The leading cause of death in CF patients is chronic, recurrent pneumonia caused by Pseudomonas aeruginosa (P. aeruginosa). Little is known about the mechanisms by which P. aeruginosa causes lung damage during pneumonia; it likely comes both from chronic inflammation as well as from direct toxic effects of the bacteria on the epithelium that lines the airways.
In our lab at UCSF, we study the contribution of the bacteria to this process by using transposon mutagenesis to create mutants of P. aeruginosa deficient in causing acute lung injury (cytotoxicity). We identify and test these mutants in an in vitro system, developed by our collaborators at UCSF, Drs. Jeanine Wiener-Kronish and Keith Mostov. This in vitro system, which uses kidney epithelial cells grown on a filter, mimics the epithelial lining of the lung. When P. aeruginosa is added to these cells, the epithelial cells die in a manner which mimics acute lung injury in human P. aeruginosa pneumonia.
Our approach differs from previous approaches in several important aspects. First, we are screening for changes in cytotoxicity rather than for changes in adherence, eliminating the potential artifacts inherent in adhesion assays with P. aeruginosa and allowing us to potentially identify novel gene products that act at steps subsequent to adherence and result in cytotoxicity. Second, the screens are not based on testing previously identified virulence factors (genes that are thought to contribute to the disease state) allowing for the possibility of identifying new virulence factors. Third, we use isogenic strains, which differ at only a single genetic locus, so as to eliminate confounding changes at other genes. Fourth, the importance of these virulence factors in contributing to the early steps of acute lung injury during P. aeruginosa pneumonia can be measured quantitatively in a rat or mouse model of P. aeruginosa pneumonia that our collaborator, Dr. Wiener-Kronish, has developed.
Using a simple, reproducible, and inexpensive screen our lab developed, we have identified mutants of P. aeruginosa that are no longer able to kill epithelial cells. The epithelial cells are stained with a blue dye that is only taken up by dead cells; bacterial mutants of interest are those that no longer kill the epithelial cells, and thus the epithelial cells are no longer stained with the dye. We are characterizing these mutants in order to learn more about the mechanisms by which P. aeruginosa injures lung cells. First, the gene in which the transposon has inserted and is therefore nonfunctional, has been sequenced. From the DNA sequence analysis and implied protein sequence, we search the DNA and protein data bases to determine if related genes, whose functions are known, have been identified in other organisms. Already, we have found mutations in several genes of P. aeruginosa that resemble genes from other bacteria that are thought to contribute to disease. Notably, these new genes that we have identified have never before been described for P. aeruginosa. We are in the process of testing these mutants of P. aeruginosa in the animal model of acute pneumonia to see if they are deficient in causing pneumonia. In the future we will also test them in animal models of CF, as these become available. Understanding the mechanism of action and the role in lung injury of these P. aeruginosa virulence factors will result in the development of novel strategies to prevent or treat Pseudomonal lung infections in CF patients, thus improving their life span and quality of life.
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