Pseudomonas aeruginosa Infection of Airway Epithelial Cells Modulates Expression of Kruppel-Like Factors 2 and 6 via RsmA-Mediated Regulation of Type III Exoenzymes S and Y

ABSTRACT Pseudomonas aeruginosa is an important opportunistic pathogen which is capable of causing both acute and chronic infections in immunocompromised patients. Successful adaptation of the bacterium to its host environment relies on the ability of the organism to tightly regulate gene expression. RsmA, a small RNA-binding protein, controls the expression of a large number of virulence-related genes in P. aeruginosa, including those encoding the type III secretion system and associated effector proteins, with important consequences for epithelial cell morphology and cytotoxicity. In order to examine the influence of RsmA-regulated functions in the pathogen on gene expression in the host, we compared global expression profiles of airway epithelial cells in response to infection with P. aeruginosa PAO1 and an rsmA mutant. The RsmA-dependent response of host cells was characterized by significant changes in the global transcriptional pattern, including the increased expression of two Kruppel-like factors, KLF2 and KLF6. This increased expression was mediated by specific type III effector proteins. ExoS was required for the enhanced expression of KLF2, whereas both ExoS and ExoY were required for the enhanced expression of KLF6. Neither ExoT nor ExoU influenced the expression of the transcription factors. Additionally, the increased gene expression of KLF2 and KLF6 was associated with ExoS-mediated cytotoxicity. Therefore, this study identifies for the first time the human transcription factors KLF2 and KLF6 as targets of the P. aeruginosa type III exoenzymes S and Y, with potential importance in host cell death.

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Modulation of Cytosolic Ca2+ Concentration in Airway Epithelial Cells by Pseudomonas aeruginosa

Infection and Immunity ◽

10.1128/iai.70.11.6399-6408.2002 ◽

2002 ◽

Vol 70 (11) ◽

pp. 6399-6408 ◽

Cited By ~ 15

Author(s):

Tobias Jacob ◽

Rebecca J. Lee ◽

Joanne N. Engel ◽

Terry E. Machen

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Type Iii Secretion ◽

Airway Epithelial Cells ◽

Ca Channel ◽

Airway Epithelial ◽

Slow Increase ◽

Type Iii ◽

Apical Chamber ◽

Type Iv

ABSTRACT Modulation of cytosolic (intracellular) Ca2+ concentration (Cai) may be an important host response when airway epithelial cells are exposed to Pseudomonas aeruginosa. We measured Cai in Calu-3 cells exposed from the apical or basolateral surface to cytotoxic and noncytotoxic strains of P. aeruginosa. Apical addition of either noncytotoxic strains or cytotoxic strains failed to affect Cai over a 3-h time period, nor were changes observed after basolateral addition of noncytotoxic strains. In contrast, basolateral addition of cytotoxic strains caused a slow increase in Cai from 100 nM to 200 to 400 nM. This increase began after 20 to 50 min and persisted for an additional 30 to 75 min, at which time the cells became nonviable. P. aeruginosa-induced increases in Cai were blocked by the addition of the Ca channel blocker La3+ to the basolateral but not to the apical chamber. Likewise, replacing the basolateral but not the apical medium with Ca-free solution prevented P. aeruginosa-mediated changes in Cai. With isogenic mutants of PA103, we demonstrated that the type III secretion apparatus, the type III-secreted effector ExoU, and type IV pili were necessary for increased Cai. We propose that translocation of ExoU through the basolateral surface of polarized airway epithelial cells via the type III secretion apparatus leads to release of Ca stored in the endoplasmic reticulum and activation of Ca channels in the basolateral membranes of epithelial cells.

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Transcriptome Analysis of Pseudomonas aeruginosa after Interaction with Human Airway Epithelial Cells

Infection and Immunity ◽

10.1128/iai.72.9.5433-5438.2004 ◽

2004 ◽

Vol 72 (9) ◽

pp. 5433-5438 ◽

Cited By ~ 67

Author(s):

Anders Frisk ◽

Jill R. Schurr ◽

Guoshun Wang ◽

Donna C. Bertucci ◽

Luis Marrero ◽

...

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Human Airway ◽

Normal Human ◽

Human Airway Epithelial Cells

ABSTRACT The transcriptional profile of Pseudomonas aeruginosa after interactions with primary normal human airway epithelial cells was determined using Affymetrix GeneChip technology. Gene expression profiles indicated that various genes involved in phosphate acquisition and iron scavenging were differentially regulated.

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Selection of reference genes for quantitative PCR: identifying reference genes for airway epithelial cells exposed to Pseudomonas aeruginosa

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00158.2020 ◽

2020 ◽

Vol 319 (2) ◽

pp. L256-L265

Author(s):

Thomas H. Hampton ◽

Katja Koeppen ◽

Laura Bashor ◽

Bruce A. Stanton

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Quantitative Pcr ◽

Reference Gene ◽

Reference Genes ◽

Primary Cultures ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Reference Gene Expression

Most quantitative PCR (qPCR) experiments report differential expression relative to the expression of one or more reference genes. Therefore, when experimental conditions alter reference gene expression, qPCR results may be compromised. Little is known about the magnitude of this problem in practice. We found that reference gene responses are common and hard to predict and that their stability should be demonstrated in each experiment. Our reanalysis of 15 airway epithelia microarray data sets retrieved from the National Center for Biotechnology Information (NCBI) identified no common reference gene that was reliable in all 15 studies. Reanalysis of published RNA sequencing (RNA-seq) data in which human bronchial epithelial cells (HBEC) were exposed to Pseudomonas aeruginosa revealed that minor experimental details, including bacterial strain, may alter reference gene responses. Direct measurement of 32 TaqMan reference genes in primary cultures of HBEC exposed to P. aeruginosa (strain PA14) demonstrated that choosing an unstable reference gene could make it impossible to observe statistically significant changes in IL8 gene expression. We found that reference gene instability is a general phenomenon and not limited to studies of airway epithelial cells. In a diverse compendium of 986 human microarray experiments retrieved from the NCBI, reference genes were differentially expressed in 42% of studies. Experimentally induced changes in reference gene expression ranged from 21% to 212%. These results highlight the importance of identifying adequate reference genes for each experimental system and documenting their response to treatment in each experiment. This will enhance experimental rigor and reproducibility in qPCR studies.

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Regulation of Human MUC7 Mucin Gene Expression by Cigarette Smoke Extract or Cigarette Smoke and Pseudomonas aeruginosa Lipopolysaccharide in Human Airway Epithelial Cells and in MUC7 Transgenic Mice~!2010-04-05~!2010-06-09~!2010-07-14~!

The Open Respiratory Medicine Journal ◽

10.2174/1874306401004010063 ◽

2010 ◽

Vol 4 (1) ◽

pp. 63-70 ◽

Cited By ~ 4

Author(s):

Hao Fan ◽

Libuse A. Bobek

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Transgenic Mice ◽

Cigarette Smoke ◽

Cigarette Smoke Extract ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Human Airway ◽

Muc7 Mucin

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The Posttranscriptional Regulator RsmA Plays a Role in the Interaction between Pseudomonas aeruginosa and Human Airway Epithelial Cells by Positively Regulating the Type III Secretion System

Infection and Immunity ◽

10.1128/iai.74.5.3012-3015.2006 ◽

2006 ◽

Vol 74 (5) ◽

pp. 3012-3015 ◽

Cited By ~ 54

Author(s):

Heidi Mulcahy ◽

Julie O'Callaghan ◽

Eoin P. O'Grady ◽

Claire Adams ◽

Fergal O'Gara

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Type Iii Secretion ◽

Posttranscriptional Regulation ◽

Rna Binding ◽

Type Iii Secretion System ◽

Airway Epithelial Cells ◽

Secretion System ◽

Airway Epithelial ◽

Type Iii

ABSTRACT Posttranscriptional regulation of certain virulence-related genes in Pseudomonas aeruginosa is brought about by RsmA, a small RNA-binding protein. During interaction with airway epithelial cells, RsmA promoted actin depolymerization, cytotoxicity, and anti-internalization of P. aeruginosa by positively regulating the virulence-associated type III secretion system.

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Regulation of Human MUC7 Mucin Gene Expression by Cigarette Smoke Extract or Cigarette Smoke and Pseudomonas aeruginosa Lipopolysaccharide in Human Airway Epithelial Cells and in MUC7 Transgenic Mice

The Open Respiratory Medicine Journal ◽

10.2174/18743064010040100063 ◽

2010 ◽

Vol 4 (1) ◽

pp. 63-70 ◽

Cited By ~ 1

Author(s):

Hao Fan ◽

Libuse A. Bobek

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Transgenic Mice ◽

Cigarette Smoke ◽

Cigarette Smoke Extract ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Human Airway ◽

Muc7 Mucin

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The Type III Toxins of Pseudomonas aeruginosa Disrupt Epithelial Barrier Function

Journal of Bacteriology ◽

10.1128/jb.01567-07 ◽

2007 ◽

Vol 190 (8) ◽

pp. 2814-2821 ◽

Cited By ~ 62

Author(s):

Grace Soong ◽

Dane Parker ◽

Mariah Magargee ◽

Alice S. Prince

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Actin Polymerization ◽

Airway Epithelial Cells ◽

Confocal Imaging ◽

Bacterial Invasion ◽

Airway Epithelial ◽

Erm Proteins ◽

Epithelial Barrier Function ◽

Type Iii

ABSTRACT The type III secreted toxins of Pseudomonas aeruginosa are important virulence factors associated with clinically important infection. However, their effects on bacterial invasion across mucosal surfaces have not been well characterized. One of the most commonly expressed toxins, ExoS, has two domains that are predicted to affect cytoskeletal integrity, including a GTPase-activating protein (GAP) domain, which targets Rho, a major regulator of actin polymerization; and an ADP-ribosylating domain that affects the ERM proteins, which link the plasma membrane to the actin cytoskeleton. The activities of these toxins, and ExoS specifically, on the permeability properties of polarized airway epithelial cells with intact tight junctions were examined. Strains expressing type III toxins altered the distribution of the tight junction proteins ZO-1 and occludin and were able to transmigrate across polarized airway epithelial monolayers, in contrast to ΔSTY mutants. These effects on epithelial permeability were associated with the ADP-ribosylating domain of ExoS, as bacteria expressing plasmids lacking expression of the ExoS GAP activity nonetheless increased the permeation of fluorescent dextrans, as well as bacteria, across polarized airway epithelial cells. Treatment of epithelial cells with cytochalasin D depolymerized actin filaments and increased permeation across the monolayers but did not eliminate the differential effects of wild-type and toxin-negative mutants on the epithelial cells, suggesting that additional epithelial targets are involved. Confocal imaging studies demonstrated that ZO-1, occludin, and ezrin undergo substantial redistribution in human airway cells intoxicated by ExoS, -T, and -Y. These studies support the hypothesis that type III toxins enhance P. aeruginosa's invasive capabilities by interacting with multiple eukaryotic cytoskeletal components.

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Somatic cell hemoglobin modulates nitrogen oxide metabolism in the human airway epithelium

Scientific Reports ◽

10.1038/s41598-021-94782-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nadzeya Marozkina ◽

Laura Smith ◽

Yi Zhao ◽

Joe Zein ◽

James F. Chmiel ◽

...

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Nitrogen Oxides ◽

Airway Epithelium ◽

Airflow Obstruction ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Human Airway ◽

Human Airway Epithelium ◽

Human Airway Epithelial Cells

AbstractEndothelial hemoglobin (Hb)α regulates endothelial nitric oxide synthase (eNOS) biochemistry. We hypothesized that Hb could also be expressed and biochemically active in the ciliated human airway epithelium. Primary human airway epithelial cells, cultured at air–liquid interface (ALI), were obtained by clinical airway brushings or from explanted lungs. Human airway Hb mRNA data were from publically available databases; or from RT-PCR. Hb proteins were identified by immunoprecipitation, immunoblot, immunohistochemistry, immunofluorescence and liquid chromatography- mass spectrometry. Viral vectors were used to alter Hbβ expression. Heme and nitrogen oxides were measured colorimetrically. Hb mRNA was expressed in human ciliated epithelial cells. Heme proteins (Hbα, β, and δ) were detected in ALI cultures by several methods. Higher levels of airway epithelial Hbβ gene expression were associated with lower FEV1 in asthma. Both Hbβ knockdown and overexpression affected cell morphology. Hbβ and eNOS were apically colocalized. Binding heme with CO decreased extracellular accumulation of nitrogen oxides. Human airway epithelial cells express Hb. Higher levels of Hbβ gene expression were associated with airflow obstruction. Hbβ and eNOS were colocalized in ciliated cells, and heme affected oxidation of the NOS product. Epithelial Hb expression may be relevant to human airways diseases.

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Individual Matrix Metalloproteinases Control Distinct Transcriptional Responses in Airway Epithelial Cells Infected with Pseudomonas aeruginosa

Infection and Immunity ◽

10.1128/iai.00799-07 ◽

2007 ◽

Vol 75 (12) ◽

pp. 5640-5650 ◽

Cited By ~ 48

Author(s):

Sean Y. Kassim ◽

Sina A. Gharib ◽

Brigham H. Mecham ◽

Timothy P. Birkland ◽

William C. Parks ◽

...

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Initial Point ◽

Global Gene Expression ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Transcriptional Responses ◽

Matrix Metalloproteinase 7 ◽

Node Network ◽

Global Gene Expression Analysis

ABSTRACT Airway epithelium is the initial point of host-pathogen interaction in Pseudomonas aeruginosa infection, an important pathogen in cystic fibrosis and nosocomial pneumonia. We used global gene expression analysis to determine airway epithelial transcriptional responses dependent on matrilysin (matrix metalloproteinase 7 [MMP-7]) and stromelysin-2 (MMP-10), two MMPs induced by acute P. aeruginosa pulmonary infection. Extraction of differential gene expression (EDGE) analysis of gene expression changes in P. aeruginosa-infected organotypic tracheal epithelial cell cultures from wild-type, Mmp7 −/−, and Mmp10 −/− mice identified 2,091 matrilysin-dependent and 1,628 stromelysin-2-dependent genes that were differentially expressed. Key node network analysis showed that these MMPs controlled distinct gene expression programs involved in proliferation, cell death, immune responses, and signal transduction, among other host defense processes. Our results demonstrate discrete roles for these MMPs in regulating epithelial responses to Pseudomonas infection and show that a global genomics strategy can be used to assess MMP function.

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