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

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 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.

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 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.

Expression of Pseudomonas aeruginosa exoS is controlled by quorum sensing and RpoS

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 843-851 ◽  
Author(s):  
Michael Hogardt ◽  
Maximilian Roeder ◽  
Anna Maria Schreff ◽  
Leo Eberl ◽  
Jürgen Heesemann
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Homoserine Lactone ◽  
Host Cells ◽  
Regulatory Control ◽  
Reasonable Assumption ◽  
Virulence Determinants ◽  
Exoenzyme S ◽  
Regulatory Cascade

In Pseudomonas aeruginosa, virulence determinants and biofilm formation are coordinated via a hierarchical quorum sensing cascade, which involves the transcriptional regulators LasR and RhlR and their cognate homoserine lactone activators C12-HSL [N-(3-oxododecanoyl)-l-homoserine lactone] and c4-hsl (n-butanoyl-l-homoserine lactone), which are produced by LasI and RhlI, respectively. The exoenzyme S regulon of P. aeruginosa, comprises genes for a type III secretion system and for four anti-host effector proteins (ExoS, T, U and Y), which are translocated into host cells. It is a reasonable assumption that this ExoS regulon should be downregulated in the biofilm growth state and thus should also be under the regulatory control of the Las/Rhl system. Therefore, an exoS′-gfp reporter construct was used, and the influence of the Las and Rhl quorum sensing systems and the effect of the stationary-phase sigma factor RpoS on regulation of the exoS gene was examined. Evidence is provided for downregulation of exoS during biofilm formation of P. aeruginosa PAO1. The rhlI mutant PDO100 and rhlR mutant PDO111, but not the lasI mutant PDO-JP1, showed approximately twofold upregulation of the exoS′-gfp reporter in comparison to PAO1. Upregulation of exoS′-gfp in the PDO100 mutant could be repressed to normal level by adding C4-HSL autoinducer, indicating a negative regulatory effect of RhlR/C4-HSL on exoS expression. As RhlR/C4-HSL is also involved in regulation of RpoS, the P. aeruginosa rpoS mutant SS24 was examined and the exoS′-gfp reporter was found to be fivefold upregulated in comparison to PAO1. For the first time evidence is reported for a regulatory cascade linking RhlR/RhlI and RpoS with the expression of the anti-host effector ExoS, part of the exoenzyme S regulon. Moreover, these data suggest that the exoenzyme S regulon may be downregulated in P. aeruginosa biofilms.

scholarly journals Revisiting the quorum-sensing hierarchy in Pseudomonas aeruginosa: the transcriptional regulator RhlR regulates LasR-specific factors

Microbiology ◽  
2009 ◽  
Vol 155 (3) ◽  
pp. 712-723 ◽  
Author(s):  
Valérie Dekimpe ◽  
Eric Déziel
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Transcriptional Regulator ◽  
Homoserine Lactone ◽  
The Other ◽  
Virulence Determinants ◽  
Late Stationary Phase ◽  
Regulatory Systems ◽  
Specific Factors

Pseudomonas aeruginosa uses the two major quorum-sensing (QS) regulatory systems las and rhl to modulate the expression of many of its virulence factors. The las system is considered to stand at the top of the QS hierarchy. However, some virulence factors such as pyocyanin have been reported to still be produced in lasR mutants under certain conditions. Interestingly, such mutants arise spontaneously under various conditions, including in the airways of cystic fibrosis patients. Using transcriptional lacZ reporters, LC/MS quantification and phenotypic assays, we have investigated the regulation of QS-controlled factors by the las system. Our results show that activity of the rhl system is only delayed in a lasR mutant, thus allowing the expression of multiple virulence determinants such as pyocyanin, rhamnolipids and C4-homoserine lactone (HSL) during the late stationary phase. Moreover, at this stage, RhlR is able to overcome the absence of the las system by activating specific LasR-controlled functions, including production of 3-oxo-C12-HSL and Pseudomonas quinolone signal (PQS). P. aeruginosa is thus able to circumvent the deficiency of one of its QS systems by allowing the other to take over. This work demonstrates that the QS hierarchy is more complex than the model simply presenting the las system above the rhl system.

scholarly journals Bacteriophage-Based Biosensing of Pseudomonas aeruginosa: An Integrated Approach for the Putative Real-Time Detection of Multi-Drug-Resistant Strains

Biosensors ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 124
Author(s):  
Liliam K. Harada ◽  
Waldemar Bonventi Júnior ◽  
Erica C. Silva ◽  
Thais J. Oliveira ◽  
Fernanda C. Moreli ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Infections ◽  
Research Effort ◽  
Acquired Resistance ◽  
Integrated Approach ◽  
Healthcare Services ◽  
Host Cells ◽  
Color Changes ◽  
Metabolic Versatility ◽  
The Matrix

During the last decennium, it has become widely accepted that ubiquitous bacterial viruses, or bacteriophages, exert enormous influences on our planet’s biosphere, killing between 4–50% of the daily produced bacteria and constituting the largest genetic diversity pool on our planet. Currently, bacterial infections linked to healthcare services are widespread, which, when associated with the increasing surge of antibiotic-resistant microorganisms, play a major role in patient morbidity and mortality. In this scenario, Pseudomonas aeruginosa alone is responsible for ca. 13–15% of all hospital-acquired infections. The pathogen P. aeruginosa is an opportunistic one, being endowed with metabolic versatility and high (both intrinsic and acquired) resistance to antibiotics. Bacteriophages (or phages) have been recognized as a tool with high potential for the detection of bacterial infections since these metabolically inert entities specifically attach to, and lyse, bacterial host cells, thus, allowing confirmation of the presence of viable cells. In the research effort described herein, three different phages with broad lytic spectrum capable of infecting P. aeruginosa were isolated from environmental sources. The isolated phages were elected on the basis of their ability to form clear and distinctive plaques, which is a hallmark characteristic of virulent phages. Next, their structural and functional stabilization was achieved via entrapment within the matrix of porous alginate, biopolymeric, and bio-reactive, chromogenic hydrogels aiming at their use as sensitive matrices producing both color changes and/or light emissions evolving from a reaction with (released) cytoplasmic moieties, as a bio-detection kit for P. aeruginosa cells. Full physicochemical and biological characterization of the isolated bacteriophages was the subject of a previous research paper.

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