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 (email@example.com)
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
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# 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
28. Shafikhani SH, Engel J: Pseudomonas aeruginosa type III-
secreted toxin ExoT inhibits host-cell division by targeting
cytokinesis at multiple steps. Proc Natl Acad Sci U S A 2006,
This paper, along with [38??], demonstrates that in addition to the pre-
viously known ability of ExoT to inhibit cell migration, each domain of
ExoT can independently inhibit cell division. The GAP activity of ExoT
inhibits Rho, which is required at early stages of cytokinesis. The ADPRT
activity, by inhibiting Crk function, prevents the final stage of cytokinesis,
daughter cell separation. These studies provide the first evidence for the
involvement of Crk and other focal adhesion proteins in cytokinesis.
29. Sun J, Maresso AW, Kim JJ, Barbieri JT: How bacterial ADP-
ribosylating toxins recognize substrates. Nat Struct Mol Biol
30. Maresso AW, Deng Q, Pereckas MS, Wakim BT, Barbieri JT:
Pseudomonas aeruginosa ExoS ADP-ribosyltransferase
inhibits ERM phosphorylation. Cell Microbiol 2007, 9:97-105.
31. DiNovo AA, Schey KL, Vachon WS, McGuffie EM, Olson JC,
Vincent TS: ADP-ribosylation of cyclophilin A by Pseudomonas
aeruginosa exoenzyme S. Biochemistry 2006, 45:4664-4673.
32. Alaoui-El-Azher M, Jia J, Lian W, Jin S: ExoS of Pseudomonas
aeruginosa induces apoptosis through a Fas receptor/
caspase 8-independent pathway in HeLa cells. Cell Microbiol
33. Jia J, Alaoui-El-Azher M, Chow M, Chambers TC, Baker H, Jin S:
c-Jun NH2-terminal kinase-mediated signaling is essential for
Pseudomonas aeruginosa ExoS-induced apoptosis. Infect
Immun 2003, 71:3361-3370.
The authors demonstrate that T3SS proteins of P. aeruginosa disrupt
epithelial junctions in airway epithelial cells, leading to increased perme-
ability and allowing bacteria to transmigrate across the epithelial barrier.
The ADPRT domain of ExoS was at least partly responsible for these
Soong G, Parker D, Magargee M, Prince AS: The type III toxins of
Pseudomonas aeruginosa disrupt epithelial barrier function. J
Bacteriol 2008, 190:2814-2821.
35. Sun J, Barbieri JT: Pseudomonas aeruginosa ExoT ADP-
ribosylates CT10 regulator of kinase (Crk) proteins. J Biol
Chem 2003, 278:32794-32800.
36. Pielage JF, Powell KR, Kalman D, Engel JN: RNAi screen reveals
an Abl kinase-dependent host cell pathway involved in
Pseudomonas aeruginosa internalization. PLoS Pathog 2008,
37. Garrity-Ryan L, Shafikhani S, Balachandran P, Nguyen L, Oza J,
Jakobsen T, Sargent J, Fang X, Cordwell S, Matthay MA et al.: The
ADP ribosyltransferase domain of Pseudomonas aeruginosa
ExoT contributes to its biological activities. Infect Immun 2004,
Shafikhani SH, Morales C, Engel J: The Pseudomonas
aeruginosa type III secreted toxin ExoT is necessary and
sufficient to induce apoptosis in epithelial cells. Cell Microbiol
This paper, along with , reveals unexpected roles for ExoT and for Crk
in host cell biology. The authors demonstrate that ExoT is necessary and
sufficient to induce apoptosis, probably through inactivation of Crk
adaptor proteins. Together with its ability to inhibit cytokinesis and cell
migration, this study demonstrates why ExoT is a potent inhibitor of
Balachandran P, Dragone L, Garrity-Ryan L, Lemus A, Weiss A,
Engel J: The ubiquitin ligase Cbl-b limits Pseudomonas
aeruginosa exotoxin T-mediated virulence. J Clin Invest 2007,
Through in vitro and in vivo studies, the authors demonstrate that by
binding Crk, ExoT recruits the ubiquitin ligase Cbl-b. ExoT becomes
ubiquinated and undergoes proteosomal degradation. Enhanced bacter-
in an ExoT-specific manner. This study represents the first identification
of a mammalian gene product that is specifically required for in vivo
resistance to disease mediated by a T3SS effector.
40. Viboud GI, Bliska JB: Yersinia outer proteins: role in modulation
of host cell signalling responses and pathogenesis. Annu Rev
Microbiol 2005, 59:69-89.
41. Miao EA, Alpuche-Aranda CM, Dors M, Clark AE, Bader MW,
Miller SI, Aderem A: Cytoplasmic flagellin activates caspase-1
and secretion of interleukin 1beta via Ipaf. Nat Immunol 2006,
Franchi L, Stoolman J, Kanneganti TD, Verma A, Ramphal R,
Nunez G: Critical role for Ipaf in Pseudomonas aeruginosa-
induced caspase-1 activation. Eur J Immunol 2007, 37:3030-
The authors show that P. aeruginosa flagellin was essential for caspase-
1-dependent activation of IL-1beta and thatneitherExoS, ExoT, norExoY
inhibited this Ipaf-dependent pathway.
Galle M, Schotte P, Haegman M, Wullaert A, Yang HJ, Jin S,
Beyaert R: The Pseudomonas aeruginosa Type III secretion
system plays a dual role in the regulation of caspase-1
mediated IL-1beta maturation. J Cell Mol Med 2008, 12:1767-
In contrast to [42?], the authors had shown that ExoS inhibits caspase-1-
dependent activation of IL-1beta by the P. aeruginosa T3SS.
aeruginosa effectors secretedbythe TypeIII secretion system
in vitro and during infection. Infect Immun 2005, 73:1695-1705.
Zhuo H, Yang K, Lynch SV, Dotson RH, Glidden DV, Singh G,
Webb WR, Elicker BM, Garcia O, Brown R et al.: Increased
mortality of ventilated patients with endotracheal
Pseudomonas aeruginosa without clinical signs of infection.
Crit Care Med 2008, 36:2495-2503.
associated withincreased mortalityinventilatedpatientsthat donotmeet
the clinical criteria for pneumonia. These patients also had higher bacter-
ial burdens than patients colonized with strains whose T3SS was non-
functional. The study suggests that colonization with T3SS-proficient
strains of P. aeruginosa is associated with increased mortality.
Lee VT, Pukatzki S, Sato H, Kikawada E, Kazimirova AA, Huang J,
Li X, Arm JP, Frank DW, Lory S: Pseudolipasin A is a specific
inhibitor for phospholipase A2 activity of Pseudomonas
aeruginosa cytotoxin ExoU. Infect Immun 2007, 75:1089-1098.
In a high-throughput cell-based assay to screen compound libraries that
protected cells against the cytotoxic effects of ExoU, the authors identi-
fied a novel inhibitor of ExoU-specific phospholipase A2 activity.
Felise HB, Nguyen HV, Pfuetzner RA, Barry KC, Jackson SR,
Blanc MP, Bronstein PA, Kline T, Miller SI: An inhibitor of gram-
negative bacterial virulence protein secretion. Cell Host
Microbe 2008, 4:325-336.
of small molecules that inhibit T3SS.
48. Sawa T, Yahr TL, Ohara M, Kurahashi K, Gropper MA, Wiener-
Kronish JP, Frank DW: Active and passive immunization with
the Pseudomonas V antigen protects against type III
intoxication and lung injury. Nat Med 1999, 5:392-398.
49. Imamura Y, Yanagihara K, Fukuda Y, Kaneko Y, Seki M,
Izumikawa K, Miyazaki Y, Hirakata Y, Sawa T, Wiener-Kronish JP
et al.: Effect of anti-PcrV antibody in a murine chronic airway
Pseudomonas aeruginosa infection model. Eur Respir J 2007,
50. Corech R, Rao A, Laxova A, Moss J, Rock MJ, Li Z, Kosorok MR,
Splaingard ML, Farrell PM, Barbieri JT: Early immune response
to the components of the type III system of Pseudomonas
aeruginosa in children with cystic fibrosis. J Clin Microbiol
Host-microbe interactions: bacteria
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