Pseudomonas aeruginosa Exotoxin Y Is a Promiscuous Cyclase That Increases Endothelial Tau Phosphorylation and Permeability

Ochoa, Cristhiaan D.; Alexeyev, Mikhail; Pastukh, Viktor M.; Balczon, Ron; Stevens, Troy

doi:10.1074/jbc.m111.301440

Cited by 84 publications

(84 citation statements)

References 80 publications

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“…This activator will likely be different from that for PaExoY, as the same extract that stimulated PaExoY did not stimulate VvExoY and indeed seemed to inhibit it. Another distinct difference between the two proteins is that PaExoY has been shown to also be efficient in the conversion of GTP to cGMP (53). We found that under the reaction conditions employed here, VvExoY did not function as a guanylate cyclase either during coculture with CHO cells or in vitro (data not shown).…”

Section: Discussionmentioning

confidence: 58%

Vibrio vulnificus Biotype 3 Multifunctional Autoprocessing RTX Toxin Is an Adenylate Cyclase Toxin Essential for Virulence in Mice

et al. 2014

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Vibrio vulnificus is an environmental organism that causes both food-borne and wound infections with high morbidity and mortality in humans. The annual incidence and global distribution of infections associated with this pathogen are increasing with climate change. In the late 1990s, an outbreak of tilapia-associated wound infections in Israel was linked to a previously unrecognized variant of V. vulnificus designated biotype 3. The sudden emergence and clonality of the outbreak suggest that this strain may be a true newly emergent pathogen with novel virulence properties compared to those of other V. vulnificus strains. In a subcutaneous infection model to mimic wound infection, the multifunctional autoprocessing RTX (MARTX) toxin of biotype 3 strains was shown to be an essential virulence factor contributing to highly inflammatory skin wounds with severe damage affecting every tissue layer. We conducted a sequencing-based analysis of the MARTX toxin and found that biotype 3 MARTX toxin has an effector domain structure distinct from that of either biotype 1 or biotype 2. Of the two new domains identified, a domain similar to Pseudomonas aeruginosa ExoY was shown to confer adenylate cyclase activity on the MARTX toxin. This is the first demonstration that the biotype 3 MARTX toxin is essential for virulence and that the ExoY-like MARTX effector domain is a catalytically active adenylate cyclase.

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Section: Discussionmentioning

confidence: 58%

Vibrio vulnificus Biotype 3 Multifunctional Autoprocessing RTX Toxin Is an Adenylate Cyclase Toxin Essential for Virulence in Mice

et al. 2014

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“…Given the shared enzymatic properties between ExoY, CyaA, and edema factor, it was reasonable to consider that ExoY generates multiple cyclic nucleotide monophosphates. Coupling this possibility with the fact that cAMP and cGMP both influence endothelial barrier integrity, Ochoa et al (2012) hypothesized that ExoY possessed both adenylyl and guanylyl cyclase activity when introduced into pulmonary microvascular endothelial cells (PMVECs). When PMVECs were infected, ExoY intoxication generated a significantly higher (~10-fold) cGMP than cAMP signal, suggesting ExoY is principally a guanylyl cyclase (Ochoa et al 2012).…”

Section: Discovery That Exoy Is a Guanylyl Cyclasementioning

confidence: 99%

“…We have seen that ExoY functions not only as an adenylyl cyclase, but rather, as a promiscuous purine and pyrimidine nucleotidyl cyclase in vascular endothelium, generating cGMP, cAMP, cUMP and, to a lesser extent, cCMP (Morrow et al 2015). The elevation of these cyclic nucleotide monophosphates causes endothelial tau hyperphosphorylation, which leads to microtubule breakdown and endothelial hyperpermeability (Balczon et al 2013; Morrow et al 2016; Ochoa et al 2012; Prasain et al 2009; Sayner et al 2011). Hyperphosphorylation appears to trigger tau oligomerization and release, contributing to a transmissible proteinopathy that perpetuates cytotoxicity during infection (Morrow et al 2016).…”

Section: Introductionmentioning

confidence: 99%

The Pseudomonas aeruginosa Exoenzyme Y: A Promiscuous Nucleotidyl Cyclase Edema Factor and Virulence Determinant

Morrow

Frank

Balczon

et al. 2016

Handbook of Experimental Pharmacology

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Exoenzyme Y (ExoY) was identified as a component of the Pseudomonas aeruginosa type 3 secretion system secretome in 1998. It is a common contributor to the arsenal of type 3 secretion system effectors, as it is present in approximately 90% of Pseudomonas isolates. ExoY has adenylyl cyclase activity that is dependent upon its association with a host cell cofactor. However, recent evidence indicates that ExoY is not just an adenylyl cyclase; rather, it is a promiscuous cyclase capable of generating purine and pyrimidine cyclic nucleotide monophosphates. ExoY’s enzymatic activity causes a characteristic rounding of mammalian cells, due to microtubule breakdown. In endothelium, this cell rounding disrupts cell-to-cell junctions, leading to loss of barrier integrity and an increase in tissue edema. Microtubule breakdown seems to depend upon tau phosphorylation, where the elevation of cyclic nucleotide monophosphates activates protein kinases A and G and causes phosphorylation of endothelial microtubule associated protein tau. Phosphorylation is a stimulus for tau release from microtubules, leading to microtubule instability. Phosphorylated tau accumulates inside endothelium as a high molecular weight, oligomeric form, and is then released from the cell. Extracellular high molecular weight tau causes a transmissible cytotoxicity that significantly hinders cellular repair following infection. Thus, ExoY may contribute to bacterial virulence in at least two ways; first, by microtubule breakdown leading to loss of endothelial cell barrier integrity, and second, by promoting release of a high molecular weight tau cytotoxin that impairs cellular recovery following infection.

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“…The method consisted of a 40-min gradient at a flow rate of 300 nl/min using a gradient from solvent A (5% acetonitrile and 0.1% formic acid in water) and solvent B (80% acetonitrile and 0.08% formic acid in water). The system includes a 300-m-by-5-mm PepMap C 18 precolumn and a 75-m-by-250-mm C 18 PepMap. MS and MS/MS data were acquired using Xcalibur (Thermo Fischer Scientific) and processed automatically using Mascot Daemon software (Matrix Science).…”

Section: Methodsmentioning

confidence: 99%

“…The main consequence of ExoS/ExoT action at the cell level is the dismantlement of the actin cytoskeleton and the focal contacts, leading to cell retraction (16). ExoY is a potent adenylate cyclase, which has no effect on cell retraction when injected alone and even induces a slight but significant spreading when cells are infected at a low multiplicity of infection (MOI) with a mutant strain secreting ExoY as the sole type 3 toxin (16,17), while ExoY induces the disruption of the microtubules at a high MOI and longer infection times (18).…”

mentioning

confidence: 99%

Pharmacological Activation of Rap1 Antagonizes the Endothelial Barrier Disruption Induced by Exotoxins ExoS and ExoT of Pseudomonas aeruginosa

2015

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Most clinical strains of Pseudomonas aeruginosa, a leading agent of nosocomial infections, are multiresistant to antibiotherapy. Because of the paucity of new available antibiotics, the investigation of strategies aimed at limiting the action of its major virulence factors has gained much interest. The type 3 secretion system of P. aeruginosa and its effectors are known to be major determinants of toxicity and are required for bacterial dissemination in the host. Bacterial transmigration across the vascular wall is considered to be an important step in the infectious process. Using human endothelial primary cells, we demonstrate that forskolin (FSK), a drug inducing cyclic AMP (cAMP) elevation in eukaryotic cells, strikingly reduced the cell retraction provoked by two type 3 toxins, ExoS and ExoT, found in the majority of clinical strains. Conversely, cytotoxicity of a strain carrying the type 3 effector ExoU was unaffected by FSK. In addition, FSK altered the capacity of two ExoS/ExoT strains to transmigrate across cell monolayers. In agreement with these findings, other drugs and a cytokine inducing the increase of cAMP intracellular levels have also protected cells from retraction. cAMP is an activator of both protein kinase A and EPAC, a GTPase exchange factor of Rap1. Using activators or inhibitors of either pathway, we show that the beneficial effect of FSK is exerted by the activation of the EPAC/Rap1 axis, suggesting that its protective effect is mediated by reinforcing cell-cell and cell-substrate adhesion. Pseudomonas aeruginosa is an opportunistic pathogen and a leading agent of nosocomial infections. The largest cohorts of P. aeruginosa-infected patients are found in three pathological settings: ventilator-associated pneumonia, bacteremia, and cystic fibrosis. In acute infections, P. aeruginosa disseminates from the primary infection site to the blood and other organs, leading to sepsis and multiple organ failure. From a clinical point of view, vascular barrier breakdown is thus considered to be a key step in the pathophysiology of infection (1).Most P. aeruginosa clinical isolates are multidrug or even extremely drug resistant to antibiotics, which explains the high fatality rates of P. aeruginosa infections. The pathogen has been recently included in a family of so-called "ESKAPE" bacterial pathogens, a group which also includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Enterobacter species, that are able to efficiently "escape" the effects of available antibacterial drugs and for which there is an urgent need for developments of novel types of drugs (2, 3). In this context, the investigation of new strategies limiting the action of the virulence factors rather than bactericidal agents has gained much interest.P. aeruginosa is extremely well equipped in virulence determinants, which are membrane-embedded protein machineries dedicated for effector/toxin export (4). The type 3 secretion system (T3SS) and its effectors are recognized as the most i...

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Pseudomonas aeruginosa Exotoxin Y Is a Promiscuous Cyclase That Increases Endothelial Tau Phosphorylation and Permeability

Cited by 84 publications

References 80 publications

Vibrio vulnificus Biotype 3 Multifunctional Autoprocessing RTX Toxin Is an Adenylate Cyclase Toxin Essential for Virulence in Mice

Vibrio vulnificus Biotype 3 Multifunctional Autoprocessing RTX Toxin Is an Adenylate Cyclase Toxin Essential for Virulence in Mice

The Pseudomonas aeruginosa Exoenzyme Y: A Promiscuous Nucleotidyl Cyclase Edema Factor and Virulence Determinant

Pharmacological Activation of Rap1 Antagonizes the Endothelial Barrier Disruption Induced by Exotoxins ExoS and ExoT of Pseudomonas aeruginosa

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