scholarly journals Quercetin attenuates the production of pro-inflammatory cytokines in H292 human lung epithelial cells infected with Pseudomonas aeruginosa, by modulating ExoS production

2020 ◽  
Author(s):  
Hye In Ahn ◽  
Ji-Won Park ◽  
Hyun-Jae Jang ◽  
Ok-kyoung Kwon ◽  
Jung Hee Kim ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mrna Level ◽  
Inhibitory Effect ◽  
Lung Epithelial Cells ◽  
Host Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa System ◽  
Type Three Secretion System ◽  
Sandwich Type ◽  
Needle Protein

Abstract Background: The type three secretion system (T3SS) is a major virulence system of Pseudomonas aeruginosa (P. aeruginosa). The effector protein Exotoxin S (ExoS) produced by P. aeruginosa is secreted into the host cells via the T3SS. For the purpose of screening the inhibitors with regard to ExoS secretion, we developed the sandwich-type enzyme-linked immunosorbent assay (ELISA) system. From the initial screening, quercetin was selected because it has the prominent effect of ExoS inhibition and also is known to have anti-inflammatory and antioxidant effects on mammalian cells.Results: In this study, we investigated the effects of quercetin on the expression and secretion of ExoS using the ELISA and Western blot analysis methods. The results showed that the secretion of ExoS was significantly decreased by 10, 20uM quercetin. Also, pscF and popD, which are composed of the T3SS needle, are reduced by quercetin at the mRNA level, and we confirmed the inhibitory effect of quercetin on cytokines in P. aeruginosa-infected H292 cells by real-time polymerase chain reaction (PCR). Conclusion: Collectively, quercetin inhibits the secretion of ExoS by reducing both ExoS production and the expression of the needle protein of T3SS. Furthermore, these results suggest that quercetin has the potential to be used as an anti-toxic treatment for the disease caused by P. aeruginosa infection.

scholarly journals PrtR Homeostasis Contributes to Pseudomonas aeruginosa Pathogenesis and Resistance against Ciprofloxacin

2014 ◽  
Vol 82 (4) ◽  
pp. 1638-1647 ◽  
Author(s):  
Ziyu Sun ◽  
Jing Shi ◽  
Chang Liu ◽  
Yongxin Jin ◽  
Kewei Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Bacterial Colonization ◽  
Mrna Level ◽  
Sos Response ◽  
Opportunistic Pathogen ◽  
Host Cells ◽  
Chronic Infections ◽  
Mobility Shift ◽  
Content Type

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen that causes acute and chronic infections in humans. Pyocins are bacteriocins produced byP. aeruginosathat are usually released through lysis of the producer strains. Expression of pyocin genes is negatively regulated by PrtR, which gets cleaved under SOS response, leading to upregulation of pyocin synthetic genes. Previously, we demonstrated that PrtR is required for the expression of type III secretion system (T3SS), which is an important virulence component ofP. aeruginosa. In this study, we demonstrate that mutation inprtRresults in reduced bacterial colonization in a mouse acute pneumonia model. Examination of bacterial and host cells in the bronchoalveolar lavage fluids from infected mice revealed that expression of PrtR is induced by reactive oxygen species (ROS) released by neutrophils. We further demonstrate that treatment with hydrogen peroxide or ciprofloxacin, known to induce the SOS response and pyocin production, resulted in an elevated PrtR mRNA level. Overexpression of PrtR by atacpromoter repressed the endogenousprtRpromoter activity, and electrophoretic mobility shift assay revealed that PrtR binds to its own promoter, suggesting an autorepressive mechanism of regulation. A high level of PrtR expressed from a plasmid resulted in increased T3SS gene expression during infection and higher resistance against ciprofloxacin. Overall, our results suggest that the autorepression of PrtR contributes to the maintenance of a relatively stable level of PrtR, which is permissive to T3SS gene expression in the presence of ROS while increasing bacterial tolerance to stresses, such as ciprofloxacin, by limiting pyocin production.

scholarly journals Peroxisome Proliferator-Activated Receptors Mediate Host Cell Proinflammatory Responses to Pseudomonas aeruginosa Autoinducer

2008 ◽  
Vol 190 (13) ◽  
pp. 4408-4415 ◽  
Author(s):  
Aruna Jahoor ◽  
Rashila Patel ◽  
Amanda Bryan ◽  
Catherine Do ◽  
Jay Krier ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Transcriptional Activity ◽  
Expression Patterns ◽  
Lung Epithelial Cells ◽  
Host Cells ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Pparγ Agonist ◽  
Lung Epithelial

ABSTRACT The pathogenic bacterium Pseudomonas aeruginosa utilizes the 3-oxododecanoyl homoserine lactone (3OC12-HSL) autoinducer as a signaling molecule to coordinate the expression of virulence genes through quorum sensing. 3OC12-HSL also affects responses in host cells, including the upregulation of genes encoding inflammatory cytokines. This proinflammatory response may exacerbate underlying disease during P. aeruginosa infections. The specific mechanism(s) through which 3OC12-HSL influences host responses is unclear, and no mammalian receptors for 3OC12-HSL have been identified to date. Here, we report that 3OC12-HSL increases mRNA levels for a common panel of proinflammatory genes in murine fibroblasts and human lung epithelial cells. To identify putative 3OC12-HSL receptors, we examined the expression patterns of a panel of nuclear hormone receptors in these two cell lines and determined that both peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) and PPARγ were expressed. 3OC12-HSL functioned as an agonist of PPARβ/δ transcriptional activity and an antagonist of PPARγ transcriptional activity and inhibited the DNA binding ability of PPARγ. The proinflammatory effect of 3OC12-HSL in lung epithelial cells was blocked by the PPARγ agonist rosiglitazone, suggesting that 3OC12-HSL and rosiglitazone are mutually antagonistic negative and positive regulators of PPARγ activity, respectively. These data identify PPARβ/δ and PPARγ as putative mammalian 3OC12-HSL receptors and suggest that PPARγ agonists may be employed as anti-inflammatory therapeutics for P. aeruginosa infections.

scholarly journals Naturally Produced Outer Membrane Vesicles from Pseudomonas aeruginosa Elicit a Potent Innate Immune Response via Combined Sensing of Both Lipopolysaccharide and Protein Components

2010 ◽  
Vol 78 (9) ◽  
pp. 3822-3831 ◽  
Author(s):  
Terri N. Ellis ◽  
Sara A. Leiman ◽  
Meta J. Kuehn
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Response Analysis ◽  
Specific Response ◽  
Protein Components ◽  
Vesicle Proteins

ABSTRACT Pseudomonas aeruginosa is a prevalent opportunistic human pathogen that, like other Gram-negative pathogens, secretes outer membrane vesicles. Vesicles are complex entities composed of a subset of envelope lipid and protein components that have been observed to interact with and be internalized by host cells. This study characterized the inflammatory responses to naturally produced P. aeruginosa vesicles and determined the contribution of vesicle Toll-like receptor (TLR) ligands and vesicle proteins to that response. Analysis of macrophage responses to purified vesicles by real-time PCR and enzyme-linked immunosorbent assay identified proinflammatory cytokines upregulated by vesicles. Intact vesicles were shown to elicit a profoundly greater inflammatory response than the response to purified lipopolysaccharide (LPS). Both TLR ligands LPS and flagellin contributed to specific vesicle cytokine responses, whereas the CpG DNA content of vesicles did not. Neutralization of LPS sensing demonstrated that macrophage responses to the protein composition of vesicles required the adjuvantlike activity of LPS to elicit strain specific responses. Protease treatment to remove proteins from the vesicle surface resulted in decreased interleukin-6 and tumor necrosis factor alpha production, indicating that the production of these specific cytokines may be linked to macrophage recognition of vesicle proteins. Confocal microscopy of vesicle uptake by macrophages revealed that vesicle LPS allows for binding to macrophage surfaces, whereas vesicle protein content is required for internalization. These data demonstrate that macrophage sensing of both LPS and protein components of outer membrane vesicles combine to produce a bacterial strain-specific response that is distinct from those triggered by individual, purified vesicle components.

scholarly journals The Novel Fibrinogen-Binding Protein FbsB Promotes Streptococcus agalactiae Invasion into Epithelial Cells

2004 ◽  
Vol 72 (6) ◽  
pp. 3495-3504 ◽  
Author(s):  
Heike Gutekunst ◽  
Bernhard J. Eikmanns ◽  
Dieter J. Reinscheid
Keyword(s):  
Epithelial Cells ◽  
Streptococcus Agalactiae ◽  
Deletion Mutant ◽  
Binding Protein ◽  
Lung Epithelial Cells ◽  
Host Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bacterial Invasion ◽  
Fibrinogen Binding ◽  
Lung Epithelial

ABSTRACT Streptococcus agalactiae is a major cause of bacterial sepsis and meningitis in human newborns. The interaction of S. agalactiae with host proteins and the entry into host cells thereby represent important virulence traits of these bacteria. The present report describes the identification of the fbsB gene, encoding a novel fibrinogen-binding protein that plays a crucial role in the invasion of S. agalactiae into human cells. In Western blots and enzyme-linked immunosorbent assay (ELISA) experiments, the FbsB protein was demonstrated to interact with soluble and immobilized fibrinogen. Binding studies showed the N-terminal 388 residues of FbsB and the Aα-subunit of human fibrinogen to recognize each other. By reverse transcription (RT)-PCR, the fbsB gene was shown to be cotranscribed with the gbs0851 gene in S. agalactiae. Deletion of the fbsB gene in the genome of S. agalactiae did not influence the binding of the bacteria to fibrinogen, suggesting that FbsB does not participate in the attachment of S. agalactiae to fibrinogen. In tissue culture experiments, however, the fbsB deletion mutant was severely impaired in its invasion into lung epithelial cells. Bacterial invasion could be reestablished by introducing the fbsB gene on a shuttle plasmid into the fbsB deletion mutant. Furthermore, treatment of lung epithelial cells with FbsB fusion protein blocked S. agalactiae invasion of epithelial cells in a dose-dependent fashion. These results suggest an important role of the FbsB protein in the overall process of host cell entry by S. agalactiae.

scholarly journals Mutations in the Pseudomonas aeruginosa Needle Protein GenepscFConfer Resistance to Phenoxyacetamide Inhibitors of the Type III Secretion System

2014 ◽  
Vol 58 (4) ◽  
pp. 2211-2220 ◽  
Author(s):  
Nicholas O. Bowlin ◽  
John D. Williams ◽  
Claire A. Knoten ◽  
Matthew C. Torhan ◽  
Tommy F. Tashjian ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Molecular Target ◽  
Secretion System ◽  
Host Cells ◽  
Content Type ◽  
Type Iii ◽  
Unknown Protein ◽  
Needle Protein

ABSTRACTThe type III secretion system (T3SS) is a clinically important virulence mechanism inPseudomonas aeruginosathat secretes and translocates effector toxins into host cells, impeding the host's rapid innate immune response to infection. Inhibitors of T3SS may be useful as prophylactic or adjunctive therapeutic agents to augment the activity of antibiotics inP. aeruginosainfections, such as pneumonia and bacteremia. One such inhibitor, the phenoxyacetamide MBX 1641, exhibits very responsive structure-activity relationships, including striking stereoselectivity, in its inhibition ofP. aeruginosaT3SS. These features suggest interaction with a specific, but unknown, protein target. Here, we identify the apparent molecular target by isolating inhibitor-resistant mutants and mapping the mutation sites by deep sequencing. Selection and sequencing of four independent mutants resistant to the phenoxyacetamide inhibitor MBX 2359 identified the T3SS genepscF, encoding the needle apparatus, as the only locus of mutations common to all four strains. Transfer of the wild-type and mutated alleles ofpscF, together with its chaperone and cochaperone genespscEandpscG, to a ΔpscF P. aeruginosastrain demonstrated that each of the single-codon mutations inpscFis necessary and sufficient to provide secretion and translocation that is resistant to a variety of phenoxyacetamide inhibitor analogs but not to T3SS inhibitors with different chemical scaffolds. These results implicate the PscF needle protein as an apparent new molecular target for T3SS inhibitor discovery and suggest that three other chemically distinct T3SS inhibitors interact with one or more different targets or a different region of PscF.

scholarly journals Prevalence of ExoY Activity in Pseudomonas aeruginosa Reference Panel Strains and Impact on Cytotoxicity in Epithelial Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Hazel Silistre ◽  
Dorothée Raoux-Barbot ◽  
Federica Mancinelli ◽  
Flora Sangouard ◽  
Alice Dupin ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Bacterial Species ◽  
Lung Epithelial Cells ◽  
Reference Panel ◽  
Host Cells ◽  
Cytotoxic Effects ◽  
Infection Models ◽  
Significant Difference

ExoY is among the effectors that are injected by the type III secretion system (T3SS) of Pseudomonas aeruginosa into host cells. Inside eukaryotic cells, ExoY interacts with F-actin, which stimulates its potent nucleotidyl cyclase activity to produce cyclic nucleotide monophosphates (cNMPs). ExoY has broad substrate specificity with GTP as a preferential substrate in vitro. How ExoY contributes to the virulence of P. aeruginosa remains largely unknown. Here, we examined the prevalence of active ExoY among strains from the international P. aeruginosa reference panel, a collection of strains that includes environmental and clinical isolates, commonly used laboratory strains, and sequential clonal isolates from cystic fibrosis (CF) patients and thus represents the large diversity of this bacterial species. The ability to secrete active ExoY was determined by measuring the F-actin stimulated guanylate cyclase (GC) activity in bacterial culture supernatants. We found an overall ExoY activity prevalence of about 60% among the 40 examined strains with no significant difference between CF and non-CF isolates. In parallel, we used cellular infection models of human lung epithelial cells to compare the cytotoxic effects of isogenic reference strains expressing active ExoY or lacking the exoY gene. We found that P. aeruginosa strains lacking ExoY were in fact more cytotoxic to the epithelial cells than those secreting active ExoY. This suggests that under certain conditions, ExoY might partly alleviate the cytotoxic effects of other virulence factors of P. aeruginosa.

scholarly journals Development and some applications of enzyme-linked immunosorbent assay system for murine macrophage inflammatory protein-2 (MIP-2)

1996 ◽  
Vol 5 (3) ◽  
pp. 206-209 ◽  
Author(s):  
H. Ochiai ◽  
S. Sakai ◽  
T. Kogure ◽  
T. Hirabaytashi ◽  
K. Nakajima ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Sandwich Elisa ◽  
Protein A ◽  
Inhibitory Effect ◽  
Raw264.7 Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Murine Macrophage ◽  
Fusion Construct ◽  
Elisa System ◽  
Inflammatory Protein

We attempted to establish an enzyme-linked immunosorbent assay (ELISA) system by preparation of recombinant murine MIP-2 and its rabbit antibodies. A fusion construct of MIP-2 to protein A was used to enable easy purification as well as the generation of a sufficiently large antibody response. The specificity of antibody was confirmed by Western blotting analysis of 20-h conditioned medium from lipopolysaccharide (LPS)-stimulated RAW264.7 cells, a murine macrophage cell line; antibody gave a single band with a molecular weight of approximately 6000, which is identical to that of murine MIP-2 reported previously. Biotin–streptavidin sandwich ELISA could detect quantitatively MIP-2 at concentration range of 20 to 1000 pg/ml. In some applications of this ELISA system, time-related production of MIP-2 and inhibitory effect of dexamethasone on its production have been demonstrated in LPS-stimulated RAW264.7 cells. Thus, ELISA system established in this study is considered to be a useful tool to study MIP-2 response in various inflammation models in mice.

4,6,6-trimethylbicyclo [3.1.1] hept-3-ene: Analysis of the Inhibitory Effect of Monoterpere on Pseudomonas aeruginosa Strain

2017 ◽  
Author(s):  
Sávio Ferreira ◽  
Ticiane Farias ◽  
Letícia de Eduardo ◽  
Siluana Ferreira ◽  
Zilka Lima
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Inhibitory Effect

scholarly journals The MarR-Type Regulator PA3458 Is Involved in Osmoadaptation Control in Pseudomonas aeruginosa

2021 ◽  
Vol 22 (8) ◽  
pp. 3982
Author(s):  
Karolina Kotecka ◽  
Adam Kawalek ◽  
Kamil Kobylecki ◽  
Aneta Agnieszka Bartosik
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Sites ◽  
Transcriptional Profiling ◽  
Mrna Level ◽  
Transcriptional Regulators ◽  
Resistance Mechanisms ◽  
Chronic Infections ◽  
Environmental Signals ◽  
Regulatory Systems

Pseudomonas aeruginosa is a facultative human pathogen, causing acute and chronic infections that are especially dangerous for immunocompromised patients. The eradication of P. aeruginosa is difficult due to its intrinsic antibiotic resistance mechanisms, high adaptability, and genetic plasticity. The bacterium possesses multilevel regulatory systems engaging a huge repertoire of transcriptional regulators (TRs). Among these, the MarR family encompasses a number of proteins, mainly acting as repressors, which are involved in response to various environmental signals. In this work, we aimed to decipher the role of PA3458, a putative MarR-type TR from P. aeruginosa. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3458 showed changes in the mRNA level of 133 genes; among them, 100 were down-regulated, suggesting the repressor function of PA3458. Concomitantly, ChIP-seq analysis identified more than 300 PA3458 binding sites in P. aeruginosa. The PA3458 regulon encompasses genes involved in stress response, including the PA3459–PA3461 operon, which is divergent to PA3458. This operon encodes an asparagine synthase, a GNAT-family acetyltransferase, and a glutamyl aminopeptidase engaged in the production of N-acetylglutaminylglutamine amide (NAGGN), which is a potent bacterial osmoprotectant. We showed that PA3458-mediated control of PA3459–PA3461 expression is required for the adaptation of P. aeruginosa growth in high osmolarity. Overall, our data indicate that PA3458 plays a role in osmoadaptation control in P. aeruginosa.

scholarly journals The Assessment of IL-21 and IL-22 at the mRNA Level in Tumor Tissue and Protein Concentration in Serum and Peritoneal Fluid in Patients with Ovarian Cancer

2021 ◽  
Vol 10 (14) ◽  
pp. 3058
Author(s):  
Aleksandra Mielczarek-Palacz ◽  
Celina Kruszniewska-Rajs ◽  
Marta Smycz-Kubańska ◽  
Jarosław Strzelczyk ◽  
Wojciech Szanecki ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Peritoneal Fluid ◽  
Tumor Tissue ◽  
Mrna Level ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
First Time ◽  
Ovarian Serous Cancer

The aim of the analysis was for the first time to assess the expression of genes encoding IL-21 and IL-22 at the mRNA level in ovarian tumor specimens and the concentration of these parameters in serum and peritoneal fluid in patients with ovarian serous cancer. The levels of IL-21 and IL-22 transcripts were evaluated with the use of the real-time RT-qPCR. Enzyme-linked immunosorbent assay (ELISA) was used to determine the concentration of proteins. Quantitative analysis of IL-21 gene mRNA in the tumor tissue showed the highest activity in the G1 degree of histopathological differentiation and was higher in G1 compared to the control group. The concentration of IL-21 and IL-22 in the serum and in the peritoneal fluid of women with ovarian cancer varied depending on the degree of histopathological differentiation of the cancer and showed statistical variability compared to controls. The conducted studies have shown that the local and systemic changes in the immune system involving IL-21 and IL-22 indicate the participation of these parameters in the pathogenesis of ovarian cancer, and modulation in the IL-21/IL-22 system may prove useful in the development of new diagnostic and therapeutic strategies used in patients, which require further research.

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