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 Pseudolipasin A Is a Specific Inhibitor for Phospholipase A2 Activity of Pseudomonas aeruginosa Cytotoxin ExoU

2006 ◽  
Vol 75 (3) ◽  
pp. 1089-1098 ◽  
Author(s):  
Vincent T. Lee ◽  
Stefan Pukatzki ◽  
Hiromi Sato ◽  
Eriya Kikawada ◽  
Anastasia A. Kazimirova ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Specific Inhibitor ◽  
Effector Proteins ◽  
Host Cells ◽  
Cytotoxic Effects ◽  
Type Iii ◽  
Secretion Pathway ◽  
Phospholipase A2 Activity

ABSTRACT A number of bacterial pathogens utilize the type III secretion pathway to deliver effector proteins directly into the host cell cytoplasm. Certain strains of Pseudomonas aeruginosa associated with acute infections express a potent cytotoxin, exoenzyme U (ExoU), that is delivered via the type III secretion pathway directly into contacting host cells. Once inside the mammalian cell, ExoU rapidly lyses the intoxicated cells via its phospholipase A2 (PLA2) activity. A high-throughput cell-based assay was developed to screen libraries of compounds for those capable of protecting cells against the cytotoxic effects of ExoU. A number of compounds were identified in this screen, including one group that blocks the intracellular activity of ExoU. In addition, these compounds specifically inhibited the PLA2 activity of ExoU in vitro, whereas eukaryotic secreted PLA2 and cytosolic PLA2 were not inhibited. This novel inhibitor of ExoU-specific PLA2 activity, named pseudolipasin A, may provide a new lead for virulence factor-based therapeutic design.

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 IL-17A-mediated expression of epithelial IL-17C promotes inflammation during acute Pseudomonas aeruginosa pneumonia

2016 ◽  
Vol 311 (5) ◽  
pp. L1015-L1022 ◽  
Author(s):  
Lisa Wolf ◽  
Sandra Sapich ◽  
Anja Honecker ◽  
Christopher Jungnickel ◽  
Frederik Seiler ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Pulmonary Inflammation ◽  
Alveolar Epithelial Cells ◽  
Lung Epithelial Cells ◽  
Immune Functions ◽  
Alveolar Epithelial ◽  
Inflammatory Protein ◽  
Interleukin 17A

Lung epithelial cells are suggested to promote pathogen-induced pulmonary inflammation by the release of chemokines, resulting in enhanced recruitment of circulating leukocytes. Recent studies have shown that the interleukin-17C (IL-17C) regulates innate immune functions of epithelial cells in an autocrine manner. The aim of this study was to investigate the contribution of IL-17C to pulmonary inflammation in a mouse model of acute Pseudomonas aeruginosa pneumonia. Infection with P. aeruginosa resulted in an increased expression of IL-17C in lung tissue of wild-type mice. Numbers of neutrophils and the expression of the neutrophil-recruiting chemokines keratinocyte-derived chemokine and macrophage inflammatory protein 2 were significantly decreased in lungs of IL-17C-deficient (IL-17C−/−) mice infected with P. aeruginosa at 24 h. Systemic concentrations of interleukin-6 (IL-6) were significantly decreased in infected IL-17C−/− mice at 24 h and the survival of IL-17C−/− mice was significantly increased at 48 h. The expression of IL-17C was reduced in infected mice deficient for interleukin-17A (IL-17A), whereas pulmonary concentrations of IL-17A were not affected by the deficiency for IL-17C. Stimulation of primary alveolar epithelial cells with IL-17A resulted in a significantly increased expression of IL-17C in vitro. Our data suggest that IL-17A-mediated expression of epithelial IL-17C amplifies the release of chemokines by epithelial cells and thereby contributes to the recruitment of neutrophils and systemic inflammation during acute P. aeruginosa pneumonia.

scholarly journals A Novel Anti-PcrV Antibody Providing Enhanced Protection against Pseudomonas aeruginosa in Multiple Animal Infection Models

2014 ◽  
Vol 58 (8) ◽  
pp. 4384-4391 ◽  
Author(s):  
Paul Warrener ◽  
Reena Varkey ◽  
Jessica C. Bonnell ◽  
Antonio DiGiandomenico ◽  
Maria Camara ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Host Cells ◽  
Content Type ◽  
Mechanically Ventilated ◽  
Potent Inhibition ◽  
Infection Models ◽  
High Risk Populations ◽  
Hospital Acquired

ABSTRACTPseudomonas aeruginosais a major cause of hospital-acquired infections, particularly in mechanically ventilated patients, and it is the leading cause of death in cystic fibrosis patients. A key virulence factor associated with disease severity is theP. aeruginosatype III secretion system (T3SS), which injects bacterial toxins directly into the cytoplasm of host cells. The PcrV protein, located at the tip of the T3SS injectisome complex, is required for T3SS function and is a well-validated target in animal models of immunoprophylactic strategies targetingP. aeruginosa. In an effort to identify a highly potent and protective monoclonal antibody (MAb) that inhibits the T3SS, we generated and characterized a panel of novel anti-PcrV MAbs. Interestingly, some MAbs exhibiting potent inhibition of T3SSin vitrofailed to provide protection in a mouse model ofP. aeruginosainfection, suggesting that effectivein vivoinhibition of T3SS with anti-PcrV MAbs is epitope dependent. V2L2MD, while not the most potent MAb as assessed byin vitrocytotoxicity inhibition assays, provided strong prophylactic protection in several murine infection models and a postinfection therapeutic model. V2L2MD mediated significantly (P< 0.0001) betterin vivoprotection than that provided by a comparator antibody, MAb166, a well-characterized anti-PcrV MAb and the progenitor of a clinical candidate, KB001-A. The results described here support further development of a V2L2MD-containing immunotherapeutic and may suggest even greater potential than was previously recognized for the prevention and treatment ofP. aeruginosainfections in high-risk populations.

scholarly journals Campylobacter jejuni Cocultured with Epithelial Cells Reduces Surface Capsular Polysaccharide Expression

2009 ◽  
Vol 77 (5) ◽  
pp. 1959-1967 ◽  
Author(s):  
N. Corcionivoschi ◽  
M. Clyne ◽  
A. Lyons ◽  
A. Elmi ◽  
O. Gundogdu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Campylobacter Jejuni ◽  
Capsular Polysaccharide ◽  
Bacterial Species ◽  
Molecular Techniques ◽  
Conditioned Media ◽  
Host Cells ◽  
Proteinase K ◽  
Surface Polysaccharides

ABSTRACT The host cell environment can alter bacterial pathogenicity. We employed a combination of cellular and molecular techniques to study the expression of Campylobacter jejuni polysaccharides cocultured with HCT-8 epithelial cells. After two passages, the amount of membrane-bound high-molecular-weight polysaccharide was considerably reduced. Microarray profiling confirmed significant downregulation of capsular polysaccharide (CPS) locus genes. Experiments using conditioned media showed that sugar depletion occurred only when the bacterial and epithelial cells were cocultured. CPS depletion occurred when C. jejuni organisms were exposed to conditioned media from a different C. jejuni strain but not when exposed to conditioned media from other bacterial species. Proteinase K or heat treatment of conditioned media under coculture conditions abrogated the effect on the sugars, as did formaldehyde fixation and cycloheximide treatment of host cells or chloramphenicol treatment of the bacteria. However, sugar depletion was not affected in flagellar export (fliQ) and quorum-sensing (luxS) gene mutants. Passaged C. jejuni showed reduced invasiveness and increased serum sensitivity in vitro. C. jejuni alters its surface polysaccharides when cocultured with epithelial cells, suggesting the existence of a cross talk mechanism that modulates CPS expression during infection.

scholarly journals Effect of Hypoxia on the Pathogenesis of Acinetobacter baumannii and Pseudomonas aeruginosa In Vitro and in Murine Experimental Models of Infection

2018 ◽  
Vol 86 (10) ◽  
Author(s):  
María Luisa Gil-Marqués ◽  
María Eugenía Pachón-Ibáñez ◽  
Jerónimo Pachón ◽  
Younes Smani
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Acinetobacter Baumannii ◽  
Experimental Models ◽  
Host Cells ◽  
Content Type ◽  
Bactericidal Activities ◽  
Bacterial Concentrations

ABSTRACT Hypoxia modulates bacterial virulence and the inflammation response through hypoxia-inducible factor 1α (HIF-1α). Here we study the influence of hypoxia on Acinetobacter baumannii and Pseudomonas aeruginosa infections. In vitro, hypoxia increases the bactericidal activities of epithelial cells against A. baumannii and P. aeruginosa, reducing extracellular bacterial concentrations to 50.5% ± 7.5% and 90.8% ± 13.9%, respectively, at 2 h postinfection. The same phenomenon occurs in macrophages (67.6% ± 18.2% for A. baumannii at 2 h and 50.3% ± 10.9% for P. aeruginosa at 24 h). Hypoxia decreases the adherence of A. baumannii to epithelial cells (42.87% ± 8.16% at 2 h) and macrophages (52.0% ± 18.7% at 24 h), as well as that of P. aeruginosa (24.9% ± 4.5% in epithelial cells and 65.7% ± 5.5% in macrophages at 2 h). Moreover, hypoxia decreases the invasion of epithelial cells (48.6% ± 3.8%) and macrophages (8.7% ± 6.9%) by A. baumannii at 24 h postinfection and by P. aeruginosa at 2 h postinfection (75.0% ± 16.3% and 63.4% ± 5.4%, respectively). In vivo, hypoxia diminishes bacterial loads in fluids and tissues in animal models of infection by both pathogens. In contrast, mouse survival time was shorter under hypoxia (23.92 versus 36.42 h) with A. baumannii infection. No differences in the production of cytokines or HIF-1α were found between hypoxia and normoxia in vitro or in vivo. We conclude that hypoxia increases the bactericidal activities of host cells against both pathogens and reduces the interaction of pathogens with host cells. Moreover, hypoxia accelerates the rate at which animals die despite the lower bacterial concentrations in vivo.

scholarly journals Roles of Chaperone/Usher Pathways of Yersinia pestis in a Murine Model of Plague and Adhesion to Host Cells

2012 ◽  
Vol 80 (10) ◽  
pp. 3490-3500 ◽  
Author(s):  
Matthew Hatkoff ◽  
Lisa M. Runco ◽  
Celine Pujol ◽  
Indralatha Jayatilaka ◽  
Martha B. Furie ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Yersinia Pestis ◽  
Pathogenic Bacteria ◽  
Lung Epithelial Cells ◽  
Host Cells ◽  
Cell Binding ◽  
Content Type ◽  
Infection Models ◽  
Virulence Attenuation ◽  
Lung Epithelial

ABSTRACTYersinia pestisand many other Gram-negative pathogenic bacteria use the chaperone/usher (CU) pathway to assemble virulence-associated surface fibers termed pili or fimbriae.Y. pestishas two well-characterized CU pathways: thecafgenes coding for the F1 capsule and thepsagenes coding for the pH 6 antigen. TheY. pestisgenome contains additional CU pathways that are capable of assembling pilus fibers, but the roles of these pathways in the pathogenesis of plague are not understood. We constructed deletion mutations in the usher genes for six of the additionalY. pestisCU pathways. The wild-type (WT) and usher deletion strains were compared in the murine bubonic (subcutaneous) and pneumonic (intranasal) plague infection models.Y. pestisstrains containing deletions in CU pathwaysy0348-0352,y1858-1862, andy1869-1873were attenuated for virulence compared to the WT strain by the intranasal, but not subcutaneous, routes of infection, suggesting specific roles for these pathways during pneumonic plague. We examined binding of theY. pestisWT and usher deletion strains to A549 human lung epithelial cells, HEp-2 human cervical epithelial cells, and primary human and murine macrophages.Y. pestisCU pathwaysy0348-0352andy1858-1862were found to contribute to adhesion to all host cells tested, whereas pathwayy1869-1873was specific for binding to macrophages. The correlation between the virulence attenuation and host cell binding phenotypes of the usher deletion mutants identifies three of the additional CU pathways ofY. pestisas mediating interactions with host cells that are important for the pathogenesis of plague.

scholarly journals Factor H Binding to PspC of Streptococcus pneumoniae Increases Adherence to Human Cell Lines In Vitro and Enhances Invasion of Mouse Lungs In Vivo

2007 ◽  
Vol 75 (8) ◽  
pp. 4082-4087 ◽  
Author(s):  
Lisa R. Quin ◽  
Chinwendu Onwubiko ◽  
Quincy C. Moore ◽  
Megumi Fujioka Mills ◽  
Larry S. McDaniel ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Human Cell ◽  
Factor H ◽  
Lung Epithelial Cells ◽  
Host Cells ◽  
Complement Factor ◽  
Human Cell Lines

ABSTRACTPneumococcal surface protein C (PspC) binds to both human secretory immunoglobulin A (sIgA) and complement factor H (FH). FH, a regulator of the alternative pathway of complement, can also mediate adherence of different host cells. Since PspC contributes to adherence and invasion of host cells, we hypothesized that the interaction of PspC with FH may also mediate adherence of pneumococci to human cells. In this study, we investigated FH- and sIgA-mediated pneumococcal adherence to human cell lines in vitro. Adherence assays demonstrated that preincubation ofStreptococcus pneumoniaeD39 with FH increased adherence to human umbilical vein endothelial cells (HUVEC) 5-fold and to lung epithelial cells (SK-MES-1) 18-fold, relative to that of D39 without FH on the surface. The presence of sIgA enhanced adherence to SK-MES-1 6-fold and to pharyngeal epithelial cells (Detroit 562) 14-fold. Furthermore, sIgA had an additive effect on adherence to HUVEC; specifically, preincubation of D39 with both FH and sIgA led to a 21-fold increase in adherence. Finally, using a mouse model, we examined the significance of the FH-PspC interaction in pneumococcal nasal colonization and lung invasion. Mice intranasally infected with D39 preincubated with FH had increased bacteremia and lung invasion, but they had similar levels of nasopharyngeal colonization compared to that of mice challenged with D39 without FH.

scholarly journals Impact of n-6 Polyunsaturated Fatty Acids on Growth of Multidrug-Resistant Pseudomonas aeruginosa: Interactions with Amikacin and Ceftazidime

2000 ◽  
Vol 44 (8) ◽  
pp. 2187-2189 ◽  
Author(s):  
E. J. Giamarellos-Bourboulis ◽  
P. Grecka ◽  
A. Dionyssiou-Asteriou ◽  
H. Giamarellou
Keyword(s):  
Fatty Acids ◽  
Pseudomonas Aeruginosa ◽  
Arachidonic Acid ◽  
Experimental Infection ◽  
Multidrug Resistant ◽  
Gamma Linolenic Acid ◽  
Infection Models ◽  
Over Time ◽  
In Vitro Interactions

ABSTRACT Twenty-six multidrug-resistant Pseudomonas aeruginosaisolates were exposed over time to 300 μg of gamma-linolenic acid or arachidonic acid per ml or to the combination of both acids at 150 μg/ml each with ceftazidime and amikacin with or without albumin to observe the in vitro interactions of the antibiotics. Antibiotics and albumin were applied at their levels found in serum. Synergy between acids and antibiotics was found against 13 isolates, and it was expressed after 5 h of growth in the presence of albumin. The results indicate that further application in experimental infection models is merited.

scholarly journals Effect of Slit/Robo signaling on regeneration in lung emphysema

2021 ◽  
Author(s):  
Jin-Soo Park ◽  
RyeonJin Cho ◽  
Eun-Young Kang ◽  
Yeon-Mok Oh
Keyword(s):  
Epithelial Cells ◽  
Mouse Model ◽  
Lung Epithelial Cells ◽  
Mouse Lung ◽  
Chronic Obstructive ◽  
Irreversible Damage ◽  
Lung Epithelial ◽  
And Migration ◽  
Proliferation And Migration

AbstractEmphysema, a pathological component of chronic obstructive pulmonary disease, causes irreversible damage to the lung. Previous studies have shown that Slit plays essential roles in cell proliferation, angiogenesis, and organ development. In this study, we evaluated the effect of Slit2 on the proliferation and migration of mouse lung epithelial cells and its role in regeneration in an emphysema lung mouse model. Here, we have shown that Slit2/Robo signaling contributes to the regeneration of lungs damaged by emphysema. Mouse epithelial lung cells treated with Slit2 exhibited increased proliferation and migration in vitro. Our results also showed that Slit2 administration improved alveolar regeneration in the emphysema mouse model in vivo. Furthermore, Slit2/Robo signaling increased the phosphorylation of ERK and Akt, which was mediated by Ras activity. These Slit2-mediated cellular signaling processes may be involved in the proliferation and migration of mouse lung epithelial cells and are also associated with the potential mechanism of lung regeneration. Our findings suggest that Slit2 administration may be beneficial for alveolar regeneration in lungs damaged by emphysema.

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