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Infection and Immunity, September 2004, p. 5433-5438, Vol. 72, No. 9
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.9.5433-5438.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Anders Frisk,1 Jill R. Schurr,2 Guoshun Wang,3 Donna C. Bertucci,3 Luis Marrero,4 Sung Hei Hwang,5 Daniel J. Hassett,5 and Michael J. Schurr1*

Department of Microbiology and Immunology, Program in Molecular Pathogenesis and Immunity, Louisiana Center for Lung Biology and Immunotherapy, Tulane University Health Sciences Center,1 Department of Genetics,2 Gene Therapy Program,3 Morphology and Imaging Core, Louisiana State University Health Sciences Center, New Orleans, Louisiana,4 Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio5

Received 11 February 2004/ Returned for modification 21 April 2004/ Accepted 14 June 2004


   ABSTRACT
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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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Interaction of Pseudomonas aeruginosa with host cells in vitro has typically been studied using a variety of immortalized mammalian cell lines (1, 7, 23). Although several of these studies have suggested that P. aeruginosa is capable of attaching and invading epithelial cells, few have examined the interaction by use of primary normal human airway epithelial (PNHAE) cells. These cells have differentiated structures (mucin and cilia) and have tight junctions, unlike their monolayer-grown counterparts (13). Recently, several studies have suggested that cell polarity and the integrity of tight junctions are important in adherence, as disruption of cell polarity increased P. aeruginosa adherence and invasion of epithelial cells (6, 16, 22). Host-pathogen interactions have been examined with use of microarrays to determine the transcriptional profiles of Salmonella enterica infecting macrophages (4) and human epithelial cells infected with P. aeruginosa (12). Recently, P. aeruginosa transcriptional profiling data were reviewed for different environmental conditions (8). Nevertheless, a lack of information exists regarding P. aeruginosa gene expression when the organism interacts with human host cells.

P. aeruginosa localizes to the basolateral surface of PNHAE cells 12 h postinfection. PNHAE cells grown on transwell inserts were chosen as an in vitro infection model since these cells have their apical surface exposed to air, become fully differentiated, produce extracellular proteins such as mucin and cilia, and form tight junctions (13). PNHAE cells were obtained from healthy organ donors, processed, and seeded at 2.5 x 105 to 5 x 105 cells/cm2 with use of collagen-coated Millicell-PCF membrane inserts (0.4-µm pore size, 12-mm diameter) in 24-well plates (Millipore) (13). The cells were cultured and maintained in Dulbecco's modified Eagle's medium-Hanks' F-12 supplement (DMEM-F-12) medium (Invitrogen) containing 0.4% glucose, 13 µM Fe2/3+, 2 mM L-glutamine, 15 mM HEPES, and 2% Ultroser G supplement, a serum substitute (Crescent Chemicals Co., Inc., Islandia, N.Y.), at 37°C in a 9% CO2 humidified atmosphere (13). After 7 to 9 days of culture, the cells were stimulated with 100 ng of keratinocyte growth factor/ml to initiate cell replication and to stimulate differentiation. Differentiation was assessed by visual examination of the cells and measurement of the transepithelial electrical resistance across the cell membrane. Transepithelial electrical resistance values of 500 to 1,000 {Omega}/cm2 were routinely observed over the membrane for differentiated cells. P. aeruginosa strain PAO1 harboring the green fluorescent protein (GFP) expression plasmid pMRP9-1 (25) was used initially for a time course infection study. The bacteria were cultured in 100 ml of Luria-Bertani broth containing 300 µg of carbenicillin/ml at 37°C and grown to an optical density at 600 nm of 0.4. The bacteria were then washed once and resuspended in the described cell culture medium, without serum supplement, and inoculated to the apical cell surface at a multiplicity of infection of 100 (5 x 107 CFU/ml). At 1, 4, 8, 12, and 16 h postinfection, the infected cells were washed once with cell culture medium, fixed, and then stained with DAPI (4',6'-diamidino-2-phenylindole) and phalloidin as previously described (26). The GFP-expressing P. aeruginosa and the stained eukaryotic cells were subsequently visualized by Zeiss Axioplan II deconvolution microscopy.

P. aeruginosa was located on the apical surface of the cells 4 h after infection (Fig. 1A). The majority of the bacteria remained on the apical surface up to 8 h postinfection (Fig. 1B). However, after 12 h of infection, P. aeruginosa localized to the edges of the airway epithelial cells and the majority of bacteria were observed at the basolateral membrane (Fig. 1C). Our 1- and 4-h observations are in agreement with a study that showed limited adherence and invasion following 3 h of incubation of P. aeruginosa with human nasal epithelial cells (6). Plotkowski et al. reported similar results on normal and cystic fibrosis-affected bronchial epithelial cells grown in thick and thin collagen gels (22). Both studies suggested that well-polarized intact epithelia are relatively resistant to P. aeruginosa infection. However, our observations at later time points (12 and 16 h) suggest that P. aeruginosa was capable of disrupting tight junctions, which may serve as a port of entry for P. aeruginosa, facilitating access to the basolateral membrane. The disruption of tight junctions in Calu-3 monolayers and cultured bovine tracheal monolayers has been reported to allow increased P. aeruginosa binding and cytotoxicity (16).



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  		FIG. 1. Interaction of P. aeruginosa PAO1-GFP with PNHAE cells for 4, 8, and 12 h (A to C, respectively). Cells were stained with DAPI and phalloidin and visualized using Zeiss Axioplan II deconvolution microscopy.

 
Differential P. aeruginosa gene expression after 4 h of interaction with PNHAE cells. The 4-h time point was chosen for identification of differentially expressed genes in P. aeruginosa PAO1 early in the infection process with use of the Affymetrix GeneChip technology. Three replicates of total RNA were obtained for the control and experimental conditions. Each replicate of the control consisted of total RNA isolated from six wells of P. aeruginosa added to the Millicell insert with DMEM-F-12 medium without PNHAE cells. For the experimental condition, each replicate consisted of total RNA pooled from six separate wells of PNHAE cells infected with P. aeruginosa PAO1. Since we used total RNA from infected eukaryotic cells, an additional control was employed to eliminate eukaryotic transcripts that hybridize to the Pseudomonas GeneChip array. For this control, total RNA was isolated from 18 independent wells of the PNHAE cells alone (RNAs from six wells were pooled for each replicate and hybridized to the Pseudomonas Affymetrix GeneChip). For the infection studies, P. aeruginosa was grown and added to the PNHAE cells as described above. After washing with DMEM-F-12 medium to remove nonadherent bacteria, total P. aeruginosa and/or eukaryotic RNA was isolated by adding lysis buffer (5 mg of lysozyme/ml in Tris-EDTA, 10 mM Tris, pH 8.0) to the Millicell inserts for 5 min and then extracted using the RNeasy Midi kit per the manufacturer's instructions (Qiagen). The RNA was treated with 2 U of DNase I for 15 min at 37°C with the DNase-free kit (Ambion) to remove any contaminating DNA and ethanol precipitated. The quality of the RNA was assessed by size chromatography with an Agilent 2100 Bioanalyzer (Fig. 2). Ten micrograms of total RNA from three replicates of bacteria alone (control 1 [4 h in medium alone], control 2 [12 h in medium alone], and control 3 [cells alone]) and P. aeruginosa interacting with eukaryotic cells was used for cDNA synthesis, fragmentation, labeling, and hybridization per the manufacturer's instructions (Affymetrix GeneChip P. aeruginosa). cDNA generated from eukaryotic RNA was hybridized to P. aeruginosa Genome Arrays in triplicate for identification and subtraction of cross-reacting background signals. Microarray data were generated and analysis was performed using the Affymetrix expression analysis protocol as described previously (17).



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  		FIG. 2. Agilent chromatogram of RNA used in microarray studies. Representative size chromatographic separation of total RNA from PNHAE cells (lane 1), from P. aeruginosa PAO1 and from PNHAE cells (lanes 2 [4 h] and 3 [12 h]), and from P. aeruginosa PAO1 (lane 4). The size ladder is shown in kilobases at left.

 
There were 41 differentially expressed P. aeruginosa genes after 4 h of infection on PNHAE cells when the transcriptional profiles were compared to those of the bacteria in medium alone (Table 1). Five of the 24 activated genes encode putative proteins involved in membrane transport (PA2019, PA4770 [lldP], PA2673, PA1540, and PA2437; Table 2). Additional activated genes encode probable transcriptional regulators (PA0163, PA4203, and PA2877), indicating that they may be important in virulence. The most interesting trend of gene expression observed at the 4-h time point was the repression of eight genes associated with the iron acquisition pyoverdine pathway including pvdA (PA2386), pvdM (PA2393), pvdFE-fpvA-pvdD (PA2396 to PA2399, respectively), pvdJ (PA2401), and PA2411 as well as pcdD encoding the pyochelin biosynthesis protein PcdD (Table 2). This result may indicate that P. aeruginosa is able to acquire ample iron from the eukaryotic cells during the initial stages of infection. Similarly, the sodM gene, which is activated only upon iron starvation in P. aeruginosa in a quorum-sensing-dependent fashion, was also repressed (11).


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  		TABLE 1. Effects of PNHAE cells on P. aeruginosa gene expression

 

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  		TABLE 2. Activated and repressed genes in P. aeruginosa following 4 h of interaction with PNHAE cells

 
Differential P. aeruginosa gene expression after 12 h of interaction with PNHAE cells. Analysis of the transcriptional profile of P. aeruginosa after 12 h of infection on PNHAE cells revealed that 121 genes were differentially expressed (Table 1). Several genes associated with phosphate acquisition were significantly activated (Table 3). One of these genes is also associated with virulence in P. aeruginosa, plcN (PA3319), encoding a nonhemolytic phospholipase C protein (3, 14, 31). P. aeruginosa has been shown to produce and secrete two phospholipase C enzymes, one hemolytic (PlcH) and one nonhemolytic (PlcN), both dependent on the twin-arginine translocation (Tat) system for their transport across the inner membrane (19, 30), and one phospholipase D (32). The enzyme activity of PlcN has been shown to hydrolyze phosphatidylcholine and phosphatidylserine (21), present in both outer and inner leaflets of eukaryotic erythrocytes, respectively. A possible role for PlcN in P. aeruginosa lung infection in vivo may be relevant since phosphatidylcholine is also an abundant constituent in lung surfactant and thus may serve as a substrate for the extracellular enzyme PlcN (34).


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  		TABLE 3. Activated and repressed genes in P. aeruginosa following 12 h of interaction with PNHAE cells

 
Other upregulated phosphate genes identified were phoA (PA3296), encoding an alkaline phosphatase protein (5); oprO (PA3280), encoding a polyphosphate-specific outer membrane porin (24); and glpQ, encoding glycerophosphoryl diester phosphodiesterase (30). GlpQ hydrolyzes deacylated phospholipids to glycerol-3-phosphate, which may facilitate the release of inorganic phosphate for alkaline phosphatases, such as PlcN (30), and/or provide a carbon source for the organism. A limited availability of phosphate during the interaction of P. aeruginosa with the PNHAE cells is likely an explanation for the increased transcription of these genes. In addition, phoA, oprO, plcN, and glpQ are induced by phosphate starvation (5, 21, 24, 30). Our study also identified uncharacterized genes that may be associated with phosphate acquisition as determined by homology to genes in other organisms and in P. aeruginosa. These include PA3910 (41% homology to phosphodiesterase-alkaline phosphatase), PA2635 (probable phosphatase), PA0688 (100% identical to PhoA), PA3909 (probable extracellular nuclease), PA4350 (potential hemolysin), and PA2331 (49% homology to macrophage infectivity protein). These data indicate that genes involved in acquisition and metabolism of phosphate may be important for P. aeruginosa infection of host cells and survival.

After 12 h of infection on the PNHAE cells, P. aeruginosa repressed the transcription of 30 genes more than sevenfold (Table 3). Surprisingly, 14 of these genes (PA4218 to PA4231) are associated with the siderophore-mediated iron acquisition pyochelin pathway. Moreover, 24 other genes that are responsive to iron (20) were repressed at this time point (Table 4). In fact, the number of repressed iron-regulated genes increased over time (10 genes at 4 h and 38 genes at 12 h [with use of fourfold change as a cutoff]), correlating with probable increased airway epithelial cell damage (15). One explanation for the observed repression of these iron-regulated genes may be that iron is made available during interaction with the PNHAE cells. The increase in the number of repressed iron-associated genes supports this possibility. The iron concentration of the medium used in this study was fairly low, approximately 13 µM. However, the effects of these iron levels were likely not observed, as the medium was identical in the control and experimental conditions. In addition, this concentration of iron in minimal medium has been shown to induce, not repress, iron-responsive genes (20). It is well documented that the pyoverdine, pyochelin, and iron-regulated genes are important during P. aeruginosa murine lung infection, and this is in stark contrast to our observations of repression of these genes (2, 9, 10, 15, 18, 27-29, 33). P. aeruginosa during in vivo infection is likely subjected to the normal clearance mechanisms of cells such as macrophages and polymorphonuclear leukocytes, cells that are absent in our epithelial cell model. However, in the study conducted by Takase et al. (27), the siderophores were not required for lung infection of immunosuppressed mice. These authors suggest that non-siderophore-mediated iron acquisition, such as heme uptake, may play an important role in P. aeruginosa infections. We did not observe a change in expression of non-siderophore-mediated iron acquisition genes in our infection model.


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  		TABLE 4. Comparison of previously reported iron-regulated genes and genes repressed in P. aeruginosa after 4 and 12 h of infection on PNHAE cells

 
The present work evaluated the transcriptional profiles of P. aeruginosa after 4- and 12-h interactions with PNHAE cells in vitro. Global expression analysis revealed activation of phosphate and repression of iron acquisition genes. The number of genes showing these trends increased over time, suggesting that P. aeruginosa may be able to acquire ample iron but not phosphate for growth from the epithelial cells during infection. Further studies are warranted to explore the role of the genes involved in phosphate acquisition during epithelial cell interaction.


   ACKNOWLEDGMENTS
 
Microarray equipment and technical support were supplied by the GeneChip Bioinformatics Core at the Louisiana State University Health Sciences Center.

This work was supported by HEF (2000-05)-06 from the State of Louisiana-Board of Regents.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Program in Molecular Pathogenesis and Immunity, Louisiana Center for Lung Biology and Immunotherapy, Tulane University Health Sciences Center, New Orleans, LA 70112-2699. Phone: (504) 988-4607. Fax: (504) 588-5144. E-mail: mschurr{at}tulane.edu. Back

Editor: V. J. DiRita


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Infection and Immunity, September 2004, p. 5433-5438, Vol. 72, No. 9
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.9.5433-5438.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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