Host-Pathogen Interactions: Pseudomonas and Cystic Fibrosis

Principal Investigator: Fred Ausubel

Overview

Pseudomonas aeruginosa, a ubiquitous Gram-negative bacterium isolated from soil, water, and plants (1), is an opportunistic pathogen that infects cystic fibrosis patients, patients with thermal burns and patients who are immunodeficient or otherwise compromised. The Ausubel laboratory has developed a novel multi-host pathogenesis system that uses non-vertebrate hosts as an adjunct to mammalian models to identify and study P. aeruginosa virulence-related genes as well as host genes that are involved in the defense response to pathogen attack (2-7). Remarkably, among 20 P. aeruginosa strain PA14 genes that are required for pathogenesis in at least one of three different invertebrate hosts (a plant, a nematode and an insect), 17 were also required for full pathogenicity in a mouse burn model. This project concerns the use of genomic approaches to extend this multi-host pathogenesis model to the pathogenic processes that occur during the pulmonary infection of mice that contain a knock-out mutation in the CFTR gene, the gene which is mutated in human cystic fibrosis (CF) patients.

The first genomic approach, involves the analysis of the PA14 genome for candidate virulence genes absent in less-pathogenic strains of P. aeruginosa, as well as the generation of a non-redundant library of P. aeruginosa mutants that contain a transposon insertion in each of the estimated 4,800 non-essential genes. This library will greatly facilitate the identification of P. aeruginosa virulence factors that play a role in the colonization of the lungs of CF patients. The second genomic approach involves the use of DNA microarray analysis to identify both P. aeruginosa and mouse genes that are specifically activated or repressed during pulmonary infection of CF mice. We predict that these latter experiments will lead to the identification of mouse genes that are involved in the innate immune response to P. aeruginosa colonization.

Project Goals:

1. Generate enhancements/refinements to the P. aeruginosa PA14 non-redundant mutation library and related resources including the PA14 genome sequence.
   1. Annotate the PA14 genome and transfer the annotated genome data to PseudoCAP (Pseudomonas Community Annotation Project).
   2. Identify and mutate 300-400 non-essential genes estimated to be missing from the PA14 library.
   3. Distribute the non-redundant PA14 mutant library to the public and transfer the responsibility for distributing the library to the Pseudomonas FLEX Gene Repository at Harvard Medical School.
2. Design and manufacture a multi-purpose spotted P. aeruginosa microarray for transcriptional profiling, transposon site hybridization (TraSH), and phylogenetic studies, and make it available to the public.
3. Identify P. aeruginosa pathogenicity-related genes, construct non-polar deletions of these genes, test them in mouse infection models, and make the resulting collection of mutants available to the public.
   1. Identify putative PA14 pathogenicity-related genes by transcriptional profiling and Trash analysis.
   2. Prioritize putative PA14 pathogenicity-related mutants for further characterization.
   3. Test putative PA14 pathogenicity-related genes for virulence-related phenotypes.
   4. Correlate phenotypes of PA14 mutants with a transposon insertion in a particular ORF.
   5. Construct non-polar in-frame deletions corresponding to selected mutants identified in Aims 3a-3d.
   6. Test mutants from Aim 3e in murine CF oropharyngeal and GI colonization models.
4. Expand the public PA14 database so that it coordinates three genomic resources: the annotated PA14 sequence, the PA14 mutant library, and the spotted oligonucleotide array. Transfer the responsibility of maintaining the database to PseudoCAP.

References:

1. Palleronic, J. N. 1984. Family I. Pseudomonadaceae. In Bergey's Manuel of systemative bacteriology. Bergey, ed, p. 141-172.
2. Rahme, L. G., E. J. Stevens, S. F. Wolfort, J. Shao, R. G. Tompkins, and F. M. Ausubel. 1995. Common virulence factors for bacterial pathogenicity in plants and animals [see comments]. Science 268:1899-902.
3. Rahme, L. G., M. W. Tan, L. Le, S. M. Wong, R. G. Tompkins, S. B. Calderwood, and F. M. Ausubel. 1997. Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors. Proc Natl Acad Sci U S A 94:13245-50.
4. Mahajan-Miklos, S., M. W. Tan, L. G. Rahme, and F. M. Ausubel. 1999. Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosa-Caenorhabditis elegans pathogenesis model. Cell 96:47-56.
5. Tan, M. W., S. Mahajan-Miklos, and F. M. Ausubel. 1999. Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis. Proc Natl Acad Sci U S A 96:715-20.
6. Tan, M. W., L. G. Rahme, J. A. Sternberg, R. G. Tompkins, and F. M. Ausubel. 1999. Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors. Proc Natl Acad Sci U S A 96:2408-13.
7. Jander, g., L. G. Rahme, and F. M. Ausubel. 2000 (submitted). Positive Correlation between Virulence in Pseudomonas aeruginosa mutants in mice and insects.

Learn More

To learn more about the Host-Pathogen Interactions: Pseudomonas and Cystic Fibrosis project, visit the Ausubel Lab Web Site.

For more information on specific sub-sections of this project, visit the following pages:

1. PA14 Genomic Sequencing Project
2. PA14 Mutant Library
3. CF Animal Models

For available resources pertaining to this project, visit the Bacterial Pathogen Resources Page.