Paraoxonase 2 is down-regulated by the Pseudomonas aeruginosa quorumsensing signal N-(3-oxododecanoyl)-L-homoserine lactone and attenuates oxidative stress induced by pyocyanin

2010 ◽  
Vol 426 (1) ◽  
pp. 73-83 ◽  
Author(s):  
Sven Horke ◽  
Ines Witte ◽  
Sebastian Altenhöfer ◽  
Petra Wilgenbus ◽  
Marion Goldeck ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Pseudomonas Aeruginosa ◽  
Homoserine Lactone ◽  
Hydrolytic Activity ◽  
Host Cells ◽  
Ionophore A23187 ◽  
Acetoxymethyl Ester ◽  
Bacterial Gene ◽  
Bacterial Gene Expression ◽  
Ros Formation

Two virulence factors produced by Pseudomonas aeruginosa are pyocyanin and N-(3-oxododecanoyl)-L-homoserine lactone (3OC12). Pyocyanin damages host cells by generating ROS (reactive oxygen species). 3OC12 is a quorum-sensing signalling molecule which regulates bacterial gene expression and modulates host immune responses. PON2 (paraoxonase-2) is an esterase that inactivates 3OC12 and potentially attenuates Ps. aeruginosa virulence. Because increased intracellular Ca2+ initiates the degradation of PON2 mRNA and protein and 3OC12 causes increases in cytosolic Ca2+, we hypothesized that 3OC12 would also down-regulate PON2. 3OC12 and the Ca2+ ionophore A23187 caused a rapid cytosolic Ca2+ influx and down-regulated PON2 mRNA, protein and hydrolytic activity in A549 and EA.hy 926 cells. The decrease in PON2 hydrolytic activity was much more extensive and rapid than decreases in protein, suggesting a rapid post-translational mechanism which blocks PON2's hydrolytic activity. The Ca2+ chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)] diminished the ability of 3OC12 to decrease PON2, demonstrating that the effects are mediated by Ca2+. PON2 also has antioxidative properties and we show that it protects cells from pyocyanin-induced oxidative stress. Knockdown of PON2 by transfecting cells with siRNA (small interfering RNA) rendered them more sensitive to, whereas overexpression of PON2 protected cells from, pyocyanin-induced ROS formation. Additionally, 3OC12 potentiated pyocyanin-induced ROS formation, presumably by inactivating PON2. These findings support a key role for PON2 in the defence against Ps. aeruginosa virulence, but also reveal a mechanism by which the bacterium may subvert the protection afforded by PON2.

scholarly journals Calcium-Induced Virulence Factors Associated with the Extracellular Matrix of Mucoid Pseudomonas aeruginosa Biofilms

2005 ◽  
Vol 187 (13) ◽  
pp. 4327-4337 ◽  
Author(s):  
S. Sarkisova ◽  
M. A. Patrauchan ◽  
D. Berglund ◽  
D. E. Nivens ◽  
M. J. Franklin
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Pseudomonas Aeruginosa ◽  
Host Cells ◽  
Matrix Composition ◽  
Confocal Laser ◽  
Extracellular Proteases ◽  
Pulmonary Tissue ◽  
Bacterial Gene ◽  
Calcium Addition

ABSTRACT Pseudomonas aeruginosa colonizes the pulmonary tissue of patients with cystic fibrosis (CF), leading to biofilm-associated infections. The pulmonary fluid of CF patients usually contains elevated concentrations of cations and may contain the P. aeruginosa redox-active pigment pyocyanin, which is known to disrupt calcium homeostasis of host cells. Since divalent cations are important bridging ions for bacterial polysaccharides and since they may play regulatory roles in bacterial gene expression, we investigated the effect of calcium ions on the extracellular matrix constituents of P. aeruginosa biofilms. For mucoid strain P. aeruginosa FRD1, calcium addition (1.0 and 10 mM as CaCl2) resulted in biofilms that were at least 10-fold thicker than biofilms without added calcium. Scanning confocal laser microscopy showed increased spacing between cells for the thick biofilms, and Fourier transform infrared spectroscopy revealed that the material between cells is primarily alginate. An algD transcriptional reporter demonstrated that calcium addition caused an eightfold increase in alg gene expression in FRD1 biofilms. Calcium addition also resulted in increased amounts of three extracellular proteases (AprA, LasB, and PrpL). Immunoblots of the biofilm extracellular material established that AprA was harbored within the biofilm extracellular matrix. An aprA deletion mutation and a mutation in gene for a putative P. aeruginosa calmodulin-like protein did not significantly affect calcium-induced biofilm structure. Two-dimensional gel electrophoresis showed increased amounts of phenazine biosynthetic proteins in FRD1 biofilms and in calcium-amended planktonic cultures. Spectrochemical analyses showed that the calcium addition causes a three- to fivefold increase in pyocyanin production. These results demonstrate that calcium addition affects the structure and extracellular matrix composition of mucoid P. aeruginosa biofilms, through increased expression and stability of bacterial extracellular products. The calcium-induced extracellular matrix of mucoid P. aeruginosa consists primarily of the virulence factor alginate and also harbors extracellular proteases and perhaps pyocyanin, a biomolecule that may further disrupt cellular calcium levels.

scholarly journals Pseudomonas aeruginosa N-3-Oxo-Dodecanoyl-Homoserine Lactone Impacts Mitochondrial Networks Morphology, Energetics, and Proteome in Host Cells

2020 ◽  
Vol 11 ◽  
Author(s):  
Henrik Josephson ◽  
Maria Ntzouni ◽  
Camilla Skoglund ◽  
Stig Linder ◽  
Maria V. Turkina ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Homoserine Lactone ◽  
Host Cells ◽  
Mitochondrial Networks

scholarly journals Paraoxonases-2 and -3 Are Important Defense Enzymes againstPseudomonas aeruginosaVirulence Factors due to Their Anti-Oxidative and Anti-Inflammatory Properties

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Eva-Maria Schweikert ◽  
Julianna Amort ◽  
Petra Wilgenbus ◽  
Ulrich Förstermann ◽  
John F. Teiber ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Defense Mechanism ◽  
Homoserine Lactone ◽  
Host Cells ◽  
Defense System ◽  
Innate Defense ◽  
Redox Active ◽  
Intracellular Ca ◽  
Anti Inflammatory ◽  
Underlying Mechanisms

The pathogenPseudomonas aeruginosacauses serious damage in immunocompromised patients by secretion of various virulence factors, among them the quorum sensing N-(3-oxododecanoyl)-L-homoserine lactone (3OC12) and the redox-active pyocyanin (PCN). Paraoxonase-2 (PON2) may protect againstP. aeruginosainfections, as it efficiently inactivates 3OC12 and diminishes PCN-induced oxidative stress. This defense could be circumvented because 3OC12 mediates intracellular Ca2+-rise in host cells, which causes rapid inactivation and degradation of PON2. Importantly, we recently found that the PON2 paralogue PON3 prevents mitochondrial radical formation. Here we investigated its role as additional potential defense mechanism againstP. aeruginosainfections. Our studies demonstrate that PON3 diminished PCN-induced oxidative stress. Moreover, it showed clear anti-inflammatory potential by protecting against NF-κB activation and IL-8 release. The latter similarly applied to PON2. Furthermore, we observed a Ca2+-mediated inactivation and degradation of PON3, again in accordance with previous findings for PON2. Our results suggest that the anti-oxidative and anti-inflammatory functions of PON2 and PON3 are an important part of our innate defense system againstP. aeruginosainfections. Furthermore, we conclude thatP. aeruginosacircumvents PON3 protection by the same pathway as for PON2. This may help identifying underlying mechanisms in order to sustain the protection afforded by these enzymes.

scholarly journals Thapsigargin blocks Pseudomonas aeruginosa homoserine lactone-induced apoptosis in airway epithelia

2014 ◽  
Vol 306 (9) ◽  
pp. C844-C855 ◽  
Author(s):  
Christian Schwarzer ◽  
Bharat Ravishankar ◽  
Maria Patanwala ◽  
Stacey Shuai ◽  
Zhu Fu ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Tight Junction ◽  
Fluorescent Protein ◽  
Homoserine Lactone ◽  
Host Cells ◽  
Early Event ◽  
Airway Epithelial ◽  
Tracheal Epithelial Cells ◽  
Junction Protein ◽  
Induced Apoptosis

Pseudomonas aeruginosa secretes N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule to regulate gene expression. Micromolar concentrations are found in the airway surface liquid of infected lungs. Exposure of the airway surface to C12 caused a loss of transepithelial resistance within 1 h that was accompanied by disassembly of tight junctions, as indicated by relocation of the tight junction protein zonula occludens 1 from the apical to the basolateral pole and into the cytosol of polarized human airway epithelial cell cultures (Calu-3 and primary tracheal epithelial cells). These effects were blocked by carbobenzoxy-valyl-alanyl-aspartyl-[ O-methyl]-fluoromethylketone, a pan-caspase blocker, indicating that tight junction disassembly was an early event in C12-triggered apoptosis. Short-duration (10 min) pretreatment of airway epithelial (Calu-3 and JME) cells with 1 μM thapsigargin (Tg), an inhibitor of Ca2+ uptake into the endoplasmic reticulum (ER), was found to be protective against the C12-induced airway epithelial barrier breakdown and also against other apoptosis-related effects, including shrinkage and fragmentation of nuclei, activation of caspase 3/7 (the executioner caspase in apoptosis), release of ER-targeted redox-sensitive green fluorescent protein into the cytosol, and depolarization of mitochondrial membrane potential. Pretreatment of Calu-3 airway cell monolayers with BAPTA-AM [to buffer cytosolic Ca2+ concentration (Cacyto)] or Ca2+-free solution + BAPTA-AM reduced C12 activation of apoptotic events, suggesting that C12-triggered apoptosis may involve Ca2+. Because C12 and Tg reduced Ca2+ concentration in the ER and increased Cacyto, while Tg increased mitochondrial Ca2+ concentration (Camito) and C12 reduced Camito, it is proposed that Tg may reduce C12-induced apoptosis in host cells not by raising Cacyto, but by preventing C12-induced decreases in Camito.

Probing bacterial gene expression within host cells

1997 ◽  
Vol 5 (9) ◽  
pp. 360-363 ◽  
Author(s):  
Raphael H. Valdivia ◽  
Stanley Falkow
Keyword(s):  
Gene Expression ◽  
Host Cells ◽  
Bacterial Gene ◽  
Bacterial Gene Expression

scholarly journals Novel Paraoxonase 2-Dependent Mechanism Mediating the Biological Effects of the Pseudomonas aeruginosa Quorum-Sensing MoleculeN-(3-Oxo-Dodecanoyl)-l-Homoserine Lactone

2015 ◽  
Vol 83 (9) ◽  
pp. 3369-3380 ◽  
Author(s):  
Sven Horke ◽  
Junhui Xiao ◽  
Eva-Maria Schütz ◽  
Gerald L. Kramer ◽  
Petra Wilgenbus ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Calcium Release ◽  
Biological Effects ◽  
Homoserine Lactone ◽  
Host Cells ◽  
Stress Signaling ◽  
Intracellular Acidification ◽  
Content Type ◽  
Biological Responses ◽  
Ph Decrease

Pseudomonas aeruginosaproducesN-(3-oxo-dodecanoyl)-l-homoserine lactone (3OC12), a crucial signaling molecule that elicits diverse biological responses in host cells thought to subvert immune defenses. The mechanism mediating many of these responses remains unknown. The intracellular lactonase paraoxonase 2 (PON2) hydrolyzes and inactivates 3OC12 and is therefore considered a component of host cells that attenuates 3OC12-mediated responses. Here, we demonstrate in cell lines and in primary human bronchial epithelial cells that 3OC12 is rapidly hydrolyzed intracellularly by PON2 to 3OC12 acid, which becomes trapped and accumulates within the cells. Subcellularly, 3OC12 acid accumulated within the mitochondria, a compartment where PON2 is localized. Treatment with 3OC12 caused a rapid PON2-dependent cytosolic and mitochondrial pH decrease, calcium release, and phosphorylation of stress signaling kinases. The results indicate a novel, PON2-dependent intracellular acidification mechanism by which 3OC12 can mediate its biological effects. Thus, PON2 is a central regulator of host cell responses to 3OC12, acting to decrease the availability of 3OC12 for receptor-mediated effects and acting to promote effects, such as calcium release and stress signaling, via intracellular acidification.

scholarly journals Early phase of effective treatment induces distinct transcriptional changes in Mycobacterium tuberculosis expelled by pulmonary tuberculosis patients

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ambreen Shaikh ◽  
Kalpana Sriraman ◽  
Smriti Vaswani ◽  
Vikas Oswal ◽  
Sudha Rao ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Effective Treatment ◽  
Early Phase ◽  
Treatment Initiation ◽  
Host Cells ◽  
New Host ◽  
Bacterial Gene ◽  
Cell Wall Assembly ◽  
Treatment Regimens ◽  
Bacterial Gene Expression

AbstractEffective treatment reduces a tuberculosis patient's ability to infect others even before they test negative in sputum or culture. Currently, the basis of reduced infectiousness of the Mycobacterium tuberculosis (Mtb) with effective treatment is unclear. We evaluated changes in aerosolized bacteria expelled by patients through a transcriptomic approach before and after treatment initiation (up to 14 days) by RNA sequencing. A distinct change in the overall transcriptional profile was seen post-treatment initiation compared to pretreatment, only when patients received effective treatment. This also led to the downregulation of genes associated with cellular activities, cell wall assembly, virulence factors indicating loss of pathogenicity, and a diminished ability to infect and survive in new host cells. Based on this, we identified genes whose expression levels changed with effective treatment. The observations of the study open up avenues for further evaluating the changes in bacterial gene expression during the early phase of treatment as biomarkers for monitoring response to tuberculosis treatment regimens and provide means of identifying better correlates of Mtb transmission.

scholarly journals Impact of Pseudomonas aeruginosa quorum sensing signaling molecules on adhesion and inflammatory markers in endothelial cells

2018 ◽  
Vol 14 ◽  
pp. 2580-2588 ◽  
Author(s):  
Carmen Curutiu ◽  
Florin Iordache ◽  
Veronica Lazar ◽  
Aurelia Magdalena Pisoschi ◽  
Aneta Pop ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Endothelial Cells ◽  
Quorum Sensing ◽  
Inflammatory Response ◽  
Adhesion Molecules ◽  
Laboratory Strain ◽  
Homoserine Lactone ◽  
Signaling Molecules ◽  
Host Cells ◽  
Infectious Process

Pseudomonas aeruginosa relies on the quorum sensing (QS) signaling system as a central regulator mechanism of virulence expression that contributes to the formation and maintenance of biofilms and tolerance to conventional antimicrobials. QS Signaling molecules (QSSMs) may be recognized and may function also within the host cells, being potentially involved in the progression of the infectious process. In this study we evaluate the expression of adhesion and inflammatory molecules in endothelial cells treated with P. aeruginosa QSSMs, in order to bring new insights on the mechanisms involved in the interaction of P. aeruginosa with host cells during the infectious process. Endothelial cells were stimulated with 20 µM of main P. aeruginosa QSSMs (OdDHL = N-(3-oxododecanoyl)-L-homoserine lactone, C4HSL = N-butyryl-L-homoserine lactone, PQS = 2-heptyl-3-hydroxy-4(1H)-quinolone and HHQ = 2-heptyl-4-quinolone). Adherence to endothelial cells, inert substratum and biofilm formation was evaluated. The expression of adhesion molecules (VE-cadherin, PECAM-1, ICAM-1, and P-selectin) and inflammatory response molecules (IL-1β, IL-6, TNFα, TGFβ, and eNOS) was assessed by qRT-PCR and flow cytometry. Our results showed that bacterial adherence to inert substratum and biofilm were decreased in the presence of all tested QSSMs. The adherence index of PAO1 laboratory strain to host cells was decreased between 10–40% in the presence of QSSMs, as compared to untreated control. Expression of eukaryotic cells adhesion molecules ICAM-1 and P-selectin was stimulated by QSSMs, whereas VE-cadherin and PECAM-1 levels were increased only by C4HSL. The inflammatory response of endothelial cells was also modulated, as observed by the modified expression of IL-1β (for C4HSL, PQS and HHQ), IL-6 (for C4HSL and HHQ), TNFα (for C4HSL and HHQ), TGFβ, and eNOS factors. Our results demonstrate that the main pseudomonadal QSSMs differentially modulate endothelial cells adhesion and proinflammatory cytokine expression. These observations provide new insights in the mechanisms by which different QSSMs activate endothelial cells and modulate the infectious process, and support the importance of recent studies aiming to develop anti-QS therapeutic strategies to fight against P. aeruginosa infections.

scholarly journals Pseudomonas aeruginosa Autoinducer Enters and Functions in Mammalian Cells

2004 ◽  
Vol 186 (8) ◽  
pp. 2281-2287 ◽  
Author(s):  
Simon C. Williams ◽  
Erin K. Patterson ◽  
Nancy L. Carty ◽  
John A. Griswold ◽  
Abdul N. Hamood ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Transcription Factors ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Homoserine Lactone ◽  
Host Cells ◽  
Human Pathogens ◽  
Coding Region ◽  
Binding Domains

ABSTRACT Quorum sensing (QS) is a cell density-dependent signaling mechanism used by many bacteria to control gene expression. Several recent reports indicate that the signaling molecules (autoinducers) that mediate QS in Pseudomonas aeruginosa may also modulate gene expression in host cells; however, the mechanisms are largely unknown. Here we show that two P. aeruginosa autoinducers, N-3-oxododecanoyl-homoserine lactone and N-butyryl-homoserine lactone, can both enter eukaryotic cells and activate artificial chimeric transcription factors based on their cognate transcriptional activators, LasR and RhlR, respectively. The autoinducers promoted nuclear localization of chimeric proteins containing the full LasR or RhlR coding region, and the LasR-based proteins were capable of activating transcription of a LasR-dependent luciferase gene. Responsiveness to autoinducer required the N-terminal autoinducer-binding domains of LasR and RhlR. Truncated proteins consisting of only the C-terminal helix-turn-helix DNA-binding domains of both proteins attached to a nuclear localization signal efficiently translocated to the nucleus in the absence of autoinducer, and truncated LasR-based proteins functioned as constitutively active transcription factors. Chimeric LasR proteins were only activated by their cognate autoinducer ligand and not by N-butyryl-l-homoserine lactone. These data provide evidence that autoinducer molecules from human pathogens can enter mammalian cells and suggest that autoinducers may influence gene expression in host cells by interacting with and activating as-yet-unidentified endogenous proteins.

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