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Infection and Immunity, January 2000, p. 403-406, Vol. 68, No. 1
0019-9567/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
ExoT of Cytotoxic Pseudomonas aeruginosa
Prevents Uptake by Corneal Epithelial Cells
Brigitte A.
Cowell,1
David Y.
Chen,1
Dara W.
Frank,2
Amy J.
Vallis,2 and
Suzanne
M. J.
Fleiszig1,*
School of Optometry, University of
California, Berkeley, California 94720,1 and
Department of Microbiology and Molecular Genetics, Medical
College of Wisconsin, Milwaukee, Wisconsin 532262
Received 29 June 1999/Returned for modification 23 September
1999/Accepted 8 October 1999
 |
ABSTRACT |
The presence of invasion-inhibitory activity that is regulated by
the transcriptional activator ExsA of cytotoxic Pseudomonas aeruginosa has previously been proposed. The results of this
study show that both ExoT and ExoS, known type III secreted effector proteins of P. aeruginosa that are regulated by ExsA,
possess this activity. Invasion was reduced 94.4% by ExoT and 96.0%
by ExoS. Invasion-inhibitory activity is not linked to ADP-ribosylation activity, at least for ExoS, since a noncatalytic mutant also inhibits
uptake by an epithelial cell line (invasion was reduced 96.0% by ExoSE381A).
 |
TEXT |
The earliest events in most
Pseudomonas aeruginosa infections occur at the epithelium.
Depending on their interaction with epithelial cells in vitro, clinical
isolates of P. aeruginosa may be classed as cytotoxic or
invasive, and these phenotypes correspond to distinct genotypes
(10). Acute epithelial cell death induced by cytotoxic
P. aeruginosa has been demonstrated to be due to the
expression of ExoU, delivered via type III secretion, that is regulated
by the transcriptional activator ExsA (7). Wild-type
cytotoxic strains do not invade epithelial cells. An exoU
mutant of P. aeruginosa
(PA103exoU::Tn5Tc) is noncytotoxic but
remains noninvasive (5, 12). A subsequent mutation in exsA results in an invasive phenotype (5). This
result suggests that there is at least one ExsA-regulated gene encoding
the inhibition of invasion in cytotoxic P. aeruginosa.
Inhibition of P. aeruginosa uptake by epithelial
cells.
YopE and YopH, secreted via type III secretion by
Yersinia spp., prevent uptake by eukaryotic cells
(15). Furthermore, YopH is essential for antiphagocytic
function (14). YopH is a protein tyrosine phosphatase
(11) that has been demonstrated to prevent uptake of other
microorganisms (26).
P. aeruginosa PA103 was used to demonstrate a similar
capacity by cytotoxic P. aeruginosa to block uptake of other
bacteria. To allow invasion levels to be quantified, a noncytotoxic
mutant, PA103exoU (Table 1),
was used for these experiments. Invasion of a rabbit corneal epithelial
(RCE) cell line was determined as previously described (9).
RCE cells were fed with modified SHEM (13), containing
bovine pituitary extract (5 µg/ml) in place of cholera toxin. For
most assays, cells were cultured on 24-well plates and used between 3 and 6 days after passage. Cells were washed with phosphate-buffered
saline (PBS) and inoculated with 200 µl of bacterial suspension (in
modified Eagle's medium with Earle's salts and
L-glutamine, buffered with 1 M HEPES-NaOH [pH 7.6],
0.35 g of NaHCO3 per liter, and 6 g of bovine
serum albumin per liter [Sigma, St. Louis, Mo.]). Infected cells were then incubated for 3 h at 37°C in 5% CO2. The
bacterial suspension was then carefully aspirated, and the wells were
washed with two sequential 0.5-ml aliquots of PBS (Sigma) to remove
nonassociated bacteria. Adherent extracellular bacteria were killed by
incubation for 1 h after the addition of 1 ml of gentamicin (200 µg/ml). The antibiotic was aspirated, and the excess was removed by
one wash of 1 ml of PBS before the cells were lysed with 0.025% Triton X-100. The number of intracellular bacteria was determined by culturing
serial dilutions of the lysate.
To demonstrate uptake inhibition by cytotoxic
P. aeruginosa,
PA103
exoU was coincubated with invasive strains of
P. aeruginosa.
The uptake of invasive strains 6294 and 6487 and an
invasive mutant
of PA103 (PA103
exoUexsA) (Table
1) was
compared to the uptake
of these strains in the presence of
PA103
exoU. An inoculum of
10
7 CFU was used for
PA103 mutants, and the inoculum for strains
6294 and 6487 was reduced
to 2 × 10
6 CFU as these strains are significantly
more invasive (
10).
As a control, the effect of
PA103
exoUexsA (which lacks invasion-inhibitory
activity) on
the invasion of 6294 was also examined. Different
strains of
internalized bacteria were distinguished in viable
counts by plating
duplicate aliquots on nonselective medium (MacConkey
agar) and
selective medium (tryptic soy agar [Difco, Detroit,
Mich.] with 100 µg of tetracycline per ml or 10 µg of streptomycin
per ml
[Sigma]).
PA103
exoU inhibited uptake of all three invasive
P. aeruginosa strains tested (
P < 0.001 for all
strains) (Fig.
1a to c).
In contrast,
PA103
exoUexsA was not able to inhibit uptake of 6294
(
P = 0.062; Mann-Whitney U test) (Fig.
1d).

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FIG. 1.
PA103exoU inhibits uptake of invasive strains
of P. aeruginosa. PA103exoU was coincubated with
invasive strains of P. aeruginosa during infection of RCE
cells, and uptake was quantified. PA103exoU ( ) inhibited
the invasion of all strains assayed when coincubated with 6294 ( )
(a), 6487 ( ) (b), and PA103exoUexsA
( ) (c).
PA103exoUexsA ( ) did not inhibit invasion when
coincubated with 6294 ( ) (d).
|
|
Levels of bacterial association with RCE cells (i.e., the sum of
adherent and invasive bacteria) were determined for both
PA103
exoU and PA103
exoUexsA as previously
described (
9). Bacteria
were incubated with RCE as described
above, and then cells were
washed vigorously three times with PBS
before the cells were lysed
with Triton X-100 and bacteria were
enumerated. There was no significant
difference between the abilities
of PA103
exoU and PA103
exoUexsA to associate with
cells (mean ± standard error, [1.40 ± 0.32]
× 10
6 CFU/well compared to [1.19 ± 0.24] × 10
6 CFU/well, respectively;
P = 0.1489 by
the Mann-Whitney U test).
This suggested that uptake inhibition by
PA103
exoU was not due
to an inability to associate with the
host cell
membrane.
A likely candidate for an invasion inhibitor might be a
Pseudomonas homolog to YopH, a type III effector that
inhibits uptake
of
Yersinia. A TBLASTN 2.0.8 search of the
published genome of
P. aeruginosa failed to find a region
homologous to YopH (
1)
or to the catalytic region that is
invariantly conserved among
protein tyrosine phosphatases such as YopH
(
11).
Of the type III secreted effector proteins that have been reported for
P. aeruginosa (
19,
20), only ExoU and ExoT are
produced by the cytotoxic strain PA103. ExoU has been shown to
be
essential for acute cytotoxicity; ExoT is not required (
7).
ExoT and ExoS possess invasion inhibitory activity.
To
investigate the role of ExoT in uptake inhibition, the ability of a
noncytotoxic mutant of PA103 with a second mutation in exoT
(PA103exoUexoT) (Table 1) to invade corneal epithelial cells
was compared to that of PA103exoU. PA103exoUexoT
invaded epithelial cells 40-fold more efficiently than
PA103exoU (Fig. 2). When
exoT was complemented back in trans (Table 1),
inhibition of invasion was restored (Fig. 2).

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FIG. 2.
Uptake of PA103exoU mutants by RCE cells.
Bacterial strains were incubated for 3 h with RCE cells before the
determination of intracellular bacteria. PA103exoU is
noninvasive, and further mutation of exsA or exoT
resulted in an invasive phenotype. Complementation with
exoT, exoS, or exoSE381A restored
invasion-inhibitory activity.
|
|
ExoT and ExoS belong to the ADP-ribosyltransferase family. Although
they have 75% amino acid identity, ExoT possesses only
0.2% enzymatic
activity compared to ExoS (
18). Interestingly,
both
exoS and a noncatalytic version of
exoS (Table
1)
complemented
the invasion-inhibitory activity that was lost by mutating
exoT in PA103
exoU (Fig.
2), suggesting that
ADP-ribosylating activity
is not required for the inhibition of uptake,
at least by
ExoS.
Actin microfilaments are involved in the ability of epithelial cells to
take up
P. aeruginosa (
8). It was recently
reported
by Vallis and coworkers (
17) that ExoT, ExoS, and
the noncatalytic
mutant ExoSE381A caused morphological changes to CHO
cells without
apparent membrane damage. In the present study, RCE cells
were
also found to display rounded morphology, but not trypan blue
staining, after infection with bacteria expressing these proteins
(data
not shown). These changes to host cell morphology suggest
that the
cytoskeleton may be affected by these proteins, and this
might explain
the cells' loss of ability to take up
bacteria.
ExoT inhibits uptake of an invasive strain of P. aeruginosa.
Unlike PA103exoU, PA103exoUexoT
was not able to block uptake of the invasive strain 6294 by RCE cells
(Table 2). Complementation with
exoT in trans restored the ability of
PA103exoUexoT to block RCE cell uptake of 6294. This result
demonstrated that ExoT in a cytotoxic strain can function to block
invasion of invasive P. aeruginosa.
The results of this study demonstrated that both ExoT and ExoS can
inhibit the uptake of a cytotoxic strain of
P. aeruginosa by
epithelial cells. Interestingly, invasive
P. aeruginosa
encodes
and secretes both of these proteins, yet these strains invade
efficiently. Furthermore, mutation of
exsA in an invasive
strain
(PAO1) does not significantly affect its ability to invade
(
10).
There are a number of possible explanations for this.
P. aeruginosa produces exoenzyme S as a heterologous
aggregate of ExoS and ExoT
(
17). Possibly, the interaction
of these two effectors results
in the loss of their invasion-inhibitory
activity. Alternatively,
invasive strains might also encode a
suppressor of ExoT and ExoS
or an effector that has a more dominant and
positive effect on
invasion than ExoT or
ExoS.
A third possibility is that the ExsA-regulated type III secretion
system is not activated by cell contact with corneal epithelial
cells
in invasive
P. aeruginosa. Vallis and coworkers
(
16) have
shown that there are differences in stimulation of
the ExsA-regulated
system between low-calcium conditions and the
presence of serum
or cell contact with CHO cells. Although ExoT and
ExoS are secreted
by invasive
P. aeruginosa under conditions
inducing growth (such
as low calcium levels), they may not necessarily
be secreted upon
contact with corneal epithelial cells in
culture.
What is the role of inhibition of invasion by ExoT in the pathogenesis
of cytotoxic
P. aeruginosa? There are considerable
similarities between the type III secretion systems of
P. aeruginosa and those of
Yersinia. In this study, we
have shown that ExoT
inhibits uptake of
P. aeruginosa,
similar to the function of YopH
of
Yersinia spp. A strain of
Yersinia pseudotuberculosis lacking
YopH remains cytotoxic
(
14), and an
exoT mutant of
P. aeruginosa also remains cytotoxic (
7). However, a
yopH deletion mutant
of
Y. pseudotuberculosis is
avirulent in a murine model of intraperitoneal
infection, while an
exoT mutant of
P. aeruginosa remains virulent
in
an acute lung infection model (
3,
7). The presence of
ExoU,
which is associated with acute cytotoxicity, may mask any
effect of
ExoT in this model. It is clear, however, that ExsA-regulated
proteins
other than ExoU are involved in virulence. An
exsA mutant
of
invasive
P. aeruginosa is avirulent or less virulent in
acute
infection models (
4,
7). Invasive
P. aeruginosa does not
encode ExoU, indicating an important role for
other effectors,
at least in this class of
P. aeruginosa
strains at some stage
of the infectious
process.
ExoT is the only type III effector protein reported to date that is
encoded by both invasive and cytotoxic
P. aeruginosa
strains.
The conservation of this gene across strains would suggest an
important role in survival; this may involve the control of
phagocytosis
by eukaryotic
cells.
 |
ACKNOWLEDGMENTS |
This work was supported by a Bausch & Lomb PostDoctoral Fellowship
to B.A.C., grants AI-31665 and AI-01289 to D.W.F. from the National
Institute of Allergy and Infectious Diseases, National Institutes of
Health, and grant RO1-EY11221 to S.M.J.F. from the National Eye
Institute, National Institutes of Health.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: School of
Optometry, 688 Minor Hall, University of California, Berkeley, CA
94720-2020. Phone: (510) 643-0990. Fax: (510) 643-5109. E-mail:
fleiszig{at}socrates.berkeley.edu.
Editor:
J. T. Barbieri
 |
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Infection and Immunity, January 2000, p. 403-406, Vol. 68, No. 1
0019-9567/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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