Heterogeneity of pulmonary endothelial cyclic nucleotide response to<i>Pseudomonas aeruginosa</i>ExoY infection

Morrow, K. Adam; Seifert, Roland; Kaever, Volkhard; Britain, Andrea L.; Sayner, Sarah L.; Ochoa, Cristhiaan D.; Cioffi, Eugene A.; Frank, Dara W.; Rich, Thomas C.; Stevens, Troy

doi:10.1152/ajplung.00165.2015

Cited by 26 publications

(28 citation statements)

References 61 publications

(77 reference statements)

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“…2A). Endothelial cell gaps visible by light microscopy were first noted after 3 h, which is consistent with previous accounts (39,41,55). PMVECs rounded and lost cell adhesions, leaving only membranous strands as a means of cell-cell communication.…”

Section: Resultssupporting

confidence: 90%

“…Our previous studies addressed ExoY's mechanism of action in the endothelium and the pulmonary microvasculature (5,41,55). Our laboratory and others have determined that ExoY is a purine and pyrimidine cyclase that synthesizes cytosolic cAMP, cGMP, and cUMP (39,41,59,72). The cAMP signal, in particular, results in endothelial cell tau hyperphosphorylation and insolubility, which result in dissociation of tau from microtubules, leading to their breakdown (5,46,55).…”

Section: Discussionmentioning

confidence: 99%

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Pseudomonas aeruginosa exoenzymes U and Y induce a transmissible endothelial proteinopathy

Morrow

Ochoa

Balczon

et al. 2016

American Journal of Physiology-Lung Cellular and Molecular Phys

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We tested the hypothesis that Pseudomonas aeruginosa type 3 secretion system effectors exoenzymes Y and U (ExoY and ExoU) induce release of a highmolecular-weight endothelial tau, causing transmissible cell injury characteristic of an infectious proteinopathy. Both the bacterial delivery of ExoY and ExoU and the conditional expression of an activity-attenuated ExoU induced time-dependent pulmonary microvascular endothelial cell gap formation that was paralleled by the loss of intracellular tau and the concomitant appearance of high-molecular-weight extracellular tau. Transfer of the highmolecular-weight tau in filtered supernatant to naïve endothelial cells resulted in intracellular accumulation of tau clusters, which was accompanied by cell injury, interendothelial gap formation, decreased endothelial network stability in Matrigel, and increased lung permeability. Tau oligomer monoclonal antibodies captured monomeric tau from filtered supernatant but did not retrieve higher-molecular-weight endothelial tau and did not rescue the injurious effects of tau. Enrichment and transfer of high-molecularweight tau to naïve cells was sufficient to cause injury. Thus we provide the first evidence for a pathophysiological stimulus that induces release and transmissibility of high-molecular-weight endothelial tau characteristic of an endothelial proteinopathy.

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Section: Resultssupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Pseudomonas aeruginosa exoenzymes U and Y induce a transmissible endothelial proteinopathy

Morrow

Ochoa

Balczon

et al. 2016

American Journal of Physiology-Lung Cellular and Molecular Phys

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“…Although these studies further supported the notion that ExoY is a promiscuous cyclase, the cell type (s) responsible for increased cyclic nucleotide monophosphates were unclear; it was suggested that both epithelial and endothelial cells contribute to this increase in cyclic nucleotide monophosphates. We examined whether ExoY + produces both purine and pyrimidine cyclic nucleotide monophosphates in lung endothelium (Morrow et al 2015). Intoxication of pulmonary endothelium at an MOI of 20:1 with ExoY + produces cGMP first, followed by cUMP, and then cAMP; cCMP concentrations increase last, and this increase is of a relatively low magnitude.…”

Section: Exoy As a Purine And Pyrimidine Cyclasementioning

confidence: 99%

“…1b). 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).…”

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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“…However, MRPs may help to detoxify cells with pathologically high cCMP or cUMP concentrations, e.g., after infection with ExoY-positive P. aeruginosa bacteria. The exotoxin ExoY is a nucleotidylyl cyclase, which causes a dramatic increase in cUMP concentration in infected tissues Morrow et al 2015). To the best of our knowledge, at the moment nothing is known about cIMP transport by MRPs.…”

Section: Inactivation Of Cnmps By Outward Transportmentioning

confidence: 99%

Inactivation of Non-canonical Cyclic Nucleotides: Hydrolysis and Transport

Schneider

Seifert

2016

Non-Canonical Cyclic Nucleotides

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This chapter addresses cNMP hydrolysis by phosphodiesterases (PDEs) and export by multidrug resistance associated proteins (MRPs). Both mechanisms are well-established for the canonical cNMPs, cAMP, and cGMP. Increasing evidence shows that non-canonical cNMPs (specifically cCMP, cUMP) are also PDE and MRP substrates. Hydrolysis of cUMP is achieved by PDE 3A, 3B, and 9A, which possibly explains the cUMP-degrading activities previously reported for heart, adipose tissue, and brain. Regarding cCMP, the only known "conventional" (class I) PDE that hydrolyzes cCMP is PDE7A. Older reports describe cCMP-degrading PDE-like activities in mammalian tissues, bacteria, and plants, but the molecular identity of these enzymes is not clear. High K and V values, insensitivity to common inhibitors, and unusually broad substrate specificities indicate that these activities probably do not represent class I PDEs. Moreover, the older results have to be interpreted with caution, since the historical analytical methods were not as reliable as modern highly sensitive and specific techniques like HPLC-MS/MS. Besides PDEs, the transporters MRP4 and 5 are of major importance for cAMP and cGMP disposal. Additionally, both MRPs also export cUMP, while cCMP is only exported by MRP5. Much less data are available for the non-canonical cNMPs, cIMP, cXMP, and cTMP. None of these cNMPs has been examined as MRP substrate. It was shown, however, that they are hydrolyzed by several conventional class I PDEs. Finally, this chapter reveals that there are still large gaps in our knowledge about PDE and MRP activities for canonical and non-canonical cNMPs. Future research should perform a comprehensive characterization of the known PDEs and MRPs with the physiologically most important cNMP substrates.

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Heterogeneity of pulmonary endothelial cyclic nucleotide response toPseudomonas aeruginosaExoY infection

Abstract: Morrow KA, Seifert R, Kaever V, Britain AL, Sayner SL, Ochoa CD, Cioffi EA, Frank DW, Rich TC, Stevens T. Heterogeneity of pulmonary endothelial cyclic nucleotide response to Pseudomonas aeruginosa ExoY infection.

Cited by 26 publications

References 61 publications

Pseudomonas aeruginosa exoenzymes U and Y induce a transmissible endothelial proteinopathy

Pseudomonas aeruginosa exoenzymes U and Y induce a transmissible endothelial proteinopathy

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

Inactivation of Non-canonical Cyclic Nucleotides: Hydrolysis and Transport

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