methods-for-detecting-cytotoxic-amyloids-following-infection

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by <em>Pseudomonas aeruginosa</em>

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

A method has recently been established for inducing the physical detachment of kinetochores from chromosomes in human HeLa cells, and was used in the studies reported here to investigate the organization and function of dissociated HeLa kinetochores. Immunofluorescence labeling demonstrated that the detached HeLa kinetochores were relatively intact, with the number of detached kinetochores being only moderately more than the diploid number of chromosomes in HeLa cells. In addition, the detached kinetochores could be labeled with antibodies specific for the inner kinetochore plate, outer kinetochore, and subjacent centromeric heterochromatin. A functional assay demonstrated that detached kinetochores retained the capacity to activate the spindle checkpoint, leading to metaphase arrest. Analysis of kinetochore DNA indicated that it consisted primarily of DNA fragments of 130-160 kb in size, while the remainder of the chromosomes were sheared into much smaller fragments during the kinetochore detachment event. Further analysis of kinetochore DNA indicated that it was first cleaved into high molecular weight DNA (>200 kb) fragments during the initial stages of the kinetochore detachment process, and then underwent further maturation following nuclear envelope breakdown to give rise to the 130-160 kb fragment in detached kinetochores. Collectively, these data indicate that detached human kinetochores will be a useful system for investigating the organization, assembly, and function of human kinetochores.

Analysis of detached human kinetochores.

The centrosome is the major microtubule organizing center in mammalian cells. During interphase, the single centrosome is duplicated and the progeny centrosomes then serve as the spindle poles during mitosis. Little is known about the signals that drive centrosome doubling. In these studies, various inhibitors and molecular approaches were used to demonstrate a role for the Stat pathway in regulating the events of centrosome doubling. Both piceatannol and a dominant negative behaving Stat3 adenovirus were able to disrupt centrosome duplication in hydroxyurea-arrested Chinese hamster ovary cells, demonstrating that Stat3 is a key signaling molecule in the events of centrosome duplication. Investigation into the role of Stat3 signaling during centrosome production demonstrated that Stat3 does not directly regulate the transcription of the centrosome genes encoding gamma-tubulin and PCM-1. Instead, Stat3 apparently regulated gamma-tubulin levels through post-transcriptional mechanisms whereas PCM-1 levels actually increased when Stat3 was inhibited, suggesting more complex mechanisms for regulating PCM-1 production. These studies demonstrate that Stat3 plays a vital role in centrosome duplication events, although the downstream targets of Stat3 activation leading to centrosome production remain to be established. The proposed signaling pathway utilizes Stat3 as a fundamental signaling molecule that directs the production of the various centrosome proteins indirectly.

Stat3 activity is required for centrosome duplication in chinese hamster ovary cells.

Soluble adenylyl cyclase toxins, such as Pseudomonas aeruginosa exoY, generate a cAMP pool that retracts cell borders. However, the cytoskeletal basis by which this cAMP signal retracts cell borders is not known. We sought to determine whether activation of chimeric, soluble adenylyl cyclase I/II (sACI/II) reorganizes either microtubules or peripheral actin. Endothelial cells were stably transfected with either green fluorescent protein-labeled alpha-tubulin or beta-actin, and then infected with adenovirus to express sACI/II. Forskolin, which stimulates both the endogenously expressed transmembrane adenylyl cyclases and sACI/II, induced cell retraction accompanied by the reorganization of peripheral microtubules. However, cortical filamentous-actin (f-actin) did not reorganize into stress fibers, and myosin light-chain-20 phosphorylation was decreased. Isoproterenol, which activates endogenous adenylyl cyclases but does not activate sACI/II, did not induce endothelial cell gaps and did not influence microtubule or f-actin architecture. Thus, sACI/II generates a cAMP signal that reorganizes microtubules and induces cell retraction, without inducing f-actin stress fibers. These findings illustrate that endothelial cell gap formation can proceed without f-actin stress fiber formation, and provide mechanistic insight how bacterial adenylyl cyclase toxins reorganize the cytoskeleton to induce cell rounding.

Soluble adenylyl cyclase-dependent microtubule disassembly reveals a novel mechanism of endothelial cell retraction.

L&ouml;sliche Fehlfunktion Guanylatcyclase Toxine, wie Pseudomonas Aeruginosa ExoY, generieren einen cAMP Pool, der Zellrahmen zur&uuml;ckzieht. Die Zytoskelett Basis, die mit der dieses cAMP Signal Zellrahmen R&uuml;ckz&uuml;ge ist jedoch nicht bekannt. Wollten wir bestimmen ob Aktivierung von Chim&auml;ren, l&ouml;sliches Fehlfunktion Guanylatcyclase ich / II (sACI/II) reorganisiert Mikrotubuli oder periphere Aktin. Endothelzellen waren stabil transfected mit gr&uuml;n fluoreszierenden Protein-beschriftet Alpha-Tubulin oder Beta-Actin, und dann infiziert mit Adenoviren sACI/II zum Ausdruck zu bringen. Forskolin, die endogen exprimierten Transmembran-Fehlfunktion bearbeitet sowohl die sACI/II stimuliert, induzierte Zelle Retraktion von der Neuordnung der peripheren Mikrotubuli begleitet. Jedoch kortikale filament&ouml;sen-Actin (f-Actin) nicht in Stress-Fasern zu reorganisieren und Phosphorylierung der Myosin-Licht-Kette-20 wurde verringert. Isoproterenol, die endogene membran&auml;rer Adenylylzyklasen aktiviert, sondern aktiviert nicht sACI/II, nicht induzieren Endothelzellen L&uuml;cken und hatten keinen Einfluss auf Microtubule oder f-Actin-Architektur. SACI/II erzeugt so ein cAMP Signal, die Mikrotubuli reorganisiert und induziert Zelle einfahren, ohne damit noch f-Actin-Spannung-Fasern. Diese Ergebnisse zeigen, dass Endothelzellen L&uuml;cke Formation kann ohne f-Actin-Spannung-Faser-Bildung fortfahren und mechanistische wie bakterielle Toxine in erschienen Guanylatcyclase Einblicke Zytoskeletts, Zelle Rundung genau zu reorganisieren.

L&ouml;sliche Fehlfunktion Guanylatcyclase abh&auml;ngigen Microtubule Zerlegung offenbart einen neuen Mechanismus der endothelial Zelle Retraktion.

Microtubules are composed of α-tubulin and β-tubulin dimers. Microtubules yield tubulin dimers when exposed to cold, which reassemble spontaneously to form microtubule fibers at 37°C. However, mammalian neurons, glial cells, and fibroblasts have cold-stable microtubules. While studying the microtubule toxicity mechanisms of the exotoxin Y from Pseudomonas aeruginosa in pulmonary microvascular endothelial cells, we observed that some endothelial microtubules were very difficult to disassemble in the cold. As a consequence, we designed studies to test the hypothesis that microvascular endothelium has a population of cold-stable microtubules. Pulmonary microvascular endothelial cells and HeLa cells (control) were grown under regular cell culture conditions, followed by exposure to an ice-cold water bath and a microtubule extraction protocol. Polymerized microtubules were detected by immunofluorescence confocal microscopy and Western blot analyses. After cold exposure, immunofluorescence revealed that the majority of HeLa cell microtubules disassembled, whereas a smaller population of endothelial cell microtubules disassembled. Immunoblot analyses showed that microvascular endothelial cells express the microtubule cold-stabilizing protein N-STOP (neuronal stable tubule-only polypeptides), and that N-STOP binds to endothelial microtubules after cold exposure, but not if microtubules are disassembled with nocodazole before cold exposure. Hence, pulmonary endothelia have a population of cold-stable microtubules.

Cold exposure reveals two populations of microtubules in pulmonary endothelia.

Transmembrane adenylyl cyclase (AC) generates a cAMP pool within the subplasma membrane compartment that strengthens the endothelial cell barrier. This cAMP signal is steered toward effectors that promote junctional integrity and is inactivated before it accesses microtubules, where the cAMP signal causes phosphorylation of tau, leading to microtubule disassembly and barrier disruption. During infection, Pseudomonas aeruginosa uses a type III secretion system to inject a soluble AC, ExoY, into the cytosol of pulmonary microvascular endothelial cells. ExoY generates a cAMP signal that disrupts the endothelial cell barrier. We tested the hypothesis that this ExoY-dependent cAMP signal causes phosphorylation of tau, without inducing phosphorylation of membrane effectors that strengthen endothelial barrier function. To approach this hypothesis, we first discerned the membrane compartment in which endogenous transmembrane AC6 resides. AC6 was resolved in caveolin-rich lipid raft fractions with calcium channel proteins and the cell adhesion molecules N-cadherin, E-cadherin, and activated leukocyte adhesion molecule. VE-cadherin was excluded from the caveolin-rich fractions and was detected in the bulk plasma membrane fractions. The actin binding protein, filamin A, was detected in all membrane fractions. Isoproterenol activation of ACs promoted filamin phosphorylation, whereas thrombin inhibition of AC6 reduced filamin phosphorylation within the membrane fraction. In contrast, ExoY produced a cAMP signal that did not cause filamin phosphorylation yet induced tau phosphorylation. Hence, our data indicate that cAMP signals are strictly compartmentalized; whereas cAMP emanating from transmembrane ACs activates barrier-enhancing targets, such as filamin, cAMP emanating from soluble ACs activates barrier-disrupting targets, such as tau.

Filamin A is a phosphorylation target of membrane but not cytosolic adenylyl cyclase activity.

Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to cause sporadic human infections with a high fatality rate. Respiratory failure due to acute respiratory distress syndrome (ARDS) is a complication among hospitalized patients. Since progressive pulmonary endothelial damage is the hallmark of ARDS, we investigated host responses following HPAI virus infection of human pulmonary microvascular endothelial cells. Evaluation of these cells for the presence of receptors preferred by influenza virus demonstrated that avian-like (α2-3-linked) receptors were more abundant than human-like (α2-6-linked) receptors. To test the permissiveness of pulmonary endothelial cells to virus infection, we compared the replication of selected seasonal, pandemic (2009 H1N1 and 1918), and potentially pandemic (H5N1) influenza virus strains. We observed that these cells support productive replication only of HPAI H5N1 viruses, which preferentially enter through and are released from the apical surface of polarized human endothelial monolayers. Furthermore, A/Thailand/16/2004 and A/Vietnam/1203/2004 (VN/1203) H5N1 viruses, which exhibit heightened virulence in mammalian models, replicated to higher titers than less virulent H5N1 strains. VN/1203 infection caused a significant decrease in endothelial cell proliferation compared to other subtype viruses. VN/1203 virus was also found to be a potent inducer of cytokines and adhesion molecules known to regulate inflammation during acute lung injury. Deletion of the H5 hemagglutinin (HA) multibasic cleavage site did not affect virus infectivity but resulted in decreased virus replication in endothelial cells. Our results highlight remarkable tropism and infectivity of the H5N1 viruses for human pulmonary endothelial cells, resulting in the potent induction of host inflammatory responses.

Human pulmonary microvascular endothelial cells support productive replication of highly pathogenic avian influenza viruses: possible involvement in the pathogenesis of human H5N1 virus infection.

Exotoxin Y (ExoY) is a type III secretion system effector found in ~ 90% of the Pseudomonas aeruginosa isolates. Although it is known that ExoY causes inter-endothelial gaps and vascular leak, the mechanisms by which this occurs are poorly understood. Using both a bacteria-delivered and a codon-optimized conditionally expressed ExoY, we report that this toxin is a dual soluble adenylyl and guanylyl cyclase that results in intracellular cAMP and cGMP accumulation. The enzymatic activity of ExoY caused phosphorylation of endothelial Tau serine 214, accumulation of insoluble Tau, inter-endothelial cell gap formation, and increased macromolecular permeability. To discern whether the cAMP or cGMP signal was responsible for Tau phosphorylation and barrier disruption, pulmonary microvascular endothelial cells were engineered for the conditional expression of either wild-type guanylyl cyclase, which synthesizes cGMP, or a mutated guanylyl cyclase, which synthesizes cAMP. Sodium nitroprusside stimulation of the cGMP-generating cyclase resulted in transient Tau serine 214 phosphorylation and gap formation, whereas stimulation of the cAMP-generating cyclase induced a robust increase in Tau serine 214 phosphorylation, gap formation, and macromolecular permeability. These results indicate that the cAMP signal is the dominant stimulus for Tau phosphorylation. Hence, ExoY is a promiscuous cyclase and edema factor that uses cAMP and, to some extent, cGMP to induce the hyperphosphorylation and insolubility of endothelial Tau. Because hyperphosphorylated and insoluble Tau are hallmarks in neurodegenerative tauopathies such as Alzheimer disease, acute Pseudomonas infections cause a pathophysiological sequela in endothelium previously recognized only in chronic neurodegenerative diseases.

Pseudomonas aeruginosa exotoxin Y is a promiscuous cyclase that increases endothelial tau phosphorylation and permeability.

Pseudomonas aeruginosa uses a type III secretion system to introduce the adenylyl and guanylyl cyclase exotoxin Y (ExoY) into the cytoplasm of endothelial cells. ExoY induces Tau hyperphosphorylation and insolubility, microtubule breakdown, barrier disruption and edema, although the mechanism(s) responsible for microtubule breakdown remain poorly understood. Here we investigated both microtubule behavior and centrosome activity to test the hypothesis that ExoY disrupts microtubule dynamics. Fluorescence microscopy determined that infected pulmonary microvascular endothelial cells contained fewer microtubules than control cells, and further studies demonstrated that the microtubule-associated protein Tau was hyperphosphorylated following infection and dissociated from microtubules. Disassembly/reassembly studies determined that microtubule assembly was disrupted in infected cells, with no detectable effects on either microtubule disassembly or microtubule nucleation by centrosomes. This effect of ExoY on microtubules was abolished when the cAMP-dependent kinase phosphorylation site (Ser-214) on Tau was mutated to a non-phosphorylatable form. These studies identify Tau in microvascular endothelial cells as the target of ExoY in control of microtubule architecture following pulmonary infection by Pseudomonas aeruginosa and demonstrate that phosphorylation of tau following infection decreases microtubule assembly. 

Pseudomonas aeruginosa exotoxin Y-mediated tau hyperphosphorylation impairs microtubule assembly in pulmonary microvascular endothelial cells.

Exoenzyme Y (ExoY) is a Pseudomonas aeruginosa toxin that is introduced into host cells through the type 3 secretion system (T3SS). Once inside the host cell cytoplasm, ExoY generates cyclic nucleotides that cause tau phosphorylation and microtubule breakdown. Microtubule breakdown causes interendothelial cell gap formation and tissue edema. Although ExoY transiently induces interendothelial cell gap formation, it remains unclear whether ExoY prevents repair of the endothelial cell barrier. Here, we test the hypothesis that ExoY intoxication impairs recovery of the endothelial cell barrier following gap formation, decreasing migration, proliferation, and lung repair. Pulmonary microvascular endothelial cells (PMVECs) were infected with P. aeruginosa strains for 6 h, including one possessing an active ExoY (PA103 exoUexoT::Tc pUCPexoY; ExoY(+)), one with an inactive ExoY (PA103ΔexoUexoT::Tc pUCPexoY(K81M); ExoY(K81M)), and one that lacks PcrV required for a functional T3SS (ΔPcrV). ExoY(+) induced interendothelial cell gaps, whereas ExoY(K81M) and ΔPcrV did not promote gap formation. Following gap formation, bacteria were removed and endothelial cell repair was examined. PMVECs were unable to repair gaps even 3-5 days after infection. Serum-stimulated growth was greatly diminished following ExoY intoxication. Intratracheal inoculation of ExoY(+) and ExoY(K81M) caused severe pneumonia and acute lung injury. However, whereas the pulmonary endothelial cell barrier was functionally improved 1 wk following ExoY(K81M) infection, pulmonary endothelium was unable to restrict the hyperpermeability response to elevated hydrostatic pressure following ExoY(+) infection. In conclusion, ExoY is an edema factor that chronically impairs endothelial cell barrier integrity following lung injury. 

The Pseudomonas aeruginosa exoenzyme Y impairs endothelial cell proliferation and vascular repair following lung injury.

We tested the hypothesis that Pseudomonas aeruginosa type 3 secretion system effectors exoenzymes Y and U (ExoY and ExoU) induce release of a high-molecular-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 high-molecular-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-molecular-weight 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. 

Pseudomonas aeruginosa exoenzymes U and Y induce a transmissible endothelial proteinopathy.

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.

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

Patients who recover from pneumonia subsequently have elevated rates of death after hospital discharge as a result of secondary organ damage, the causes of which are unknown. We used the bacterium , a common cause of hospital-acquired pneumonia, as a model for investigating this phenomenon. We show that infection of pulmonary endothelial cells by  induces production and release of a cytotoxic amyloid molecule with prion characteristics, including resistance to various nucleases and proteases. This cytotoxin was self-propagating, was neutralized by anti-amyloid Abs, and induced death of endothelial cells and neurons. Moreover, the cytotoxin induced edema in isolated lungs. Endothelial cells and isolated lungs were protected from cytotoxin-induced death by stimulation of signal transduction pathways that are linked to prion protein. Analysis of bronchoalveolar lavage fluid collected from human patients with  pneumonia demonstrated cytotoxic activity, and lavage fluid contained amyloid molecules, including oligomeric τ and Aβ. Demonstration of long-lived cytotoxic agents after  infection may establish a molecular link to the high rates of death as a result of end-organ damage in the months after recovery from pneumonia, and modulation of signal transduction pathways that have been linked to prion protein may provide a mechanism for intervention.-Balczon, R., Morrow, K. A., Zhou, C., Edmonds, B., Alexeyev, M., Pittet, J.-F., Wagener, B. M., Moser, S. A., Leavesley, S., Zha, X., Frank, D. W., Stevens, T.  infection liberates transmissible, cytotoxic prion amyloids.

Article	Year
Analysis of detached human kinetochores. Chromosoma\| PubMed ID: 12883946	2003
Stat3 activity is required for centrosome duplication in chinese hamster ovary cells. The Journal of biological chemistry\| PubMed ID: 15294906	2004
Soluble adenylyl cyclase-dependent microtubule disassembly reveals a novel mechanism of endothelial cell retraction. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 19395666	2009
Cold exposure reveals two populations of microtubules in pulmonary endothelia. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 20971804	2011
Filamin A is a phosphorylation target of membrane but not cytosolic adenylyl cyclase activity. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 21478251	2011
Human pulmonary microvascular endothelial cells support productive replication of highly pathogenic avian influenza viruses: possible involvement in the pathogenesis of human H5N1 virus infe... Journal of virology\| PubMed ID: 22072765	2012
Pseudomonas aeruginosa exotoxin Y is a promiscuous cyclase that increases endothelial tau phosphorylation and permeability. The Journal of biological chemistry\| PubMed ID: 22637478	2012
Pseudomonas aeruginosa exotoxin Y-mediated tau hyperphosphorylation impairs microtubule assembly in pulmonary microvascular endothelial cells. PloS one\| PubMed ID: 24023939	2013
The Pseudomonas aeruginosa exoenzyme Y impairs endothelial cell proliferation and vascular repair following lung injury. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 24705722	2014
Pseudomonas aeruginosa exoenzymes U and Y induce a transmissible endothelial proteinopathy. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 26637633	2016
The Pseudomonas aeruginosa Exoenzyme Y: A Promiscuous Nucleotidyl Cyclase Edema Factor and Virulence Determinant. Handbook of experimental pharmacology\| PubMed ID: 28181005	2017
infection liberates transmissible, cytotoxic prion amyloids. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 28314768	2017

Ron Balczon

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