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(Chest. 2002;121:36S-40S.)
© 2002 American College of Chest Physicians

Pseudomonas-Epithelial Cell Interactions Dissected With DNA Microarrays*

Stephen Lory, PhD and Jeffrey K. Ichikawa, PhD

* From the Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston MA.

Correspondence to: Stephen Lory, PhD, Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave, Boston, MA 02115; e-mail: stephen_lory{at}hms harvard.edu

Respiratory infections by the opportunistic pathogen Pseudomonas aeruginosa in immunocompromised patients or in patients with cystic fibrosis (CF) are the result of unique conditions that allow this Gram-negative bacterium to colonize the lung. This interaction between the pathogen and host leads to responses in both cells that ultimately lead to severe lung degeneration in individuals with CF. Recently, several new resources and technologies have become available to investigators, which should greatly accelerate our understanding of the mechanism of P aeruginosa pathogenesis, as well as of host defenses. Foremost among these are DNA microarrays, which allow transcriptional profiling of the partial or entire genetic repertoires of the pathogen and its host. A comprehensive compendium of expression profiles will serve in understanding the complex interplay between pathogen and host during respiratory infections.


   Use of Genomics in Infectious Disease
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The term systems biology describes the recent shift from studying single genes at a time to a more global study of whole genomes. This focus change has been made possible by the recent advent of a number of high-throughput methods and technologies, and has significantly influenced the understanding of the complex interactions between living cells, particularly as it applies to the interaction of bacteria with their hosts. Whole genome sequencing projects have provided the key resources for experimental and in silico analysis of genetic repertoires related to disease. As of June 2001, 51 complete microbial genomes have been published, with significant emphasis on pathogens (http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/micr.html). Another impressive advance is the rapid buildup of genome sequences of higher organisms, including that of the human genome.1 2 Thus, it is now possible to examine, in a truly global sense, the mechanisms employed by bacteria to cause disease in humans and at the same time identify pathways that are mobilized by the host to eliminate the infecting organism.

Infections of immunocompromised humans by pathogenic bacteria provide interesting models to study the mechanisms of defense and adaptation in both pathogen and host. When bacteria normally found in the environment encounter a mammalian host, they must adapt in order to survive against challenges such as the immune system and changes in the availability of certain nutrients. These adaptations include synthesis of a capsule to avoid phagocytosis, changes to the outer membrane protein profile such as phase variation, initiation of type III secretion systems to alter host signaling, and induction of systems required for uptake of limiting nutrients such as iron. These genome-wide changes are critical for invading pathogens to survive in a hostile environment.

P aeruginosa is a ubiquitous Gram-negative bacterium. It is a major nosocomial pathogen, infecting burn, cancer, and other compromised patients. One of the most prevalent infections occurs in individuals with CF, the most common inherited disease among whites. Although several different mutations can lead to CF, 70% of these patients have a three-base pair deletion in the CF transmembrane conductance regulator (CFTR) gene resulting in the disruption of the normal processing of the protein product. While the loss of CFTR function, which normally serves as a chloride channel in epithelial cells, can directly account for many symptoms and manifestations of CF, complications from the chronic colonization of the respiratory tract by particular opportunistic bacterial pathogens, predominantly P aeruginosa, is the single most important cause of morbidity and mortality in such patients. Several studies have provided clues to the basis for the susceptibility of individuals with mutant CFTR to bacterial colonization. Some intriguing results include the following: (1) impairment in the activity of bactericidal mechanisms in the respiratory tract due to the unique composition of the airway fluid,3 (2) utilization of CFTR as a receptor for bacterial internalization by epithelial cells,4 (3) altered signaling by epithelial cells to the host defenses following binding of bacteria,5 and (4) influence of bacterial signaling pathways as they grow in biofilms within the respiratory tract.6 These insights deepen our appreciation of the undoubtedly complex interplay of host and bacterial factors during the initial colonization process. However, these findings do not fully explain the basic paradox of this unusual respiratory disease: why do patients succumb to overwhelming colonization by a common environmental bacterium in spite of a functional immune system and a robust inflammatory response? We may be closer to answering this question by application of research tools that examine the global nature of host-pathogen interactions by monitoring the combined activities of the virulence factors produced by P aeruginosa and their impact on the different cell types that this pathogen encounters in the respiratory tract.


   Signalling Pathways in Respiratory Epithelial Cells Activated by P aeruginosa
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Early detection of infecting bacteria by the host is crucial for effective mobilization of innate and specific defense mechanisms. Inhalation of P aeruginosa by susceptible individuals brings the bacteria in contact with airway epithelial cells, which should signal these cells to mobilize the appropriate immune responses. The epithelial cells of the respiratory tract have traditionally been considered a passive barrier to infection, with their role in antimicrobial defense restricted to mucociliary clearance functions. There is growing evidence that epithelial cells contribute more significantly to host defense than previously appreciated. Normal epithelial cells can function as nonprofessional phagocytes and can serve in antigen-presentation to T cells.7 Additionally, they participate in the innate defense response by producing and responding to numerous cytokines.5 8 9 Finally, epithelial cells have a potential for direct killing of microorganisms by producing oxidants and antimicrobial peptides.10 11

The chronic respiratory disease of CF patients is characterized by extensive bacterial proliferation in the respiratory tract, and neutrophil-dominated inflammation.12 Increased concentrations of proinflammatory cytokines have been readily demonstrated in sputa of CF patients and in bronchial lavage fluids.13 Similarly, the levels of anti-inflammatory cytokine interleukin (IL)-10 were significantly reduced in CF patients compared to healthy control subjects.14 While these studies showed that alveolar macrophages are the likely source of the cytokines in the bronchoalveolar fluid, they have not ruled out the possibility that early events in CF colonization involve epithelial cell recognition of pathogens and their triggering in the cytokine synthesis. Two studies have further demonstrated that epithelial cells, in response to exposure to P aeruginosa, produce enhanced levels of IL-8. Massion et al15 reported that human transformed bronchial epithelial cells secrete IL-8 following stimulation by secreted P aeruginosa products. Furthermore, DiMango et al8 identified a number of cell-associated and secreted gene products of P aeruginosa, which are capable of inducing IL-8 synthesis from respiratory epithelial cells.

Interaction of P aeruginosa with epithelial cells can result in rapid cytotoxicity. Bacterially induced apoptosis has been demonstrated in a variety of cells, including epithelial cells, macrophages, and T lymphocytes.5 16 17 18 19 A variety of differentially regulated bacterial products have been implicated in this process. The environment of the respiratory tract may provide the necessary signals that control the expression of specific virulence factors. Furthermore, rapid response of the host to the presence of the bacterium may be needed to prevent widespread tissue destruction. This race between activation of pathogenic factors in the bacterium and immune responses in the host most likely involve many changes in the transcriptional profiles of each. Tools of functional genomics, specifically global expression profiling, should allow a comprehensive analysis of the flow of signals generated by bacterial-host interactions. These findings should allow us to understand the basis of an effective defense against P aeruginosa in healthy lungs and the mechanisms that are defective in individuals susceptible to infections by this pathogen.


   Contact-Dependent Activation of the Interferon Signalling Pathway by P aeruginosa
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P aeruginosa is capable of adhering to a variety of epithelial cells, which is believed to be a critical initial step in colonization of mucosal surfaces. Although a number of bacterial ligands mediate this interaction, the best characterized are the type IV pili.20 These bacterial organelles have been implicated in the enhancement of IL-8 production by cultured respiratory epithelial cells, by binding to asialo-GM1 ganglioside, and mobilization of nuclear factor-{kappa}B.21 Pilus-mediated signaling may be widespread among mucosal pathogens. For example, in cultured bladder epithelial cells, Escherichia coli induces synthesis of IL-6 and IL-8,22 and a renal carcinoma cell line responds to stimulation by E coli S-fimbria by synthesizing IL-6.23 The P-pilus–mediated attachment was specifically attributed to the activation of the ceramide signaling pathway24 and leads to enhanced neutrophil migration.25 Furthermore, in cervical epithelial cells, pathogenic Neisseria strains expressing type IV pili have been shown to increase [Ca2+] in through CD46 interactions.26

In order to investigate the host responses during initial contact, we carried out microarray analysis on A549 lung epithelial cells during a time course of exposure to P aeruginosa. RNA was extracted from A549 cells that were coincubated with P aeruginosa briefly or for 3 h. Each RNA sample was used to prepare complementary DNA probes, which were labeled with different fluorescently modified nucleotides during synthesis. These probes were combined and hybridized to the same microarray to determine the relative expression levels in the two samples. Approximately 25% of human genes represented on the array gave a detectable signal. Analysis of the bacterially responsive genes revealed that exposure of A549 cells to P aeruginosa resulted in alteration in messenger RNA levels of a number of signaling molecules and transcriptional factors (Fig 1 ). Most notably, the gene encoding interferon regulatory factor (IRF)-1 was activated approximately twofold in epithelial cells by P aeruginosa (Fig 2 ). The response was verified by Northern blot analysis and by reverse transcriptase-polymerase chain reaction (RT-PCR). The activation of IRF-1 was independent of serum components, such as lipopolysaccharide (LPS) binding protein and CD14, indicating that it did not involve LPS.



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  		Figure 1. Microarray analysis of signaling and transcription factor genes during exposure to P aeruginosa. The messenger RNA samples isolated from an infection time-course (3 h vs 0 h postinfection) were differentially labeled and pair-wise hybridized to a microarray. The microarray was scanned and the images were analyzed using custom software developed at the University of Washington.29 The false-color images of several hybridization spots and differential ratios corresponding to genes involved in signal transduction are shown. Those genes with increased hybridization signal in the 3-h sample are shown in red, those higher in the 0-h sample are green, and those with equal signal are yellow. The ratios are calculated as 3-h/0-h, such that induced genes have a ratio > 1. The relative error is shown in parentheses.

 


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  		Figure 2. Verification of microarray data by RT-PCR and Northern blot analysis. Top, panel A: Close-up image of the IRF-1 (arrow) containing region of a microarray is shown. The IRF-1 gene was induced 2.57-fold in the 3-h sample relative to the 0-h sample.29 Center, panel B: The microarray data were verified using RT-PCR. The messenger RNA was used in a reverse transcription reaction to produce complementary DNA. Different concentrations of the complementary DNA was then used in a polymerase chain reaction to amplify IRF-1. The polymerase chain reaction products were analyzed by gel electrophoresis, and the relative ratio from each sample was calculated. The gene for actin was also amplified as a normalization control. Bottom, panel C: Northern blot analysis was also used to verify the microarray data using a radiolabeled probe to either IRF-1 or actin.

 
In addition to IRF-1, interferon-activated signaling pathway leads to enhanced expression of several of genes that control multiple functions, ranging from antiviral response to cancer and development.27 28 However, no activation of genes previously shown to be interferon responsive were detected on the microarray (Fig 3 ), including the {gamma}-subunit of the p48 transcriptional regulator, interferon-stimulated gene factor 3-{gamma}. These findings suggest that the pathway of activation of IRF-1 by P aeruginosa is different from the normal signaling pathway initiated by the binding of interferon to their cognate receptors.



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  		Figure 3. Microarray analysis of interferon-regulated genes. All genes represented on the microarray that are regulated by interferon are shown. The ratios are calculated as described in Figure 1 . ISGF3-{gamma} = interferon-stimulated gene factor 3-{gamma}; ND = not detected.

 
In order to define the most likely bacterial ligand that is responsible for activation of IRF-1 in respiratory epithelial cells exposed to P aeruginosa, we examined the levels of IRF-1 messenger RNA in cells exposed to wild-type PAK and an isogenic mutant P aeruginosa strain PAK-NP. The mutation in this strain is the disruption of the pilA gene, encoding the major subunit of the type IV pili, which serves as an adhesin to epithelial cell. Higher IRF-1 messenger RNA levels were detected in those A549 cells that were exposed to adhering P aeruginosa. Furthermore, purified LPS from P aeruginosa did not increase the level of IRF-1 messenger RNA, suggesting that the adhesin-mediated signaling is due to the interaction of type IV pili with the receptor on A549 cells, and not merely enhancing the stimulatory activity of LPS by anchoring bacteria to the epithelial cell.29

Application of microarray technology to study the response of human cells to infecting microorganisms promises to be an important tool for the discovery of new pathways that operate in disease. The key to understanding bacterial respiratory infections is to elucidate the highly effective defense mechanisms in the healthy lung and apply this information to the disease state. Comprehensive transcriptional profiling can be exploited in two ways. First, using infection model systems, such as cell or organ cultures, new signaling pathways can be discovered that may include known and previously uncharacterized host factors. Second, an investigator using a combination of microarray analysis and various bacterial pathogens carrying mutations in genes encoding virulence factors should be able to characterize the role of individual virulence factors in pathogen recognition and subsequent mobilization of host responses. The transcriptional profiles of both host and pathogen can be combined to provide a truly comprehensive picture of communication during this intricate interaction. The use of microarray technology will therefore remain among the workhorse tools of research in bacterial pathogenesis.


   Footnotes
 
Abbreviations: CF = cystic fibrosis; CFTR = cystic fibrosis transmembrane conductance regulator; IL = interleukin; IRF = interferon regulatory factor; LPS = lipopolysaccharide; RT-PCR = reverse transcriptase-polymerase chain reaction

Supported by a research grant from the Cystic Fibrosis Foundation.

Dr. Ichikawa is a Cystic Fibrosis Foundation Fellow.


   References
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 Contact-Dependent Activation of...
 References
 

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