J.B., et al. (2000) Disruption of signaling by Yersinia
effector YopJ, a ubiquitin-like protein protease. Science
290: 1594–1597.
Patel, J.C., Hueffer, K., Lam, T.T., and Galan, J.E. (2009)
Diversification of a Salmonella virulence protein function by
ubiquitin differential localization. Cell 137: 283–294.
Phillips, R.M., Six, D.A., Dennis, E.A., and Ghosh, P. (2003)
In vivo phospholipase activity of the Pseudomonas aerugi-
nosa cytotoxin ExoU and protection of mammalian cells
with phospholipase A2 inhibitors. J Biol Chem 278: 41326–
Pier, G.B., and Ramphal, R. (2005) Pseudomonas
aeruginosa.InPrinciples and Practice of Infectious Dis-
eases, 6th edn. Mandell, G.L., Bennett, J.E., and Dolin, R.
(eds). Philadelphia: Elsevier Churchill Livingstone, pp.
Rohde, J.R., Breitkreutz, A., Chenal, A., Sansonetti, P.J., and
Parsot, C. (2007) Type III secretion effectors of the IpaH
family are E3 ubiquitin ligases. Cell Host Microbe 1: 77–83.
Rytkonen, A., Poh, J., Garmendia, J., Boyle, C., Thompson,
A., Liu, M., et al. (2007) SseL, a Salmonella deubiquitinase
required for macrophage killing and virulence. Proc Natl
Acad Sci USA 104: 3502–3507.
Sato, H., and Frank, D.W. (2004) ExoU is a potent intracel-
lular phospholipase. Mol Microbiol 53: 1279–1290.
Sato, H., Frank, D.W., Hillard, C.J., Feix, J., Pankhaniya, R.,
Moriyama, K., et al. (2003) The mechanism of action of the
Pseudomonas aeruginosa-encoded type III cytotoxin,
ExoU. EMBO J 22: 2959–2969.
Sato, H., Feix, J.B., Hillard, C.J., and Frank, D.W. (2005)
Characterization of phospholipase activity of the
Pseudomonas aeruginosa type III cytotoxin, ExoU.
J Bacteriol 187: 1192–1195.
Sato, H., Feix, J.B., and Frank, D.W. (2006) Identification of
superoxide dismutase as a cofactor for the Pseudomonas
type III toxin, ExoU. Biochemistry 45: 10368–10375.
Sayner, S.L., Frank, D., King, J., VandeWaa, J., and Stevens,
T. (2004) Paradoxical cAMPinduced endothelial hyperper-
meability. Circ Res 95: 196–203.
Schmalzer, K.M., Benson, M.A., and Frank, D.W. (2010) Acti-
vation of ExoU phospholipase activity requires specific
C-terminal regions. J Bacteriol 192: 1801–1812.
Schnupf, P., Portnoy, D.A., and Decatur, A.L. (2006) Phos-
phorylation, ubiquitination and degradation of listeriolysin
O in mammalian cells:role of the PEST-like sequence. Cell
Microbiol 8: 353–364.
Shames, S.R., Auweter, S.D., and Finlay, B.B. (2009)
Co-evolution and exploitation of host signaling pathways
by bacterial pathogens. Int J Biochem Cell Biol 41: 380–
Stirling, F.R., Cuzick, A., Kelly, S.M., Oxley, D., and Evans,
T.J. (2006) Eukaryotic localization, activation and ubiquiti-
nylation of a bacterial type III secreted toxin. Cell Microbiol
8: 1294–1309.
Sun, J., and Barbieri, J.T. (2003) Pseudomonas aeruginosa
ExoT ADP-ribosylates CT10 regulator of kinase (Crk)
proteins. J Biol Chem 278: 32794–32800.
Sweet, C.R., Conlon, J., Golenbock, D.T., Goguen, J., and
Sliverman, N. (2007) YopJ targets TRAF proteins to inhibit
TLR-mediated NF-kappaB, MAPK and IRF3 signal
transduction. Cell Microbiol 9: 2700–2715.
Yahr, T.L., Mende-Mueller, L.M., Friese, M.B., and Frank,
D.W. (1997) Identification of type III secreted products of
the Pseudomonas aeruginosa exoenzyme S regulon.
J Bacteriol 179: 7165–7168.
Yahr, T.L., Vallis, A.J., Hancock, M.K., Barbieri, J.T., and
Frank, D.W. (1998) ExoY, an adenylate cyclase secreted
by the Pseudomonas aeruginosa type III system. Proc Natl
Acad Sci USA 95: 13899–13904.
Ye, Z., Petrof, E.O., Boone, D., Claud, E.C., and Sun, J.
(2007) Salmonella effector AvrA regulation of colonic epi-
thelial cell inflammation by deubiquitination. Am J Pathol
171: 882–892.
Zhang, Y., Higashide, W.M., McCormick, B.A., Chen, J., and
Zhou, D. (2006) The inflammation-associated Salmonella
SopA HECT-like E3 ubiquitin ligase. Mol Microbiol 62: 786–
Zhou, H., Monack, D.M., Kayagaki, N., Wertz, I., Yin, J., Wolf,
B., and Dixit, V.M. (2005) Yersinia virulence factor YopJ
acts as a deubiquitinase to ihibiti NF-kappa B activation.
J Exp Med 202: 1327–1332.
Supporting information
Additional supporting information may be found in the online
version of this article.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials supplied
by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the article.
Ubiquitin activation of ExoU
© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 82, 1454–1467
    • "This may be an important reason why the majority of T3Es show no apparent phenotype when expressed in yeast. Against this hypothesis are examples like ExoU, which requires binding to ubiquitin or ubiquitin-modified proteins and is functional in both animal and yeast cells, which still contain the required ubiquitylated proteins [111,112]. Second, the specific process affected by the T3E may not be sufficiently conserved across kingdoms, for example, because budding yeast lacks key proteins in a pathway, or their structure and/or function is not sufficiently conserved to be targeted by T3Es. For instance, regulation of growth and cell cycle progression in yeast depends on cell-size checkpoints, whereas mammalian cells respond to extracellular growth factors [113]. "
    [Show abstract] [Hide abstract] ABSTRACT: Type III effectors (T3E) are key virulence proteins that are injected by bacterial pathogens inside the cells of their host to subvert cellular processes and contribute to disease. The budding yeast Saccharomyces cerevisiae represents an important heterologous system for the functional characterisation of T3E proteins in a eukaryotic environment. Importantly, yeast contains eukaryotic processes with low redundancy and are devoid of immunity mechanisms that counteract T3Es and mask their function. Expression in yeast of effectors from both plant and animal pathogens that perturb conserved cellular processes often resulted in robust phenotypes that were exploited to elucidate effector functions, biochemical properties, and host targets. The genetic tractability of yeast and its amenability for high-throughput functional studies contributed to the success of this system that, in recent years, has been used to study over 100 effectors. Here, we provide a critical view on this body of work and describe advantages and limitations inherent to the use of yeast in T3E research. "Favourite" targets of T3Es in yeast are cytoskeleton components and small GTPases of the Rho family. We describe how mitogen-activated protein kinase (MAPK) signalling, vesicle trafficking, membrane structures, and programmed cell death are also often altered by T3Es in yeast and how this reflects their function in the natural host. We describe how effector structure-function studies and analysis of candidate targeted processes or pathways can be carried out in yeast. We critically analyse technologies that have been used in yeast to assign biochemical functions to T3Es, including transcriptomics and proteomics, as well as suppressor, gain-of-function, or synthetic lethality screens. We also describe how yeast can be used to select for molecules that block T3E function in search of new antibacterial drugs with medical applications. Finally, we provide our opinion on the limitations of S. cerevisiae as a model system and its most promising future applications.
    Full-text · Article · Feb 2016
    • "ExoU is held inactive inside the bacterium and is unable to exert toxicity from outside the host cells. To cause cytotoxicity, ExoU requires to be translocated into the cytoplasm of the host cell, where it is activated by the contact with three cofactors: the eukaryotic cytoplasmic superoxide dismutase [88], ubiquitin or ubiquitin-modified proteins [89] [90], and PI(4,5)P 2 [91]. Distinct residues of ExoU are critical for its activation by ubiquitin and by PI(4,5),P 2 , indicating that these factors activate ExoU by discrete mechanisms [91]. "
    [Show abstract] [Hide abstract] ABSTRACT: Bacterial sphingomyelinases and phospholipases constitute a heterogeneous group of surface associated or secreted esterases, produced by pathogenic bacteria. Those enzymes might favor in different ways the colonization of the infected tissue, the establishment and progression of the infection or the evasion of the immune response by both intracellular and extracellular pathogens. This chapter presents an overview of the main physiopathological activities of the membrane-damaging and cytotoxic bacterial sphingomyelinases and phospholipases, providing examples of their roles as virulence factors in several human and animal diseases.
    Chapter · Jun 2015 · Critical Care
    • "Site-directed spin-labeling electron paramagnetic resonance spectroscopy revealed that the addition of SOD1 induced conformational changes in ExoU [65] . PLA 2 activity of ExoU was demonstrated by using ubiquitinated yeast SOD1 and other ubiquitinated mammalian pro- teins [66]. Therefore, it seems that ubiquitinated SOD1 works as a ubiquitin donor and that ubiquitination of the ExoU C-terminal activates the PLA 2 activity of ExoU. "
    [Show abstract] [Hide abstract] ABSTRACT: Pseudomonas aeruginosa uses a complex type III secretion system to inject the toxins ExoS, ExoT, ExoU, and ExoY into the cytosol of target eukaryotic cells. This system is regulated by the exoenzyme S regulon and includes the transcriptional activator ExsA. Of the four toxins, ExoU is characterized as the major virulence factor responsible for alveolar epithelial injury in patients with P. aeruginosa pneumonia. Virulent strains of P. aeruginosa possess the exoU gene, whereas non-virulent strains lack this particular gene. The mechanism of virulence for the exoU + genotype relies on the presence of a pathogenic gene cluster (PAPI-2) encoding exoU and its chaperone, spcU. The ExoU toxin has a patatin-like phospholipase domain in its N-terminal, exhibits phospholipase A2 activity, and requires a eukaryotic cell factor for activation. The C-terminal of ExoU has a ubiquitinylation mechanism of activation. This probably induces a structural change in enzymatic active sites required for phospholipase A2 activity. In P. aeruginosa clinical isolates, the exoU + genotype correlates with a fluoroquinolone resistance phenotype. Additionally, poor clinical outcomes have been observed in patients with pneumonia caused by exoU + -fluoroquinolone-resistant isolates. Therefore, the potential exists to improve clinical outcomes in patients with P. aeruginosa pneumonia by identifying virulent and antimicrobial drug-resistant strains through exoU genotyping or ExoU protein phenotyping or both.
    Full-text · Article · Dec 2014
Show more