Characterization of Phospholipase Activity of the
            <i>Pseudomonas aeruginosa</i>
            Type III Cytotoxin, ExoU

Satō, Hiromi; Feix, Jimmy B.; Hillard, Cecilia J.; Frank, Dara W.

doi:10.1128/jb.187.3.1192-1195.2005

Cited by 55 publications

(82 citation statements)

References 21 publications

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“…1B) and substantially diminished activation of ExoU (Fig. 1C), indicating that ubiquitin was necessary for membrane fraction activation of ExoU (14). We next tested whether protease-treated membrane fraction could activate ExoU in the presence of small amounts of exogenous ubiquitin (i.e., concentrations of ubiquitin below those needed for detectable activation).…”

Section: Resultsmentioning

confidence: 99%

“…Due to the potent in vitro activation of ExoU by PI(4,5)P2 in combination with ubiquitin, we investigated the biological relevance of this effect in eukaryotic cells. The budding yeast Saccharomyces cerevisiae is a useful model for studying ExoU, as ExoU kills yeast similarly to mammalian cells (14,22). Additionally, both ubiquitin and PI (4,5)P2 are found in all eukaryotes, including yeast (21,31).…”

Section: Resultsmentioning

confidence: 99%

“…The N-terminal 101 residues of ExoU comprise the secretion and SpcUbinding domains, although SpcU binds to other portions of ExoU as well (13). Residues 102 to 471 encompass the catalytic PLA 2 domain of ExoU, which is responsible for cleavage of a wide variety of phospholipids following injection of ExoU into host cells (13,14). Like the other P. aeruginosa T3SS effectors, ExoU requires a host cell cofactor to manifest this catalytic activity (15).…”

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confidence: 99%

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Phosphatidylinositol 4,5-Bisphosphate Is a Novel Coactivator of the Pseudomonas aeruginosa Cytotoxin ExoU

Tyson

Hauser

2013

Infect Immun

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ExoU is a potent phospholipase A 2 effector protein secreted by the type III secretion system of Pseudomonas aeruginosa. By cleaving plasma membrane phospholipids, it causes rapid lysis of eukaryotic cells. However, ExoU does not exhibit activity on its own but instead requires eukaryotic cell cofactors for activation. Ubiquitin and ubiquitinated proteins have been shown to activate ExoU, but previous work suggested that other cofactors are also involved. In this study, we demonstrate that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is another important coactivator of ExoU. PI(4,5)P2 works synergistically with ubiquitin to greatly enhance the phospholipase A 2 activity of ExoU. Distinct residues of ExoU were critical for activation by PI(4,5)P2 and by ubiquitin, indicating that these factors activate ExoU by discrete mechanisms. In support of the biological relevance of PI(4,5)P2 coactivation, a yeast mutant with reduced PI(4,5)P2 levels was less susceptible to the cytotoxic activity of ExoU. Together, these findings further elaborate the molecular mechanism of ExoU.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Phosphatidylinositol 4,5-Bisphosphate Is a Novel Coactivator of the Pseudomonas aeruginosa Cytotoxin ExoU

Tyson

Hauser

2013

Infect Immun

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“…It has been shown that ExoU exhibits a patatin-like phospholipase activity (Sato et al, 2003;Sato and Frank 2004;Sato et al, 2005) in combination with host factors, e.g. superoxide dismutase (Sato et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Mutation of the phospholipase catalytic domain of the Pseudomonas aeruginosa cytotoxin ExoU abolishes colonization promoting activity and reduces corneal disease severity

Tam

Lewis

et al. 2007

Experimental Eye Research

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We have previously shown that ExoU, a type III secreted cytotoxin of Pseudomonas aeruginosa, causes acute cytotoxicity towards corneal epithelial cells in vitro, and contributes to corneal disease pathology and ocular colonization in vivo. Subsequently, we reported that ExoU represses phagocyte infiltration of infected corneas in vivo. ExoU has patatin-like phospholipase activity that is required for cytotoxic activity in vitro (mammalian cell injury and death) and for disease in a murine model of pneumonia. We hypothesized that the phospholipase activity was required for ExoU-mediated corneal disease and ocular colonization. Using the murine scarification model, corneal disease pathology was examined after inoculation with ~10 6 cfu of a P. aeruginosa effector mutant (PA103ΔexoUexoT::Tc) complemented with either exoU (pUCPexoU), phospholipase-inactive exoU (pUCPexoUD344A) or a plasmid control (pUCP18). Eyes were photographed and disease severity scored at 24 and 48 h post-infection. Viable bacteria colonizing infected eyes were quantified at 6 and 48 h. Complementation with exoU caused significantly more pathology (increased disease severity scores) and enabled bacteria to better colonize (bỹ 1000-fold) at 48 h as compared to phospholipase-inactive exoU which did not differ from plasmid control. Surprisingly, exoU did not contribute to early (6 h) colonization. In-vitro assays confirmed that the phospholipase domain of exoU was required for cytotoxicity towards human corneal epithelial cells. Taken together these data show that the phospholipase activity of the P. aeruginosa cytotoxin, ExoU, plays a role in the pathogenesis of corneal infection via mechanism(s) occurring after initial colonization of a susceptible cornea.

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“…Upon activation, the type III secretion apparatus translocates effector molecules into the cytoplasm of host cells, resulting in cell rounding and lifting and cell death by necrosis or apoptosis (Finck-Barbancon et al, 1997;Pederson et al, 1999;Kaufman et al, 2000). There are four known effector molecules, including ExoS and ExoT, two homologous toxins with both ADP-ribosyltransferase and GTPaseactivating protein activities, an acute cytotoxin, ExoU, with lipase activity, and an adenylate cyclase, ExoY (Yahr et al, 1996(Yahr et al, , 1998Finck-Barbancon et al, 1997;Hauser et al, 1998;Sato et al, 2005). It is well known that ExoS preferentially ADP-ribosylates several Ras family members (GTP-binding proteins) required for the regulation of intracellular vesicle transport, cell proliferation and differentiation (Coburn & Gill, 1991;Ganesan et al, 1998).…”

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confidence: 99%

Factors triggering type III secretion in Pseudomonas aeruginosa

et al. 2005

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The type III secretion system of Pseudomonas aeruginosa is tightly regulated by various environmental signals, such as low calcium and contact with the host cell. However, the exact signals triggering type III secretion are unknown. The present study describes the finding that secretion of P. aeruginosa type III effector molecules requires protein factors from serum and L broth, designated type III secretion factors (TSFs), in addition to the low-calcium environment. In the absence of TSF or calcium chelator EGTA, basal levels of type III effector molecules are accumulated intracellularly. Addition of TSF and EGTA together effectively triggers the secretion of pre-existing effector molecules in a short time, even before the active expression of type III genes; thus, active type III gene expression does not seem to be a prerequisite for type III secretion. A search for TSF molecules in serum and L broth resulted in the identification of albumin and casein as the functional TSF molecules. Although there is no clear sequence similarity between albumin and casein, both proteins are known to have a low-affinity, high-capacity calcium-binding property. Tests of well-studied calcium-binding proteins seemed to indicate that low-affinity calcium-binding proteins have TSF activity, although the requirement of low-affinity calcium-binding ability for the TSF activity is not clear. P. aeruginosa seems to have evolved a sensing mechanism to detect target cells for type III injection through host-derived proteins in combination with a low-calcium signal. Disruption of the bacterial ability to sense low calcium or TSF might be a valid avenue to the effective control of this bacterial pathogen. INTRODUCTIONThe remarkable ability of Pseudomonas aeruginosa to adapt to and thrive in a wide variety of environments contributes significantly to the ability of this bacterium to cause various human infections (Bodey et al., 1983;Holder, 1993;Pier, 2002). Unlike most human pathogens possessing highly restricted host ranges, P. aeruginosa is pathogenic not only to humans, but also to Caenorhabditis elegans, Drosophila and Arabidopsis thaliana (Rahme et al., 1995;D'Argenio et al., 2001;Aballay & Ausubel, 2002). This broad host range is contributed in part by the numerous virulence factors encoded by P. aeruginosa, including exotoxin A, phospholipase C, alkaline protease, elastase, alginate, pyocyanin, pili and non-pilus adhesins (Ramphal et al., 1991;Deretic et al., 1995;Lory & Strom, 1997;Vasil & Ochsner, 1999). P. aeruginosa, like many other Gram-negative animal and plant pathogens, also encodes a type III secretion machinery, where over 30 proteins assemble into a complex designed to deliver effector molecules directly into the cytoplasmic compartment of eukaryotic cells (Yahr et al., 1995;Frank, 1997). Injection of the bacterial effector molecules into the host cells results in various physiological changes, all of which seem to confer a survival advantage on the bacterial pathogen within the host environment (Hueck, 1998;Muller et al...

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Characterization of Phospholipase Activity of the Pseudomonas aeruginosa Type III Cytotoxin, ExoU

Cited by 55 publications

References 21 publications

Phosphatidylinositol 4,5-Bisphosphate Is a Novel Coactivator of the Pseudomonas aeruginosa Cytotoxin ExoU

Phosphatidylinositol 4,5-Bisphosphate Is a Novel Coactivator of the Pseudomonas aeruginosa Cytotoxin ExoU

Mutation of the phospholipase catalytic domain of the Pseudomonas aeruginosa cytotoxin ExoU abolishes colonization promoting activity and reduces corneal disease severity

Factors triggering type III secretion in Pseudomonas aeruginosa

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